article
stringlengths 0
682k
| abstract
stringlengths 146
3.69k
|
|---|---|
primary sjgren 's syndrome (pss), also known as autoimmune epithelitis, is a common chronic autoimmune disease characterised by the inflammation of exocrine glands and the clinical signs of xerostomia and keratoconjunctivitis sicca. a combination of environmental, genetic, and possibly hormonal factors leads to the dysregulation of the glandular epithelium, mononuclear cell infiltration, and abnormal lymphocyte activation and proliferation [1, 2 ]. aberrant humoral autoimmune responses, b cell hyperactivity, and autoantibody production are the hallmarks of pss [35 ]. follicular helper t (tfh) cells are specialized subsets of effector t cells that provide essential help to antigen specific b cells in the secondary lymphoid organs. tfh cells are originated from naive cd4 t cells which are activated by dendritic cells (dcs) in the interfollicular or t cell zones [6, 7 ]. as a result of the initial interaction with dcs, primed cd4 t cells migrate to the border of t and b cell areas and become pre - tfh cells. this follicular homing process is directed by bcl-6, which coordinates the downregulation of t cell zone homing c - c chemokine receptor type 7 (ccr7) in parallel with the upregulation of b cell region homing c - x - c chemokine receptor 5 (cxcr5) [812 ]. at the border of t and b cell areas, the interaction between pre - tfh cells and activated b cells is crucial for both the generation of antibody - producing extrafollicular plasmablasts and the formation of germinal centers (gcs). in order to enter gcs, pre - tfh cells require mutual signals from activated b cells via cd28-cd86, icos - icosl, cd40l - cd40, programmed cell death protein-1 (pd-1)-pd-1l, and ox40-ox40l as well as signaling lymphocytic activation molecule (slam) family members [1317 ]. in gcs, the interplay between tfh and gc b cells is bidirectional ; survival signals, completed with interleukin (il)-21, are important not only for b cell survival, proliferation, and differentiation but also for the maturation of tfh cells [18, 19 ]. the upregulation of bcl-6 in activated gc b cells supports their survival and extremely high proliferation rate and additionally leads to the activation - induced cytidine deaminase (aid) mediated somatic hypermutation (shm) in the dark zone of gcs. through the subsequent stimulation of cd40 by tfh cells, centroblasts differentiate into centrocytes and follicular dcs (fdcs) and tfh cells promote the positive selection and possible immunoglobulin class - switch recombination (csr) of several centrocytes resulting in their differentiation into high - affinity memory b cells and long - lived plasma cells. recent studies highlighted the role of tfh cells in the pathogenesis of different autoimmune conditions, including systemic lupus erythematosus, sjgren 's syndrome, rheumatoid arthritis, juvenile dermatomyositis, myasthenia gravis, and autoimmune thyroid disorders [2328 ]. in our previous work, we demonstrated elevated circulating tfh cell percentages in pss and revealed the importance of this cell type in the pathogenesis of the disease. despite the increased research activity in this field, the molecular mechanisms and the function of tfh cells are still not known in detail. in order to extend the current knowledge, in the present study we focused on the site of the inflammation and assessed the composition of lymphocyte infiltration in labial salivary gland (lsg) biopsies with a special emphasis on the presence and potential importance of tfh cells at the time of disease onset. in the present study, we enrolled ten female patients (mean age sd : 57.2 11.4) with pss, who had been diagnosed and followed up regularly in the outpatient clinic for systemic autoimmune diseases at the division of clinical immunology, university of debrecen. the diagnosis of pss was established according to the european - american consensus group criteria (aecg). the diagnosis of the patients was confirmed with positive lsg biopsy at the disease onset. none of them had evidence of malignant lymphoma or showed egms at the time of the pathological sampling. three individuals, who complained of only mild sicca symptoms without fulfilling diagnostic criteria, served as controls for the histological evaluation. all patients underwent extensive clinical and serological examinations during the follow - up. data were obtained retrospectively from their records which contained detailed information on symptoms, physical conditions, and laboratory and other findings. anti - ssa / ro and anti - ssb / la autoantibodies were determined by elisa technique with autostat ii kits (hycor biomedical, indianapolis, in, usa) according to the manufacturer 's instructions. the titers of serum immunoglobulin (ig)g, iga, and igm were analyzed by turbidimetric immunoassay (dialab gmbh, neudorf, austria). at the end of the follow - up, circulating tfh - like cells were determined by cd4, cxcr5, icos, and pd-1 cell surface molecules and were assessed using bd facs calibur flow cytometer (becton dickinson, franklin lakes, nj), as described previously. informed written consent was given by patients for their clinical records and archived biopsy samples to be used in this investigation. the study has been approved by the local ethics committees (debrecen, hungary) in 2012 (reference number : ix - r-052/00016 - 22/2012.). formalin - fixed, paraffin - embedded (ffpe) tissue blocks were obtained from the archives of the department of pathology, university of debrecen, which had been previously collected for routine diagnostic purposes in years 20012010. four-m thick serial sections of lsg tissue specimens were prepared and stained with haematoxylin - eosin (he) for conventional histopathological examination. the determination of focus score (fs) was based on the degree of lymphocytic infiltration in the whole biopsy. the focus score was defined as the group of inflammatory cell aggregates containing at least 50 mononuclear cells per 4 mm of tissue area. it was classified as fs = 0 : no lymphocytic infiltration ; fs = 1 : less than 1 lymphocytic focus per 4 mm ; fs = 2 : less than 2 lymphocytic foci per 4 mm ; fs = 3 : two or more lymphocytic foci per 4 mm. immunohistochemical (ihc) staining was performed on serial sections of tissue blocks using standard methods. briefly, 4 m thick ffpe sections were deparaffinized, rehydrated on descending ethanol dilutions, and treated with 3% h2o2 to block endogenous peroxidase. for antigen retrieval, sections were heated in boiling citrate buffer (ph 6.0) or tris / edta buffer (ph 9.0) for 3 min using a pressure cooker. after cooling, the slides were incubated with primary antibodies for 1 hour at room temperature. the following monoclonal antibodies were (mab) used in the procedure : cd4, clone 1f6 mouse mab (novocastra, leica biosystems, nussloch, germany) ; cd5, clone 4c7 mouse mab (novocastra) ; cd20, clone l26 mouse mab (dako, glostrup, denmark) ; cd84, clone epr8325 rabbit mab (abcam, cambridge, uk) ; cd138, clone mi15 mouse mab (dako) ; pd-1, clone nat mouse mab (abcam) ; bcl-6, clone pg - b6p mouse mab (dako). biotin - free envision / hrp (dako) system as secondary ab with very intense purple (vip) peroxidase substrate (vector laboratories, peterborough, uk) was used for detection. the stained tissue samples were digitalized using pannoramic midi digital slide - scanner (3d - histech co., budapest, hungary) utilizing zeiss plan - apochromat objective (magnification : 20x/0.8 numerical aperture) and hitachi (hv - f22cl) 3ccd progressive scan color camera (resolution : 0.2325 m / pixel). if applicable, at least 4 (ranging from 2 to 6) lymphocytic foci were selected randomly in each specimen per patient for analytic measurements and photodocumentation. field area (fa ; overall field area in mm) and mask area (ma ; overall mask area in mm) were computed by the software. the fa represents the whole area of the marked infiltrates, while the ma indicates the cell - specific marker positive area. the relative ma (rma) values were calculated as ma / fa multiplied by 100. the organizational levels of each lymphocytic infiltrate were graded by ihc staining of serial sections using cd4 and cd20 cell markers. a small number of distributed perivascular and intraepithelial lymphocytes were graded as (1) ; mild lymphocytic aggregates without clear organization of separate t and b cell zones were defined as grade (2) ; more organized lymphoid follicles were classified as grade (3) ; aggregates with the highest level of arrangement, which displayed distinct t and b cell regions, were graded as (4). the latter organization was also characterised by an extensive fdc network detected with cd21 marker in the center of the lymphoid aggregates, whose pattern corresponded to ectopic gc structures. double immunofluorescence (if) staining for bcl-6 in combination with cd3 (clone ln10, mmab, novocastra) or cd20 was carried out with sequential immunostaining, as described previously. after 1-hour treatment with anti - bcl-6 primary ab, the slides were incubated using anti - mouse igg(fab)2 as secondary ab coupled to polymer - hrp (dako), followed by a tetramethylrhodamine- (tmr-) conjugated tyramide reagent of the fluorescent amplification kit (tsr - tmr system, perkin elmer life science, boston, ma, usa) to visualize the red nuclear fluorescence. the second layer of the double if staining was applied with anti - cd3 or anti - cd20 primary abs plus biotinylated anti - mouse secondary igg f(ab')2 followed by streptavidin - fluorescein isothiocyanate (fitc). images were obtained using a zeiss axio imager z2 microscope (carl zeiss microscopy gmbh, jena, germany) equipped with the following objectives : 10x/0.3 na ; 20x/0.5 na. for transferring and editing images, isis software (metasystems group inc., newton, ma, usa) and adobe photoshop cs5 version 12.0 were used. the mean age at the time of the diagnosis was 50.80 10.34 and the total duration of follow - up was 7.40 3.10 years. we evaluated their clinical and serological features retrospectively and assessed the relation between laboratory results, disease course, and the early histopathological findings. three patients formed the group of pss with fs = 2 and 7 patients belonged to the group of pss with fs = 3. peripheral tfh - like cell percentages were tendentiously elevated at the end of follow - up in patients with higher fs at disease onset (mean percentages of pss with fs = 3 versus controls : 0.86% 0.38 versus 0.32% 0.12, and pss with fs = 3 versus pss with fs = 2 : 0.86% 0.38 versus 0.33% 0.08, resp.). importantly, systemic features such as polyarthritis (n = 3), raynaud 's syndrome (n = 2), lymphadenopathy (n = 1) and fibrosis pulmonum (n = 1), and associated diseases including primary biliary cirrhosis (n = 1) or primary sclerosing cholangitis (n = 1) developed later in the disease course only in patients with fs = 3. when studying the morphology of lsg specimens in patients with pss, we identified different organizational levels of inflammatory mononuclear cell infiltrates. the whole lsg specimen was characterized based on the fs, while the extension and the structural arrangement level of each periductal cellular infiltrate were graded within the biopsy section. as displayed in figure 1(a), four distinct categories could be identified. in our study, the biopsy samples with fs = 2 consisted of lymphocytic aggregates only graded as 1 or 2. more organized follicles as grade 3 or 4 were exclusively found in pss with fs = 3. figure 1(b) presents the distributions of the four distinguished levels of cellular arrangement in the two groups of patients. in many cases, biopsy specimens included cellular aggregates of different kinds of grades, particularly in higher organizational levels. in the biopsy samples of patients with fs = 2 in pss with fs = 3, the infiltrations were extensive and penetrated the ductal epithelia with occasional destruction of the acini. furthermore, three patients from pss group with fs = 3 also had ectopic gc formation in lsg samples. serial immunostainings for the incidence and densities of inflammatory cell - specific markers within the infiltrates of the subgroups are demonstrated in figure 2. as shown, cell surface markers including cd4, cd5, cd20, cd138, cd84, and pd-1 were found in both groups, albeit in different arrangements and densities. in the aggregates of pss group with fs = 2, mainly the t helper cell marker cd4, the pan - t cell and b1 cell marker cd5, and the pan - b cell marker cd20 were detected, while the tfh - related markers cd84 and pd-1 were less evident. the cd138 plasma cells were dispersed throughout the whole lsg samples and found mostly outside the aggregates. in pss group with fs = 3, the distribution of specific cell markers showed a different pattern along with more organized structures. cd5 were detected mainly in the t cell regions at the periphery of mononuclear cell infiltrates and only a few cells in the b cell area were positive for it. similar to pss group with fs = 2, cd138 plasma cells were also displayed as a scattered distribution outside the infiltrates ; however, some of them were observed at the border of b cell zone as well. the expression of cd84 cell surface molecule was diffused throughout the inflammatory infiltrate but accumulated at the inner area. in addition, the expression of pd-1 was solely found in the location of cd20 b cells. after analysing the pss group with fs = 3, intragroup variances were discovered ; at grade 4 organization level bcl-6 cells were clustered in the central region and expressed with higher intensity, while in grade 3 aggregates bcl-6 cells were scattered and showed much lower expression (data not shown). digitalized slide imaging allowed us to make numerical comparisons for marker expressions between the two groups. 117 slides were digitalized in total, and the studied proteins were analyzed in randomly selected lymphocytic aggregates. the average size of the aggregates in pss with fs = 3 was larger than those in pss with fs = 2 [0.3114 mm (0.0950.642) versus 0.1927 mm (0.0580.566), resp. ]. as shown in figure 3, distribution of the expression of cell - specific molecules varied according to the focus scores of biopsy samples. the expression of markers which participate in the tfh - b cell interaction were tendentiously higher in pss with fs = 3. the last question of this study was whether bcl-6 expression was limited to cd20 b cell infiltrates of lsg or whether it could be demonstrated in cd3 t cells as well. to prove that cd3bcl-6 t cells were involved in the formation of gc - like structures in lsg, we stained sections by double if for bcl-6 and cd3 or cd20 expressions. figure 4(a) shows the double labeling of cd3 pan - t cell marker with the transcription factor bcl-6 in lesional lymphocytes, indicating that a few t cells in the infiltrates were positive for bcl-6. bcl-6 b cells with the typical formation of conventional gcs have also been detected in the central area of the lymphoid follicle demonstrated in figure 4(b). obtaining lsg biopsy is part of the routine diagnosis procedures in pss according to the aecg, and it provides an excellent opportunity to reveal the severity of autoimmune inflammatory processes in the early stage of the disease [33, 34 ]. previous studies revealed the presence of t and b cells with fewer macrophages and dcs in lsg of pss patients [3537 ]. the distribution of b cells, dcs, and fdcs correlates positively with the severity of inflammation. additionally, foxp3 cells and il-17 and il-21-producing cells were also detected in the infiltrates of lsg tissues [3941 ]. in a recent study, kang. demonstrated the coexpression of il-21 and cxcr5 in lsg infiltrates which raised the question about the presence of tfh cells. maehara. focused on infiltrating t lymphocyte subsets and described that the expression of t helper 2 and certain tfh - related molecules was associated with robust lymphocytic accumulation and ectopic gc formation. moreover, gong. recently demonstrated the ability of epithelial cells to induce the differentiation of tfh cells in salivary glands. however, before our present investigations, tfh cells were not studied in glandular lymphocytic infiltrates with different organizational levels. in our study, we classified lsg specimens according to the severity of inflammatory cell infiltrates not only with focus scoring but also with grading of the lymphoid aggregates. to determine the fs and the grades of aggregates, we examined the entire tissue section. we observed that the biopsy samples contained different grades of mononuclear cell infiltrates, and the periductal lymphoid structures showed a higher level of organization in pss group with fs = 3 than in pss group with fs = 2. ectopic gc structures with peripheral positioned t cells, centrally localized b cell area, and a reticular pattern of fdc network were only observed in fs = 3 with grade 4 aggregates. when examining the expression of tfh - related molecules, such as cd84, pd-1, and bcl-6 in the infiltrates, we found a pronounced expression in pss with fs = 3. cd84, which is a member of slam family, is responsible for the maintenance of stable t - b conjugates to achieve a complete interaction and helper function by tfh cells. pd-1 receptor, which regulates the selection and survival of b cells in the gcs, is also an important phenotypic determinant of tfh cells. marked bcl-6 expression was detected only in grade 4 aggregates with the colocalization of b cell zone. in grade 3 infiltrates, its expression was significantly weaker. it is known that bcl-6 is specially expressed by gc b cells during the centroblast phase and usually, but not consistently, in centrocytes as well. according to experimental studies, bcl6 gene defect resulted in disturbed gcs formation, with the lack of shm and csr, which highlights the role of bcl-6 in gc responses. human studies also demonstrated the requirement of bcl-6 in the establishment of gcs and found that, in contrast with aggregates, only real ectopic gcs express detectable amount of bcl-6 [47, 48 ]. for that purpose, we paid a special attention to the presence and localization of tfh and gc b cells in the mononuclear cell infiltration. with double if staining, we demonstrated that, close to b cell area, a certain subset of infiltrating t cells expressed both cd3 and bcl-6 markers, which suggests that the presence of tfh cells was adjacent to gc b cells in lsg lesions. however, real gc - like structures with tfh cells were merely found in those lymphoid follicles that belong to pss group with fs = 3 and showed more severe inflammatory lesions. our findings are in correlation with a previous study which revealed the presence of aid in lymphocytic aggregates with higher organizational level in pss patients. aid is expressed in gc b cells undergoing shm and csr, following the upregulation of bcl-6. we summarized the possible role of tfh cells in lymphoid aggregates in the labial salivary glands of pss patients in a graphical figure (figure 5). it is important to emphasize that our investigations were performed on lsg biopsies which were collected at the time of the diagnosis, when only the initial symptoms developed in patients. the retrospective evaluation, of both laboratory and clinical data recorded during the follow - up period, revealed associations between the formation of gcs with the presence of tfh cells in lsgs at disease onset and the development of egms and associated diseases during the disease course. additionally, patients, who have tfh cells in their salivary gland infiltrations already at the time of diagnosis, seem to also have an elevated peripheral tfh cell ratio later in the disease course. it must be considered that the limitation of the present study is related to its small patient sample ; thus, the correlations between the local presence of tfh cells and the development of systemic clinical features can not be justified statistically. nevertheless, the present findings are in line with our earlier observations that the higher proportions of tfh cells are associated with higher fs in glandular biopsies and the presence of extraglandular manifestations. in the present study we demonstrated that tfh cell markers, including cd84, pd-1, and bcl-6, occur predominantly in more organized inflammatory cell infiltrates developed in lsgs with higher focus scores. our results indicate that the presence of tfh cells in lsgs at the time of disease onset may predict a more pronounced clinical course of pss ; nevertheless, this observation should be confirmed in a larger patient population as well. we expect that the further understanding of molecular and cellular regulation of tfh cells will provide new potential therapeutic targets in the treatment of pss patients with systemic manifestations. | recently, we revealed the importance of follicular helper t cells (tfh) in the pathogenesis of primary sjgren 's syndrome (pss). in the present study, we focused on the site of the inflammation and determined the composition of lymphocyte infiltration in labial salivary gland (lsg) biopsies with special emphasis on tfh and germinal center b cells. we selected tissue blocks obtained from ten patients at the time of disease onset. detection of cell specific markers was performed with immunohistochemical and immunofluorescence stainings. we evaluated patients ' clinical and laboratory features retrospectively and assessed the relation between disease course and early histopathological findings. lsg biopsies were graded based on the extension and arrangement level of periductal inflammatory cell infiltrates. tfh cell markers (cd84, pd-1, and bcl-6) occurred predominantly in more organized structures with higher focus scores. the coexpression of cd3 and bcl-6 markers clearly identified tfh cells close to bcl-6 + b cells with the typical formation of germinal centers. systemic features were developed later in the disease course only in patients with highly structured infiltrates and the presence of tfh cells. our observations suggest that the presence of tfh cells in lsgs at the disease onset may predict a more pronounced clinical course of pss. |
peptide mass spectrometry provides a powerful method to analyze proteome expression in cell lysates. at the core of this method, experimental mass spectra of fragmented peptides the accuracy of peptide matching depends critically on the effectiveness of algorithms that match the theoretical and experimental spectra. we present here a method to evaluate algorithms to obtain high - confidence interrogation of proteomes. in typical experiments, proteins in cell lysates are digested with an endoprotease, typically trypsin, to obtain peptides in size ranges that can be successfully analyzed by tandem mass spectrometry (ms / ms). trypsin fragments with pronounced peaks in the first ms are selected for collision induced dissociation (cid) in the second ms. cid causes fragmentation of the trypsin peptides, typically at amide bonds, producing n - terminal b ions and c - terminal y ions that give rise to a set of detected mass to charge (m / z) peaks. peptide - searching algorithms compare experimental m / z spectra with theoretical ion ladders derived from tryptic fragments of an input sequence database of all proteins in the proteome. each spectrum - matching algorithm, however, is different in its design and structure. the best peptide - spectrum matches are determined by techniques such as cross - correlation (e.g., sequest) or by model - based approaches using statistical significance (omssa, mascot). each algorithm has multiple parameter settings, including mass tolerances between theoretical and observed trypsin fragments (precursor mass tolerance) or theoretical and cid fragments (fragment mass tolerance) ; which and how many optional mass modifications to allow per peptide ; and which cid ion series (e.g., a, b, y) to assess. it is important to choose appropriate parameter settings of algorithms for accurate peptide identifications. given the many combinations of choices, what are good strategies to determine parameter settings ? do different parameter settings of algorithms applied to a given data set provide different samplings of the proteome, and are these samplings of high quality ? the effectiveness of an algorithm is typically assessed through decoy analysis. for each forward protein sequence in the sequence database, the algorithm is also presented with the reverse of that sequence. the forward and reverse sequences are processed blindly together, including a theoretical trypsin digestion, and the frequency that decoy reverse peptides are chosen by the algorithm indicates the false discovery rate (fdr) for that particular choice of parameter settings. given that the algorithms output quality scores for each peptide - spectrum match (psm), scoring thresholds can be computed and used to filter the output data to ensure an fdr below a desired level (e.g., 1% or 5%). decoy analysis is an excellent strategy to assess algorithm parameter settings, especially since the approach is inherently independent of any particular algorithm. we wished to develop additional approaches to assess algorithm performance : methods to be used alongside decoy analysis in order to build confidence in peptide matches by different standardized parameter settings of algorithms. expanding on a protocol suggested by park., we present a strategy that evaluates peptide - spectrum matches by assessing the masses of parent proteins prior to trypsin digestion, an approach that can be applied to any algorithm using the spectra sets presented here (supporting information) or new data sets if desired. this parent - protein profiling approach uses a gel slice strategy to partition cell lysates according to parent - protein mass. application of this approach suggests that different algorithms can provide different, yet valid, samplings of the proteome, and that it can also be extremely productive to run algorithms multiple times with different parameter settings, an approach that is becoming increasingly possible given the availability of increased computational power. we first focus our discussion on the sequest and omssa algorithms and then present equivalent analysis of the mascot algorithm, which revealed similar results. conformance to parent proteins before digestion with trypsin was used to assess algorithm matches of spectra to tryptic peptides. yeast cell lysates were partitioned into gel slices of known molecular weight size ranges (2537, 3750, and 5075 kda). although the algorithms had no knowledge of the parent - protein sizes before trypsin digestion, peptide matches would be expected to conform to the correct parent - protein size ranges if the algorithm was matching successfully. this has been shown previously in ms / ms - based gel - band analysis of the proteome of pseudomonas putida bacteria. protein samples were prepared as follows : 100 ml of ysh474 cells were grown to mid - log phase in ypd and lysed with ripa buffer (150 mm nacl, 1% igepal, 0.1% sds, 50 mm tris ph 8.0), and acid - washed glass beads. to prevent degradation, protease inhibitors (roche) and pmsf were added, and samples were chilled on ice during lysis. the lysate was spun at 5,000 rpm, and samples of 500 or 1,000 g were run alongside protein standard markers (bio - rad) on 420% sds - page gels (bio - rad). protein standard bands served as a guide for the excision of gel slices of various molecular weight size ranges (2537, 3750, and 5075 kda ; figure 1). samples were subjected to reduction and alkylation followed by overnight in - gel trypsin digestion. extracted peptides were resuspended in 0.1% tfa, loaded onto a c18 packed (michrom) nanospray column (polymicro), and run with a 180-min gradient on a lcq deca xp (thermo - scientific) coupled to a high - performance liquid chromatography (hplc) system (agilent 1100 series) and a nanoelectrospray (nano - esi) ion source. preliminary tests indicated that gels with visible degradation had limited conformance to parent - protein masses. hence, gels were discarded and not analyzed if their appearance suggested visible degradation. peptide matches were identified using the sequest algorithm (proteome discoverer v.1.2) run on a dell alienware aurora r4 server, the open mass spectrometry search algorithm (omssa) run on a 90-node cluster, and the mascot algorithm run on a dell xps 8300 server. algorithm parameters were set up to search for either the standard b and y ions following cid or with the addition of a ions. optional mass increases to peptides included dynamic modifications of + 42 da for acetylation of any n - terminal amino acid residue and + 16 da for oxidation of methionine residues, and static modifications included + 57 da for carbamidomethyl modification of cysteine residues. the sequest algorithm parameter file included a precursor mass tolerance of 3.0 da and a fragment mass tolerance of 1.0 da, while the omssa algorithm was run using five different standard sets of parameters (table 1) and mascot was run using four parameter sets (figure 6a) ; the sequest parameter set is similar to that reported for peptideatlas yeast data (http://www.peptideatlas.org/) ; the omssa and mascot parameter sets are similar to the algorithm default parameters. precursor peptides for liquid chromatography ms / ms (lc ms / ms) analysis were prepared by trypsin digestion, which cuts after arginine or lysine, except when flanked by proline. for the sequest, omssa, and mascot analysis, we required trypsin - cleavage sites at both ends of the precursor peptides (or one end if a terminal peptide). a sequence database file containing protein translations of annotated and downstream open reading frames (dnorfs) in fasta format was constructed as described previously. output data from the three algorithms were uploaded to a relational database and analyzed with stored procedures written in ms - sql to compute decoy false discovery rates (fdrs) and parent - protein conformance scores. reverse - sequence decoy analysis was performed as described previously, and peptide matches were filtered to give a target fdr of 5%. before computing decoy score thresholds, for each lc ms / ms run, we excluded matches with internal trypsin sites and matches with an initial ranking (rank) > 1 if a sequest or mascot matched peptide. the decoy score thresholds were then applied to the output data after first excluding omssa matches (which are not ranked) where multiple nondecoy peptides matched to the same spectrum. as discussed in section 3.2 below, the peptide matching by omssa is stringent, and the false detection rate was typically below 5% after application of these filters (1.65.1% depending on parameter settings and cid ions assessed). for all three algorithms, we also excluded peptides that mapped to multiple parent proteins ; although these were likely correct identifications, these peptides were excluded because they could not be assigned to unique parent proteins. parent - protein conformance scores were computed from forward peptide matches classified as conforming or nonconforming peptides according to the known molecular weight size range of the gel slice (2537, 3750, and 5075 kda). ms / ms run, even if detected by multiple spectra. to account for aberrant protein travel through the gel or possible post - translational modifications, mass tolerances of 10% of the molecular weight size range for example, peptide matches from the 2537 kda gel slice range were categorized as conforming if the parent proteins were between 22.5 and 40.7 kda. conformance scores for individual lc ms / ms runs were computed as follows : an overall conformance score (for a single set of parameter settings for the algorithm) was computed by summing the number of conforming peptides and nonconforming peptides across all 22 lc ms / ms runs counting each matched peptide a maximum of once per run : peptide - spectrum matching algorithms score individual peptide matches according to how well the masses of expected cid fragments of a tryptic peptide match the m / z peaks in a detected spectrum. the approach is based on the fact that algorithms have no knowledge of the masses of parent proteins prior to trypsin digestion. by partitioning parent proteins into known molecular weight size ranges (2537, 3750, and 5075 kda) using sds - page and processing individual gel slices for assessment through lc ms / ms (figure 1), we investigated whether peptide matches by algorithms conformed to the expected parent - protein size range. allowing a mass tolerance of 10% to compensate for experimental variations inherent in the gel slice approach, we computed conformance scores indicative of the effectiveness of an algorithm parameter set (table 2a.). fdr threshold values were (a) sequest : 0.081 ; omssa parameter sets 0 to 4 : 1.0. (b) sequest : 0.164025 ; omssa parameter sets 0, 1, 3, 4 : 1.0 ; omssa parameter set 2 : 0.9. a total of 2,842 distinct peptides were detected when counted only once regardless of which algorithm, parameter set, ion screen, or gel - slice range. of these, 10.5% (298 distinct peptides) were detected in more than one gel - slice range, and 5.9% (168 distinct peptides) were scored as both conforming and nonconforming depending on size range. we ran individual lc ms / ms runs for each of the 22 gel slices and assessed spectra from the runs using a standard parameter set for the sequest algorithm and five different parameter sets for the omssa algorithm (table 1). after employing decoy analysis to ensure false discovery rates (fdrs) below 5%, we used the convention that even if a peptide were detected with multiple spectra, each peptide was counted only once per lc (the same peptide was counted more than once if detected in multiple runs, which in some cases were from gel slices with different size ranges.) in a standard b / y ion screen, 84.4% of the 4,480 peptides detected by sequest had parent proteins conforming to the expected size range. using the same spectra, omssa detected between 2,134 and 3,644 peptides with conformance scores ranging from 87.6% to 88.8% depending on the particular parameter set (table 2a). the conformance scores provide a relative measure of the peptide matching accuracy of each set of parameter settings of an algorithm. for example, the standardized omssa parameter sets have somewhat higher conformance scores than sequest. however, although correlated, the conformance score is not numerically equal to the matching efficiency of the algorithm due to several factors : parent proteins may run aberrantly during gel electrophoresis. post - translational modifications of parent proteins, such as glycosylation or proteolytic cleavage, may substantially change their molecular weights. parent proteins of peptides randomly matched by the algorithm may be randomly assigned to the correct size range ; for example, 25% of annotated yeast proteins have masses between 22.5 and 40.7 kda, so 25% of random matches would conform to this size range. indeed, the overall conformance scores for the decoy reverse peptides are 20.6% (table 2a). because these contributing factors likely apply equivalently across all parameter settings of algorithms analyzing the same spectrum data sets, differences in conformance scores nevertheless provide an excellent assessment of the relative accuracies of the different algorithms and can be used to assess new algorithms or new parameter settings of current algorithms. the five parameter sets of omssa all showed higher parent - protein conformance scores compared with sequest. indeed, the conformance score distributions from bootstrap analysis indicated that these differences are significant (supplementary figure 2). given that the data sets were filtered to give a 5% fdr, the difference in conformance scores indicates that the detection of reverse - sequence decoys by the two algorithms was not equivalent. indeed, even before applying a 5% fdr scoring filter, the standard implementation of omssa returned decoys at rates of 1.63.9% depending on the parameter set, indicating that the omssa algorithm is quite stringent in its interpretation of acceptable psms. moreover, unlike sequest, omssa does not standardly output a ranking of psms ; if only the best - scoring psms for each spectrum are considered, then the decoy rates are even lower for the omssa algorithm (0.61.7%). for comparison, we reassessed the sequest output using a 0.6% fdr threshold instead of 5%. this resulted in fewer matches (3,398 instead of 4,480) and a significantly higher parent - protein conformance rate (88.2% instead of 84.4%) comparable to those of omssa (supplementary figure 2). however, for the analysis that follows we used standard implementations of both algorithms with 5% fdr thresholds and filters as described in methods. counting peptides once even if seen with multiple omssa parameter sets, we classified peptides according to the number of standardized parameter sets (i.e., 15) for which a peptide was detected. although conformance scores for the individual parameter sets indicated high confidence in peptide matches, only 42.9% of the 3,922 peptide matches were detected by all five omssa standardized parameter sets (figure 2a.). the different samplings of peptides from different parameter settings suggest that using multiple standardized settings of omssa can increase the yield of high - confidence detected peptides. indeed, when probing the same spectrum data set, the union of the outputs from five omssa parameter sets gave 3,922 detected peptides at an overall conformance score of 86.2%. furthermore, we found that peptides detected by only one of the five standardized parameter sets of omssa had a considerably lower conformance score (61.1%) and accounted for only 5.0% of the detected peptides (figure 2a). this suggests that when using multiple settings of omssa it may be appropriate to exclude any peptides detected by only one of the standardized parameter sets given that this class of orphan peptides is found to be of lower confidence based on the parent - protein profiling approach. union of outputs from multiple omssa parameter sets increases the yield of detected peptides. conformance scores calculated from distinct peptides detected in the b / y (a) or a / b / y (b) ion screens in omssa ; peptides were counted once even if seen with multiple standardized parameter sets. collision induced dissociation (cid) most commonly cleaves peptides at the amide bonds (between the c and n atoms) to give b and y ions. these are the two ion types typically assessed by the matching algorithms (b / y ion screen). however, a ions can also be produced if the cleavage position is shifted by one carbon, and algorithms can be configured to screen for a ions in addition to the b and y ions (a / b / y ion screen). since assessment of a ions is sometimes included in specialized screens (e.g., of glutaraldehyde modified peptides), we tested whether inclusion of the a ions might increase peptide detections. we performed an a / b / y ion screen on the same spectra data sets from the parent - protein profiling experiments above and examined the conformance scores (table 2b). using sequest, we detected 5,016 peptides, counting a peptide once per lc ms / ms run whether seen in one or both of the b / y and a / b / y ion screens (figure 3b). this corresponds to 2,261 unique peptides, of which 1,752 (77.5%) peptides were detected by both the b / y and a / b / y ion screens. bootstrap analysis (figure 3c, p 1 parameter set and (ii) from sequest to those that are detected in both b / y and a / b / y ion screens. the above results suggest that low protein expression is associated with poor conformance to parent proteins. indeed, subsets of sequest spectrum matches with progressively lower protein expression reveal that parent - protein conformance ranges from 59.4% (protein expression 10 molecules per cell) (table 3a). interestingly, proteins in the undetected set (protein expression value = 0) have high conformance rates, suggesting that they were undetected for experimental reasons (e.g., inaccessible epitope tag) rather than low protein expression. protein expression (pe ; estimated protein molecules per cell) based on large scale western analysis. ms / ms run even if detected by both the b / y and a / b / y ion screens and, in the case of omssa, even if detected by multiple parameter sets. scoring confidence d = log10(psm_score / fdr_threshold). for omssa, implemented in proteome discoverer 1.2, we used the probability outputs to compute a psm score = 10. conformance scores are significantly depressed for lower abundance proteins (chi - squared : sequest, p < 6.18 10 ; omssa, p < 3.46 10) and for lower d scores (sequest, p < 7.23 10 ; omssa, p < 6.25 10) this result indicates that parent - protein conformance is not a uniform property within data sets but instead varies with protein expression levels : the algorithms are less effective at matching spectra for lower abundance proteins as might be expected. we examined the relationship between protein expression and the algorithm peptide - spectrum match (psm) scores (sequest probability score, omssa e - value score) and found that higher - confidence matching scores tend to be associated with higher expression proteins, whereas lower - confidence matching scores are common for both high and low expression proteins (supplementary figure 5). for this analysis we used a distance measure : which measures the relative distance between an algorithm psm score and the 95% confidence threshold for each experimental series. for example a d - score of 2 implies that the spectrum match is 10 fold better than the fdr threshold, which is the least stringent acceptable score for inclusion in the data set based on decoy analysis. not surprisingly, given this relationship between protein expression and algorithm psm scores, we found that conformance to parent proteins tends to be higher for subsets of matches with higher - confidence algorithm psm scores. we examined parent - protein conformance for subsets of spectrum matches with progressively better score ranges (table 3b). this revealed that for both algorithms, matches with scores within 10-fold of the fdr threshold have parent conformance scores of only 6164%, whereas score bins with greater distances from the fdr threshold have conformance scores that increase progressively up to 9193%. this analysis suggests that a given parent - protein conformance rate for a data set represents an average rate for the set of spectrum matches, and that the conformance rate is much lower for matches close to the fdr threshold and correspondingly higher for those further from the threshold. similarly, as discussed previously, fdrs measure the average values for a data set and subsets of data with algorithm scores closer to the fdr threshold have elevated rates of false identification and hence are of lower confidence compared to matches with scores further from the fdr threshold. we note that although decoy analysis can be employed to assess subsets of poor - scoring psms as discussed above, it would not be practical to use decoy analysis to assess algorithm performance with the other special subsets of detected peptides presented above, such as the outputs from different ion screens or different algorithms, due to difficulties in determining appropriate subsets of decoys for computing fdrs. the parent - protein profiling data set analyzed in this study provides a useful resource for assessing spectrum - peptide matching algorithms. ms / ms runs from the parent - protein profiling approach may be used as a benchmark data set for future yeast proteomic studies that are based on cid fragmentation and a mass spectrometer of similar resolution and sensitivity to the lcq deca xp (supplementary materials). for example, the spectra from the 22 gel slice experiments were used to evaluate several parameter settings of the mascot algorithm, which is commonly used by many groups (figure 6a). peptide - spectrum matches identified by mascot (figure 6b, c) showed similar parent - protein conformance rates as sequest and omssa and a similar graded dependence of conformance on protein expression (figure 6f) and scoring confidence (figure 6 g). like omssa, peptides detected with only one set of parameter settings of mascot had poorer conformance compared to those detected by multiple parameter sets (figure 6e). however, the behavior of the b / y and a / b / y ion screens was somewhat different for mascot in that all matches detected by the a / b / y ion screen were also detected by the b / y screen, and the few matches detected by the b / y ion screen alone had poor parent - protein conformance (figure 6d). (a) the mascot algorithm was run using similar parameter settings to those used with omssa. (c) parent protein conformance scores for a / b / y ion screen. (d) few peptides were detected by the b / y ion screen alone, and these had relatively lower conformance. (e) peptides detected by only one of the mascot standardized parameter sets have lower conformance compared to those detected with multiple parameter sets. (f) conformance scores are depressed for detected proteins with low expression (p < 1.05 10 ; chi - squared) ; for example, the set of proteins where protein molecules per cell < 1000 (i.e., log(pe) < 3) have a conformance level of 53.9%. (g) similarly, conformance scores are depressed for peptide matches that score close to the decoy fdr threshold. this is the case for the subsets of psms with scores below 77.6%, the 1% score threshold from bootstrap analysis. in this assessment, the scoring confidence, d = log10(psm_prob_score / fdr_threshold), is computed using psm probabilities from the mascot output : score = 10log10(psm_prob_score). with the ongoing improvements in the design of currently available peptide - spectrum matching algorithms, as well as the development of new algorithms, our parent - protein profiling approach provides an unbiased and valid evaluation for assessing different algorithms and choosing effective parameter settings to obtain high confidence peptide matches. in particular, conformance rates provide a relative measure for comparing different algorithms and different standardized parameter settings. ms / ms runs also calls for the use of multiple algorithms and parameter settings to increase the yield of identified peptides. in the case of sequest, taking the intersection of the output from the b / y and a / b / y ion screens may increase confidence in peptide matches. in the case of omssa, taking the union of the outputs from multiple parameter sets and excluding psms detected by a single parameter set may increase confidence in peptide matches. in the case of mascot, combining both of these filters (taking the intersection of b / y and a / b / y matches and excluding psms detected by only one parameter set) may increase confidence. as expected, assessment of psms in the context of known protein expression levels of the detected parent proteins indicates that confidence in spectrum matching by an algorithm varies within a data set and is lower for matches to low abundance proteins and matches with low - confidence algorithm psm scores. | peptide mass spectrometry relies crucially on algorithms that match peptides to spectra. we describe a method to evaluate the accuracy of these algorithms based on the masses of parent proteins before trypsin endoprotease digestion. measurement of conformance to parent proteins provides a score for comparison of the performances of different algorithms as well as alternative parameter settings for a given algorithm. tracking of conformance scores for spectrum matches to proteins with progressively lower expression levels revealed that conformance scores are not uniform within data sets but are significantly lower for less abundant proteins. similarly peptides with lower algorithm peptide - spectrum match scores have lower conformance. although peptide mass spectrometry data is typically filtered through decoy analysis to ensure a low false discovery rate, this analysis confirms that the filtered data should not be considered as having a uniform confidence. the analysis suggests that use of different algorithms and multiple standardized parameter settings of these algorithms can increase significantly the numbers of peptides identified. this data set can be used as a resource for future algorithm assessment. |
lupus nephritis (ln) is one of the most frequent manifestations of systemic lupus erythematosus (sle) and represents a major determinant of disease morbidity and mortality. its clinical course is often characterized by flares of activity alternated with periods of quiescence, generally induced by therapy. the identification of noninvasive biomarkers may help to predict the renal involvement at diagnosis and monitor relapses of ln during the follow - up. many studies have tested the value of a number of autoantibodies for predicting or confirming the diagnosis of renal flares with contrasting results. some [35 ] but not all studies have demonstrated that anti - dsdna antibodies (anti - dsdna) and complement fractions may be useful in assessing the disease and the renal activity. one paper and a recent review concluded that anti - nucleosome antibodies have high prevalence in severe ln but are of limited help in differentiating active from inactive ln. a number of cross sectional studies found that antic1q antibodies (antic1q) have a significant association with renal involvement [915 ]. in our previous paper on a large cohort of sle patients evaluated prospectively for 6 years, we demonstrated that renal exacerbations seem to be quite improbable in the presence of normal values of c3, c4, anti - dsdna, anti - c1q, and that anti - c1q was slightly better than the other tests to confirm the clinical activity of ln. noteworthy, in the vast majority of studies the diagnosis of ln flares relies on variable clinical definitions based on activity of urine sediment, amount of proteinuria, and deterioration of renal function, whilst the gold standard for the diagnosis of renal activity is represented by renal biopsy. in this prospective study, serum samples at renal biopsy and after the induction therapy of 107 ln patients were tested for a panel of autoantibodies (including anti - dsdna, anti - c1q, anti - nucleosome, anti - ribosome antibodies, and c3 and c4 complement fractions) to investigate their association with the clinical and histological data. one hundred and seven patients with sle, diagnosed according to the american college of rheumatology criteria (94 females, 13 males) at admission in two italian renal units (fondazione ospedale maggiore and azienda ospedaliera ospedale san carlo borromeo, milano) to undergo renal biopsy for assessment of ln, entered the study. sera at renal biopsy were tested for a panel of auto antibodies including anti - dsdna and anti - c1q, anti - nucleosome, and anti - ribosome antibodies as well as c3 and c4 complement fractions. we have obtained an informed consent to participate in the study from all the patients involved. the aim of this study was to assess the performance of these tests in predicting : the histological classes of lupus nephritis, the activity and chronicity index at renal biopsy, the clinical feature of ln at renal biopsy, the response of lupus nephritis at 3, 6, and 12 months after the beginning of the induction therapy. the histological classes of lupus nephritis, the activity and chronicity index at renal biopsy, the clinical feature of ln at renal biopsy, the response of lupus nephritis at 3, 6, and 12 months after the beginning of the induction therapy. anti - dsdna antibodies were measured by a commercial quantitative elisa (varelisa anti - dsdna antibodies, phadia gmbh, freiburg, germany) and c3 and c4 plasma levels by nephelometry (nephelometer analyser ii, behring, marburg gmbh, germany). anti - c1q antibodies were detected using a home - made elisa as described by sinico.. anti - nucleosome antibodies were measured by elisa according to manufacturer instructions using quanta lite chromatin assay (inova diagnostics, inc., san diego, ca, usa).. anti - ribosome p antibodies were measured by elisa according to manufacturer instructions using quanta lite ribosomal p assay (inova diagnostics, inc., san diego, ca, usa). at each clinical examination the activity of ln was classified as follows : 0 = complete renal remission : normal renal function for at least 6 months, proteinuria 10 red blood cells / hpf, cellular casts) with or without an increase in proteinuria ; 3 = proteinuric flare : increase of proteinuria of at least 2 g / day in patients with non nephrotic syndrome or the doubling of nephrotic proteinuria with stable renal function ; 4 = persistent renal activity : the lack of achievement of remission after induction therapy. 0 = complete renal remission : normal renal function for at least 6 months, proteinuria 10 red blood cells / hpf, cellular casts) with or without an increase in proteinuria ; 3 = proteinuric flare : increase of proteinuria of at least 2 g / day in patients with non nephrotic syndrome or the doubling of nephrotic proteinuria with stable renal function ; 4 = persistent renal activity : the lack of achievement of remission after induction therapy. mean and standard deviation, together with median and interquartile (iq) range (2575 percentile) were used as descriptive statistics. for continuous variables, the nonparametric wilcoxon test was used for assessing any difference between the two groups of patients, while the chi - square test was used for dichotomized variables. multivariate logistic regression analysis has been used to find predictors of histological classes of lupus nephritis and for the predictors of complete renal response after the beginning of induction therapy. odds ratios (or) and their 95% confidence interval (ci) for the covariates were derived as the antilogarithm of the regression coefficients. the statistical package s - plus (mathsoft inc.) was used for all the analyses and plots. the mean age at diagnosis of sle was 35.3 14.2 years, (median 34) and that at renal biopsy was 36.4 13.9 years (median 36). the mean time between the diagnosis of sle and that of renal involvement was 5.1 6.5 years, (median 3 years). in 45 patients, considering that a preliminary analysis demonstrated no significant differences in the mean values of c3, c4, anti - dsdna, anti - c1q, anti - nucleosome, and anti - ribosome antibodies between class ii and class v and between class iii and class iv ln (data not shown), the subsequent analysis was performed comparing class ii plus class v (nonproliferative forms ; 26 patients) versus class iii plus class iv (proliferative forms ; 85 patients). at renal biopsy, high titers of anti - dsdna were present in 77.5% of cases, high titers of anti - c1q in 70.5% of cases, high titers of anti - nucleosome antibodies in 80.3% of cases, and high titers of anti- ribosome antibodies in 14% of cases ; c3 were low in 82% of cases and c4 in 74% of cases. table 2 reports the comparison at time of renal biopsy of clinical data and of the panel of autoantibodies between proliferative forms (class iii plus class iv) and nonproliferative forms (class ii plus class v) of ln. at univariate analysis, among the clinical parameters, proteinuria (p = 0.02) and hemoglobin (p = 0.0008) and among the immunological tests, c3 (p = 0.02) and c4 (p = 0.02) complement fractions, anti - dna (p = 0.001), anti - c1q (p = 0.0005), and high titers of anti - c1q antibodies or of anti - dsdna antibodies (p = 0.0000) and anti - nucleosome antibodies (p = 0.04) were able to differentiate proliferative from nonproliferative forms of ln. at multivariate analysis, hemoglobin (p = 0.008, or = 0.68, ci : 0.520.9) and anti - c1q antibodies (p = 0.03, or = 1.004, ci : 1.00031.007) were the independent predictors to discriminate between proliferative versus nonproliferative lupus nephritis. excluding clinical parameters, at multivariate analysis, logarithm of erythrocyte sedimentation rate (esr) (p = 0.03, or = 1.9, ci : 1.083.42) and high titers of anti - c1q antibodies or of anti - dsdna antibodies (p = 0.005, or = 8.67, ci : 2.0337.3) are the independent predictors which are able to discriminate proliferative from nonproliferative lupus nephritis. among patients with proliferative forms of lupus nephritis, 95% have high titers of anti c1q or of anti - dsdna (66.2% have high titers of both anti - c1q and of anti - dsdna) while 5% have the results of both tests in a normal range. among patients with nonproliferative forms, 64% have high titers of anti c1q or of anti dna while 36% have the results of both tests in a normal range (p = 0.000). the correlations among the basal clinical and the immunological data and the activity and the chronicty index at renal biopsy are reported in table 3. all the clinical and immunological parameters evaluated with the exception for c reactive protein (crp) and anti - ribosome antibodies showed a significant correlation with activity index. at multivariate analysis proteinuria (p = 0.0013), low c4 (p = 0.0010), and high esr (p = 0,037) were the independent predictors of the activity index. excluding the clinical variables, low c4 (p = 0.0004) and high esr (p = 0,0025) were the independent predictors of activity index. in contrast, serum creatinine was the only parameter among those evaluated that showed a direct correlation with the chronicity index (r : 0.4, p = 0.0000). no correlation was found at time of renal biopsy between serum creatinine and the panel of autoantibodies, c3 and c4 complement fractions, and esr and crp. among these tests, anti - c1q only showed a significant direct correlation with the amount of proteinuria (r = 0.2, p = 0.03). in addition, patients with high titers of anti - c1q or of anti - dsdna had significant higher proteinuria (median 2.7 g / day, iq 1.64.6) than those with both tests in normal range (1.8 g / day, iq 1.02.2, p = 0.05). anti - c1q, anti - dsdna, and esr were inversely correlated with hemoglobin (r-0.22, p = 0.02, r-0,24, p = 0.01, r-0.32, p = 0.002, resp.), while c3 and c4 were correlated with hemoglobin (r 0.36, p = 0.0002 and r 0.25, p = 0.01). after renal biopsy and the beginning of induction therapy, 104 patients had a second evaluation between 3 and 12 months. table 4 reported the results of clinical and immunological tests in patients reevaluated at 3 months, at 6 months, and at 12 months. at 3 months, serum creatinine was unchanged and proteinuria did not show a significant improvement, while esr, c3, anti - dna, anti - c1q, and anti - nucleosome antibodies showed a significant improvement in the median values. at 6 and at 12 months, proteinuria significantly improved together with all immunological tests with the exception of esr at 6 months and anti - ribosome antibodies at 12 months. altogether, during the observation period, 39 patients (37.5%) achieved and 65 (62.5%) did not achieve complete renal remission (46 were in partial remission, 13 had persistent renal activity, and 6 had persistent nephrotic syndrome). clinical and immunological tests at the time of renal biopsy have been tested as predictors of complete renal remission (table 5). at univariate analysis, none of the immunological tests were predictive of complete remission. at multivariate analysis, proteinuria (p = 0.015, or : 0.76 ci 0.620.95) and the duration of therapy (p = 0.03 or : 1.19 ci 1.0171.39) were the independent predictors of complete renal remission. in this study, we have investigated the prevalence and the value of a panel of autoantibodies (anti - dsdna, anti - c1q, anti - nucleosome, and anti - ribosome antibodies) as well as c3 and c4 complement fractions in predicting the activity of ln at the time of renal biopsy. the most important difference of our study compared to many previous studies is the timing of blood sampling in relation to renal activity. as a matter of fact, in the majority of the studies evaluating the predictive values of autoantibodies [11, 15, 2123 ], the renal activity of ln at the time of blood sampling was judged by clinical parameters but not confirmed by renal biopsy. to the best of our knowledge, only a few studies have evaluated the association of some autoantibodies with activity of ln at the time of renal biopsy [2427 ]. reported that all but one out of 36 patients with proliferative lupus were positive for anti - c1q at the time of renal biopsy compared with 35% of patients with inactive ln. in 136 chinese patients, anti - c1q and anti - dsdna were more closely correlated with histological activity of ln at the time of renal biopsy than anti - extractable nuclear antigen antibodies, anti - c protein antibodies, anti cardiolipin, and anti beta2 glycoprotein antibodies. the combination of anti - c1q and anti - dsdna indicates higher renal disease activity and predicts poor long term renal outcome. another paper investigated the clinical and pathological association of anti - c1q in ln and found a higher prevalence of the autoantibody in class iv than in the other histological classes. among the clinical variables low haemoglobin was associated with anti - c1q positivity. in this paper, we have shown that there was a significant difference in the autoantibodies profile between proliferative forms (class iii plus iv) and the other forms of ln (class v and class ll). all the autoantibodies evaluated, with the exception of anti - ribosome antibodies, had significant higher prevalence and higher titres in proliferative than in nonproliferative forms of ln. c3 and c4 complement fractions too were significantly lower in proliferative than in nonproliferative ln. at multivariate analysis, considering clinical and immunological tests, only low haemoglobin and high anti - c1q were the independent predictors of proliferative ln. excluding the clinical variables, high esr and positive anti - c1q or anti - ninety - five percent of patients with proliferative ln had high titers of anti c1q or of anti - dsdna (66.2% had high titers of both tests) while 4 patients only had the results of both tests in normal range. the increasing power of the combination of anti - c1q or anti - dsdna positivity in predicting the activity of ln has been reported by other studies [25, 28 ]. this higher predictive value of anti c1q for proliferative ln confirmed our findings in a previous study in which we demonstrated that 80% of flares that developed in patients with proliferative forms were associated with high titres of anti - c1q in comparison to only 54% of those that occurred in the nonproliferative forms. instead, other cohort studies did not show differences in the prevalence of antic1q between proliferative and nonproliferative lupus nephritis [2830 ]. this discrepancy could be due to the fact that in these studies the diagnosis of renal activity was done on clinical grounds and not confirmed by renal biopsy. again, anti - c1q, alone or associated with anti - dsdna, was the only test among the immunological parameters that significantly correlated with the amount of proteinuria. none of the immunological tests correlated with serum creatinine but the majority of our patients had normal renal function, and many tests correlated with hemoglobin, a manifestation not specific for ln but an expression of the general activity of sle. low c4 and high esr were the independent predictors of a high activity index at multivariate analysis while none of the tests of the panel correlated with chronicity index., in our cohort, none of the immunological tests at the time of renal biopsy was predictive of the renal response, at least in the short term. however, we have shown that, three months after the start of the induction therapy and prior to the improvement of proteinuria, a significant reduction of the mean value of anti - c1q, anti - dsdna, and anti - nucleosome antibody occurred. the progressive and significant drop in autoantibodies titres continued at 6 and at 12 months together with a clinical improvement as reported in other studies [24, 26, 31 ]. anti - c1q antibodies can be detected by different methods (reviewed in [32, 33 ]). in the early 1980s, a solid - phase assay using purified c1q, immobilized on plastic assay plates, was used for the detection of circulating immune complexes in sle patients. to differentiate between immune complexes and anti - c1q antibodies, high - salt concentrations (0.51.0 m sodium chloride) the binding of the globular heads of c1q to immune complexes is prevented, whereas anti - c1q antibodies can still interact with the coated c1q. subsequently, to eliminate the need to use high - ionic strength buffer, assays have been developed that utilize only the c1q collagen - like region. however, additional exposed epitopes, by cleaving of the c1q molecule, might interfere with the results obtained with this assay. more recently, peptides derived from c1q that have the properties to detect a major linear epitope in a high percentage of the patients in the absence of high - ionic strength buffer has been proposed. unfortunately, systematic studies comparing anti - c1q antibody detected by different assays are not available and different studies have used different methods. in our study, we have used the classic assay which has been used in the majority of published clinical studies because it is more readily available. we found a significantly different autoantibodies profile between proliferative and nonproliferative forms of ln at the time of renal biopsy. among the panel of autoantibodies evaluated in this study, anti - c1q alone or in combination with anti - dsdna emerged as the most reliable in differentiating proliferative and nonproliferative ln and anti - c1q is the only test correlated with the clinical presentation of ln. after the beginning of therapy, the titer of the autoantibodies progressively and significantly reduced, but none of them was predictive of complete renal remission. the results of this work, which outlines the role of autoantibodies and in particular of anti - c1q, in defining the activity of lupus nephritis at the time of renal biopsy, confirm their utility in diagnosing the acute exacerbations of ln made on clinical grounds only. | few studies have correlated serum biomarkers with renal histology, the gold standard for renal activity, in lupus nephritis (ln). we tested a panel of autoantibodies and complement at the time of kidney biopsy and after treatment. anti - dsdna, anti - nucleosome, anti - ribosome p, and anti - c1q antibodies and c3/c4 were measured in 107 patients with ln at the time of renal biopsy and after 612 months and were correlated with clinical / histological parameters. at multivariate analysis, high titers of anti - c1q antibodies or of anti - dsdna antibodies (p = 0.005, or = 8.67, ci : 2.0337.3) were the independent predictors that discriminate proliferative from nonproliferative ln. all the immunological parameters, except anti - ribosome, showed a significant correlation with activity index but not with chronicity index. only anti - c1q showed a significant correlation with the amount of proteinuria (r = 0.2, p = 0.03). none of the immunological parameters were predictive of remission at 6 and 12 months. we found that anti - c1q alone or in combination with anti - dsdna emerged as the most reliable test in differentiating proliferative and nonproliferative ln. anti - c1q was the only test correlated with the clinical presentation of ln. after treatment, the titre of the autoantibodies was significantly reduced, but none was predictive of remission. |
giardiasis causes significant worldwide morbidity with an estimated 184 million symptomatic cases annually (pires., 2015) and an associated 171,100 disability - adjusted life years (dalys) (kirk., 2015 while giardiasis is more prevalent in the developing world it is also a burden in developed countries, with hospital based treatments in the united states of america costing $ 34.4 million (usd) annually (collier., 2012) giardiasis is commonly associated with clinical symptoms including nausea, vomiting and acute diarrhoea (nash., 1987, farthing, 1996). however it can manifest as a chronic disease and cause malabsorption, weight loss and failure to thrive in children (al - mekhlafi., 2005, al - mekhlafi., 2013, bartelt., there is also mounting evidence that giardia infection may be linked to irritable bowel syndrome, food allergies and obesity (di prisco., 1998, hanevik., 2009, as there is no currently available vaccine for humans, the control of giardiasis is dependent on chemotherapy. current chemotherapeutic options are limited to a small number of compounds which are associated with treatment failures and clinical resistance (reviewed in ansell., 2015). the 5-nitroimidazole class of compounds, typically metronidazole, are the most commonly used treatment agents (watkins and eckmann, 2014). however, these compounds have reported clinical failure rates of up to 40% (oren., 1991, farthing, 1996 ; reviewed in watkins and eckmann, 2014, nabarro., 2015) and can also cause significant side - effects including neurological disorders and sudden death (escobedo and cimerman, 2007). however, the efficacy of these drugs varies widely (e.g. hall and nahar, 1993, escobedo., in addition, the benzimidazole drugs appear particularly susceptible to the development of drug resistance, with data suggesting that parasite resistance can be easily selected in vitro (gardner and hill, 2001). new anti - giardia agents with improved efficacy and toxicity a number of low to high throughput in vitro assays have been developed to identify new compounds active against giardia. however, most rely on metabolic indicators or manual cell counting. activity assays that rely on manual cell counting via microscopy have the advantage of permitting the assessment of growth at multiple time - points and provide useful morphological information, but are time consuming and may be subjective. while the more automated assays that make use of growth indicators including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide (xtt), resazurin (alamarblue) h - thymidine, atp content or the assessment of glucuronidase activity in transgenic parasites (mller., 2009) are more rapid, they inherently increase assay cost, provide limited activity / morphology information and permit only single time - point of assessment. activity assays reliant on transgene expression are also limited to assessing activity against genetically manipulated parasites. efforts to improve current growth assay methods have included combining microscopy with automated image analysis software to decrease time limitations associated with manual enumeration methods (bonilla - santiago., 2008, faghiri., 2011, gut., 2011). (2011) parasites are stained with 4,6-diamidino-2-phenylindole (dapi) to automatically distinguish and enumerate living trophozoites without bias. while this significantly reduces assay evaluation time, parasites must still be fixed and stained which necessitates extra handling and eliminates the possibility of multiple time - point evaluations. in this study, we developed an automated live - cell digital phase - contrast microscopy assay to assess the activity of compounds against giardia trophozoites in vitro. the perkin - elmer operetta, with its associated harmony and phenologic software, was used to exploit the power of automated digital phase - contrast microscopy and image analysis as a mechanism to identify and enumerate parasites based on their morphology without the need for a cell marker. a particular advantage of this approach is the ability to assess parasite growth at multiple time - points. this assay was used to assess the anti - giardia activity of compounds from the malaria box. the malaria box, a set of compounds with known activity against mammalian cells (kaiser., 2015) multiple parasite species including p. falciparum (spangenberg., 2013), toxoplasma gondii, entamoeba histolytica (boyom., 2014), cryptosporidium parvum (bessoff., 2014), leishmania major (khraiwesh., 2016) and trypanosoma spp. (kaiser., 2015) has never previously been assessed for anti - giardia activity. g. duodenalis (strain bris/91/hepu/1279 ; metronidazole sensitive ; assemblage b (upcroft., 1995, nolan., 2011)) was grown axenically (3% o2 5% co2, in n2 at 37 c) in kiesters - modified tyi - s-33 media in 8 ml borosilicate vials (pyrex glass, no. 9825 ; vwr) as previously described (keister, 1983, meloni and thompson, 1987). media was prepared on a weekly basis and stored at 4 c. when required for use, aliquots were supplemented with 10% foetal bovine serum, 100 units / ml penicillin and 100 g / ml streptomycin. albendazole, metronidazole and furazolidone were obtained from sigma - aldrich, usa and prepared in 100% dmso to stock concentrations of 1050 mm. malaria box compounds were obtained from the medicines for malaria venture (mmv ; www.mmv.org) as 10 mm stocks prepared in 100% dmso. giardia parasites were grown in stock 8 ml borosilicate tubes (section 2.1) to 80% confluence. after detachment, parasites were collected, counted using a haemocytometer and seeded in 96-well micro titre plates (corning costar 3596 ; total volume 200 l ; 2 10 to 5 10 cells / well). outside wells of plates contained phosphate - buffered saline to reduce evaporation (pbs ; 200 l). plates were incubated at 37 c in sealed, activated anaerocult c mini bags as per manufacturer instructions as previously described (upcroft and upcroft, 2001). growth of parasites seeded in triplicate wells on two separate occasions was assessed at 24 and 48 h by digital phase - contrast microscopy, enumerated using harmony and phenologic software (section 2.6) and by the manual counting of bright - field images. data from all experiments were combined (mean parasite count/1.7 mm sd) and manual versus automated counts were compared using a student 's t - test (graphpad prism 7). after detachment, parasites were collected, counted using a haemocytometer and seeded in 96-well micro titre plates (corning costar 3596 ; total volume 200 l ; 6 10 to 5 10 cells / well). however, as a reliable source of anaerocult c mini bags (merck, millipore) could not be obtained, microaerophilic conditions were established by incubating plates at 37 c in air - tight chambers filled with 3% o2 5% co2 in n2 as previously described (gut., 2011). growth of parasites was assessed at 24 and 48 h by digital phase - contrast microscopy and enumerated using harmony and phenologic software (section 2.6). data are presented as mean trophozoite count sd of 4 separate experiments, each carried out in triplicate wells. the average doubling time between 24 and 48 h for each seeding concentration was calculated using the equation, t = (24) log (2)/log (c / c) where t = doubling time, c was the average 24 h count and c was the average 48 h count. as assay plates were outside of anaerobic conditions during imaging (section 2.6 ; 20 min) and then returned to culture post - imaging for further incubation and assessment, the impact of imaging on parasite growth was assessed. in these assays two identical 96-well micro titre plates were prepared. one plate was imaged at 24 and 48 h and the other only at 48 h. in brief trophozoites were seeded into 96-well plates (3 10 to 3.75 10 parasites / well in 200 l) and incubated in 3% o2 5% co2, in n2 at 37 c until imaging (section 2.6). the 24 and 48 h imaged plate was returned to culture conditions after imaging at 24 h and re - imaged again at 48 h whereas the 48 h only plate remained in microaerophilic conditions until imaging at 48 h. each cell seeding concentration was plated in six technical replicates on a single plate and each assay was repeated on three separate occasions. data are presented as mean parasite count/1.7 mm sd and cell counts were compared using a student 's t - test (graphpad prism 7). each compound was serially diluted in triplicate wells (100 l ; 8 point dilution series for albendazole and furazolidone and 15 point dilution series for metronidazole), and all wells except media only controls, were seeded with 1.5 10 giardia trophozoites (100 l ; 200 l final volume). plates were incubated in 3% o2 5% co2, in n2 at 37 c until imaging and growth analysis (section 2.6) at 24 and 48 h. each assay included no drug with vehicle (0.2% dmso) and no vehicle controls and in each case three independent assays were carried out. the concentration of dmso in drug dilutions was kept constant at 0.2% and as previously shown (johns., 1995) mean percentage growth inhibition compared to vehicle (0.2% dmso) and background controls was determined for each assay. ic50 values were calculated using log - linear interpolation (huber and koella, 1993). all malaria box compounds were screened for activity against g. duodenalis bris/91/hepu/1279 at a final concentration of 10 m in singlicate, in two independent experiments. each plate included, background media, vehicle (0.2% dmso), no vehicle and albendazole (10 m) controls. assays were performed under the same conditions as those used to assess the activity of control anti - giardia compounds (section 2.4 ; 1.5 10 parasites / well in final volume 200 l ; imaged at 24 and 48 h). compounds demonstrating greater than 50% inhibition at this concentration were assessed for activity at 5 m in duplicate (n = 2). z - factors were calculated for each plate of each screening assay as previously described (zhang., 1999). compounds showing 50% inhibition at 5 m were further investigated to determine ic50 values as described for control compounds (section 2.4 ; each titration was performed in duplicate on three occasions ; 8 point dilution series ; compound concentration range 10,00078 nm). plates were removed from incubation and each well was imaged using brightfield and phase - contrast microscopy (total area imaged 1.7 mm 50% inhibition at 24 or 48 h when assessed at 10 m (fig. 3a and b ; table s1). the z factor of all assays plates in the 10 m screen was > 0.5 (average sd ; 0.74 + 0.11). further analysis of the 122 compounds identified 22 with > 50% growth inhibition at 5 m (fig. the z factor of all assays plates in 5 m assays was also > 0.5 (average sd ; 0.73 + 0.10). further dose response analysis of the 22 compound with > 50% inhibition at 5 m identified three compounds (mmv007384, mmv019690 and mmv006203) with sub-m ic50 values (table 2, table s1 & fig. we have developed an in vitro medium throughput assay that permits giardia drug susceptibility testing in real - time without any need to stain parasites. this assay is unique in that it harnesses the power of digital phase - contrast microscopy and dedicated analysis software to identify and count parasites thereby permitting speedy, multi - time point analysis of live parasite numbers. a comparison of automatically generated parasite counts with manual counts demonstrates that the system can quickly and reliably assess trophozoite numbers (fig. 1). as this assay permits the activity of compounds to be assessed at multiple time - points without any impact on parasite growth (fig. additional information regarding the time course of compound activity and morphological effects, which can be derived from acquired images, may aid in compound triage and mechanism of action studies. further reductions in cost and additional data acquisition may also be possible given that the assay is likely to be amenable to miniaturization and longer assessment periods (up to 72 h (upcroft and upcroft, 2001, kulakova. while a potential limitation of the current assay may be in its assessment of parasite number rather than a metabolic parameter linked to viability, compounds with static activity can be of use therapeutically (pankey and sabath, 2004). in addition, metabolic assays can be associated with the same liability in the case of dormancy or when the compounds assessed interfere with the metabolic process used to quantitate inhibition (collier and pritsos, 2003, ulukaya., 2004). indeed, as a result of continued growth and the enhanced metabolic activity of controls over time, both assay types are likely to identify compounds with static activity as inhibitors. more specialized methods designed to assess mode of action are therefore more adequately placed to examine the nature of compound activity, post - identification. an additional limitation of the current assay that should be considered is its assessment of parasite number based on adherence. while this is an inherent limitation of other assays including those that require the removal of culture media and well - washing prior to activity assessment, this would mean that the assay is likely to identify compounds that effect attachment in addition to compounds that effect replication. although the consequences of this anti - attachment activity in the in vivo setting may be limited, more specialized assays would be required to discriminate between compounds that effect attachment versus those that inhibit replication. nevertheless, the ability of the current image - based assay to effectively examine the activity of compounds against giardia parasites was demonstrated by assessing the activity of control anti - giardia compounds albendazole, metronidazole and furazolidone, with ic50 values generated by the automated imaging and enumeration system being within the range of previously published studies (table 1). the suitability of the assay as a mechanism to identify compounds with activity against giardia parasites was also demonstrated by assessing the malaria box compound set for potential anti - giardia activity. the mean z factor for all plates in these assays (0.74 in 10 m and 0.73 in 5 m assays) suggest that the assay is of excellent quality (zhang., 1999) and a promising new tool for giardia parasite drug discovery. of interest a previously described image - based giardia assay which is dependent on parasite staining and hence an end - point assay, reported a z factor of 0.54 (gut., 2011). the identification of anti - giardia compounds within the malaria box set that have structural similarities to known anti - giardia compounds provides additional evidence that the current assay is suitable for compound activity assessment. mmv007384, the most potent of the anti - giardia hits identified (fig. s1, table 2 ; 24 h ic50 0.8 m and 48 h ic50 0.6 m) is a benzimidazole. in addition mmv667492 (table s1 ; 24 h ic50 3.7 m and 48 h ic50 2.6 m) is a napthoquinone similar to menadione that has been shown to have promising efficacy against g. duodenalis trophozoites and cysts in vitro (paget., 2004). two malaria box compounds, in addition to mmv007384 were identified to have sub m ic50 values against giardia parasites in the current study. these compounds were mmv019690 (table 2 ; 24 h ic50 2.8 m and 48 h ic50 0.9 m) and mmv006203 (table 2 ; 24 h ic50 3.1 m and 48 h ic50 0.7 m). while the selectivity index for mmv019690 (4.8 ; table 2), generated using ic50 data against mrc-5 fibroblasts (kaiser., 2015) suggest this compound may be associated with toxicity, the selectivity index of mmv006203, (25.7 ; table 2), was more favourable, falling within recently published lead criteria range (katsuno., 2015). importantly, the identification of cell debris in images acquired during the assessment of mmv006203 and mmv019690 (fig. the current study has validated digital - phase contrast microscopy and automated parasite enumeration as a method to investigate giardia drug susceptibility and identified new chemical scaffolds with anti - giardia activity that may warrant further investigation. unlike previously published image - based assessment giardia assays, the method described in this study negates the need for cell staining and permits multiple - time point activity assessment which can improve screening costs and only add value to current drug discovery efforts. | giardia duodenalis is an intestinal parasite that causes giardiasis, a widespread human gastrointestinal disease. treatment of giardiasis relies on a small arsenal of compounds that can suffer from limitations including side - effects, variable treatment efficacy and parasite drug resistance. thus new anti - giardia drug leads are required. the search for new compounds with anti - giardia activity currently depends on assays that can be labour - intensive, expensive and restricted to measuring activity at a single time - point. here we describe a new in vitro assay to assess anti - giardia activity. this image - based assay utilizes the perkin - elmer operetta and permits automated assessment of parasite growth at multiple time points without cell - staining. using this new approach, we assessed the malaria box compound set for anti - giardia activity. three compounds with sub-m activity (ic50 0.60.9 m) were identified as potential starting points for giardiasis drug discovery. |
the prognostic potential of different laboratory tests was evaluated in 15 naturally infected animals, of which 12 dogs were presented to the clinic for small animal internal medicine at the vetsuisse faculty, university of zurich, and 3 dogs to private veterinary practices in switzerland in the years 2011 to 2013. inclusion criteria were the presence of acute clinical signs consistent with canine babesiosis at admission and the identification of large babesia species by microscopic evaluation of giemsastained blood smears. in each dog, b. canis diagnosis was confirmed by pcr27 and direct sequencing of the amplicons.1 at time of admission, blood samples were collected, and all animals were treated with antibabesial therapy (a single dose of 36 mg / kg body weight [bw ] imidocarb diproprionate i m or combined with 10 mg / kg bw doxycycline po q12h for at least 10 days, and a second dose of imidocarb diproprionate after 14 days). the animals were categorized into 2 groups according to clinical outcome, which was defined as survival (survivor, n = 7 dogs) or death (nonsurvivor, n = 8 dogs). six of the nonsurvivors died spontaneously within 24 hours of admission and 2 dogs had to be euthanized within 48 hours because of clinical deterioration within 48 hours. survivors were considered to be cured based on the absence of parasites 14 days after first admission on evaluation of giemsastained blood smears and pcr. three facilityhoused adult beagles (of which 1 was 4 years and 2 were 6 years old) were inoculated iv with approximately 1 10 parasitized erythrocytes from an isolate stored in liquid nitrogen. the parasite isolate originated from a naturally infected bernese mountain dog from switzerland that had travelled to hungary. the experiments were terminated at the very first signs of acute crisis (which was defined as weak pulse, shallow breathing, somnolence, and any clinical signs of acute shock or central nervous depression). experiments with dogs were conducted according to swiss animal rights and regulations standards and approved by the cantonal veterinary office of zurich (permission number 122/2012) before the study. venous blood samples from the naturally infected dogs were collected into tubes with and without ethylenediaminetetraacetic acid (edta) at the time of first admission and before any treatment. serum and edtapreserved blood samples were collected through an indwelling catheter from the experimentally inoculated dogs at different times. in addition, citrated plasma samples were collected from these dogs at the end of the experiments. parasitemia was expressed as the percentage of infected erythrocytes in giemsastained blood smears by manually scanning at least 5000 erythrocytes. exposure to ehrlichia canis and anaplasma phagocytophilum was tested by an immunofluorescence antibody test (ifat).2 3 complete blood cell counts were performed using edtaanticoagulated blood in an automated analyzer.4 hematologic analysis included total white blood cell (wbc), thrombocyte and red blood cell (rbc) counts and rbc indices. serum biochemical profiles were performed using an automated analyzer.5 laboratory reference intervals are stated as 5% and 95% quantiles. portable handheld devices for rapid inclinic testing were used to measure concentrations of lactate6 and glucose7 immediately in freshly collected edta samples.28, 29 serum crp concentration was determined using a caninespecific immunoturbidimetric assay8 and serum amyloid a (saa) concentration was measured using a latex agglutination turbimetric immunoassay on an automated analyzer.5 9 in the citrated samples from the experimentally infected animals, fibrinogen concentrations were measured using the clauss method and a semiautomated coagulometer.10 ddimer concentrations were measured on an automated analyzer.5 11 results of the 2 groups (survivor and nonsurvivor) of naturally infected dogs were compared by the mann the initially significant variables then were analyzed with receiver operator characteristic (roc) curves for which the area under the curve (auc) was calculated. the roc analysis was used for determining a prognostic cutoff value for best differentiating between survivors and nonsurvivors with a maximal youden 's index.30, 31 if the cutoff value fell within the normal reference range, it was set at the corresponding border of the reference. statistical analyses were performed using a statistical software package.12 a pvalue <.05 was considered statistically significant. the hematologic and serum biochemical profiles from the samples collected at private practices were excluded from the analysis because these variables were measured with other analytical instruments. for parasitemia, variables from handheld devices, and the acute phase response, all of the naturally infected dogs were included in the analysis (7 survivors and 8 nonsurvivors). the prognostic potential of different laboratory tests was evaluated in 15 naturally infected animals, of which 12 dogs were presented to the clinic for small animal internal medicine at the vetsuisse faculty, university of zurich, and 3 dogs to private veterinary practices in switzerland in the years 2011 to 2013. inclusion criteria were the presence of acute clinical signs consistent with canine babesiosis at admission and the identification of large babesia species by microscopic evaluation of giemsastained blood smears. in each dog, b. canis diagnosis was confirmed by pcr27 and direct sequencing of the amplicons.1 at time of admission, blood samples were collected, and all animals were treated with antibabesial therapy (a single dose of 36 mg / kg body weight [bw ] imidocarb diproprionate i m or combined with 10 mg / kg bw doxycycline po q12h for at least 10 days, and a second dose of imidocarb diproprionate after 14 days). the animals were categorized into 2 groups according to clinical outcome, which was defined as survival (survivor, n = 7 dogs) or death (nonsurvivor, n = 8 dogs). six of the nonsurvivors died spontaneously within 24 hours of admission and 2 dogs had to be euthanized within 48 hours because of clinical deterioration within 48 hours. survivors were considered to be cured based on the absence of parasites 14 days after first admission on evaluation of giemsastained blood smears and pcr. three facilityhoused adult beagles (of which 1 was 4 years and 2 were 6 years old) were inoculated iv with approximately 1 10 parasitized erythrocytes from an isolate stored in liquid nitrogen. the parasite isolate originated from a naturally infected bernese mountain dog from switzerland that had travelled to hungary. the experiments were terminated at the very first signs of acute crisis (which was defined as weak pulse, shallow breathing, somnolence, and any clinical signs of acute shock or central nervous depression). experiments with dogs were conducted according to swiss animal rights and regulations standards and approved by the cantonal veterinary office of zurich (permission number 122/2012) before the study. the prognostic potential of different laboratory tests was evaluated in 15 naturally infected animals, of which 12 dogs were presented to the clinic for small animal internal medicine at the vetsuisse faculty, university of zurich, and 3 dogs to private veterinary practices in switzerland in the years 2011 to 2013. inclusion criteria were the presence of acute clinical signs consistent with canine babesiosis at admission and the identification of large babesia species by microscopic evaluation of giemsastained blood smears. in each dog, b. canis diagnosis was confirmed by pcr27 and direct sequencing of the amplicons.1 at time of admission, blood samples were collected, and all animals were treated with antibabesial therapy (a single dose of 36 mg / kg body weight [bw ] imidocarb diproprionate i m or combined with 10 mg / kg bw doxycycline po q12h for at least 10 days, and a second dose of imidocarb diproprionate after 14 days). the animals were categorized into 2 groups according to clinical outcome, which was defined as survival (survivor, n = 7 dogs) or death (nonsurvivor, n = 8 dogs). six of the nonsurvivors died spontaneously within 24 hours of admission and 2 dogs had to be euthanized within 48 hours because of clinical deterioration within 48 hours. survivors were considered to be cured based on the absence of parasites 14 days after first admission on evaluation of giemsastained blood smears and pcr. the course of laboratory test results was evaluated in experimentallyinfected animals. three facilityhoused adult beagles (of which 1 was 4 years and 2 were 6 years old) were inoculated iv with approximately 1 10 parasitized erythrocytes from an isolate stored in liquid nitrogen. the parasite isolate originated from a naturally infected bernese mountain dog from switzerland that had travelled to hungary. the experiments were terminated at the very first signs of acute crisis (which was defined as weak pulse, shallow breathing, somnolence, and any clinical signs of acute shock or central nervous depression). experiments with dogs were conducted according to swiss animal rights and regulations standards and approved by the cantonal veterinary office of zurich (permission number 122/2012) before the study. venous blood samples from the naturally infected dogs were collected into tubes with and without ethylenediaminetetraacetic acid (edta) at the time of first admission and before any treatment. serum and edtapreserved blood samples were collected through an indwelling catheter from the experimentally inoculated dogs at different times. in addition, citrated plasma samples were collected from these dogs at the end of the experiments. parasitemia was expressed as the percentage of infected erythrocytes in giemsastained blood smears by manually scanning at least 5000 erythrocytes. exposure to ehrlichia canis and anaplasma phagocytophilum was tested by an immunofluorescence antibody test (ifat).2 3 complete blood cell counts were performed using edtaanticoagulated blood in an automated analyzer.4 hematologic analysis included total white blood cell (wbc), thrombocyte and red blood cell (rbc) counts and rbc indices. serum biochemical profiles were performed using an automated analyzer.5 laboratory reference intervals are stated as 5% and 95% quantiles. portable handheld devices for rapid inclinic testing were used to measure concentrations of lactate6 and glucose7 immediately in freshly collected edta samples.28, 29 serum crp concentration was determined using a caninespecific immunoturbidimetric assay8 and serum amyloid a (saa) concentration was measured using a latex agglutination turbimetric immunoassay on an automated analyzer.5 9 in the citrated samples from the experimentally infected animals, fibrinogen concentrations were measured using the clauss method and a semiautomated coagulometer.10 ddimer concentrations were measured on an automated analyzer.5 11 results of the 2 groups (survivor and nonsurvivor) of naturally infected dogs were compared by the mann the initially significant variables then were analyzed with receiver operator characteristic (roc) curves for which the area under the curve (auc) was calculated. the roc analysis was used for determining a prognostic cutoff value for best differentiating between survivors and nonsurvivors with a maximal youden 's index.30, 31 if the cutoff value fell within the normal reference range, it was set at the corresponding border of the reference. statistical analyses were performed using a statistical software package.12 a pvalue <.05 was considered statistically significant. the hematologic and serum biochemical profiles from the samples collected at private practices were excluded from the analysis because these variables were measured with other analytical instruments. hence, for these variables 6 survivors and 6 nonsurvivors were included. for parasitemia, variables from handheld devices, and the acute phase response, all of the naturally infected dogs were included in the analysis (7 survivors and 8 nonsurvivors). at admission, all of the naturally infected dogs had diverse clinical signs consistent with canine babesiosis, including lethargy (all 15 dogs), pale mucous membranes (all 15 dogs), pigmenturia (10 of 15), icterus (6 of 15), pyrexia (5 of 15), anorexia (4 of 15), vomiting (4 of 15), water hammer pulse (4 of 15), and epistaxis (3 of 15). although babesia infection was assumed and antibabesial treatment initiated shortly after admission, 8 of the 15 dogs died or had to be euthanized within 2 days of admission. all of the dogs were positive for b. canis in giemsastained blood smears and by pcr, and none of these dogs reacted serologically to e. canis or a. phagocytophilum on ifat. data on characteristics of the individual dogs (animal description, travel history, and clinical signs) are summarized in supplemental file 1. no statistical difference in age, sex, and clinical signs was identified between survivors and nonsurvivors. the parasitemia ranged between 0.5 and 3.1% (median, 1.2% ; interquartile range [iqr ], 0.831.63), but no statistical difference was identified in the level of parasitemia between the survivors and nonsurvivors. results of laboratory findings as well as comparison between outcome groups are summarized in table 1. in both groups of dogs, mild to moderate normochromic normocytic nonregenerative anemia, mild to severe hyperbilirubinemia, mild to moderate azotemia, mild to moderate hypoalbuminemia, mildly increased alkaline phosphatase (ap) activity, moderate to severe hyponatremia, moderate hypocalcaemia, and a mild to moderate increase in crp concentration were observed commonly..001), triglycerides (p <.01), and phosphate (p <.05), and significantly lower hematocrit (p <.05), wbc counts (p <.01), total serum protein concentrations (p <.05), and thrombocyte counts (p <.05) than survivors. median values of various variables (minimum maximum value) in dogs with naturally acute babesia canis infections : a comparison between survivors and nonsurvivors these 7 initially identified prognostic factors were further analyzed by roc analysis (table 2). of all variables studied, lactate concentrations and wbc counts showed the best prognostic sensitivity and specificity (both 100%) to differentiate between survivors and nonsurvivors. all nonsurvivors (8 of 8) had moderate to severe hyperlactatemia (median, 8.35 mmol / l ; iqr, 7.189.13), whereas most survivors (6 of 7) had concentrations within the reference range (median, 1.6 mmol / l ; iqr, 1.052.3). the wbc counts for all of the survivors (6 of 6) were within the reference range (median, 6.85 10/l ; iqr, 6.038.2) unlike the group of nonsurvivors, which had mild to moderate leukopenia (6 of 6 ; median, 2.65 10/l ; iqr, 1.73.53). results of the roc analysis with prognostic cutoff values of significantly altered variables and respective sensitivity, specificity, area under the curve (auc), and standard error (se) associated with the outcome in babesia canis infected dogs set at the border of the reference range (calculated cutoff at 4.8 10/l). median and interquartile range for significant prognostic markers (p <.05) recorded at admission in naturally infected dogs that did or did not survive an acute b. canis infection. dots correspond to the data from individual dogs ; the shaded grey areas represent the reference intervals. the course of the prognostic variable, parasitemia, and the acute phase response was followed in the 3 dogs experimentally inoculated with b. canis. the 3 infected dogs became lethargic and showed signs of hemolysis (pale mucous membranes and pigmenturia) 105, 120, and 119 hours postinoculation (on days 45), respectively. they had a low grade parasitemia with a maximum of 1.75% of the erythrocytes infected at the end of the experiment, and during the course 2 of the 3 dogs had episodes of pyrexia (fig 2a). an acute phase response could be observed with a moderate increase in crp concentration and a moderate decrease in serum albumin concentration (fig 2b), whereas saa concentrations remained below the diagnostic limit (data not shown). the hematologic variables leukocytes, thrombocytes, and hematocrit was found before the identification of parasites in stained blood smears, and resulted in moderate leukopenia, severe thrombocytopenia, and decreased hematocrit. in general, changes in lactate, triglyceride, and phosphate concentrations corresponded to the first appearance of parasites, and they only exceeded the prognostic threshold at the first observation of acute crisis. in addition, thrombocytopenia was a common finding and platelet counts exceeded the prognostic threshold toward the end of the experiment. total serum protein concentrations also decreased over time but passed the threshold only in 2 of the 3 dogs before first signs of an acute breakdown. at the end of the experiment, the 3 dogs showed mildly increased levels of fibrinogen of 2.6 mg / l, 0.44 mg / l, and 0.92 mg / l (reference range, < 0.4 mg / l), respectively. dog 1 : solid line ; dog 2 : broken line ; dog 3 : dotted line. (a) body temperature (left yaxis) and parasitemia (right yaxis ; lines with dots). (b) crp (left yaxis) as a marker for positive acute phase response and albumin (right yaxis ; lines with dots) as a marker for negative acute phase response. dog 1 : solid line ; dog 2 : broken line ; dog 3 : dotted line. (a) blood lactate, (b) wbc, (c) triglycerides, (d) phosphate, (e) thrombocyte count, (f) total protein, (g) hematocrit. in this study, several variables were shown to be associated with poor outcome in acute babesia canis infections. by including 2 rapid inclinic tests, standard hematologi and biochemical variables, and acute phase proteins, we found the variables lactate, wbc, triglycerides, phosphate, thrombocytes, total serum protein, and hematocrit to be significant prognostic markers. thus, nonsurvivors at admission had more severe anemia, leukopenia, and thrombocytopenia in addition to alterations in their serum biochemical profile results. lactate concentrations were significantly lower in survivors and showed a clear difference from the nonsurvivors. this finding is similar to what is observed in dogs infected with babesia rossi, the agent of severe canine babesiosis in south africa, where serum lactate concentration is used for posttreatment monitoring,21 and high blood lactate concentrations correlate with poor outcome.32, 33, 34 nevertheless, the pathogenesis of hyperlactatemia in dogs with acute babesiosis is not well established, and it might not be caused by hypoxia as a consequence of anemia, which remains mild to moderate in most b. canis infected animals.34 hence, hypoxia in canine babesiosis may be the consequence of alterations in the macro and microcirulation triggered by protozoal sepsis, hypotension, dic, and sirs, all of which are well known in b. canis infections18, 35. indeed, increased lactate concentrations have prognostic value in sirs caused by various conditions.36, 37 the second variable that clearly differentiates between the 2 studied groups was wbc count. nonsurvivors had mild to moderate leukopenia in contrast to the survivors with wbc counts in the reference range. although the wbc count was a significant marker for outcome in our study, leukopenia was reported in 60% of mild cases of acute canine babesiosis.17 indeed, the wbc count fell below the prognostic cutoff before any clinical signs were observed in the experimentally inoculated dogs. severely affected dogs had mild to moderate neutropenia, with an overall degenerative tendency and lacking a left shift (see supplemental file 2). furthermore, lymphopenia seems to be a hallmark of acute canine babesiosis.17, 19 a markedly increased serum cortisol concentration was found in dogs with lethal b. rossi infections, indicating a potential immunosuppressed state in these animals, which also is indicated by an unexpected mild to moderate regenerative response of lymphocytes in dogs that survived.24, 38 furthermore, studies in humans with acute malaria infections with plasmodium falciparum and p. vivax, which are related to babesia spp., identified mechanisms that could explain a depletion of lymphocytes from the peripheral blood by acute sequestration of the cells in the lymph nodes or other parts of the body or by immune cell exhaustion and abnormal cell death through parasiteinduced apoptosis.39, 40 similarly, toxic parasitic factors have been shown to be involved in canine b. gibsoni infection.41 hemolytic anemia and thrombocytopenia are the most frequent abnormalities associated with a diagnosis of b. canis in naturally infected dogs and thrombocytopenia usually is the most dramatic hematologic abnormality in the course of babesiosis.12, 42, 43, 44 our data indicate that severe thrombocytopenia is associated with poor outcome by a prognostic cutoff of 27,500 thrombocytes per l, although a sensitivity and specificity of 83.3% for each indicates limited prognostic value. presumably, several factors are involved in the origin of thrombocytopenia in canine babesiosis including increased platelet activation and consumption by a sirs (hypercoagulable state), increased platelet sequestration and aggregation, and a decreased platelet production.19, 45, 46 comparable in b. rossi infections, poor outcome was associated with a consumptive coagulopathy, although even severe thrombocytopenia was not accompanied by apparent bleeding diathesis and hemorrhage.25, 47, 48 increased phosphate concentrations often are associated with metabolic acidosis characterized by tissue hypoxia and high blood lactate concentrations, although the underlying mechanisms have not been completely explained.49 hemorrhage, hypovolemia, and shock as cause or consequence of tissue hypoperfusion could further explain changes in altered variables, also including azotemia and potential proteinlosing nephropathy caused by hypoxic renal damage.35 complications related to hemolytic anemia, coagulation disorders and hypotension, sirs, and secondary impaired renal function likely account for the severe outcome of the infection.6, 9, 10, 12 furthermore, in other studies, acute respiratory distress syndrome, renal failure, immunemediated hemolytic anemia, cerebral syndrome, and dic were associated with increased mortality in acute b. canis infections.17, 50 acute phase proteins were used as prognostic factors for different inflammatory processes,51 and an acute phase response also was observed in acute b. canis infections.12, 13, 14, 20 we measured the acute phase proteins crp and saa, because they are considered major app in dogs52 and are not significantly affected by hyperbilirubinemia, which is commonly present in acute babesiosis.51 we found an increase in crp before parasite detection as previously observed,12 without any significant difference between the outcome groups. this finding is in accordance with findings in b. rossi infections in which no prognostic value for crp concentrations was observed.53 furthermore, the saa concentrations did not increase significantly in naturally and experimentally infected animals. this finding is in contrast to other observations of increased saa concentrations in dogs with babesiosis on the day of admission.14 as another indicator, serum albumin concentration could serve as a negative app.51 with the onset of acute infection, we observed a moderate decrease in serum albumin concentration and it had no prognostic relevance. although differences between survivors and nonsurvivors were absent for an acute phase response, app (among other variables) could serve as important variables for monitoring response to therapy.14, 54 in the course of validating prognostic markers in 3 experimentally inoculated dogs, we observed low grade parasitemia with a maximum of 1.75% of infected erythrocytes, which was comparable to the group of naturally infected animals. even in infections with serious clinical signs, low parasitemia is a common finding in b. canis infections.1, 6, 12 the course in the infected dogs highlights the prognostic value of lactate, triglycerides, and phosphate concentrations, and thrombocyte counts, because these factors only crossed the prognostic threshold in an acute crisis. missing data about the course of disease before admission and the time point of infection in the naturally infected dogs is a limitation of this study. generally, practitioners inquire about the duration of illness and the appearance of the first clinical signs, and they can estimate the time of the infection in affected dogs. in this respect, the prognostic markers are helpful for guiding clinical decision making. to get an overall picture of individual cases, a systematic collection of clinical, laboratorial, and other individual factors must be emphasized. for example, in our cohort of infected dogs, circulatory disturbances were detected in 4 relatively young dogs (7 month to approximately 3 years), of which 3 dogs died (see supplemental file 1). such clinical variables could affect outcome in the laboratory test results and the likely progression of a patient 's infection.55 in any case, outcome depends on a rapid diagnosis and early treatment. mortality in the investigated group of dogs was higher as compared to an endemic area.5 this finding reflects a typical situation for nonendemic areas such as switzerland, where dogs became infected from local babesia outbreaks or have traveled to an endemic area. these dogs likely never have had contact with the parasite and therefore did not develop partial immunity.46, 56 nonetheless, findings on mortality rates should not be over interpreted because of the small sample size. in our cohort unfortunately, we did not have precise data about infection rates in dogs in switzerland. however, during the sampling period, 2 indigenous outbreaks were reported in 44 dogs, of which 10 died.57, 58 most indigenous cases in our cohort originated from these areas (4 survivors and 1 nonsurvivor), whereas 1 dog originated from geneva, a known endemic region in switzerland.59 the remaining 9 infected dogs had a positive travel history. for example, from 2011 to 2013, the diagnostic unit of the institute of parasitology in zurich (which offers a travel screening panel) identified 2.1% of 804 samples as positive on blood smears for large babesia species (f. grimm, personal communication). this observation is in agreement with observed cases in dogs in germany that have travelled, with 3.7% (19/508) of animals positive for large babesia spp. in giemsastained blood or buffy coat smears.60 hence, to compensate for the small sample size, prognostic markers were crossvalidated in the course of experimental babesiosis. although a significant prognostic marker is not necessarily clinically relevant, the pathophysiologic reason for death would be of interest. with this in mind, additional studies should include postmortem examination, and more prognostic factor studies should be conducted including other nonroutine variables. this study focused on rapid inpractice tests (e.g. lactate and glucose determined by handheld analyzers) and routine laboratory variables, and the associated findings summarize the prognostic value of these variables. nevertheless, additional research is needed to evaluate what additional evaluation and intensive care is needed for dogs with a poor prognosis. in this context, several markers have been demonstrated as good variables for followup and posttreatment monitoring after antibabesial therapy, such as app, lactate, thrombocytes, and leukocytes.13, 14, 21, 43, 54 supplemental file 1. characteristics of the individual dogs (animal description, travel history, and clinical signs). differential wbc count in the course of 3 experimentally inoculated dogs, and in dogs that did or did not survive a naturally acquired acute babesia canis infection. | backgroundcanine babesiosis, caused by babesia canis, is a prevalent and clinically relevant disease in europe. severe acute babesiosis is characterized by a high mortality but prognosis is not always correlated with clinical signs nor with the level of parasitemia.objectivethis study evaluated prognostic markers associated with poor outcomes in acute babesia canis infections.animals and methodswe compared the results of routine laboratory profiles, handheld lactate and glucose analyzer, and the acute phase response in 2 groups of naturally infected dogs (7 survivors and 8 nonsurvivors). samples were collected at the time of first admission and before any treatment. subsequently, the course of prognostic markers was followed in 3 dogs experimentally inoculated with b. canis.resultsnonsurvivors showed significantly higher concentrations of lactate, triglycerides and phosphate and lower hematocrit, leukocyte counts, total serum protein concentrations, and thrombocyte counts when compared to survivors. all nonsurvivors (8/8) had hyperlactatemia, whereas most survivors (6/7) had values within the reference range. all survivors had leucocyte counts within the reference range, unlike the nonsurvivors, which showed leukopenia. during the course of acute babesiosis, the variables serum lactate, triglyceride, and phosphate concentrations, and thrombocyte count only exceeded a prognostic threshold during acute crisis.conclusions and clinical importancepoor outcome in acute b. canis infection is indicated by changes in the laboratory profile. intensive care should be considered for dogs presenting with moderate anemia, severe thrombocytopenia, mild to moderate leukopenia, hyperlactatemia, moderately increased serum phosphate, and triglyceride concentrations, and moderately decreased total serum protein concentrations. |
it is usually due to congenital dysplasia, trauma, strain, or other causes of abnormalities in the bony connection between adjacent vertebrae, leading to partial or complete slippage of one vertebrae on adjacent vertebrae. the typical symptoms of this condition are neurological deficits, including low back pain, nerve root irritation, and neural dysfunctions. a variety of surgical fusion techniques, such as anterior interbody fusion, posterior interbody fusion, posterolateral fusion, repair of the pars interarticularis, and reduction and fusion have been applied to stabilize the spine, relieve pain, and improve the patients life quality. radiological investigation is the key component of evaluation of lumbar spondylolisthesis to determine its anatomical abnormalities, etiologies, severity, and possible pathogenic mechanisms to guide the clinical management and assess the prognosis. for this purpose, a number of x - ray, ct, and mri techniques have been employed to analyze anatomy of vertebrae, lumbar lordosis (ll), and the facet joints associated with the occurrence of slippage. in contrast to the abundant radiological data on spondylolisthesis of single vertebral bodies, data for consecutive lumbar spondylolisthesis are absent, although multilevel lumbar spondylolisthesis does occur and accounts for up to 11% of spondylolisthesis. importantly, multilevel segmental involvement is of considerable significance for the occurrence of cauda equina syndrome. we identified a correlation between the forward displacement of the involved vertebrae and pelvic sagittal parameters. our findings suggest that pelvic compensatory mechanisms play a role in maintaining the overall sagittal spinal and pelvic stability. a total of 967 patients were diagnosed with and treated for spondylolisthesis at our hospital from june 2005 to march 2012. among them, 17 consecutive spondylolisthesis cases (1.75%) were identified in 5 males and 12 females with a median age of 56 years. out of these 17 patients, 7 had spondylolisthesis involving l3l4, and 10 had spondylolisthesis involving l4l5. the enrollment criteria for these patients were as follows : no spinal fractures, or scoliosis history ; no history of spinal surgery ; diagnosis of consecutive spondylolisthesis on a lateral lumbar spine x - ray. the lateral x - ray images for individual patients were retrieved from our picture archiving and communication systems (pacs). the taillard index was defined as the relative displacement distance between the involved vertebrae divided by the horizontal length of the upper vertebral body (fig. the pelvic incidence (pi) was denoted as the angle between the vertical line of s1 endplate and the line connecting midpoint of s1 endplate to midpoint of the femoral heads. the sacrum slope (ss) was defined as the angle between the s1 endplate and the horizontal line, while the pelvic tilt (pt) was the angle formed by the vertical line and the line connecting the midpoint of s1 endplate to the midpoint of the femoral heads (fig. the displacement between the upper and lower intervertebral space was determined on flexion extension dynamic x - ray radiographs (fig. 3). the measurement of taillard index : the forward displacement distance of upper vertebral body / the length of the upper vertebral body 100%. the measurement of the lumbar lordosis (ll), pelvic incidence (pi), pelvic tilt (pt), and sacrum slope (ss). the angular displacement of the upper intervertebral space was calculated as (a1b1) ; the angular displacement of lower intervertebral space was determined by calculating (a2b2). student t test was used to analyze the relative anterior displacement of the vertebrae and the angular displacement of the intervertebral spaces. pearson correlation analysis was applied to investigate the correlation between ll and pelvic sagittal parameters and the correlation between taillard index and angular displacements. this study was approved by the ethical committee of the third hospital of hebei medical university, shijiazhuang, hebei, china. a total of 967 patients were diagnosed with and treated for spondylolisthesis at our hospital from june 2005 to march 2012. among them, 17 consecutive spondylolisthesis cases (1.75%) were identified in 5 males and 12 females with a median age of 56 years. out of these 17 patients, 7 had spondylolisthesis involving l3l4, and 10 had spondylolisthesis involving l4l5. the enrollment criteria for these patients were as follows : no spinal fractures, or scoliosis history ; no history of spinal surgery ; diagnosis of consecutive spondylolisthesis on a lateral lumbar spine x - ray. the lateral x - ray images for individual patients were retrieved from our picture archiving and communication systems (pacs). the taillard index was defined as the relative displacement distance between the involved vertebrae divided by the horizontal length of the upper vertebral body (fig. 1). the ll was the angle between the l1 endplate and s1 endplate. the pelvic incidence (pi) was denoted as the angle between the vertical line of s1 endplate and the line connecting midpoint of s1 endplate to midpoint of the femoral heads. the sacrum slope (ss) was defined as the angle between the s1 endplate and the horizontal line, while the pelvic tilt (pt) was the angle formed by the vertical line and the line connecting the midpoint of s1 endplate to the midpoint of the femoral heads (fig. the displacement between the upper and lower intervertebral space was determined on flexion extension dynamic x - ray radiographs (fig. 3). the measurement of taillard index : the forward displacement distance of upper vertebral body / the length of the upper vertebral body 100%. the measurement of the lumbar lordosis (ll), pelvic incidence (pi), pelvic tilt (pt), and sacrum slope (ss). the angular displacement of the upper intervertebral space was calculated as (a1b1) ; the angular displacement of lower intervertebral space was determined by calculating (a2b2). the data were analyzed with spss13.0. student t test was used to analyze the relative anterior displacement of the vertebrae and the angular displacement of the intervertebral spaces. pearson correlation analysis was applied to investigate the correlation between ll and pelvic sagittal parameters and the correlation between taillard index and angular displacements. this study was approved by the ethical committee of the third hospital of hebei medical university, shijiazhuang, hebei, china. twenty isthmic (upper and lower vertebrae) and 14 (upper and lower vertebrae) degenerative consecutive spondylolisthesis were identified in 34 vertebral bodies in 17 patients (table 1). among the above mentioned 7 patients with l3l4 consecutive spondylolisthesis. out of the 10 patients with consecutive spondylolisthesis, the average taillard index of the upper vertebrae was 17.6 4.1%, whereas its value for the lower vertebrae was 22.4 4.1% (t = 7.672, p the average angular displacement of the upper vertebrae was 10.8 2.6, whereas that of the lower vertebrae was 18.6 5.5 (t = 5.251, p 0.05). for example, isthmus crack spondylolisthesis can be due to a congenital isthmus defect, an acute lumbar trauma leading to an isthmus fracture, stress - related fractures caused by chronic fatigue on the basis of congenital isthmus dysplasia. this condition is commonly detected in the 5th lumbar vertebral body in 30- to 40-year - old adults, as males and females are affected approximately equally. on the other hand, degenerative spondylolisthesis is usually secondary to intervertebral disc degeneration and commonly affects l4l5 in 50- to 60-year - old women. although the diverse causative factors usually lead to single - level spondylolisthesis, we found that consecutive spondylolisthesis is not a rare condition, especially in individuals performing long - term heavy physical labor, such as the subjects enrolled in the present study. we also discovered that pelvic compensation mechanisms play a key role in maintaining the overall sagittal spinal and pelvic stability in this condition. to the best of our knowledge, due to its distinct anatomical nature, isthmic spondylolisthesis is considered to be inherently more instable than degenerative spondylolisthesis, a fact reflected by the difference in the angular displacement existing between these 2 types. in the present study, identical types of spondylolisthesis in the 2 levels of vertebrae were present in each individual. therefore, we believe that the effects exerted by the type of spondylolisthesis on angle displacement were minimal in the studied patients. interbody gravity force is transmitted and divided into a compression force perpendicular to the shift force that is parallel to the vertebral endplates. in general, lower vertebral bodies bear greater shearing forces with respect to the upper vertebrae, which can accelerate intervertebral degeneration in the disc and facet joints. consequently, from an anatomical standpoint, the range of movement in the discs that are closer to the lumbosacral region is greater than that in those located away from the lumbosacral region. in line with this mechanism, we found that the angular displacement in the lower vertebral bodies was much more prominent than that in the upper vertebrae in the clinical setting of consecutive spondylolisthesis. it is worth noting that the antishearing force mechanisms are compromised in lumbar spondylolisthesis. to compensate for this defect, a series of compensatory changes these adaptive changes are usually reflected by the alteration of spine pelvis sagittal parameters, such as pi. a number of studies have shown that the extent of increase of pi values in patients with single - level lumbar spondylolisthesis is positively correlated with the severity of displacement of the involved vertebrae. barrey has further suggested that pi value is to some extent predictive for displacement of affected vertebrae in 1-level spondylolisthesis. we calculated the pi values in 2-level spondylolisthesis and found that the average pi value in consecutive spondylolisthesis was higher than the reported ones in single - level segment spondylolisthesis (68.7 vs 66.3), and not surprisingly either, higher than that in healthy individuals. we postulated that the high pi values in consecutive spondylolisthesis might be a risk factor for displacement of the affected vertebrae. nevertheless, to evaluate the biomechanical relevance of an increased pi value in spondylolisthesis requires further prospective studies. any change in orientation within an anatomical spine segment will cause adaptive changes in adjacent segments in order to maintain the stability of the body. for example, vialle have reported that ss values in single - vertebral lumbar spondylolisthesis gradually increase along with the degree of displacement in spondylolisthesis grade i although our results clearly demonstrated that consecutive spondylolisthesis resulted in a modification of pelvic parameters, our data indicated that the values of ss (37.2) and pt (31.6) in patients with consecutive spondylolisthesis were higher than those observed in the healthy population (pt = 25.1 and ss = 30.7) in the same age group. in the present study, all the taillard indexes were below 50%, classifying the grade of spondylolisthesis as i or ii. we hypothesize, that the compensatory mechanisms in the spine pelvis sequence in 2-segment spondylolisthesis might be different from that in 1-level spondylolisthesis. another set of data supporting this notion was the failure of identification of the existence of a significant correlation between pt and ss, which was compatible with the reported finding indicating that pi = ss + pt, and there are opposite trend shifts of pt and ss in spondylolisthesis. first, consecutive spondylolisthesis may cause a distinct pelvic spine compensation which leads to a discordant change in pt and ss. second, the small sample size and selection bias in the present study might have led to the occurrence of this discrepancy. as a limitation of the current investigation, we have to point out that the data collected in the present study are derived from x - ray images only. since whole - spine imaging was not employed, we were unable to conduct an analysis of the whole - spine sequence. in summary, our study showed that in the spondylolisthesis of 2 adjacent lumbar segments both the degree of the vertebral slip and the angular displacement of the lower vertebrae were greater, than those of the upper vertebrae, indicating that the compensatory mechanism of the pelvis plays an important role in maintaining the sagittal balance. | abstractradiographic features of consecutive lumbar spondylolisthesis were retrospectively analyzed in a total of 17 patients treated for this condition at the third hospital of hebei medical university from june 2005 to march 2012.to investigate the radiographic features, pelvic compensatory mechanisms, and possible underlying etiologies of consecutive lumbar spondylolisthesis.to the best of our knowledge, there is no previous report concerning the characteristics of consecutive lumbar spondylolisthesis.the taillard index and the lumbar lordosis (ll), pelvic incidence (pi), sacrum slope (ss), and pelvic tilt (pt) were determined on lateral x - ray images, and the angular displacement was analyzed on flexion extension x - ray images. correlation between ll and various pelvic parameters and correlation between taillard index and angular displacement were assessed by pearson correlation analysis.a total of 20 cases of isthmic spondylolisthesis and 14 of degenerative spondylolisthesis were retrospectively studied in 17 patients. the taillard index and the angular displacement in the lower vertebrae were both larger than those in the upper vertebrae. statistical analysis revealed that ll was correlated with pi and pt, whereas pi was correlated with pt and ss. however, no correlation was identified between taillard index and angular displacement.in consecutive lumbar spondylolisthesis, the degree of vertebral slip and the angular displacement of the lower vertebrae were both greater than those of the upper vertebrae, indicating that the compensatory mechanism of the pelvis plays an important role in maintaining sagittal balance. |
sample size was calculated to measure a difference of greater than 2 mm of hg between the two instruments with an estimated standard deviation of 4 mm, for 80% power and a type 1 error of 5%. we needed 34 patients to measure this difference and recruited 40 consecutive adult subjects attending a glaucoma prevalence study for the trial. all subjects who could undergo visual acuity measurement, refraction, slit - lamp examination and applanation tension measurement were eligible. corneal pathology including astigmatism of 2 diopter (d) or greater, inability to measure iop or history of allergy to proparacaine or fluorescein were reasons for exclusion. intraocular pressure was measured by one of two examiners, using the zeiss at 030 (gatz) (carl zeiss, jena, germany) and the inamil-5110 (gati) (inami and co., tokyo, japan) goldmann type applanation tonometers. at the start of the day the instruments were calibrated as per the manufacturers instructions and were used for examination only if they were accurately calibrated. to minimize the influence of any iop - lowering effect induced by applanation tonometry on the results, the sequence of measurements was randomized. applanation tonometry was performed first on one randomly selected instrument followed immediately after by measurement on the second instrument. the iop was measured after anesthetizing the cornea with sterile 0.5% proparacain eye drops (paracain, sunways, mumbai, india) and staining the tear film with fluorescein strips. the tonometer was set to the zero mark prior to the start of the examination. measurements on both instruments for a subject were performed by a single observer who was blinded to the actual readings, which were read and recorded by the second examiner who then reset the applanation tonometer to the zero mark. the iop was measured two consecutive times for each eye on each instrument, and the mean of the two readings was used for analysis ; if there was a difference of greater than 2 mm of hg between the readings, a third measurement was taken and the median of three readings was taken. by convention, the right eye was examined first for every patient. the iop was measured on the second instrument, placed in the same examination room, almost immediately after completing recording on the first instrument. intraocular pressure was compared using the paired t test and agreement was assessed by the altman and bland plot.6 forty eyes (40 subjects, mean age : 53.3 sd 7.9 years, 22 males, 18 females) were included. mean iop (sd) on gatz was 15.32 (6.80) mm hg (range:9 mm hg - 36 mm hg) and on gati 13.52 (5.65) mm hg (range:7 mm hg - 30 mm hg) (p<0.001, 95% ci of the difference : -2.48 to -1.11). the bland and altman plot [figure 1 ] revealed a tendency for higher iop recordings on the gatz (95% limits of agreement:-2.47 to 6.16 mm hg). the absolute level of iop may not be as important to glaucoma diagnosis as was once thought, however, the trend of iop measurements is relevant in the management of glaucoma patients. we attempted to minimize their influence on the study results. in order to avoid an observer bias, two observers were used for the study - however, the same observer made iop measurements on both instruments for a single patient. to minimize the effect of a possible transient lowering of iop following applanation tonometry, on the results, we randomly allocated the order of iop measurement by either machine. the iops on the second instrument were measured within a few minutes of the measurement on the first machine to minimize any temporal variation in iop. lower iop readings were consistently recorded on the inami at. in 70% of subjects iop differences were within the clinically acceptable range of 2 mm. with both instruments calibrated accurately as per the manufacturers recommendations better agreement between the instruments would be expected. since manometric measurements were not made in any of our subjects it is difficult to comment on which instrument was giving erroneous results. the clinical implications of this variation on disease diagnosis are considerable since both instruments are widely used in asia. it could result in potential misdiagnosis of normal tension glaucoma (ntg) or primary open angle glaucoma (poag). follow - up may not be affected as significantly as long as all measurements are made on a single instrument by a single observer keeping other factors such as calibration and the time of measurement constant. however, with increasing cross - referrals and the use of multiple instruments in various clinics the possibility of lack of agreement between devices has to be kept in mind. with the large number of similar applanation tonometers available, agreement between other commercially available devices needs to be assessed. in this study if this was indeed the case there should not have been a significant difference between devices and certainly not a difference of the magnitude that was found. perhaps, the manufacturers need to reassess the calibration procedures for these devices. in conclusion, we demonstrate yet another variable in iop measurements, the effect of which could be minimized by performing baseline and follow - up iop measurements on a single instrument. additionally, therapeutic decisions should be made keeping in mind the possibility of an inter - instrument variability. | the aim of the study was to assess agreement between two commercially available applanation tonometers for the measurement of intraocular pressure (iop). forty subjects underwent iop measurement on two accurately calibrated goldmann type applanation tonometers (zeiss at 030 (gatz) and inami l-5110(gati)). the order of examination was randomized and observers were masked to the iop recorded. the mean of two consecutive readings, from a randomly selected eye for each subject, was used for analysis. agreement was assessed using the altman and bland plot. the mean (sd) iop readings on gatz was 15.32 (6.80) mm hg and on gati was 13.52 (5.65) mm hg (p<0.001, 95% ci of the difference : -2.48 to -1.11). the 95% limits of agreement on the altman and bland plot were:-2.47 to 6.16 mm hg). there was significant inter - instrument variability between the two accurately calibrated goldmann type applanation tonometers studied. |
decentration can lead to undesired complications such as decreased visual acuity, astigmatism, glare, and monocular diplopia, which would overcome the purposes of the surgery (1, 2). to minimize the risk of decentration measurement of the angle kappa between the visual and papillary axes plays an important role in this process. positive and negative angle kappa values correspond to the reflection of corneal light nasally and temporally, respectively (3). a positive angle kappa 5 is physiologic, whereas values > 5 can lead to pseudostrabismus (4). hyperopic eyes show large angle kappa in comparison to myopic eyes ; therefore, a small decentration in these eyes can lead to severe complications. the angle kappa is commonly measured in cases of strabismus and plays an important role in the preoperative assessment. one method that can be used to measure the angle kappa is the orbscan ii device, which is widely used preoperatively in photoablation surgeries. the purpose of this study was to determine the mean angle kappa and its intercepts among normal young adults, who comprise the target population for keratorefractive surgery. in a non - random simple sampling, a total of 977 cases among the patients, who were referred to the khatam eye center (a specialized eye hospital in mashhad, northeast of iran) from march 2011 to february 2012 and claimed to be healthy, were enrolled in this observational cross - sectional study. exclusion criteria were as follows : history of any deviation or strabismus, with or without orthoptic or surgical treatment ; any intraocular, corneal, or keratorefractive surgery ; using contact lens ; any corneal anomaly ; any ophthalmic or systemic drug consumption ; and hyperopic spherical refraction > + 3.00 diopters (d), myopic spherical refraction 2.00 d. these criteria were defined to exclude cases with refractive errors that tend to induce pathologies. this study was registered with the ethics committee of mashhad university of medical sciences, and clearance was obtained. considered ethical codes for this study were 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 14, 17, and 20. complete ophthalmologic and orthoptic examinations were performed, including slit lamp biomicroscopy and dilated pupil fundoscopy. the orbscan ii device (bausch and lomb, technolas, ny) one acquisition was done per eye, unless an unacceptable result was obtained that would mandate repetition. for measurements with the orbscan ii device the examiner used the joystick of the device to make adjustments such that the final result would be a sharply focused image of the eye. descriptive analysis was used to determine the mean values and the student 's t - test was used to compare differences. in a non - random simple sampling, a total of 977 cases among the patients, who were referred to the khatam eye center (a specialized eye hospital in mashhad, northeast of iran) from march 2011 to february 2012 and claimed to be healthy, were enrolled in this observational cross - sectional study. exclusion criteria were as follows : history of any deviation or strabismus, with or without orthoptic or surgical treatment ; any intraocular, corneal, or keratorefractive surgery ; using contact lens ; any corneal anomaly ; any ophthalmic or systemic drug consumption ; and hyperopic spherical refraction > + 3.00 diopters (d), myopic spherical refraction 2.00 d. these criteria were defined to exclude cases with refractive errors that tend to induce pathologies. this study was registered with the ethics committee of mashhad university of medical sciences, and clearance was obtained. considered ethical codes for this study were 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 14, 17, and 20. complete ophthalmologic and orthoptic examinations were performed, including slit lamp biomicroscopy and dilated pupil fundoscopy. the orbscan ii device (bausch and lomb, technolas, ny) one acquisition was done per eye, unless an unacceptable result was obtained that would mandate repetition. for measurements with the orbscan ii device the examiner used the joystick of the device to make adjustments such that the final result would be a sharply focused image of the eye. descriptive analysis was used to determine the mean values and the student 's t - test was used to compare differences. a total of 977 healthy young adults, aged 18 to 45 years, participated in this observational cross - sectional study. the study population consisted of 614 females and 363 males (p < 0.001). figure 1 shows the distribution of angle kappa in degrees in different ages according to sex. the mean angle kappa was 4.97 1.38, with a mean of 5.00 1.36 in males and 4.96 1.30 in females (p = 0.63). the means of angle kappa in the right and left eyes were 5.16 1.44 and 4.78 1.31, the means of horizontal (x - axis) angle kappa were -0.44 0.28 and 0.37 0.25 in the right and left eyes, respectively (p = 0.012). the means of vertical (y - axis) angle kappa were -0.11 0.31 and -0.07 0.34 in the right and left eyes, respectively (p = 0.01). the mean of horizontal angle kappa was -0.02 0.49, with a mean of -0.02 0.50 in males and -0.02 0.49 in females (p = 0.93). the mean vertical angle kappa was -0.09 0.32, with a mean of -0.09 0.33 in males and -0.09 0.32 in females (p = 0.74) (table 2). we measured the variables in the right and left eyes separately (tables 3 and 4). the angle kappa plays an important role in refractive surgery, and inaccurate preoperative measurements of this angle can lead to serious surgical complications associated with decentration (1, 5). in patients with strabismus, precise measurement of the angle kappa prevents the under - calculation or over - calculation of the degree of deviation (3). in patients with a large angle kappa, the lens of prescription glasses can result in a large prismatic effect, which can disturb the best - corrected visual acuity (6). angle kappa is increased with age (7). to achieve more accurate results in the preoperative assessment, we measured the angle kappa in a large sample of a normal population. hashemi. previously measured the angle kappa distribution in a population of tehran residents with a wide age range (6). their findings encouraged us to restrict our sampling to the population that was most likely to undergo refractive surgery (age range, 18 - 35 years). therefore, our results might be more reliable for estimating the angle kappa in this population (8). we used the orbscan ii device because of its very precise measurements and excellent reproducibility with only one acquisition per eye. to reduce the risk of systematic bias, a single operator used the orbscan ii. the orbscan ii finds the center of the pupil and finds where the perpendicular axis to the pupil center intercepts the cornea. in the reports, the plus or cursor sign (+) shows the corneal apex and the small " k " shows the intercept of the pupillary axis. the cornea periphery is assumed 360, initiating at the 3-oclock position and rotate counterclockwise. for example, the locus is at 346.47. therefore, the kappa angle is 4.59 at (@) 346.47. the third and fourth are the intercepts in x - axis and y - axis, respectively. the dimension of the intercept is divided into x - axis (horizontal) and the y - axis (vertical). in our example, it is + 0.19 mm in the left of the corneal apex and -0.11 mm below the corneal apex. among the 977 individuals (1954 eyes) who were examined, there were more women than men (p < 0.001), and the values obtained from women would have more effect on the overall results. the angle kappa for both men and women decreased with age, according to the linear regression test. we observed a significant difference between the mean angle kappa in the right and left eyes. no previous study had obtained this result, which did not seem to be accidental due to the large number of cases. further investigations are needed to resolve this difference. in comparison to the reported value of 4.96 1.38 in this study, hashemi. reported a mean angle kappa of 5.46 previously (6) ; however, we used a different age range with a smaller refractive error than they did. because a higher refractive error can result in larger angle kappa, lower angle kappa results were expected in our study. (4) used an age range similar to ours (20 - 40 years) and observed an average angle kappa of 5.22. in wachler. the angle kappa was reportedly greater in patients with hyperopia (8) while hashemi. unfortunately, we did not divide patients into groups on the basis of their refraction and hence, we could not comment on the angle kappa distribution among different refractive groups. in another recently published study, performed in our center, the angle kappa changes after photorefractive surgery were evaluated in a population different from this study s and it was found that in 96 eyes, the angle kappa had not changed significantly after the surgery (4.97 1.24 preoperatively and 4.99 1.10 at six months postoperation). findings of the current study were the same as our previous population (9). moreover, we found just another document about the measurement of kappa angle in normal population. basmak. in 100 normal individuals, compared synaptophore with the orbscan, and the effect of refraction on the kappa angle. they found that a correlation exists between positive refractive errors and large positive angle kappa values. moreover, data gathered by orbscan showed a little bit higher values in comparison to synaptophore ; however, the orbscan system provides angle kappa values with quantitatively established precise measurements (10). park. have reviewed the concept of angle kappa, its measurement and distribution in normal populations, and its implications in refractive surgery and concluded that evidences about the kappa angle effects on refractive surgery were growing. decentered treatment might be induced by ignoring it and visual symptoms might aggravate the vision. in treating refractive errors in modern era, compensating kappa angle is important in achieving optimal correction (11). although a large sample size in this study could assume a good evaluation of the amount of angle kappa in refractive surgery cases, one of our limitations was ignoring the spherical equivalent of patients for more precise classification of angle kappa, which is known to be affected by this variable. | background : the angle kappa is important in proper centration of corneal ablation in keratorefractive surgery. orbscan ii device is widely used preoperatively in photoablation surgeries and can be used to measure the angle kappa.objectives:this study aimed to determine the mean angle kappa and its intercepts in healthy young iranian adults.patients and methods : in this cross - sectional study, orthotropic patients (age range, 18 - 35 years) who were referred to the khatam eye hospital (mashhad, iran) were included. exclusion criteria were as follows : history of any eye deviation or strabismus with or without orthoptic or surgical treatment ; any intraocular, corneal, or keratorefractive surgery ; contact lens use ; any corneal anomaly ; any ophthalmic or systemic drug consumption ; and hyperopic spherical refraction > + 3.00 diopters (d), spherical refraction > -5.00 d, or cylindrical refraction > 2.00 d. all of the parameters were measured by the same operator through an orbscan ii device.results:a total of 977 healthy participants who aged 18 to 45 years were included consecutively. the study population consisted of 614 females and 363 males. the average angle kappa was 5.00 1.36 at 240.21 97.17 in males and 4.97 1.30 at 244.22 94.39 in females (p = 0.63). the average horizontal (x - axis) angle kappa was -0.02 0.49, with a mean of -0.02 0.50 in males and -0.02 0.49 in females (p = 0.93). the average vertical (y - axis) angle kappa was -0.09 0.32, with a mean of -0.09 0.33 in males and -0.09 0.32 in females (p = 0.74).conclusions : by using the normal angle kappa determined in this study, pseudodeviations can be identified more precisely in those who might undergo keratorefractive surgery. |
in 1951, eldon j. gardner (1909 1989), a college teacher of genetics, first described the syndrome, as a rare autosomal, dominant, inherited disorder with a high degree of penetrance, characterized by a triad of multiple osteomas, colonic polyposis, and mesenchymal tumors of the skin and soft tissues. it is a variant of the familial adenomatous polyposis syndrome, known to be caused by the mutation in the adenomatous polyposis coli (apc) gene located on chromosome 5q21 (band q21 on chromosome 5). the incidence of gardner 's syndrome in the general population has been estimated as one in 14,025 live births. the mandible is the most common location, however, osteomas may occur in the skull, paranasal sinuses, and the long bones. osteomas precede the clinical and radiographic evidence of colonic polyposis or gardner 's syndrome, therefore, may be insightful markers for the disease. it is also proposed that radiography of the jaws may serve as a valuable tool for the early detection of carriers of gardner 's syndrome. the epidermoid cyst is the most common cutaneous finding in gardner 's syndrome and is present in multiple forms in 50 60% of the patients and may occur on the face, extremities, and / or the scalp. desmoid tumor is benign fibrous proliferation that occurs under the skin predominantly in the abdominal wall. our patient was a 25-year - old female with a history of multiple swellings in the lower jaw that had developed over a period of 11 years. an extraoral examination revealed gross facial asymmetry, because of a diffuse swelling on the left side of the mandible measuring about 3 cm 4 cm in size, extending from the parasymphysis to the posterior border of the mandible [figure 1 ]. similar swellings, three in number, were present on the inferior aspect on the right side of the mandible. on palpation, all the swellings were bony hard in consistency, non - tender, and were fixed to the mandible. a 25-year - old female patient with diffuse bilateral swellings on the lower third of face along the mandibular border. intraoral examination revealed a diffuse swelling, obliterating the right buccal vestibule, measuring about 2 cm 2 cm in size, and extending from the mesial aspect of tooth 46 to the distal aspect of tooth 48 anteroposteriorly. on palpation, all the swellings were bony hard in consistency and non - tender. there were clinically missing teeth 21 and 23 (no history of extraction revealed) and over - retained tooth 63. patient had a small swelling measuring about 1 cm 1 cm on the scalp, which was fluctuant and cystic in nature (epidermoid / sebaceous cyst). the patient did not have any symptoms of rectal bleeding, cramping abdominal pain, weight loss, diarrhea or small bowel or colonic obstruction, and was reluctant to get a colonoscopy done to rule out the presence of polyps in the intestine, colon, and rectum. the clinical and radiographic findings, of osteomas, odontomes, impacted teeth, and sebaceous cyst suggested a diagnosis of gardner 's syndrome. our patient 's need was aesthetic improvement, for which the osteomas were surgically excised and sent for histopathological examination. the patient was followed up for one year and has remained asymptomatic, with no relapse of the lesions [figure 2 ]. postoperative radiograph after removal of osteomas, impacted teeth, and implant placement, with respect to tooth 21. the patient underwent maxillary and mandibular cross - sectional occlusal radiograph, orthopantomograph (opg), and posteroanterior radiograph [figure 3 ]. the opg revealed impacted teeth 21 and 23 and homogenous radiopaque masses over the right and left inferior aspect of the mandible, suggestive of osteomas [figure 4 ]. also, multiple small, homogeneous radiopaque masses surrounded by a radiolucent halo could be seen throughout the maxilla and mandible, suggestive of complex odontomes. diffuse sclerotic masses were present throughout the body of the mandible giving it a mottled appearance. impacted teeth 21 and 23 and the complex odontomes in the near vicinity were better visualized in the maxillary cross - sectional occlusal radiograph [figure 5 ]. opg demonstrates impacted teeth 21 and 23, homogenous radiopaque masses over the right and left inferior aspect of the mandible suggestive of osteomas (large arrows). also multiple small, homogeneous radiopaque masses surrounded by a radiolucent halo could be seen throughout the maxilla and mandible suggestive of complex odontomes (small arrows).diffuse sclerotic masses are present throughout the body of mandible giving it a mottled appearance maxillary occlusal radiograph shows impacted teeth 21, 23 (large arrows) and odontomes (small arrows) in the near vicinity. the histopathological report revealed the presence of compact lamellar bone with a haversian canal, lacunae, histiocytes, and reversal and resting lines suggestive of osteoma [figure 6 ]. photomicrograph shows the presence of compact lamellar bone with haversian canal, lacunae, histiocytes and reversal and resting lines suggestive of osteoma. it is an autosomal dominant condition with an incidence between 1 in 8300 and 1 in 14,025 live births, affecting both genders equally, with a uniform worldwide distribution. symptoms are usually present by the end of the second decade of life, but they may present anytime between two months and 70 years. as the clinical and radiological features in the maxillofacial region, such as osteomas, skin cysts, atypical skin pigmentation, and abnormal dental findings or radiographical lesions can precede frequent asymptomatic intestinal polyps for many years, dental practitioners and radiologists should be familiar with the manifestations of this syndrome. unlike most patients with gardner 's syndrome who present with gastrointestinal symptoms such as bloody diarrhea and pain in the abdomen, our case showed the classical clinical features associated with the syndrome : multiple osteomas and odontomes, epidermoid cyst, and impacted teeth. skeletal abnormalities appear in approximately 90% of the patients with gardner 's syndrome, and osteoma is considered to be the most common. supernumerary teeth, compound odontomes, and / or impacted teeth are seen in 30% of the patients with this disease. the gastrointestinal manifestations of gardner 's syndrome include colonic adenomatous polyps (tubular, villous, tubulovillous), gastric and small intestinal adenomatous polyps (12% of the patients), and periampullary carcinomas (2% of the patients). in our patient patients may also suffer from congenital hypertrophy of the retinal pigment epithelium, although the finding was absent in our case. panoramic radiography by the dentist can be useful for early detection of gardner 's syndrome, because the components of this entity, like osteoma, odontoma, supernumerary, and impacted teeth, can be detected. however, panoramic radiography is of limited value in the evaluation, localization, and extension of the tumor mass, considering the superimposition of the bony structures and the two - dimensional image. therefore, dental professionals should be familiar with the significance of the syndrome as a pre - cancerous condition. dental management includes removal of the impacted teeth, cysts of the jaw or face, as well as, resection of osteomas for functional or cosmetic reasons. tooth extraction can be difficult because of generalized increased alveolar bone density and complete absence of periodontal space caused by hypercementosis. osteomas of the skull, angle of the mandible and its inferior surface are classic bony features, in addition to intestinal ployps, in patients with gardner syndrome. a practicing dental surgeon should be aware of this entity in which intestinal polyps have 100% chance of malignant transformation. | gardner 's syndrome is an autosomal dominant disease and is a subtype of familial adenomatous polyposis. it is characterized by adenomatous intestinal polyps, multiple osteomas in the skull, maxillae, mandible, and multiple cutaneous and subcutaneous masses (epidermoids and desmoid). intestinal polyps, if not treated, have 100% chance of becoming malignant. we report a case of a 25-year - old female patient with gardner 's syndrome, with clinical manifestations including impacted supernumerary teeth, odontomes, sebaceous cyst on the scalp, and osteomas. it is important for the general dental practitioners to be aware of the clinical and radiological characteristics of gardner 's syndrome. |
tuberculosis (tb) is one of the most common infections worldwide, and in 2012, an estimated 8.6 million people developed tb and 1.3 million died from the disease (including 320,000 deaths among hiv - positive people) [1, 2 ]. mycobacterium tuberculosis (mtb) is an intracellular pathogen capable of infecting and surviving within the host s mononuclear cells particularly macrophages. elimination of the microorganism is through a combination of various killing mechanisms including apoptosis of host macrophages. these responses are orchestrated by t helper 1-type (th1) pro - inflammatory cytokines, which are synthesized by phagocytes upon recognition of pathogen - associated molecular patterns (pamps) on mtb by pattern recognition receptors (prrs). mtb is usually transmitted via aerosols and establishes a stable infectious state in the respiratory system. there, mtb is engulfed by macrophages and dendritic cells (dcs), which serve as host cells for mtb survival and propagation. binding of mtb ligands to tlr-2, -4 and -9 initiates release of inflammatory mediators, expression of adhesion molecules and further recruitment of macrophages, dcs and pmn to the mtb infected area. although the host s innate immune response to mtb infection is critical for the initial defense against bacteria, the adaptive immune response is ultimately required for containment of the infection in the chronic stage of disease. adaptive immunity to mtb infection is characterized by the appearance of antigen specific cd4 + t - cells that secrete ifn-, which, in turn, activates macrophages and other antigen presenting cells (apc) to kill intracellular bacteria. cd8 + t - cells are also important cells for controlling mtb during the chronic phase of infection [7, 8 ]. in addition, th17 cells and il-17 have been reported to be involved in the pathogenesis of mtb. il-17 is a proinflammatory cytokine produced by th17 cells and by airway structural cells, which provides ifn--dependent or ifn--independent protection to mtb infection (see figs. 1 and 2) [1012].fig. 1schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria. abbreviations : clr c - type lectin receptors, mtb mycobacterium tuberculosis, tlrs toll like receptors, nlrs nod - like receptorsfig. 2the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells. abbreviation : tlr toll like receptor, mtb mycobacterium tuberculosis schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria. abbreviations : clr c - type lectin receptors, mtb mycobacterium tuberculosis, tlrs toll like receptors, nlrs nod - like receptors the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells. abbreviation : tlr toll like receptor, mtb mycobacterium tuberculosis indeed, in models of mtb infection, il-17 and th17 cells were first implicated in the protective immune response to rapidly growing extracellular bacteria in the lung and gut mucosal surfaces through efficient induction of neutrophil recruitment and tissue repair [1315 ]. il-17 and th17 cells are important during the initial stages of infection and act upon hematopoietic and non - hematopoietic cells to promote the secretion of antimicrobial peptides such as g - csf and cxc chemokines. as a consequence of this, dcs migrate to the local lymph nodes and induce the differentiation of both th1 and th17 cells. the increased levels of chemokines in the infected lung also promote recruitment of other protective cells such as macrophages and pmn and the formation of mononuclear granulomas. moreover, an accumulation cytokines such as il-6 and il-23 in the lungs can further induce the differentiation and activation of th17 cells and accelerate the pathogenesis of tb. in this review we focus on the role of signal transduction pathways which have an impact on the pathogenesis of tb. among these, the generation of ros and the later activation of pprs including tlrs and of the inflammasome are highlighted. reactive oxygen species (ros) and reactive nitrogen species (rns) are considered to play important role in the pathogenesis of various inflammatory diseases [17, 18 ]. under physiologic conditions ; ros are generated as byproducts of oxygen metabolism. ros are found in all biological systems and originate from the metabolism of molecular oxygen (o2). under physiological conditions o2 undergoes reduction by accepting four electrons which results in the formation of water. during this process, reactive intermediates such as the superoxide anion (o2), hydrogen peroxide (h2o2) and hydroxyl (oh) radicals are formed. activated macrophages express two major enzymes, phagocyte oxidase (nox2/gp91) and inducible nitric oxide synthase (nos2), which are able to generate reactive oxygen intermediates (roi) and reactive nitrogen intermediates (rni), respectively. upon phagocytosis, the preformed nox2 subunits assemble into an enzymatically active enzyme complex that transfers electrons across the membrane from cytosolic nadph to molecular oxygen. this produces o2 which dismutate into hydrogen peroxide (h2o2) and thus generate oh radicals which are toxic to mtb. following inhalation of mtb, alveolar macrophages engulf the bacilli and initiate their killing using a number of mechanisms including the generation of roi and rni [21, 22 ]. the rapid generation of ros is critical in host defense against many bacteria and fungi, and ros has broad signaling functions. for example, the nadph oxidase protein complexes generate the superoxide anion and downstream ros. thus, nadph oxidase has an important role in host defense against mtb and any patients with a loss of function mutation in genes encoding components of this enzyme complex could be deficient in killing bacilli. indeed, mutations in the cybb gene encoding the gp91 (phox) subunit of the phagocyte nadph oxidase is associated with mtb. in addition, a hemizygous splice mutation in intron 5 of cybb was linked to the concomitant occurrence of chronic granulomatous disease (cgd) with mtb. ifn- induces nos2 and its product nitric oxide (no) which in turn can be broken down to nitrite and nitrate. under acidic conditions, such as within the phagosomes of ifn- activated macrophages, nitrite forms nitrous acid, which dismutates to no and the toxic radical, nitrogen dioxide. no can synergize with superoxide, produced by the macrophage or generated as byproduct of respiratory metabolism by the pathogen, to form the highly poisonous peroxynitrite (onoo) radical. these roi and rni react with a wide range of molecules, including nucleic acids, proteins, lipids and carbohydrates, resulting in the killing of mtb. to counteract these actions, mtb uses a variety of molecules to either detoxify roi and rni before they can inflict damage or to repair the damage they cause. in particular, the presence of mtb results in glucose-6-phosphate (g6p) being oxidised by nadp - dependent and f420-dependent (fgd1) dehydrogenases to generate nadph, an important source of electrons, and thereby overcome oxidative stress. in addition, mtb uses a combination of its cell surface alpha - ketoacid dehydrogenase complexes to form a nadh - dependent peroxidase and peroxynitrite reductase. pattern recognition receptors (prrs) are a group of receptors which sense the presence of bacteria, fungi and viruses. prrs are also responsible for recognizing endogenous molecules released from damaged cells, which are named damage associated molecular patterns (damps) [30, 31 ]. to date these families include transmembrane proteins such as the tlrs and c - type lectin receptors (ctlrs), as well as cytoplasmic proteins such as the retinoic acid - inducible gene (rig)-i - like receptors (rlrs) and nod - like receptors (nlrs). tlrs are a family of single membrane - spanning receptors of which 10 have been characterized in man and 13 in mouse [3436 ]. tlrs play a critical role in both innate resistance and the initiation of adaptive immunity to infectious agents [3740 ]. they act by recognizing pathogen - associated molecular patterns (pamps) or endogenous inflammation - associated molecules [36, 41, 42 ]. these are distinct molecular structures on microbes and different sets of tlrs have been associated with the response to different classes of microorganisms e.g. recognition of viruses by tlr3, tlr7, tlr8 and tlr9 [36, 41, 4346 ]. bacterial dna which contains unmethylated cpg oligonucleotides (odn) motifs also acts as important regulators of human neutrophil functions via tlr9. for example, stimulation of the tlr9 pathway by cpgodn induces cxcl8 production by human neutrophils via the generation of onoo [47, 48 ]. tlr - ligand binding can induce two signaling pathways, the myeloid differentiation primary response gene 88 (myd88)-dependent and myd88-independent pathways, which induce the production of both pro - inflammatory cytokines and type i ifns [36, 49, 50 ]. these two distinct responses are mediated via the selective use of adaptor molecules recruited to the toll / il-1 receptor (tir) domains of tlrs after ligand binding. four adaptor molecules have been identified to date : myd88, tir - associated protein (tirap), tir domain - containing adaptor protein - inducing ifn- (trif) and trif related adaptor molecules (tram). myd88 and tirap are responsible for the induction of pro - inflammatory genes, and trif and tram induce ifns. in myd88-dependent signaling, myd88 is recruited to, and associates with, the cytoplasmic domain of the tlrs upon ligand binding. then il-1r - associated kinase 4 (irak-4) and irak- 1 are subsequently recruited and activated by phosphorylation. activated irak-4 phosphorylates irak-1, which then, in turn, associates with tumor necrosis factor receptor (tnfr)-associated factor 6 (traf6). traf6 activates transforming growth factor (tgf) activating kinase 1 (tak1), which, in turn, phosphorylates ikk- and mitogen - activated protein kinase (mapk) kinase6 (mkk6), leading to degradation of i-b, nuclear translocation of nf-b and induction of inflammatory genes. as a result tlr activation upregulates the transcription of proinflammatory cytokines including il-1, tnf- and il-6 which are essential for the recruitment of immune cells to the site of infection and controlling mtb infection [5355 ]. activation of the myd88-dependent pathway also results in the activation of mitogen - activated protein (map) kinases (mapk) such as p38 and jnk, which leads to the activation of ap-1. during myd88-independent signaling tlr4 activation triggers the induction of a type 1 ifn response, leading to the induction of ifn- and ifn - inducible genes. the tlrs known to be involved in recognition of mtb are tlr2, tlr4, tlr9, and possibly tlr8 [36, 41, 4346 ]. four primary immunodeficiencies (pids) involving mutations in myd88, irak4, nemo and ikba are associated with altered susceptibility to m. tuberculosis [5759 ]. these heterodimers have been implicated in the recognition of mycobacterial cell wall glycolipids including lipoarabinomannan (lam), lipomannan (lm), 38-kda and 19-kd mycobacterial glycoproteins, phosphatidylinositol mannoside (pim), triacylated (tlr2/tlr1) or diacylated (tlr2/tlr6) lipoproteins [49, 60, 61 ]. tlr2 and tlr1 act together to mediate responses to m. tuberculosis [62, 63 ] and the role of tlr1/2 gene variants in the predisposition to tuberculosis has been investigated. most studies have focused on tlr2 variants and only weak and non - replicated associations have been reported to date [62, 63 ]. tlr2 is believed to be important in the initiation of the innate host defense against mtb [61, 64 ]. in addition, il-1 production is dependent upon tlr2 and tlr6, but not tlr4 or tlr9, stimulation. tlr2 mice show defective granuloma formation following mtb infection and have a greatly enhanced susceptibility to infection compared to the wt mice [53, 67 ]. in addition, tlr2 mice are unable to control chronic infection with mtb [67, 68 ]. mice lacking tlr9 also succumb earlier to mtb infection than wild - type animals [61, 66, 6972 ]. the role of other tlrs, such as tlr4 and tlr9 in the pathogenesis of mtb has not studied in such detail [65, 73, 74 ]. mice deficient in the tlr / il-1r family receptor adaptor molecule myd88 have been shown to be highly susceptible to infection with mtb, which suggests a major role for this pathway in the innate defense against the mtb [68, 7586 ]. in addition, tlr2-induced ros production plays a crucial role in the expression of cxcl8 and ccl2 in human monocytes requiring the activation of both p38 and erk1/2 mapk pathways. overexpression of both tlr2 and tlr4 are important for viable mtb infection in human cell lines. other studies in mice with inactivated tlr genes indicated that tlr2 is important in controlling and surviving mtb infection [68, 73 ]. however, other studies suggested that tlr4 is critical for surviving mtb infection [80, 87 ]. the importance of tlr4 may depend on the dose of mtb used for challenge or the mouse strain used. human studies show that polymorphisms in both tlr2 and tlr4 are associated with increased susceptibility to microbial infections possibly by changing the th1/th2 response [8285 ]. reported that the expression of tlrs in tb lung granulomas related to the presence or absence of immunohistologically detectable il-4. changes in tlr expression and/or their down - stream activation state might represent useful markers of the immunological status of tb patients and their contacts. the tlr distribution in tb granulomas lesions indicates that tlr1, tlr2, and tlr4 are expressed in both immune cells and non - immune cells ; however tlr9 is only detectable in immune cells. furthermore, in an animal model of tb, tlr8-deficient mice succumb more rapidly to infection with m. tuberculosis, despite efficiently controlling the number of viable bacilli in different organs. although no changes in cd4 + and cd8 + t - cells were observed there were increases in lung neutrophils and macrophages. exaggerated mortality was due to massive liver necrosis and was reversed by a combination of blocking antibodies to il-1 and tnf-. thus, in this model of mtb infection, tlr8 plays a key role in dampening inflammation and tissue damage. overall, recognition of mtb by tlrs triggers various intracellular signaling cascades ultimately resulting in the production of cytokines, chemokines and antimicrobial molecules [91, 92 ]. in humans, different polymorphisms in the human tlr2 gene were reported to associate with increased susceptibility to tb in some studies [9397 ] but not others [98101 ]. furthermore, a mal / tirap functional variant, affecting signaling through tlr2, was shown to be protective in tb. genetic polymorphisms in tlr4 were linked to an increased susceptibility and severity of pulmonary tb in an asian population in india but not in indian or chinese tb patients in gambia [101, 102, 104 ]. this discrepancy might be due to a dynamic host - pathogen interplay between genetic and pathogen phenotypes. pentraxin 3 (ptx3), or tnf - stimulated gene 14 (tsg-14), is a 42-kda soluble pattern - recognition receptor produced by phagocytes and non - immune cells at sites of inflammation or injury and plays an important role in female fertility and vascular biology. ptx3 shows up to 28 % sequence identity to human c - reactive protein (crp) and serum amyloid p - component (sap) [104, 105 ]. it is a member of the pentraxin family which are involved in the acute phase response to injury, trauma and infection. ptx3 is rapidly secreted into the serum of mice and humans from extra - hepatic sources after lps, il-1 or tnf- stimulation. ptx3 binds to the complement component c1q and to microorganisms, including pseudomonas aeruginosa, salmonella typhimurium and aspergillus fumigates to induce innate immune responses [109, 110 ] and to drive a protective adaptive immunity. since whole mycobacteria and mycobacterial lipoarabinomannan strongly induce ptx3 production by human mononuclear phagocytes a role for ptx3 in the immunobiology of mycobacterial infection has been inferred. interestingly, ptx3 receptor gene variants are associated with an increased risk of pulmonary tuberculosis in west africans. furthermore, ptx3, levels are significantly correlated with the severity of clinical presentation at diagnosis and of lung involvement in disease and may represent a good biomarker for inflammation and disease activity during mtb infection. there are two classes of innate immune receptors described : a) tlrs, located on cell membranes or intracellularly, and b) nlrs located in the cytoplasm [114118 ]. both classes of receptors are programmed to recognize microbial pamps and danger - associated molecular patterns (damps) and switch cells for activation to releasing of proinflammatory and chemokines. the importance of two receptors in pathogenesis of chronic lung disease has elicited much attention [119, 120 ]. in the next section, we describe the regulation of inflammasome signaling and discuss whether abnormalities in nlrp3 inflammasome function may be associated with mtb. the inflammasome consists of a multimeric cytosolic complex comprising the adaptor protein apoptosis associated speck - like protein containing a caspase recruiting domain (asc), a sensor protein such as nrlp3 together with the effector proteins caspase-1 and caspase 5. several nlrs function in immunity through the formation of a multi - protein complex known as an inflammasome which play critical roles in the 0 pathogenesis of chronic disorders [119121 ]. nlrs exist in three families ; the nods, the nlrps and the (ipafs). stimulation of cells with pamps, or by damps, leads to increased expression of il-1 and other il-1 cytokine family members, such as il-18 and il-33. proinflammatory cytokines of the il-1 family may play an important role in anti - mycobacterial host defense mechanisms. moreover, mtb stimulates inflammatory cells to release il-1 through pathways involving tlr2/tlr6 and nod2 receptors. recognition of mtb by tlr and nod2 leads to increased transcription of pro - il-1 through mechanisms involving erk, p38 and rip2, but not jnk. interestingly, although caspase-1 is necessary for the processing of pro - il-1, activation of caspase-1 is not dependent on the stimulation of cells by mtb. in human thp-1 macrophages, mtb activation results in secretion of il-1 in an asc / nlrp3-dependent manner. in addition, mycobacterium marinum activates il-1 production in an nlrp3- and caspase-1-dependent manner in vitro highlighting the potential importance of inflammasome signaling in the pathogenesis of mbt [125, 126 ]. inflammasome - mediated il-1 secretion is triggered by a combination of signal transduction pathways activated via tlrs and purinergic (p2x7) receptors. in turn, il-1 induces the release of gm - csf which leads to the activation and increased survival of monocytes / macrophages and enhanced oxidative burst in the lungs, thus maintaining and prolonging inflammatory reactions. the purinergic p2x7 receptor is the key driver of atp - mediated inflammasome maturation and release of il-1 [122, 128, 129 ]. pro - inflammatory cytokine regulation by the inflammasome may be critical to long - term survival of mtb infection since experiments in il-1/, il-1r and il-18 knockout (ko) mice have shown that these cytokines play a role in limiting bacterial burden in the lung, in regulating the subsequent expression of other cytokines, in controlling no production and in the formation of granulomas [122, 128, 129 ]. caspase-1 independent il-1 production may also be critical for host resistance to mtb and this occurs independently of tlr signaling in vivo. furthermore, although il-1 induction by mtb in vitro depends on tlr triggering and the inflammasome, both triggers are dispensable for il-1 production in mice infected with the pathogen in vivo. thus, although recent data established that il-1 plays a critical component in innate resistance to mtb, the pathways involved in the expression and regulation of il-1 induction following mtb infection in vivo are complex and may involve mechanisms that do not fit the classical paradigms of tlr recognition and inflammasome - mediated caspase-1 processing seen with other infections or in the response to mtb observed in vitro. mtb - induced il-1 secretion in human and mouse macrophages in vitro and this process was dependent on asc, caspase-1, and nlrp3, but not nlcr4. in vivo, murine asc helps protect the host from death during chronic mtb infection whilst the effects of casp-1 and nlp3 were negligible. the inability of asc ko mice to form organized granulomas and the reduced presence of lung dendritic cells indicates a breakdown in host defense against mtb. thus, asc was identified as a critical protein involved in the host response to mtb infection in an inflammasome - independent manner. other cytokines activated by the inflammasome have also been reported to play a role in the pathogenesis of mtb. thus, il-12 and il-18 produced by dendritic cells and macrophages induce nk - cell activity and skew the immune response towards an ifn--dependent th1 response, which is considered critical for protection against mtb. data in myd88-deficient mice which are highly susceptible to mtb and succumb very rapidly to infection supports a role for myd88 in regulating mtb infection [75, 131 ]. myd88, however, plays a role in both inflammasome and tlr signaling and this raises the possibility that a lack of il-1 or il-18 is responsible for the heightened susceptibility of myd88 ko mice to mtb infection. reported that il-1r - signalling is important for protection against mtb whilst il-18r - signalling is not. in contrast, schneider has reported a similar degree of susceptibility to mtb infection to that observed in myd88 ko mice in il-18 ko mice. il-18 ko mice succumbed much more readily to experimental mtb infection than wt or tlr-2/-4 double ko (tlr-2/-4 dko) mice. in the absence of il-18, immunity to mtb was hampered by decreased th1 responses and pmn - dominated lung immunopathology concomitant with unrestrained growth of the tubercle bacilli. thus, some controversy still remains as to the precise role of il-18 in the protective immunity against mtb infection. tb remains one of the leading causes of death from a single infectious agent worldwide. in order to generate better protective strategies we need to further define the pathological mechanisms underlying the immune response to mtb. whilst inflammasome and tlr cross talk does not seem to be essential for the primary control of mtb infection, recent data suggests a critical role of these pathways in the persistence of mtb. activation of these pathways results in the release of inflammatory mediators that recruit protective cells to the infected area. however, there is a down side to this effect. excessive production of il-23 and il-17 causes pathology due to excessive recruitment and phenotypic changes in inflammatory cells. hence, there is a fine balance between th1 and inflammasome / tlrs responses that is central in defining the outcome of mtb infection. the role and mechanisms underpinning ptx3 and other pprs in the immune response to mtb still requires further elucidation however. in addition, it is critical to further define the mechanisms associated with the cross talk between tlrs and the inflammasome and to use this knowledge to generate rational protective strategies that promote a balanced acquired immune response with minimal collateral damage. determination of key nodes within the pathways involved in the pathogenesis of mtb may provide new therapeutic targets to prevent the persistence of disease. | tuberculosis (tb) is considered a major worldwide health problem with 10 million new cases diagnosed each year. our understanding of tb immunology has become greater and more refined since the identification of mycobacterium tuberculosis (mtb) as an etiologic agent and the recognition of new signaling pathways modulating infection. understanding the mechanisms through which the cells of the immune system recognize mtb can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. a great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the mtb. several classes of pattern recognition receptors (prrs) are involved in the recognition of mtb including toll - like receptors (tlrs), c - type lectin receptors (clrs) and nod - like receptors (nlrs) linked to inflammasome activation. among the tlr family, tlr1, tlr2, tlr4, and tlr9 and their down - stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of tb. the inflammasome pathway is associated with the coordinated release of cytokines such as il-1 and il-18 which also play a role in the pathogenesis of tb. understanding the cross - talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. abnormalities in prr signaling pathways regulated by tb will affect disease pathogenesis and need to be elucidated. in this review we provide an update on prr signaling during m. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities. |
a 44-year - old, para 2, female with chronic pelvic pain was admitted for microlaparoscopic conscious pain mapping to evaluate the cause of her pain. the patient had undergone a cesarean hysterectomy in 1979 during her last delivery secondary to having multiple large leiomyomata. over the next decade, she suffered from chronic right - sided pelvic pain that was unrelieved with conservative medical therapy including hormonal therapy, nonsteroidal anti - inflammatory drugs, and narcotic medications. a laparotomy with bilateral salpingo - oophorectomy and appendectomy were performed, which revealed endometriosis., the patient underwent 14 laparoscopic surgeries at various centers with minimal to no improvement. although many of the operative reports describe fulguration of endometriosis and lysis of adhesions, others reported normal findings. after an extensive history and physical examination were obtained and a review of the available medical records and videotape of her most recent laparoscopy was conducted, she was offered the option of undergoing micro - laparoscopic conscious pain mapping. prior to surgery, the patient was familiarized with the conscious pain mapping procedure and pain scoring system. she was asked to rate her pain on a scale of 0, no pain, to 4, severe pain. the procedure was conducted in the outpatient surgery center of a private community hospital. with the patient in the supine position, conscious sedation (atropine 0.2 mg, ondansetron hydrochloride 4 mg, midazolam hydrochloride 1 mg, and fentanyl citrate 350 g given slowly and titrated at 50 g increments to effect) was administered intravenously. periumbilical and suprapubic blocks were administered at operative sites of 10 ml of 1% lidocaine with epinephrine 1:100,000 buffered with sodium bicarbonate (10:1 dilution). a pneumoperitoneum was created with carbon dioxide using the insuflow device (georgia biomedical inc., macon, ga) after inserting a veress needle and 2-mm cannula. after advancing a 2-mm microlaparoscope (minisite, us surgical corp., norwalk, ct) into the peritoneal cavity, a second 2-mm cannula was placed in the suprapubic region and a 2-mm manipulating probe directed into the pelvic cavity. diagnostic microlaparoscopy and conscious pain mapping were performed by systematic probing of the pelvic cavity and obtaining intraoperative patient feedback regarding the presence or absence of pain. inspection of the pelvis confirmed a previous hysterectomy, bilateral salpingo - oophorectomy and appendectomy. the posterior cul - de - sac had a few small powder - burn endometriosis lesions that produced a minimal pain score of 1 (out of 4). however, the patient had old sutures with granulation tissue (figure 1) over the right pelvic brim with a pain mapping score of 4 (figure 2). this exquisitely painful area was probed a second time to authenticate its role as the pain focus. fulguration of the site was performed with 2-mm monopolar cautery scissors at a power setting of 40 w spray coagulation (figure 3). she tolerated both the diagnostic and operative components of her surgery under local anesthesia with conscious sedation. at the scheduled 2-week and 6-month postoperative visits, she reported being completely pain - free without the use of analgesics. chronic pelvic pain accounts for approximately 10% to 15% of a woman 's visits to the gynecologist, up to 50% of diagnostic laparoscopies, and many hysterectomies each year. interestingly, a variable percentage of patients have no pathologic findings with the traditional laparoscopic approach under general anesthesia. physical examination sometimes provides confusing and minimally useful data for evaluating chronic pelvic pain. in a large study of 1194 patients with chronic pelvic pain, normal pelvic examinations were found in 749 patients. not surprisingly, in 479 patients with chronic pelvic pain and a normal examination, 63% had abnormal findings during diagnostic laparoscopy. however, 17.5% of the study patients with an abnormal pelvic examination had a normal diagnostic laparoscopy. it is for this latter subgroup of patients that conscious pain mapping may provide more clinical information than with traditional laparoscopy under general anesthesia. she subsequently underwent a bilateral salpingo - oophorectomy and appendectomy without symptom relief. over a period of 12 years, she underwent 14 laparoscopies, all performed under general anesthesia. although some of these operative reports describe the presence of endometriosis, adhesions, or both of these, none addressed the sutures in the right pelvic brim. further confusing the patient 's clinical picture is the fact that no consistent relationship existed between the severity of endometriosis and pelvic pain. the burned out endometriosis lesions seen during conscious pain mapping produced a low pain score of 1. once electrosurgery to fulgurate the lesion over the right pelvic brim was performed, the patient 's pain score dropped from a severe 4 (out of 4) to an insignificant score of 1. it is critical to perform conscious pain mapping in patients with chronic pelvic pain who have undergone unsuccessful surgical attempts to resolve their pain. | chronic pelvic pain is a debilitating, life - altering syndrome that negatively affects a woman 's quality of life and personal relationships. many women continue to suffer with pelvic pain despite having undergone multiple medical and surgical treatments. unfortunately, some women are incorrectly labeled as having psychological illness when organic disease may be present. i report a case of a woman who underwent multiple pelvic and abdominal surgeries before the cause of her pain was identified through microlaparoscopic conscious pain mapping. |
the classical or canonical model of signaling by cytokines such as the interferons (ifns) involves ligand interaction with receptor extracellular domain, followed by allosteric changes in the receptor cytoplasmic domain that results in autophosphorylation of the relevant janus tyrosine kinases (jaks) and subsequent tyrosine phosphorylation of receptor cytoplasmic domain(s) [14 ]. the climatic event is the association and tyrosine phosphorylation of the appropriate signal transducer and activator of transcription (stats) factors. the beauty and frailty of the model lies in the simplicity of the stats being responsible for the specific functional effects attributed to the ifns. for example, ifn signaling via a heterodimeric receptor results in the activation of stat1, by receptor - associated jak1 and jak2, to form an asymmetric dimer which undergoes nuclear translocation to specific promoters of genes that are activated by ifn. in the case of the family of the 15 or more type 1 ifn subtypes, all acting through the same heterodimeric receptor, jak1 and tyk2 kinases activate stat1 and stat2 in conjunction with the receptor cytoplasmic domains. stat1 and stat2 form a trimeric complex with ifn regulatory factor 9 (irf9), known as isgf3, followed by nuclear translocation and association with promoters of genes specifically activated by the type i ifns. the canonical model of ifn signaling is remarkably lacking in specificity mechanisms, probably attributable to its attractive simplicity and skewed focus on stats. in the case of ifns, it has never been shown that activation of the corresponding stats independent of the ifns or their receptors has resulted in the induction of an antiviral state. the discovery of a novel member of the type i ifn family, called ifn, places a particular strain on the canonical model of ifn signaling. ovine ifn was originally identified not as an ifn but as a pregnancy recognition hormone in ruminants. structurally, the amino acid sequence of ifn shows 30 to 70 percent homology to other type i ifns. ifn operates via the same heterodimeric receptor as all the type i ifns, is as potent antiviral and antiproliferative agent as ifn, and is equally effective in induction of (2-5) oligoadenylate synthetase but unlike ifn it is relatively nontoxic at high doses. it is noteworthy that ifn and ifn had similar specific antiviral activities but ifn is bound to receptor at a 10-fold higher binding affinity. antibodies to ifn c - terminus blocked binding of both ifn and ifn to the receptor but antibodies to ifn n - terminus only blocked ifn binding, suggesting that they recognized the receptor similarly at their c - terminus but differently at their n - terminus. the findings suggested that maximal ifn antiviral activity required only fractional occupancy of receptor by the ifns while toxicity was associated with maximal receptor occupancy. consistent with similar antiviral activity, ifn and ifn phosphorylated jak kinase tyk2 and stat1 and stat2 transcription factors similarly, suggesting that phosphorylation of these signal transduction molecules was associated with antiviral activity and not toxicity. the similar and differential effects of these two type i ifns operating through the same receptor complex are not readily explained by the canonical model of type i ifn signaling. we have discovered, developed, and characterized a noncanonical ifn signal transduction pathway that is remarkably similar to steroid / steroid receptor signaling (reviewed in [8, 9 ]). it is our contention that the pathway not only provides the mechanism of genetic and epigenetic signaling by ifns but also provides insight into other cytokine, growth factor, and hormone signaling pathways. the central players of ifn signaling via the classical or canonical pathway involve the ifn, receptor subunits ifngr1 and ifngr2, tyrosine kinases jak1 and jak2, and stat1 transcription factor [14 ]. type i ifn signaling involves a type i ifn, receptor subunits ifnar1 and ifnar2, tyrosine kinases jak1 and tyk2, and stat1 and stat2 transcription factors. figure 1 illustrates the sequence of events from ifn / receptor binding to the presence of activated stats at the response elements of genes that are specifically activated by the ifns. proponents of these classical pathways point out the interaction of the activated stats with coactivators. stats in the nucleus, for example, interacting with epigenetic players such as p300 and creb binding protein (cbp) where creb means camp response element binding protein. p300 and cbp are acetyl - transferases that are involved in chromatin remodeling [10, 11 ]. ifn also activates other players and pathways, including mapk (mitogen - activated protein kinase), pi-3k (phosphoinositide 3-kinase), and nf - kb (nuclear factor kb) [2, 12 ]. all of these and other pathways activated by ifn signaling and stat activation of the canonical model are generic in the sense that a host of different cytokines, hormones, and growth factors also activate them. there is to date no known specificity - determining orchestration or coordination center identified for any of the stat or non - stat aspects of the canonical model. so, identification of these ifn induced events is simply in agreement with that which is known to occur with a plethora of other cytokines that have functions different from that of ifns. the report of activated jak2 in the nucleus is, in our view, a very important event that is a game changer as far as the specific mechanism of cytokine signaling at the level of gene activation including the associated epigenetic events. we briefly describe here and will explore in some depth later why this finding is important in specific gene activation by cytokines. it was shown in 2009 that leukemic cells with gain - of - function mutated jak2, and jak2v617f, which is constitutively activated, performs a key epigenetic function in the nucleus. phosphorylated h3y41, h3py41, causes the dissociation of heterochromatin protein 1 (hp1) from histone h3, resulting in transcription of genes repressed by hp1. wild - type jak2 that is activated by growth factors such as platelet - derived growth factor (pdgf) undergo nuclear translocation and similarly phosphorylate histone h3 at y41, causing dissociation of hp1, resulting in chromatin remodeling for gene activation. the presence of activated jaks in the nucleus and activated stats, both activated by the same cytokine, raises the question as to how this fits into events associated with specific gene activation by the cytokine. we show that both types i and ii ifn activation of tyk2 and jak2, respectively, via the noncanonical jak / stat pathway provides a mechanism for specific gene activation by these ifns including the associated epigenetics of h3y41 phosphorylation. there are a number of interesting observations concerning ifn activity that are beyond rational explanation or understanding in the context of the canonical jak / stat model of ifn signaling. specifically, human ifn delivered by a liposome vector induced an antitumor effect in murine macrophages, expression of nonsecretable human ifn in murine fibroblasts induced antiviral activity, and microinjected human ifn induced ia antigen expression in murine macrophages [1416 ]. these findings are at odds with the well - known species preference of exogenous human ifn which has no activity on murine cells. these reports suggest that ifn can induce function via an intracellular mechanism that is not species restricted. the fact that these observations were essentially ignored by the ifn community would suggest that they were considered to be of no significance in the context of the canonical jak / stat model of ifn signaling. the ifn molecule is an asymmetric dimer and the ifn receptor consists of a noncovalent linked tetramer consisting of two subunits called ifngr1 and ifngr2. according to the canonical model, ifn cross - links the ifngr1 subunit, resulting in allosteric changes to the receptor cytoplasmic domain. this allosteric change initiated from the ifn / receptor extracellular interaction is not specified but merely assumed in the context of the model. the intervening hydrophobic transmembrane sequence of the receptor, separating receptor extracellular and cytoplasmic domain, is similarly ignored as to its role in these allosteric events. given the stasis of the canonical model in its lack of predictive appeal, we approached ifn signaling by first doing binding studies of ifn and ifngr1. specifically, we carried out ifn bindings to intact soluble receptor subunit ifngr1 consisting of both extracellular and cytoplasmic domains. using intact ifn, overlapping ifn peptides, and overlapping ifngr1 extracellular and cytoplasmic peptides along with site specific antibodies, we discovered that the n - terminus of ifn is bound to ifngr1 extracellular domain and that the c - terminus of ifn is bound to receptor cytoplasmic domain. murine ifn c - terminus peptide, ifn(95 - 132), and the corresponding sequence of human ifn are bound to residues 253 - 287 of ifngr1 cytoplasmic domain. this binding was adjacent to the binding site of jak2 on ifngr1 and was specifically blocked by anti-(253 - 287) specific antibodies in fixed / permeabilized cells [17, 18 ]. it was observed that when cells were treated with ifn jak2 binding shifted from receptor subunit ifngr2 to ifngr1, presumably as a result of the allosteric changes referred to above [2, 12 ]. by comparison, we showed that sepharose coupled jak2 (seph - jak2) bound our soluble radiolabeled ifngr1 and that such binding was enhanced by both intact ifn and its c - terminal peptide, ifn(95 - 132), but not by the receptor extracellular domain - binding peptide ifn(1 - 39). the enhanced binding of jak2 was blocked by the ifngr1 peptide, ifngr1(253 - 287), that corresponded to the ifngr1 binding site for ifn c - terminus, showing specificity of enhanced binding. a receptor peptide corresponding to the jak2 binding site, ifngr1(283 - 309), also blocked jak2 binding, while a peptide to an adjacent site had no effect on enhanced jak2 binding, providing further evidence of specificity. ifn c - terminus enhancement of jak2 binding to ifngr1 cytoplasmic domain would seem to be consistent with the well - known law of mass action, shifting the equilibrium between ifngr1 and ifngr2, rather than by allosteric changes evoked by the canonical jak / stat model. functionally, treatment of murine macrophage cell lines with either murine ifn c - terminal peptide, ifn(95 - 132), or its human counterpart resulted in upregulation of mhc class ii molecules and induction of an antiviral state similar to ifn. the peptides were internalized via pinocytosis by the macrophages and were not effective against nonpinocytotic fibroblasts, presumably because they could not access ifngr1 cytoplasmic domain. this lack of effect on fibroblasts was overcome by attachment of a palmitate to ifn(95 - 132), resulting in cell penetration and induction of an antiviral state and upregulation of mhc class ii molecules in fibroblasts. these investigators also showed that cells with ifngr1 gene knockout were refractive to the effects of the ifn peptide which was evidence that the peptide acted through receptor subunit ifngr1. the challenge was to show that ifn interaction with receptor on intact cells finds its way to the ifngr1 cytoplasm binding site, as per ifngr1(253 - 287) peptide, during the process of endocytosis. first, specific binding to a murine cell line (p388d1) was established using radiolabeled murine ifn, i - ifn. binding was carried out at 4c to prevent endocytosis. unlabeled ifn blocked i - ifn binding by competing for receptor while ifngr1(253 - 287) that corresponds to the ifngr1 cytoplasmic binding site for ifn in soluble receptor binding had no effect on extracellular receptor binding (figure 2(a)). to assess intracellular binding to the sequence 253 - 287 of ifngr1, p388d1 cells were incubated at 37c to facilitate intracellular loading of the ifngr1(253 - 287) peptide. cells were then incubated at 37c for a short period with i ifn after which they were lowered to 4c and surface i - ifn was removed by acid treatment. importantly, ifngr1(253 - 287) loaded cells blocked binding of i ifn to the corresponding site on ifngr1. an added caveat to this experiment is that blockage of ifn binding to ifngr1 cytoplasmic domain resulted in the absence of activation of stat1 as assessed by phosphorylation of tyrosine 701 by jak2 (figure 2(c)). taking together, these binding studies show that ifn binds extracellular receptor domain and traverses to the cytoplasmic domain of ifngr1 which is coupled to stat1 activation. historically, the smallpox virus has been responsible for billions of deaths and has been estimated to have wiped out as many as 90% of the south american population as a result of european introduction of the virus. a central reason for the potent virulence is probably due to the remarkable refractiveness of the virus to ifns as a result of the induction of ifn decoy receptors. the vaccinia virus, for example, codes for secreted, soluble proteins b18r and b8r that are truncated such as to retain only the extracellular, ligand binding domain of the receptor that competes with type i and type ii ifn, respectively [23, 24 ]. the ifn based c - terminus mimetics by comparison are potent inhibitors of vaccinia virus because they are not recognized by virus decoy receptors [25, 26 ]. thus, the decoy receptors can neutralize the intact ifns and not the c - terminal peptides that are devoid of the domain involved in the extracellular binding of the decoy receptor. it should be noted that these mimetics are the result of the noncanonical pathway of ifn signaling. the abovementioned scenario with ifn and ifn receptor subunit ifngr1 is applicable with variations to type i ifn signaling system. in ifn signaling, the receptor subunit ifngr2 remains on the plasma membrane during endocytosis, while ifngr1 is endocytosed with ifn [27, 28 ]. for a type i ifn like ifn, both receptor subunits ifnar1 and ifnar2 are endocytosed with the ifn. in this section, we address issues of complex formation in the cytoplasm and movement to specific genes in the nucleus. the observation that ifn translocates to the nucleus in receptor - expressing cells with kinetics similar to those of activated stat1 is not considered in the context of the canonical model of jak / stat signaling, perhaps because of the central role ascribed to stat (reviewed in [8, 9 ]). however, such observations are of potential importance in the context of the noncanonical model of ifn signaling. we showed that ifn nuclear translocation was driven by a polycationic nuclear localization sequence (nls), rkrkrsr, in its c - terminus that is similar to that of the prototypical sv40 large tumor antigen (t - ag) nls(pkkkrkv). mutations of the ifn nls resulted in loss of biological activity which was restored by t - ag prototypical nls. efficient nuclear transport via polycationic nlss involves high affinity recognition by members of the importin (imp) superfamily of nuclear transport molecules [3234 ]. ifn is actively transported by the heterodimeric imp/ complex in the cytoplasm where imp binds the ifn nls and imp mediates the interaction with the nuclear pore and ran, with atp / gtp as energy source. the imp association of ifn was established by immunoprecipitation with antibody to imp (anti - npi-1) and western blotting. related to this, t - ag which binds to imp competitively inhibited ifn function in cells. we showed above that endocytosed ifn interacted with the cytoplasmic domain of receptor subunit ifngr1 at residues 253 - 287 which is adjacent to jak2 binding site. immunofluorescent confocal microscopy and cell fractionation studies showed that receptor in lipid microdomains on the cell surface played an important role in the endocytosis and that receptor subunit ifngr2 did not undergo endocytosis but remained on the cell surface [27, 28 ]. the function of ifngr2 may be to serve as reservoir for jak2 that binds to ifngr1 with higher affinity as a result of ifn binding. given the central emphasis that has been placed on stats in specific gene activation by cytokines, it seems reasonable in the case of ifn to determine what proteins are associated with stat1 at the gas element in genes activated by this ifn. accordingly, we used the combination of immunoprecipitation with western blotting, nuclear confocal immunofluorescence, chromatin immunoprecipitation (chip) followed by pcr, and other focused techniques to show that ifn, ifngr1, jak1 and jak2, and stat1 were all present at the gas element of genes activated by ifn. we initially focused on the role of the ifn nls in translocation of ifngr1 into the nucleus [8, 9, 31, 35 ]. we established that cells treated with ifn or the internalized ifn c - terminus peptide ifn(95 - 132) resulted in ifngr1 translocation to the nucleus and that the ifn nls was required [8, 9, 31, 35 ]. we next showed that activated stat1 (pstat1) and the activated jaks, pjak1 and pjak2, also required the nls of ifn for nuclear translocation, all as a complex of ifn/ifngr1/pstat1/pjak1/pjak2 [8, 9, 31, 35 ]. we showed that the complex played an essential role in the coordinated events of specific gene activation and the associated epigenetics. thus, stat1 is but one of the collection of key players in specific gene activation by cytokines such as ifn. see figure 3(a) for an illustration of the ifn events. we similarly examined type i ifn system for noncanonical signaling. using the same approach with particular use of chip followed by pcr, immunoprecipitations, and confocal microscopy, we showed the association of pstat1, ifnar1, ifnar2, and tyk2 with the isre element of the oligoadenylate synthetase 1 (oas1) promoter in ifn2 treated cells. such association was not shown at the -actin promoter after ifn treatment as type i ifn does not activate the -actin gene. see figure 3(b) for an illustration of the ifn events. as indicated earlier, both wild - type and gain - of - function mutated jak2 were shown to be present in the nucleus. specifically, constitutively activated jak2v617f was shown to phosphorylate histone h3 on tyrosine 41 (h3py41) which caused dissociation of the inhibitor hp1 from h3. the key result of this was chromatin remodeling to euchromatin which led to gene activation. jak2v617f mutation is associated with particular hematologic disorders, suggesting that the epigenetic effect involves interaction with the relevant hematological receptor. it is our view that the power of a model is that it both explains and predicts mechanisms. in this regard, it was shown that jak2v617f association with a homodimeric type i cytokine receptor, the erythropoietin receptor (epor), the thrombopoietin receptor, or the granulocyte colony - stimulating factor receptor, was necessary for the induction of the transforming leukemic phenotype [36, 37 ]. the question of whether receptor / jak2v617f complexes were present at the promoters of genes that were activated in cancers caused by or associated with jak2v617f was not addressed. it is our view that hematopoietic receptor activation of jak2v617f in the cytoplasm is not sufficient to induce the h3py41 phosphorylation specifically at the genes that are activated by jakv617f as we are not aware that jak2v617f possesses such intrinsic properties. thus, in addition to activation of jak2v617f, the receptor may also colocalize with the kinase at the specific promoter in the nucleus. these results with mammalian systems were preceded by similar observations in a drosophila model of hematopoietic tumors, including the suppressive effects of drosophila hpi on the mutant jak in conjunction with inhibition of tumor. perhaps the most intriguing finding of our noncanonical approach to ifn signaling was the association of h3py41 with nuclear pjak2 and pstat1 at the irf-1 promoter in cells treated with ifn, and the association of h3py41 with nuclear tyk2 and pstat1 at the oas1 promoter in cells treated with ifn. genes such as -actin, which are not activated by ifns, were negative for the relevant jaks and stats [29, 35 ]. the fact that ifn-associated tyk2 can phosphorylate y41 on h3 is evidence that the phosphorylation is not restricted to jak2. in this regard, the mutated jakv617f appears to be a special case of a more general process. the key is not just the particular jak but also the particular cytokine, growth factor, or hormone that is the activator of the jak. the question arises as to the significance of jak induction of h3py41 to nucleosome transient unwrapping so that factors such as the complexes of our noncanonical studies can become involved in dna transcription. specifically, it was shown that h3py41 increased nucleosome unwrapping and access to transcription factor binding by severalfold [39, 40 ]. h3 at lysine 56, h3k56, is located at the same dna - histone interface as h3y41. however, the combination of h3py41 and h3k56ac had a multiplicative effect and increased unwrapping by 17-fold. it was concluded that the combination of phosphorylation with acetylation significantly increased dna accessibility to regulatory transcription complexes. the movement of membrane receptors to the nucleus following endocytosis is not a rare anomaly just limited to some odd ifn result. on the contrary, there are a plethora of plasma membrane receptors that translocate to the nucleus following ligand / receptor interaction. most of the membrane receptors that traffic to the nucleus tend to signal via the jak / stat pathway. receptor tyrosine kinases (rtks) such as epidermal growth factor receptor (egfr), fibroblast growth factor receptor (fgfr), vascular endothelial growth factor (vegfr), growth hormone receptor (ghr), and insulin receptor are well - known growth factors that have been shown to similarly undergo nuclear translocation with ligand [42, 43 ]. g protein - coupled receptors (gpcr) involving peptide ligands such as angiotensin also have been found in the nucleus. considerable work has also been done on the mechanism of nuclear transport of membrane receptors. we showed that internalized ifn is bound to the cytoplasmic domain of ifngr1 but the challenge is to decipher the mechanism (figure 2). we have addressed this issue in part where we showed that the presence of ifngr1 and ifngr2 in the lipid microdomain was central to the endocytosis that is linked to the noncanonical signaling pathway. the cytoplasmic domain of ifngr1 in the endocytic vesicle is exposed to the cytoplasm as intracellular injected antibodies specific to ifn c - terminus blocked nuclear translocation as well as stat1 activation while the antibodies had no effect on nuclear accumulation of stat1 in cells treated with ifn. retrograde trafficking of the receptor tyrosine kinase egfr from the cell surface into the nucleus has been studied extensively [46, 47 ]. following egf induced endocytosis, the endocytic vesicles with the egfr fuse with early endosomes which traffic to golgi. retrograde trafficking was blocked by brefeldin a or dominant negative mutants of the small gtpasearf (adp - ribosylation factor). both treatments resulted in disassembly of the copi (coat protein complex i) which was interpreted as copi regulation of retrograde vesicular trafficking of egfr from the golgi to the er (endoplasmic endothelium). the sec61 translocon was shown to be required for trafficking of egfr from the er into the nucleus. epigenetically, egfr has been shown to modulate dna synthesis and repair through phosphorylation of tyrosine on histone h4 at residue h4y72 which is connected to enhanced methylation at h4 k20. thus, there are specific mechanistic data on a key epigenetic event associated with activation of egfr. mechanisms of nuclear translocation and some epigenetic effects have similarly been reported for other rtks [48, 49 ]. receptor - associated tyrosine kinases such as the jaks and rtks such as egfr in the nucleus are probably key players in normal and abnormal cellular activity. our noncanonical model of ifn signaling where jaks, receptors, and stats are physically linked significantly demystifies genetic and epigenetic aspects of cytokine signaling. jak2v617f associated hematopoietic cancers are much better understood in the context of jak2v617f linkage with receptors such as epor for both activation and specific function in the nucleus. the same pertains to rtks where the kinase is part of the receptor. the noncanonical signaling model should thus provide insight into regulation of both homeostatic and nonhomeostatic cellular processes. | the canonical model of cytokine signaling via the jak / stat pathway dominates our view of signal transduction but provides no insight into the significance of the simultaneous presence of activated jaks and stats in the nucleus of cells treated with cytokines. such a mechanistic shortcoming challenges the usefulness of the model in its present form. focusing on the interferon (ifn) cytokines, we have developed a noncanonical model of ifn signaling that naturally connects activated jaks and stats at or near response elements of genes that are activated by the ifns. specifically, cells treated with ifn showed association of activated stat1 and jak2 at the gas element of genes activated by ifn. for ifn treated cells, the association involved activated stat1 and tyk2 jak kinase at the isre promoter. the power of the noncanonical model is that it provides mechanistic insight into specific gene activation at the level of the associated epigenetics, akin to that of steroid / steroid receptor signaling. |
due to industrialization and urbanization, the standard of living continues to rise particularly in developing countries. this has led to weight gain and obesity, which are posing a threat to the health of citizens. obesity is perhaps the most prevalent form of malnutrition in developing countries, both among adults and children. studies have demonstrated that obesity is related to elevated systolic blood pressure (sbp) and diastolic blood pressure (dbp) elevation, dyslipidemia, diabetes, etc. obesity, its attendant health consequences and consequent health burden, is expected to reach epidemic proportions in developing countries like india. an increase in the dimension of this problem has been reported in the high socio - economic group in india. a study in delhi revealed even higher prevalence (32 - 50%) of overweight (body mass index (bmi) > 25) among adults belonging to high income group as compared with 16.2 - 20% in those belonging to middle income group. bmi, calculated as weight in kg / height in meters squared, is most widely used to estimate the prevalence of obesity or underweight within a population. the relationship between bmi and blood pressure india in a process of rapid economic development and modernization with changing life style factors has an increasing trend of hypertension especially among urban population. it is important from a public health perspective to have data on the characteristics and health of a population and of different subgroups in the population because of the racial / ethnic disparities in terms of long - term health consequences. the present study was therefore undertaken to examine the prevalence of overweight and obesity among punjabi adults on the basis of bmi and to analyze the relation between anthropometric measures and blood pressure. cross - sectional survey of all the people belonging to punjabi community residing in roshanara area and jaina building in delhi, for the past 20 years was conducted. a total of 117 males and 123 females aged from 18 to 50 years were included in the present study. ethical approval for this study was taken from the ethical committee of the department of anthoropology, university of delhi. anthropometric measurements including height, weight, circumferences (upper arm, calf), skinfold thicknesses (at triceps, biceps, subscapular, and suprailiac) and physiological dimensions like blood pressure were taken on each subject. bmi was classified according to the proposed criteria of world health organization (who) (ced iii 160 mmhg (sbp) and > 100 mmhg (dbp) were classified as stage ii hypertension (jnc2003). body fat percentage is total body fat expressed as a percentage of total body weight. descriptive statistics of mean and standard deviation, standard error were used to examine the data. regression analysis and cross tabulation was also carried out to see relationship between the variables. the effect of age was controlled statistically to find out the relationship between blood pressure and other variables. multiple linear regression was performed to quantify the effect of individual variables to sbp and dbp. sbp and dbp in separate models were the dependent variables ; the independent variables were bmi, etc. descriptive statistics of mean and standard deviation, standard error were used to examine the data. regression analysis and cross tabulation was also carried out to see relationship between the variables. the effect of age was controlled statistically to find out the relationship between blood pressure and other variables. multiple linear regression was performed to quantify the effect of individual variables to sbp and dbp. sbp and dbp in separate models were the dependent variables ; the independent variables were bmi, etc. table 1 shows the basic measurements of males and females and the difference between the two genders for the same. mean values of height, weight, upper arm circumference, calf circumference, pulse rate, sbp, and dbp were found to be significantly higher in males as compared with females. the mean values of all the skinfold thicknesses, that is, biceps, triceps, subscapular, and suprailiac were higher among females than males significance of the gender difference between various measurements age was found to have positive and statistically significant correlation with both sbp (r = 0.21, p 18 years living in chennai, the prevalence of prehypertension was reported as 47%. even in the rural population in assam, 54% of subjects had prehypertension and one - third had hypertension. the prevalence of prehypertension among punjabis of the present study was higher as compared with other studies, 47.6% in the baniyas of delhi, 28.5% in uruguay, 20.0% in australia, 31% in the united states, and 34% in taiwan. in the present study, the males mostly belonged to businessmen category, involved in transport business. the higher prevalence of prehypertension and hypertension among punjabis may be attributable to differences in dietary habits, socio - economic status, sedentary life style, intake of alcohol, and rates of obesity. they did jobs that involved more of mental strain in spite of the fact that they were more or less sedentary (they have drivers and helpers to carry out the various jobs), than the other categories of occupation such as professionals and those doing office work. they were also found to have higher mean values of weight, almost all anthropometric measurements and skinfold thicknesses. the lower levels of blood pressure among women may be attributable to a protective effect of estrogen, smoking, and alcoholic status ; most of the women were premenopausal and all of them were nonsmokers and nondrinkers. the prevalence of hypertension has been increasing in india, both in rural and in urban regions. the public health burden of people with prehypertension is worthy of serious evaluation as these subjects are unaware of their condition and if a population approach to disease prevention is applied, we could expect that a small reduction in mean population blood pressure will result in relatively large reduction in overall disease risk. in the present study, prevalence of high blood pressure was greater in those with high bmi, which was also reported by other studies. relationship between prehypertension and overweight and obesity as observed in the present study has also been observed in other studies. individuals in the urban environment did not only show higher prevalence of obesity but also more elevated blood pressure level. explained obesity - associated hypertension as an inadequate vasodilatation in the presence of increased blood volume and cardiac output, which are natural consequences of an increased mass. among both males and females, overweight / obesity has been found to be risk factor, more for dbp, which is more dependent on peripheral resistance. since, dbp is closely correlated with sbp, the factors that increase dbp may thereby also increase sbp. the overall findings suggest obesity to be important risk factor for prehypertension and hypertension as prehypertension and hypertension are more prevalent among overweight and obese subjects as compared with other categories. classification as prehypertensive or even at risk for hypertension may cause obese subjects to take notice. as bmi is a reflection of life style, addressing it would be appropriate when subjects are in that range. an elevated bmi being associated with prehypertension may suggest that such individuals are at increased risk of progressing to frank hypertension. therefore weight management programs are more important for these punjabis than the life style modification programs targeted at hypertension. but we can not draw causal relationships at this stage because of cross - sectional nature of our study. | background : the blood pressure and anthropometric measurements are important for evaluating the health of children, adolescents as well as adults.aim:the aim is to study the blood pressure and body dimensions and to find out the prevalence of overweight / obesity and hypertension among adults.materials and methods : a cross - sectional study was conducted of all the people belonging to the punjabi community, residing in roshanara area and jaina building in delhi, for the past 20 years and aged 18 - 50 years. the men were engaged in transport business and women were mainly housewives.results:mean values of all the measurements, that is, height, weight, upper arm circumference, pulse rate, systolic blood pressure (sbp), and diastolic blood pressure (dbp) were higher among males as compared with females, except skinfold thicknesses. body mass index (bmi) and fat percentage was found to be higher among females as compared with males. there was a significant positive correlation between bmi, fat percentage, and blood pressure both sbp as well as dbp. odds ratio showed that overweight / obese subjects were more likely to have hypertension than those with normal bmi.conclusion:prevalence of prehypertension among overweight / obese suggested an early clinical detection of prehypertension and intervention including life style modification, particularly weight management. |
a 74-year - old man with infective endocarditis and valvular vegetations was scheduled to undergo aortic and mitral valvular replacement surgery. he had a previous medical history of laparoscopic radical prostatectomy with pelvic lymph node dissection for prostatic cancer, and had received hormonal and radiation therapy with subsequent episodes of rectal bleeding due to radiation - induced prostatitis and telangiectasia. other than these previous problems, he had no esophageal or gastric related diseases or coagulation abnormalities. in the operation room, the patient was monitored with electrocardiography, and the continuous arterial blood pressure and noninvasive peripheral oxygen saturation were also assessed. five minutes after preoxygenation with 50% to 100% oxygen, anesthesia was induced with 3 mg of midazolam, 20 mg of etomidate and 10 mg of vecuronium. anesthesia was maintained with isoflurane at an inspired oxygen fraction of 1.0 and a continuous infusion of 0.8 ug / kg / min vecuronium. thereafter, a transesophageal echocardiography (tee) probe was inserted into the patient 's esophagus through the mouth. the tee probe was in place throughout the operation. before initiating the cardiopulmonary bypass (cpb, the tee probe was manipulated to visualize both the mitral and aortic valves for both insufficiency and vegetations. during the cpb period, after weaning the patient off cpb, the tee was reexamined by a cardiologist to assess the function of the prosthetic aortic and mitral valves. the mid - esophageal five chamber view, two chamber view and long axis view, the transgastric short axis and long axis view and the deep transgastric long axis view were all visualized. after the operation, the patient was transferred to the cvicu (cardiovascular intensive care unit) without any event. four hours after admission to the cvicu, the patient had hematemesis amounting to up to 2 l of blood. the egd finding was a 3 - 4 cm sized deep, linear mallory - weiss laceration at the cardia, just below the gastroesophageal junction (fig. the bleeder was clipped successfully with no remnant bleeding at the site. on the second postoperative day, eight days after operation, egd was performed again and there was no trace of bleeding at the lesion (fig. 2), and was concluded as healing status. the patient was discharged without any further complications related to the mallory - weiss laceration. intraoperative tee is widely used because it is relatively noninvasive and safe to apply, and it visualizes the cardiac function and hemodynamic status without interfering with an operation. it supplements transthoracic echocardiography (tte) before the operation and this modality can estimate the result of the operation on a real time basis. perioperative tee examination is recommended in cases of infective endocarditis and doing this is strongly supported by the evidence of improved clinical outcomes. however, tee can sometimes induce direct and indirect oropharyngeal, esophageal and gastric complications. most of these complications are mild, but sometimes they can be lethal, although this is rare. there are a few reports of injuries outside the alimentary tract (e.g., the spleen). kallmeyer. studied 7,200 cardiac surgery patients who underwent intraoperative tee at a single center. in this study, the tee - associated complication rate was 0.2% and the most common complication was oropharyngeal trauma. odynophagia occurred in 0.1% of the patients and this accounted for 50% of all the complications. gastrointestinal trauma associated with tee was seen in seven patients (0.1%), and two of them had significant upper gastrointestinal (ugi) hemorrhage. examined 10,419 patients who underwent tee in a multicenter survey and 0.18% of the population had tee - associated complications. the incidence of tee - associated direct ugi trauma shows a low risk rate (0.1 - 1.2%). however, direct ugi trauma can induce serious complications. in a study by lennon., six of 516 patients who underwent tee showed major gastrointestinal (gi) complications. three patients had esophageal and/or gastric tear, two had gastric perforation and one had gastric ulcer. three of them underwent laparotomy, two received endoscopic treatment and one was treated with transfusion only. the patient in the present case had a deep, linear laceration at the cardia of the stomach. we supposed that the laceration may have developed while attempting to obtain the deep transgastric long - axis view. the deep transgastric long - axis view is obtained by anteflexion of the tee probe, and the operator needs to advance or withdraw the probe to achieve more accurate visualization. during this procedure, many operators do not unfold the anteflexed probe, and this may render the esophagus and stomach more susceptible to injury. thermal and sustained - pressure induced injury can also occur. in a study by orihashi., the placement of the tee probe for obtaining the transgastric short - axis view was reviewed in 24 patients. the probe tip was in the stomach in 72.7% of the patients, at the cardia in 13.6% and in the esophagus in 13.6% of the patients. operators should take caution while searching for the transgastric view, and not just while searching for the deep transgastric view. also the operators should always be careful not to move the probe unless it is straightened, and not to fold the probe tip for long time so as to avoid a possible thermal or pressure - induced injury. it is riskier to insert the tee probe while the patient is anesthetized because the patients can not respond to pain. cpb - applied surgery like the present case has a higher risk of gi trauma because cpb requires full heparinization and it also may induce coagulopathy such as platelet dysfunction, fibrinolysis and coagulation factor consumption. to decrease the complications, the operators should be careful not to advance the probe more than 30 cm from the incisor.. reported on 201 cases of failure to insert a tee probe and 98.5% of which was due to the lack of operator 's experience. rigid laryngoscope - assisted tee insertion can also decrease injury to the oropharyngeal mucosal in anesthetized patients. as discussed above, not moving the probe while folded and straightening the probe right after the procedure will lower the complication rate. maintaining the tee probe in a freeze status while not in use will help to avoid any thermal injury. tee examinations should be avoided or only performed with great caution in patients with significant esophageal or gastric pathology. esophageal web, stricture or rings, esophageal perforation and obstructive esophageal neoplasm are classified as contraindications for conducting a tee examination. cervical spine instability is also a contraindication because adequate neck flexion and extension are needed while inserting the probe. conditions such as esophageal diverticulum, large hiatal hernia, recent esophageal or gastric surgery, esophageal varices, a history of odynophaiga or dysphagia, cervical arthritis, a history of mediastinal irradiation, oropharyngeal deformities, and severe coagulopathy are also thought to be the relative contraindications. in the present case, the patient had a history of radiation therapy and radiation - induced fragility of the lower intestine. therefore, the mallory - weiss laceration is not thought to be due to the patient 's condition or prior treatment. intraoperative tee is a highly useful modality, especially for reconfirming before surgery the existence of vegetation on heart valves with infective endocarditis, and its usage has become more frequent. anesthesiologists need to be trained on how to safely manipulate the tee probe and they should always check the patient 's preoperative state for whether the patient has esophagogastric pathologies or coagulopathy. even if the patient is without pathology, clinicians should always remember that the tee probe can cause injuries on the gi tract and try to run the probe in a smooth and gentle manner. | transesophageal echocardiography (tee) is a relatively noninvasive and highly valuable diagnostic modality to monitor cardiac surgery. tee is utilized to estimate the results of the surgical correction or the cardiac function on a real time basis. accordingly, the frequency of tee usage is increasing. previous studies have shown low risk of tee - associated complications ; nonetheless, major gastrointestinal trauma can occur on a rare occasion. we herein present a case of mallory - weiss laceration after an intraoperative tee examination. |
since standard preclinical models have notoriously overestimated the clinical potential of her tkis, we challenged the traditional approach to evaluating tkis in these models. traditionally, signaling inhibitors are thought to have a continuous suppressive effect through rapid and sustained inhibition of their direct molecular targets and downstream signaling events. this notion of drug therapy may be too simplistic. clearly, continuous exposure to a growth factor stimulus does not produce continuous high output downstream signaling. rather, it leads to a sequelae of signaling events, programmed by negative and positive feedback signaling, until establishment of a new steady state in the presence of continued stimulus. we find here that continuous exposure to tkis similarly leads to a sequence of signaling events that manifest over time, until a new steady state is reached. with this new perspective, we report that her3/pi3k / akt signaling is not effectively inhibited by current tkis. in particular, the allocation of kinase and signaling functions to different members within the her family allows the signaling substrate her3 to restore signaling activity despite significant inhibition of her2 kinase, in effect buffering her3/pi3k / akt signaling against an incomplete loss of her2 kinase function. this inherent signal buffering capacity allows tumor cells to evade the pro - apoptotic effects of tkis resulting in a significant loss of their anti - tumor activity. the highly effective treatment of her - driven cancers may require drugs with much higher potency or drugs that completely inactivate her kinase function. irreversible tkis, although more potent, are subject to similar limitations. due to their reactive groups and reduced selectivity, future highly selective irreversible inhibitors may turn out to be more effective. until highly specific and fully inactivating drugs can be designed, combination treatment strategies designed to undermine the resiliency of her family signaling may offer the most promising approach in the near future. in addition, inhibition of autophosphorylation activity deceptively overstates the efficacy of tkis and is a poor in vivo biologic marker. the signal buffering capacity endowed by the separation of kinase and signaling functions to different family members in the her kinase family attests to an evolutionary advantage conferred by the loss of catalytic activity in the her3 protein kinase. this can shed light on why approximately 10% of the human kinome appears to be catalytically inactive 28. reagent sources are detailed in supplementary materials. for immunofluorescence studies, cells grown on fibronectin coated cover slips were treated as indicated, fixed in 4% paraformaldehyde, permeabilized, and stained with the indicated primary antibodies and fitc conjugated secondary antibodies. apoptotic cells were identified and quantified by analysis of annexin v binding using the annexin v - fitc apoptosis detection kit (calbiochem) according to the manufacturer s instructions, or by their sub - g1 dna content and quantified by facs analysis as previously described 30. all experimental arms were done in duplicate and displayed as averages with standard of deviation error bars. cells were seeded at a density of 300,000 cells per well in 12-well plates and transfected the following day. for sirna transfections 100300nmol of sirna (dharmacon) was premixed with lipofectemine2000 in opti - mem media and then added to each well. for plasmid transfections, 2 ug of plasmid dna was premixed with lipofectamine2000 in opti - mem media and added to wells for 6 hours. freshly prepared sulfo - nhs - ss - biotin was added to the final concentration of 0.5 mg / ml in pbs. following 45min incubation at 4c cells cells were rinsed twice with pbs and incubated with 10 m of freshly prepared h2dcfda in phenol - red free media for 45 min at 37c. reagent sources are detailed in supplementary materials. for immunofluorescence studies, cells grown on fibronectin coated cover slips were treated as indicated, fixed in 4% paraformaldehyde, permeabilized, and stained with the indicated primary antibodies and fitc conjugated secondary antibodies. apoptotic cells were identified and quantified by analysis of annexin v binding using the annexin v - fitc apoptosis detection kit (calbiochem) according to the manufacturer s instructions, or by their sub - g1 dna content and quantified by facs analysis as previously described 30. all experimental arms were done in duplicate and displayed as averages with standard of deviation error bars. cells were seeded at a density of 300,000 cells per well in 12-well plates and transfected the following day. for sirna transfections 100300nmol of sirna (dharmacon) was premixed with lipofectemine2000 in opti - mem media and then added to each well. for plasmid transfections, 2 ug of plasmid dna was premixed with lipofectamine2000 in opti - mem media and added to wells for 6 hours. freshly prepared sulfo - nhs - ss - biotin was added to the final concentration of 0.5 mg / ml in pbs. following 45min incubation at 4c cells cells were rinsed twice with pbs and incubated with 10 m of freshly prepared h2dcfda in phenol - red free media for 45 min at 37c. | oncogenic tyrosine kinases have proven to be promising targets for the development of highly effective anticancer drugs. however her family tyrosine kinase inhibitors (tkis) show only limited activity against her2-driven cancers despite effective inhibition of egfr and her2 in vivo 18. the reasons for this are unclear. signaling in trans is a key feature of this multimember family and the critically important pi3k / akt pathway is driven predominantly through transphosphorylation of the kinase - inactive her3 9,10. we report that her3 and consequently pi3k / akt signaling evade inhibition by current her family tkis in vitro and in tumors in vivo. this is due to a compensatory shift in her3 phosphorylation - dephosphorylation equilibrium driven by increased membrane her3 expression driving the phosphorylation reaction and reduced her3 phosphatase activity impeding the dephosphorylation reaction. these compensatory changes are driven by akt mediated negative feedback signaling. although her3 is not a direct target of tkis, her3 substrate resistance undermines their efficacy and has thus far gone undetected. the experimental abbrogation of her3 resistance by sirna knockdown restores potent pro - apoptotic effects to otherwise cytostatic her tkis, re - affirming the oncogene - addicted nature of her2-driven tumors and the therapeutic promise of this oncoprotein target. however, since her3 signaling is buffered against an incomplete inhibition of her2 kinase, much more potent tkis or combination strategies are required to effectively silence oncogenic her2 signaling. the biologic marker to guide her tkis should be the transphosphorylation of her3. |
substance abuse poses a major political, social and health challenge worldwide. besides being a personal risk, addiction is a social problem and imposes harmful and permanent effects on society. at present, 3.6%6.9% of adults (1564-years - old individuals) are under the influence of illicit substances. according to the world drug report 2013, since 2008, the number of illicit drug users has shown 18% increase ; rise in population and ease of availability appears to be the two common reasons. substance abuse in iran, one of the middle east countries, has a historical origin. during world war i, among 250,000 individuals of the tehran municipality, 25,000 were reported to be addicted to opium. in recent centuries these traditions are stronger among rural populations owing to a lack of access to trained physicians. the usage of opium had been so common in rural areas that mothers often exposed babies to opium in an effort to calm them and sleep better. the past two centuries have seen several changes with respect to handling illicit substances in iran, from open access to death penalty for carrying illicit drugs. at present, iran policy against illicit drugs is considered to be a combination of war on drugs and harm reduction strategies. with regards to opium use, iran ranks among the top three countries in the world. therefore, epidemiological studies in iran can provide better understanding for national policy makers as well as for other researchers in the world. although several studies on the prevalence of substance abuse in iran have been reported, the majority have been limited to urban areas [5, 6 ] ; studies in rural areas were limited or often included small sample size. approximate one third of the iran population lives in rural areas ; hence, it is critical to evaluate the substance abuse pattern in rural areas. in addition, the majority of epidemiological studies focussed on the trends of substance abuse because drug use behaviours are often dynamic, and with time are affected by various factors such as drug availability. the present study aimed to investigate the trends of substance abuse among the rural communities of the kerman province, the largest province in iran, during a 12-year period by comparing data for the years 2000 and 2012. first, kerman as an eastern province, due to neighborhood with afghanistan, the largest oipioid producing country in the world, is at higher risk of substance use, especially in youth group. a previous study reported that the usage of waterpipe, cigarette and alcohol, among high school boys, in this area was 51.5%, 34.6% and 7.27%, respectively. historically, kerman had highest of opium usage in iran ; second, accessibility of data from earlier years from this region makes it easy to compare and analyse the changes with time. to the best of our knowledge, a household survey was conducted in 2012 in dashtkhak, a northeastern village of kerman province with a total population of 4416. the research was conducted under the approval of the ethical committee of kerman university of medical sciences (approval code : k/90/516). following a brief meeting with the village council, details regarding the aim of the study, protocols and information regarding the questionnaire confidentiality agreement and informed consent form were provided before the study. a researcher along with community health workers distributed the questionnaires to each house. one participant above 12 years of age per household was chosen to complete the questionnaire. upon completion of the questionnaire community health workers are employed by the ministry of health and provide primary health care in rural areas. because of their acquaintance with the residents, their involvement in this research provided a great benefit to acquire data in a timely manner. the validity and reliability of the questionnaire used in this study has been confirmed in previous studies. it focussed primarily on three areas : the demographic features, details on substance abuse and the availability of substance. for details on substance abuse, substance abused throughout the lifetime, the last 30 days and every day were accounted. considering the pattern of substance abuse in iran [5, 10 ], the questionnaire included the following substances : cigarette, waterpipe, opium, shireh (opium residues), heroin, cannabis, shisheh, alcohol and sedatives. substance availability was counted using a four - degree likert scale. shireh comprises opium residue obtained after opium consumption that is boiled and concentrated ; this is more potent than opium. the chi - square test was used to compare the frequency of substances abused between the two time points. two separate multivariate logistic regression models for tobacco and the other drugs were included to assess the association between baseline characteristics and substance abuse. among the 1200 distributed questionnaires, 900 participants responded and completed the questionnaires (response rate = 75.0%). the average age and sex of non - respondents did not differ from those of respondents. the majority of respondents (61.8%) were below 30 years of age, and 54.4% among them were male (table 1). majority of the substances were consumed high among men compared with women (table 2). cigarette, opium and sedative usage was higher among the participants above 30 years compared with younger groups, whereas waterpipe consumption revealed a reverse pattern (table 3). from the participant point of view, cigarette, opium and waterpipe were easily accessible compared with other substances (table 4). moreover, cigarette, opiates, heroin and sedatives consumption increased significantly in 2012 (table 5). tobacco smoking was prevalent among male (or = 10.8, ci 95% : 6.218.6) and unemployed participants (or = 2.4, ci 95% : 1.44.3). other drugs such as opium, heroin, cannabis, sedatives, alcohol and amphetamines were common among males (or = 2.7, ci 95% : 1.94.0) and married (or = 4.5, ci 95% : 2.48.5) and unemployed (or = 2.4, ci 95% : detailed analysis on the trend and pattern of substance abuse is necessary to develop preventive measures. the present study illustrates a substantial increase in substance abuse in a rural area over a 12-year period. our study evaluates two different time periods in the same area using similar method, with a gap of 12 years ; this offers the additional advantage for comparing the effect of substance abuse over time. household surveys are considered as the gold standard for estimating the number of substance abusers, provided the participants are ensured of privacy and that they trust those collecting their information. such mutual trust between village inhabitants and community health workers governs the rural regions in iran. the primary drawback of this study was its limitation to a single rural area, which requires caution in generalizing results. in addition, although response rates less than 70% were considered acceptable, non - participation of 25% of subjects in our study may limit data interpretation. kerman province, because of its borders with afghanistan, is in the transit path for opium and heroin from eastern borders to other parts of the country. since the early 19th century, heavy opium use among the natives has been connected to its easy access. the participant population in the year 2000 was younger compared with that in 2012 (p < 0.05). this is probably because of an increase in average life span in iran in recent years. the lifetime substance abuse pattern showed a minor difference compared with that of the near - daily pattern. opium, waterpipe and cigarette had highest prevalence of substance abuse throughout life and opium, shireh and cigarette among the daily abused substances (table 2). this study reveals that opium abuse is endemic in this region with lifetime prevalence higher than that of cigarette smoking (table 2). in comparison with the data from 2000 showed that, in 2012, except for cannabis and alcohol, abuse of all other substances increased significantly. shireh and heroin abuse enhanced 7 - 8 times. based on anecdotal data, one of the reasons for the consumption of substances, such as waterpipe and shisheh, was not investigated in 2000 ; these substance gained popularity during the last few years and are now considered as re - emerging drugs [11, 12, 17 ]. although methamphetamine consumption was low compared with that of opiates, in urban areas, a gradual switch from traditional substances such as opium to synthetic substances such as methamphetamine is observed. however, according to the current study, substance abuse patterns in rural areas differ from those in urban areas. sedative consumption increased 6-fold, which is probably caused by easy availability of prescription drugs. an earlier study conducted in one of the rural regions of northern iran reported that 13.5% of the participants abused opium almost daily and 28.1% smoked cigarette daily. in another study, which analysed urine samples through anonymous unlinked testing, 14.5% showed opioid consumption, that is, similar to the rate of daily abuse of opium in our study (15.7%). a comparison of substance abuse among urban and rural population showed that opioid abuse is higher among rural population, whereas other substances such as alcohol waterpipe and cannabis were prevalent among urban population. substance consumption, except waterpipe, was higher among participants above 30 years of age compared with the younger age groups, indicating that waterpipe is regaining popularity among youth. in united states, it has been reported that except ecstasy and amphetamines, drug usage in the past year is relatively similar among adolescents in rural and urban regions. although new studies are in favour of higher usage of tobacco, cannabis and alcohol in u.s. the substance abuse was also much higher among men compared with that among women (table 3) ; the most obvious difference was observed for cigarette, suggesting the social stigma associated with cigarette smoking in women in iranian culture, while the stigma associated with waterpipe consumption is significantly less. sedative consumption was similar among both men and women probably because of similar and easy access to prescription drugs and the prevalence of anxiety and insomnia in general population. approximately three quarters of rural population had easy access to cigarette and water pipe, and half of them had easy access to opium. despite the law against usage of tobacco, sale of cigarette and waterpipe tobacco in public places is common in iran. waterpipe usage is common among youth and has increased in recent years, which can be often observed in coffee shops, parks and college dormitories. because alcohol was prohibited by religion, it was expected that access to alcohol be most difficult. the logistic regression analysis showed that higher the difficulty towards access to tobacco, the less the probability of tobacco smoking (table 6). in iran, there is no close monitoring on tobacco sales to youth and cigarette smoking is socially well accepted in metropolitan areas than rural areas. in rural areas, cultural barrier appears to play a role than geographical barrier, and the difficulty in access may be related to factors such as family supervision and cultural taboo on smoking by youth and women. the higher probability of tobacco smoking among men was in - line with the earlier results from both the urban [20, 21 ] and rural areas of iran. the differences among men and women regarding drug abuse (or = 2.7) were less prominent than tobacco smoking (or = 10.8) (table 6). it may be because of the fact that except for tobacco all other substance abuse is illegal in iran ; while drug abuse is considered a taboo among men and women, usage of tobacco is socially accepted among men, whereas it is stigmatized among women [20, 21 ]. in contrast to drug abuse, age had no effect on tobacco smoking, perhaps because of the higher age at onset of drug abuse comparing with that of tobacco abuse [10, 21 ]. drug abuse was more common among unemployed ; a finding that may not imply causation because of an ambiguous order of variables (i.e. unemployment and drug abuse). in conclusion, the present study shows increased prevalence of substance abuse among rural population in iran. the results of the present study emphasize the necessity of immediate multi - dimensional preventive measures to regulate substance abuse among rural communities. | introduction and aim. substance abuse imposes hazards on human health in all biopsychosocial aspects. limited studies exist on epidemiology of substance abuse and its trend in rural areas. the present study aimed to compare substance abuse in one of the rural areas of southeast iran, in a 12-year period (2000 and 2012). design and methods. in a household survey conducted in 2012, in dashtkhak / kerman, 1200 individuals above 12 years of age completed a questionnaire to determine their frequency of substance abuse. the questionnaire included the following three areas : demographic characteristics, frequency of substance abuse and ease of access to various drugs. results. among 900 completed questionnaires, majority of the participants (61.8%) were below 30 years of age and among them 54.4% were male. cigarette (17.0%), opium (15.7%) and opium residue (9.0%) were the most frequent substances abused on a daily basis. based on the participant 's opinion, we conclude that the ease of access to cigarette, waterpipe and opium contributed to their increase in consumption compared with earlier years. discussion and conclusion. the steady rise in substance abuse in rural communities demands immediate attention and emergency preventive measures from policy makers. |
periodontitis is a common local inflammatory disease of tooth - supporting tissues initiated by microorganisms present in the dental plaque. as the disease progresses, there is periodontal pocket formation with increased attachment loss, alveolar bone destruction, and increased tooth mobility that ultimately result in the loss of teeth. association between various systemic diseases and periodontitis has long been established and it is the focus of most researches in the recent past. diabetes mellitus (dm) is a commonly prevalent systemic disease and has been proved to have a bidirectional relationship with periodontitis. diabetes is characterized by hyperglycemia (elevated blood glucose) that results from defects in the secretion of the hormone insulin or from impaired insulin action or both. chronic hyperglycemia is associated with long - term dysfunction and damage to numerous end - organs, with marked effects on the eyes, kidneys, heart, nerves, and blood vessels. although periodontitis is a recognized complication of diabetes, people with well - controlled diabetes, having good oral hygiene, are not at an increased risk of periodontitis. however, their susceptibility to periodontitis is significantly increased when their diabetes is poorly controlled, particularly smokers. national diabetes fact sheet, 2011, presented by the centres for disease control and prevention (cdc) reported that adults aged 45 years or older with poorly controlled diabetes were 2.9 times more likely to develop severe periodontitis than nondiabetics. in the successful treatment and prevention of both these commonly prevalent diseases, knowledge about their mutual influence among the dentists, general practitioners, and patients plays a critical role. in addition, general practitioners and health - care providers for diabetic patients should also possess basic dental knowledge to find out the signs and symptoms of dental diseases to provide appropriate treatment or advice to visit a dentist. hence, the present study aimed to assess the levels of awareness about the relationship between diabetes and periodontitis among high - risk age group of the general population of nellore. in addition, the attitude of general physicians in suggesting the diabetic patients to visit a dentist has also been assessed. a structured, closed - ended questionnaire was prepared in english and in the local language (telugu) for evaluating the main aim of the study. the study was designed and conducted at the department of periodontics, narayana dental college & hospital, nellore, ap. the questionnaire was distributed among patients attending the outpatient unit of the department and satisfying the following selection criteria : patients of age 4055 years ; patients without any condition that limits their ability to brush their teeth ; patients with not < 20 teeth ; and patients who can read and write either in english or in the local language (telugu). patients who readily agreed to participate in the study were provided with the self - constructed questionnaire [table 1 ]. prior to study, a questionnaire was pretested and validated. the questionnaire was validated for construct and content validity, reliability, and ease of use. questionnaire showed high degree (0.89) of agreement during test - retest of questionnaire. demographic data such as age, sex, educational status, and occupation have been recorded and documented. two hundred and three participants were included in the study and the completed questionnaire forms from the participants were collected and analyzed. the collected data were imported into the statistical package for social sciences version 21 (ibm spss statistics for windows, version 21.0. chi - square test was used for assessing the association between diabetic population and knowledge about the mutual relationship. demographic data of the study population including response to questionnaire were represented in table 2. nearly, 4.4% were from health - related professions such as pharmacy, nursing, and paramedical. a composition of 30% of the studied population were reportedly first - time visitors to a dentist. nearly, 29.6% of the studied population were diabetic and 44.8% had a family history of diabetes, of which, 37.1% are nondiabetic but having a family history of diabetic. in spite of being at a high risk (based on age 40 years) for diabetes, 37.9% of the studied population did not undergo any blood test for screening dm in the past 6 months and 13.79% had not undergone any blood test knowing the family history of diabetes. only 49.8% of the sample population knew about the mutual relationship between diabetes and periodontitis and the percentage of knowledge about mutual relationship between diabetic and nondiabetic population is shown in table 3 and the source of information regarding mutual relationship is shown in graph 1. general physicians have suggested only 46% of their diabetic patients to visit a dentist [table 4 ]. nearly, 21.2% of the studied population had no idea that diabetes would cause periodontal problems, 60% of the diabetic population agreed that their current oral status is related to diabetes, and 46.66% felt that they had periodontal problems. response to questionnaire percentage of diabetic and nondiabetic population knowledge about mutual relationship source of information regarding mutual relationship percentage of physician suggesting study population to visit a dentist both periodontitis and dm are frequent chronic diseases and generate enormous costs for the public health - care system. numerous studies, review articles, and meta - analyses indicated a mutual influence between periodontitis and dm. grossi and genco in 1998 proposed that the relation between periodontitis and diabetes is bi - directional, this was further supported by taylor. various factors have been reported that deter patients with dm from seeking dental care, including financial factors, the fear of dental treatment, and lack of knowledge of the need for dental check - up. type 2 dm patients were 2.8 times more likely to have destructive periodontal disease and 4.2 times more likely to have alveolar bone loss progression. mealey and oates in 2006 have shown the prevalence of periodontitis in diabetic subjects that is estimated to be double or even triple the number in the normal population. the world dental federation and international diabetes federation have pointed out that the key for the prevention of periodontitis in patients with dm lies in close collaboration between dentists and physicians. however, in the present study, an attempt has been made to estimate the knowledge about the established bi - directional relationship between diabetes and periodontitis by questionnaire study. we limited the age group to be between 40 and 55 years based on the national diabetes fact sheet, 2011, by cdc. education qualification of minimum of 10 class was set as a limit so that participants can read and write either english or local language (telugu). occupation of the participants was categorized as health - related and nonhealth - related professions. number of participants related to health profession was insignificant, i.e., 4.4%, which has a negligible impact on the study. according to a study by aggarwal and panat, only 10.8% of the patients with dm visit a dentist for regular check - ups and indians with type 2 diabetes reported suboptimal oral hygiene behavior. reported that a total of 56% of the participants of their study had an insufficient knowledge about the mutual influence between diabetes and periodontitis, which correlates with the current study (50.2%). among the diabetic study population, lin. stated that endocrinologists and dentists are not equally equipped with the knowledge about the relationship between dm and periodontitis. in our study, only 46% (p < 0.0001) of the general physicians suggested their patients to visit a dentist. these findings suggest that there is a significant need for increased knowledge of mutual relationship and adoption of preventive oral hygiene behaviors that would improve oral health among diabetics. smaller sample size too early to generalize the resultsstratification of education levels was not done in the study. higher level of education may influence the knowledge about relationshipfurther studies with larger sample size and inclusion of level of education might potentiate the study. smaller sample size too early to generalize the results stratification of education levels was not done in the study. higher level of education may influence the knowledge about relationship further studies with larger sample size and inclusion of level of education might potentiate the study. smaller sample size too early to generalize the resultsstratification of education levels was not done in the study. higher level of education may influence the knowledge about relationshipfurther studies with larger sample size and inclusion of level of education might potentiate the study. smaller sample size too early to generalize the results stratification of education levels was not done in the study. higher level of education may influence the knowledge about relationship further studies with larger sample size and inclusion of level of education might potentiate the study. there is an insufficient knowledge among the high - risk age group individuals about the mutual relationship between diabetes and periodontitis. to promote proper oral health and to reduce the risk of oral diseases, health professionals in both the dental and medical fields need to take responsibility for educating the public about the oral manifestations of diabetes and its complications | aim : the study aimed to assess the levels of awareness about the mutual relationship between diabetes and periodontitis among high - risk age group of the general population and to assess the attitude of general physicians in suggesting diabetic patients to visit a dentist.materials and methods : a structured, closed - ended questionnaire either in english or in local language (telugu) was distributed and collected from 203 patients who were willing to participate in the study attending the department of periodontics, narayana dental college & hospital, nellore, ap. data were statistically analyzed and represented in percentages and number.results:only 49.8% of the sample population knew about the mutual relationship between diabetes and periodontitis and only 46% of the diabetic study population was suggested to visit a dentist by the physician.conclusion:there is an insufficient knowledge among the diabetic population regarding the mutual relationship. only few physicians have suggested their diabetic patients to visit a dentist. as diabetic patients tend to visit a physician earlier than a dentist, it is their responsibility to educate and motivate their patients to seek dental treatment. |
the risk of occupational exposure to blood borne pathogens (including hepatitis b, hepatitis c and hiv) via sharp injuries such as needle stick injuries (nsis) among health care workers, especially dental, nursing and midwifery students, is a challenging issue,,,,,. inadequate staff, lack of experience, insufficient training, duty overload and fatigue may lead to occupational sharp injuries,. it is estimated that approximately 600,000 to 800,000 nsis occur each year among health care workers in the united states (one injury every ten seconds). the risk of pathogen transmission from infected persons through an injury with a sharp object have been estimated to be 630% for hbv in non - immune individuals, 510% for hcv, and 0.3% for hiv. administration of pre - exposure vaccination or post - exposure prophylaxis is effective in preventing hbv and hiv infections, respectively, but is not available or not effective in preventing hcv infection. the aim of this study was to evaluate the frequency of nsis in iranian dental, nursing and midwifery students and their knowledge, attitude and practices regarding the use of protective strategies against exposure to blood borne pathogens). a cross - sectional survey of 264 dental and 435 nursing and midwifery students during their under graduate clinical training at the shiraz university of medical sciences, iran, was conducted. a questionnaire that included demographic data, frequency and reporting of nsi, protective practices and knowledge, attitude, and concerns regarding blood - borne pathogens was administred to all participants. the questionnaire was pre - tested for reliability on 9.2% of the 55 sample population and found to have a high (r=0.812) test - retest reliability. the data was entered in to a computer using the software package epi - info (version 2000). descriptive and significant tests, duncan test, spearman s correlation coefficient and student t - test were performed using spss - version10. the survey was completed by 137 (51.9%) dental students and 208 (47.8%) nursing and midwifery students. there was a significant female predominance among the nursing and midwifery students when compared to dental students (67.3% vs. 50% ; p=0.002). the majority of the students (85%) reported to have received information about standard precautions. since entering their clinical year, 72.1% (150/208) of the nursing and midwifery students, and 73.7% (101/137) of the dental students experienced a total of 424 and 268 nsis, respectively, giving a ratio of 1:1.9 and 1:2 nsis among students in their undergraduate clinical practice over an average of a 12 months period (maximum 17 months). differences were noted in the situation of injury among nursing and midwifery students : 31.4% (133/424, p<0.005) of all nsi occurred at the delivery room, 26.9% in patient rooms, 22.9% in the operation theatre, 17.9% in the emergency room, and 0.9% at unknown instances. in dental students, 53% (142/268, p<0.001) of all nsis occurred during patient treatment, 9.3% in surgical wards, 3.3% in the emergency room, 1.1% in the operation theater, and 3.3% at unknown instances. the clinical activities most frequently associated with injuries involved a hollow - bore needle used during venous sampling or iv injection in both groups, followed by wound suturing, in nursing and midwifery, and recapping in the dental students. the 3 most common activity associated with nsis in nursing and midwifery students was recapping (7.6%) and wound suturing (17%) in dental students (table 1 (tab. 1)). the majority of the last injury recalled involved the students injuring themselves : 93% nursing and midwifery students and 96% dental students, respectively. 75% of nursing and midwife students, and 85% of dental students did not report there injury. the reason for not reporting in decreasing frequency included not knowing the reporting mechanism, did not realize that all nsis required reporting and evaluation or they did not know to whom to report the injury. the majority of students (85%) reported to have received information about standard precautions, yet most had not been encouraged by clinical staff to double glove while using needles. glove use behaviors were examined by clinical activities and almost all (95.2% ; 198/208) students reporting to routinely wear gloves for wound suturing. glove use for all other activities was poor with 24.1% of nursing and midwifery students wearing gloves while performing venous sampling or iv injection, 2% always wearing double gloves while scrubbed in the or, 1% double gloved while in the er, and 1% during iv injection. for dental students, glove use was slightly better while performing venous sampling or iv injection, 9.5% always wore double gloves while in the er. the reasons given by students who did not routinely wear double gloves was decreased tactile sensitivity during manipulation, followed by inadequate facilities not providing gloves at the point of care. among nursing and midwifery students the use of sharp containers was higher (59.1%) when compared to dental students (35%) with more than 2/3 of all students practicing needle recapping. eye protection in the operating room was not used by the majority (97%) of nursing and midwifery students while 47% of the dental students used eye protection. 95% of all dental students and 75% of all nursing and midwifery students were vaccinated. 44.2% of nursing and midwife students, and 66.4% of dental students stated, that they are extremely or very concerned about the risk of contracting a blood borne virus infection. students reported patient risk factors on a five - point scale, ranging from extreme to no concern, with aids (9199%), hbsag - positive (9197%) and injecting drug use (76.496%) causing extreme concerns by the majority of students. concerns that the type of surgery was a risk factor were not significantly (p=0.37) different for the extreme (27.7%) to moderate (23.0%) levels of concern. students report risk factors associated with gender (32.1%), race (0.4%) and age (44.7%) at the very concerned to moderately concerned levels. this is the first survey of nsi among midwife, nursing and dental students in shiraz, iran. nsis are a recognized source of exposure to blood borne pathogens for health care workers in high risk occupations,. turkish nurses asked on how many times they had been injured by a needle or another sharp object in the past 12 months, reported an average of 1.7 (range 012) nsis. most of the nurses (52.5%) reported that they had experienced nsis more than once in the last year. of those, 186 nurses had been injured more than twice in the past year. 61.9% of students in taiwan had at least one nsi and the majority (70.1%) of these nsi occurred in the patient room. lee. reported 56% of emergency medicine students having one or more nsis, 31% of which were due to hollow - bore needles. 30% of medical students in washington had sustained at least one nsi and that these most commonly (72.1%) occurred in the operating room. in our study, 73% of our students reported at least one nsi, most of which occurred among nursing and midwifery students in the delivery room and patient room during venous blood sampling or iv injection. lack of experience in many procedures, insufficient training, duty overload and fatigue lead to occupational sharp injuries,,,,. the high level of non - reporting nsis (80%) suggests that the students need a center of prevention which address the importance of reporting all nsis,,,,,,. furthermore, regarding that a frequently stated reason for not reporting was not knowing the reporting mechanism or not knowing to whom to report indicates the need for a better reporting management. our study showed that 84.8% of the students reported receiving information about standard precautions and bloodborne pathogen exposure. this is in contrast to a study performed by patterson., which evaluated nsis among medical students in developed country and found 98% of them reported receiving information about this topic. these data indicate that students need to be provided structured education on standard precautions for the improvement of occupational safety,,. the risk of hbv infection can be avoided by ensuring adequate hepatitis b vaccination. 16% of taiwanese student nurses had not been vaccinated against hbv, a preventable measure. 32.4% (45 of 139) of turkish nurses had not been vaccinated against hbv. our study revealed that dental students had higher hbv vaccination when compared to nursing and midwifery students (95% vs. 75%). one of the protective strategies against nsi is use of gloves by health care workers,. in this study, most students reported gloving during wound suturing. although some studies showed that one feasible protection strategy is the use of double gloves,, 50% of medical students in a study conducted in strasbourg did not use gloves. in our survey, 59.1% of nursing and midwifery students used sharps container and only 5.7% of them did not recap needles after use. our analysis indicates the nursing and midwifery students are at high risk for sharps injuries and bloodborne pathogen exposure. in agreement with ayranci et we believe that the prevention of nsis through the increase knowledge regarding transmission of bloodborne infections, education about standard precautions and protection strategies against bloodborne pathogens such as wearing gloves, using eye protection, using sharps containers to dispose of needles and not recapping needles. furthermore, an optimization of the management for reporting in warranted. this study was funded by the deputy for research at the shiraz university of medical science (grant no. this study was funded by the deputy for research at the shiraz university of medical science (grant no. | background : the risk of occupational exposure to blood borne pathogens (including hepatitis b, hepatitis c and hiv) via sharp injuries such as needle stick injuries (nsis) among health care workers, especially dental, nursing and midwifery students is a challenging issue. inadequate staff, lack of experience, insufficient training, duty overload and fatigue may lead to occupational sharp injuries. the aim of this prospective cross - sectional study was to evaluate the frequency of nsis in iranian dental, nursing, and midwifery students and their knowledge, attitude and practices regarding prevention of nsis.methods : a questionnaire was provided to 264 dental and 435 nursing and midwifery students during their under graduate clinical training. 52% of dental students and 48% of nursing and midwifery students responded to the questionnaire. the questionnaire was pre - tested for reliability on 9.2% of the 55 sample population and found to have a high (r=0.812) test - retest reliability.results : 73% of students reported at least one nsi during the past year. activities most frequently associated with injuries involved use of a hollow - bore needle during venous sampling or iv injection in both groups, followed by wound suturing in nursing and midwifery students and recapping in dental students. nsis and non - reporting of nsis were highly prevalent in these participants. the reason for not reporting injuries included not knowing the reporting mechanism or not knowing to whom to report.conclusion : education about transmission of blood borne infections, standard precaution and increasing availability of protective strategies must be enforced. furthermore, an optimization of the management for reporting is warranted. |
breast milk provides an abundance of nutrients in bioavailable forms that are crucial for the infant 's normal growth and development. exclusive breastfeeding provides strong protection against lower respiratory tract infections, gastroenteritis, middle ear infections, and childhood obesity [25 ]. currently only 14.1% of infants in the us are breastfed exclusively through 6 months, below the target rate of 25.5% in the healthy people 2020 objectives. breastfeeding mothers may encounter cultural and commercial barriers that make it difficult for them to sustain exclusive breastfeeding for the recommended duration. due to escalating acceptance of infant formula use by doctors and hospitals, breastfeeding can become something people feel they can opt in or out of and may lose its place as an essential part of infant development [8, 9 ]. following the birth of a baby, information given to the mother can influence her confidence and adaptation to breastfeeding. hospital practices that avoid formula supplementation and encourage early maternal contact with the newborn (e.g., holding baby skin - to - skin right after birth) and rooming - in support breastfeeding. however, by distributing commercial gift bags containing formula samples, hospitals are inadvertently endorsing formula feeding, an action that has been associated with reducing exclusive breastfeeding rates [7, 10 ]. this subtle endorsement appears to increase early formula supplementation leading to untimely termination of breastfeeding, particularly with mothers who have unclear or short breastfeeding goals [11, 12 ]. in earlier findings, researchers recommended that materials consistent with the world health organization code (no free samples to mothers, and no promotion products in health care facilities including the distribution of free or low - cost supplies) replace commercial discharge materials for breastfeeding women. for example, the launch of a major breastfeeding initiative in 2006 by the new york city department of health and mental hygiene led to the elimination of formula sample packs from all 11 public hospitals operated by the new york city health and hospitals corporation. however, most hospitals in the us continue to distribute commercial discharge bags packaged as smart diaper bags containing various coupons, advertisements, baby products, and infant formula samples. in 2008, merewood. examined 21 eastern states and washington dc for their gift pack distribution practice. they found that formula sample distribution was still a common practice in eastern states ; that is, 94% of hospitals distributed formula sample packs to new mothers at discharge, ranging from 70% to 100%. new jersey was one of four states in which all maternity hospitals distributed discharge bags containing sample formulas. increasing number of hospitals was discontinuing the practice, with regional differences ; for example, distribution was least prevalent in new england and was most prevalent in dc and neighboring states. though a number of studies have examined the effect of commercial discharge bags containing formula samples on the duration of breastfeeding, studies that examine the impact of varying content of hospital discharge bags on breastfeeding duration are few and outdated [1619 ]. in order to promote exclusive breastfeeding, a closer examination of the usage of hospital discharge bags is warranted. the purpose of this study was to compare the effects of innovative discharge gift bags on the exclusivity and duration of breastfeeding compared to commercial discharge bags. the specific research hypotheses were that (1) the content of discharge gift bags will have an impact on the exclusivity and duration of breastfeeding, and (2) mothers who receive the innovative discharge gift bags will likely to breastfeed longer than mothers who receive traditional commercial discharge bags. the commercial gift bags (commercial) that hospitals routinely gave to postpartum mothers contained infant formula samples and industry coupons for formula, baby food and bottles, and industry - printed guide to breastfeeding. two types of innovative gift bags were used in this study : (1) bf - info gift bags containing breastfeeding information and nursing supplies and (2) pump gift bags containing a manual breast pump as well as breastfeeding information and nursing supplies. common contents selected to include in the two innovative gift bags (see table 1) were designed to support the breastfeeding mothers. for example, we provided disposable and reusable / washable breast pads for leaking milk [20, 21 ], water bottle for hydration, sample nipple cream to ease any soreness [2225 ], and a dvd showing correct latching [26, 27 ] in addition to breastfeeding information and resources for local lactation supports. the intervention in this study was the provision of innovative discharge gift bags for mothers when they are discharged from the hospital. a cohort of breastfeeding mothers was invited to participate in the study from three maternity hospitals in southern new jersey during 2009 and 2010. the eligibility criteria to participate in this study were breastfeeding mothers who spoke english or spanish, 18 years or over in age, and delivered a full - term baby. when breastfeeding mothers volunteered to participate in the study, they were assigned to groups. for example, we recruited the cohort for commercial first and then the cohort for bf - info followed by the cohort for pump. once the projected sample size for the first cohort (commercial) was reached, we recruited the second cohort (bf - info) followed by the third cohort (pump). participants received either the industry gift bag or one of the innovative discharge gift bags in the hospital. mothers were then followed over 12 postpartum weeks to query their infant feeding methods, exclusive or partial breastfeeding, at three contact points. mothers who were feeding the baby breast milk exclusively, without any supplementation (e.g., infant formula, other fluids, or solid food), were recorded as exclusive breastfeeding, that is, nursing at the breast or bottle - feeding with expressed milk. mothers who supplemented their breastfeeding with infant formula or any other food / fluid were recorded as partial breastfeeding. on the day of the discharge (at baseline) the contents of the bag were reviewed with minimal commentary in a question and answer format. upon receiving gift bags at baseline, participating mothers completed a survey that included demographic information and asked about their intended duration of breastfeeding, as well as obtaining contact information for followup. at 2, 4, and 12 weeks postpartum, the mothers were contacted via email / phone / postal mail to inquire about their infant feeding methods as well as their perception of the innovative gift bags. inquiry followed a structured questionnaire composed of multiple - choice and open - ended questions. the institute of review board at the university and the three maternity hospitals approved the study protocol. the mean duration of exclusive or partial breastfeeding was compared between groups using the analysis of variance. the tukey 's hsd test was performed to detect which specific means were significantly different from one another. the proportion of mothers who maintained exclusive or partial breastfeeding through 2, 4, or 12 weeks was compared between groups at each point using the chi - square analysis. demographic characteristics were then compared between groups to test the equivalency of intervention and control groups. for demographic characteristics (i.e., marital status) that are significantly different (p 0.05). however, the marital status was statistically different between groups (p = 0.04). a higher proportion of mothers in commercial group were single, compared to bf - info and pump groups : 16.1% versus 6.6% versus 9.4%, respectively. therefore, additional comparison of the mean duration of exclusive breastfeeding was performed to determine the relationship between the marital status and exclusive breastfeeding. participant loss in followups (nonrespondents) was largely due to their change of contact information or absence of response to phone messages / mails. the demography of non - respondents compared to respondents who completed the study was different (p 0.05) in breastfeeding duration and exclusivity between groups. on the other hand, bliss. reported that receiving a manual pump in the discharge bags helped mothers breastfeed exclusively during early postpartum period, at 6 weeks. the current study provides the most recent evidence of the positive impact of using discharge bags that contain breastfeeding information plus a manual breast pump on the duration and exclusivity of breastfeeding. the role of the manual pump in early postpartum is made clearer from the mothers ' descriptions of their use in this study. it is important to remember that previous studies that tested the content of hospital gift bags were conducted more than a decade ago. the positive impact of receiving the gift bag containing a manual pump in addition to breastfeeding information in the current study could be a combination of evolution in attitudes toward breastfeeding, staff support during hospital stay, and the technological advancement of the manual pump over the past decade. married mothers in the current study consistently breastfed longer and maintained exclusivity more than single mothers, regardless of the type of gift bag they received. when comparing mothers in bf - info group, the difference of the mean exclusive breastfeeding duration in this study was large and statistically significant between marital statuses (p = 0.042). it may be that single mothers may lack the social support needed to utilize the breastfeeding information to its fullest, for example, unable to utilize the local lactation resources, or readily discuss with partners about lactation challenges. in addition, married mothers maybe able to stay at home with their babies and breastfeed while single mothers are more likely to need to go to work as the sole household provider. when comparing mothers in pump group, the difference of the mean duration of exclusive breastfeeding between marital statuses was minor and statistically insignificant (p = 0.653). this supports the potential role of the manual pump in enabling single mothers to maintain breastfeeding exclusivity. limitations of this study include the lack of diversity in participants, for example, non - wic participants, primarily white, and relatively highly educated women participated in the study. future studies with more diverse and vulnerable populations such as wic eligible women and working mothers can provide further understanding of needs for exclusive breastfeeding. other limitations include having a long gap between follow - up contacts (2nd and 3rd) and relying on mother 's report on infant feeding practice. more frequent follow - up contacts (e.g., every 2 weeks) could improve the completion rate. future studies could incorporate additional methods (e.g., review of medical chart) to determine infant feeding practice to cross - validate mothers ' reports. moreover, studies that examine the rate of exclusive breastfeeding through 6 months are needed to confirm the findings of the current study. one of the most noteworthy findings from the current study is that the rate of exclusive breastfeeding for mothers provided with a manual pump was far above the rate in previous studies : 62.2% versus less than 30% at 12 weeks. hospitals that continue to provide commercial gift bags while also supporting breastfeeding during mothers ' hospital stay are sending confusing signals to breastfeeding mothers. by providing breastfeeding - friendly innovative gift bags, hospitals can practice a uniform promotion strategy for breastfeeding and encourage mothers to breastfeed their babies. recent studies have shown that hospitals have become more open to distributing alternative gift bags for ethical or health - based reasons. these alternative bags commonly contain breast pads, baby blankets, and water bottles for the mother. adding a manual breast pump in the alternative gift bags is worth consideration for which we could provide mothers with environmental as well as technical and practical support. hospitals, government institutions, and private industries should work together to support strategies and practices that reinforce our national effort to promote exclusive breastfeeding. | the type of gift bags given to new mothers at the time of discharge from the hospital can influence their confidence in breastfeeding. most hospitals in the us continue to distribute commercial gift bags containing formula samples despite the reported negative influence of commercial bags on the duration of breastfeeding. this study compared breastfeeding outcomes in women receiving three different kinds of gift bags at discharge. a prospective intervention study was conducted during 2009 - 2010 in new jersey. three breastfeeding cohorts were recruited and assigned to three groups : commercial received discharge bags containing formula samples, bf - info received breastfeeding information and supplies, and pump received breastfeeding information / supplies plus a manual breast pump. follow - up contacts were at 2, 4, and 12 postpartum weeks to determine breastfeeding outcome. the mean durations of exclusive (ebf) and partial breastfeeding were compared between groups using anova. a total of 386 participants completed the study. the mean ebf duration (weeks) in the pump (n = 138, 8.28 4.86) and bf - info (n = 121, 7.87 4.63) were significantly longer (p < 0.01) than commercial (n = 127, 6.12 4.49). the rate of ebf through 12 weeks in pump was most consistent. the mean duration of partial breastfeeding showed similar results : significantly longer in pump and bf - info than commercial (p < 0.01). |
the inrhythm study is a prospective study of af and its relationship to psychosocial and ci. participants with symptomatic af were recruited and followed at 1 of 3 ambulatory clinics associated with the university of massachusetts medical center af treatment clinic (heywood hospital, marlborough hospital, and the university of massachusetts medical center university campus). the present analysis includes 218 inrhythm participants with symptomatic af and available clinical, psychosocial, cognitive, and qol data. all participants gave informed consent, and all inrhythm protocols were approved by the university of massachusetts medical school review board. all inrhythm study participants underwent a history, physical examination, and laboratory evaluation as part of their routine clinical evaluation for af. the demographic, clinical, and treatment characteristics of inrhythm participants were abstracted from the electronic medical record by trained study staff. information abstracted from the medical record included information about participant s age, sex, race, type of af, cardiovascular comorbidities (eg, myocardial infarction, diabetes, hypertension, and heart failure), noncardiovascular comorbidities (eg, anemia, renal failure), and prior antiarrhythmic drug exposure status. participants were classified as having af if the arrhythmia was present on a 12-lead electrocardiogram obtained during an af treatment center clinic visit or an encounter with an outside health care provider, on a holter or cardiac event monitor, or if af was noted in any hospital record. the moca is a 10- minute, 30-item screening tool that was designed to assist physicians in detecting mild ci. the moca is the currently recommended screening test for ci in patients with cardiovascular disease by the national institute for neurologic disorders and stroke and the canadian stroke institute. a cutoff score of 27 (range, 030) has been shown to have a high sensitivity (0.90) and specificity (0.87) for detecting mild ci, and was used as a cutoff to define ci (scores of 26 and below were considered as impaired). depression was assessed using the 9-item version of the patient health questionnaire (phq-9). using a cut - point range of 10 (range, 027), the phq-9 has high sensitivity (0.88) and specificity (0.88) for detecting major depression. anxiety was assessed using the generalized anxiety disorder-7 scale, a revised version of the anxiety module from the phq that consists of diagnostic and statistical manual of mental disorders, 4th edition (dsm - iv) criteria for generalized anxiety disorder over the past 2 weeks. the generalized anxiety disorder-7 scale score ranges from 0 to 27 with a score 10 having high sensitivity (0.89) and specificity (0.82) for psychiatrist - diagnosed anxiety disorder. disease - specific qol was measured during a clinic visit using the atrial fibrillation effect on quality - of - life (afeqt) questionnaire, which includes subscales for symptom severity, global well - being, af burden, and impact on health care utilization. the afeqt questionnaire consists of 20 questions separated into 4 domains : symptoms, treatment concerns, daily activities, and treatment satisfaction. each question is graded on a 1- to 7-point scale, and the total raw score is transformed to a 0100 scale, with 100 points indicating the best possible af - related qol and 0 points indicating the poorest possible af - related qol. we compared the characteristics of inrhythm participants according to number of psychosocial and cognitive comorbidities (count of anxiety, depression, and ci ; range, 03) and compared baseline patient characteristics by number of comorbidities using analysis of variance for continuous variables and the test for categorical variables. we used a linear regression model to examine associations between baseline burden of psychosocial / cognitive comorbidity and af - specific qol score at 6 months, adjusting for all factors associated with psychosocial / cognitive comorbidity (p 0.2) in univariate analyses. covariates included in multivariable models included age, sex, white race, education, smoking status, heart failure, prior stroke, anemia, renal failure, and total number of cardiac comorbidities. the inrhythm study is a prospective study of af and its relationship to psychosocial and ci. participants with symptomatic af were recruited and followed at 1 of 3 ambulatory clinics associated with the university of massachusetts medical center af treatment clinic (heywood hospital, marlborough hospital, and the university of massachusetts medical center university campus). the present analysis includes 218 inrhythm participants with symptomatic af and available clinical, psychosocial, cognitive, and qol data. all participants gave informed consent, and all inrhythm protocols were approved by the university of massachusetts medical school review board. all inrhythm study participants underwent a history, physical examination, and laboratory evaluation as part of their routine clinical evaluation for af. the demographic, clinical, and treatment characteristics of inrhythm participants were abstracted from the electronic medical record by trained study staff. information abstracted from the medical record included information about participant s age, sex, race, type of af, cardiovascular comorbidities (eg, myocardial infarction, diabetes, hypertension, and heart failure), noncardiovascular comorbidities (eg, anemia, renal failure), and prior antiarrhythmic drug exposure status. participants were classified as having af if the arrhythmia was present on a 12-lead electrocardiogram obtained during an af treatment center clinic visit or an encounter with an outside health care provider, on a holter or cardiac event monitor, or if af was noted in any hospital record. the moca is a 10- minute, 30-item screening tool that was designed to assist physicians in detecting mild ci. the moca is the currently recommended screening test for ci in patients with cardiovascular disease by the national institute for neurologic disorders and stroke and the canadian stroke institute. a cutoff score of 27 (range, 030) has been shown to have a high sensitivity (0.90) and specificity (0.87) for detecting mild ci, and was used as a cutoff to define ci (scores of 26 and below were considered as impaired). depression was assessed using the 9-item version of the patient health questionnaire (phq-9). using a cut - point range of 10 (range, 027), the phq-9 has high sensitivity (0.88) and specificity (0.88) for detecting major depression. anxiety was assessed using the generalized anxiety disorder-7 scale, a revised version of the anxiety module from the phq that consists of diagnostic and statistical manual of mental disorders, 4th edition (dsm - iv) criteria for generalized anxiety disorder over the past 2 weeks. the generalized anxiety disorder-7 scale score ranges from 0 to 27 with a score 10 having high sensitivity (0.89) and specificity (0.82) for psychiatrist - diagnosed anxiety disorder. disease - specific qol was measured during a clinic visit using the atrial fibrillation effect on quality - of - life (afeqt) questionnaire, which includes subscales for symptom severity, global well - being, af burden, and impact on health care utilization. the afeqt questionnaire consists of 20 questions separated into 4 domains : symptoms, treatment concerns, daily activities, and treatment satisfaction. each question is graded on a 1- to 7-point scale, and the total raw score is transformed to a 0100 scale, with 100 points indicating the best possible af - related qol and 0 points indicating the poorest possible af - related qol. we compared the characteristics of inrhythm participants according to number of psychosocial and cognitive comorbidities (count of anxiety, depression, and ci ; range, 03) and compared baseline patient characteristics by number of comorbidities using analysis of variance for continuous variables and the test for categorical variables. we used a linear regression model to examine associations between baseline burden of psychosocial / cognitive comorbidity and af - specific qol score at 6 months, adjusting for all factors associated with psychosocial / cognitive comorbidity (p 0.2) in univariate analyses. covariates included in multivariable models included age, sex, white race, education, smoking status, heart failure, prior stroke, anemia, renal failure, and total number of cardiac comorbidities. a total of 218 adults with symptomatic af who were willing to complete a study assessment of psychosocial factors and cognition comprised our study sample. the cohort was comprised mostly of middle - aged and older adults with a modest burden of cardiovascular and noncardiovascular risk factors. the cohort was predominantly female (64.9%) and white (93.9%) with an average age of 64 years. the majority of participants had paroxysmal af (81%) and 68% had a college degree. most patients carried a diagnosis of hypertension (77%) and the majority were taking an antiarrhythmic medication for rhythm control (82%). just over one - half of participants used an antiplatelet agent (56%) and 3 out of 4 were prescribed an oral anticoagulation (56% on warfarin, 24% on a target - specific oral anticoagulant). depression was the most commonly observed psychosocial or ci among inrhythm participants, noted in almost half of patients (45%). nearly one - third (30%) of participants were cognitively impaired and 29% were noted to have anxiety (fig. only 35% of participants were free from any psychosocial or ci. psychosocial and cognitive comorbidities clustered frequently. approximately one - third (34%) of the participants had a single psychosocial or ci, one - quarter (23%) had 2 impairments, and 7% were affected by all 3 impairments (fig. percentage values refer to the percent of the total study population (n = 218). 1). for example, almost one - fifth (17%) of all participants experienced both depression and anxiety. ci was most likely to occur on its own, but nevertheless almost half of participants with ci also had impairment in another domain (48%). participants with 1 or more psychosocial or cis were older (p < 0.001), had less formal education (p < 0.001), and were more likely to be current smokers (p = 0.01). with the exception of heart failure, which was more prevalent among patients with multiple psychosocial and cis (p = 0.03), the prevalence of comorbidities, such as hypertension, diabetes, stroke, etc. women were less likely than men to have multiple impairments (p = 0.02). characteristics of participants by burden of psychosocial (depression, anxiety) or cognitive impairments out of the 218 patients who completed baseline interviews, 180 (82.5%) completed the afeqt questionnaire 6 months after enrolment. based on their responses, greater burden of psychosocial and ci at baseline was associated with poorer af - specific qol at 6 months. in multivariable models adjusting for potential confounders (referent group = 0 impairments), impairment in 1 domain was associated with a 5-point (p = 0.236) lower score on the 100-point afeqt, impairment in 2 domains with a 14-point (p < 0.001) lower score, and impairment in 3 domains was associated with a 15-point lower afeqt score (p = 0.02). linear regression of afeqt score compared with psychosocial or cognitive burden reported as adjusted b coefficient depression was the most commonly observed psychosocial or ci among inrhythm participants, noted in almost half of patients (45%). nearly one - third (30%) of participants were cognitively impaired and 29% were noted to have anxiety (fig. only 35% of participants were free from any psychosocial or ci. psychosocial and cognitive comorbidities clustered frequently. approximately one - third (34%) of the participants had a single psychosocial or ci, one - quarter (23%) had 2 impairments, and 7% were affected by all 3 impairments (fig. percentage values refer to the percent of the total study population (n = 218). 1). for example, almost one - fifth (17%) of all participants experienced both depression and anxiety. ci was most likely to occur on its own, but nevertheless almost half of participants with ci also had impairment in another domain (48%). participants with 1 or more psychosocial or cis were older (p < 0.001), had less formal education (p < 0.001), and were more likely to be current smokers (p = 0.01). with the exception of heart failure, which was more prevalent among patients with multiple psychosocial and cis (p = 0.03), the prevalence of comorbidities, such as hypertension, diabetes, stroke, etc. women were less likely than men to have multiple impairments (p = 0.02). out of the 218 patients who completed baseline interviews, 180 (82.5%) completed the afeqt questionnaire 6 months after enrolment. based on their responses, greater burden of psychosocial and ci at baseline was associated with poorer af - specific qol at 6 months. in multivariable models adjusting for potential confounders (referent group = 0 impairments) impairment in 1 domain was associated with a 5-point (p = 0.236) lower score on the 100-point afeqt, impairment in 2 domains with a 14-point (p < 0.001) lower score, and impairment in 3 domains was associated with a 15-point lower afeqt score (p = 0.02). linear regression of afeqt score compared with psychosocial or cognitive burden reported as adjusted b coefficient in our study of 218 middle - aged and older adults with symptomatic af, we observed that anxiety, depression, and ci were common comorbid illnesses and frequently co - occurred. certain patterns of psychosocial and cognitive comorbidity were noted, with anxiety and depression frequently being observed together. finally, we showed that impairment in 2 or more cognitive or psychosocial domains was associated with lower af - specific qol at 6 months in adjusted regression models. depression or anxiety have been shown to affect up to one - half of patients with af, and patients with af are at a 2-fold higher risk of ci than same - aged patients without af. thus, the rates of anxiety, depression, and ci observed in our study were high, but remarkably similar to those reported in prior, albeit smaller studies of patients with af. the current study validates these findings in a larger sample of well - characterized ambulatory patients with symptomatic af and extends current knowledge by illustrating that these psychosocial and/or cis frequently co - occurred in individual patients (fig. furthermore, impairment in multiple domains was associated with poorer af - specific qol at 6 months. importantly, we observed that psychosocial and cognitive multimorbidity exerts a powerful and negative effect on af - specific qol. differences in qol scores between participants with multiple impairments as compared with participants with ci, anxiety, and depression alone were clinically meaningful and statistically significant. our finding that af patients with an increasing burden of psychosocial or cognitive comorbidities also reported that lower qol scores reflects greater symptom severity and likely relates to the fact that depressed and anxious af patients utilize health resources at higher rates than do patients free from these mood disorders (eg, greater number of clinic and emergency room visits). indeed, several previous studies have examined the negative impact of individual psychosocial impairments on qol in patients with af. however, only one of these prior studies assessed af - specific qol, and none examined how the co - occurrence of multiple impairments collectively affected af - specific qol. as mentioned above, depression, anxiety, and ci are common in patients with af ; they are also common in other chronic cardiovascular pathologies. as illustrated by the 2008 statement of the american heart association s council on clinical cardiology, mood and cognition are becoming increasingly recognized for their role in the risk, management, and prognosis of cardiovascular disease, including in the context of stable coronary artery disease, heart failure, af, and myocardial infarction. impairments in mood and cognition have myriad adverse effects, including higher rates of morbidity, symptom severity, health care utilization, and even mortality. although there is a growing appreciation for the importance of mood and cis in the natural history of af, no prior studies have examined their co - occurrence in af patients. especially in light of the fact that anxiety and depression so frequently co - occur in individuals with other chronic medical conditions, and given their negative effects on cognitive performance, it is not surprising, but nonetheless significant, that we observed a high rate of multimorbidity across distinct psychosocial and cognitive domains in patients with symptomatic af. our findings point to the profound impact of disease clusters on symptom severity, suggesting that assessments of mood and cognition should be performed in concert to best characterize af patients at the greatest risk for complications. despite the increasing recognition of the importance of cognitive and psychosocial characteristics as determinants of response to cardiovascular treatments, including rhythm control for af, contemporary af treatment guidelines do not recommend routine assessment for ci, depression, or anxiety. our findings would suggest that such assessments might be useful in patients with symptomatic af given their close relations with af - specific qol, symptom burden, and rehospitalization, which are major targets for contemporary rhythm control therapies, especially catheter ablation. the strengths of the present investigation include its prospective design, the enrollment of af patients recruited from 3 different academic and community ambulatory clinics, the indepth characterization of participants af history, as well as the use of validated instruments to characterize each participant s psychosocial status, cognitive function, and qol. our study has several limitations, however, that should be considered when interpreting our results. first, all inrhythm study participants had symptomatic af and were identified from cardiology clinics, so our findings may not be generalizable to older asymptomatic af patients who are evaluated in other settings. also, the inrhythm cohort was comprised mostly of white individuals of european descent and those with paroxysmal af, thereby limiting generalizability of our primary findings to other racial and ethnic groups or those with different types of af. finally, we did not adjust for factors that may have confounded or mediated relations between psychosocial and cognitive predictors and af - specific qol (eg, adherence to medications, cardioversions, emergency department visits, or baseline symptom severity). the strengths of the present investigation include its prospective design, the enrollment of af patients recruited from 3 different academic and community ambulatory clinics, the indepth characterization of participants af history, as well as the use of validated instruments to characterize each participant s psychosocial status, cognitive function, and qol. our study has several limitations, however, that should be considered when interpreting our results. first, all inrhythm study participants had symptomatic af and were identified from cardiology clinics, so our findings may not be generalizable to older asymptomatic af patients who are evaluated in other settings. also, the inrhythm cohort was comprised mostly of white individuals of european descent and those with paroxysmal af, thereby limiting generalizability of our primary findings to other racial and ethnic groups or those with different types of af. finally, we did not adjust for factors that may have confounded or mediated relations between psychosocial and cognitive predictors and af - specific qol (eg, adherence to medications, cardioversions, emergency department visits, or baseline symptom severity). our findings suggest that cognitive and psychosocial impairments are common among patients with symptomatic af and often co - occur, imparting risk for poorer af - specific qol. knowledge of psychosocial and cis may help guide patients, families, and physicians in appropriate screening and making informed af treatment decisions. | background : impairments in psychosocial status and cognition relate to poor clinical outcomes in patients with atrial fibrillation (af). however, how often these conditions co - occur and associations between burden of psychosocial and cognitive impairment and quality of life (qol) have not been systematically examined in patients with af.methods:a total of 218 patients with symptomatic af were enrolled in a prospective study of af and psychosocial factors between may 2013 and october 2014 at the university of massachusetts medical center. cognitive function, depression, and anxiety were assessed at baseline and af - specific qol was assessed 6 months after enrollment using validated instruments. demographic and clinical information were obtained from a structured interview and medical record review.results:the mean age of the study participants was 63.5 10.2 years, 35% were male, and 81% had paroxysmal af. prevalences of impairment in 1, 2, and 3 psychosocial / cognitive domains (eg, depression, anxiety, or cognition) were 75 (34.4%), 51 (23.4%), and 16 (7.3%), respectively. patients with co - occurring psychosocial / cognitive impairments (eg, > 1 domain) were older, more likely to smoke, had less education, and were more likely to have heart failure (all p < 0.05). compared with participants with no psychosocial / cognitive impairments, af - specific qol at 6 months was significantly poorer among participants with baseline impairment in 2 (b = 13.6, 95% ci : 21.7 to 5.4) or 3 (b = 15.1, 95% ci : 28.0 to 2.2) psychosocial / cognitive domains.conclusion:depression, anxiety, and impaired cognition were common in our cohort of patients with symptomatic af and often co - occurred. higher burden of psychosocial / cognitive impairment was associated with poorer af - specific qol. |
subscapularis tendon is one of the four rotator cuff tendons which in addition include supraspinatus, infraspinatus, and teres minor. subscapularis inserts onto the lesser tuberosity of the humerus, whereas the other rotator cuff tendons insert into the greater tuberosity.1 in 1834, smith was credited as describing the first case series of subscapularis repair.1 tears, involving the subscapularis tendon, have not been considered as common. autopsy and cadaveric studies have shown the incidence of subscapularis tears to be between 3% and 13%.2 more recently, there has been a renewed interest in understanding the subscapularis muscle tears. in just the past decade, there have been several articles dedicated to diagnosing and treating subscapularis tendon tears.34 as our understanding of the shoulder has increased, so as the rate at which subscapularis tendon tears have been identified. clinical examination supported by radiological investigations has helped in identifying subscapularis tendon tears.5 subscapularis tears can be isolated,2345 part of the anterosuperior rotator cuff tear,678 or a continuum of large and massive rotator cuff involvement.9 however, the authors could not find from literature the incidence of isolated subscapularis or combined subscapularis and other rotator cuff tendon tears (2007) grading system from grade 1 to 5.10 there are several articles published showing the short term results measuring the outcome of open repair and arthroscopic subscapularis repair.1112 however, the midterm and long term outcome studies for arthroscopic subscapularis repair are few. we studied the midterm functional results of subscapularis tendon repair in association with repair of other cuff muscles. 35 patients who underwent arthroscopic rotator cuff repair between may 2008 and june 2012 were included in this retrospective study. there were 22 males and 13 female patients with mean age of 58.2 years (range 41 - 72 years). rotator cuff tears were diagnosed on clinical examination and magnetic resonance imaging (mri) findings [figures 1 and 2 ]. clinical tests included the gerber 's lift - off test, bear - hug test, and belly - press test along with an assessment of other rotator cuff tendons. radiological evaluation was done with 1.5 t mri by an experienced musculoskeletal radiologist reporting on the scans. inclusion criteria were (1) all patients who underwent arthroscopic isolated subscapularis or combined subscapularis and other rotator cuff tear repairs (2) minimum 2 years followup postoperatively. the exclusion criteria were (1) rotator cuff tears treated by open methods (2) massive irreparable rotator cuff tears (3) cases of glenohumeral arthritis (4) cases with severe cuff tear arthropathy (hamada. classification grade 3 or more and above).13 magnetic resonance imaging axial cut showing the subscapularis tear magnetic resonance imaging saggital view showing subscapularis tear all operations were performed by the senior authors together (dr and ks). only patients with confirmed partial to full thickness subscapularis tears were included in this study. all patients gave informed consent to participate in this study. in the followup, all patients were subjected to thorough physical examination, including lift - off test, belly - press test, and bear - hug sign. visual analogue scale (vas) and modified university of california at los angeles (ucla) score were calculated. all patients were asked to list all of their current activities and state of their satisfaction with their shoulders. the physical examination also evaluated their active range of motion and graded their strength throughout their range of motion on a scale from 0 to 5 according to mrc. all the data were analyzed statistically using paired t - test and p value was calculated using spss 18 software [ibm, chicago, usa ]. we start with repair of the subscapularis tendon before addressing any other pathology in shoulder as recommended in literature.69 all surgeries were performed with the patients in beach chair position. subscapularis tendon edges were freshened using shaver, and the lesser tuberosity surface was made raw using a rasp. a good debridement of tendon edges and medial and inferior release is necessary for completely retracted subscapularis tendon tears for optimum repair. all subscapularis repairs were performed using 5 mm metal suture anchors (double loaded, arthrex) [figure 3 ]. subscapularis tendon insertion is 2.5 cm in width and generally one double loaded suture anchor suffices if 50% of tendon insertion is torn two suture anchors are used. upon completion of the repair, the arm is internally and externally rotated to confirm the completeness of the repair [figure 4 ]. arthroscopic view showing the insertion of the suture anchor into the lesser tuberosity during subscapularis repair. the viewing is through a 70 arthroscope arthroscopic view showing complete subscapularis repair and the complete coverage of the lesser tuberosity by the subscapularis tendon. the area is being viewed through a 70 arthroscope subscapularis tendon tears were classified according to lafosse 's. classification.13 associated supraspinatus and infraspinatus tendon tears were also documented and classified according to the thickness of the involved tendons and were repaired. all patients were given a sling with a wedge, applied in the operating room. the sling is worn full time for 6 weeks ; however, the wedge was removed after 4 weeks. during the first 6 weeks, patients are instructed to perform active elbow flexion and extension with arm at the side. after 6 weeks, patients begin a passive stretching program that includes overhead stretches with a rope and pulley. a 70 arthroscope was also used while performing subscapularis repair for better visualization. subscapularis tendon edges were freshened using shaver, and the lesser tuberosity surface was made raw using a rasp. a good debridement of tendon edges and medial and inferior release is necessary for completely retracted subscapularis tendon tears for optimum repair. all subscapularis repairs were performed using 5 mm metal suture anchors (double loaded, arthrex) [figure 3 ]. subscapularis tendon insertion is 2.5 cm in width and generally one double loaded suture anchor suffices if 50% of tendon insertion is torn two suture anchors are used. upon completion of the repair, the arm is internally and externally rotated to confirm the completeness of the repair [figure 4 ]. arthroscopic view showing the insertion of the suture anchor into the lesser tuberosity during subscapularis repair. the viewing is through a 70 arthroscope arthroscopic view showing complete subscapularis repair and the complete coverage of the lesser tuberosity by the subscapularis tendon. the area is being viewed through a 70 arthroscope subscapularis tendon tears were classified according to lafosse 's. classification.13 associated supraspinatus and infraspinatus tendon tears were also documented and classified according to the thickness of the involved tendons and were repaired. all patients were given a sling with a wedge, applied in the operating room. the sling is worn full time for 6 weeks ; however, the wedge was removed after 4 weeks. during the first 6 weeks, patients are instructed to perform active elbow flexion and extension with arm at the side. after 6 weeks, patients begin a passive stretching program that includes overhead stretches with a rope and pulley. 25 (71.4%) patients presented with lafosse grade 2 tears and 7 (20%) patients with grade 1 tear [table 1 ]. combined rotator cuff tears were more common (71.4%) than isolated subscapularis tear (28.6%) [table 2 ]. the grading of subscapularis tears found in this study the pattern of rotator cuff tears mechanism of injury was most commonly traumatic fall (70%) in the cases of isolated subscapularis tears (7 out of 10). however, combined rotator cuff cases did not give a history of significant trauma in most cases and were degenerative in origin (76% cases, 19 out of 25 cases). isolated subscapularis tear also tends to happen at a younger age (mean 52.3 years) as compared to the occurrence of subscapularis tears along with other rotator cuff tendon tears (mean age 63.4 years). the mean vas score decreased significantly from 8.03 in preoperative period to 1.44 in the postoperative period., there were 22 excellent, 11 good, and 2 fair results among 35 patients. around elevation increased from 73.68 to 170.88. external rotation increased from 23.09 to 71.18, and internal rotation increased from l5, s1 to t7 in the followup [figures 5 and 6 ]. clinical photograph showing full abduction on right side after two years followup clinical photograph showing internal rotation after two years followup power of cuff muscles increased from a mean of 2.5 to 4.74 in elevation, 2.24 to 4.79 in external rotation, and 2.29 to 4.82 in internal rotation. persistent belly - press and lift - off positive postsurgery were found in 14.28% cases (5 out of 35). thus, the postoperative results of both isolated and combined rotator cuff tears were comparable after arthroscopic repair as 2 years postoperative vas and ucla scores were not significantly different in both groups p = 0.23 and 0.12, respectively. the present study represents the largest series to date in patients with rotator cuff injury that have been treated with an all arthroscopic subscapularis tendon repair. several authors have reported on clinical outcomes after open repair of isolated subscapularis tendon tears.511 we identified only a few studies reporting the results of arthroscopic repair of subscapularis tendon tears.1415 the present study shows the good results after arthroscopic repair of subscapularis tears (mean post of ucla 33.15 and mean vas 1.44). the results of both isolated subscapularis and combined subscapularis and other tendon tears were comparable [table 3 ]. the preoperative and 2 years postoperative vas and ucla scores and range of movements mechanism of injury was most commonly traumatic fall (70%) in the cases of isolated subscapularis tears, whereas combined rotator cuff cases were degenerative in origin (76% cases). isolated subscapularis tear also tends to happen at a younger age (mean 52.3 years). all these data indicate that the etiopathogenesis of isolated subscapularis tear may be different to that of the occurrence of subscapularis tear along with other rotator cuff tendon tears. bennett9 (2003) published a series of 8 patients with 2 years followup after arthroscopic repair of isolated subscapularis tendon tears. the postoperative constant score was 74 points, but no information was given about postoperative clinical subscapularis tests or structural integrity of the repair. adam. (2000)14 reported excellent results after arthroscopic repair of isolated subscapularis tears in 7 patients after a minimum followup of 3 years. lafosse.15 (2007) studied 17 patients, the first series with detailed information on postoperative results of arthroscopic subscapularis repair for isolated tears. they found good clinical results with a postoperative constant score of 84 points in 17 patients after a followup of 29 months, a rerupture rate of 12% evaluated by computed tomography arthrograms, and a rate of persistent positive or weakened belly - press tests in 24% of patients. in a multicenter study, edwards.16 reported the results of open repair in 84 isolated subscapularis tendon tears. the constant score averaged 79.5 points after 45 months. marked fatty infiltration of subscapularis muscle negatively influenced the postoperative belly - test, but postoperative constant score was not influenced by fatty degeneration. additional tenodesis or tenotomy of long head of the biceps had a significant positive influence on the outcome. gerber and krushell6 (1991) reported on 16 patients with an average followup of 43 months and a score of 82% in the age and gender matched constant score after open repair of an isolated subscapularis tear. gerber.7 and edwards.16 each reported persistent positive lift - off and belly - press tests in the postoperative course representing a partial subscapularis muscle insufficiency at a rate of 31% and 20%, respectively. it was shown that positive postoperative subscapularis tests were not indicative of a poor clinical outcome, as their patients did not achieve lower constant scores, but edwards.16 stated that the community used shoulder scores do not adequately reflect subscapularis muscle strength. persistent belly - press and lift - off positive postsurgery were found in 14.28% cases (5 out of 35) in this series..7 and edwards.16 that positive postoperative subscapularis tests were not indicative of a poor clinical outcome, as their patients did not achieve lower shoulder scores. in a study by burkhart and tehrany,17 (2002) the authors were first to describe the technique and preliminary results of arthroscopic repair of the subscapularis tendon. this study evaluated 25 patients with a mean age of 61 years who had either anterosuperior or isolated subscapularis tendon tears repaired with an arthroscopic surgical technique. the mean followup in this study was 10.7 months with 23 good to excellent results, 1 fair result, and 1 poor result. warner.3 (2001) evaluated 19 patients with a mean age of 58 years in whom they did anterosuperior and subscapularis tendon repairs. the mean followup in their study was approximately 3.3 years with 5 had an excellent result, and 3 had good results. flury.18 (2006) evaluated 63 patients with a mean age of 56 years who had either anterosuperior or isolated subscapularis tendon tears repaired with an open surgical technique. the mean followup in their study was 2.9 years with 98% patient being satisfied with the operation. this series is one of the largest series of arthroscopic subscapularis repair and goes on to show the good results of undertaking such a repair. the surgeon attempting to repair subscapularis would greatly benefit if he also has a 70 arthroscope in his setup. the limitations of study are that this study is a case series (level 4 evidence). the ideal study design to see however, we can not undertake such a study because we tend to repair all our diagnosed subscapularis tears. we conclude that the midterm results of arthroscopic subscapularis tendon repair are good, and it remains a good option for the management of subscapularis tendon tear along with other rotator cuff muscle repair. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | background : rotator cuff tears are a common cause of shoulder pain and dysfunction. more recently, there has been a renewed interest in understanding the subscapularis tears. there are multiple articles in the literature showing the short term results of isolated subscapularis tendon repair. however, the midterm and long term outcome studies for arthroscopic subscapularis repair are few. this study evaluates the functional outcome after arthroscopic subscapularis repair.materials and methods : the records of 35 patients who underwent an arthroscopic subscapularis repair between may 2008 and june 2012 were included in this retrospective study. the records of all patients were reviewed. there were 22 males and 13 female patients with mean age of 58.2 years (range 41 - 72 years). all patients had a complete history, physical examination, and radiographs of their shoulders. visual analogue scale (vas), range of movements, power of cuff muscles, and modified university of california at los angeles (ucla) score were assessed.results:the mean followup was 2.8 years (range 2 - 4 year). functional outcome after arthroscopic subscapularis repair has an excellent outcome as analysed by clinical outcome, vas score and ucla score. results were analyzed and had statistically significant values. the vas for pain improved significantly (p < 0.001), and the mean modified ucla score improved significantly (p < 0.001) from 14.24 4.72 preoperatively to 33.15 2.29 at 2 years postoperative. according to the ucla system, there were 22 excellent, 11 good, and 2 fair results. around 95% of patients returned to their usual work after surgery.conclusion:at a median followup of 2 years, 95% of patients had a good to excellent result after an arthroscopic subscapularis tendon repair. we conclude that the midterm results show that arthroscopic subscapularis repair remains a good option for the treatment of patients with subscapularis tendon repair. |
however spinal tumors constitute only 1 - 10% of all pediatric central nervous system tumors. they are firm encapsulated benign tumors and total surgical removal can be achieved in most of the cases with an excellent outcome. the study included 21 patients in the pediatric age group (< 18 years) of spinal schwannomas who underwent surgery over 10 years from january 1998 to april 2008 in the department of neurosurgery, national institute of mental health and neurosciences, bangalore. the demographic profile, clinical features, radiological findings, operative procedures, postoperative complications, and outcome were noted and analyzed. there were a total of 21 patients with an age range of 6 - 18 years with a median age of 16 years. the duration of symptoms ranged from 1 month to 36 months with average duration of 5 months. six patients had bladder disturbances (29%) and six patients had associated neurofibromatosis. among these, five had neurofibromatosis type i with multiple caf - au - lait spots and multiple subcutaneous swellings, and one had neurofibromatosis type ii with bilateral vestibular schwannomas. this patient underwent emergency decompression of the tumor and was put on ventilator. gradually the patient was weaned off from the ventilator support and made significant improvement. the predominant location was the cervical region in 10 cases (47%), followed by dorsal in 8 cases (38%). in three patients, four of the 21 patients had dumbbell schwannomas. among the 21 patients, 18 underwent laminectomy, 3 underwent laminotomy and microsurgical excision of the tumor. twenty patients underwent gross total excision and one underwent partial excision in view of tight adherence to multiple nerve roots in cervical region. the follow - up period ranged from 4 months to 56 months with average follow - up period of 38 months. motor deficits improved by at least one grade in 6 patients and by 2 grades in 10 patients. of the six patients who had bladder symptoms, four patients made improvement in urinary symptoms and two patients were on intermittent self - catheterization. x - rays of the spine was performed at follow - up and showed no signs of instability in any of the patients. follow - up mri at last follow - up revealed no recurrence in any of the patients. there were a total of 21 patients with an age range of 6 - 18 years with a median age of 16 years. the duration of symptoms ranged from 1 month to 36 months with average duration of 5 months. six patients had bladder disturbances (29%) and six patients had associated neurofibromatosis. among these, five had neurofibromatosis type i with multiple caf - au - lait spots and multiple subcutaneous swellings, and one had neurofibromatosis type ii with bilateral vestibular schwannomas. this patient underwent emergency decompression of the tumor and was put on ventilator. gradually the patient was weaned off from the ventilator support and made significant improvement. the predominant location was the cervical region in 10 cases (47%), followed by dorsal in 8 cases (38%). in three patients, among the 21 patients, 18 underwent laminectomy, 3 underwent laminotomy and microsurgical excision of the tumor. twenty patients underwent gross total excision and one underwent partial excision in view of tight adherence to multiple nerve roots in cervical region. the follow - up period ranged from 4 months to 56 months with average follow - up period of 38 months. motor deficits improved by at least one grade in 6 patients and by 2 grades in 10 patients. of the six patients who had bladder symptoms, four patients made improvement in urinary symptoms and two patients were on intermittent self - catheterization. x - rays of the spine was performed at follow - up and showed no signs of instability in any of the patients. follow - up mri at last follow - up revealed no recurrence in any of the patients. spinal cord tumors are a relatively rare diagnosis and account for 1% to 10% of all pediatric central nervous system tumors. this is in contrast with adults where they constitute about 25% of primary intradural tumors. however, some studies indicate a higher incidence of spinal schwannomas in males, as reported by hori. (57%), and this percentage corresponds to our study in which 67% of schwannomas appeared in the female population. in our study, the majority of the patients presented with progressive compressive myelopathy (86%). the frequency of motor involvement and localized pain was much higher in our study compared with the available literature where it has ranged from 40% to 60%. the autonomic (bowel and bladder) involvement in our study was 29% compared to 10 - -20% in the literature. (five had nf i and one had nf ii.) the most common spinal tumors in nf i are neurofibromas. paraspinal neurofibromas including dumbbell - shaped tumors are commonly found in asymptomatic patients, especially at younger age groups. the most common location was the cervical region (47%) followed by the dorsal region in 38% of the cases [figure 1 ]. this differs from the available literature in which the majority of the tumors are located in the cervical and lumbar regions. according to the available literature 70 - -80% of spinal schwannomas are intradural in location and those extending through the dural aperture as the dumbbell mass involving both intradural as well as extradural space account for another 15%. intramedullary schwannomas are extremely rare. in our study, 17 patients had intradural schwannomas (80%) and 4 patients (20%) had dumbbell schwannomas. preoperative magnetic resonance imaging cervical spine (post contrast- sagittal) showing well - defined contrast enhancing intradural extradural lesion all our patients underwent surgery and excision of the tumor (gte in 20 and ste in 1) ; [figures 2 and 3 ] the outcome is comparable to the available literature and correlates with the preoperative neurological status of the patient. preoperative magnetic resonance imaging (mri) cervical spine (post contrast - axial) showing well - defined contrast enhancing intradural extradural lesion with the cord pushed to the left (arrow) postoperative magnetic resonance imaging (mri) cervical spine (t2-weighted) showing complete excision spinal cord tumors are a relatively rare diagnosis and account for 1% to 10% of all pediatric central nervous system tumors. this is in contrast with adults where they constitute about 25% of primary intradural tumors. however, some studies indicate a higher incidence of spinal schwannomas in males, as reported by hori. (57%), and this percentage corresponds to our study in which 67% of schwannomas appeared in the female population. in our study, the majority of the patients presented with progressive compressive myelopathy (86%). the frequency of motor involvement and localized pain was much higher in our study compared with the available literature where it has ranged from 40% to 60%. the autonomic (bowel and bladder) involvement in our study was 29% compared to 10 - -20% in the literature. (five had nf i and one had nf ii.) the most common spinal tumors in nf i are neurofibromas. paraspinal neurofibromas including dumbbell - shaped tumors are commonly found in asymptomatic patients, especially at younger age groups. the most common location was the cervical region (47%) followed by the dorsal region in 38% of the cases [figure 1 ]. this differs from the available literature in which the majority of the tumors are located in the cervical and lumbar regions. according to the available literature 70 - -80% of spinal schwannomas are intradural in location and those extending through the dural aperture as the dumbbell mass involving both intradural as well as extradural space account for another 15%. intramedullary schwannomas are extremely rare. in our study, 17 patients had intradural schwannomas (80%) and 4 patients (20%) had dumbbell schwannomas. preoperative magnetic resonance imaging cervical spine (post contrast- sagittal) showing well - defined contrast enhancing intradural extradural lesion in our study, the majority of the patients presented with progressive compressive myelopathy (86%). the frequency of motor involvement and localized pain was much higher in our study compared with the available literature where it has ranged from 40% to 60%. the autonomic (bowel and bladder) involvement in our study was 29% compared to 10 - -20% in the literature. (five had nf i and one had nf ii.) the most common spinal tumors in nf i are neurofibromas. paraspinal neurofibromas including dumbbell - shaped tumors are commonly found in asymptomatic patients, especially at younger age groups. the most common location was the cervical region (47%) followed by the dorsal region in 38% of the cases [figure 1 ]. this differs from the available literature in which the majority of the tumors are located in the cervical and lumbar regions. according to the available literature 70 - -80% of spinal schwannomas are intradural in location and those extending through the dural aperture as the dumbbell mass involving both intradural as well as extradural space account for another 15%. intramedullary schwannomas are extremely rare. in our study, 17 patients had intradural schwannomas (80%) and 4 patients (20%) had dumbbell schwannomas. preoperative magnetic resonance imaging cervical spine (post contrast- sagittal) showing well - defined contrast enhancing intradural extradural lesion all our patients underwent surgery and excision of the tumor (gte in 20 and ste in 1) ; [figures 2 and 3 ] the outcome is comparable to the available literature and correlates with the preoperative neurological status of the patient. preoperative magnetic resonance imaging (mri) cervical spine (post contrast - axial) showing well - defined contrast enhancing intradural extradural lesion with the cord pushed to the left (arrow) postoperative magnetic resonance imaging (mri) cervical spine (t2-weighted) showing complete excision | objective : the objective was to analyze the demography, clinical presentation, and management of spinal intradural schwannomas in pediatric population.materials and methods : this retrospective study includes 21 pediatric patients (under 18 years of age) who underwent surgery for spinal intradural schwannomas from january 1998 to april 2008. the medical records were reviewed retrospectively and the information regarding clinical presentation, tumor location, operative findings, and postoperative status and functional outcome were analyzed.results:a total of 21 patients (14 females and 7 males) were operated for spinal schwannomas. six patients had associated neurofibromatosis (five were nf i and one was nf ii) at presentation. the most common presenting symptom was progressive myelopathy (86%). the tumor location was either cervical or dorsal in 18 cases. all patients underwent surgery. gross total excision was achieved in 20 cases. the median follow - up was 38 months. all the patients had neurological improvement in both power and bladder symptoms.conclusion:pediatric spinal neurofibromas / schwannomas are an uncommon but completely treatable group of tumors. complete surgical excision gives excellent outcome. |
an agilent 1200 series hplc system equipped with a diode array detector and a phenomenex c18 column (5 m, 250 21.2 mm) was used for preparative hplc. for hplc - ms analysis, an agilent hplc system equipped with a diode array detector and a 6130 series quadrupole mass spectrometer was used with a phenomenex c18 (5 m, 100 4.6 mm) column. the following gradient was used for hplc - ms analysis : 05 min, isocratic 10% ch3cn + 0.1% formic acid ; 525 min, linear gradient from 10% ch3cn + 0.1% formic acid to 100% ch3cn + 0.1% formic acid. nmr spectra were recorded in cd3od (for compounds 14) or cd2cl2 (for compound 5) at 600 mhz and referenced to the internal solvent peak at h 3.30 and c 49.0 or h 5.32 and c 53.8, respectively. high - resolution mass spectrometry (hr - ms) was performed at the university of illinois urbana an ant colony of a. dentigerum was collected from pipeline road, panama, on may 29, 2008, and placed in a sterile petri dish with moist cotton. after allowing the nest to stabilize for a few days, the pseudonocardia symbiont from this colony was isolated directly from the mesoternal lobe of a worker by scraping bacteria off the cuticle of the ant using a sterile scalpel and plating on chitin media following the methods of caldera and currie, and identified as pseudonocardia sp. ec080529 - 01 were grown on solid isp-2 medium (per liter : yeast extract, 4 g ; malt extract, 10 g ; glucose, 4 g) in 12 petri plates (150 20 mm, 1.2 l total) for 7 d at 30 c. the solid agar was cut into small cubes and soaked in etoac (1.2 l) overnight. the etoac was filtered and dried in vacuo to give the crude etoac extract. the solid agar was re - extracted overnight with meoh (1.2 l), and the meoh was filtered and dried in vacuo to give the crude meoh extract. the crude etoac extract was dissolved in 90% meoh h2o (20 ml) and passed through a c18 column, eluting with additional 90% meoh the eluent from this column was diluted with h2o to give a final meoh concentration of 60%. this solution was passed through another c18 column and washed with additional 60% meoh h2o fraction was purified by preparative hplc using the following gradient : 05 min, isocratic 20% ch3cn h2o ; 560 min, linear gradient from 20% ch3cn the 100% meoh fraction from this c18 column was purified by preparative hplc using the following gradient : 010 min, isocratic 50% ch3cn h2o ; 1060 min, linear gradient from 50% ch3cn the crude meoh extract was dissolved in h2o and passed through an hp-20 column. the hp-20 column was washed with water to remove polar components, and the compounds of interest were then eluted with 100% meoh. the eluent from this column was diluted with h2o to give a final meoh concentration of 30%. this solution was passed through another c18 column and washed with additional 30% meoh the 100% meoh fraction from this c18 column was purified by reversed - phase hplc using the following gradient : 010 min, isocratic 10% ch3cn h2o + 0.1% formic acid ; 1060 min linear gradient from 10% ch3cn h2o + 0.1% formic acid to 100% ch3cn + 0.1% formic acid to give pure 1 (2.0 mg), 2 (0.9 mg), and 3 (1.8 mg). colorless solid (2.0 mg) ; []d 11 (c 0.02, meoh) ; uv (meoh) max (log) 310 nm (3.11), 279 (sh), 271 nm (3.59) ; h nmr (600 mhz, cd3od) and c nmr (150 mhz, cd3od), see table 1 ; (+) -hresi m / z 497.1438 [m + h ] (calcd for c26h25o10, 497.1448). yellow solid (0.9 mg) ; []d 2 (c 0.02, meoh) ; uv (meoh) max (log) 377 nm (3.52), 305 nm (3.87) ; h nmr (600 mhz, cd3od) and c nmr (150 mhz, cd3od), see table 1 ; (+) -hresi m / z 495.1295 [m + h ] (calcd for c26h23o10, 495.1291). orange solid (1.8 mg) ; []d 3 (c 0.09, meoh) ; uv (meoh) max (log) 403 nm (3.53), 314 nm (3.96) ; h nmr (600 mhz, cd3od) and c nmr (150 mhz, cd3od) see table 1 ; (+) -hresi m / z 511.1229 (calcd for c26h23o11, 511.1240). the appropriate test organism was grown in a 5 ml culture overnight in either lb medium (for e. coli and b. subtilis) or ypd medium (for c. albicans and s. cerevisiae) at 30 c. in each case, the overnight culture was diluted with additional sterile medium (lb or ypd) to an od600 of 0.01. compounds 15 were dissolved in dmso to give a concentration of 5 mg / ml and 2-fold serially diluted. these solutions (1 l) were added to the wells of a 96-well plate, followed by the diluted culture of the test organism (99 l) to give a final compound concentration ranging from 50 to 0.1 g / ml. the cultures were allowed to grow for 24 h at 30 c before the od600 was measured using a plate reader. the mic was defined as the lowest concentration that gave less than 5% of the maximum od600. dynemicin a was used as a positive control and gave mic values against e. coli, b. subtilis, c. albicans, and s. cerevisiae of 313, 0.16, 156, and 156 ng / ml, respectively. compounds were tested for activity against hepg2 human hepatoma cells (atcc) that were maintained in dmem (invitrogen), 10% fbs (sigma), and 1% antibiotic antimycotic (invitrogen) in a standard tissue culture incubator (37 c, 5% co2). for assays, compounds 15 (in dmso) the final concentration of dmso was 1%, and compounds varied from 0 to 50 g / ml. cells were incubated with the compounds for 2 days at 37 c, and then liver cell viability was assessed with celltiter - glo (promega). the relative signal intensity of each sample was evaluated with an envision (perkinelmer) system. liver - stage p. berghei assays were performed using a luciferase - expressing sporozoite strain of p. berghei anka. parasites were obtained from dissection of plasmodium - infected anopheles stephensi mosquitoes (new york university langone medical center insectary). malaria parasites (4000 sporozoites) were used to infect hepg2 cells (15 000 cells) in a 384-well plate in the presence of compounds 15 in triplicate. the final concentration of dmso was 1%, and compounds varied from 0 to 50 g / ml. cells were incubated with the compounds for 2 days at 37 c, and then relative parasite load was determined after addition of bright - glo (promega). data analysis for hepg2 toxicity and liver - stage malaria activity was carried out using graphpad prism, and curves were fit with a standard inhibition dose all statistical results are the mean ic50 value averaged from two independent experiments. atovaquone was used as a positive control and gave an ic50 in blood stage assays of 0.3 nm. | three new members of the angucycline class of antibiotics, pseudonocardones a c (13), along with the known antibiotics 6-deoxy-8-o - methylrabelomycin (4) and x-14881 e (5) have been isolated from the culture of a pseudonocardia strain associated with the fungus - growing ant apterostigma dentigerum. compounds 4 and 5 showed antibiotic activity against bacillus subtilis 3610 and liver - stage plasmodium berghei, while 13 were inactive or only weakly active in a variety of biological assays. compound 5 also showed moderate cytotoxicity against hepg2 cells. |
the novel h1n1 influenza outbreak in april 2009 was unprecedented in several respects including the new virus, the season of origin (spring, not late fall), and the cohort at risk for infection and death (children and young adults, not infants and the elderly) [14 ]. the pandemic h1n1 2009 virus is currently the dominant influenza strain in most parts of the world [58 ]. by march 2010, almost all countries had reported cases of infection and over 17,700 related deaths had been reported to the world health organization [912 ]. posttraumatic stress disorder (ptsd) is a severe anxiety disorder that can result in severe disability across several domains of functioning [1315 ]. ptsd usually occurs after exposure to any event that results in psychological trauma ; however, most people so exposed do not develop ptsd. to the best of our knowledge only 1 study has explored ptsd public during an epidemic. that study reported the prevalence of probable ptsd cases was significantly higher in older people and in residents of severe acute respiratory syndrome (sars)-prevalent regions, indicating that exposure degree and age might be significant predictors of ptsd. the h1n1 influenza pandemic was unusual in that most people could not avoid being affected in some way, since television news, the internet and newspapers were filled with the reports of infection and death related to it. the outbreak of h1n1 influenza caused not only extraordinary public health concerns but also tremendous psychological distress, particularly among youth, as they are among the most easily affected group during a global event. universities are mass gathering places for youth, which has a high risk of spreading infectious diseases, particularly the 2009 pandemic influenza h1n1. the present study was designed to explore 2 research questions regarding stress symptoms among chinese university students during november - december 2009. second, are there predictors of distress in this sample ? regarding this question, it was hypothesized that greater overall stress symptom severity would be predicted by female gender, residence in north china (where more influenza h1n1 cases were reported in china), lower university grade, having less knowledge about h1n1 influenza, not receiving the vaccine, having h1n1 influenza, having family members, friends or acquaintances having h1n1 influenza, contacting people infected with h1n1 influenza, and being afraid of h1n1 influenza. the findings of this study will be important in planning for future outbreaks of emerging infectious diseases, especially in university students. the subjects were recruited from 4 provinces of china, including heilongjiang, beijing, shanghai and sichuan during november - december 2009. the 4 provinces provide an adequate representation of socioeconomic status and geographical location of mainland china (figure 1). in north china, similarly, in south china, shanghai is a developed area, while sichuan is a developing. university students attending various classes in one of the major classroom buildings on campus in the above 4 provinces were recruited in this study to complete a questionnaire designed by the primary authors. the sample sizes from heilongjiang, beijing, shanghai and sichuan were 455, 106, 419, and 102, respectively. demographics for the study samples from north china and south china are presented in table 1. the procedure for sampling classes was not random because availability of university students depended on instructor permission and scheduling considerations. all participants completed a 17-item self - reported questionnaire, the ptsd checklist - civilian version (pcl - c), which assesses the intrusive, avoidant, and arousal diagnostic and statistic manual of mental disorders - iv ptsd symptom clusters. the frequency of stress symptoms occurrence during the past 4 weeks was rated on a 5-point scale ranging from 1 not at all, through 3 moderately, to 5 extremely. this measure can be scored continuously by summing the values of each response or dichotomously using an algorithm that considers a corresponding response of moderately or greater as a symptom and follows the diagnostic and statistic manual of mental disorders - iv diagnostic rules requiring at least 1 symptom from the intrusive symptoms cluster, 3 symptoms from the avoidance symptoms cluster and 2 symptoms from the hyper - arousal symptoms. in the present study, the prevalence of symptoms consistent with a diagnosis of ptsd was ascertained using the recommended algorithm, whereas the relationship between participants characteristics and the burden of psychologic symptoms was ascertained using the pcl score as a continuous variable. the difference of gender composition was analyzed with the test while age was sampled with the two - sample t test. reliability was assessed by using intraclass correlation coefficients and pearson correlation coefficients. the scores of ptsd reaction index were expressed as meansd. in preparation for regression analyses examining predictors of stress symptoms, correlations (pearson r) were calculated between hypothesized predictors and the pcl - c total score. the possible predictors were area (0=in south china ; 1=in north china), gender (0=female;1=male), university grade (1=first year ; 2=second year ; 3= third year ; 4= fourth year), having knowledge about h1n1 influenza (1= a lot ; 2= some ; 3= a little ; 4= none), received vaccine (0= yes ; 1= no), having h1n1 influenza (1= yes, still in treatment ; 2= yes, but recovered ; 3= no), having family members, friends or acquaintances having h1n1 influenza (1= family members ; 2= friends ; 3= acquaintances ; 4= no), contacting people infected with h1n1 influenza (0= yes ; 1= no), being afraid of h1n1 influenza (1= very ; 2= somewhat ; 3= not). the significantly correlated variables for the pcl - c total score were entered simultaneously as predictors in the regression analyses. the subjects were recruited from 4 provinces of china, including heilongjiang, beijing, shanghai and sichuan during november - december 2009. the 4 provinces provide an adequate representation of socioeconomic status and geographical location of mainland china (figure 1). in north china, similarly, in south china, shanghai is a developed area, while sichuan is a developing. university students attending various classes in one of the major classroom buildings on campus in the above 4 provinces were recruited in this study to complete a questionnaire designed by the primary authors. the sample sizes from heilongjiang, beijing, shanghai and sichuan were 455, 106, 419, and 102, respectively. demographics for the study samples from north china and south china are presented in table 1. the procedure for sampling classes was not random because availability of university students depended on instructor permission and scheduling considerations. all participants completed a 17-item self - reported questionnaire, the ptsd checklist - civilian version (pcl - c), which assesses the intrusive, avoidant, and arousal diagnostic and statistic manual of mental disorders - iv ptsd symptom clusters. the frequency of stress symptoms occurrence during the past 4 weeks was rated on a 5-point scale ranging from 1 not at all, through 3 moderately, to 5 extremely. this measure can be scored continuously by summing the values of each response or dichotomously using an algorithm that considers a corresponding response of moderately or greater as a symptom and follows the diagnostic and statistic manual of mental disorders - iv diagnostic rules requiring at least 1 symptom from the intrusive symptoms cluster, 3 symptoms from the avoidance symptoms cluster and 2 symptoms from the hyper - arousal symptoms. in the present study, the prevalence of symptoms consistent with a diagnosis of ptsd was ascertained using the recommended algorithm, whereas the relationship between participants characteristics and the burden of psychologic symptoms was ascertained using the pcl score as a continuous variable. the difference of gender composition was analyzed with the test while age was sampled with the two - sample t test. the scores of ptsd reaction index were expressed as meansd. in preparation for regression analyses examining predictors of stress symptoms, correlations (pearson r) were calculated between hypothesized predictors and the pcl - c total score. the possible predictors were area (0=in south china ; 1=in north china), gender (0=female;1=male), university grade (1=first year ; 2=second year ; 3= third year ; 4= fourth year), having knowledge about h1n1 influenza (1= a lot ; 2= some ; 3= a little ; 4= none), received vaccine (0= yes ; 1= no), having h1n1 influenza (1= yes, still in treatment ; 2= yes, but recovered ; 3= no), having family members, friends or acquaintances having h1n1 influenza (1= family members ; 2= friends ; 3= acquaintances ; 4= no), contacting people infected with h1n1 influenza (0= yes ; 1= no), being afraid of h1n1 influenza (1= very ; 2= somewhat ; 3= not). the significantly correlated variables for the pcl - c total score were entered simultaneously as predictors in the regression analyses. there were 1082 people who participated in this study : 521 from south china and 561 from north china. overall, the proportion of university students enrolled in this study who met symptomatic criteria for ptsd was 2% (22 students). the mean pcl - c total score in the sample was 22.09, with a standard deviation of 8.01 and scores ranging from 17 to 58 (minimum possible score= 17 ; maximum= 58). as exposure degree and age have been shown to be related to the prevalence of ptsd in public, the potential predictors explored in this study include area, university year (first year, second year, etc.), having h1n1 influenza, having family members, friends or acquaintances having h1n1 influenza, and contacting people infected with h1n1 influenza. in addition, gender, having knowledge about h1n1 influenza, and being afraid of h1n1 influenza were also included. the correlational analyses revealed a significant positive relationship between the pcl - c total score and area, and university grade (p0.05, table 2). the regression analyses revealed that in north china, female gender, having h1n1 influenza, having family members, friends or acquaintances having h1n1 influenza, and being afraid of h1n1 influenza were significant predictors of the stress symptoms among chinese university students during the 2009 h1n1 influenza (p<0.01, table 3). children and young adults are the cohort at highest risk for h1n1 infection and death. in addition, they are the most easily affected group during a global event. however, no information is available about the stress symptoms among youth during the 2009 h1n1 influenza pandemic. the university environment has a high risk of spreading infectious diseases, particularly the 2009 pandemic influenza h1n1, as it is a mass gathering place for youth. to the best of our knowledge, the present study is the first to report stress symptoms among university students during the 2009 h1n1 influenza pandemic. in general, many students experienced a variety of stress reactions to the 2009 h1n1 influenza, and about 2.0% of students developed ptsd. as predicted, in north china, female gender, having h1n1 influenza, having family members, friends or acquaintances having h1n1 influenza and being afraid of h1n1 influenza were significant predictors of stress symptoms in the current study. this result is in line with the facts that stress symptoms are related to the degree of exposure to a stressful event. in addition, as reported, females are in general 2.382.49 times more likely to develop lifetime ptsd than men after exposure to similar traumas. however, having knowledge about h1n1 influenza, receiving the vaccine, and contacting people infected h1n1 influenza were not predictors of the stress symptoms. university students of higher grade had more stress symptoms, which was contrary to our previous prediction. it is possible that university students of higher grades may be much more worried about the potential for 2009 h1n1 influenza to affect them directly when they begin their careers. first, the participants were not recruited by random sampling procedures, and therefore there may be some bias in the sample that reduces the ability of the results to be generalized. second, the measures of stress symptoms used in the present study may be vulnerable to various types of inherent bias as it is a self - report instrument. however, the pcl - c has been widely proven to be a well validated measure of stress symptoms [1921 ]. the current study supports other work indicating that individuals exposed to disasters only through media reports can also experience significant distress. despite this study s methodological shortcomings, it extends research in this area by showing the stress symptoms and the significant predictors of the symptoms among chinese university students during the 2009 h1n1 influenza pandemic. moreover, this study may contribute to understanding stress reactions among university students who were involved in the h1n1 influenza outbreak. most importantly, the predictors found in this study may be extremely effective in defining the high risk group of the university students in similar influenza epidemics. organizations will need to develop an integrated administrative and psychosocial response to the psychological challenges that are caused by future outbreaks of this nature. in conclusion, in north china, female, having h1n1 influenza, having family members, friends, or others known having h1n1 influenza, and afraid of h1n1 influenza were significant predictors of the stress symptoms. | summarybackgroundthe university environment poses a high risk of spreading infectious diseases, particularly the 2009 pandemic influenza h1n1, as it is a mass gathering place for youth. this study aimed to evaluate the predictors of stress symptoms among chinese university students during the 2009 h1n1 influenza pandemic.material/methodswe used a self - reported questionnaire, the ptsd (posttraumatic stress disorder) checklist - civilian version (pcl - c) to evaluate the stress symptoms among chinese university students from heilongjiang (n=455), beijing (n=106), shanghai (n=419) and sichuan (n=102). we then analyzed the predictors of stress symptoms.resultsthe proportion of university students enrolled in this study who met symptomatic criteria for ptsd was 2% (22 students). the mean pcl - c total score in the sample was 22.098.01. the correlational analyses revealed a significant positive relationship between the pcl - c total score and area, and university grade (p<0.01). moreover, a negative relationship was found between the pcl - c total score and gender, having h1n1 influenza, having family members, friends or acquaintances having h1n1 influenza, and being afraid of h1n1 influenza (p<0.01). the regression analyses showed that in north china, female gender, having h1n1 influenza, having family members or acquaintances with h1n1 influenza, and being afraid of h1n1 influenza were significant predictors of the stress symptoms.conclusionsin north china, female gender, having h1n1 influenza, having family members, friends, or acquaintances with h1n1 influenza, and being afraid of h1n1 influenza were significant predictors of the stress symptoms. |
polymyalgia rheumatica (pmr) is a clinical syndrome characterized by muscle pain and stiffness in the neck, shoulders and pelvic girdle. it is often associated with giant cell arteritis (gca), suggesting that these diseases represent different clinical manifestations of the same disease process. only a few cases of renal aa amyloidosis have been reported as renal involvement of pmr [4, 5 ]. a 70-year - old man complained of muscle pain and stiffness of the neck, shoulders, pelvic girdle and thighs at the end of september 2011. he also had a low - grade fever (< 38.0c) with general fatigue and appetite loss. although he took a non - steroidal anti - inflammatory drug, namely loxoprofen (180 mg / day), muscle pain and the other clinical symptoms did not improve. he suspended the use of loxoprofen and consulted a physician at a local hospital. upon further examination at the hospital, blood analysis showed that white blood cells had mildly increased to 12,000/l, c - reactive protein (crp) level had increased to 8.7 mg / dl, hemoglobin (hb) level had decreased to 9.2 g / dl and creatine kinase (ck) level was in the normal range (20 mu / ml). urinalysis showed proteinuria (2 +) and hematuria (+), and urinary sediments showed red blood cells (1015/field). since the high fever, general fatigue, appetite loss and muscle pain continued with these blood and urinary abnormalities, based on the clinical course and the increase of acute phase reactants, bacterial infection was suspected as the etiology of this case at that time. therefore, he received an antibiotic, namely meropenem (1 g / day), for 10 days ; however, these symptoms as well as hematuria and proteinuria did not improve at all. therefore, the patient was transferred to our department for diagnosis and treatment at the end of november 2011. upon examination at admission, he had muscle pain in the neck, shoulders, pelvic girdle and thighs. he also had symptoms such as low - grade fever (37.8c), general fatigue, appetite loss, morning stiffness in the hands and edema of the face and legs. blood analysis showed that white blood cells mildly increased to 13,400/l, erythrocyte sedimentation rate increased to 81 mm / h, crp level increased to 10.3 mg / dl and plasma interleukin (il)-6 level increased to 30.5 pg / ml (normal range < 4.0 pg / ml). hb level decreased to 8.7 g / dl (mean corpuscular volume (86 fl) and mean corpuscular hb (29.4 pg) were in the normal range), while ck level was in the normal range (23 mu / ml). urinalysis showed proteinuria (5.2 g / day) and hematuria (3 +), and urinary sediments showed red blood cells (2030/field) with red blood cell cast (34/all fields) and granular cast (12/all fields). table 1 shows other blood analysis and urinalysis results upon admission. as shown in table 1, no autoantibody suggestive of autoimmune diseases was detected in blood analysis, and no bacterial or viral infection, including streptococcus, staphylococcus aureus, parvovirus b19, herpes simplex virus, cytomegalovirus and epstein - barr virus, was detected in sputum, blood or urinalysis specimens. the computerized tomography (ct) scan of the brain, chest and abdomen also did not show any lesions suggestive of infections or malignancies. blood analysis showed normocytic normochromic anemia, and upper gastrointestinal endoscopy and colonoscopy did not show any lesions that caused anemia such as ulcers and malignancies. the abdominal ct scan showed that the kidneys were normal shape and not atrophied (left kidney : 10.2 5.9 cm ; right kidney : 10.6 5.3 cm). the patient 's extrarenal symptoms and blood analysis results led to the diagnosis of pmr because they fulfilled three different sets of diagnosis criteria for pmr [6, 7, 8 ]. the renal biopsy specimen showed glomeruli that were large and cellular, with the infiltration of polymorphonuclear leukocytes and focal and segmental increased mesangial matrix and proliferation of mesangial cells by light microscopic analysis (fig. immunofluorescence analysis showed the deposition of iga and c3c in the mesangial area (fig. electron microscopy analysis showed dense deposits in the mesangial area and foot process effacement (fig. taken together, these extrarenal symptoms, the results of blood and urinary analysis and the renal histological analysis led to the diagnosis of diffuse endocapillary proliferative glomerulonephritis associated with pmr. low - dose prednisolone (psl ; 10 mg / day) was administered as treatment. erythrocyte sedimentation rate decreased to 5 mm / h, crp decreased to 0.55 mg / dl, body temperature was normalized, muscle pain, fatigue and appetite loss disappeared at day 4, hematuria decreased to (< + 1) and proteinuria decreased to 0.4 g / day at 2 weeks after psl treatment. there were also no data suggestive of bacterial or viral infections, including streptococcal and parvovirus b19 infections, which were reported to cause endocapillary proliferative glomerulonephritis [9, 10 ]. the extrarenal symptoms and blood analysis results of this patient fulfilled three different sets of diagnosis criteria for pmr [6, 7, 8 ]. taking these results together, we diagnosed this case as diffuse endocapillary proliferative glomerulonephritis with renal involvement of pmr. glomerulonephritis markedly improved with low - dose psl associated with the improvement of extrarenal symptoms and acute phase reactants of pmr. although the clinical course, laboratory data, and good and rapid response to low - dose psl of this patient may support that glomerulonephritis could be a renal involvement of pmr, the possibility that acute post infectious glomerulonephritis occurred primarily and reactive arthritis was consequently developed should be considered too. the relatively low level of compliments and self - limiting clinical course may support this speculation. reported a case of renal aa amyloidosis that showed nephrotic range proteinuria and rapidly deteriorating renal function within 18 months of the onset of pmr symptoms. they suggested that the patient might have had a longstanding, uncharacterized inflammatory process, and might have developed aa amyloidosis earlier on. we did not detect amyloid deposition in the kidney of the patient in the present case. although the cause of pmr remains unknown, inflammation has been considered to contribute to pmr. mild synovitis characterized by a predominance of macrophages and cd4 + t lymphocytes has been described in specimens of shoulder synovial membranes in pmr patients. shintani. reported that igg, iga and fibrinogen were deposited in the perifascicular area of the perimysium of pmr patients. in addition to these lines of evidence, the infiltration of dendritic cells and inflammatory cytokines derived from macrophages, such as il-1 and il-6, are detectable in histologically normal temporal arteries in pmr patients. these activated immune cells may have contributed to glomerulonephritis in the present case because deposition of iga and c3c in the mesangial area and the infiltration of polymorphonuclear leukocytes in the endocapillary space were observed. increased plasma il-6 level in this patient may also have contributed to glomerulonephritis because il-6 has been reported to increase mesangial matrix and proliferation of mesangial cells [14, 15 ]. it was reported that pmr could be inherited in genetically susceptible individuals whose immune system can be stimulated by pathogen. it has also been reported that there is an association between the genetic factors and several glomerular nephritis [17, 18 ]. although we could not perform a genetic analysis in the present case, the patient 's genetic factor might have contributed to the development of pmr and glomerulonephritis. further studies will be required to investigate the mechanism and factors of glomerulonephritis associated with pmr. after low - dose psl treatment, diffuse endocapillary proliferative glomerulonephritis was markedly improved in association with the improvement of extrarenal symptoms and acute phase reactants of pmr. in conclusion, clinical observations, laboratory data and the result of a renal biopsy specimen suggested a case of diffuse endocapillary proliferative glomerulonephritis associated with pmr, which was successfully treated by low - dose psl. the renal complication of pmr is rare but important to be considered early in the right clinical context. | a 70-year - old man complained of muscle pain in his neck, shoulders and pelvic girdle. proteinuria and hematuria subsequently developed. blood analysis showed increased acute phase reactants. the histology of renal biopsy showed diffuse endocapillary proliferative glomerulonephritis. there were no signs of autoimmune diseases, malignancies and bacterial or viral infections. his extrarenal symptoms and the results of blood analysis fulfilled three different criteria of polymyalgia rheumatica (pmr). therefore, diffuse endocapillary proliferative glomerulonephritis associated with pmr was diagnosed. after low - dose prednisolone (10 mg / day) treatment, the muscle pain disappeared, acute phase reactants decreased and hematuria and proteinuria improved. the renal complication of pmr is rare but important to be considered early in the right clinical context. |
numerous proton magnetic resonance spectroscopy (1h - mrs) studies have linked cytosolic choline levels to the neuropathology of mood disorders (17). increases in choline in depressed subjects have been noted in adults (1,3) and in pediatric samples (4,5). auer and others (8) did not note any significant difference in choline levels in the cingulate in their study of mdd subjects and controls. the choline resonance derived by 1h - mrs arises mainly from phosphocholine and glycerophosphocholine (9,10). children and adolescents have been relatively understudied with regard to the neurobiology of mood disorders. this is despite the fact that the prevalence of depression increases during adolescence, rising from 1% up to adult levels (68%). mood disorders in youth are associated with impairment in social, family and academic functioning, are highly predictive of future episodes and are significantly related to suicide (11). despite the prevalence and outcomes associated with the mood disorders, there is a paucity of information about the etiology. hence, studies of children and adolescents are necessary in order to determine if abnormalities noted in adult populations occur across the life span, how early they present during development, and whether they can be used as predictors for future risk of developing depression. studies early in the course of an illness also avoid the potential confounds of illness chronicity, hospitalization and long - term medication use. this study endeavors to uncover neurobiological correlates of juvenile depression. based on the adult literature (13,6,7) and the reports on pediatric depression (4,5), we hypothesized that youth with mdd and those without psychiatric illness will demonstrate differences with regard to cytosolic choline in the prefrontal cortex. subjects were recruited through either of two methods : (1) advertisements, or (2) as participants in clinical treatment (combined pharmacological and psychotherapy) programs. all subjects and one of their parents / guardians signed informed consents for the study after a full explanation of the procedures in accordance with the research ethics board approval provided by the iwk health centre. each subject completed the children s depression rating scale (cdrs) (mood disorder group mean = 66.083 + 14.042) at the time of scan. diagnosis based on kiddie - schedule for affective disorders and schizophrenia - present and lifetime version (k - sads - pl) criteria were established by a board - certified psychiatrist (primarily vk). twelve subjects (5 males, 7 females) aged 10 to 18 years old with mdd and twelve age and sex - matched healthy controls participated in this study. exclusion criteria for participation in this study included a history of neurological illness, serious medical illness, claustrophobia, age greater than 18 years, or the presence of a ferrous implant or pacemaker. scans were conducted at the queen elizabeth ii health sciences centre using a siemens magnetom vision 1.5 tesla scanner. multi - slice scout images (axial, coronal and sagittal planes) were used for voxel orientation. special care was taken to ensure adequate coverage of the right prefrontal gray matter by the voxel. a long echo proton magnetic resonance spectroscopic imaging (1h - mrs) parameters were as follows : te = 135 ms, tr = 1500 ms, acquisitions 256, voxel = 4cc, time = 8 minutes (see figure 1 for sample spectra). a trained mrs analyst (fpm) analyzed data in a blind manner. mrs metabolites that can be visualized with this technique are n - acetyl - aspartate (naa, 2.02ppm), creatine / phosphocreatine (cr, 3.03ppm) and choline compounds (cho, 3.20ppm). following a convention that minimizes variations in the magnetic field homogeneity, metabolite concentrations are expressed as ratios of peak areas (i.e. naa / cr, cho / cr). a repeated measures anova was used to determine group differences in choline levels between mdd patients and healthy age and sex - matched controls. we expected that close age and sex- matching allows for a reduction in the potential confounds of sex and age on the data set. correlations (two - tailed) between metabolites and age and, in the psychiatric group, depressive symptom severity were also conducted. subjects were recruited through either of two methods : (1) advertisements, or (2) as participants in clinical treatment (combined pharmacological and psychotherapy) programs. all subjects and one of their parents / guardians signed informed consents for the study after a full explanation of the procedures in accordance with the research ethics board approval provided by the iwk health centre. each subject completed the children s depression rating scale (cdrs) (mood disorder group mean = 66.083 + 14.042) at the time of scan. diagnosis based on kiddie - schedule for affective disorders and schizophrenia - present and lifetime version (k - sads - pl) criteria were established by a board - certified psychiatrist (primarily vk). twelve subjects (5 males, 7 females) aged 10 to 18 years old with mdd and twelve age and sex - matched healthy controls participated in this study. exclusion criteria for participation in this study included a history of neurological illness, serious medical illness, claustrophobia, age greater than 18 years, or the presence of a ferrous implant or pacemaker. scans were conducted at the queen elizabeth ii health sciences centre using a siemens magnetom vision 1.5 tesla scanner. multi - slice scout images (axial, coronal and sagittal planes) were used for voxel orientation. special care was taken to ensure adequate coverage of the right prefrontal gray matter by the voxel. a long echo proton magnetic resonance spectroscopic imaging (1h - mrs) parameters were as follows : te = 135 ms, tr = 1500 ms, acquisitions 256, voxel = 4cc, time = 8 minutes (see figure 1 for sample spectra). a trained mrs analyst (fpm) analyzed data in a blind manner. mrs metabolites that can be visualized with this technique are n - acetyl - aspartate (naa, 2.02ppm), creatine / phosphocreatine (cr, 3.03ppm) and choline compounds (cho, 3.20ppm). following a convention that minimizes variations in the magnetic field homogeneity, metabolite concentrations are expressed as ratios of peak areas (i.e. naa / cr, cho / cr). a repeated measures anova was used to determine group differences in choline levels between mdd patients and healthy age and sex - matched controls. we expected that close age and sex- matching allows for a reduction in the potential confounds of sex and age on the data set. correlations (two - tailed) between metabolites and age and, in the psychiatric group, depressive symptom severity were also conducted. the two groups did not differ with regard to age (control group mean = 14.917 years + 2.575, mood disorder group mean = 14.750 years + 2.454, t11 = 1.000, p = 0.339). cho / cr ratios were increased (39%) in right prefrontal cortex in the mdd group as compared to matched healthy controls (control mean = 0.782 + 0.170, mdd group mean = 1.088 + 0.368, f1, 11 = 10.741, p = 0.007). right prefrontal naa / cr did not differ between the healthy controls and mdd subjects (control mean = 2.135 + 0.378, mdd group mean = 2.408 + 0.490, f1, 11 = 2.702, p = 0.129). in the control group, cho / cr demonstrated a trend for inverse correlation with age in years (r = 0.516, p = 0.087) but not in the mdd group (r = 0.343, p = 0.284). in the mdd group, cho / cr demonstrated a weak trend for positive correlation with cdrs symptom severity score (r = 0.480, p = 0.117) we report a significant increase in cho / cr in youth with mdd as compared to age- and sex - matched healthy controls in a voxel located in the right prefrontal cortex. this finding is consistent with previous reports of abnormalities in the choline resonance in mood disorders (17). choline acts as a precursor of the neurotransmitter acetylcholine and the membrane lipids, phosphatidylcholine and sphingomyelin. as these lipids are typically bound in the membrane, they contribute little to the mrs choline resonance, while the cytosolic choline compounds, such as glycerophosphocholine (gpc) and phosphocholine (pc) contribute as much as 50% to the choline signal. free choline, acetylcholine, and cytidine diphosphate choline make smaller contributions to the resonance (1214). based on this information, it has been postulated that changes in the choline resonance may be reflective of changes in local neuronal metabolism. these changes in metabolism may be required for the incorporation of cytosolic choline compounds into phospholipids. a previous study has shown changes in pet measures of metabolism to be inversely correlated with the 1h - mrs choline resonance (15). steingard and others (4) have also found increases in prefrontal choline in adolescent depression. this is an interesting finding given that the prefrontal cortex has been repeatedly implicated in metabolic neuroimaging studies in mood disorders (16,17). it is also a possibility that the choline increase demonstrated in the prefrontal cortex of mdd subjects as compared to the controls in this study may reflect differences in glucocorticoid activity. one of the most - replicated findings in biological psychiatry has been the alteration in the endocrine system in mood disorders. many mood disorder patients demonstrate hypothalamic - pituitary - adrenal (hpa) axis over activation (18). in conditions that result in excess glucocorticoids, such as graves disease, bhatara and others (19) glucocorticoids likely adversely affect phosphatidylcholine (ptd - cho) metabolism in the brain by inhibiting the activity of enzymes such as phospholipase a2 and c (21). ptd - cho plays a role in the second messenger system and signal transduction, as it is a source for diaglycerol (2225). as an increase in choline has been shown in this report, a primary role for glucocorticoids in this instance may be unlikely. in emotional processing, tone to a stimulus while the prefrontal cortex provides inhibitory modulation (26). prefrontal cortex modulation of amygdala function may be diminished in mood disorders, and hence a lack of control over negative cognition may result. should critical prefrontal inputs to the amygdala be rendered impotent, the inhibitory control of the prefrontal cortex would be lost. most likely, however, is that the emotional homeostatic mechanisms of both the prefrontal and limbic regions are dysfunctional in mood disorders (27). previous work has demonstrated that the prefrontal choline resonance decreases with increasing age in healthy children and adults (28). in our study, only the cho / cr levels in controls were inversely correlated with age in years. as changes in the choline resonance during childhood and adolescence are typically thought to reflect changes in brain development, the lack of a correlation in the mood disorder sample may be indicative of a differential developmental trajectory for these subjects. alternatively, the change in choline may be wrought by changes in metabolism, signal transduction, or glucocorticoids. the principle limitations of this study are the small sample size and the heterogeneity of clinical presentation of the mdd group. a further limitation of this study is the lack of absolute quantification of the prefrontal metabolites. however, absolute quantifications are themselves subject to magnetic inhomogeneities and partial volume effects within the region of interest. the use of ratios minimizes the error introduced by variable tissue composition and instrumental instability while allowing the evaluation of relative alterations in metabolites. further experiments are required to elucidate the exact mechanism of the increase in the cho / cr peaks and its relation to depressive symptomotology. we report a significant increase in cho / cr in youth with mdd as compared to age- and sex - matched healthy controls in a voxel located in the right prefrontal cortex. this increase in the cho / cr ratio may be the result of reductions in metabolism in the prefrontal cortex. | purpose of study : the prefrontal cortex has been previously implicated in the neuropathology of major depressive disorder (mdd). hence, we used proton magnetic resonance spectroscopy (1h - mrs) to examine choline levels in the prefrontal cortex of youth with major depression. basic procedures : twelve age- and sex - matched case - control pairs were examined (age range 10 to 18 years, 7 females and 5 males in each group). all subjects were treatment naive at the time of the scan. a long echo 1h - mrs scan was acquired from the right prefrontal cortex (4cc) in all subjects. main findings : right prefrontal choline / creatine ratios were elevated in the youth with mood disorders (f1, 11 = 10.741, p = 0.007) as compared with healthy controls. principal conclusions : these findings suggest that prefrontal cytosolic choline may be increased in youth with mdd in comparison with healthy controls. this is consistent with reported findings in both adults and adolescents and suggests that mdd in youth is associated with alterations in choline metabolism in the prefrontal cortex. |
the apicomplexan parasite cryptosporidium parvum is a waterborne enteric protozoan with significant veterinary importance (1). cryptosporidiosis in the immunocompetent host is a self - limiting diarrheal illness, but in immunosuppressed individuals such as those with human immunodeficiency virus (hiv) infection, the disease can be serious and life threatening (1, 2). in addition to humans, c. parvum has been reported as a common serious primary cause of diarrheal outbreaks in farm animals, especially newborn ruminants such as cows that result in significant economic losses (3). to date, due to the lack of effective drugs and approved vaccines, the prevention and treatment of cryptosporidiosis remains a big problem (4)., have the capacity to invade and replicate within the cells of their vertebrate hosts (5). all of these genera possess three kinds of secretory vesicles consisting of the apical organelles (rhoptries, micronemes and dense granules), which secrete substances that enable parasites to adhere selectively to and invade host cells ; once within a host, they cause further modifications and eventually escape (6). rhoptries are perhaps the most unusual organelles found within apicomplexan parasites (7, 8). they are the largest of the apical organelles, and their name alludes to their club - like shape that is visible in the apicomplexan parasites (6). each zoite has two rhoptries in plasmodium merozoites and sporezoites, babesia caballi has three (9), theileria parva merozoites have six (10), and t. gondii has eight or more in every sporozoite. rhoptry proteins have been linked with both the adhesion and invasion processes of the parasites and with intracellular pathways such as the delivery of signals to the host cell nucleus that is achieved by crossing the parasitophorous vacuole membrane (pvm) (12) or through evacuoles (13). furthermore, some rhoptry proteins were shown to be important virulence factors (14). although c. parvum contains rhopty, few rhoptry proteins in c. parvum have been reported to date. in the present study, subcellular fractionation was coupled with proteomic techniques to identify novel rhoptry components in c. parvum. the c. parvum iowa isolate used in this study was purchased from waterborne inc. and maintained by passage in newborn cryptosporidium - free holstein bull calves (15). oocysts were isolated from calf feces using sucrose density gradient centrifugation (16) and 0.5% (wt / vol) hypochlorite treated immediately at 4c for 10 min. oocyts excystation was conducted in 0.75% (wt / vol) nataurocholate (sigma, hercules, ca, usa) at 37c for 2 h until it exceeded 90% (15). a total of 1 10 sporozoites were collected by centrifugation at 1,300 g for 10 min at 4c. the sporozoites were washed once in phosphate - buffered saline (pbs) and in r buffer (250 mm sucrose, 10 mm mops, ph 7.2, 2 mm dithiothretol, 1 mm ethylenediaminetetraacetic acid [edta ], complete protease inhibitor mixture), respectively. the pellet was resuspended in r buffer (5 10 sporozoites / ml) and then disrupted by passage through a french type pressure cell disrupter (stansted fluid power ltd.) as previously described (18). after being centrifuged at 1,300 g for 20 min, the organellar pellet was created by centrifugation at 25,000 g for 25 min and resuspended in r buffer containing 30% (wt / vol) percoll. the mixture was centrifuged at 61,500 g for 25 min and a brownish band was collected. the band was diluted in r buffer and pelleted again at 100,000 g for 90 min to remove the percoll. the mixture (after percoll removal and r buffer dilution) was overlaid with steps of sucrose (36%, 39%, 42%, 45%, 48%, and 60%) in s buffer (10 mm mops, 2 mm dithiothreitol, 1 mm edta, and complete protease inhibitor mixture) and centrifuged at 150,000 g for 18 h. fractions in each gradient were collected, diluted in r buffer, and centrifuged at 100,000 g for 90 min. pooled gradient fractions were respectively fixed in 2.5% glutaraldehyde, 4% sucrose, and 0.05 m phosphate buffer (ph 7.4) for 2 h, washed three times with ice - cold pbs, and centrifuged at 14,000 rpm for 10 min. total protein was extracted using a readyprep protein extraction kit (total protein) (bio - rad, usa) according to the manufacturer s instructions. the extraction process could solubilize many types of cell proteins, including both soluble and membrane fractions. briefly, the purified rhoptry fraction was added to 1 ml of sample buffer (7 m urea, 2 m thiourea, 1% [wt / vol ] asb-14 detergent, 40 mm tris base, and 0.001% bromophenol blue) in a 2-ml microcentrifuge tube and sonicated on ice for 30-sec bursts (typically 34 times). rhoptry proteins were separated in 15% sds - page and visualized using coomassie blue staining. the entire protein gel profile was excised from the gel and maintained in the new tube with milliq water until the protein was digested. the entire 1-d sds - page gel profile was hydrolyzed to cleave the peptides and then analyzed using lc / ms - ms as described below. first, the sample was minced and washed twice with milliq water, decolorized twice with 50% meoh for 30 min and subsequently washed with milliq water, 50% acetonitrile and 100% acetonitrile. after the sample was reduced with 10 mm dtt for 1 h at 55 c, alkylated 55 mm iodoacetamide was added for 45 min in the dark and the sample was washed again with milliq water, 50% acetonitrile and 100% acetonitrile. second, the dried sample was digested with 3 l of 12.5 ng/l trypsin (promega v5280) overnight at 37c and centrifuged, and the supernatant was acidified using 1% formic acid. digested peptides were dissolved using 25 mm nh4hco3 (0.1% formic acid and 2% acetonitrile) were analyzed using lc / ms - ms on an ltq - orbitrap mass spectrometer (thermo electron, bremen, germany). the peptides were separated on a biobasic c18 picofit column (100 m, 10 cm long, 3 m resin ; michrom bioresources, auburn, ca, usa) at a flow rate of 300 nl / min. water and acetonitrile added with 0.1% formic acid were used as solvents a and b, respectively. the gradient was started at 5% solvent b and increased to 35% solvent b for 120 min. peptide ions were analyzed in data - dependent ms experiments and detected in a survey scan from 400 to 1700 amu (3 scans) followed by five data - dependent ms / ms scans (5 scans each ; isolation width, 3 amu ; 35% normalized collision energy ; dynamic exclusion for 3 min). bioworks 3.2 software was used to process the data and convert it to an mgf file. the search was performed using the mascot in the ncbi, cryptodb v5.0, and eupathdb v2.16 databases. the c. parvum iowa isolate used in this study was purchased from waterborne inc. and maintained by passage in newborn cryptosporidium - free holstein bull calves (15). oocysts were isolated from calf feces using sucrose density gradient centrifugation (16) and 0.5% (wt / vol) hypochlorite treated immediately at 4c for 10 min. oocyts excystation was conducted in 0.75% (wt / vol) nataurocholate (sigma, hercules, ca, usa) at 37c for 2 h until it exceeded 90% (15). a total of 1 10 sporozoites were collected by centrifugation at 1,300 g for 10 min at 4c. the sporozoites were washed once in phosphate - buffered saline (pbs) and in r buffer (250 mm sucrose, 10 mm mops, ph 7.2, 2 mm dithiothretol, 1 mm ethylenediaminetetraacetic acid [edta ], complete protease inhibitor mixture), respectively. the pellet was resuspended in r buffer (5 10 sporozoites / ml) and then disrupted by passage through a french type pressure cell disrupter (stansted fluid power ltd.) as previously described (18). after being centrifuged at 1,300 g for 20 min, the organellar pellet was created by centrifugation at 25,000 g for 25 min and resuspended in r buffer containing 30% (wt / vol) percoll. the mixture was centrifuged at 61,500 g for 25 min and a brownish band was collected. the band was diluted in r buffer and pelleted again at 100,000 g for 90 min to remove the percoll. the mixture (after percoll removal and r buffer dilution) was overlaid with steps of sucrose (36%, 39%, 42%, 45%, 48%, and 60%) in s buffer (10 mm mops, 2 mm dithiothreitol, 1 mm edta, and complete protease inhibitor mixture) and centrifuged at 150,000 g for 18 h. fractions in each gradient were collected, diluted in r buffer, and centrifuged at 100,000 g for 90 min. pooled gradient fractions were respectively fixed in 2.5% glutaraldehyde, 4% sucrose, and 0.05 m phosphate buffer (ph 7.4) for 2 h, washed three times with ice - cold pbs, and centrifuged at 14,000 rpm for 10 min. total protein was extracted using a readyprep protein extraction kit (total protein) (bio - rad, usa) according to the manufacturer s instructions. the extraction process could solubilize many types of cell proteins, including both soluble and membrane fractions. briefly, the purified rhoptry fraction was added to 1 ml of sample buffer (7 m urea, 2 m thiourea, 1% [wt / vol ] asb-14 detergent, 40 mm tris base, and 0.001% bromophenol blue) in a 2-ml microcentrifuge tube and sonicated on ice for 30-sec bursts (typically 34 times). rhoptry proteins were separated in 15% sds - page and visualized using coomassie blue staining. the entire protein gel profile was excised from the gel and maintained in the new tube with milliq water until the protein was digested. the entire 1-d sds - page gel profile was hydrolyzed to cleave the peptides and then analyzed using lc / ms - ms as described below. first, the sample was minced and washed twice with milliq water, decolorized twice with 50% meoh for 30 min and subsequently washed with milliq water, 50% acetonitrile and 100% acetonitrile. after the sample was reduced with 10 mm dtt for 1 h at 55 c, alkylated 55 mm iodoacetamide was added for 45 min in the dark and the sample was washed again with milliq water, 50% acetonitrile and 100% acetonitrile. finally, the sample was dried in vacuo for 30 min. second, the dried sample was digested with 3 l of 12.5 ng/l trypsin (promega v5280) overnight at 37c and centrifuged, and the supernatant was acidified using 1% formic acid. digested peptides were dissolved using 25 mm nh4hco3 (0.1% formic acid and 2% acetonitrile) were analyzed using lc / ms - ms on an ltq - orbitrap mass spectrometer (thermo electron, bremen, germany). the peptides were separated on a biobasic c18 picofit column (100 m, 10 cm long, 3 m resin ; michrom bioresources, auburn, ca, usa) at a flow rate of 300 nl / min. water and acetonitrile added with 0.1% formic acid were used as solvents a and b, respectively. the gradient was started at 5% solvent b and increased to 35% solvent b for 120 min. peptide ions were analyzed in data - dependent ms experiments and detected in a survey scan from 400 to 1700 amu (3 scans) followed by five data - dependent ms / ms scans (5 scans each ; isolation width, 3 amu ; 35% normalized collision energy ; dynamic exclusion for 3 min). bioworks 3.2 software was used to process the data and convert it to an mgf file. the search was performed using the mascot in the ncbi, cryptodb v5.0, and eupathdb v2.16 databases. the disrupeted sporozoite composition was subjected to sucrose gradient floatation using a gradient of 36 60% sucrose. fractions of each gradient (36%, 39%, 42%, 45%, 48%, and 60% sucrose) were processed for electron microscopy, and those that were enriched with rhoptry proteins were identified. the only fraction between 36% and 39% sucrose was enriched with rhoptries as indicated by its black tadpole - like appearance (fig. electron microscopy of sucrose graction of rhoptry fraction other sucrose concentrations did not find any similar structures. the crystalline objects in the background may be the leaving sugars. based on these results, pooled gradient fractions of c. parvum different protein compositions were observed among the subcellular fractions in 1-d sds - page (fig. the molecular weights of some of the proteins were > 260 kda (a), while the others were 25260 kda (b). these two sections were excised and identified using lc / ms - ms. a total of 22 proteins (four from part a and 18 from part b) were identified, including kinases, secret proteins, large proteins, membrane protein, atpase, peptidase, dikinase, transmembrane protein, and hypothetical proteins (table 1). among these proteins, a serine threonine (ser / thr) protein kinase of c. parvum (cgd7_590) possessed 40% amino acid sequence identity with t. gondii me49 rop17 (tgrop17) and 34% identity with n. caninum rop20 (ncrop20). 1-d sds - page resolution of the rhoptry fraction a : the proteins with molecular weight more than 260 kda ; b : the proteins with molecular weight between 25 kda to 260 kda identification of rhoptry proteins by lc / ms - ms mascot ms protein score, the probability score was 260 kda (a), while the others were 25260 kda (b). these two sections were excised and identified using lc / ms - ms. a total of 22 proteins (four from part a and 18 from part b) were identified, including kinases, secret proteins, large proteins, membrane protein, atpase, peptidase, dikinase, transmembrane protein, and hypothetical proteins (table 1). among these proteins, a serine threonine (ser / thr) protein kinase of c. parvum (cgd7_590) possessed 40% amino acid sequence identity with t. gondii me49 rop17 (tgrop17) and 34% identity with n. caninum rop20 (ncrop20). 1-d sds - page resolution of the rhoptry fraction a : the proteins with molecular weight more than 260 kda ; b : the proteins with molecular weight between 25 kda to 260 kda identification of rhoptry proteins by lc / ms - ms mascot ms protein score, the probability score was 260 kda. due to protein abundance and other reasons, however, the present work provided a starting point for the study of the repertoire and function of c. parvum rhoptry proteins. nonetheless, further studies must be conducted to confirm their characteristics, localization within rhoptries, synergistic effect with other proteins, and functional role in the c. parvumlife cycle. twenty - two potential novel rhoptry proteins were detected from cryptosporidium parvum rhoptry - enriched fractions by one - dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by liquid chromatography coupled with mass spectrometry analysis. these novel candidate proteins may be considered targets for researching the invasion pathway of c. parvum and the pathogenic mechanisms of rhoptry proteins. 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 : rhoptries are unique secretory / excretory organelles that are found exclusively in the apicomplexa, and their contents are discharged at the time of invasion and are critical in the establishment of productive infection. several rhoptry proteins have been identified in toxoplasma gondii, plasmodium falciparum and neospora caninum and have been linked not only with the parasites adhesion and invasion processes but also with their intracellular pathways. to date, only one cryptosporidium parvum rhoptry protein candidate related to tgron1 of t. gondii and pfasp of p. falciparum has been reported.methods:subcellular fractionation of sporozoites was performed to obtain highly purified organelles. one - dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by liquid chromatography coupled with mass spectrometry was applied for fraction analysis, and 22 potential novel rhoptry proteins were detected by protein domain analysis using online softwares.results:twenty-two potential novel rhoptry proteins were detected. a protein with t. gondii and n. caninum rhoptry protein homologs and some proteins with domains similar to that of t. gondii rhoptry proteins were identified.conclusion:these novel candidate proteins may be considered targets for researching the invasion pathway of c. parvum and the pathogenic mechanisms of rhoptry proteins. the present work provides a starting point towards the elucidation of the repertoire and function of c. parvum rhoptry proteins. |
an ideally aligned posture is regarded as one in which there is perfect alignment of the weight - bearing segment, and it is commonly described by the vertical line of gravity passing anterior to the knee, posterior to the hip, through the bodies of vertebrae in both the cervical and lumbar spine, through the shoulder joint, and through the external auditory meatus2,3,4. proper posture is achieved by maintaining the musculoskeletal balance associated with minimal stress on the body and is considered an important factor in assessment of health condition. among many factors, including vision, vestibular function, the somatosensory system, and the musculoskeletal system, proprioception is considered an essential factor for the maintenance of balance1, 5, 6. however, several factors, including neck pain and/or shoulder pain, can disrupt this balance, leading to development of a postural problem5, 7, 8. forward head posture (fhp), one of the most common abnormal head postures, is a postural head - on - trunk misalignment, which is defined as a head that is positioned anterior to a vertical line of gravity7,8,9,10. it is commonly quantified by measurement of craniovertebral (cv) angle, which assesses the head posture2, 11, 12. fhp can lead to development of several musculoskeletal problems, including neck pain, cervicogenic headache, temporomandibular disorder, and muscular dysfunction7, 13. the close relation of fhp to chronic neck and shoulder pain has been well documented12. however, only a few studies investigating the correlation between fhp and proprioceptive function have been reported. therefore, the question of whether there is a correlation between head posture and proprioceptive function in the cervical region was investigated in the current study. seventy - two subjects (35 males and 37 females) with no history of fracture, neuromuscular disorder, or pain in the cervical region, participated in this study. their mean age, height, and weight were 22.26 (2.10) years, 167.98 (11.89) cm, and 62.56 (11.89) kg, respectively. the purpose and procedures of this study were explained to all subjects, and they provided written informed consent prior to participation. all subjects were instructed to stand in a self - selected comfortable upright posture. then, the skin overlying the spinous process of the seventh cervical vertebra (c7) and tragus of the ear was marked. the cv angle was calculated based on the angle between a horizontal line passing through c7 and a line extending from the tragus of the ear to c7. the x - axis values for the craniovertebral angle were measured prior to movement. regarding measurement of fhp, a previous assessment of the test - retest reliability of the cv angle measurement revealed an icc of 0.880.9815. for assessment of proprioceptive function, joint position sense was evaluated using a dual digital inclinometer (acumar, lafayette instrument, lagatette, in, usa), which used to measure the joint position error between the starting standard position and the returned standard position. notably, joint position sense becomes more inaccurate as the degree of position sense error increases. subjects were instructed to stand upright and to memorize their head position as a neutral start position. while keeping their eyes closed, the main unit of the dual digital inclinometer was placed on the top of their head in the sagittal plane, and the companion unit of the inclinometer was placed on the c7 spinous process. then, subjects performed a maximal cervical range of the movement (flexion / extension) for approximately 2 seconds and returned to their memorized neutral position. to measure the reposition error value, this was repeated three times, and the sequence of movement (flexion / extension) was assigned randomly. demographic data (age, weight, and height) spearman s correlation coefficient was used to examine the correlation between the joint position sense and cv angle. subjects had an average cv angle of 53.70 (5.05) degrees, an average position sense error for flexion of 2.86 (1.74) degrees, and an average position sense error for extension of 2.65 (1.58) degrees. in addition, there was no significant difference between genders and head posture (p=0.734). significant negative correlation was observed between the cv angle and position sense error for flexion (r=0.655, p=0.000) and extension (r=0.557, p=0.000). a summary of the statistical values is shown in table 1table 1.correlation between craniovertebral angle and cervical joint position errorcraniovertebral angle (degree)correlation coefficient (r)cervical joint position error (degree)flexion0.665extension0.557p<0.05. the present study investigated the cv angle and joint position error in the cervical region to elucidate the correlation between head posture and proprioceptive function. in this study, the position - reposition error in the cervical region after cervical flexion and extension was measured. as a result since fhp can be quantified by the cv angle2, 11, 12, this result implies that severe fhp might be relevant to poor proprioceptive function. use of visual display terminals is associated with an increase of musculoskeletal disorders accompanying posture problems. in particular, the combination of extension in the upper cervical region and flexion in the lower cervical region appears in patients with fhp because of a misalignment in head posture. changes in the cervical region, induced by sustained poor head posture, cause excessive joint and muscle loading, and subsequently influencing weakness of the deep cervical muscles8, 20, 21. among many body structures located in the cervical region, the muscle is regarded as a main element for position sense through its receptors, such as muscle spindles22. mechanoreceptors, including muscle spindles, are densely concentrated in the cervical region, and therefore play a key role in providing proprioceptive information23. furthermore, several studies have reported that precise movement requires proper input from the muscle spindle24, 25. muscle imbalance, including weakness of cervical flexors and shortening of cervical extensors, has been reported in patients with fhp8, 26. these abnormal changes in the muscles can lead to disruption of afferent input from the muscle spindles, which may have an adverse effect on joint position sense27. thus, it is suggested that fhp may influence joint position sense via muscle spindles influenced by muscle conditions. consequently, the current study concluded that fhp is correlated with greater repositioning error than a more upright posture. our results imply that changes of muscle condition following fhp can lead to disruption of afferent input from the muscle spindles. thus, it is suggested that this alteration in the muscle spindle plays a major role in the poor proprioceptive function shown in fhp. firstly, as it did not report any measured values of muscle conditions such as muscle length and activity, the results of the current study might not be sufficient to demonstrate the effect of muscle spindles on position - reposition error. secondly, there are various types of receptors that receive sensory information in the human body. since the muscle spindle is just one of the sensory receptors, it is necessary to investigate whether there are other receptors that influence repositioning error. thus, it is thought that further study is needed to elucidate the effects of the other sensory receptors. also, further research is needed to determine whether correction of forward head posture has any impact on repositioning error. | [purpose ] the aim of the present study was to investigate correlation between head posture and proprioceptive function in the cervical region. [subjects and methods ] seventy - two subjects (35 males and 37 females) participated in this study. for measurement of head posture, the craniovertebral angle was calculated based on the angle between a horizontal line passing through c7 and a line extending from the tragus of the ear to c7. the joint position sense was evaluated using a dual digital inclinometer (acumar, lafayette instrument, lafayette, in, usa), which was used to measure the joint position error for cervical flexion and extension. [results ] a significant negative correlation was observed between the craniovertebral angle and position sense error for flexion and extension. [conclusion ] forward head posture is correlated with greater repositioning error than a more upright posture, and further research is needed to determine whether correction of forward head posture has any impact on repositioning error. |
we report a case of 18-year - old boy who presented with vomiting, backache, and fever for 1-month, diagnosed to have anaplastic large cell lymphoma of urinary bladder with hypercalcemia and metastatic calcification in multiple viscera. his computed tomography scan was suggestive of soft tissue lesion in the urinary bladder and multiple lytic skeletal lesions. bone scan showed unusual visceral uptake in lungs, liver, spleen, and myocardium in addition to osseous lesions. the clinical laboratory test revealed functional impairment of visceral organs. the patient died 3 months later. |
|
cardiovascular malformations (cvm) are one of the most prevalent groups of birth defects and include congenital heart defects and vessels anomalies. in the populations covered by the european surveillance of congenital anomalies (eurocat), the total prevalence of cvm in 2000 - 2005 was 7.97 per 1000 births (including stillborn and terminations of pregnancy due to fetal anomaly). the reported prevalence in other countries cardiovascular malformations are considered an important public health issue, being the main cause of infant deaths in developed countries. in the united states, cvm represented about 1/3 of all infant deaths, which were due to congenital anomalies. on the other hand, recent advances in medical care for this group of patients it has been estimated that the worldwide adult population with a cvm is growing by 5% per year. no administrative units are members of eurocat, and only a register in the moscow oblast is a member of international clearinghouse for birth defects surveillance and research. according to federal methodology, systematic registration is obligatory for only two forms of cvm : transposition of great vessels and hypoplasia of left heart. as far as northern territories of rf are concerned, sources of information about cvm prevalence are limited, and no studies are yet available. the russian institute of public health estimated the prevalence in north - west russia to be in the range 2.7 - 3.8 per 1000 children (0 - 13 years) in 2006 - 2008. in the population - based birth register in monchegorsk, which was the first of its kind in the rf, all anomalies diagnosed prenatally were systematically registered in contrary to the national norm. these data provide a possibility to quantify and analyse the perinatal prevalence and survival of newborns with cvm in a population of northern russia, which we have made use of in this study. the mortality rate in the rf from cardiovascular diseases and cvm among children 0 - 14 years was 14.4 per 100,000 children in year 2000. according to official figures of the rf, about 50% of children with cvm die neonatally without receiving specialised medical aid, and an additional 25% die later in infancy. the heart develops in the period 3 - 11 weeks of gestation, and hence the critical period for exposure to teratogens. among the birth defects observed to co - occur with chromosomal anomalies, neural tube defects are more common than malformations of the heart. an increased risk of cvm has been observed when maternal age is above 41 or under 16 years, and when the mother smokes, drinks alcohol or uses drugs in early pregnancy. maternal diseases, such as diabetes mellitus, obesity and overweight, and viral infections during pregnancy (rubella, coxsackie virus, respiratory viruses) are also reported to be risk factors. in addition some occupational exposures (e.g. dyes, lacquers or paints) appear to increase the risk. the aim of our study was to investigate the perinatal prevalence and structure of cvm among newborns in monchegorsk, and the mortality among the affected newborns. monchegorsk had 47,975 inhabitants in 2010 and is one of the largest cities in murmansk oblast (mo) in north - west russia (census-2010 official results). the study population was all newborns in the township in the period 1973 - 2008, a total of 28,650 live and stillborn. the maternal care and maternal benefits in russia, as well as the study population, have been described earlier in some detail. the study was register - based using data from the kola birth register (kbr) and murmansk county birth register. information about all births in monchegorsk from march 1973 through 2005 was compiled in a population - based birth register, a total of 26,841 births. the register was set up to study the association between occupational exposures and reproductive health and pregnancy outcome. data about all live births and stillbirths from 28 weeks of pregnancy were collected from general medical journals, the hospital gynaecology records and delivery records. we included all registered newborns in the study, except those for whom the source chart containing diagnoses at birth was missing or the interpretation of the recorded diagnosis was uncertain. hence, 26,711 newborns from the years 1973 - 2005 were included in the study. the murmansk county birth register (mcbr) includes all births in mo since january 1, 2006. the registered data have been systematically collected in all 15 obstetrics departments in mo from five sources : the mother s medical history, obstetric journal and delivery record, the newborn s birth record, and through an interview with the mother conducted by a physician or midwife. in the period 2006 - 2008, of these, nine records were missing data about birth defects and excluded from the study. the population coverage of the register was estimated to 98.9% in 2006. in both registries, the diagnoses of congenital malformations were registered based on the international classification of disease (icd) coding system. according to the tenth version (icd-10), the diagnoses were made before the newborns left the birth clinic. from the late 1990s the use of echocardiography supplemented routine examinations and prenatal indications of cvm needed confirmation by examination after birth. in case of fetal deaths, the study included 28,511 newborns. to analyse the structure of cvm, the prevalence and proportional distribution of the different forms newborns with more than one cvm - diagnosis were included in the numerator for each diagnosis. newborns with multiple malformations were included in the numerator, if cvm was present among the diagnoses. in addition, the stillbirth, early neonatal and perinatal mortality rates among newborns with cvm were estimated. the time trends of the prevalence and mortality were estimated using six 6-year time intervals from 1973 to 2008, and tested using chi - square for trend (medcalc 12.0 software). the prevalence of selected, possible risk factors was estimated for newborns with and without cvm, respectively, and statistically compared using the chi - square or t - test. the comparison between the two groups was carried out for the following potential risk factors : mean maternal age, parity, torch infections (i.e., toxoplasmosis, rubella, cytomegalovirus, herpes simplex, syphilis) during pregnancy, alcohol abuse during pregnancy, tobacco smoking during pregnancy, endocrine disease during pregnancy, paternal or maternal employment in the production departments of the nickel factory at the onset of the pregnancy, maternal occupation with exposure to organic solvents, and maternal body mass index (bmi) at the first antenatal visit. the adjusted risk of a cvm associated with the studied risk factors was analysed by multiple logistic regression, with cvm as a binary outcome (ibm spss 17.0 software package). the significance level in all analyses the data in the birth registers have been approved for research purposes by the murmansk regional health administration and the committee for research ethics at the university in troms. monchegorsk had 47,975 inhabitants in 2010 and is one of the largest cities in murmansk oblast (mo) in north - west russia (census-2010 official results). the study population was all newborns in the township in the period 1973 - 2008, a total of 28,650 live and stillborn. the maternal care and maternal benefits in russia, as well as the study population, have been described earlier in some detail. the study was register - based using data from the kola birth register (kbr) and murmansk county birth register. information about all births in monchegorsk from march 1973 through 2005 was compiled in a population - based birth register, a total of 26,841 births. the register was set up to study the association between occupational exposures and reproductive health and pregnancy outcome. data about all live births and stillbirths from 28 weeks of pregnancy were collected from general medical journals, the hospital gynaecology records and delivery records. we included all registered newborns in the study, except those for whom the source chart containing diagnoses at birth was missing or the interpretation of the recorded diagnosis was uncertain. hence, 26,711 newborns from the years 1973 - 2005 were included in the study. the murmansk county birth register (mcbr) includes all births in mo since january 1, 2006. the registered data have been systematically collected in all 15 obstetrics departments in mo from five sources : the mother s medical history, obstetric journal and delivery record, the newborn s birth record, and through an interview with the mother conducted by a physician or midwife. in the period 2006 - 2008, of these, nine records were missing data about birth defects and excluded from the study. the population coverage of the register was estimated to 98.9% in 2006. in both registries, the diagnoses of congenital malformations were registered based on the international classification of disease (icd) coding system. according to the tenth version (icd-10), the diagnoses were made before the newborns left the birth clinic. from the late 1990s the use of echocardiography supplemented routine examinations and prenatal indications of cvm needed confirmation by examination after birth. in case of fetal deaths, information about all births in monchegorsk from march 1973 through 2005 was compiled in a population - based birth register, a total of 26,841 births. the register was set up to study the association between occupational exposures and reproductive health and pregnancy outcome. data about all live births and stillbirths from 28 weeks of pregnancy were collected from general medical journals, the hospital gynaecology records and delivery records. we included all registered newborns in the study, except those for whom the source chart containing diagnoses at birth was missing or the interpretation of the recorded diagnosis was uncertain. hence, 26,711 newborns from the years 1973 - 2005 were included in the study. the murmansk county birth register (mcbr) includes all births in mo since january 1, 2006. the registered data have been systematically collected in all 15 obstetrics departments in mo from five sources : the mother s medical history, obstetric journal and delivery record, the newborn s birth record, and through an interview with the mother conducted by a physician or midwife. in the period 2006 - 2008, of these, nine records were missing data about birth defects and excluded from the study. the population coverage of the register was estimated to 98.9% in 2006. in both registries, the diagnoses of congenital malformations were registered based on the international classification of disease (icd) coding system. according to the tenth version (icd-10), all cvm were coded in the range q 20-q 28. the diagnoses were made before the newborns left the birth clinic. from the late 1990s the use of echocardiography supplemented routine examinations and prenatal indications of cvm needed confirmation by examination after birth. in case of fetal deaths, the diagnosis was based on autopsy results. in total to analyse the structure of cvm, the prevalence and proportional distribution of the different forms were estimated based on the registered two- and three - digit level icd-10 code. newborns with more than one cvm - diagnosis were included in the numerator for each diagnosis. newborns with multiple malformations were included in the numerator, if cvm was present among the diagnoses. in addition, the stillbirth, early neonatal and perinatal mortality rates among newborns with cvm were estimated. the time trends of the prevalence and mortality were estimated using six 6-year time intervals from 1973 to 2008, and tested using chi - square for trend (medcalc 12.0 software). the prevalence of selected, possible risk factors was estimated for newborns with and without cvm, respectively, and statistically compared using the chi - square or t - test. the comparison between the two groups was carried out for the following potential risk factors : mean maternal age, parity, torch infections (i.e., toxoplasmosis, rubella, cytomegalovirus, herpes simplex, syphilis) during pregnancy, alcohol abuse during pregnancy, tobacco smoking during pregnancy, endocrine disease during pregnancy, paternal or maternal employment in the production departments of the nickel factory at the onset of the pregnancy, maternal occupation with exposure to organic solvents, and maternal body mass index (bmi) at the first antenatal visit. the adjusted risk of a cvm associated with the studied risk factors was analysed by multiple logistic regression, with cvm as a binary outcome (ibm spss 17.0 software package). the significance level in all analyses the data in the birth registers have been approved for research purposes by the murmansk regional health administration and the committee for research ethics at the university in troms. of the 28,511 newborns, 436 were from multiple - birth deliveries, 274 were stillborn (1.0%), and 555 (1.9%) died during the first 7 days after birth. the total number of newborns with one or more anomalies was 1029 (36.1 per 1000 newborns), of these 86 had one or more cvm [3.0 (95% ci : 2.1, 3.9) per 1000 newborns ]. one newborn had three cvm, three newborns had two and the rest had one cvm ; one in this last group was born as a twin. the mean gestational age for newborns with cvm was significantly lower in comparison with the reference group. the women who delivered a baby with a cvm were ten times more likely to have had a previous stillbirth than the mothers of the newborns without a diagnosed cvm. additional characteristics of mothers, pregnancies and newborns in the study groups are presented in table 1. the prevalence of cvm among stillborn was 51/1000 (95% ci : 25/1000, 78/1000), compared to 2.5/1000 (95% ci : 1.4/1000, 3.1/1000) among live born. the prevalence of cvm among term and preterm stillbirths did not differ : 54.5/1000 (95%ci : 11.4/1000, 97.7/1000) and 49.4/1000 (95%ci : 15.7/1000, 83.1/1000). the prevalence of newborns with cvm, and among live- and stillborn, in the different time periods is presented in table 2. the 72 live newborns with cvm, 25 were delivered preterm and 24 died during the first seven days (333 per 1000 live births). all newborns with chamber defects and 86% of newborns with anomalies in arteries and veins died perinatally. the perinatal mortality rate with isolated cvm or combined with other congenital malformations was 442 per 1000 newborns with cvm. table 3 shows the structure of cvm among all newborns and those who died perinatally. septal defects constituted 27% of all cvm and were the most prevalent of the verified diagnoses, while among stillborn and perinatal deaths, chamber defects were the most prevalent (24.1%). in total, 46.1% of the cvm diagnoses were unspecified, but this percentage was smaller among those who died perinatally. of the two types of cvm that have been obligatory to register in the federal system in the rf, there was one diagnosed case (q 20.3). the adjusted odds ratio (or) for smoking during pregnancy was 4.09 (95% ci : 1.75, 9.53). none of the other risk factors we studied were associated with the risk of cvm (table 4). a relatively high average stillbirth rate (10/1000 births) with a slight decline in the last decade was observed in monchegorsk. in most european countries, it was less than 6 per 1000 newborns in the same period. in a similar study period in hungary (1971 - 2010), the prevalence, observed in our study, was also higher than the officially reported average rate from russia as a whole, which declined from 6.8 to 4.7 during 2000 - 2010. we found no difference in prevalence of cvm between preterm and term stillbirths. according to the data of the russian state statistics service, the most common cause of stillbirth has been intrauterine hypoxia and birth asphyxia, which suggests that the main explanation of the higher stillbirth rate in the rf was insufficient health care service during delivery. at the same time, live births before 28 weeks in the rf were until 2011 defined as stillborn unless they survived the first seven days, which inflates the stillbirth rate compared to most other european countries. interestingly, the mothers who delivered a child with cvm were ten times more likely to have experienced a previous stillbirth. another study reported the same picture for ebstein s anomaly, and suggested that genetic risk factors lead to defects that are non - compatible with life. this hypothesis is supported by the data in our study. when we excluded stillbirths from the analysis, the relative likelihood of previous stillbirth among the mothers who delivered a child with cvm fell from factor ten to factor three. the overall prevalence was lower than that reported in eurocat and from studies in two populations in other parts of the rf. compared to the eurocat averages, the prevalence was lower for all 16 forms of cvm that the eurocat monitors. the most prevalent group of cvm was septal defects, but almost one - half of all registered cases were recorded with an unspecified cvm diagnosis. there were no registered cases of the severe defects : hypoplastic left heart and coarctation of aorta. also the prevalence of transposition of great vessels (the other diagnosis that is obligatory to report in the russian system of surveillance) was lower than that reported from neighbouring regions of mo and from moscow. in the rf, a prenatal diagnosis of severe cvm in regional districts may prompt a transfer of the delivery to regional centres or to moscow for early surgical correction. according to the data obtained from the paediatric polyclinic in monchegorsk, two deliveries of newborns with hypoplastic left heart were transferred to moscow in 2002 - 03. records of births which took place elsewhere were not filed at the local hospital and were not registered in either the kbr or the mcbr. the absence of obligatory ultrasound screening for fetal anomalies until 2000 could be another possible explanation low prevalence rate. in any birth surveillance system, both inside and outside the rf the estimated rates are a function of the degree of prenatal screening and of early neonatal diagnostic measures, while the true rates also include children who have cvm that reveals itself later in life. a study in bosnia - herzegovina found that the average age for cvm diagnosis was between the first and the second year of life, and that most of the late diagnoses were minor cvm. thus, the prevalence of small septal and valves defects without haemodynamic problems in our study was likely an underestimation, but the registered number of chamber defects can be assumed to be close to the true frequency. in provincial clinics in the rf, the lack of diagnostic tools, such as echocardiography, at the neonatal stage of care also suggests that some forms of cvm were under - diagnosed perinatally in most of the studied period. to achieve a better assessment of the prevalence among newborns in monchegorsk we did a chart review at the local paediatric polyclinic. the findings revealed 16 children born in 2006 - 2008 who had a diagnosis of cvm at the age of one year, while our perinatal figures based on the mcbr included four of these (25%). thus, the true perinatal prevalence was likely at least four times higher than the perinatal diagnostic procedures, recording, reporting and registration in the mcbr revealed. although birth - registers data provide gross underestimations of the true prevalence of cvm and are not very comparable between systems, the data may reveal interesting trends over time within a system, especially in terms of mortality. we observed no increase in prevalence over time, despite the technological improvements for perinatal diagnostics that have taken place in the rf during the last twenty years. this finding may suggest that the true prevalence has decreased, or that the improved detection also led to more pregnancies being terminated in the second trimester. our study revealed that the perinatal mortality among newborns with cvm has decreased in monchegorsk (from 23 per 1000 newborns in the 1970s to 9.3 per 1000 in 2003 - 06), and that the reduction mainly occurred in the early neonatal period. this finding suggests that the capability of the neonatal care has improved, or that a larger proportion of severe cases are transferred. the absence of perinatal mortality observed among newborns with cvm in monchegorsk in the last time period may be explained by the small number of newborns in that period (only half as many per year as in the 1970 - 80s) and transfer of women with a severe pathology of the fetus to another clinic (one such newborn with cvm died in moscow in 2002). based on data from the monchegorsk paediatric polyclinic, five cases of cvm were eliminated by induced abortion after 20 - 25 weeks of gestation in the period 2000 - 2006 (there were six newborns with cvm in this time period). data concerning miscarriages and abortions before 28 weeks are not logged in the birth registers, and therefore pregnancies are not included in our prevalence estimates. there was a tendency towards an elevated risk of cvm associated with all the factors studied except alcohol abuse, but only an association with smoking during pregnancy was statistically significant [or : 4.09 (95%ci:1.75, 9.53) ]. one of the studies investigated the association between smoking and 22 different categories of congenital malformations, and found a causal association only with cvm. birth registers have limitations when it comes to assessment of outcomes that are not readily diagnosed in the perinatal period. our findings suggest that most cases of cvm were not revealed in the perinatal period, and that some were transferred prenatally, and thereby not registered in the birth register on which our study population was based, which constitutes a weakness. in addition, the small numbers due to the rarity of cvm and the size of the study population lowered the precision of the estimates. nevertheless, the limitations we have found concerning monitoring and studies of cvm are important findings in themselves. the study also revealed new knowledge about the distribution of different cvm diagnoses, the degree of diagnostic specification, and the distribution and level of perinatal mortality associated with cvm in a population in the rf. our study was the first in the rf that estimated the perinatal prevalence of cvm through the use of data from population - based birth registers. the diagnosed perinatal prevalence in monchegorsk was relatively low and we did not observe a change over time (1973 - 2008). mothers of newborns with cvm were ten times more likely to have a history of previous stillbirth. one out of three infants born alive with cvm died during the first week of life, but the perinatal survival increased over time. all newborns with chamber defects and 86% of newborns with anomalies in arteries and veins died perinatally. the adjusted risk of giving birth to an infant with cvm was higher among smoking mothers than among non - smoking. | backgroundcardiovascular malformations (cvm) are one of the most prevalent groups of birth defects. knowledge about the prevalence, distribution and survival in russia has been limited. the aim of our study was to assess the perinatal prevalence, structure and risk factors for cvm among newborns in monchegorsk (murmansk oblast, russia) and the mortality among the affected newborns in the period 1973-2008.design and methodsa register - based study on data from the kola and murmansk county birth registers. the study included 28,511 births.resultsthe registered perinatal prevalence was 3.0 per 1000 new - borns, with septal defects as the most prevalent. cvm was twenty times more prevalent among stillborn than live born, and one - third of the live born with a cvm died during the first week of life. the perinatal mortality rate with cvm was 442 per 1000 newborns. this indicator decreased over time. the mothers of newborns with a cvm were ten times more likely to have stillbirth in their anamnesis. the adjusted odds ratio between maternal smoking during pregnancy and cvm was 4.09 [95% confidence interval : 1.75 - 9.53].conclusionsthe diagnosed perinatal prevalence was relatively low. a previous stillbirth by the mother was highly associated with being born with a cvm. an adjusted elevated risk was also observed among smoking mothers. perinatal survival increased over time, but varied to a large extent between the different types of cvm.significance for public healthcardiovascular malformation is one of the most common groups of birth defects. it is considered an important public health issue, as these malformations are the main cause of infant deaths in developed countries. precise estimates about the prevalence and perinatal survival are needed to organise and plan health care for such newborns. our study is the first report from the russian federation based on data from population - based birth registers. |
it is the most common dermatological disease, affecting approximately 80% of teenagers between 12 and 18 years of age. it is predominant in female adolescents, where it first appears around 14 years of age, and is less severe than in males who start showing symptoms at approximately 16 years of age.1 acne is the most common diagnosis made by dermatologists but is also commonly made by physicians other than dermatologists. diagnosis is confirmed by clinical observation, and is based on the patient s age at the time that the lesions first appear, and the type of polymorphism, lesions, and their location.2,3 the pathogenesis and existing treatment strategies for acne are complex. moreover, acne interferes with quality of life and requires both therapeutic and psychological support.4,5 studies suggest that the emotional impact of acne is comparable to that experienced by patients with systemic diseases, such as diabetes and epilepsy.6,7 the right treatment for the right patient is key to treating acne safely. numerous published guidelines, recommendations, and scientific reviews on therapeutic management of acne exist today. the global alliance to improve outcomes in acne published an algorithm for acne management in 2003, and updated it in 2009;1,8,9 in the same year, brazilian dermatologists ramos - e - silva and carneiro published an article with recommendations for treating different types of acne severity.1 additionally, although not yet included in the treatment algorithms, dermocosmetic products are often prescribed as part of acne treatment regimens.10 despite acne treatment guidelines being regularly communicated and updated, it is not known if dermatologists actually follow the recommendations in private practice. it is important to investigate the practices that dermatologists adopt to better understand the reality of therapeutic approaches. in this context, we performed a survey to assess the therapeutic choices made by brazilian dermatologists for grade i, ii, iii, and iv acne. for each level of acne severity, we recorded population characteristics, the product prescribed, and whether or not this treatment was associated with dermocosmetic prescription. this survey was conducted between january 2014 and february 2014, with 596 dermatologists in private practice in 12 states of brazil, including so paulo, minas gerais, rio de janeiro, paran, rio grande do sul, esprito santo, santa catarina, distrito federal, gois, rio grande do norte, paraba, and amap participating. doctors received a questionnaire with six questions concerning the characteristics of the population of acne patients seen in consultation, the most frequent acne grades received (i, ii, iii, and iv), and doctors therapeutic choices for each grade. acne classification was based on the brazilian society of dermatology description : grade i, comedones, without inflammatory lesions ; grade ii, comedones, papules, and pustules with variable intensity and few to numerous inflammatory lesions with some erythema ; grade iii, comedones, papules, and pustules with intense inflammatory reactions leading to nodule formation, which may contain pus (cysts) ; grade iv, comedones, papules, pustules, and larger cystic fistulas forming lesions. for each severity level, the doctors were asked if they would choose : 1) topical drug treatment (ie, benzoyl peroxide, isotretinoin / tretinoin, erythromycin / clindamycin, azelaic acid, combination benzoyl peroxide plus adapalene, combination benzoyl peroxide plus clindamycin, combination erythromycin plus isotretinoin, combination clindamycin plus tretinoin, and others : salicylic acid, beta - lipohydroxy acid, nicotinamide, sulfur, zinc gluconate, or glycolic acid) ; 2) systemic drug treatment (ie, isotretinoin or cyclins, [lymecycline, tetracycline, minocycline, doxycycline, or azithromycin / erythromycin ], oral contraceptives [ethinyl estradiol plus second and third generation progestogens ], or antiandrogens [cyproterone acetate, chlormadinone acetate, dienogest, trimegestone, spironolactone, drospirenone, or flutamide ]) ; or 3) dermocosmetic treatment (associated with drug treatment ; either at the same time or in maintenance or in monotherapy). data analysis was performed by insider inteligncia de mercado for all questionnaires completed by dermatologists. variables were expressed by percentage (%) ; quantitative data were expressed by mean standard deviation (sd ; minimum maximum) and median. as this study corresponded only to doctors interviews, ethical approval was deemed not to apply. regarding the acne patients received by the brazilian dermatologists questioned, 52% of dermatologists treated only adolescents (under 18 years of age), 41% of dermatologists treated both adolescents and adults, and 7% of dermatologists treated only adults. the most common acne grade was grade ii, followed by grades i, iii, and iv, respectively (figure 1). the doctors could choose more than one type of treatment for each patient, and treatment choices varied according to acne severity (table 1). considering grade i acne, 94% of brazilian dermatologists chose topical treatment with drugs and for 76% of dermatologists at night - time), and only 28% of dermatologists chose systemic treatments (table 1). for grade ii acne, the standard for treatment option for 98% of dermatologists was topical drugs mainly once daily, at night - time (table 1). the choice for systemic drugs increased significantly compared to grade i acne ; 64% of the dermatologists interviewed also chose an oral treatment for grade ii acne patients (table 1). iii and iv acne, systemic treatment was the first option for 96% and 98% of dermatologists, respectively (table 1). interestingly, for grade iii acne patients, 83% of dermatologists also considered a topical drug (in monotherapy or in association) and for grade iv only, 56% of dermatologists considered a topical drug (table 1). the most frequently prescribed drugs for grades iii and iv are presented in figures 4 and 5, respectively. the prescription of dermocosmetics for acne patients is considered for all acne grades, but its prescription frequency decreases as acne severity increases (table 2). considering grade i acne, among the 75% of dermatologists prescribing dermocosmetics, 57% prescribe them only as adjunctive therapy and 20% only as maintenance therapy. as described in table 1, 75% of dermatologists answered that they prescribe a dermocosmetic for grade 1 acne. after that, they could choose more than one option ; in adjunctive therapy, in maintenance therapy, and in monotherapy. for grades ii, iii, and iv acne, the use of dermocosmetics was considered by 67%, 56%, and 44% of the surveyed dermatologists, respectively (table 2). according to treatment option chosen (table 1), the percentage of dermatologists prescribing dermocosmetics as adjunctive therapy with topical medication decreased from grade i to grade iv and was higher in adjunctive therapy with systemic medication than for topical ones (table 2). the choice of including a dermocosmetic in therapy has also varied largely from region to region : in so paulo and in the south of brazil, dermocosmetic use was higher than in rio de janeiro / esprito santo, minas gerais, and in the northern states of the country (table 3). although the lack of validated measures was a limitation, the present survey provides a better understanding of how different grades of acne are being treated and how treatment is aligned with the standards established by international expert recommendations, such as from the global alliance acne treatment algorithm and the gilea (grupo ibero - latinoamericano de estudio del acn) algorithm (figure 6).8,9,11,12 both the global alliance acne treatment and gilea classify acne severity into mild, moderate, and severe categories, which differs from the present study classification in acne grades i, ii, iii, and iv (the brazilian society of dermatology [sbd ] classification). therefore, to compare these study results with other acne treatment algorithms, we considered the equivalence measures described in table 4. in a previous study with 5,809 acne patients, it was observed that european dermatologists do not usually prescribe medical treatments to treat grade i acne (in 44% of cases, they prescribed only a dermocosmetic product to patients with very mild acne) ; one drug treatment, usually a topical one, to treat grade ii acne (in 44% of cases, one treatment was prescribed to patients with mild acne) ; and two treatments (a combination of topical and systemic therapy) to those with grades iii or iv acne.13 topical medical treatment is recommended in both global alliance acne treatment algorithm and in the gilea algorithm as the first choice of prescription for grade 1 patients (topical retinoid or benzoyl peroxide [bpo ], or bpo plus retinoid), and this recommendation is closer to actual brazilian dermatologists practices as observed in the present survey (figure 2). although systemic drugs are not considered for patients with grade i acne in either the global alliance acne treatment or gilea algorithms, 28% of brazilian dermatologists also prescribed a systemic therapy (and 16% of dermatologists use isotretinoin ; figure 2) for that population. this percentage increased significantly for grade ii acne, when 64% of dermatologists chose a systemic treatment for their patients (and 22% of dermatologists chose isotretinoin ; figure 3). for grades iii and iv acne, systemic treatment became the first option for 96% and 98% of brazilian dermatologists, respectively, which is in line with the literature. however, for them, the drug of choice for both grades of acne was isotretinoin (for 77% of dermatologists for grade iii and for 95% of dermatologists for grade iv acne ; figures 4 and 5). oral isotretinoin is a teratogenic drug, so its use is contraindicated in pregnant women and can be a first choice for grade iv or nodular / conglobate lesions in adolescents. furthermore, it has been suggested that some patients may experience depression while using oral isotretinoin, but studies have not shown a decisive relationship.7,14,15 the development of bacterial resistance and ensuing recommendations from health authorities has limited the use of local and systematic antibiotics, favoring their use within the framework of combined therapy regimens.16,17 nevertheless, in 12% of cases, only one local or systematic antibiotic has been noted to be prescribed by european dermatologists, which contradicts the published recommendations and indicates that dermatologists should not use antibiotics alone.13 similarly, in our current survey, local and systematic antibiotics were heavily prescribed for all acne grades, either alone or in association, by brazilian dermatologists (topical antibiotic was prescribed by 6% of dermatologists for grade i acne, by 17% for grade ii acne, and by 11% and 7% of dermatologists for grades iii and iv acne, respectively ; oral antibiotic was prescribed by 11% of dermatologists for grade i acne, by 29% for grade ii acne, and by 26% and 20% of dermatologists for grades iii and iv acne, respectively). however, interestingly, brazilian dermatologists favored topical and oral antibiotic use within the framework of combined therapy regimens, because for all grades of acne they frequently chose fixed combinations such as bpo plus adapalene, bpo plus clindamycin, or clindamycin plus tretinoin more than european dermatologists, perhaps because this recommendation is more clearly indicated in the gilea algorithm (figure 6). the current survey data indicate an indiscriminate use of drugs by brazilian dermatologists, particularly of isotretinoin, for mild to moderate acne, and highlights overuse of local and systematic antibiotics, practices that contradict the published recommendations and indicate that dermatologists should not use oral medication for mild to moderate acne or antibiotics alone in order to avoid bacterial resistance. these data also indicate that it is necessary to favor topical treatments with one application a day to ensure patients use the treatment regularly.8,9,11,12 furthermore, only 7% of brazilian dermatologists recommended dermocosmetics as monotherapy for grade i patients in the current survey, indicating the low consideration of active dermocosmetic treatment (ie, keratolytic products) for acne by brazilian dermatologists, although dermocosmetics effectiveness has been demonstrated,13 and they are recommended in both algorithms.8,9,11,12 although the use of skin care products (cleanser, skin barrier repair, sunscreen, etc) is recommended in the gilea algorithm in combination with drug therapy to improve patient tolerance of treatments and patient comfort while maintaining and/or strengthening the overall efficacy of the regimen, we noted in the current survey that a decreasing percentage of brazilian dermatologists prescribed skin care products as the severity of acne increased (table 2). nevertheless, the prescription of skin care products in adjunctive therapy increased with the acne grade, particularly with the prescription of systemic medications well known for their secondary side effects (eg, skin dryness, irritation, photo - sensitization, etc). this result indicates that dermocosmetic products play an important role in acne treatment and should be considered by more dermatologists in future acne treatment protocols. differences in dermocosmetic prescription practices between regions in brazil were noted in the current survey, with a greater number of dermatologists prescribing dermocosmetics in the southern states compared to the northern ones (table 3) ; this difference may be explained by climate differences (lower temperatures and humidity similar to european countries may explain the higher use of dermocosmetics in the southern states of brazil). in conclusion, the present survey shows that brazilian dermatologists frequently prescribe several treatments for acne, a practice that diverges on many points from published recommendations, especially the global alliance acne treatment and gilea algorithms. furthermore, in spite of published recommendations contraindicating their use, isotretinoin plus local antibiotics, or systematic antibiotics alone, are often prescribed, even for mild / moderate acne. finally, dermocosmetic products are rarely prescribed in monotherapy by brazilian dermatologists in private practice, and should be considered in acne treatment protocols ; unfortunately, these protocols have not yet been developed. in future, we propose the development of a brazilian acne treatment algorithm to meet the specific needs of dermatologists in brazil. | backgroundacne is a chronic disease of the pilosebaceous unit that mainly affects adolescents. it is the most common dermatological problem, affecting approximately 80% of teenagers between 12 and 18 years of age. diagnosis is clinical and is based on the patient s age at the time the lesions first appear, and on its polymorphism, type of lesions, and their anatomical location. the right treatment for the right patient is key to treating acne safely. the aim of this investigational survey was to evaluate how brazilian dermatologists in private practice currently manage acne.materials and methodsdermatologists practicing in 12 states of brazil were asked how they manage patients with grades i, ii, iii, and iv acne. each dermatologist completed a written questionnaire about patient characteristics, acne severity, and the therapy they usually prescribe for each situation.resultsin total, 596 dermatologists were interviewed. adolescents presented as the most common acneic population received by dermatologists, and the most common acne grade was grade ii. the doctors could choose more than one type of treatment for each patient, and treatment choices varied according to acne severity. a great majority of dermatologists considered treatment with drugs as the first alternative for all acne grades, choosing either topical or oral presentation depending on the pathology severity. dermocosmetics were chosen mostly as adjunctive therapy, and their inclusion in the treatment regimen decreased as acne grades increased.conclusionthis survey illustrates that brazilian dermatologists employ complex treatment regimens to manage acne, choosing systemic drugs, particularly isotretinoin, even in some cases of grade i acne, and heavily prescribe antibiotics. because complex regimens are harder for patients to comply with, this result notably raises the question of adherence, which is a key factor in successful treatment. |
ovarian hyperstimulation syndrome (ohss) is an iatrogenic complication of assisted reproductive technology (art) with development of multiple follicles. ohss is characterized by cystic enlargement of the ovaries and an acute fluid shift from the intravascular compartment to the third space, which may result in ascites, pleural and/or pericardial infusion, and even generalized edema. ohss patients suffer from lower abdominal discomfort, nausea, and vomiting. in severe cases of ohss, thromboembolic events, acute respiratory distress syndrome (ards), and renal failure clinical practitioners are unsure whether ohss and subsequent treatments, such as incessant pleural or abdominal punctures, volume expansion, and diuretics, would have an adverse effect on pregnancy outcomes of ohss patients. previous research on this topic had a lack of an appropriate contemporaneous control group, and there were potential confounders in the research, thus making interpretation of such data unclear. in the current study, based on consistent age and count of mature ii (m - ii) oocytes, we compared pregnancy outcomes of patients with and without ohss, and examined the possible effects of ohss on pregnancy outcomes. the in vitro fertilization (ivf) database was set up and maintained by research faculty members in our department. ohss patients except for mild ohss patients diagnosed and treated in our hospital from 2002 to 2012 were included, and basic information was recorded in the database., we identified 190 ivf patients with ohss. in a total population of 5487 ivf fresh cycles, 197 contemporaneous non - ohss cycles matched for age and count of m - ii oocytes were selected as the unexposed group. the amount discrepancy of age and count of m - ii oocytes between the two or three matching patients was no more than 2. the corresponding non - ohss cycle occurred in the same or near month with the ohss cycle. while in our study, we excluded the mild ohss patients since they were treated outpatient. the severity of ohss was defined according to the criteria proposed by golan. and navot. moderate ohss was characterized by abdominal distension and discomfort, nausea, vomiting or diarrhea, enlarged ovarian size (512 cm), and ultrasonic evidence of ascites. severe ohss was characterized by variable ovarian enlargement ; massive ascites hydrothorax ; hematocrit > 45% ; white blood cell count > 15,000/ml ; oliguria ; creatinine 1.01.5 mg / dl ; liver dysfunction ; and anasarca. critical ohss was characterized by variable ovarian enlargement ; tense ascites hydrothorax ; hematocrit > 55% ; white blood cell count > 25,000/ml ; oliguria ; creatinine 1.6 mg / dl ; creatinine clearance 45% ; white blood cell count > 15,000/ml ; oliguria ; creatinine 1.01.5 mg / dl ; liver dysfunction ; and anasarca. critical ohss was characterized by variable ovarian enlargement ; tense ascites hydrothorax ; hematocrit > 55% ; white blood cell count > 25,000/ml ; oliguria ; creatinine 1.6 mg / dl ; creatinine clearance < 50 ml / min ; renal failure ; thromboembolic phenomena ; and ards. hematocrit, white blood cell count, and liver and kidney function indices were dynamically monitored. changes in ovarian size and abdominal or pleural fluid were monitored by ultrasound when necessary. based on the status of disease, liver - protecting, anti - infection, and diuretic treatments, as well as drainage of abdominal and pleural fluid, were administered. pregnancy outcomes included clinical pregnancy, miscarriage, miscarriage of one twin, fetal intrauterine death, gestational age at birth, delivery mode, neonatal birth weight, and neonatal deformity. early miscarriage occurred before 12 gestational weeks, and late - term miscarriage was between 13 and 28 gestational weeks. premature delivery was defined as birth before 37 and after 28 completed weeks of pregnancy. low birth weight (lbw) was defined as birth weight below 2500 g, and small - for - gestational age (sga) was defined as a birth weight lower than the tenth percentile of the same gestational age of neonatal birth weight. data were expressed as mean standard deviation (sd), median (interquartile range), or n (%). continuous variables were compared using student 's t - test or mann whitney u - test. odds ratios (ors) and 95% confidence intervals (cis) were calculated after adjustment for controlled ovarian hyperstimulation (coh) protocol, gonadotropin (gn) dosage, human chorionic gonadotropin (hcg) dose protocol on hcg day, luteal supporting protocol, polycystic ovary syndrome (pcos), and anovulation. we identified 39 moderate (20.5%), 141 severe (74.2%), and 10 critical (5.3%) ohss patients. the incidence of ohss among 5487 fresh ivf cycles was 3.46%, and the rates of serious adverse events and thromboembolism in ohss patients were 2.63% and 1.58%, respectively. the median duration of hospitalization was 11 days (273 days) and the mean number of abdominal and plural punctures was 3 (range : 018). comparison of ivf data between ivf patients with or without ohss is shown in table 1. the mean dosage of gn used for ovulation induction for ohss patients was lower than that of non - ohss (p = 0.007). the clinical characteristics, including age, body mass index, diagnosis of infertility, and duration of infertility, were not significantly different between the two groups [table 1 ]. additionally, no significant difference was found in basal follicle - stimulating hormone or serum estradiol (e2) levels on hcg day between the groups. comparison of ivf data between ohss and non - ohss groups data were showed as mean sd, median (iqr), or n (%). ohss : ovarian hyperstimulation syndrome ; bmi : body mass index ; ivf : in vitro fertilization ; pcos : polycystic ovary syndrome ; fsh : follicle - stimulating hormone ; coh : controlled ovarian hyperstimulation ; gn : gonadotropin ; hcg : human chorionic gonadotropin ; rhcg : recombinant human chorionic gonadotropin ; m - ii : mature - ii ; iqr : interquartile range ; e2 : estradiol ; sd : standard deviation. seven patients with ohss canceled embryo transfer (et) because of early - onset severe ohss, and 13 patients without ohss canceled et for a high risk of ohss. among the 183 ohss patients who did undergo et, 168 patients achieved clinical pregnancy with a clinical pregnancy rate of 91.8%, which was significantly higher than that in the control group (43.5%, p < 0.001). the rates of multiple pregnancy and miscarriage were not significantly different between the two groups, and all the triplets and quadruplets were surgically reduced to twins during 912 weeks of gestational age. comparison of pregnancy outcomes between ohss and non - ohss group data were showed as mean sd or n (%). lbw : low birth weight ; sga : small - for - gestational age ; ohss : ovarian hyperstimulation syndrome ; sd : standard deviation. the delivery outcomes of 138 ohss live births (84 singletons, 54 twins) were compared with those of the control group, which were 63 live births (41 singletons, 22 twins). we found no significant differences in the rates of live birth (82.1% vs. 78.8%), preterm delivery (20.9% vs. 17.5%), preterm birth before 34 weeks gestation (8.6% vs. 7.9%), singleton lbw (9.5% vs. 4.9%), and singleton sga (7.1% vs. 7.3%) between the two groups. pregnancy and maternal outcome of ohss patients with major complications ards : acute respiratory distress syndrome ; ohss : ovarian hyperstimulation syndrome. thereafter, we compared ivf data of moderate ohss, severe / critical ohss with that of non - ohss patients, respectively [table 4 ]. the proportion of different coh protocol was statistically different between severe / critical ohss and non - ohss patients (p = 0.039). the proportion of short protocol was comparatively higher in non - ohss than severe / critical ohss patients. after controlling for coh protocol, gn dosage, hcg dose on hcg day, luteal supporting protocol, pcos, and anovulation, ohss was associated with increased probability of clinical pregnancy. the adjusted ors of moderate ohss and severe / critical ohss for clinical pregnancy were 4.65 (95% ci, 1.8611.61) and 5.83 (95% ci, 3.459.86), respectively. comparison of ivf data among moderate, severe / critical ohss and non - ohss patients data were showed as mean sd, median (iqr), or n (%). p1 represents moderate ohss compared with non - ohss patients and p2 represents severe / critical ohss compared with non - ohss patients. ohss : ovarian hyperstimulation syndrome ; bmi : body mass index ; ivf : in vitro fertilization ; pcos : polycystic ovary syndrome ; fsh : follicle - stimulating hormone ; coh : controlled ovarian hyperstimulation ; gn : gonadotropin ; hcg : human chorionic gonadotropin ; rhcg : recombinant human chorionic gonadotropin ; m - ii : mature - ii ; iqr : interquartile range ; e2 : estradiol ; sd : standard deviation. clinical practitioners have always been committed to improving ovarian stimulation protocols to keep the incidence of ohss no more than 5%. however, critical ohss occasionally occurs, including acute renal failure, thrombosis, stroke, pulmonary edema, myocardial infarction, ards, and even maternal death. considering ohss - associated complications, clinical practitioners and patients need to determine whether to terminate pregnancy because this would substantially alleviate the condition of ohss patients. while most patients choose to continue pregnancy because this disease is self - limited, they are also wondering whether ohss would bring adverse impact to pregnancy. a previous study has demonstrated that ohss is more likely to occur at a younger age and in treatment cycles with the highest ovarian response to stimulation. additionally, infertility is an independent factor that appears to be involved with a poor obstetric outcome. the oocyte yield, independent of age, shows a linear relationship with live births with up to 15 oocytes in ivf cycles. therefore, to exclude potential bias, we matched age and count of m - ii oocytes. body mass index, causes of infertility, length of infertility, and basal ovarian function in the two groups were assessed. we observed that the dosage of gn for ovarian stimulation and hcg for luteal support was significantly lower in ohss patients than in non - ohss patients, which suggested that ohss patients were more sensitive to stimulatory drugs. multiple studies have reported that the clinical pregnancy rate in ohss patients is significantly higher than that in general ivf patients or non - ohss patients. pregnancy triggers and aggravates ohss, and this is called late - term onset ohss. late ohss is triggered by endogenous hcg release in the event of pregnancy, generally occurring after 910 days following hcg injection. early ohss is caused by administration of exogenous hcg, which appears to be associated with an excessive ovarian response to gn stimulation, generally occurring before the 9 and 10 day after hcg injection. because we performed a retrospective study, it is difficult to define the onset model according to patients subjective recall. in terms of pregnancy outcome, the rates of miscarriage and perinatal complications including preterm birth, sga, pregnancy - induced hypertension and/or stillbirth were significantly higher in ohss group than non - ohss group, as reported in literature. we analyzed that it was probably the relatively higher rate of multiple pregnancy that induced massive perinatal complications [table 5 ]. similar miscarriage rates between groups were observed after excluding this confounder in our study and courbiere 's series. comparison of pregnancy outcome between ohss and non - ohss group, literature review : data not mentioned. : compared with non - ohss patients, the multiple pregnancy rate of ohss patients increased 58%86% ; the rate of perinatal complication including preterm birth, lbw and stillbirth, of ohss patients increased 2631%. : compared with non - ohss patients, the clinical pregnancy rate of ohss patients increased 98%168%. pih : pregnancy - induced hypertention ; lbw : low birth weight ; ohss : ovarian hyperstimulation syndrome. some authors have postulated that systemic vascular dysfunction and microthromboembolic events might affect trophoblastic invasion, leading to placental insufficiency. thromboembolic events occurred in four of the 40 ohss pregnant patients in courbiere 's study, characterized by increased thromboembolic events up to 10%, with a comparably higher rate of preterm than non - ohss pregnant patients. we may hypothesize that this seemingly higher rate of preterm may be due to thrombosis. not all of the thrombosis in ivf patients was correlated with ohss, and ivf pregnant patients complicated by ohss had an increased risk of arterial thrombosis. therefore, ohss and pregnancy could be viewed as precipitating factors for thrombosis in ivf. supraphysiological ovarian stimulation results in e2 levels greater than those in natural conception (nc) cycles and causes e2 levels in the early stage to be similar to those in the late stage of the first trimester of nc. previous studies showed that the high maternal e2 environment in the first trimester was correlated with increased risks of lbw and sga. additionally, the birth rates of singleton lbw and singleton sga of fresh et were significantly higher than those of frozen et and nc (6.3%, 4.4%, and 3.6%, and 6.9%, 5.0%, and 4.8%, respectively). large - scale studies in china on the epidemiology of sga and lbw are still lacking. some hospital - based studies and regional investigations have described that the rate of preterm birth ranges from 3.1% to 5.8%, lbw is 1.6%, and sga is 2.9%. in the aggregate series, the rate of singleton preterm birth was 8.8%, singleton lbw was 8.0%, and singleton sga was 7.2%. generally, rates of preterm birth and lbw or sga were higher in ivf cycles than in nc. in conclusion, ohss, which occurs in the luteal phase or early pregnancy of ivf patients and represents transient abnormal hemodynamics, was not found to exert any obviously adverse effect on the subsequent pregnancy. however, whether ohss would exert adverse effect on the offsprings of ivf mothers in the long - term, required further studies. | background : the effect of ovarian hyperstimulation syndrome (ohss) on pregnancy outcomes of in vitro fertilization (ivf) patients is still ambiguous. this study aimed to analyze pregnancy outcomes of ivf with or without ohss in chinese patients.methods:a retrospective cohort study was undertaken to compare pregnancy outcomes between 190 women with ohss and 197 women without ohss. we examined the rates of clinical pregnancy, multiple pregnancies, miscarriage, live birth, preterm delivery, preterm birth before 34 weeks gestation, cesarean delivery, low birth weight (lbw), and small - for - gestational age (sga) between the two groups. odds ratios (ors) and 95% confidence intervals (cis) of measure of clinical pregnancy were also analyzed.results:the clinical pregnancy rate of ohss patients was significantly higher than that of non - ohss patients (91.8% vs. 43.5%, p < 0.001). after controlling for drug protocol and causes of infertility, the adjusted ors of moderate ohss and severe / critical ohss for clinical pregnancy were 4.65 (95% ci, 1.8611.61) and 5.83 (95% ci, 3.459.86), respectively. there were no significant differences in rates of multiple pregnancy (4.0% vs. 3.7%) and miscarriage (16.1% vs. 17.5%) between the two groups. with regard to ongoing clinical pregnancy, we also found no significant differences in the rates of live birth (82.1% vs. 78.8%), preterm delivery (20.9% vs. 17.5%), preterm birth before 34 weeks gestation (8.6% vs. 7.9%), cesarean delivery (84.9% vs. 66.3%), lbw (30.2% vs. 23.5%), and sga (21.9% vs. 17.6%) between the two groups.conclusion:ohss, which occurs in the luteal phase or early pregnancy in ivf patients and represents abnormal transient hemodynamics, does not exert any obviously adverse effect on the subsequent pregnancy. |
in recent time we are observing in the context of oncological breast surgery, an increasing trend towards the more effective treatment with minimum invasiveness, with the intent to combine the local control of the disease with the respect of patient s quality of life. the introduction of conservative breast surgery, the debate on axillary nodes dissection in cases of sentinel node micrometastasis can be deemed to belong to this behavior. the widespread use of accelerated partial - breast irradiation (apbi) after breast conservative surgery as an alternative to whole breast irradiation (wbi) can be considered also part of this approach (1). the procedure consists in the irradiation on the breast limited to the tissues surrounding the resected tumor, following the evidence of the higher incidence of local recurrence rate near the site of tumor bed with respect to the rest of the breast (2, 3). a particular type of apbi is the intraoperative radiotherapy (iort) that can be provided by electrons or with 50 kv x - rays (intrabeam system) (4). iort allows the patient with particular personal and tumor characteristics to conclude the oncological treatment in one operating session with many consequent advantages ; in particular allows a better comfortability for the patient avoiding delays related to logistical difficulties and above all permitting patients to forget shortly to be sick. the selection criteria for the application of apbi were defined by the two working groups of the american society for radiation oncology (astro) and the groupe europen de curiethrapie - european society for therapeutic radiology and oncology (gec - estro) which delineated the risk categories and warned to apply the techniques in the context of clinical trials (5, 6, 7, 8). two randomized phase iii trials, eliot trial (9) and targit - a trial (10), have been developed regarding the use of iort with results at a medium follow - up of 5.8 and 2.4 years respectively. a heated debate is going on concerning the question of applying iort instead of postoperative wbi after breast conservative treatment following the strict selection criteria dictated by astro and gec - estro. in targit - a local recurrence rate at 5 years in the iort group was 3.3%, while in eliot trial 4.4%. although the findings of higher recurrence rate with respect to wbi, the pre - specified equivalence margins were respected. therefore indication for iort might be restricted to patients with low risk of local events and respecting very strict selection criteria (11). in the clinical experience of vinh - hung. (12) 52 women received iort after conservative surgery, but only the 65% as unique treatment. the rest of patients received further postoperative external radiotherapy, because inclusion criteria were not met for different unexpected findings. despite the favorable result of no incidence of local events at 1 year follow - up in all patients treated, the short follow - up does not allow to make definitive and optimistic conclusions, considering the long - term local recurrences rate reported in the two randomized trials. in vinh - hung study, patients following the preoperative selection criteria underwent iort ; however 35% of patients resulted to be not suitable for the treatment after receiving the definitive histopathological analysis, therefore required subsequent wbi. the frozen sections of resected tumor allow to evaluate during surgery if selection criteria regarding tumor characteristics are still respected such as sufficient resection margins and intraductal component and to plan the final appropriate radiotherapic treatment (13). mammographic and ultrasonographic exams prior to surgery are useful for non palpable lesions, however in accordance with our experience, an appropriate magnetic resonance imaging (mri) examination is required in some cases before surgery to make a control of whole breast excluding the presence of multifocality. it has been reported a 9.6% of cases in which a variation in patients selection for apbi was necessary after performing an mri, because of the identification of unexpected additional disease (14). the greater total radiation dose delivered due to the addition of iort boost to wbi is supposed to lead to an increased risk of radiation toxicity on breast tissues. radiation toxicity was investigated in vinh - hung study using the lent - soma scale, resulting a not significant difference (p=0.631) on the grades of toxicity in the 18 patients requiring wbi in addition to iort, considering 90 days time interval between the two treatments. however follow - up was almost 1 month in all patients, therefore there are no data regarding possible increasing in the fibrosis rate in the long time period. there are evidences that subcutaneous fibrosis tends to increase with a longer follow - up. the eortc 22881- 10882 trial showed a statistically significant higher rate of fibrosis after 10 years follow - up for the boost group rather than the no - boost group (15), however there are no evidences at 5 years (16). furthermore the rate of fibrosis and late toxicities, such as edema, telangiectasia, breast retraction, hyperpigmentation and pain, seem to be related to the time interval of adjuvant wbi delivery after iort boost, with data showing a high toxicity rate within 36 days (17) and possible no incidence of tissue toxicity after 56 or more weeks of delay (18). higher frequency of postoperative wound seroma was found after iort in vinh - hung study, in accordance with the evidences of targit - a, however it was not significantly associated with the rate of fibrosis, that was observed to be higher in the iort boost group together with toxicity grade 3/4. the minor rate of fibrosis in the iort group can be correlated to the evidences of less breast and arm symptoms (19). two cases of grade 4 skin toxicity requiring re - operation were found after iort in the study of vinh - hung ; no heart toxicity was found, though lung symptoms were diagnosed in six cases after iort and one after further wbi. however the short follow - up does not allow a clear evaluation of long - term effects of radiation. in targit - a, more cases of pulmonary fibrosis occurred after wbi (38 out of 83) than after iort (4 out of 95). regarding patients treated with iort respect to wbi in terms of skin side effects, no significant differences were seen in targit - a trial (20), while in eliot trial is reported a significative higher incidence in the wbi group. another evidence to emphasize is the higher rate of subcutaneous fibrosis seen in patients treated with iort boost using the intrabeam system rather than external electrons or intraoperative boost with electrons (21, 22, 23, 24, 25). concerning the use of iort, at present time it is not clear about what happens after application of a single high dose of 2021 gy. this dose might correspond to a fractionated dose of 65 gy, therefore a greater incidence of severe fibrosis should be expected ; on the contrary there are not definitive results supporting this hypotesis (26). the incidence of fibrosis might influence the final cosmetic result and the subsequent psychological comfort of the patient. a cosmetic result and patient s satisfaction analysis should be performed when considering the application of postoperative wbi or iort. techniques of reduction mammoplasty can be associated to conservative surgery and iort in cases of mammary hypertrophy or ptosis with the great advantage of obtaining a good cosmetic result in the same surgical session of oncologic treatment without the need to re - operate the patient afterwards. the application of oncoplastic techniques allows a greater surgical access with the advantage of performing a more comfortable wide excision of the tumor and applying a greater shielding disk when there is necessity to protect underlying vital structures, with deliver of radiotherapy on more extent of glandular tissue. subsequently glandular flaps can be harvested and mobilized to refill the defect and recreate the volume of the breast (13). after surgery the rearrangement of the mammary gland should be taken under control by expert radiologists in order to distinguish benign findings benign calcifications or fat necrosis from malignancies (27). the removal of a larger extent of glandular tissue and the gland remodeling in cases of application of oncoplastic techniques is associated with a more favorable aesthetic global judgement either with iort or wbi (28). therefore in the clinical cases of lower risk of recurrence and respecting the strict selection criteria, iort should be taken into account, considering also the advantage of a good quality of life and the less chronic skin toxicities especially after iort alone identified in iort patients (20). furthermore, if patients conditions result favorable, oncoplastic techniques should be offered after a direct and precise preoperative conversation with the objective to get the patient involved with the surgical options. the complexity in surgical techniques and oncologic treatment involves a multidisciplinary team and require a continuous dynamic communication between oncologic surgeon, plastic surgeon and radiotherapist to carry out a successful breast cancer treatment that satisfy the patient and ensure an adequate local control of the tumor. | after the results obtained in the two randomized clinical trial, the eliot trial and the targit - a trial, a heated debate is going on concerning the question of applying intraoperative radiotherapy (iort) instead of postoperative whole breast irradiation (wbi) after breast conservative treatment. currently, many centers are applying the iort following the strict selection criteria dictated by the working groups american society for radiation oncology (astro) and groupe europen de curiethrapie - european society for therapeutic radiology and oncology (gec - estro) and monitoring the oncological outcome together with radiation toxicity on breast tissue. the clinical experience of the geneva university hospital regarding the use of the intrabeam system is evaluated and compared with current evidences. |
the definition of ' elderly ' may vary according to chronological, biological, psychological, and social aspects, however, most developed countries have accepted the chronological age of 65 years as a definition of ' elderly ' or older person, as defined by the world health organization (who). advances in medical science have led to an increase in life expectancy, and, consequently, the proportion of elderly people among the total population. in 2013, approximately 12.2% of the korean population was aged over 65 years.11) with continued expansion of the aging population, and the development of less invasive and high - quality neuroimaging modalities, more elderly patients are expected to develop intracranial aneurysms. as supportive intensive critical care and neurointerventional treatment options become more advanced, more aggressive treatment of elderly aneurysm patients is being encouraged.1) aggressive treatment for many conditions is becoming more common, resulting in more positive outcomes.5)16)20) the aim of our study was to describe intracranial aneurysm treatments in all elderly patients treated in our institute and to observe their clinical course and immediate prognosis under aggressive therapy. the medical records of all patients treated at our hospital from september 2008 to december 2013 were reviewed for selection of patients aged 65 years or older who underwent microsurgical clipping or endovascular management for treatment of an intracranial aneurysm. to reduce variability, a single vascular neurosurgeon performed all microsurgical clippings and endovascular coilings in these patients. clinical information obtained included age, sex, hunt and hess grade (hhg), aneurysm location, fisher grade (fg), and treatment (microsurgical clipping, endovascular coiling). to determine which risk factors affected the functional outcome, all eligible patients were placed in the microsurgical clipping group or the endovascular coiling group. clinical outcome at the time of discharge was assessed for each patient using the glasgow outcome scale (gos) score : favorable outcome (good recovery, gos 5 ; moderate disability, gos 4) or poor outcome (severe disability, gos 2 - 3 ; death, gos 1). statistical analysis was performed using commercially available software (spss version 18, spss inc., univariate comparison of continuous variables with a normal distribution was performed using two sample t - tests. logistic regression was used to test bivariate associations between variables of interest and good versus poor outcome. a comparison of the clinical characteristics of patients treated by endovascular coiling or microsurgical clipping is shown in table 1. a total of 183 (18%) aneurysms were treated in 159 (18.6%) elderly patients, including 134 women (84.3%) and 25 men (15.7%) with a mean age of 71.6 years ; 108 (67.9%) patients experienced subarachnoid hemorrhage (sah) and 51 (32.1%) patients had unruptured intracranial aneurysms (uia) ; 101 (63.5%) patients were treated by coiling and 58 (36.5%) patients were treated by microsurgical clipping. fifty three patients presented with a high - grade sah (hhg iv or v) and 55 patients presented with a sah of hhg ii or iii. the average age of patients with aneurysms coiled and clipped was 72.3 (65 - 93) and 70.2 (65 - 84) years, respectively (p < 0.01). no significant differences based on gender, risk factors, hhg, and aneurysm size were observed between the coiling group and the clipping group. there were 164 (89.6%) anterior circulation aneurysms and 19 (10.4%) posterior circulation aneurysms. forty eight (26.2%) aneurysms occurred in the posterior communicating artery, 43 (26.2%) in the middle cerebral artery bifurcation, and 36 (19.7%) in the anterior communicating artery. ninety three (56.7%) of the anterior circulation aneurysms and 18 (94.7%) of the posterior circulation aneurysms were treated by coiling. seventy one (43.3%) anterior circulation aneurysms and one (5.3%) posterior circulation aneurysm were treated by microsurgical clipping. of the 113 individuals with gos scores of favorable outcome (i.e., independence in activities of daily living), 34 (58.6%) individuals belonged to the clipping group and 79 (78.2%) belonged to the coiling group (fig. 1a). in the ruptured population, 62 (57.4%) patients achieved a favorable outcome. in the population with unruptured aneurysms, 50 (98%) and one (2%) patients achieved an excellent and good outcome, respectively. in the clipping group, there were 46 patients with sah. nine (19.6%) patients had an excellent outcome and 13 (28.3%) patients had a good outcome. fifteen (32.6%) patients experienced moderate and severe disability. in the coiling group, twenty three (37.1%) patients were discharged in excellent status and 17 (27.4%) patients had a good outcome. thirteen (21%) patients experienced moderate disability or required hospitalization (tables 3, 4, fig. despite aggressive treatment, upon discharge, the overall mortality rate was 11.3% (n = 18). statistical analysis using the binary outcome scale was performed for comparison of favorable outcome with poor outcome in terms of hhg. hhg was divided into binary variables as either good (hhg i, ii, iii) or not. analysis for other variables including age, co - existence of other medical diseases such as hypertension and diabetes mellitus, and medications taken such as antiplatelet or anticoagulants was performed using the same method. as shown in table 2, high grade hhg, advanced age, and ivh were strong predictors of poor outcome. table 5 shows the causes of mortality in patients with ruptured aneurysms, with the two most common causes being initial hemorrhage (52.6% ; 10 of 19 deaths) and pneumonia (26.3% ; five of 19 deaths). sepsis / multiorgan failure (three deaths), myocardial infarction (one death) were other causes of death for patients who experienced ruptured aneurysms. there was no occurrence of death in the unruptured aneurysmal group. a comparison of the clinical characteristics of patients treated by endovascular coiling or microsurgical clipping is shown in table 1. a total of 183 (18%) aneurysms were treated in 159 (18.6%) elderly patients, including 134 women (84.3%) and 25 men (15.7%) with a mean age of 71.6 years ; 108 (67.9%) patients experienced subarachnoid hemorrhage (sah) and 51 (32.1%) patients had unruptured intracranial aneurysms (uia) ; 101 (63.5%) patients were treated by coiling and 58 (36.5%) patients were treated by microsurgical clipping. fifty three patients presented with a high - grade sah (hhg iv or v) and 55 patients presented with a sah of hhg ii or iii. the average age of patients with aneurysms coiled and clipped was 72.3 (65 - 93) and 70.2 (65 - 84) years, respectively (p < 0.01). no significant differences based on gender, risk factors, hhg, and aneurysm size were observed between the coiling group and the clipping group. there were 164 (89.6%) anterior circulation aneurysms and 19 (10.4%) posterior circulation aneurysms. forty eight (26.2%) aneurysms occurred in the posterior communicating artery, 43 (26.2%) in the middle cerebral artery bifurcation, and 36 (19.7%) in the anterior communicating artery. ninety three (56.7%) of the anterior circulation aneurysms and 18 (94.7%) of the posterior circulation aneurysms were treated by coiling. seventy one (43.3%) anterior circulation aneurysms and one (5.3%) posterior circulation aneurysm were treated by microsurgical clipping. of the 113 individuals with gos scores of favorable outcome (i.e., independence in activities of daily living), 34 (58.6%) individuals belonged to the clipping group and 79 (78.2%) belonged to the coiling group (fig. 1a). in the ruptured population, 62 (57.4%) patients achieved a favorable outcome. in the population with unruptured aneurysms, 50 (98%) and one (2%) patients achieved an excellent and good outcome, respectively. in the clipping group, there were 46 patients with sah. nine (19.6%) patients had an excellent outcome and 13 (28.3%) patients had a good outcome. fifteen (32.6%) patients experienced moderate and severe disability. in the coiling group, there were 62 sah patients. twenty three (37.1%) patients were discharged in excellent status and 17 (27.4%) patients had a good outcome. thirteen (21%) patients experienced moderate disability or required hospitalization (tables 3, 4, fig. despite aggressive treatment, upon discharge, the overall mortality rate was 11.3% (n = 18). statistical analysis using the binary outcome scale was performed for comparison of favorable outcome with poor outcome in terms of hhg. hhg was divided into binary variables as either good (hhg i, ii, iii) or not. analysis for other variables including age, co - existence of other medical diseases such as hypertension and diabetes mellitus, and medications taken such as antiplatelet or anticoagulants was performed using the same method. as shown in table 2, high grade hhg, advanced age, and ivh were strong predictors of poor outcome. table 5 shows the causes of mortality in patients with ruptured aneurysms, with the two most common causes being initial hemorrhage (52.6% ; 10 of 19 deaths) and pneumonia (26.3% ; five of 19 deaths). sepsis / multiorgan failure (three deaths), myocardial infarction (one death) were other causes of death for patients who experienced ruptured aneurysms. there was no occurrence of death in the unruptured aneurysmal group. in this study, the primary finding was that patients with high grade hhg, increasing age, and intraventricular hematoma (ivh, fg4) had poor clinical outcomes as compared to patients without these factors, which were as follows. the initial clinical presentation of patients is the highest predictive power of the outcome. in our series, the final mortality rate and percentage of severely disabled patients with high grade hhg (hhg iv, v) was 28.3% and 24.5%, respectively. a favorable outcome was achieved in 31% of treated patients, higher than the result from previously published studies.5)7) in patients with a poor clinical grade with acute hydrocephalus and significant ivh (fg4), early placement of an external ventricular drainage (evd) prior to aneurysm securing intervention improved the level of consciousness and short - term outcome.15)17)19) this study also demonstrated that placement of an evd improved the clinical grade and outcome. the secondary finding is that both microsurgical clipping and coiling yielded excellent results in elderly patients with unruptured aneurysm. the overall morbidity and mortality rates were 2% and 0% in each respective treatment modality, similar to those found in the literature.3)8)12)14) currently, there are no established guidelines for ideal management of uias in elderly patients. in the distribution of aneurysm sizes, larger sized unruptured aneurysms were more common in the older age group and the annual incidence of sah was noted to be three times higher.2)6)21) thus, some researchers have suggested that elderly patients with uias should be considered for active surgical intervention to improve their outcomes. advanced age alone has been reported to increase the risk of complications in elderly patients with unruptured aneurysm undergoing microsurgical clipping or endovascular coiling.4)10) in the current study, more patients with uias were treated by endovascular coiling (76.5%) and the mean age was significantly older than that of the microsurgical clipping group. to our surprise, favorable results have been obtained from both treatments along with our results. our study supports the idea that surgical treatment of uias in the elderly should be advocated in making decisions on the optimal treatment strategy after careful assessment of each individual 's circumstances. in the ruptured aneurysm group, nine (47.4%) patients died from ' other ' causes unrelated to sah. among the eight patients who died, five died of pneumonia and three died of sepsis and/or multiorgan failure. according to these results, more than one - third of the patients died within the acute period of the sah, even if it was not due to the hemorrhage. clinical care of elderly patients is challenging because of a greater chance of pharmacological complications, cardiopulmonary dysfunction, and so on. elderly patients who survive sah often face prolonged management in an intensive care unit (icu), including treatment for multiple potential neurological and medical complications.13)18) therefore, for proper co - management of these patients, in order to handle multiple issues, it is necessary to employ a more specialized neurointensivist dedicated to the icu.9) this study is limited by the fact that it was a retrospective review, with a limited number of patients at a single institution and no strictly defined selection criteria for the treatment procedure along with a lack of long - term follow up results. lack of standard treatment guidelines and sufficient prognostic domestic data concerning treatment of aneurysms in the elderly make it difficult for clinicians when it comes to deciding on the proper treatment modality in such patients. we believe that our study may provide a valuable guide to help in decision making regarding treatment enrollment of elderly patients who could benefit from treatment, despite the known challenges in the management of this age group. according to our data, elderly patients with high grade hhg and ivh are truly associated with poor outcome, so that the indication for aggressive therapy should be considered carefully. to improve outcomes in elderly sah patients, co - management by specialized neurointensivists who can handle patients with multiple conditions should be considered. according to our data, all elderly patients with uias had an excellent outcome, regardless of the treatment modality. | objectivethe aim of this study is to evaluate the clinical course of intracranial aneurysm in patients aged 65 years and older and the immediate outcome after its aggressive management.materials and methodswe performed a retrospective analysis using the medical records of 159 elderly patients managed at our institute from september 2008 to december 2013. obtained clinical information included age, sex, hunt and hess grade (hhg), aneurysm location, fisher grade (fg) and the treatment modality. concomitant clinical data aside from cerebrovascular condition (hypertension, diabetes, previous medication) were evaluated to determine risk factors that might affect the functional outcomes.resultsa total of 108 patients (67.9%) presented with subarachnoid hemorrhage (sah), and 51 (32.1%) with unruptured intracranial aneurysms (uias). coiling was performed in 101 patients and 58 patients underwent clipping. in the sah population, 62 patients (57.4%) showed favorable outcomes, with a mortality rate of 11.3% (n = 18). in the uias population, 50 (98%) patients achieved ' excellent ' and one (2%) achieved ' good ' outcome. factors including high - grade hhg (p < 0.001), advanced age (p = 0.014), and the presence of intraventricular hematoma (ivh) (p = 0.017) were significant predictors of poor outcome.conclusionsah patients with high grade hhg and ivh are associated with poor outcome with statistical significance, all the more prominent the older the patient is. therefore, the indication for aggressive therapy should be considered more carefully in these patients. however, as the outcomes for elderly patients with uias were excellent regardless of the treatment modality, aggressive treatment could always be considered in uias cases. |
at present, it is accepted that natural killer (nk) cells are a subset of lymphocytes with an important role in the early response to tumors [1, 2 ]. they destroy tumor cells by two main cytotoxic pathways : a perforin / granzyme - mediated secretory mechanism and a tnf - family ligand - mediated apoptotic killing. while the former mechanism acts mainly against cultured leukemia cell targets, the second is the way for which nk - cells act against most tumor cell targets [36 ]. furthermore, the finding that the number of intratumoral cd57 nk - cells influences the survival of the patients has been described for patients with gastric carcinoma or squamous lung cancer supporting the biological effect of immunological defense mechanism mediated by nk - cells. on the other hand, it is well - known that immunologic manipulation, in an early phase of carcinogenesis, can modulate the tumor development. since nk - cells represent a defense against tumors, it is logical to suppose that the capacity to act against tumors through nk - cells will determine the biggest or smaller easiness with which a tumor developed. therefore, if a patient shows strong immunological defense mechanisms by means of nk - cells in the tissue of a brain metastasis, it is logical to suppose that the tumor recurrence or the development of a new cerebral metastasis will be more difficult. in a previous study, we concluded that the number of cd57 nk - cells in the tumor stroma of brain metastases does not correlate with the number of apoptotic tumor cells. this finding suggests that, in brain metastases, apoptosis related to immune response is mainly mediated by activated tumor - infiltrating mononuclear cells other than cd57 nk - cells. we studied here if the number of cd57 nk - cells within the tumor tissue of brain metastases influences the clinical behavior, in terms of influencing the capacity of the brain to be protected for the development of a new metastasis or tumor recurrence. for this study we selected twenty male patients operated on because of a single cerebral metastasis from lung adenocarcinoma and that developed local recurrence or new brain metastases after surgery. in all cases tumor resection was considered complete, and the patients received holocraneal radiotherapy (3040 gy in 1020 fractions) after surgery. the age at time of surgery ranged between 42 and 78 years (mean : 64 years). paraffin - embedded specimens from the resected tumors were studied. a first study (unpublished data) showed that in all resected tumors, a variable number of cells expressed cd95 (fas / apo1). from each tumor, a histological slice was processed by haematoxylin - eosin (he) technique for studying both the histological pattern of the tumor and the degree and distribution of lymphocytic infiltration. histological sections from paraffin - embedded samples were mounted on glass slides and were deparaffinized by treatment in xylene for 15 minutes. the sections were rehydrated in a graded ethanol series and rinsed in phosphate - buffered saline (pbs) at ph 7.4. the slides were then washed in citrate - buffered solution (ph 6.0) for 10 minutes on microwave and then placed in hydrogen peroxide 3% in methanol for 15 minutes in order to block endogenous peroxidase activity, and the sections were immersed in pbs. for detection of nk - cells, primary monoclonal antibody to cd57 (1 : 100, master diagnostica, granada, spain) was used. monoclonal antibody was added overnight at 4c on wet chamber, and the histological slices were again rinsed in pbs. a 30-minute incubation with biotinyled secondary antibody at 37c was followed by a standard pbs rinse. another 30-minute incubation with streptavidin - peroxidase complex, at 37c, was carried out, and then chromogen solution was added (diaminobenzidine). after it, the slices were stained with hematoxylin, mounted, and examined microscopically. in all cases, negative controls were performed using rabbit normal serum as primary antibody. for each tumor, the number of cd57-immunostained cells was counted at least on 10 randomized histological fields, at 200x, and then averaged. in all cases, the evaluation of the number of nk - cells per field was conducted by two investigators with no previous knowledge of the case from each sample obtained. generally a high grade of agreement between the observers was obtained, but, in any case, the means of values recorded by these investigators were recorded as final values. these cells did not show a uniform distribution in the tumor but were usually grouped around blood vessels or within the tumor stroma (figure 1). after a randomized study of different fields from each tumor, at 200x, a number of 8.4 4.8 (mean standard deviation) cd57 nk - cells was estimated as an averaged value (figure 2(a)) in the series. on the other hand, the period of time free of new cerebral metastases or local tumor recurrence in the patients of the series ranged between 10 and 52 weeks (mean standard deviation : 22.7 11.9) (figure 2(b)). lastly, we analyzed for each case the correlation between the time free of local recurrence or new intracranial mestastases after surgery and the degree of cd57 nk - cells infiltrating the resected brain metastasis. after this analysis, a correlation between these two variables could not be found (p = 0.63 ; correlation coefficient (r) : 0.1128 ; 95% confidence interval : 0.52 to 0.34) (figure 3). for the present study, we have identified cd57 nk - cells in the tissue of resected brain metastases, in a homogeneous series of metastatic brain tumors developed as a result of the spread of a lung adenocarcinoma. furthermore, in each case, we have recorded the time free of local tumor recurrence or appearance of new cerebral metastases after treatment. although cytotoxic efficacy of nk - cells in tumor tissue may not be judged by their numerical presence, it is accepted that these cells play an important role in the immunological defense against tumor cells. thus, it seems logical to assume a relationship between the degree of nk - cells infiltration in tumor tissue and the effectiveness of this type of immunological defense. on the other hand, it is accepted that the main mechanism for which nk - cells act is through inducing apoptosis in the tumor cells [36 ], and we previously obtained in all the tumors of the series (data not shown) variable expression of cd95 (fas / apo1), a 48-kd transmembrane glycoprotein, at present identified as an important mediator in the apoptotic process mediated by nk - cells. although this finding suggested the possible susceptibility of tumors to the action of infiltrating nk - cells, in a previous study, we concluded that the number of nk - cells that are present in the stroma of brain metastases does not correlate with the number of apoptotic tumor cells. therefore, it is possible that nk - cells do not play an important role in the immunological defense against brain metastases. the purpose of the present study is to add new data to this hypothesis, verifying if the degree of local immunological response against a metastatic brain tumor, measured by the degree of nk - cell infiltration within the tumor tissue, influences the clinical behavior, in terms of influencing the capacity of the brain to be protected for the development of new metastases or local tumor recurrence. our present results showed that the time free of new cerebral affectation for metastatic dissemination or recurrence of the previously resected tumor is not related with the degree of immunological response mediated by the presence of nk - cells. although it is obvious that our present series has scarce number of cases and that multiple factors, mainly the evolution of the systemic disease, can influence the clinical behavior of patients suffering metastatic brain disease, we think that our present analysis represents a new argument supporting that in brain metastases, the immune response mediated by cd57 nk - cells plays a doubtful role. this consideration should be kept in mind in therapeutic trials based on the hypothetical defensive action of the nk - cells against metastatic brain tumors. our present findings suggest that clinical behavior in metastatic brain disease is not influenced by the immunological response mediated by cd57 nk - cells. | objectives. the purpose of the present study is to verify if the degree of immunological response against metastatic tumors, measured by the number of cd57 + nk - cells in the tissue of a brain metastasis, influences the later development of new brain metastases or tumor recurrence. patients and methods. cd57 + nk - cells were immunohistochemically identified in the resected tumor, in a series of twenty patients operated on by a single brain metastasis secondary to lung adenocarcinoma. in each case, the degree of cd57 + nk - cells infiltration within the tumor tissue and the period free of new intracranial disease after brain surgery were recorded. results. all the studied tumors showed variable number of cd57 + nk - cells (mean standard deviation : 8.4 4.8 per microscopical field, at 200x). the period free of intracranial disease ranged between 10 and 52 weeks (mean standard deviation : 22.7 11.9). statistical analysis showed that there was no correlation between the degree of nk - cells infiltration within the resected tumor and the period free of intracranial disease after surgery (p > 0.05). conclusion. this finding supports that clinical behavior in metastatic brain disease is not influenced by the immunological response mediated by cd57 + nk - cells. |
an increased risk of als is associated with certain populations who have a history of extensive physical contact such as varsity athletics, professional soccer players, and military veterans [13 ]. motor nerve injury as a trigger to degeneration has been proposed in these populations, but the underlying mechanism remains elusive. in order to investigate this hypothesis, we utilized the motor nerve injury (facial nerve axotomy (fna)) in the als mouse model (sod1 mice) to evaluate the impact of fna on motoneuron survival after injury. we found that fna - induced motor neuron loss is significantly increased in sod1 mice relative to wt mice. importantly, the increased motor neuron loss in sod1 mice can be prevented by adoptive transfer of immune cells from wild - type mice. these data suggest that individuals with a genetic susceptibility to als are more vulnerable to nerve injury - induced neurodegeneration. because such vulnerability is impacted by the immune system, we hypothesize that fna may induce a more pronounced proinflammatory response in sod1 mice than in wt mice, which in turn impairs the function of neuroprotective immune responses. as the pivotal cell of immunoregulation, the cd4 t cell has been of a great interest in the investigation of the pathogenesis of als. cd4 t cells have several subsets with distinct immunoregulatory functions. in late - stage als patients, the total number of nave cd4 t cells is decreased and cd4 t cell infiltration in the spinal cord and brain is significantly increased [5, 6 ]. in addition, elevated th1 cells in cerebrospinal fluid and elevated il-17 and th17-related cytokines (il-6, tnf-, il-1, and il-23) [79 ] in the serum have also been observed in als patients. furthermore, the level of antineuroinflammatory subsets, th2 and treg cells [10, 11 ], appear to regulate the speed of disease progression. however, the roles of proneuroinflammatory subsets, th1 and th17 cells, in promoting als development has yet to be established due to two challenges : late diagnosis and the chronic nature of the disease. the late diagnosis makes it difficult to conclude whether observed abnormal immune response and inflammation are the cause or the result of the disease. the chronic nature of als also makes it difficult to determine the best timing for the detection of such autoimmune responses. we have previously demonstrated that fna is capable of inducing a readily detectable immune response in a predictable time period (714 days). in the current study, we performed fna in presymptomatic b6sjl sod1 mice (8-week - old) and examined cd4 t responses in a time course after fna. we found that abnormal cd4 t cell activation with increased th17 cells is present in sod1 mice prior to the onset of neurological symptoms. these results suggest that sod1 mice have impaired immunoregulatory mechanisms that normally dampen injury - induced inflammatory responses and that th17 cell - promoted inflammation might contribute to the increase in injury - induced motoneuron death in sod1 mice. six - week - old female b6sjl sod1 and wild - type female b6sjlf1/j mice were obtained from jackson laboratory (sacramento, ca, usa). all mice were housed and surgery was performed as previously described [4, 12 ]. all surgical procedures were completed in accordance with national institutes of health guidelines on the care and use of laboratory animals for research purposes. the right cervical lymph nodes (draining cervical lymph node (dcln)) were collected from uninjured (control) or axotomized mice (n = 4/group) at 7, 9, and 14 days postaxotomy (dpa). cd4 t cells were isolated via automacs using anti - cd4 magnetic beads as previously described [4, 12 ]. cd4 t cells separated from the draining cervical lymph node preparation were incubated for 6 hours with phorbol myristate acetate (pma, 50 ng / ml) and ionomycin (500 ng / ml, p / i, sigma, st. louis, mo) with brefeldin a (bfa, 10 g / ml, sigma, st. louis, mo) added during the final 2 hours. for the intracellular staining, cd4 t cells were permeabilized with saponin (1 mg / ml, sigma, st. louis, mo) and double - stained with two of the following antigens : anti - cd4-efluor 450, anti - ifn--alexa fluor-488, anti - il-17a - pe - cy7, anti - tnf--pe, anti - il-10-percp - cy5.5 and anti - il-4-apc, or anti - cd8-fitc. for the immune cells subsets identification, freshly collected dcln cells were stained with anti - cd3-pe, anti - b220-apc - cy7, anti - cd4-fitc, and anti - cd8-apc. for identification of activation, cells were stained with anti - cd4-efluor 450, anti - cd69-percp - cy5.5, anti - cd44-apc, and anti - cd62l - fitc. a one - way anova with the bonferroni post hoc test was used for comparisons of two specific groups. head injury is associated with an increased risk for developing als [13 ], leading us to hypothesize that inappropriate activation of the immune system from prior injury may underlie the development of als. therefore, in the current study, we used the fna model of motor neuron injury to compare immune responses in wt versus sod1 mice which serve as a mouse model of als to examine underlying alterations in immune activation and implications for disease development in sod1 mice (presymptomatic, 8-week - old b6sjl). as shown in figure 1(a), basal numbers (prior to the fna) of total cells recovered from one dcln wt mouse were 6.13 0.44 (10) versus 12.1 0.99 (10) for sod1 mice, suggesting that sod1 mice have greater baseline number of lymphocytes than do wt mice. following fna, a transient increase in the number of total cells recovered was noted in wt mice and returned to basal levels at 14 days after fna. in contrast, sod1 mice showed a progressive and sustained increase in total cell numbers in the dcln. differences in cell counts correlated with the size of dcln in these mice (figure 1(b)). to further differentiate t cell subsets, we analyzed the percentage of cd4 versus cd8 t cells (figures 1(c)1(e)). prior to the fna, both wt and sod1 mice had a ratio of cd4 : cd8 that was approximately 2 : 1. although the ratio of cd4 : cd8 remained close to 2 : 1 in wt mice following fna, it decreased to a ratio approaching 1 : 1 in sod1 mice (figures 1(c)-1(d)). however, because total cell numbers in the dcln increased after fna, the absolute number of both cd4 and cd8 increased in both wt and sod1 (figure 1(e)). these data suggest that an enhanced basal level of inflammation in sod1 mice may impair immunoregulatory mechanisms that normally dampen injury - induced inflammatory responses before the onset of neurological symptoms. accordingly, we examined further the activation status of cd4 t cell responses at 7 days after fna, the peak time of cd4 t cell response. in the wt mice, t cells at both the early activation stage (cd69, figures 1(f)1(h)) and effector stage (cd62lcd44, figures 1(i)1(k)) were increased after fna, as reflected by frequency (figures 1(g) and 1(j)) and total number (figures 1(h) and 1(k)). the same pattern was also found in sod1 mice, but at a higher magnitude. in addition, fna - induced activation levels of cd4 t cells in wt mice were comparable to that of sod1 mice prior to the fna in terms of both percentage and total number, suggesting that the fna - induced activation of t cells in wt mice may occur in a similar manner as t cell activation in sod1 mice, prior to disease onset. cd4 t subsets have distinct functions in terms of neuroprotective or neurodestructive effects [1015 ]. therefore, we examined dcln cd4 t subsets in wt and sod1 mice at 7 days after fna by staining intracellularly for ifn- (th1 cells), il-17 (th17 cells), tnf- (pro - inflammatory cells), il-4 (th2 cells), and il-10 (treg cells). following fna, the frequency of th1 cells in the wt mice was decreased, although the total number did not significantly change. in contrast, both the frequency and total number of th1 cells were significantly increased in sod1 mice (figures 2(a)-2(b), first column). in addition, th17 cells in the wt mice did not change in frequency and increased only slightly in total number. in contrast, sod1 mice possessed both a greater frequency and more total th17 cells than did wt mice, regardless of fna state. in fact, the frequency of th17 cells was 3-fold higher (0.65 0.05% versus 0.13 0.03%) prior to injury and was 4-fold higher (0.77 0.02% versus 0.16 0.02%) after fna (figure 2(b), middle column) in sod1 mice, whereas the numbers of total th17 cells were 9-fold higher [2.8 0.31 versus 0.28 0.03 (10) ] prior to injury and 5-fold higher [5.39 0.61 versus 0.89 0.14 (10) ] after fna (figure 2(c) middle column) in sod1 mice. in the wt mice, the frequency and total number of th1th17 cells were similar prior to and after fna. in contrast, the frequency and total number of th1th17 cells in sod1 mice decreased after fna. we did not find significant difference of th2 and treg cells between wt and sod1 mice prior to or after fna (data not shown). further analysis of tnf- expression in cd4 t cells revealed that both frequency and percentage of tnf--single - positive cells were significantly increased in the wt mice following fna. however, in sod1 mice, though the percentage did not change after fna, the total number of tnf- cells significantly increased (figures 2(e) and 2(f), left column). tnf--expressing th17 (tnf-th17) in wt mice was low in both frequency and total number and did not significantly change after fna. in contrast, tnf-th17 in sod1 mice had a higher basal frequency and total number which was further increased in response to fna (figures 2(e) and 2(f), right column). these data suggest that tnf--expressing th17 cells might be an important subset of autoimmune cells involved in injury - induced inflammatory damage in sod1 mice. we have previously demonstrated that cd4 t cells mediate neuroprotection after nerve injury [4, 16 ]. in als, cd4 t cells also play an important role in restricting disease progression [13, 14 ] ; however, this neuroprotection is in a context- and subset - dependent manner [1015 ]. for example, anti - inflammatory subsets of cd4 t cells are generally thought to be the types of neuroprotective immune cells which support facial motoneuron survival after nerve injury and may therefore slow down the disease progression in als [10, 11 ]. our previous studies revealed that multiple subsets of cd4 t cells develop following fna in wt mice, including both anti - inflammatory and proinflammatory subsets of cd4 t cells. we hypothesize that the balance between these subsets of cd4 t is critical for the resolution of necessary and beneficial inflammation as well as induction of repair tissue and support mechanisms for survival of damaged motoneurons. in the current work, we used the sod1 mice, an als mouse model, to show that fna induces significant motoneuron loss relative to wt mice and that this loss is similar to that of immunodeficient mice (rag2 mice) [4, 16 ]. exacerbation of fna - induced motoneuron loss in sod1 mice may result from a poorly controlled inflammatory response to injury, increased basal levels of inflammation inherent to this model or perhaps to failure of development of neuroprotective cd4 t subsets as the current study revealed that there are significant differences of fna - induced immune responses between wt and sod1 mice. first, we found that while wt mice mounted a well - controlled immune response in the draining lymph nodes following fna, sod1 mice had an enhanced and persistent immune response as indicated by the enlarged size of the draining lymph nodes as well as an increase in total cell numbers. further analysis for t cell subset responses revealed that the ratio of cd4 : cd8 t cells in wt mice did not change in response to fna, whereas the ratio decreased in sod1 mice. because cd4 t cells regulate cd8 t cell responses and cd4 t cells are the major type of t cells that impact the neuronal survival after fna, we focused on cd4 t cell responses in the analysis of t cell activation and subsets. our data indicate that activation of cd4 t cells is higher in sod1 than wt mice. regarding the subsets, we did not find significant differences in anti - inflammatory t cell subsets (th2 and treg cells) between wt and sod1 mice either prior to or after fna ; however, significant differences were noted in the proinflammatory subsets (th1 and th17 cells) between these two types of mice. without nerve injury, th1 cell frequency and total number were similar in wt and sod1 mice ; however, fna induced an increase in th1 total number in sod1 mice but not in wt mice. in the uninjured state, both th17 cell frequency and total number were higher in sod1 mice than in wt mice. further increases in th17 cell frequency and total number were noted in sod1 mice but not in wt mice under fna injury conditions. importantly, th17 cells are also tnf--expressing cells in sod1 mice but not in wt mice. these data collectively indicate that feed - forward proinflammatory response to injury occurs in sod1 mice. we recently demonstrated that whole splenocytes, but not isolated cd4 t cells, from wt mice can reduce fna - induced facial motor nucleus (fmn) loss in sod1 mice. in addition, isolated cd4 t cells, but not whole splenocytes collected from sod1 mice, are capable of supporting fmn survival in immunodeficient mice (rag2). these data suggest that the microenvironment in sod1 mice may direct cd4 t cell differentiation into a neurodestructive subset. consistent with this hypothesis, the data from the current study suggest that fna - induced th17 cell responses in sod1 mice may exacerbate neuroinflammation, without a concomitant induction of the neural repair phase, which in turn results in increased motoneuron death. enhanced cd4 t cell activation and th17 cell responses in sod1 mice exist prior to the onset of overt neurological symptoms. motor nerve injury further increases cd4 t cell activation and th17 cell responses in sod1 mice. we therefore propose that cell - promoted inflammation may be involved in the motoneuron death during als disease onset and progression. | an increased risk of als has been reported for veterans, varsity athletes, and professional football players. the mechanism underlying the increased risk in these populations has not been identified ; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of als. accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of als, but the specific immune cells involved have not been clearly defined. to understand how nerve injury and immune responses may contribute to als development, we investigated responses of cd4 + t cell after facial motor nerve axotomy (fna) at a presymptomatic stage in a transgenic mouse model of als (b6sjl sod1g93a). sod1g93a mice, compared with wt mice, displayed an increase in the basal activation state of cd4 + t cells and higher frequency of th17 cells, which were further enhanced by fna. in conclusion, sod1g93a mice exhibit abnormal cd4 + t cell activation with increased levels of th17 cells prior to the onset of neurological symptoms. motor nerve injury exacerbates th17 cell responses and may contribute to the development of als, especially in those who carry genetic susceptibility to this disease. |
collapsing focal segmental glomerulosclerosis (fsgs), also known as collapsing glomerulopathy (cg), was first described in association with hiv infection. subsequently the clinical course of cg is characterized by rapid progression to end - stage renal disease and a dismal response to medical therapy. a survey of observational studies reported complete and partial remission rates of 9.6 and 15.2%, respectively, despite multiple therapies that included steroids, calcineurin inhibitors (cnis) and cyclophosphamide. there have been two reports suggesting a favourable response to rituximab in children with cg [2, 3 ] a 19-year - old - male presented in may 2007 with swelling of the lower limbs and periorbital puffiness of 2 months duration. evaluation at his primary health centre had revealed albuminuria on dipstick analysis, prompting referral to our institution. he gave no history of recent infections, arthralgia, oral ulcers, cough or haemoptysis. the blood pressure was 110/70 mmhg, pulse rate was 90 b.p.m. and respiratory rate 14/min. mol / l (0.8 mg / dl) ; serum total protein and albumin were 55 and 17 g / l (5.5 and 1.7 g / dl), respectively, and aspartate and alanine aminotransferases were 12 and 14 iu / l, respectively. mmol / l (400 mg / dl), triglyceride 2.8 mmol / l (250 mg / dl), low - density lipoprotein 7.2 mmol / l (280 mg / dl) and high - density lipoprotein 1.3 mmol / l (50 mg / dl). he tested negative for hepatitis b surface antigen, anti - hcv antibody and human immunodeficiency virus i / ii. kidney biopsy (figure 1a) revealed 10 glomeruli, which were normal on light microscopy (lm), as were the tubulointerstitial and vascular compartments. immunofluorescence did not reveal any immune deposits and electron microscopy (em) showed diffuse foot process effacement. 1.photomicrograph showing (a) normal glomeruli by lm (pas, 40), and complete foot process effacement by em (not shown), consistent with minimal - change disease ; (b) segmental collapse with overlying proliferation of visceral epithelial cells consistent with collapsing glomerulopathy (masson 's trichrome, 40), (c) electron microscrograph shows hypertrophied podocytes with vacuoles over a collapsed glomerular tuft (uranyl acetate, 62 000) and (d) proliferating visceral epithelial cells stained with ki67 (ihc, 40) photomicrograph showing (a) normal glomeruli by lm (pas, 40), and complete foot process effacement by em (not shown), consistent with minimal - change disease ; (b) segmental collapse with overlying proliferation of visceral epithelial cells consistent with collapsing glomerulopathy (masson 's trichrome, 40), (c) electron microscrograph shows hypertrophied podocytes with vacuoles over a collapsed glomerular tuft (uranyl acetate, 62 000) and (d) proliferating visceral epithelial cells stained with ki67 (ihc, 40) a diagnosis of minimal - change disease (mcd) was made and the patient was started on oral prednisolone at 1 mg / kg / day. however, the proteinuria did not show any reduction. at the end of 4 months, the daily urinary protein excretion remained at 5 g. he was deemed to be steroid resistant and treated first with oral cyclophosphamide at 2 mg / kg for 3 months, followed by oral cyclosporine (4 mg / kg / day, average c0 120 ng / ml) and oral prednisolone (0.1 mg / kg / day) for 6 months, mycophenolate mofetil 2 g / day for 6 months and finally a combination of tacrolimus (0.1 mg / kg / day, average tac c0 7 ng / ml) and oral prednisolone (0.1 mg / kg / day). < 20 g / l (2.0 g / dl) and the serum creatinine 6280 mol / l (0.70.9 mg / dl) (figure 2). during the entire course of his illness, he was continued on supportive therapy with torsemide 1020 mg twice daily, atorvastatin 20 mg / day and losartan 150 mg / day. temporal evolution of the clinical and laboratory parameters. at this time, the kidney biopsy was repeated. lm revealed 13 glomeruli, 2 of which showed segmental collapse of the capillary tuft with an overlying crown of podocytes along with synechiae formation, 2 showed segmental endocapillary cellularity with foam cells, 2 were globally sclerosed and the remaining 7 did not show any significant abnormality (figure 1b). tubules showed abundant protein absorption droplets along with patchy tubular atrophy occupying 20% of the biopsy area. tubules close to the medulla showed marked nucleomegaly along with nuclear smudging associated with tubular necrosis, interstitial oedema and mild - to - moderate patchy plasma cell - rich interstitial inflammation occupying 10% of the biopsy area. there was diffuse and extensive foot process effacement, and no reticular aggregates were seen. ki 67 immunostain showed nuclear positivity in the podocytes of glomeruli showing collapse (figure 1d). the patient 's serum tested negative by polymerase chain reaction for cytomegalovirus and parvovirus b-19. in view of his resistance to the above therapies and a persistently severe nephrotic state with the biopsy showing many plasma cells, the patient was treated with 4 weekly injections of rituximab (375 mg / m / dose). his oedema subsided and he stopped requiring the diuretic 3 months after the last dose. six months after the last dose of rituximab, the 24-h urine protein was 1.8 g with a serum albumin of 37 the patient has now been followed up for 18 months and remains asymptomatic. on his last follow - up, his 24-h urine protein is 1.2 g, serum creatinine 80 mol / l (0.9 mg / dl) and serum albumin 47 figure 2 shows the evolution of his clinical and laboratory parameters during the course of the treatment. we report the first case of adult - onset collapsing fsgs successfully treated with rituximab. the prognosis of cg not associated with hiv infection has been uniformly dismal [68 ]. based upon a survey of the observational studies that have reported the response to treatment of collapsing fsgs, the rates of durable complete and partial remission were 9.6 and 15.2%, respectively. valeri. had reported steroid resistance in all of the 26 (100%) cg patients treated with steroids. one out of six (17%) of the patients with steroid - resistant cg treated with cyclophosphamide achieved partial remission and two out three patients with steroid - resistant cg treated with cyclosporine achieved remission (one complete and one partial). even though the evidence base is weak, aggressive treatment seems to be associated with higher remission rates, especially in patients with serum creatinine < 177 mol / l (< 2 mg / dl) and < 20% interstitial fibrosis on kidney biopsy [9, 10 ]. rituximab has been successfully used in the management of several glomerular diseases including idiopathic membranous glomerulonephritis, steroid - dependent and -resistant childhood nephrotic syndrome (mcd and fsgs) and vasculitides. the efficacy of rituximab in idiopathic cg was shown in two case reports in the paediatric age group. described a 2-year - old boy with steroid- and cyclosporine - resistant cg who went into complete remission with four doses of rituximab. however, the child had a relapse after 4 months and required re - treatment with methyl prednisolone to achieve remission. reported a favourable response to rituximab in a young patient with cg with dominant c1q containing mesangial immune deposits and cd 20 + interstitial infiltrates, who was initially resistant to steroids. other interesting aspects of our case were that the initial kidney biopsy showed features of mcd, failure to respond to steroids, cyclophosphamide, cnis and mycophenolate mofetil, and the disease being found to have evolved into cg. he tested negative for parvovirus b19, which has been implicated in some cases of cg. the lack of response suggests the possibility of fsgs. in a series of 11 patients with steroid - resistant mcd in whom another possibility is of unsampled fsgs reported as mcd in the first biopsy, which had 10 glomeruli, whereas ideally 25 glomeruli should be evaluated to avoid missing this focal disease. rituximab, tried in view of a persistent severe nephrotic state, led to partial remission, which has now been sustained for 18 months. a less than complete response is not surprising in view of the histological changes on biopsy. compared with the report of the successful use of rituximab in c1q nephropathy with cg by bitzan., our patient lacked cd 20 + infiltrates in the interstitium, but still responded to rituximab. recently, it has become clear that b cells display a variety of functions other than antibody production, which could contribute to autoimmunity. rituximab depletes b - lymphocytes and blocks t - cell activation by b - lymphocytes and other b - cell - derived factors. our case is unique by the fact that it is the first case of adult - onset collapsing fsgs that has responded favourably to rituximab after failing all other treatments. in conclusion, we show a favourable effect of rituximab in an adult with cg, a difficult - to - treat condition. we declare that the results presented in this paper have not been published previously in whole or in part, except in abstract format. | collapsing focal segmental glomerulosclerosis (fsgs), or collapsing glomerulopathy (cg), responds poorly to commonly employed therapies, with a high proportion of patients progressing to end - stage renal disease. we report an adult in a nephrotic state, diagnosed with minimal - change disease on biopsy, who failed to respond to steroids, calcineurin inhibitors (cnis), mycophenolate mofetil and cyclophosphamide. repeat biopsy showed cg. treatment with 4 weekly doses of rituximab led to sustained remission of his nephrotic state. this is the first report of adult - onset cg that has responded favourably to rituximab. rituximab could be a treatment option for patients with this difficult - to - treat condition. |
body fat content is a well - established risk factor for the development of hypertension (1, 2), and recent studies have suggested that abdominal visceral fat has a stronger association with hypertension incidence than does total fat mass (35). although bmi is significantly related to an individual s body fat content (6, 7), some studies have suggested that the wc is a better predictor than bmi of the amount of abdominal visceral fat determined using computed tomography ; furthermore, the wc can be easily measured and interpreted (8, 9). the impact of changes in the wc or bmi on the incidence of hypertension has been observed in previous clinical studies but not in cohort studies (1012). on the population level, the association between changes in the wc or bmi and incident hypertension is unclear, and such an association may be impacted by a change in lifestyle or a targeted intervention. the aim of this study was to compare the impact of changes in the wc or bmi on incident hypertension utilizing data from the prevention of multiple metabolic disorders and ms in jiangsu province (pmmjs) project. a multi - stage sampling method was employed for the present study. in stage one ; we randomly selected 3 districts from 13 urban districts and 9 counties from the 52 counties of jiangsu province based on the economic conditions in different regions. in the second stage, one community (such as a street district or a residential community) from each city and one rural township from each county were sampled randomly. in the final stage, households were randomly chosen from the selected communities and townships ; only one participant was randomly selected, without replacement, from each household. the local public health administrative institutes possess the household registrations, which include the addresses and telephone numbers for all participants, allowing the health status of each participant to be tracked easily in follow - up assessments. individuals who suffered from cancer, severe disability, or a severe psychiatric disturbance were excluded. data on demographic characteristics, lifestyle risk factors, personal medical history and family history of hypertension for all participants were obtained using a standard questionnaire administered by trained staff. three sitting blood pressure (bp) measurements were taken at 30-second intervals by trained observers using a standard mercury sphygmomanometer after the subjects had been resting for 5 min according to a standard protocol. the first and fifth korotkoff sounds were recorded as the systolic (sbp) and diastolic (dbp) blood pressures, respectively. the mean of the three bp measurements was used in the analysis. body weight and height were measured by using standard methods (13), and the bmi was calculated as the weight in kilograms divided by the square of the height in meters. the wc was measured two times at 1 cm above the umbilicus at minimal respiration by trained observers with the subjects standing and breathing normally during the physical examination. all plasma and serum samples were frozen at -80 c until laboratory testing was performed. the concentrations of hdl cholesterol and triglycerides were assessed enzymatically using an automatic biochemistry analyzer (hitachi inc, tokyo, japan) and commercial reagents. the friedewald equation (14) was used to calculate the ldl - c from the total cholesterol, hdl cholesterol, and triglycerides. the study was approved by the soochow university ethics committee. of the 5888 subjects eligible for follow - up at 2 years, a total of 4582 participants (77.82%) completed the first follow - up questionnaire and completed the wc re - measurement between january 2002 and august 2003. the contents and methods of the questionnaire and wc measurements in the first follow - up investigation were the same as those at baseline. in addition, data on participant s blood pressure and development of hypertension and cardiovascular diseases were collected at the first follow - up. of the 4582 subjects eligible for follow - up at 5 years, a total of 3847 participants (83.96%) completed the second follow - up questionnaire between march 2006 and november 2007. in this survey, we mainly collected information on participant s current blood pressure and the incidence of hypertension and cardiovascular diseases over the past five years. those subjects who did not attend the second follow - up examination were similar to those who did complete the second follow - up in age, sex, smoking, alcohol usage, family history of hypertension and metabolic variables. in total, 3847 participants completed the two follow - ups. after the exclusion of subjects who were found to have hypertension at baseline and the first follow - up investigation (n=882), cvd (n=32), or a bmi 0 signified that the wc or bmi increased between baseline and the first follow - up, and a greater d - value implied a larger wc or bmi increase. a d - value 0 signified that the wc or bmi increased between baseline and the first follow - up, and a greater d - value implied a larger wc or bmi increase. a d - value 0.5, vif 0.05). (table 3 and 4) table 5 shows the incidence rate and rrs of hypertension for participants stratified by whether wc and/or bmi were categorically increasing. the lowest cumulative incidence rate of hypertension was 19.1% in males and 16.0% in females in populations in which both wc and bmi were modified ; the highest hypertension incidence was 45.1% in males and 35.8% in females in populations in which wc was not modified and bmi was modified. in both sexes, the hypertension risk was higher in populations in which wc was categorically increasing but bmi was modified than that of subjects in which both wc and bmi were modified (p 0.05). cumulative incidence and hazard ratio of hypertension stratified by wc at baseline and the first follow - up cumulative incidence and hazard ratio of hypertension stratified by bmi at baseline and the first follow - up cumulative incidence and hazard ratio of hypertension stratified by whether wc and/or bmi were modified the results of this study showed that for every 1.0 kg / m increase in bmi, the odds of developing hypertension increased by 9.0% and 3.0% in males and females, respectively, and that for every 1.0 cm increase in wc, the odds of developing hypertension increased by 4.0% and 3.0% in males and females, respectively. regardless of whether the wc or bmi was abnormal at baseline, compared with subjects with a normal wc or bmi at the first follow - up, the incidence rate of hypertension was significantly higher for subjects with an abnormal wc or bmi at the first follow - up. these results indicate that the hypertension risk of obese subjects would decrease if they reduce their wc or bmi and that the hypertension risk of subjects with a normal wc or bmi would increase if their wc or bmi becomes abnormal. during follow - up in cohort studies, the wc or bmi of subjects can change due to lifestyle modification or a targeted intervention. the wc or bmi of obese subjects can return to normal, and subjects with a normal wc or bmi at baseline can become obese between baseline and the first follow - up. the impact of such a change on hypertension incidence, however have only been shown in clinical trials (1012). the changes in wc and bmi reflect changes in the distribution and composition of body fat. in cohort populations, the wc could decrease when bmi increases and the wc could increase when bmi decreases, or both the wc and bmi could be maintained at a normal level similar to the values found at baseline. therefore, when we evaluate the impact of obesity control on blood pressure, it should be determined whether wc or bmi is the more practical and sensitive indicator. the results from most of the cross - sectional studies have shown a stronger association of hypertension prevalence with central obesity (as measured by wc) than with general obesity (as measured by bmi) in different ethnic groups (1821). a previous study suggested that wc was indeed a better predictor of visceral adipose tissue than bmi (22). (23) reached the conclusion that the relationship between wc and bp was unaffected by adjustment for bmi ; in contrast, the correlation between bmi and bp was no longer significant when wc was modified. (24) with 1183 type 2 diabetes patients showed that the metabolic disease risk was higher in subjects with a normal bmi but an abnormal wc than that in subjects with an abnormal bmi but a normal wc. janssen (26) indicated that there was no difference in the health risk between overweight and obese subjects with same wc, but that the health risk of subjects with the same bmi increased with wc ; these studies confirmed that the impact of wc on hypertension risk factors was greater than that of bmi. in the current study, a low level of multicollinearity was found between the wc d - value and the bmi d - value (the tolerance value was 0.63 and the vif was 1.58) ; therefore, when the wc d - value and the bmi d - value were included as continuous variables in the same regression model, the change in wc was better correlated with hypertension than the change in bmi. indeed, the association between the bmi d - value and hypertension was no longer statistically significant when the wc d - value was modified. the hypertension risk decreased significantly if the wc changed from abnormal to normal, but a decreased risk of hypertension was not observed when the bmi changed from abnormal to normal. there was no significant difference in the hypertension risk between the group in which both wc and bmi were modified and the group in which wc was modified but bmi was categorically increasing. however, the hypertension risk in subjects whose wc was categorically increasing but whose bmi was modified was higher than that found in subjects whose wc and bmi were both modified. these results suggest that the impact of changes in the wc on hypertension risk was greater than that of changes in the bmi dynamic. the ratio of men to women was not ideal : there were more women than men included in the analysis. in addition, it would have been prudent to stratify the population into ten - year age groups and to consider the length of time subjects were obese for. thirdly, data on serum levels of uric acid and creatinine (or estimated glomerular filtration rate) are not available in our study, so we can not adjust these variables in the multivariate analysis. in addition, the diet and physical activity are the important factors for incident hypertension, but these factors were not included in the analysis. we discussed the impact of increases or decreases in wc and bmi on hypertension risk. based on our data, there is evidence that changes in wc are better predictors of the risk for hypertension than changes in bmi. intervention programs designed to reduce wc through lifestyle modifications including exercise and diet may have significant public health significance in reducing the incidence of hypertension. 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. | abstractbackgroundcurrently, obesity has become a worldwide health problem and yet little is known about the impact of changes in obesity indicator on incident hypertension. the aim of this study was to compare the impact of changes in the wc and bmi on incident hypertension in a cohort population.methodsafter a baseline investigation, we conducted the first and the second follow - up assessments for subjects after 2 and 5 years, respectively. the associations between the changes in the wc and bmi (measured as the d - value, i.e., the value at the first follow - up minus the value at baseline) and the hazard ratio (hr) of incident hypertension were analyzed with a multilevel cox proportional hazards regression model.resultsamong 2778 participants without hypertension, 660 developed hypertension between the first and the second follow - up assessments. when both the bmi and wc d - values were included in the regression model, the wc d - value was a predictor of hypertension incidence in both sexes (or= 1.03 and p values 0.05). in both sexes, hypertension risk was higher for subjects whose bmi was modified but wc was categorically increasing than for subjects whose wc and bmi were both modified.conclusionsboth wc and bmi changes were associated with hypertension, but a change in the wc was a better predictor of the hypertension. |
primary ovarian failure (pof) is a syndrome composed of amenorrhea, estrogen deficiency and follicular stimulating hormone (fsh) of menopausal ranges in young women. in this article a 29-year old woman with a history of primary amenorrhea attended hospital with full term pregnancy. she had experienced a few episodes of withdrawal bleeding on hormonal treatment initially and she had conceived spontaneously. this case was presented to emphasize the real chances of spontaneous conceptions due to intermittent and unpredictable ovarian function in patients with poi. nevertheless, egg donation is still considered the best option for infertility in such women. fuller albright first described a condition he termed primary ovarian insufficiency (poi) in 1942 when he reported a syndrome of amenorrhea, estrogen deficiency and menopausal follicular stimulating hormone (fsh) levels in young women (1). herein, we report such a case that had primary amenorrhea and presented with full term pregnancy. a 29-year - old woman attended antenatal out - patient department of north eastern indira gandhi regional institute of health and medical sciences (neigrihms), shillong, meghalaya, india with full term pregnancy in october 2009. she gave a very interesting history of primary amenorrhea with a few episodes of withdrawal bleeding upon hormonal treatment in the past. for the past 3 4 years she had not been receiving any treatment and had remained amenorrhoeic. after 4 years of marriage, she went to a local doctor for nausea and vomiting and, to her surprise, her urine pregnancy test was positive. her expected date of delivery (edd) was calculated from the first trimester ultrasound, which confirmed the gestational maturity. on examination,, uterus was found to be in the form of a full - term pregnancy. the fetus was in cephalic presentation and fetal heart rate (fhr) was 136 beats per minute and regular. per vaginal examination revealed a closed internal os with soft, short and central cervix. caesarean section was performed on maternal request and a healthy male baby weighing 2.5 kg was delivered. primary ovarian insufficiency has varied manifestations of amenorrhea, oligomenorrhea or dysfunctional uterine bleeding. overall, approximately 10% of women with poi present with primary amenorrhea (2, 3). a similar case of spontaneous pregnancy has been reported in a 27-year old woman with hypergonadotropic ovarian failure (4). we suspected her to be a case of primary ovarian insufficiency on the basis of history alone and in the absence of any laboratory test of increased fsh, since we admitted her for the first time in full term pregnancy. this case was reported to emphasize the real chances for a spontaneous conception because of the intermittent and unpredictable ovarian function in these patients. primary ovarian insufficiency occurs in only 1% of women it can still result in spontaneous pregnancy in 5 - 10% of the cases. women with poi should be educated on the nature of the disease and the current research efforts. it is important to be aware of the condition and the options for future treatment. | introductionprimary ovarian failure (pof) is a syndrome composed of amenorrhea, estrogen deficiency and follicular stimulating hormone (fsh) of menopausal ranges in young women. in this article, we report a case of primary amenorrhea that presented with full term pregnancy.case presentationa 29-year old woman with a history of primary amenorrhea attended hospital with full term pregnancy. she had experienced a few episodes of withdrawal bleeding on hormonal treatment initially and she had conceived spontaneously. subsequently, she had uneventful pregnancy and caesarean delivery on maternal request.conclusionthis case was presented to emphasize the real chances of spontaneous conceptions due to intermittent and unpredictable ovarian function in patients with poi. nevertheless, egg donation is still considered the best option for infertility in such women. |
since 1978, when the first baby conceived in vitro was born, in vitro fertilization (ivf) has become a major method for treating infertility. although ivf procedures have been greatly improved over the years, their efficiency, as defined by live birth rate, is still only about 30 - 40%. consequently, many couples have to undergo treatment several times before they succeed and this increases emotional and financial costs, as well as additional health risks for women. the efficiency of ivf can be increased by transferring multiple embryos in a single cycle, but this may lead to multiple - pregnancies, and consequently serious health complications for mothers and babies. there is, therefore, a strong need for single embryo transfers, and so the development of reliable methods of selecting embryos is crucial. here, we provide a short review of current techniques for embryo assessment, with a focus on new ways of distinguishing high - quality embryos based on recent advances in time - lapse imaging technology. visual assessment of embryo morphology is the most traditional and popular method of embryo selection. several parameters can be assessed at different developmental stages, providing valuable information about the quality of embryos. embryos can be graded according to the morphology of their pronuclei on day 1 after fertilization [4 - 6 ], the number and shape of blastomeres and degree of fragmentation on day 2 or 3 [7 - 9 ], or by the morphology of the blastocyst at day 5 or 6 [10 - 12 ]. depending on the procedure, embryos are evaluated at one or several developmental stages. although morphological assessment is inexpensive and easy to implement in a clinical environment, it has its drawbacks. moreover, even when such expertise is available, the technique is not always accurate : low - graded embryos often prove to have high developmental potential and can develop to term. therefore, there has been a drive to develop an alternative method of embryo evaluation that provides more detailed information about the developmental status of the embryo and, most importantly, is quantitative and objective. it uses polymerase chain reaction (pcr)-based techniques to diagnose specific genetic mutations or fluorescence in situ hybridization (fish) to diagnose chromosomal abnormalities or to sex the embryos for patients carrying x - chromosome - linked diseases [14 - 16 ]. preimplantation genetic diagnosis can be applied to embryos at different stages : zygotes (polar body biopsy), cleavage stage embryos (blastomere biopsy) or blastocysts (trophectoderm biopsy). an alternative approach is preimplantation genetic screening, which was developed to improve ivf outcomes in mothers of advanced age, those presenting with repeated miscarriages or implantation failure, or in cases of severe male factor infertility. the classic form of preimplantation genetic screening, involving fish assessment of a limited number of chromosomes in one blastomere of a cleavage stage embryo, has been shown to be ineffective and has been gradually replaced by technologies based on comparative genomic hybridization (cgh) or single nucleotide polymorphism (snp) arrays. both of these approaches allow the whole embryonic genome to be analysed and, as for preimplantation genetic diagnosis, used at different embryonic stages. changes in pyruvate or glucose concentration in the culture medium can reflect the energy metabolism of the embryo, although their usefulness as a tool to predict the embryo 's quality is not clear [19 - 23 ]. on the other hand, oxygen consumption [24 - 27 ] and amino - acid turnover [28 - 30 ] recently, the analysis of single metabolites has been gradually replaced by a broader approach : metabolomic or protein secrotome profiling, which can provide a complete picture of an embryo 's metabolism and gene expression patterns. although many reports have shown a correlation between metabolic status of the embryo and its viability, establishing its potential value for the ivf clinics, for the moment these techniques remain difficult to implement in a clinical environment. this is because metabolomic or secreted protein analyses involve spectroscopic / spectrometric and chromatographic techniques, which currently require expensive equipment and highly skilled staff. the method described by wong. involves time - lapse imaging of embryos every 5 min for several hours (from 1- to 4-cell stage). in comparison, the method established by ajduk. requires a much shorter period of recording (2.5 hours for mouse embryos) but with images taken every 10 seconds. due to recent advances in non - invasive time - lapse imaging established in mouse embryos, we can now follow the dynamics of embryo divisions and other fertilization - triggered events and correlate them with the developmental potential of the embryos. the last two years have seen two studies employing this technique in a very different way. a team at stanford university has shown that the timing and synchrony of the first two embryonic divisions are predictive of developmental potential of human embryos (figure 1). the authors reported that embryos with a very long first cytokinesis, with a prolonged or an atypically short interval between first and second division or with highly asynchronous divisions of two - cell blastomeres, fail to reach the blastocyst stage. this accords with previous observations, in which timely pronuclear formation and subsequent first cleavage correlated with higher quality of human embryos [34 - 36 ]. a completely different approach has been developed by our team at university of cambridge working collaboratively with teams at oxford and cardiff universities (figure 2). in this work, we showed that fertilization of mouse eggs triggers abrupt, repetitive cytoplasmic movements that correlated with ca oscillations (also triggered by sperm) and depended on the functionality of the cytoskeleton. embryos that showed very frequent increases in cytoplasmic movements (indicating very frequent ca oscillations) and low cytoplasmic speeds in the intervening periods (reflecting low quality of the actin cytoskeleton) were three - fold less successful in developing to pups than embryos displaying average values of these parameters. although both methods still have to be tested in a clinical environment and subjected to randomised controlled trials to demonstrate improved live birth outcomes, they offer great hope for more reliable assessment of embryonic quality. mouse eggs are subjected to time - lapse imaging (1 frame every 10 seconds for 2.5 hours) immediately after fertilization. acquired images are analysed by the particle image velocimetry method that follows patterns of contrasts between subsequent images and calculates how they move. the sum of all displacement vectors calculated for the zygote in a given time - point (i.e. mean cytoplasmic speed) is plotted over time. mean interval between the fast movements (in red) and mean speed in periods inbetween the fast movements (mean basal speed, in blue) are indicative of developmental potential of the embryo. research carried out over the past three decades has provided a broad repertoire of possible improved embryo selection methods. some of them, such as evaluation of pronuclear morphology, preimplantation genetic diagnosis and preimplantation genetic screening, others (e.g. analysis of metabolites, secreted proteins or cytoplasmic flows) concentrate on the cytoplasmic component, analysing the quality of embryo metabolism, the cytoskeleton or ca homeostasis. this can be achieved for instance by following the timing of embryonic cell divisions, as their duration and synchrony can be affected by improper chromosome segregation, cytoskeletal properties and energy levels. an alternative approach could combine preimplantation genetic screening with the analysis of embryo metabolism or cytoplasmic movements. combination of genetic testing of the polar body and examination of the cytoplasmic flows is especially promising, as it would provide information about embryo quality within several hours after fertilization, and, therefore, significantly quicken the selection process (figure 1). indeed, recent publications suggest that a shorter period between in vitro fertilization and embryo transfer may be beneficial, as prolonged in vitro culture of embryos alters their epigenetic modifications and gene expression. through a combination of these new methods, it is reasonable to expect that embryo selection for ivf will become much more reliable in the coming years. the method of assessing embryo developmental potential based on analysis of the cytoplasmic movements is the subject of a patent application filed in the u.s. by magdalena zernicka - goetz and anna ajduk (us | despite many recent advances in the field of reproductive biology and medicine, the efficiency of in vitro fertilization procedures remains relatively low. there is a need for a reliable and non - invasive method of embryo selection to ensure that only embryos with the highest developmental potential are chosen for transfer to mothers - to - be. here, we compare various methods currently used for assessing embryonic viability, such as examination of embryonic morphology, quality of the genetic material, or metabolism. additionally, we discuss novel procedures for embryonic assessment based on advanced time - lapse imaging techniques, which show great promise and may lead to increased in vitro fertilization efficiencies. |
avaliar os efeitos da administrao sistmica precoce e tardia de metilprednisolona nos pulmes em um modelo de morte enceflica em ratos. vinte e quatro ratos wistar machos foram anestesiados e randomizados em quatro grupos (n = 6 por grupo) : sham, somente morte enceflica (me), metilprednisolona i.v. (30 mg / kg) administrada 5 min aps a morte enceflica (mp5) e 60 min aps a morte enceflica (mp60). os grupos me, mp5 e mp60 foram submetidos morte enceflica por insuflao de um balo no espao extradural. foram determinadas variveis hemodinmicas e gasomtricas, relao peso mido / seco, escore histolgico, thiobarbituric acid reactive substances (tbars, substncias reativas ao cido tiobarbitrico), atividade de superxido dismutase (sod) e de catalase, assim como contagem diferencial de clulas brancas, protena total e nvel de desidrogenase ltica no lba. a atividade da mieloperoxidase, peroxidao lipdica e nveis de tnf- foram avaliados no tecido pulmonar. no foram observadas diferenas significativas nas variveis hemodinmicas e gasomtricas, relao peso mido / seco, anlises do lba, escore histolgico, sod, mieloperoxidase e catalase entre os grupos. os nveis de tbars foram significativamente maiores nos grupos mp5 e mp60 do que nos grupos sham e me (p < 0,001). os nveis de tnf- foram significativamente menores nos grupos mp5 e mp60 do que no grupo me (p < 0,001). neste modelo de morte cerebral, a administrao precoce e tardia de metilprednisolona apresentou efeitos semelhantes sobre a inflamao e a peroxidao lipdica no tecido pulmonar. lung transplantation is an established therapeutic option for patients with end - stage lung disease. in the last decade, there has been a significant increase in the number of lung transplant centers. as a result in contrast, the donor pool has remained fairly constant, the proportion of available lungs that are suitable for transplantation ranging from 4.9% to 27%. recently, with the use of lungs from marginal donors and the encouraging results of ex vivo lung perfusion for lung reconditioning, there has been a trend toward an increase in the number of lung transplants. brain - dead donors still represent the major source of organs for transplantation. in the icu, the lungs of potential donors are exposed to direct damage caused by the process related to the occurrence of brain death. in addition, the lungs are at risk for acute lung injury secondary to trauma, prolonged mechanical ventilation, transfusion, ischemia, aspiration, and infection. brain death is defined as the death of all central neurological tissue, resulting in the loss of cerebral function. the pathophysiology of brain death is complex, involving sympathetic, hemodynamic, and inflammatory mechanisms that can injure the lung. significant endocrine changes result in a decrease in the plasma levels of adrenocorticotropic hormone, cortisol, triiodothyronine, thyroxine, insulin, and vasopressin. currently, there is a consensus that organ grafts should not be considered immunologically inert. donor risk factors, such as previous diseases, age, cause of death, donor management, and, most importantly, brain death itself, can reprogram the graft, making it an immunologically active organ. new strategies for the treatment of potential brain - dead donors represent a promising approach to reducing the immunogenicity of the graft and improving the quality of the organ before transplantation. the administration of systemic corticosteroids to brain - dead donors is known to be beneficial mostly because of its ability to modulate the systemic inflammatory response caused by brain death. this modulation improves graft viability by reducing the release of pro - inflammatory molecules and the production of leukocyte adhesion molecules, thus increasing alveolar fluid clearance. the optimal timing for the administration of corticosteroids in the setting of brain death is controversial. the aim of the present study was to evaluate the effects that early and late systemic administration of methylprednisolone have on the lungs in a rat model of brain death. the animal care committee and the research ethics committee of the porto alegre hospital de clnicas, federal university of rio grande do sul, porto alegre, brazil, approved the protocols used in the present study. twenty - four male wistar rats, weighing 170 - 200 g, underwent general anesthesia, induced by intraperitoneal administration of ketamine (100 mg / kg) and xylazine (15 mg / kg). anesthesia was followed by a tracheostomy with an indwelling 14-gauge cannula (abbocath # 14 ; abbott laboratories, abbott park, il, usa). the rats were ventilated with room air at 70 - 80 breaths / min with a tidal volume of 10 ml / kg (harvard rodent ventilator, model 683 ; harvard apparatus co., millis, ma, usa). the right carotid artery was dissected and cannulated with a 24-gauge cannula (becton dickinson, franklin lakes, nj, usa) for mean arterial pressure and hr recordings on an ink - jet recorder (sirecust 730 ; siemens - elema, solna, sweden). the right jugular vein was dissected and cannulated in the same fashion. normal saline was used to flush the lines, with a total volume of 5 ml / kg per hour in all animals, and a heated surgical table was used to maintain the body temperature at 37c during the procedure. after the initial procedures, the animals were randomly allocated to one of the following four groups (6 animals per group) : sham (sham - operated) : a craniotomy was performed, but no balloon catheter was introduced into the extradural space.bd (brain death only) : brain death was induced ; at 5 min after brain death had been confirmed, 0.2 ml of normal saline was administered intravenously. mp5 (methylprednisolone after 5 min of brain death) : brain death was induced ; at 5 min after brain death had been confirmed, an intravenous bolus of methylprednisolone (30 mg / kg) diluted in 0.2 ml of normal saline was administered mp60 (methylprednisolone after 60 min of brain death) : brain death was induced ; at 60 min after brain death had been confirmed, an intravenous bolus of methylprednisolone (30 mg / kg) diluted in 0.2 ml of normal saline was administered sham (sham - operated) : a craniotomy was performed, but no balloon catheter was introduced into the extradural space. bd (brain death only) : brain death was induced ; at 5 min after brain death had been confirmed, 0.2 ml of normal saline was administered intravenously. mp5 (methylprednisolone after 5 min of brain death) : brain death was induced ; at 5 min after brain death had been confirmed, an intravenous bolus of methylprednisolone (30 mg / kg) diluted in 0.2 ml of normal saline was administered mp60 (methylprednisolone after 60 min of brain death) : brain death was induced ; at 60 min after brain death had been confirmed, an intravenous bolus of methylprednisolone (30 mg / kg) diluted in 0.2 ml of normal saline was administered the brain death model has previously been described in detail. in brief, through a frontolateral trepanation (1 1 mm with a dental drill), a 14 g fogarty balloon catheter (baxter healthcare corp., irvine, ca, usa) was introduced into the extradural space with the tip pointed caudally. the balloon was inflated with 1.5 ml of water for 1 min, producing a sudden rise in intracranial pressure, which resulted in rapidly progressive brain injury, leading to immediate brain death. a sharp rise and then a subsequent drop of blood pressure and hr defined the initiation of brain death. the state of brain death was confirmed by the absence of corneal reflexes and by the apnea test. the animals were monitored for 120 min, during which the pre - procedure ventilation parameters were maintained. arterial blood samples were drawn for blood gas analyses : at the time of the insertion of the arterial line (baseline) ; and at 60 and 120 min after the procedures. upon completion of the assessment, the heart - lung blocks were excised, the right main bronchus was clamped, and the left lung was submitted to bal three times (3 ml of normal saline each time). the right lung was then separated from the heart - lung block and divided into two parts. most of the right lung was snap frozen in liquid nitrogen and stored at 80c for subsequent analyses of lipid peroxidation, catalase activity, superoxide dismutase activity, and tnf- levels. the mediastinal lobe was excised and weighed (wet weight) on a precision scale. samples were then placed in a vacuum oven (at 70c for 72 h) until a stable dry weight had been achieved. the wet - to - dry ratio of the lung weight was then calculated and used as an indicator of lung edema. the bal fluid (balf) was centrifuged at 300 g for 5 min, and the resulting cell pellet was separated from the supernatant. the total protein concentration was determined using the biuret method (modular analytics ; roche diagnostics, penzberg, germany), whereas the lactate dehydrogenase concentration was determined using the uv kinetic method (modular analytics). after the samples had thawed, a 96-well plate was coated with monoclonal antibody for the determination of tnf- levels. the wells were filled with either 100 l of homogenized lung (dilution 1:2), 100 l of positive control, 100 l of negative control, or 100 l of recombinant tnf-, in concentrations established by the manufacturer of the antibody (creative biomart, new york, ny, usa). to each well, we then added 100 l of polyclonal anti - tnf- conjugated with peroxidase and incubated the plates for 3 h at room temperature. after the incubation period, the plate was washed four times with a detergent solution. a color change was then induced by adding hydrogen peroxide (0.02%) and tetramethylbenzene (2%). color intensity was measured in an automated elisa reader (titertek multiskan ; flow laboratories, mclean, va, usa), at a wavelength of 450 nm, and is expressed as optical density. the tnf- concentration in the homogenized lung was calculated from the results obtained with a standard curve. the products generated by lipid peroxidation were quantified by the thiobarbituric acid reactive substance (tbars) method using 3 mg of protein per sample. the samples were incubated at 90c for 30 min, and 500 l of 0.37% thiobarbituric acid and 15% trichloroacetic acid were then added to the samples, which were centrifuged at 2,000 x g at 4c for 15 min. the activity of superoxide dismutase was determined by pulse radiolysis based on the auto - oxidation of epinephrine in accordance with the method described by misra & fridovich. myeloperoxidase activity was defined as the quantity of enzyme degrading 1 mol of peroxide / min at 37c and was expressed in mu / g of wet tissue. the fragment of lung fixed in formalin was embedded in paraffin, cut into 3-mm sections and stained with h&e. a pathologist blinded to the experimental protocol performed the quantitative examination by light microscopy. each lung sample was examined at low and high magnifications, and 20 fields were randomly selected and analyzed. the severity of histological lesions was assessed using a five - parameter score that included intra - alveolar edema ; hyaline membrane formation ; hemorrhage ; focal alveolar collapse or consolidation ; and epithelial desquamation or necrosis of airways or alveoli. each parameter was evaluated semi - quantitatively using the following scale : 0 = absent ; 1 = mild ; 2 = moderate ; and 3 = prominent. for each animal, all of the data collected were coded, recorded, and analyzed using the statistical package for the social sciences, version 16.0 (spss inc., when anova showed a significant difference, tukey 's post hoc test for multiple comparisons was applied in order to evaluate that difference. we used repeated measures anova for intergroup comparisons related to dependent variables that were measured more than once during the observation period. for each test, the data were expressed as means and standard errors, and the level of significance was set at p < 0.05. there were no differences among the groups regarding the duration of the procedure ; wet / dry weight ratio ; arterial blood gas measurements ; myeloperoxidase activity ; superoxide dismutase activity ; catalase activity ; or (in the balf) differential white cell count, total protein content, and lactate dehydrogenase levels. nor were there any differences among the groups in terms of mean arterial pressure during the observation period. the levels of tbars were significantly higher in the two groups treated with methylprednisolone than in the sham and bd groups (p < 0.001 ; figure 1). there were no significant differences in the histological scores among the groups (figure 2). lung injury, represented by intra - alveolar hemorrhage or hyaline membrane formation, was not observed in any samples. the predominant finding was mild focal alveolar collapse, which was present in the majority of the samples, at similar intensity across the groups (figure 2). the levels of tnf- were significantly lower in the mp5 and mp60 groups when compared with the bd group (p < 0.001 for both ; figure 3). there was a significant increase in lipid peroxidation using the thiobarbituric acid reactive substance method in the groups that received methylprednisolone at 5 and 60 min after brain death (mp5 and mp60, respectively) when compared with the sham and brain death (bd) groups. 0.001. figure 2histological score and microscopic optical analysis (h&e ; magnification, 400). groups : sham ; brain death (bd) ; methylprednisolone at 5 min after brain death (mp5), and methylprednisolone at 60 min after brain death (mp60). there was a significant increase in tnf- levels in the brain death (bd) group when compared with the methylprednisolone at 5 min after brain death (mp5) and methylprednisolone at 60 min after brain death (mp60) groups. the vast majority of experimental studies have shown a significant increase in inflammatory molecules and interleukins immediately after the induction of brain death. the pro - inflammatory cytokine tnf- has been shown to be significantly up - regulated in the lung tissue after brain death and plays an important role in the development of lung injury. in the present study, brain death resulted in a systemic inflammatory response and the administration of methylprednisolone was associated with reduced tnf- expression in lung tissue. mclean. obtained similar results in a study of the effects that a glucocorticoid has on myocardial function in a porcine model of brain death. to our knowledge, the present study is the first to investigate the role of the administration of methylprednisolone in a rat model of brain death and its influence on tnf- expression and oxidative stress in the lungs. our study demonstrated that the administration of methylprednisolone has no effect on arterial blood gas and hemodynamic parameters. the lack of differences in these findings might be related to the short period of observation. the administration of methylprednisolone immediately after brain death in a rat model of lung transplantation has been shown to modulate the inflammatory injury of the donor lung, resulting in better graft performance after transplantation. however, various clinical studies using different hormone resuscitation protocols have shown promising results. nevertheless, a standard protocol has yet to be established, especially with respect to lung retrieval. the use of glucocorticoids in brain - dead donors probably has dual beneficial effects, because it reduces inflammatory activity and replaces cortisol levels in the blood. although we studied tnf- activity after brain death, it would have been interesting to have also measured the cortisol levels after brain death in this model. in a baboon model, faropoulos & apostolakis found that cortisol levels rise within 5 min after the induction of brain death, declining steadily thereafter (over the next 15 - 45 min) and becoming undetectable at 4 h after brain death. in our experiments, we found no differences in the differential white cell count in balf or in histology. we suppose that lung inflammatory changes can be assessed up to 120 min of ventilation after brain death induction in lungs that are still suitable for transplantation. however, there have been reports based on other experimental models of lung injury and brain death that advocate for the analysis of pulmonary inflammation within the first 6 h after brain death. the present study has limitations, and care must be taken when extrapolating its results to a clinical situation. we used a standardized, small animal model of brain death that includes a sudden rise in intracranial pressure, which differs from what is often found in patients evolving to brain death, in whom intracranial pressure rises at a slower pace. another difference is that the time from brain death to organ harvest is typically much longer in the clinical scenario than it was in our model. a brain death model in which intracranial pressure is gradually induced by the inflation of a subdural balloon catheter has been developed. this improved model allows for the study of donor organ quality without the use of an inotropic infusion. regarding corticosteroid administration, there is evidence that methylprednisolone given every 24 h (at 15 mg / kg i.v.) during brain death improves the effectiveness of the subsequent lung transplantation. therefore, we decided to use a higher dose of methylprednisolone in an attempt to potentiate the anti - inflammatory effects. the ideal period for the administration of the corticosteroid is still controversial in the literature. in the clinical scenario, it is expected that treatment with corticosteroids will be introduced as soon as possible in order to reduce the inflammatory activity. we considered 60 min after brain death to be late administration, given that, in a controlled animal model of brain death, the inflammatory activity at 60 min after brain death would be considerably higher than at 5 min after brain death. the results of the present study show that the early administration of methylprednisolone after acute brain death does not improve oxygenation indices or hemodynamic parameters in lungs ventilated for 120 min. we also demonstrated an increase in the generation of lipid peroxidation products in the groups treated with methylprednisolone after 5 and 60 min of brain death. as previously demonstrated, traumatic brain injury significantly increases lipid peroxidation levels in lung tissue only 24 h after the trauma, which could explain our lipid peroxidation results. however, the exact causes of these findings can not be determined from our experiments. we conclude that, in this model of brain death, early and late administration of methylprednisolone have similar effects on inflammatory and lipid peroxidation activity in lung tissue. we suggest that late administration of methylprednisolone can be used in settings of brain death, because of its potential anti - inflammatory effect. further studies investigating the effects of late administration of methylprednisolone in the setting of lung transplantation following brain death are needed in order to confirm its beneficial effects. | objective : to evaluate the effects that early and late systemic administration of methylprednisolone have on lungs in a rat model of brain death. methods : twenty - four male wistar rats were anesthetized and randomly divided into four groups (n = 6 per group) : sham - operated (sham) ; brain death only (bd) ; brain death plus methylprednisolone (30 mg / kg i.v.) after 5 min (mp5) ; and brain death plus methylprednisolone (30 mg / kg i.v.) after 60 min (mp60). in the bd, mp5, and mp60 group rats, we induced brain death by inflating a balloon catheter in the extradural space. all of the animals were observed and ventilated for 120 min. we determined hemodynamic and arterial blood gas variables ; wet / dry weight ratio ; histological score ; levels of thiobarbituric acid reactive substances (tbars) ; superoxide dismutase (sod) activity ; and catalase activity. in bal fluid, we determined differential white cell counts, total protein, and lactate dehydrogenase levels. myeloperoxidase activity, lipid peroxidation, and tnf- levels were assessed in lung tissue. results : no significant differences were found among the groups in terms of hemodynamics, arterial blood gases, wet / dry weight ratio, bal fluid analysis, or histological score - nor in terms of sod, myeloperoxidase, and catalase activity. the levels of tbars were significantly higher in the mp5 and mp60 groups than in the sham and bd groups (p < 0.001). the levels of tnf- were significantly lower in the mp5 and mp60 groups than in the bd group (p < 0.001). conclusions : in this model of brain death, the early and late administration of methylprednisolone had similar effects on inflammatory activity and lipid peroxidation in lung tissue. |
neuropsychiatric manifestations are included : p0 eripheral neuropathy, myeloneuropathy, cerebellar ataxia, optic atrophy, mood disorders, psychosis, personality changes, loss of memory, depression, dementia, confusion and more rarely reversible manic and schizoferniform status and obsessive compulsive disorder (ocd).[18 ] psychiatric manifestations of b12 deficiency seldom precede anemia.[68 ] we present a case of b12 deficiency in which ocd precedes anemia. a 29-year - old female came with anxiety and history of ocd since 11 years ago. general blood chemistries including thyroid function tests, liver function tests, renal function tests, cbc diff, hemoglobin level and iron profile had been performed 5 months ago and all were in normal ranges [table 1 ], but further investigations in recent visit showed mild anemia (hb=11.8 g / dl, mcv=89 fl) and markedly diminished serum cobalamine level to < 30 pg / ml and also iron deficiency with significant decreased ferritin level to 1.28 ng / ml [table 2 ]. laboratory findings of first visit laboratory findings of the recent visit the association between b12 deficiency and iron deficiency in this case was our explanation to her normocytic anemia. diagnosis of b12 deficiency with ocd manifestation and concurrent iron deficiency was made and parenteral b12 and oral iron replacement therapy initiated. in this 29-year - old female, ocd was the early manifestation of b12 deficiency. although, it was rarely reported, but psychiatric and mood disorders may be the first manifestation of b12 deficiency and precede anemia.[68 ] we recommend checking serum b12 and folate level in any case with psychiatric disorder such as ocd, even in the absence of anemia and other hematologic manifestations of b12 and/or folate deficiencies. b12 replacement therapy can resolve symptoms of psychiatric disorders in patients with b12 deficiency.literature review shows that patients with ocd have dysregulation in serotoninergic system and efficacy of serotonin reuptake inhibitors (sris) in the treatment of ocd was demonstrated.[912 ] neurotransmitters (dopamine, serotonin and melatonin) are necessary for a normal balanced mood, emotions and also sleeping. folic acid and vitamin b12 act as cofactors in synthesis of neurotransmitters such as serotonin and norepinephrine. although it is rare but psychiatric manifestations of b12 deficiency may precede anemia as we saw in this case. | b12 acts as a cofactor in synthesis of neurotransmitters such as serotonin and dopamine, thus b12 deficiency affects mood, emotions and sleeping and can lead to psychiatric disorders. psychiatric manifestations of b12 deficiency are varied. they seldom precede anemia. we want to present a case of b12 deficiency which was presented with obsessive compulsive disorder. |
the general population is exposed to palladium (pd) due to inhalation and contact with jewelry and especially dental restorations.1,2 although the use of palladium in dentistry had been described already in 1933,3 its wide - scale use started in the early 70 's due to the increasing gold price demanding the use of other metals as lower - cost alternatives.4 since then, the palladium content in dental alloys has exponentially increased. for dental applications, palladium is alloyed with other metals such as gold (au), silver (ag), copper (cu), gallium (ga) and zinc (zn).5 among other alloys, palladium - silver based (pd - ag) and palladium - copper based (pd - cu) alloys became attractive for restorative dentistry. dental restorations are in situ for many years and are exposed to many factors such as mechanical load and the conditions of the oral environment that lead to substantial metal ion release with possible local and systemic adverse reactions as a result.2,5,6 health concerns about palladium are mainly due to its effect on the immune system. palladium is ranked, after nickel (ni), as the second most frequent reacting skin sensitizer within metals in epidemiological studies with a prevalence of 7.4% in dental patients.1 furthermore, ni allergic individuals are more susceptible to pd, and the majority of the people with pd allergy are also sensitive to ni due to cross reactivity.2,7 clinically, signs of allergic contact dermatitis and allergic contact granuloma, contact stomatitis with gingival hyperplasia and oral lichen planus are described in case reports on pd sensitization after exposure to pd from dental restorations. also, general symptoms such as swelling of the lips and cheeks, burning mouth, dizziness, asthma, chronic urticaria have been described. besides, occupational exposure to pd may occur in dental technicians, miners and workers of the electronics and chemical industries.1,2,7 it has been shown that alloys containing palladium may release up to 33.7 gcmweek metallic ions in a solution of sodium chloride and lactic acid at a ph of 2.3.8 of note, these in vitro experiments used alloys that were industrially cast, standardized shaped, and highly polished and therefore do not resemble the in vivo situation. many factors influence corrosion of the dental alloys such as the composition9,10 and the microstructure of the alloy,5,11,12 but also the surrounding environment6,13,14 and the manufacturing process.15 during the casting process impurities might be included in the alloys, resulting in a product with different composition from what manufacturer claimed for an " as - received " alloy. this may result either from the material transformation during the casting procedure or from the inclusion of residual metals from previous work. this requires caution since it was shown that recasting can reduce the corrosion resistance of pd - based alloys.15 all together, these factors might influence the corrosion of the final product, leading to different corrosion properties from what manufacturer claimed. nevertheless, the studies on the electrochemical properties of these alloys after casting reported satisfactory corrosion behavior.11,15 also, when testing high - palladium alloys, a spontaneous passive behavior in electrochemical conditions similar to those in the oral environment was shown16 and a ag - based pd alloy could be recast up to 4 times with little effect on its corrosion susceptibility in artificial saliva.17 the aim of the present study was to evaluate whether the surface treatment and shape of the dental alloy would influence the elemental release of cast standardized shapes and crowns, compared to the " as received " pd - ag and pd - cu alloys. the corrosion behavior of two crowns, two disks and the " as - received " alloys of a pd - cu (orion vesta, elephant dental b.v., hoorn, netherlands) and a pd - ag (orion argos, elephant dental b.v., hoorn, netherlands) alloy was evaluated. the disks (d = 10.0 mm ; h = 1.4 0.1 mm thick) and the crowns (polycarbonate incisor shaped temporary crowns (p-101), 3m espe, st. paul, mn, usa), were cast according to manufacturer 's instructions using the lost - wax technique, phosphate - bonded graphitefree casting investment and individual ceramic crucibles per alloy ; melting was done by means of a gas - oxygen torch. the two crowns and two disks were cast at the same time on one sprue / base to ensure that the variation due to the casting procedure is minimized. the final surface areas of the specimens were : 2.06 cm for the disks, 2.57 cm for the crowns and 1.17 cm for the " as - received " alloys. one crown and one disk from each alloy type were wet - ground with 600-grit and 1200-grit grinding paper. the other pair and the " as - received " alloy from each alloy type remained unpolished. to submerse the specimens in the test medium, a suspension point of composite (filtek supreme xt, 3 m espe) with a nylon wire the specimens were ultrasonically cleaned with alcohol 99.9% (emsure, merck, germany) for 5 minutes. the plastic vials were rinsed with distilled water and filled with 1% hno3 (merck suprapur) for 24 hours after which, they were rinsed again with distilled water. the specimens were submersed for 7 days in 5 ml at 37. the solution used consisted of a lactic acid / sodium chloride solution from the iso 10271 standard (dental metallic materials - corrosion test methods)18 : ph = 2.3 0.1, [nacl ] = 8.845 g (merck suprapur), [lactic acid 90% ] = 9.2 the samples were diluted 10x with 1% hno3 (merck suprapur) and analysed by inductively coupled plasma mass spectroscopy (icp - ms)(elan 6100, sciex, toronto, canada). the palladium concentration was measured with a quantitative analysis method (no. of replicates = 8) using a blank and standard solutions of palladium of 10.0 ppb and 100.0 ppb (vwr international ltd. the detection limit, defined as 3x the standard deviation of the samples, was 0.7 ppb or 0.035 gcmweek for palladium for the current test procedure. the concentrations of au, ag, cu, sn, in and ga were measured using semi - quantitative analysis method (totalquant ; no. of replicates = 1). totalquant, being a semi - quantitative program, gives quantitative results typically within + /-25% of the real value in simple matrices. the following settings were used for all analysis : rf power 1100w ; nebulizer gas flow rate 0.92 l / min ; fluid peri pump rate 24 rpm. control samples were also analysed and the values for each ion were subtracted from each analysed sample. these controls were the experimental media (i.e. lactic acid / sodium chloride solution) without contact with the specimens. before and after submersion in the lactic acid / sodium chloride solution the specimens were also examined with scanning electron microscopy and energy dispersive x - ray spectrometry (sem / edax ; xl20, philips / fei, eindhoven, the netherlands) for surface structure characterization using the secondary electrons detector and the mapping mode of the edax. furthermore, the composition of the alloys (table 1) was analysed by edax with a detection limit of 0.2 wt%. the results of the quantitative analysis method of the pd ion release (n=8) were statistically analysed using oneway anova and tukey post - hoc tests at a p - level of.05. the software used was sigmastat 3.1 (systat software, inc., point richmond, ca, usa). it should be noted that all data, experimental and literature, are converted to gcmweek, using the given experiment conditions (volume of the medium, specimen surface area, observation period). the metal ion release from the 5 specimens of the two alloys studied is summarized in table 2. one - way anova (p.05), and (ii) the " as received " pd - ag alloy (orion argos) and the polished crown of the pd - ag alloy (orion argos) (p>.05). casting, surface treatment, and shape of the specimen had a significant effect on the palladium release from both alloys. casting the pd - cu alloy (orion vesta) followed by polishing never resulted in the pd release of the " as received " alloy (0.69/0.25 vs. 0.11 gcmweek). casting the pd - ag alloy (orion argos) followed by polishing resulted in the same pd release of the " as received " alloy but only for the crown specimen (0.34/0.06 vs. 0.07 gcmweek). the unpolished disks released higher amounts of palladium for both types of alloy, where pd - cu alloy (orion vesta) released the highest amounts. 2 shows the sem and edax mapping images (pd and ag) of the surface of the polished specimens of pd - ag alloys (orion argos), before and after immersion in the lactic acid / sodium chloride solution and fig. 3 the sem and edax mapping images (pd and ag) of the surface of the unpolished specimens. the edax maps of pd and ag showed that the microstructure of the pd - ag alloy was homogenous. unpolished specimens presented a coarser surface than their polished pairs. after immersion in the lactic acid / sodium chloride solution medium no or little differences in the surface appearance could be seen. the sem / edax images of orion vesta were similar (data not shown). the typical components of palladium - based alloys are silver (ag), palladium (pd), gallium (ga) and copper (cu).5 besides, our specimens also contained gold (au), tin (sn), indium (in), and trace amounts of zinc (zn), ruthenium (ru) and iridium (ir). these are thought to improve the mechanical properties of the dental alloys.19 the release of trace amounts of cu from the pd - ag alloy (orion argos) and of sn from the pd - cu alloy (orion vesta) was detected by icp - ms analysis of the immersion solutions. since the casting process was done as contamination - free as possible (e.g. use of a phosphate - bonded graphite - free casting investment and individual ceramic crucibles per alloy), the presence of this elements on the solution is likely due to the presence of trace amounts of these elements in the original alloy, under the detection limit of the edax (< 0.5 wt%). despite the presence of gold (au) in both types of alloys, for all specimens, pd - cu alloy (orion vesta) released higher amounts of pd than pd - ag alloy (orion argos) did. even though the wt% of pd was higher than that of other metals, the release of silver (ag) from the pd - ag alloy (orion argos) and copper (cu) from the pd - cu alloy (orion vesta) was higher than the release of pd itself from each type of alloy. this is in line with the theory that the less noble metal within the alloy is released more easily according to the liabilities of each metal. it is also consistent with earlier findings that the pd release is not proportional to the content of pd in the composition of the dental alloy.10,20 an explanation for the corrosion behavior of pd - cu and pd - ag alloys was proposed by sarkar.21 in which intraoral corrosion of high - pd alloys was associated with dealloying of base metals. further, their proposition on the protective nature of ag in limiting pd release might also have been observed in our findings. the pd - ag alloy released less pd compared to the pd - cu alloy, even after accounting for the higher pd content in the composition of the pd - cu alloy (i.e. the release of pd from the pd - cu alloy was always at least double than that of pd - ag even though its pd content was not). besides the main components of the alloys studied (pd, ag, and cu), sn, in and ga also showed a variable amount of corrosion products. the influence of those products in the cytotoxicity and biocompatibility of these alloys deserves further studies. comparison of the unpolished disk and the unpolished crown showed that the corrosion of the disk was 3 - 4 times higher. the same observation was made for the polished specimens and also in both alloys. to our knowledge, there are no previous reports on the influence of shape on the corrosion of pd - based dental alloys and an explanation is not straightforward. besides shape, the difference between the crown and the disk is the layer thickness : the crown is generally slightly thinner, with approximately 1 mm, which may account for the different release. furthermore, the corrosion resistance of these alloys can also be influenced by manipulation of the alloys, i.e. the casting procedure and finishing and porcelain - firing heat treatment.15,22,23 during the casting procedure, thin margins are affected differently by heat / cooling procedures, which can result in different microstructure and subsequently influence properties such as corrosion resistance.24,25,26 analysis of the surface structure under the sem showed a contrast between the homogeneous surface of the polished specimens and the rough, coarse appearance of the unpolished ones. independently of the shape, polished specimens released much lower amounts of pd than their unpolished pairs for both types of alloys, which is in line with previously reported results.22 although the rough surface of the unpolished specimens has a bigger surface area, this will not account for the 4 to 64 fold increase in the corrosion rate. the most important factor in the increase in corrosion might therefore be the crevice corrosion in the coarse surface instead of the enhancement surface area due to the roughness. the polishing process can also create a homogeneous surface which may enable the formation of the protective layer, minimizing corrosion to some extent. it was reported that high noble cast alloys containing gold (au) have the highest corrosion resistance, followed by au - based and pd - based noble alloys.27 au - pd alloys were considered to have the highest corrosion resistance compared to other au, ag, pd and ni - based alloys.13 in our study, the lower pd release from pd - ag alloy (orion argos) is in accordance with this. furthermore, au - pd, pd - ag and au - pt - pd alloys were reported to release minimal amounts of pd (< 10 gcmweek).8 in our study, only " as - received " alloys (and the polished crown) released low amounts of pd, while the disk and crown specimens released much higher amounts of pd. this suggests that the casting process and manipulation to shape the specimen introduce modifications in the alloy that play an important role in the corrosion resistance of the resultant product. for the clinical implication it is interesting to compare the pd release to the release of nickel (ni), especially because it was shown that these molecules have a cross reactivity for hypersensitivity.28,29,30 the release rate for ni assemblies that are inserted into pierced ears and other pierced parts of the human body should be less than 0.2 gcmweek (eu nickel directive 94/27/ec). for pd we may estimate that its release rate should be less than 0.4 gcmweek considering its molecular mass. while the " as received " alloys are well below this limit, cast pd - ag is slightly below the limit and cast pd - cu alloys exceeds it. it should be noted that these values are obtained in a lactic acid solution as determined by the iso standard for corrosion testing. however, these circumstances are also possible in the oral environment. therefore, since the element release is fundamental for adverse reactions, we recommend manufacturers to include the element release from the dental alloys after iso standard corrosive tests in their material 's safety data sheet. shape and surface treatment influence the metallic ion release from pd - based dental alloys with polishing being a determinant factor. the release rate of pd from cast and polished pd alloys is between 0.06 - 0.69 gcmweek ; pd - cu alloy released more pd than pd - ag alloys. these values are close to or exceed the eu nickel directive 94/27/ec compensated for the molecular mass of pd (0.4 gcm week) finally, since the element release is fundamental for adverse reactions and the composition of the alloy does not determine the element release, we recommend manufacturers to include the element release from the dental alloys after iso standard corrosive tests beside the original composition of the as - received alloy in their material 's safety data sheet. | purposethe purpose of this study was to evaluate the effects of the surface treatment and shape of the dental alloy on the composition of the prosthetic work and its metallic ion release in a corrosive medium after casting.materials and methodsorion argos (pd - ag) and orion vesta (pd - cu) were used to cast two crowns and two disks. one of each was polished while the other was not. two as - received alloys were also studied making a total of 5 specimens per alloy type. the specimens were submersed for 7 days in a lactic acid / sodium chloride solution (iso standard 10271) and evaluated for surface structure characterization using sem / edax. the solutions were quantitatively analysed for the presence of metal ions using icp - ms and the results were statistically analysed with one - way anova and a tukey post - hoc test.resultspalladium is released from all specimens studied (range 0.06 - 7.08 gcm-2week-1), with the pd - cu alloy releasing the highest amounts. for both types of alloys, ion release of both disk and crown pairs were statistically different from the as - received alloy except for the pd - ag polished crown (p>.05). for both alloy type, disk - shaped pairs and unpolished specimens released the highest amounts of pd ions (range 0.34 - 7.08 gcm-2week-1). interestingly, in solutions submerged with cast alloys trace amounts of unexpected elements were measured.conclusionshape and surface treatment influence ion release from dental alloys ; polishing is a determinant factor. the release rate of cast and polished pd alloys is between 0.06 - 0.69 gcm-2week-1, which is close to or exceeding the eu nickel directive 94/27/ec compensated for the molecular mass of pd (0.4 gcm-2week-1). the composition of the alloy does not represent the element release, therefore we recommend manufacturers to report element release after iso standard corrosion tests beside the original composition. |
the human intestinal microbiota is a complex community composed of at least several hundred different species of bacteria with approximately 1010 cells per gram of feces [14 ]. the intestinal microbiota plays a critical role in human health including colonization resistance, nutrition, metabolism of nondigestible dietary components and xenobiotics, proliferation and differentiation of intestinal mucosal epithelial cells, and homeostasis of the immune system [57 ]. direct analysis of the intestinal microbiota in the human colon is inherently difficult for routine experiments. therefore, most studies are conducted with human fecal specimens, animal models, in vitro batch culture, and continuous culture systems that mimic the human gastrointestinal tract. however, recently, a study using high - throughput anaerobic culture techniques reported that 56% of human fecal microbiota belongs to readily cultured species, over 40% of gut microbiota was uncultured species to date [3, 8, 9 ]. one of the reasons for this limitation generates from the difficulty of providing all of the appropriate nutrients and conditions for growth of the complex intestinal microbiota community. therefore, research to provide more information on in vitro culture conditions as the study by goodman. would enhance the evaluation of perturbation of the intestinal microbiota by factors that might adversely affect human health. molecular techniques that target the 16s rrna gene and other genetic markers have been used to analyze microbial community ecology in the human intestine. denaturing gradient gel electrophoresis (dgge) has been used to monitor differences and changes in the overall microbial community from fecal samples [1012 ], and quantitative real - time pcr has provided numerical abundance data for fecal microbiota [13, 14 ]. recently, application of high - throughput techniques such as pyrosequencing and the human intestinal tract chip (hitchip) microarray have been used to obtain deep phylogenetic analysis of intestinal microbiota [12, 1517 ]. in the present study, dgge, real - time pcr, and pyrosequencing were used to profile the abundance and diversity of the bacterial community from human fecal inoculum grown under different culture conditions. the aim of this study was to compare various batch culture conditions for activating and maintaining a complex fecal microbiota community to mimic growth conditions of the gastrointestinal tract. the culture conditions developed in this investigation can be applied for future research to determine the impact of antimicrobial agents, food contaminants, xenobiotics, probiotics, and dietary supplements on the human intestinal microbiota. each fecal sample was coded individual a, b, c, and d. fecal samples were cultured in brain heart infusion (bhi) broth, modified high - concentration carbohydrate medium (hcm), or low - concentration carbohydrate medium (lcm) [18, 19 ]. the composition of high- and low - carbohydrate media is described in table 1. feces were diluted with anaerobic maximum recovery diluent (mrd ; labm idg, bury, uk) buffer to a final concentration of 25% (w / v). to compare the difference of microbiota grown in three media, fecal suspensions were diluted to give an inoculum concentration of 1% (w / v), then inoculated in each medium (10 ml of final volume), and cultured anaerobically at 37c. the growth was analyzed by optical density (od) and flow cytometry on an accuri c6 fcm (accuri cytometers, ann arbor, mich, usa) following the manufacturers instruction with collected samples at each time point. to determine the optimal incubation time and check the metabolic activity of the microbiota, 1% fecal suspension decolorization of gentian violet indicates the metabolic activity of fecal microbiota [20, 21 ]. fecal supernatants were assessed as medium supplements to determine whether unknown growth factors affect in vitro growth of intestinal microbiota. individual fecal supernatant was prepared from 25% diluted fecal specimens with anaerobic mrd buffer after centrifugation at 11,000 rpm for 30 minutes. autoclaved fecal supernatant was added to each medium with a final concentration 1% (v / v). the optimal fecal inoculum concentration was also determined by the inoculating 0.1 to 5% of inoculum to low carbohydrate medium. the growth of each inoculum was analyzed by optical density at 600 nm and by flow cytometry (fcm). genomic dna was extracted from 1 ml of samples at each time point using the dna elution accessory kit of the rna power soil total rna isolation kit (mobio laboratories, carlsbad, calif, usa) by following the manufacturer 's protocol. preliminary experiments showed that this kit had the best extraction efficiency (produce high concentration of dna from same amount of fecal material) among several kits (data not shown). to conduct dgge analysis, 16s rrna gene fragments of the v3 region were amplified using primers gc - clamp-340f (5-tcc tac ggg agg cag cag-3) and 518r (5-att acc gcg gct gct gg-3) as described [22, 23 ]. the pcr reaction was performed using a mastercycler gradient instrument (eppendorf, hauppauge, ny, usa), in a final volume of 50 l with 10x taq buffer, dntp mixture (takara, shiga, japan), 10 m of each primer (mwg - biotech, ebersberg, germany), 2 u of taq polymerase (ex taq ; takara), and 1 l of template. after initial denaturation at 94c for 5 minutes, amplification consisted of 30 cycles of denaturation (30 seconds, 94c), primer annealing (30 seconds, 55c), and primer extension (30 seconds, 72c) and a final extension step of 7 minutes at 72c. the pcr product was checked by using 2% agarose gel electrophoresis and visualized using a gel doc system (biorad, hercules, calif, usa). pcr products were concentrated and purified with the qiaquick pcr purification kit (qiagen inc., valencia, calif, usa). dgge was conducted using a d - code system (biorad) with 8% (w / v) polyacrylamide gels contained 40%65% denaturant gradient, 1 mm thick, in 1x tae buffer. the equal amounts of purified pcr products were loaded on gel, and electrophoresis was performed at 25 v for 15 minutes then at 70 v for 16 hours and 30 minutes at 60c. the gel was stained in 250 ml of running buffer containing ethidium bromide (50 g ml) for 15 minutes and then rinsed in 250 ml of running buffer for 20 minutes. the sequences were identified using blast search on the genbank database and the database of type strains at eztaxon server. the profile of dgge gel was analyzed with the bionumerics program, version 6.0 (applied maths, st.- martens - latem, belgium). cluster analysis of the band pattern was performed using the unweighted pair group method using arithmetic averages (upgma) and the similarity between lanes was calculated based on the band position. the dice coefficient was used to create dendrograms of the dgge profiles obtained from different samples. real - time pcr were performed in a final 25 l volume containing 12.5 l of 2x iq sybr green supermix (biorad), 10 m of each primer (mwg - biotech), 1 l of template dna (tenfold dilution series of standard and samples dna) or distilled water (negative control). bact349f (5-agg cag cag tdr gga at-3) and bact518r (5-att acc gcg gct gct gg-3) were used to quantify total bacteria, btr275f (5-cga tgg ata ggg gtt ctg-3) and btr555r (5-ccc ttt aaa ccc aat raw tcc gg-3) were used for bacteroidetes, firm350f (5-ggc agc agt rgg gaa tct tc-3) and firm814r (5-aca cyt agy act cat cgt tt-3) were for firmicutes [2628 ]. the quantifications were performed with three independent real - time pcr runs using the cfx96 real - time pcr detection system (biorad), associated with cfx manager interface software (version 1.0.1035.131 ; biorad). the amplifications were carried out with the following steps : 50c for 2 minutes, 95c for 10 minutes, and 40 cycles of 95c for 10 seconds and 60c for 30 seconds. melting curve data were obtained from 60c to 95c at a rate of 0.5c sec with continuous measurements of the sybr green i signal intensities. dnas from cultures of escherichia coli atcc25922, bacteroides eggerthii atcc27754, and clostridium butyricum atcc19398 were used to construct standard curves for quantification by plotting the ct values obtained from amplification of dilution series. for pyrosequencing, amplification of genomic dna was performed using barcoded primers, which targeted the v1 to v3 region of the bacterial 16s rrna gene. the amplification, sequencing, and basic analysis were performed according to the methods described by chun. and completed by chunlab inc. (seoul, korea) using a 454 gs flx titanium sequencing system (roche, branford, conn, usa). briefly, analyzed sequencing reads of each sample were separated by unique barcode and filtered to remove reads, which was shorter than 300 bp or the average quality score of read was less than 25 or containing 2 more ambiguous nucleotides (ns), and then removed chimera products for further analyses [29, 30 ]. the extended eztaxon database (http://www.eztaxon-e.org/), which contains representative sequences of both cultured and uncultured bacteria with hierarchical taxonomic classification, was used for taxonomic assignments. the pyrosequencing reads were compared with sequences in the eztaxon - e database using blastn search and obtained similarity using pairwise comparison, and then the sequences were assigned a taxonomic classification through using the criteria of 97% sequence identity for species, 94% identity for genus, 90% identity for family, 85% identify for order, 80% identity for class, and 75% identity for phylum. if the sequence identity was below the cutoff value, the sequence was assigned to the unclassified group at each phylogenetic level. the diversity index and statistical analysis were performed using mothur program with the cutoff value of 97% similarity for assigning phylotypes. bacterial sequences from excised dgge bands were submitted to the genbank database under accession numbers from hq645054 to hq645071. the sequence reads from pyrosequencing are available in the embl sra database under the study accession number erp000433 (http://www.ebi.ac.uk/ena/data/view/erp000433). brain heart infusion (bhi), low - concentration carbohydrate (lcm), and high - concentration carbohydrate media (hcm) were used for intestinal microbiota growth. previous studies used hcm in human intestinal continuous culture [18, 19 ]. however, the digestible carbohydrate concentrations in the large intestine are lower than carbohydrate concentration in high carbohydrate medium. therefore, we wanted to compare hcm, lcm, and bhi media under the same inoculum and growth conditions. diluted feces (1%) were inoculated in the different media, and the intestinal microbiota growth was analyzed by spectrophotometer and quantitative real - time pcr (supplementary figure 1 available at doi : 10.1155/2011/838040). the growth of intestinal microbiota showed maximum od at 18 hours in lcm and hcm, while the maximum in bhi medium was earlier in the incubation period. the 16s rrna genes of cultured bacteria in each medium increased over the incubation period. the cell number of inoculum was 4.8 10 cells / ml (mean value of cell numbers in inoculum of three media). the highest cell number was detected at 18 hours in lcm (1.85 10 cells / ml) and hcm (1.19 10 cells / ml), whereas bhi reached maximum cell numbers (1.64 10 cells / ml) after 18 hours. to determine the metabolic activity of cultured bacteria, the fecal microbiota cultures were dosed with gentian violet, and the activity was monitored by measuring color disappearance with time. the microbiota completely decolorized gentian violet after 18 hours of incubation (supplementary figure 2). eighteen hours was chosen, because the residence time of readily digestible compounds in intestinal tract is generally within a day. the growth of intestinal microbiota in different media showed similar maximum od and the total bacterial 16s rrna gene increased over time (supplementary figure 1). however, this result did not correlate with the cell numbers determined by flow cytometry. this difference was most likely caused by the difference of rrna gene copy numbers in each species. dgge fingerprinting was used to evaluate the ability of each medium to maintain the initial fecal microbiota. the dgge banding patterns derived from the initial cultures were compared to those from the 18 hours cultures, and similarity between inoculum and cultured sample was used as the measure of microbiota stability. overall, the number of bands and the dominant bands were different in each medium (figure 1(a)). the band numbers from the in vitro cultures were fewer than the fecal inoculum and formed different profiles from the inoculum on the dgge gel. the sequences of bands were assigned to the firmicutes, bacteroidetes, and actinobacteria phyla. firmicutes and bacteroidetes were major phyla in both the fecal inoculum and the in vitro culture. bands affiliated to bacteroidetes were more dominant in the in vitro culture after 18 hours (band number 1, 6, and 11) than at zero time. the dominant firmicutes bands (band 2, 4, 5, 10, and 13) at zero time were less dominant in cultured samples. this result was supported by previous studies that the ratio of bacteroidetes / firmicutes was different between the in vitro intestinal model and the inoculum [12, 34 ]. band number 5 contained pairs of dna fragments, because similar sequences had similar denaturant gradient and the length of amplified fragment for dgge (ranged from 150 to 180 bp) was insufficient to distinguish similar sequences completely. bhi and lcm had relatively similar numbers of bands (2022 bands) and hcm had fewer bands (14 bands). the lower number of bands on hcm may be due to the high carbohydrate content in the medium. high carbohydrate promotes the proliferation of bacteroidetes, which possess a larger glycobiome than firmicutes [34, 35 ]. cluster analysis showed that the microbiota of lcm was similar to inoculum population (figure 1(a)). although the number of bands in bhi medium was similar to that of lcm, their profiles and dominant bands were more different from the inoculum than those in lcm. moreover, the profile of minor bands in lcm was similar to the profile of inoculum, and the number of minor bands was more abundant than bhi medium. profiles of lcm after 18 hours displayed the highest similarity (78.94% ; mean value of triplicate samples) with profiles of original inoculum at zero time. therefore, the lcm medium was used as basal medium for intestinal microbiota growth culture conditions. furthermore, they are unique in each individual because of interindividual differences of intestinal microbiota population, dietary habits, and metabolism. three different culture conditions (1% of fecal inoculum, 1% of fecal inoculum with 1% fecal supernatant, or 2% fecal inoculum) were compared using dgge profile (figure 1(b)) and revealed relatively similar band patterns. however, the inoculum with 1% fecal supernatant added to lcm was more similar to original inoculum than cultures without fecal supernatant in population cluster analysis (80.37% similarity). the inoculum concentration of feces is a significant factor for in vitro culture conditions, because the number and diversity of bacteria can affect growth. therefore, we determined using dgge and real - time pcr, the optimal inoculum concentration for use in this type of in vitro human fecal culture experiments. different fecal inoculum suspensions (0.1%, 0.5%, 1%, 2%, 3%, 4%, and 5%) were used to compare the bacterial communities at each concentration. we did not test concentrations over 5% because of the difficulty with handling the dense and viscous fecal samples. the dgge profiles of different inoculum concentrations were relatively similar to each other and as expected the interindividual variation of microbial community was found (figure 2). although profiles were relatively similar among different inoculum concentrations, small variations of band intensity were observed. cluster analysis of profiles showed that 1%, 2%, or 3% inoculum cultures were the most similar to original inoculum in individual b and c (figure 2). we investigated the change of bacterial communities at each incubation time (supplementary figure 3). the communities of original fecal bacteria were similar to those of inoculum and the profiles of bacterial communities were stable after 6 hours of incubation in every inoculum concentration. in addition, the batch culture was reproducible, as determined by the cluster analysis of triplicate cultures in dgge analysis (data not shown). the real - time pcr - based quantification was used to enumerate total bacteria, bacteroidetes and firmicutes in cultures with 1%5% inoculum concentrations of individual - coded a, b, and c samples at 0 and 18 hours of incubation (table 3). different numbers of bacteria, bacteroidetes and firmicutes were found in each sample at the varied inoculum concentrations, and their growth was different in the same medium. the 16s rrna gene copy number of total bacteria increased to 10 copies ml for all cultured samples, and bacteroidetes and firmicutes reached to 1010 copies ml. the 16s rrna gene copies of total bacteria and bacteroidetes in cultured fecal materials from individual c were higher than individual a and b. firmicutes were more abundant in 18 hour cultured samples of individual b than individuals a and c. these results indicated that the different community composition affected the growth of each phylum in fecal microbiota. the increased ratio of bacteroidetes (2.29 fold ; mean value of increased copy number ratio) was more abundant than that of firmicutes (1.90 fold). we compared the 16s rrna gene copies of total bacteria in 0.1%3% inoculum cultures of individual d (supplementary figure 4) over time. the increased numbers of total bacterial 16s rrna gene copies were higher in low concentration of inoculum (0.1% and 0.5%) than the high concentration of inoculum (1%3%). the batch culture with higher cell numbers has limited nutrients and there would be more competition for obtaining nutrients. however, the high concentration of inoculum (1%3%) added to the batch culture could provide more fecal material to facilitate and personalize the cultivation of indigenous microbiota as fecal supernatant [3638 ]. therefore, 3% inoculum concentration of fecal materials was chosen for in vitro culture conditions since this level would maintain a high cell number of intestinal microbiota and grow a variety of indigenous microbiota. a comparison of the intestinal microbiota of each individual fecal sample before and after culturing was determined by pyrosequencing. a total of 45,674 reads were obtained from pyrosequencing and 5,843 sequences were removed by filtering process of chimera check, length cutoff, ambiguous base call and average quality check. therefore, a total of 39,831 sequences were analyzed (ranging from 3,402 to 8,898 per sample) after the filtering process (table 4). the average length of sequences was 385.39 bp and the observed number of phylotypes ranged from 590 to 1,287 with 92% to 97% good 's coverage. the richness of samples was investigated by rarefaction curves (supplementary figure 5). the changed numbers of observed phylotype and diversity indices of samples from three individuals were different at zero time and after 18 hours of incubation. samples from individual b had the most similar observed number of phylotypes and shannon indices between the zero time culture and the 18 hour culture among the three individuals. the dominant phyla (firmicutes, bacteroidetes, actinobacteria, proteobacteria, and verrucomicrobia) from fecal samples of each person were maintained in improved culture condition of this study (figure 3). the abundance of firmicutes decreased after 18 hours (average from 72.39% to 44.95%), while bacteroidetes increased from 17.14% to 39.11% in cultured samples. although the proportion of each phylum was changed in the in vitro cultures, the dominant phyla were maintained after 18 hours of incubation. these trends are similar to those seen in a previous gut model system analyzed using phylogenetic microarray. they reported that the abundance of bacteroidetes increased from 52.49% to 75.50% (ascending model), 80.59% (transverse model) and 75.60% (descending model), while the firmicutes decreased from 44.57% to 16.81% (ascending), 10.56% (transverse), and 13.23% (descending). the abundances of actinobacteria (average 4.91%) and verrucomicrobia (0.21%) in the present study were higher than observed in the previous model system. at the genus level, a total of 210 genera (read number 0.01% of total analyzed reads) were retrieved from the zero time fecal cultures of individual a, b, and c, and 173 genera were obtained from the 18 hour cultured samples (figure 3). the dominant genera were bacteroides, subdoligranulum, faecalibacterium, parabacteroides, bifidobacterium, ruminococcus, eubacterium, blautia, roseburia, alistipes, clostridium, escherichia, and dorea (read number 1% of total analyzed reads). the community profiles of the microbiota from individuals a and c were more similar to each other than to that for individual b, both at zero time and after the 18 hour incubation. therefore, the bacterial community of each in vitro cultured sample reflected the interindividual uniqueness of the fecal microbiota. we tested a variety of conditions for the human intestinal microbiota growth in short - term in vitro batch cultures. the combination of dgge, real - time pcr, and pyrosequencing was sufficient to compare communities of intestinal microbiota in the different cultures. of the combinations tested, low - concentration carbohydrate medium (lcm) supplemented with 1% fecal supernatant and inoculated with a fecal suspension to a final concentration of 3% performed best in maintaining a metabolically active diverse population of bacteria over the 18 hour incubation. the culture conditions developed in this investigation should be suitable for use in future studies on the impact of xenobiotics on the human intestinal microbiota. | a stable intestinal microbiota is important in maintaining human physiology and health. although there have been a number of studies using in vitro and in vivo approaches to determine the impact of diet and xenobiotics on intestinal microbiota, there is no consensus for the best in vitro culture conditions for growth of the human gastrointestinal microbiota. to investigate the dynamics and activities of intestinal microbiota, it is important for the culture conditions to support the growth of a wide range of intestinal bacteria and maintain a complex microbial community representative of the human gastrointestinal tract. here, we compared the bacterial community in three culture media : brain heart infusion broth and high- and low - carbohydrate medium with different growth supplements. the bacterial community was analyzed using denaturing gradient gel electrophoresis (dgge), pyrosequencing and real - time pcr. based on the molecular analysis, this study indicated that the 3% fecal inoculum in low - concentration carbohydrate medium with 1% autoclaved fecal supernatant provided enhanced growth conditions to conduct in vitro studies representative of the human intestinal microbiota. |
to investigate potential movements of ssb on ssdna, we employed single molecule fluorescence resonance energy transfer (smfret)8,9. fret efficiencies e from individual immobilized partial duplex dna with a 3 (dt)n tail (64 n 131) bound to ssb were acquired using total internal reflection fluorescence microscopy9. surface immobilization and fluorescent labelling have no measurable effect on the dynamics of ssb binding mode transitions5. owing to the closed wrapping in the (ssb)65 binding mode favoured under our conditions (500 mm nacl or 10 mm mg)7, when ssb is bound to ssdna of 65 - 70 nt with its two ends labelled with donor (cy3) and acceptor (cy5) fluorophores, singular high fret distributions were observed5. however, when a (dt)69 tail is further extended by an additional 12 nt of sequence complementary to the overhanging cohesive end of l - strand of phage dna, individual ssb - ssdna complexes display large fret fluctuations in the millisecond time scale (fig. these fluctuations were dramatically suppressed when the 12 nt extension is hybridized to a cohesive end of a dna (fig. 1b). to exclude binding and dissociation of additional ssb molecules as the cause of fluctuations, unbound ssb was removed by a buffer wash before measurements. dna unwrapping / rewrapping dynamics, occurring in tens of microseconds in high salt3,4, is completely averaged out within our 10 - 30 ms time resolution5. we also ruled out local melting of the duplex portion as a source of fluctuations (supplementary materials, sm1). therefore, these fluctuations must arise from additional conformational states enabled by the 12 nt extension. to test whether the fret fluctuations are caused by transient excursions of ssb to the extension, we varied the length of the extension ((dt)n, n= 0 - 18) while keeping the ssdna between cy3 and cy5 at 69 or 70 nt. if an ssb tetramer binds randomly and remains fixed at the initial site of binding undergoing only transient interactions with ssdna outside the binding site, each complex will generate a fret distribution that is unique to the initial site of binding. however, all complexes for each construct displayed similar fret time trajectories (supplementary fig. furthermore, if ssb migrates along the dna, larger excursions away from the high fret state are expected for longer extensions. indeed, average fret values decreased for longer extensions while the high fret state was still transiently visited (supplementary fig. 1). the fret distribution and the time scale of fluctuations are relatively independent of the salt concentration (supplementary fig. 2), arguing against these fret changes arising from binding mode transitions which display a strong salt dependence10 - 12. hence, these fluctuations likely reflect ssb 's diffusional migration on ssdna with the different fret values corresponding to different ssb locations. to make unbiased assignments of fret states, we employed a hidden markov model (hmm) based statistical approach that determines the most likely time sequence of fret states (fig. the result is further reduced to a transition density plot (tdp)13,14, that allows the number of distinct fret states, their fret values, and the transition rates to be estimated (fig. we analyzed ssb migration on dna molecules with several 3 dt tail lengths (0 to 12 nt extension beyond 65 nt binding site size) at 13 c to slow down migration (fig. 3). for (dt)69 + 8 (12 nt extension from the 65 nt binding site size with 69 nt separation between fluorophores), 2b) with transitions occurring between nearest neighbours. we assigned the highest fret value (e ~ 0.8) to the state with ssb closest to the ss - dsdna junction and lower fret values for positions away from the junction. the rates of transition, or the stepping rates, were independent of the beginning and ending state of transition (supplementary fig. 4) and ranged between 3.0 and 4.5 s (fig. 2c). similar analysis yielded 5, 3 and 2 states for dna with 8, 2 and 0 nt extensions, respectively (supplementary fig. 3). therefore, every 2 - 4 nt of dna extension provides an additional configuration, yielding an apparent step size of about 3 nt. because fret fluctuations became too fast for hmm analysis above 13c, we used autocorrelation analysis of fret efficiency e for the temperature dependence studies (fig. the averaged auto - correlation function plots of the ssb-(dt)69 + 8 complexes were best fit by bi - exponential decays. the shorter lifetime was equal to the time resolution independent of temperature and is ascribed to photophysical or detection noise the longer lifetime, long, displayed a monotonic temperature dependence and was attributed to ssb diffusion. combined with the stepping rate of ~ 4 s at 13 c, we can then estimate a stepping rate of ~ 60 s at 37 c. assuming a 3 nt step size, the diffusion coefficient of an ssb tetramer along ssdna at 37 c is estimated to be 270 (nt)/s. as a further test of ssb migration on the ssdna, we employed single molecule 3 color fret9,15 using a donor - labelled ssb mutant (a122c labelled with ~1 alexa555 per ssb tetramer) and two different acceptors, cy5 and cy5.5, attached to the two ends of a (dt)130 (fig. the large separation between the two acceptors eliminates any significant fret between them. if a single ssb tetramer diffuses on the long ssdna, high fret events to either acceptor will be mutually exclusive. indeed, we observed rapid and anti - correlated fluctuations of apparent fret efficiencies to the two acceptors, eapp,5 and eapp,5.5, demonstrating that ssb truly diffuses on the dna (fig. 3a and b). to ensure single ssb molecules on dna, 1 min incubation with sub - saturating concentrations of ssb (< 100 pm) was followed immediately with a buffer wash and only traces displaying single donor photobleaching events were analyzed. at higher ssb concentration (10 nm), much slower fret fluctuations were observed likely due to binding of additional ssb (supplementary figure 5). to probe how far ssb can move on a long ssdna, we placed cy5 and cy5.5 on the two ends of a (dt)130 and cy3 in the middle (named (dt)65 + 65). this 3-color fret scheme allows us to determine at which end the ssb was present by following the closed wrapping of that dna segment and high fret to the corresponding acceptor (fig. both the dye pairs display transient high fret states that are anti - correlated indicating that the same ssb molecule was capable of migrating to either end of the dna (fig. therefore, ssb can move at least 65 nt via diffusion and is not constrained to its initial binding site. ssb modulates the interaction between the reca protein and ssdna in the sos response and recombinational repair pathway2,16 - 19 and mutations in the ssb gene cause inefficient recombinational repair and homologous recombination1,20 - 22. a reca filament can readily displace ssb from the dna if assisted by recfor, -modified recbcd or a preassembled nucleation cluster14,23 - 27. however, the mechanism of efficient ssb displacement by reca was unclear given the tight binding of ssb to ssdna. our estimated diffusion step size of ssb, ~ 3 nt, is the same as the binding site size of a reca monomer which is the unit of filament extension14,28. we, therefore, hypothesized that a monomer - by - monomer addition of reca to the dna segment freed up by ssb diffusion might convert the random walk of ssb into unidirectional movement (supplementary movie 1). to test this idea, we devised a 3-color fret assay using a dna with a 96 nt 3 tail, (dt)30 + 65, labelled at positions 0, 30 and 95 with cy5.5, cy3 and cy5, respectively (fig. the apparent fret efficiencies of dna only are low for both acceptors (~0.1), and drops to zero upon reca - atps filament formation (fig 4a and b). ssb addition after flushing out excess reca and atps removes the reca - atps filament from the ssdna tail, but not from the duplex dna14, and the ssdna wraps around ssb, displaying higher fret with a broad distribution that reflects ssb diffusion (fig. the reca - atps filament remaining on the duplex serves as the nucleation cluster for filament elongation on the 3 ssdna tail14 such that upon addition of reca and atp, the elongating filament rapidly replaces ssb on the ssdna (eapp = 0) (fig. 4f shows the real time 3-color fret trajectories of ssb displacement by an elongating reca filament. before elongation upon addition of reca and atp, eapp,5.5 drops first as a reca - atp filament initiates at the ss / dsdna junction. since the cy3 and cy5 are separated by 65 nt at the distal dna end, we attribute this increase in eapp,5 to the repositioning of ssb to the distal end, pushed by the elongating reca filament. we used exponential fits of average fret curves to estimate the rates of three distinct events following the addition of reca and atp (fig. (i) eapp,5.5 drops from 0.3 to 0 at a rate of k1,ssb = 0.24 0.02 s. we assign this to reca filament initiation from the reca - atps nucleation site since once initiated, the decrease in eapp,5.5 is nearly instantaneous. (ii) eapp,5 increases from 0.3 to 0.75 at the rate of k2,ssb = 0.2 0.01 s. we assign this to reca filament initiation and elongation by ~ 10 reca monomers on 30 nt and ssb movement to the distal dna end. the time intervals between the drop in the eapp,5.5 and the rise of eapp,5, place a lower limit of ~0.6 s for the reca elongation on a 30-mer of ssb - bound ssdna (supplementary fig. 6). (iii) the decrease of eapp,5 (traces synchronized when the high eapp,5 state is obtained) that we assign to ssb dissociation occurs at a much lower rate of 0.07 0.01 s. the rates of filament initiation and elongation without ssb are comparable to those obtained with ssb (fig. 4h and supplementary materials, sm2). furthermore, the rate of reca elongation on bare ssdna is about 20 s per monomer at 1 m reca14 and is similar to the lower limit we determined here with ssb (~ 6 s per monomer), suggesting that any hindrance to reca elongation by ssb is minimal. similar rates were observed on longer dna where up to two ssb tetramers can bind (supplementary fig. overall, the rate of ssb removal from the dna end is ~ 10 fold slower than what is expected from filament elongation alone. this observation suggests that ssb diffusion is important for reca filament elongation on ssb coated dna. this is because before ssb hits the dna end, its diffusion is isoenergetic and therefore is rapid, while its further diffusion at the 3 end is energetically costly. this model of rectifying the ssb diffusion by the directional growth of a reca filament does not require any direct interaction of the two proteins29 and hence could provide a general mechanism for displacement of ssb by proteins moving directionally on the ssdna. ssb inhibits reca filament formation at low salt and high ssb concentrations23,29, but stimulates reca filament formation in high salt29, likely by disrupting dna secondary structures30,31. tetrameric ssbs can in fact destabilize a dna duplex possessing a single strand tail that is shorter than the ssb binding site size32 but no significant duplex disruption was observed for a tail length equal or greater than the binding site size (fig. 1b and supplementary materials, sm1). we therefore investigated whether ssb can disrupt a physiologically more relevant structure, that is, a hairpin flanked by two single stranded regions. the melting of a hairpin with a 7 bp stem and 3 nt loop, hp, (fig. 5a) is monitored via fret between cy3 and cy5 attached to the ends of the hairpin in two different constructs, (dt)65+hp+3 and (dt)6+hp+65. a single high fret population for an intact hairpin is partially replaced by lower fret populations with ssb, signifying different states of hairpin unzipping (fig. 5c) with a majority displaying two - step unzipping with rate constants of ~ 1.1 - 1.5 s (fig. 5c and d ; details in supplementary materials, sm3). hence, a single ssb tetramer transiently disrupts dna secondary structures as stable as a 7 bp stem by repositioning itself on and off the hairpin segment. finally, we tested if such transient melting of a dna hairpin by ssb promotes reca filament formation on the hairpin. starting from a pre - nucleated reca - atps complex, a reca - atp filament was formed on (dt)6+hp+65 dna (fig. 5e) giving rise to a eapp ~ 0 population representing filament formation over the melted hairpin (supplementary materials, sm4). remarkably, filament formation over the hairpin occurred 40 fold faster when ssb is present, demonstrating that ssb stimulates filament elongation over ssdna that can form stable secondary structures (fig. 5f and supplementary materials, sm4). interestingly, for our second construct (dt)65+hp+3, filament formation over the hairpin remained slow even with ssb (fig. this dependence on hairpin position further indicates that transient hairpin disruption by ssb is necessary for efficient filament elongation for the following reason. reca filament elongation towards the 3 end decreases the length of ssdna available for ssb binding, forcing it to eventually dissociate. however, ssb dissociation occurs before the filament elongates to the hairpin region of (dt)65+hp+3 such that ssb - induced hairpin melting is reversed prior to filament growth over the hairpin segment (supplementary materials, sm4). based on these results, we propose that ssb diffusion along ssdna in the low cooperative (ssb)65 mode, where ssdna is populated mostly with single or two tetramers33, stimulates reca filament elongation by transiently removing dna secondary structures ahead of the filament, and that filament elongation via reca monomer addition in turn directionally biases ssb diffusion (fig. 5 g ; supplementary movie 1). for long ssdna bound by multiple ssb tetramers, directional migration of an ssb tetramer caused by reca filament elongation may increase the local ssb concentration and promote transitions to other binding modes from which ssb dissociation may be much more rapid3,5. if so, the findings made here may also be relevant for the removal of multiple ssbs from longer ssdna. ssb rolling would occur via partial unwrapping of one end segment of ssdna from an ssb tetramer followed by re - wrapping of the other end in its place (supplementary fig. 8 and supplementary movie 1), although our results are consistent with the rolling model, a definitive conclusion awaits further investigations. our work represents the first demonstration of any protein diffusing on ssdna. by facilitating the redistribution of a tightly bound ssb tetramer along the ssdna without full dissociation, ssb diffusion may be utilized in a variety of cellular processes, for example, stabilization of specific denaturation sites on superhelical dna35,36 and facilitation of primase activity by positioning the ssb on g4 phage type priming systems37. the c - terminal region of eco ssb interacts with a variety of dna repair enzymes and facilitates localization of these enzymes in the vicinity of ssdna38,39, raising the possibility that ssb acts as a mobile platform on the ssdna for the repair and recombination machinery. the presence of homologous ssb proteins even in metazoans suggests that similar diffusion mechanism might operate over a wide range of species40. partial duplex dna (18 bps dsdna) with 3 (dt)n tails (n ranging from 64 to 131 nucleotides, nts) carrying one donor (cy3) and up to two acceptors (cy5 for two - color fret, cy5 and cy5.5 for three - color fret) were immobilized at the duplex end on polyethylene glycol coated surface using biotin - neutravidin and incubated with 100 pm - 1 nm ssb in imaging buffer for 1 min before flushing and single molecule data was acquired using wide - field total - internal - reflection (tir) fluorescence microscopy9 with 8 - 100 ms time resolution. all single molecule measurements were performed at 231c unless specified otherwise in imaging buffer (10 mm tris (ph 8.0), 500 mm nacl, 0.1 mm na3edta, 0.1mg / ml bsa, oxygen scavenging system (0.5% w / v glucose, 1.5 mm trolox41 or 1% -mercaptoethanol, 165u / ml glucose oxidase and 2170u / ml catalase). reca - ssb experiments were conducted in 1 m reca (or 10 nm ssb), 1 mm atp (or 1 mm atps) in 25 mm tris acetate (ph 7.5), 50 mm sodium acetate, 10 mm magnesium acetate and 0.1 mg / ml bsa in combination with the oxygen scavenging system. details of dna sequences with modifications, annealing, reagents, experimental set - up and analysis indicated in the text are reported in supplementary methods. | single stranded (ss)dna generated in the cell during dna metabolism is stabilized and protected by binding of single stranded dna binding (ssb) proteins. e. coli ssb, a representative homotetrameric ssb, binds to ssdna by wrapping the dna using its four subunits. however, such a tightly wrapped, high affinity protein - dna complex still needs to be removed or repositioned quickly for unhindered action of other proteins. here, we show, using single molecule two and three - color fret, that tetrameric ssb can spontaneously migrate along ssdna. diffusional migration of ssb helps in the local displacement of ssb by an elongating reca filament. ssb diffusion also melts short dna hairpins transiently and stimulates reca filament elongation on dna with secondary structure. this first observation of diffusional movement of a protein on ssdna introduces a new paradigm for how an ssb protein can be redistributed, while remaining tightly bound to ssdna during recombination and repair processes. |
the goals of our course are two - fold : one goal is to expose students to research in major areas of psychology and a second goal is to present students with the many facets of being a scientist, with emphasis on the particular challenges experienced by women in science (see table 1 for a synopsis of topics covered). to achieve these two goals, students read about the professional and personal experiences of female psychologists and read their original research. we selected women based on their exemplary scientific contributions in the major research areas of psychology such as neuroscience, cognitive science, and developmental psychology. our main mechanism for illustrating their personal and professional experiences was through biographical information obtained from e - mail correspondence. this discussion - based course has been taught four times and is usually offered every second year. for this eight - week course, students receive two - credit hours in either psychology or women s studies. typically junior or senior psychology majors or minors and enrollment has ranged from five to 12 students who are almost exclusively female students with little to no academic background in women s studies or women s issues. this course is team - taught and both instructors actively participate in each class period. we profile one or two female psychologists who conduct research in one of the major research areas of psychology. we start with a review and discussion of a scientific article she wrote followed by a lecture that contextualizes the significance of her research. following this, students examine women s issues by reading and discussing a biography written by this female psychologist describing her professional and personal experiences. other materials pertaining to women s experiences in science are also read and discussed (table 2). students grades are based on written assignments about course readings, a group project, a written and oral test, and class participation. the group project requires interviewing male and female academics about their professional and personal experiences and making a group presentation. the presentations highlight the challenges and rewards of an academic career and the variety of ways gender impacts these experiences. prominent female psychologists were asked questions over e - mail (table 3) about the development of their career, challenges they experienced and issues facing women scientists, their experience with mentorship, and advice they would give to aspiring women scientists. we asked a dozen women who were either our acquaintances or friends to respond to our questions. we received seven responses that varied from one paragraph to, more typically, several pages in length. while we were concerned about drawing general conclusions from biographies, themes emerged across biographies such as the struggle to maintain balance between professional and personal life and to gain respect from students and colleagues. a diversity of views was evident in, for example, how single and married women maintain personal identity. also, women realized women s issues at different stages in their careers and reflected on the impact and importance of these issues differently. when i began graduate school and was told by very well - meaning feminists that i had lived my life being disadvantaged by virtue of my gender i was clearly taken aback. that had nt been my experience at all my successes and failures never seemed to be related to my gender and i d never even given such notions a thought. i was not fully aware of discrimination until i entered central administration... overall we feel that the biographies we used are an effective method for highlighting general as well as unique experiences and they reflect a variety of perspectives on women issues. to illustrate the many facets of being a scientist, we focus on the intrapersonal and interpersonal dynamics that are part of science and affect all scientists. intrapersonal dynamics refer to the changes that occur within a person, such as developing an identity as a scientist. below we will discuss the dynamics we address in our course and include examples of how biographies are used to highlight these issues. science is very much a social enterprise, yet undergraduate students often do not have this appreciation and educators may fail to reveal this reality (national academy of science, 1995 ; ramirez, 2003). many of the topics in our course, life of a psychologist, address the importance of interpersonal dynamics in scientific progress such as communicating between co - investigators and research assistants, reporting research findings at conferences and in scientific papers, and reviewing the work of others (pinner scott, 1990). interpersonal dynamics also extend to collaboration and mentorship, social experiences essential to professional growth and development. many of the women we profile addressed this issue as the following quotes from three different biographies indicate. to get an academic job you need to do the obvious very good research that gets published and the less obvious find connections and get the respect of people who are established in your chosen field. several people in my department have served a mentor - type role with me this is very important because no matter how good you are, you have to know how the system works in order to get funding to conduct your research. you can turn to your women colleagues those who have successfully negotiated the minefields and learn with them how to find your way, make sure your needs are met, and feel good about yourself along the way. of special consideration for example, women s contributions may be underappreciated by members on the research team and by others in the academic community (pinner scott, 1990 ; massachusetts institute of technology, 1999). she said that she saw the way that women s cvs can be misread to give credit to male co - authors irrespective of whether the woman is first or other author. finding a mentor that can appreciate one s unique situation is important for support and guidance. this may be more difficult for female students because of a lowered likelihood for faculty to encourage, promote, and guide female students and the scarcity of women in some areas of science (murray, 2000 ; wasserman, 2000 ; rosser, 2004 ; paul, 2005). one woman s biography reflected this experience by noting there were only a few mentors to help me initially. at university this same woman notes, in reference to two women she knew later in her career, that these were both women i respected tremendously as academics and who also showed personal attributes that were close to what i wanted to be most of the female psychologists we contacted, however, did indicate that there were people along the way to provide some form of mentorship and noted the value of this relationship in guiding their careers. for women decisions about if and when to have children exaggerate this challenge because the tenure clock often corresponds to women s biological clock (wasserman, 2000). this point is addressed in a number of biographies and is reflected in the following quote, i think one of the big issues facing women and it has been a problem for a long time is striking a balance between career and family. finding the right time to start a family can be challenging. given that women are often expected to be primary caregivers, caring for children may compromise continued professional accomplishment and success (murray, 2000 ; wasserman, 2000 ; american society for cell biology, 2002). we are fortunate to have a number of biographies address this issue from multiple perspectives. one woman writes : i began my first job as an assistant professor when i was seven months pregnant... i was not entitled to maternity leave because i had not been at the university long enough. i brought my baby into work with me while i was getting my lab set up but it was not an ideal arrangement. another woman provided a wonderfully detailed example of how she and her husband shared childcare responsibilities over the years, and how parenting responsibilities affected her career decisions. some selections from her biography include : the challenges of balancing first school, and later an academic position with two children were huge. you simply can not know in advance how you will react emotionally, and you have to be flexible to ensure that your needs as a parent and as an academic are met we (my husband and i) took turns for about 15 years in who was the primary parent it was not until the kids left home for university that i agreed to be on national committees that involved a lot of travel, or that i agreed to any administrative positions. murray (2000) describes how academics define themselves as researcher, teacher or both, but that female academics were more inclined to identify themselves as teachers. one of the women we profile in our course reflected on this issue in the following way : somewhere in the process of my deciding between psychotherapy and research careers, a wise friend advised me by saying it s all about where you get your energy from. after surprisingly little thought it became clear to me that i got my energy from doing research. women s career trajectories are impacted because of their commitments to other people (e.g., children, aging parents, spouses) and this can impact their perceptions of success (murray, 2000). while there are many paths to success, these paths narrow for women (wasserman, 2000). lack of mentoring, isolation, difficulty gaining credibility and respect, challenges balancing committee responsibilities with teaching and research are challenges to success (massachusetts institute of technology, 1999 ; rosser, 2004). these variables, along with gender inequities in salaries, can negatively affect job satisfaction. when discussing gender inequities in salary, we provide students with a number of statistics reflecting that, as a rule, male academics earn more money than female academics (american association for women in science statistics, n.d. ; this is made more salient to our students when they read the biography of an assistant professor who wrote the biggest shock came, however, when a male faculty member was hired after me but at a significantly higher salary. when i questioned why someone with less experience was being paid more than me, i heard some outrageous things such as i did not need to make as much money as a man because i did not have a family to support. a voice entails being listened to, receiving respect, and having influence. pertaining to the issue of having a voice, i experienced the disdain some powerful men have for vocal women ; and i saw and experienced the hundreds of thousands of little ways one s self esteem can be undermined by repeated experience with not being listened to, not being credited, being falsely blamed, etc. other examples of women struggling to have their voice heard and respected can be seen in the following quotes : i had a male colleague that began to treat me in a very unprofessional manner. the behavior disrupted what could have been a good colleague - to - colleague relationship. i have had difficulty with getting my superiors to take me seriously... one of my graduate school professors told me that i would never be a successful academic because i do not look or sound like a typical academic. some individuals, however, did not express challenges with gaining respect and finding a voice, and even considered that their careers were unaffected by their gender. we have evaluated course effectiveness by one indirect and two direct measures of student learning. these assessment data are limited because qualitative measures have been administered to relatively small class sizes. the indirect measure consisted of a survey that was completed by eleven students four months after completing the course. students were asked what was the most memorable part of this course ? and their most frequent responses were class discussions on the biographies and the dynamics of science. also frequently mentioned were interviews conducted with male and female academics as part of the students group project. students indicated that the biographies provided a realistic view of academia and acknowledged that scientists have a regular life and face the same challenges as everyone else. one student even commented on the value of learning about the lives of contemporary women. one advantage of using biographies of successful contemporary women scientists as opposed to women scientists with extraordinary achievements is that these women seem more accessible and real. in fact, it was clear from student responses that the issues presented in the course became real through the biographies and brought awareness that women s issues exist today. students appreciated learning about the many ways for coping with challenges and the many paths to success. together the results of the survey showed that biographies are an effective and memorable way to teach about science and women s issues. a direct measure of course effectiveness is the written and oral test students take at the end of the course. in previous course offerings, the final test included a number of multiple - choice questions on the research articles and other psychological findings we covered in class to determine if students learned about the scientific areas we covered. an essay question in which students were to summarize the results of one of the research articles they read and discuss the value of the work was also required. students responses were graded according to their accuracy in recalling the research, understanding the limitations of it, and being able to contextualize the significance of the results within the broader research area. to assess student s learning about the dynamics of science and issues facing women in science, students were required to answer three essay style questions on the exam and to engage in a group discussion in an oral component of the exam. though questions were modified for different offerings of the course, some examples include i) what does it mean to have a voice ? in what way might women s voices be minimized in academia ? iii) summarize the data you were given on gender differences in academia. describe your reaction to these data. iv) based on the readings and the interviews of faculty members, describe four qualities of a successful career. while students could provide anecdotal evidence in their answers, students performance on the exam indicates that students learned about both the scientific findings we covered in this course and the dynamics of science. our second direct measure of course effectiveness was a pre / post - course written assignment. in our most recent course offering five students were asked to write about the process of becoming an academic on the first day of class (pre - course) and then to re - address this question in an assignment they handed in on the last day of class (post - course). on the pre - course assignment, some students revealed a superficial understanding of academia. other students had a deeper understanding and noted the importance of working with others, balancing work and personal responsibilities, and gaining status and recognition. on the post - course assignment, for instance, most students addressed inequity in the workplace and issues of achieving balance and recognition. students who entered the course with a deeper understanding applied course material to their lives by describing personal strategies for balancing family and career and creating their own supportive network. comparing pre- and post - course assignments indicated that all students benefited from the course and demonstrated an understanding of the intrapersonal and interpersonal dynamics of science. we were impressed by what students took away from our course as exemplified by the following two comments. i hope that in my future positions i will be knowledgeable about workplace alternatives for balance and look forward to today s research being used to implement increasingly family friendly policies.but most important of all, psychology 384 [this course ] has prepared me for the long journey ahead ; it has helped me learn the peaks and valleys that happen on the road to a life in academics. knowing these things, i am a better and more prepared person.these quotes indicate that our course may be promoting students confidence and preparation for pursuing a career in science (campbell & skoog, 2004). i hope that in my future positions i will be knowledgeable about workplace alternatives for balance and look forward to today s research being used to implement increasingly family friendly policies. but most important of all, psychology 384 [this course ] has prepared me for the long journey ahead ; it has helped me learn the peaks and valleys that happen on the road to a life in academics. knowing these things, i am a better and more prepared person. the approach used in this course incorporates the learning of psychology with learning about the social dynamics of science. this approach allows students to learn about women s issues while focusing on women s scientific contributions. students also learn about social dynamics that will affect their career in science or any profession. one concern about using biographical materials is that information contained in these materials could be generalized when it is not appropriate to do so or discredited as a single person s issue when it is a more general phenomenon. the use of other published sources, such as qualitative and descriptive studies, should minimize this concern. team - teaching has allowed for drawing on a greater number of professional and personal experiences and points of view and for facilitating small group discussions. by team - teaching this course although these are advantages to team - teaching, we do not feel that team - teaching is necessary for successfully teaching a course using this approach. what we have noticed after having taught this course multiple times is that students become knowledgeable about women s issues in general, but are more affected by conversations about salary inequity and balancing personal and professional lives. they are less reflective about the impact of interpersonal dynamics in the workplace, particularly pertaining to issues of recognition and status. in the future, we will direct more conversation and assign more readings on the importance of recognition and status for job and life satisfaction. biographies can be a useful approach to teach an entire course or portions of a course. in our course, discussions of biographies and supplemental readings are equally balanced with learning about psychological research. other courses might focus primarily on scientific research and use biographies only occasionally to illustrate women s issues or other social issues such as the effect of ethnicity, age, sexual orientation and physical disability on career development. biographies can also be useful in highlighting and making real any issue in science such as the social responsibility of scientists, the impact of political decisions on science, and scientific integrity. we had a very positive experience asking our female colleagues to respond to our biographical questions. many of our colleagues replied with very lengthy responses that revealed not only their own personal experiences, but also helped to mentor our students. we have noticed that many students even consider some of the women that we have interviewed as role models. this suggests that biographies can be used to provide additional role models for students interested in science. | we describe using biographies in teaching a course about the intrapersonal and interpersonal dynamics of science, with an emphasis on the professional and personal experiences of women in science. in our course, life of a psychologist : experiences of women in science, students examine biographies and scientific research written by female psychologists across the main research areas of psychology. biographies by these female psychologists and research on the experiences of women scientists are used to highlight the intrapersonal and interpersonal dynamics of science. intrapersonal dynamics refer to the changes that occur within a person, such as developing an identity as a scientist, having a voice, and achieving success. interpersonal dynamics refer to exchanges between people, such as collaborating on research, mentorship, and balancing personal and professional lives. qualitative data support using biographies in teaching about the dynamics of science. suggestions for using biographies in other courses are provided. |
walnut tree (juglans regia l.), belongs to family juglandaceae (1). walnut contains up to 62% - 68% oil, containing a high amount of monounsaturated and polyunsaturated fatty acids (2). walnut leaves are considered as a source of healthcare compounds and are intensely used in traditional medicine to treat venous insufficiency and haemorrhoidal symptoms ; it is also used for its antidiarrheic, antihelminthic, depurative and astringent properties (3 - 5). this valuable tree has a long history of medicinal use to treat a wide range of health complaints. dry seeds (nuts) are very popular and largely consumed as royal food in iran. in addition, green walnuts, shells, bark, green husks (epicarps), and leaves are used in the cosmetic and pharmaceutical industries (6). the stem bark is reported to be alterative, anthelmintic, astringent, bactericidal, depurative, digestive, diuretic, laxative, detergent, stimulant, tonic and insecticidal (7). walnut oil is a component of dry skin creams, anti - wrinkle and anti - aging products, because it presents moisturizing properties as well as free radical scavenging capacity (8). in an experimental study, treatment of j. regia extracts in the experimental animal samples resulted in a significant decrease in blood glucose, glycosylated hemoglobin, low - density lipoprotein (ldl), triglyceride and total cholesterol and a significant increase in insulin and high - density lipoprotein (hdl) level (9). dm as well as its fatal complications is an important cause of death all over the world (10). for a very long time, plants have played an important role in the treatment of many chronic diseases, including dm (11). world health organization (who) has recommended the evaluation of effective plants for diseases such as diabetes, for which there are few safe modern drugs. iranian people with diabetes extensively use the extract of walnut and its hydrosol to control their blood sugar (bs). some of these patients are satisfied with this herbal product and tolerate it well. however, there are few data about its effectiveness and safety (12, 13). therefore, more studies are required to determine the effects and side effects of walnut on diabetes. the only study on the effect of walnut extract on glycemic control in patients with type 2 diabetes showed a significant increase of insulin level and decrease of glycosylated hemoglobin (13). according to the literature research, for this reason, authors conducted a pilot study on the efficacy and safety of walnut extract (wle) in patients with type 1 diabetes. walnut hydrosol was obtained from a local market in meymand, fars province, iran. the essential oil of the sample was extracted using a liquid extractor in two stages. at first, 500 ml of the sample was mixed with 500 ml of petroleum - ether as a solvent. the solvent was heated to 45c for 150 minutes and the essential oil was transformed from an aqueous phase to a petroleum - ether phase. the remains of the organic phase from the first stage were removed and additional amount (500 ml) of fresh petroleum - ether was added to the system. this stage was carried out to increase the yield of the essential oil in the organic phase. the extracted essential oil 500 ml of petroleum - ether containing the essential oil was heated to 40c and vacuumed by rotation of a pump motor at 60 rpm, evaporating the petroleum - ether and leaving the extract. this process produced approximately 50 ml of the sample dissolved in a small amount of organic phase. the concentrated essential oil was dehydrated and subjected to gas chromatography mass spectrometry (gc / ms) for analysis of the constituents (agilent technologies 7890 gas chromatograph coupled with an agilent technologies model 5975c mass detector, palo alto, ca) (14). the apparatus was equipped with a hp-5ms capillary column [phenyl - methylsiloxane, length 30 m inner diameter 0.25 mm, agilent technologies (60 - 325/350c) ]. the oven temperature increased from 60c (0 minute) to 220c in increments of 5c / minute and held at that temperature for 10 minutes. helium was selected as the carrier gas and the flow rate was adjusted at a rate of 1 ml / minute. the mass spectrometer operated in ei mode at 70 ev. the interface temperature was 280c and the mass range was 30 - 600 m / z. retention indices were determined by using retention time of n - alkanes injected after the essential oil under the same chromatographic conditions. the components of the oil were identified by calculation of kovat s indices (ki) and comparison of their mass spectra with those of wiley library or the published ones (15, 16). as a pilot study, inclusion criteria were type 1diabetes, age over 12 years old, completed puberty, having diabetes for more than two years and use of insulin analogs including lantus solostar pen and novorapid flexpen to control diabetes. subjects who used medicines other than insulin, those afflicted with other associated diseases and those who used insulin types other than lantus solostar pen and novorapid flexpen were excluded from the study. they were advised to measure their bs level at least four times daily by glucometer and record their bs level and insulin doses for at least two weeks. then, the patients were advised to drink 250 ml wle after meals twice a day. they continued to measure and record their bs level and insulin doses. during the study, the subjects were called frequently to evaluate compliance and any complications and also to adjust the dosage of insulin injection. after four weeks, the use of wle was stopped, subjects were examined by a physician, and the data including bs levels and injected insulin doses were collected. the subjects continued measuring and recording their bs levels and insulin doses for two more weeks. at that time insulin doses and bs levels were compared for each patient before and after consuming wle. walnut hydrosol was obtained from a local market in meymand, fars province, iran. the essential oil of the sample was extracted using a liquid extractor in two stages. at first, 500 ml of the sample was mixed with 500 ml of petroleum - ether as a solvent. the solvent was heated to 45c for 150 minutes and the essential oil was transformed from an aqueous phase to a petroleum - ether phase. the remains of the organic phase from the first stage were removed and additional amount (500 ml) of fresh petroleum - ether was added to the system. this stage was carried out to increase the yield of the essential oil in the organic phase. the extracted essential oil was concentrated by rotary evaporator equipped with a vacuum pump. in this process, 500 ml of petroleum - ether containing the essential oil was heated to 40c and vacuumed by rotation of a pump motor at 60 rpm, evaporating the petroleum - ether and leaving the extract. this process produced approximately 50 ml of the sample dissolved in a small amount of organic phase. the concentrated essential oil was dehydrated and subjected to gas chromatography mass spectrometry (gc / ms) for analysis of the constituents (agilent technologies 7890 gas chromatograph coupled with an agilent technologies model 5975c mass detector, palo alto, ca) (14). the apparatus was equipped with a hp-5ms capillary column [phenyl - methylsiloxane, length 30 m inner diameter 0.25 mm, agilent technologies (60 - 325/350c) ]. the oven temperature increased from 60c (0 minute) to 220c in increments of 5c / minute and held at that temperature for 10 minutes. helium was selected as the carrier gas and the flow rate was adjusted at a rate of 1 ml / minute. the mass spectrometer operated in ei mode at 70 ev. the interface temperature was 280c and the mass range was 30 - 600 m / z. retention indices were determined by using retention time of n - alkanes injected after the essential oil under the same chromatographic conditions. the components of the oil were identified by calculation of kovat s indices (ki) and comparison of their mass spectra with those of wiley library or the published ones (15, 16). as a pilot study, eight patients with type 1 dm were enrolled in the study. inclusion criteria were type 1diabetes, age over 12 years old, completed puberty, having diabetes for more than two years and use of insulin analogs including lantus solostar pen and novorapid flexpen to control diabetes. subjects who used medicines other than insulin, those afflicted with other associated diseases and those who used insulin types other than lantus solostar pen and novorapid flexpen were excluded from the study. they were advised to measure their bs level at least four times daily by glucometer and record their bs level and insulin doses for at least two weeks. then, the patients were advised to drink 250 ml wle after meals twice a day. they continued to measure and record their bs level and insulin doses. during the study, the subjects were called frequently to evaluate compliance and any complications and also to adjust the dosage of insulin injection. after four weeks, the use of wle was stopped, subjects were examined by a physician, and the data including bs levels and injected insulin doses were collected. the subjects continued measuring and recording their bs levels and insulin doses for two more weeks. at that time insulin doses and bs levels were compared for each patient before and after consuming wle. the constituents of the essential oil of juglans regia l. are presented in table 1. several compounds were identified in the oil and the rate of monoterpenoid and sesquiterpenoid compounds were 53.45% and 5.95%, respectively ; also, the rate of phenolic compound was 51.34%. the main constituents of the oil were carvacrol (33.21%), thymol (16%), and homoveratrole (15.83%). abbreviations : ki, kovat s indices ; two of them were male and six were females with the age range of 13 - 25 years, and the mean age of 19.7 years. table 2 shows the effect of wle on average daily bs level and insulin dose of each subject. two other subjects showed a decrease only in average daily bs level, but because their bs level was in a favorite range, they did not decrease the insulin doses. in one other subject, the decrease in the average daily bs level was only 5 mg / dl, which was insignificant. however, a few days after wle consumption, two subjects called one of the authors and complained about development of generalized pruritic erythematous rashes. the rashes were mild and non - progressive in one of them ; therefore, she continued wle consumption, but the other subject stopped using wle after two weeks due to progressive and troublesome skin rashes. she did not have any illness before the attack of hypoglycemia, and overall, there was no other risk factor for hypoglycemia for her. the level of consciousness was improved in the subject after a few hours and she was discharged from hospital without any side effects. two weeks after stopping wle use, insulin doses and average bs level of all subjects increased again to nearly the level before using wle. it includes three species : j. nigra, j. cinerea and j. regia although only j. regia grows in iran (17). the key chemical composition of walnut is juglone (5-hydroxy-1, 4-naphthoquinone), the toxic compound which is found only in green and fresh walnuts, but such property disappears in dried leaves (18). several other phenolic compounds with antioxidant properties are identified in j. regia leaves (19). walnut (juglans regia l.) is a plant with a significant economic value and medicinal importance for human health. it is consumed in large quantities by people ; therefore, it has a very important place in the public nutritive habits (20). leaves of j. regia are widely used in folk medicine to treat venous insufficiency and haemorrhoidal symptoms, for their antidiarrheic, antihelmintic, depurative, and astringent properties ; mix of leaves and stored - grains are also used as fungicide and insecticide (21, 22). other properties such as antibacterial, human cancer cell antiproliferative, antioxidant, keratolytic, antifungal, hypoglycemic, hypotensive, anti - scrofulous and sedative are also reported for this plant (19, 23 - 25). the beneficial effect of walnut consumption against many diseases is reported, including protection from diabetes (26) or cardiovascular diseases (27). researches have also shown that eating walnuts can improve the blood lipid profile (28). other investigations showed that j. regia extract contains ellagitannins, which is an anti - cancer agent with anti - inflammatory effects (17). examples are phytochemical analysis of the leaf volatile oil of walnut tree (29), studying about protein fractionations, amino acid composition, molecular weight distribution and gel electrophoresis of juglans regia l. (30), surveys on antioxidant and antibacterial activities of the leaf essential oil of juglans regia l. and its constituents (31), determination of mineral contents of juglans regia l. flowers and its anti - hemolytic activity. also, another study reported that j. regia improves glycemic level in the patients with type 2 diabetes, without any adverse effects on the kidney and hepatic function (14). in another study (29), the volatile oils of all 28 j. regia populations were analyzed by gas chromatography flame ionization detectors (gc / fid) and gas chromatography mass spectrometry (gc / ms). major components of the essential oils in this research were (e)-caryophyllene (1.4% - 47.9%), -pinene (4.5% - 39.5%), germacrene - d (5.0% - 23.3%), -pinene (1.5% - 18.1%), -humulene (1.1% - 11.8%), -zingiberene (0.1% - 11.3%), -copaene (0.0% - 10.1%), limonene (0.8% - 8.6%), caryophyllene oxide (0.1% - 8.6%), ar - curcumene (0.0 - 7.2%), -cadinene (0.3% - 6.7%), (e)--farnesene (0.0% - 5.9%), 1, 8-cineole (< 0.0 - 5.4%), -curcumene (0.0 - 4.2%), methyl salicylate (0.1% - 4.0%), (e)-myrtanol acetate (0.0 - 3.8%), (e)-muurola-3, 5-diene (0.0 - 3.8%), (e)--ocimene (0.6% - 3.9%), -longipinene (0.0 - 3.0%), myrcene (0.2% - 2.6%), -muurolene (0.1% - 2.5%), spathulenol (0.0 - 2.2%) and -cadinol (0.1% - 2.0%) (29). a single report on volatile compounds (head space analysis) of j. regia leaves from egypt showed the presence of germacrene - d (28.6%), and methyl salicylate (16.8%) as the main constituents (32). -pinene (30.5%), -caryophyllene (15.5%), -pinene (15.1%), germacrene - d (14.4%) and limonene (3.6%) were identified as the principal components of the essential oil of walnut leaves from kashmir (31). in another research, the essential oil was obtained by headspace method and the volatile compounds were pentanal (0.07 - 0.12%), hexanal (0.26 - 0.80%), nonanal (0.34 - 0.89%) and 2-decenal (0.25 - 0.68%) and hexanol (0.21 - 1.58%) (33). this research indicated that the percentage of aldehyde compound is higher than that of alcoholic compound. these differences can be due to ecological factors or species variations. according to the current study searches in database, this is the first pilot study to determine the effect of wh on glycemic control in subjects with type 1 diabetes. previously, only one study was conducted on the effect of walnut extract on patients with type 2 diabetes and several similar studies on animals with diabetes. wle caused improvement of glycemic control both in rats with diabetes and patients with type 2 diabetes (14, 34). in the present study, glycemic level controlled in most of the subjects with type 1 diabetes. they concluded that walnut may cause increased release of insulin from beta cells, increased insulin sensitivity, or may interfere with absorption of dietary carbohydrate (34, 35). the pathophysiology of t1 dm is completely different from that of type 2 diabetes. in t1 dm, most of beta cells are destroyed ; therefore, many agents that can help patients with type 2 diabetes are not efficacious in t1 dm. the key compounds responsible for anti - hyperglycemic effect of wle may be phenolic compounds. phenolic acids and flavonoids are two major groups of phenolic compounds existing in walnut leaves. as it was mentioned, seven components were found in wh. according to the current study investigation on these components, therefore, carvacrol or thymol or both of them may have an anti - hyperglycemia role but further studies are needed to investigate the cases. in an animal study, carvacrol in combination with rosiglitazone caused improvement in blood glucose and glycosylated hemoglobin high fat diet induced type 2 diabetes in mice (36). another animal study revealed the anti - hyperglycemic effect of methanol extract of otostegia persica boiss (labiatae) on rats with type 1 diabetes. more extensive studies are needed to determine the exact mechanism of anti - hyperglycemic effect of wle. walnut extract was not associated with any complications in animals and patients with type 2 diabetes (14, 34), but its side effects were important among the current study subjects. two of the subjects showed drug reactions as skin rashes, and one of them developed severe hypoglycemia. drug reaction at least partially may be due to technical problems in providing the extract leading to an impure extract. therefore, in leading studies, this process should be performed with more caution to prevent adding impurities. no other etiology was found for hypoglycemia in this subject and, therefore, hypoglycemia was probably the result of wh consumption. this complication can be an important limiting factor for extended use of the extract in human. wh may control glycemic level in human, but it can be associated with minor and major side effects. seven compounds were identified in walnut oil. according to the current study search, among these components two of them (thymol and carvacrol) may have an anti - hyperglycemia effect mentioned in other studies. therefore, wh should not be advised extensively to people with diabetes until the knowledge is increased about its mechanism of action and potential side effects. the most important limitation of this study was the low number of participants. according to the severe side effects in one of the subjects, it is not suggested to perform this study on larger population. however, new in vitro studies are recommended to determine the mechanism of wh effect on blood sugar in subjects with type 1 diabetes mellitus. wh may control glycemic level in human, but it can be associated with minor and major side effects. seven compounds were identified in walnut oil. according to the current study search, among these components two of them (thymol and carvacrol) may have an anti - hyperglycemia effect mentioned in other studies. therefore, wh should not be advised extensively to people with diabetes until the knowledge is increased about its mechanism of action and potential side effects. the most important limitation of this study was the low number of participants. according to the severe side effects in one of the subjects, it is not suggested to perform this study on larger population. however, new in vitro studies are recommended to determine the mechanism of wh effect on blood sugar in subjects with type 1 diabetes mellitus. | backgroundwalnut hydrosol (wh) is used extensively by iranian people with diabetes in order to control blood sugar (bs). there are few data regarding the effect of walnut on controlling diabetes.objectivesa pilot study to determine the efficacy and safety of wh in patients with type 1 diabetes.materials and methodseight patients with diabetes mellitus (dm) type 1 were enrolled in the study. they did not use any medicine except insulin. they were advised to drink 250 ml wh after meals twice a day for four weeks. their bs level was measured and their insulin dose was changed according to their bs. after four weeks, they discontinued wh use and their bs level was checked for two weeks. descriptive statistics was used to analyze the data. also, the essential oil of the sample was extracted using a liquid extractor and then analysis of the constituents was performed.resultsthe average daily bs level and insulin dose decreased in seven subjects. two subjects developed generalized pruritic erythematous skin rash. one patient presented hypoglycemic coma. she had no other coma risk factor. seven compounds were identified in the walnut essential oil and the rate of monoterpenoid and sesquiterpenes hydrocarbons were 53.45% and 5.95%, respectively. the main constituents of the oil were carvacrol (33.21%), thymol (16%) and homoveratrole (15.83%).conclusionswh may control the glycemic level in people with diabetes, but it may be associated with minor and major side effects. further in vitro studies, using these seven compounds, are recommended to determine the efficacy and complications of wh in people with diabetes. |
for many years stock raising has been an important part of livelihood and culture in sub - saharan africa [13 ]. the economic burden of livestock diseases and the declining provision of conventional veterinary services in this continent have undermined the efficiency of livestock production, especially by fulani pastoralists. many people in developing countries still rely on medicinal plants and traditional healing practices for daily healthcare needs of their animals, in spite of the advancement in conventional medicine. conventional medical system, also called western medicine, modern medicine, and biomedicine, used by most medical and veterinary doctors, focuses on disease as an enemy to be conquered. the conventional veterinary practitioner prescribes medications, uses the latest diagnostic tools, and follows peer - reviewed studies that could impact or change the way certain injuries or illnesses are treated. on the other hand holistic veterinary medicine includes such unconventional modalities as acupuncture, chiropractic, homeopathy, flower essences, raw diets, nutraceuticals (the use of concentrated doses of vitamins, minerals, and enzymes to treat disease), chinese medicine, and herbs [6, 7 ]. there is abundant undocumented traditional knowledge of medicinal plants used to treat diseases in most cultures. different traditional healing practices worldwide are designed for either therapeutic or prophylactic use in human or animal diseases [911 ]. in nigeria, pastoralists are known to treat animal diseases with herbs and other traditional medical practices before the advent of conventional medicine. traditional medical and veterinary practices remain relevant and vital in almost all cultures in nigeria due to absence or inadequate provision of modern medical services especially in hard - to - reach rural areas. ethnoveterinary medical practice is widespread among pastoral herdsmen and village livestock keepers in northern nigeria where most of the country 's livestock are concentrated. for most of these livestock owners, conventional veterinary inputs and services are not readily available and, where available, are relatively expensive. therefore, they are left with traditional choices which are locally available and affordable, with the held belief that they are more efficacious. in recognition of the fact that fulani pastoralists possess considerable existing veterinary knowledge and traditional oral history of herbal and nonherbal remedies and their application in livestock disease management, veterinarians, recently, have intensified efforts towards harnessing this knowledge for authentication and preservation. there is no record so far giving ethnoveterinary practices documentation in niger state and there is likelihood that the practices are at the verge of extinction, especially among the fulani pastoralists. this survey was therefore aimed at assessing, in nonexperimental way, the ethnoveterinary practices used by fulani pastoralists in niger state to traditionally manage contagious bovine pleuropneumonia (cbpp) and other common cattle disease conditions in their herds. also, herbal and nonherbal materials are to be identified, validated by consensus, and documented to add useful new remedies to the traditional veterinary pharmacopoeia. niger state is located in the north - central geopolitical zone, at the northern guinea savannah ecological zone of nigeria, between latitudes 820n and 1130n and longitudes 330e and 720e. it is one of the 36 states of nigeria, a gateway between northern and southwestern and south - southern parts of the country, and provides transit routes for pastoral nomads on seasonal movements from the northern parts of nigeria to the southern parts and back. the state covers a land area of about 76,363 square kilometers (29,484 square miles) or about 9% of nigeria 's total land area, making it the largest in terms of land mass in the country. the state has an estimated cattle population of about 2.4 million cattle, 1.7 million sheep, and 2.3 million goats in 2012. the state shares a common international boundary with the republic of benin at its western border and has three agroecological zones, a (bida zone), b (minna zone), and c (kontagora zone), which are based on different climatic conditions in the state (figure 1). the research was conducted in the following pastoral communities : lapai (gps coordinate n09.0102 and e006.61729) ; eyagi (n09.13506 and e006.00618) ; lemu (n09.17155 and e006.01972) in agrozone a ; paiko (n09.43533 and e006.60745) ; kuta (n09.84643 and e006.71782) ; bosso (n09.66275 and e006.47691) in agrozone b ; wushishi (n09.69760 and e006.05682) ; bobi grazing reserve (n09.16715 and e005.91701) ; borgu (n09.91455 and e004.33400) in agrozone c. participatory epidemiology (pe) exercise was conducted to collect qualitative data from the fulani pastoralists in the nine communities using participatory rural appraisal (pra) tools. the study focused on contagious bovine cbpp and other common disease conditions that frequently affect cattle in these communities and traditional remedies used to manage them. fulani pastoralists in lapai, eyagi, lemu, paiko, kuta, bosso, wushishi, bobi grazing reserve, and borgu pastoral communities were the population studied. only adult male fulani pastoralists were considered because of their long time historical and sociocultural relationship with their cattle herds. three key informants were conveniently allocated to each of the nine pastoral communities for the purpose of the participatory exercises. since nine pastoral communities were purposively selected, the number (sample size) of the key informants for the survey was, therefore, 27. the state was divided into three sampling areas based on the existing three agroecological zones : a (bida zone), b (minna zone), and c (kontagora zone) in the state. in stage two, three fulani pastoral communities were conveniently selected in each agroecological zone by purposive sampling method. in addition to the key informants participation in the pe exercises for historical information about existing ethnoveterinary knowledge and practices on cattle diseases management, other pastoralists also participated in each community. however, the number of other participants in the exercises was not restricted since there was no size limit of attendance by others in each session. the participatory rural appraisal (pra) tools of key informants, checklist, semistructured interview, probing, transect, and triangulation [1820 ] were used to discuss and collect information. (transect) observations guided by key informants to identify and collect plant species, where necessary, for documentation. during participatory appraisal activities, informants were asked specific questions about the use of botanical and nonbotanical medicinal materials, methods of preparations, and applications. the key informants ' consensus factor on each plant or nonplant material used for a particular cattle disease condition gave indication of agreements on the usefulness of the material for such disease condition. an outline of the participants ' initial ethnoveterinary remedies was drafted during each participatory session and further probed and discussed extensively in order to confirm the information provided. for every specimen identified the vernacular names were also recorded. the collected specimens were preserved and identified in the herbarium of niger state ministry of agriculture and natural resources, minna, nigeria. the collected ethnobotanical data and other ethnoveterinary information on cbpp and other cattle disease conditions were analyzed using the method of friedman. that expresses a plant 's botanical efficacy by fidelity level. the fidelity level (key informants consensus) presents the most important plant species used for treating a particular cattle disease / condition as expressed by the key informants who are considered most knowledgeable elders possessing existing veterinary knowledge and traditional oral history on livestock in the pastoral communities. in this study, the fidelity level analytical approach was also used in evaluating the nonplants and prophylactic data generated during the participatory exercises. the fidelity level is mathematically expressed as fl = (ip / iu) 100, where fl is the fidelity level of each plant or nonplant material, ip is the number of key informants who mentioned that a plant or nonplant material has specific ethnoveterinary uses against a particular disease condition, and iu is the total number of key informants who independently suggested that the same plant or nonplant material has any ethnoveterinary uses. the traditional botanical and nonbotanical ethnoveterinary practices used in managing cbpp and other cattle disease conditions as well as the modes of their preparation and administration are presented in table 1. the traditional botanical and nonbotanical ethnoveterinary practices used in managing other cattle disease conditions as well as the modes of their preparation and administration are presented in table 2. the local names of plants and nonplant materials in hausa, fulfulde, and nupe were obtained for easy identification and documentation (table 3). traditional preventive practices in use specifically for prophylaxis against cbpp and some cattle disease conditions are shown in table 4. cattle - rearing is the main occupation of fulani pastoralists in nigeria and these herdsmen use medicinal plant remedies to manage their stocks. this study indicates that 50 medicinal materials and seven preventive practices are in use by fulani pastoralists to traditionally manage cbpp and other cattle disease conditions in niger state. this agrees with earlier reports on the relevance of different traditional healing practices in nigeria as well as other parts of the world [9, 10, 22 ]. the reliance of pastoralists on herbal and nonherbal materials for both therapeutic and prophylactic purposes in nigeria has been reported [13, 22 ]. the fulani pastoralists exhibited good existing veterinary knowledge of the pathology of various probed cattle diseases and conditions and the corresponding ethnoveterinary remedies which are mostly acquired from their parents and during grazing. this is in consonance with an observation that the understanding of animal diseases by pastoralists is partly due to experiences gathered during grazing. a. digitata (baobab) is commonly found in the northern part of nigeria and fulani group frequently uses it in treating cbpp and diarrhea cases in cattle. the study found commonly used medicinal plants by the fulani pastoralists in the treatment of cbpp cases to include adansonia digitata, anogeissus leiocarpus, stachytarpheta angustifolia, striga hermonthica, and terminalia macroptera. however, it was observed that ethnobotanical management of cbpp is not very effective as indicated by their fidelity levels of the mentioned plants. except for a. digitata and terminalia macroptera that have high fidelity levels, others have very low fidelity levels which may indicate low efficacy of the plants against the disease. the survey revealed that the preventive measures involve the use of lung tissues from infected dead cattle with cbpp (believed to be rich in infective agents) soaked in fresh milk and briefly placed on the nasal area of the healthy ones or wrapped in a rag and hung on a tree very close to the herd site. also revealed is the application of ground dried infected lungs, by spread of granules in the herd. this traditional immunization finding agrees with earlier reports that livestock keepers are aware of the fact that the principle of vaccination consists of introducing a mild form of the disease. long ago, many pastoral societies of africa, such as maasai, mauritanian moors, somali, and wodaabe, invented their own vaccines for contagious bovine pleuropneumonia, rinderpest, foot - and - mouth disease, and bovine brucellosis. they used lung tissues, urine, faces, milk, material from the feet, and tongue of the infected animals and material from the aborted fetus to vaccinate other healthy animals. the mention of other preventive practices by the pastoralists agrees with reports that, in other ethnoveterinary medical practices with surgical implications, wounds, joint conditions, and swellings are treated by applying a red - hot iron over them, with the belief that as the burnt skin heals, the ailment is healed along with it. some of the nonplant materials observed in this survey to be used by the pastoralists include wood ash, honey, oils, kerosene, kaolin, potassium, local soap, and spent engine oil which they believe are effective in ethnoveterinary management. they use spent engine oil in the management of wounds, kerosene for foot rot, and local soap as disinfectant in animals. some authors have contrast views with the findings as they reported most of these nonplant materials to be carrier mechanisms with no known medicinal values but can cause perceived improvement in performance through their effects on feed efficiency. further, these authors also observed that the use of a carrier mechanism in ethnoveterinary medical practices involves arbitrary quantities of the carrier which may dilute the drug or reduce its relative potency unlike in conventional veterinary medicine where variability in the quantity of the carrier materials is not much prominent as in ethnoveterinary medicine. the study found honey to be used in wound healing, oils (especially vegetable oil) for managing poisons and bloats, cow butter for wound healing, cattle fats for burns, and salts for preservation and appetite promotion., while poonam and singh reported some of them, such as honey, cow / goat 's milk, sugar, ghee, salt, and butter milk, to be appetizers media to improve palatability and medicinal property of certain herbal remedies. the fulani pastoralists ' methods for ethnoveterinary preparation vary and include grinding or pounding dried or fresh parts, followed by boiling or soaking in water to obtain solutions that are administered orally and sometimes mixed with feed. however, ground plant portions could also be mixed with potash or salt and given for licking. these practices of medicinal herbal preparations and administration have been agreed upon by observations of some researchers [14, 22, 28 ]. the dosage administered often varied with the parts of the plant used and the mode of preparation. however most fulani pastoralists administer the preparations once or twice daily for a week or keep treating until the animal recovers. full recovery is confirmed when the animals resume feeding and other physical activities. in a similar observation, alawa. indicated that the duration of treatment for a particular disease in ethnoveterinary practices varied and depends largely on the herdsmen, with clinical improvement of affected animals usually considered as end of that disease condition when they start feeding, leaving the possibility that those causative agents might not be completely eliminated at the beginning of improvement. this contrasts the conventional veterinary medical practices where treatment might continue to complete the dosage even after the clinical signs of a disease have disappeared. also, these findings indicate that ethnoveterinary practices are readily available and can complement conventional veterinary medical practices, but there is need to standardize modes of preparation and application of the traditional practices. further research on the active ingredients and their quantities in the ethnoveterinary materials becomes scientifically necessary so as to guide their usage. the information obtained from fulani pastoralists on ethnoveterinary practices in this study will form a basis for further ethnoveterinary research especially in studies dealing with efficacy, dosage, quality, and toxicology. those plants that are found to be effective empirically can be used in the preparation of commercial local - based veterinary pharmaceuticals, which will consequently lead to protection of the important ethnoveterinary phytotherapeutics. since some of the plants used in ethnoveterinary management of cattle by this group of pastoralists are likely to be threatened species, especially with desert encroachment into the state, conservation of such plants is recommended. the fulani pastoral communities in niger state are potential beneficiaries of such conservation effort and should be involved in such efforts in the spirit and goal of participatory epidemiology. | ethnoveterinary practices are locally available and affordable to fulani pastoralists in niger state, nigeria, to whom conventional veterinary services are often not readily available and are relatively expensive. this study was designed to identify and document medicinal plant and nonplant materials used by this group in the management of cattle diseases. participatory rural appraisal tools of checklist, semistructured interview, probing, transect, and triangulations were used to assess fulani pastoralists existing knowledge on traditional veterinary practices in nine pastoral communities spread across the state. fifty medicinal materials and seven traditional preventive practices are in use against cbpp and other cattle disease conditions. of these, 38 (76.0%) are medicinal plants and 12 (24.0%) are nonplant materials (edible earth materials and minerals). family fabaceae was most commonly mentioned while leaves were the most common parts used. most of these materials are administered by drenching with few others mixed with feed. proportions of plant parts used include leaves (47.4%), barks (31.6%), roots (10.6%), and 2.6% of each of rhizomes, fruits, seeds, and whole plants. of recently used ingredients are kerosene and spent engine oil. further research into the active ingredients of ethnoveterinary materials and dosages is necessary to guide their usage. |
the birth of a baby with a tail can cause tremendous psychological disturbance to the parents. isolated case reports of various types of human tails have been described in the literature. the main features and imaging findings of six patients have been summarised in table 1. the plain radiographs showed spina bifida in three patients at l 5 and s 1. summary of main features and imaging findings of all patients other than patient number 5 (lumbar lipomeningomyelocele), all other patients underwent surgical excision. the parents of patient number 5 refused to give consent for the operation and the patient was lost to follow - up. on histopathological examination, the specimen of sacrococcygeal teratoma showed mature tissues that included bones, cartilage, fat and neural tissues, whereas other tails showed mature adipose tissue, blood vessels and nerves. a vestigial tail describes a remnant of a structure found in embryonic life or in ancestral forms. during the 5 to 6 week of intrauterine life it contains adipose tissue, connective tissue, central bundles of striated muscle, blood vessels and nerves and is covered by skin. it can move and contract and occurs twice as often in males as in females. none of our patients showed any movement of the tail. unlike the tail of other vertebrates, human tails do not contain vertebral structures. the additional lesions found with pseudo tails are lipomas, teratomas, chondromegaly and gliomas, and there may be elongated parasitic fetus. human tail usually occurs in the lumbosacral region, but it has also been reported in the cervical region. teratoma in the human tail has also been reported. in a review series of 48 skin - covered lumbosacral masses the preoperative assessment includes a complete clinical examination including neurology, plain radiographs of the spine and computed tomography or magnetic resonance imaging. an occult spinal lesion has been reported in around 50% of the cases. in another review by lu, tail in the midline of lumbar region tail in the left side of the buttock, away from the midline tail in the midline of lumbar region tail in the midline of lumbar region tail in the midline of lumbar region (lipomyelomeningocele) sacrococcygeal teratoma with tail, away from the midline | human tail is a curiosity, a cosmetic stigma and presents as an appendage in the lumbosacral region. six patients of tail in the lumbosacral region are presented here to discuss the spectrum of presentation of human tails. the embryology, pathology and treatment of this entity are discussed along with a brief review of the literature. |
alprazolam has antidepressant activity and has been shown to be similar in efficacy to imipramine in the treatment of unipolar depression in humans. thus, alprazolam may be particularly useful in patients with mixed anxiety / depression. however, its general acceptance as an antidepressant awaits further study. deficiency of serotonin, noradrenaline and dopamine is implicated as a causal factor in depression [2, 3 ]. however, since the 1960s there has been a strong emphasis on the role of norepinephrine in both the pathogenesis of effective disorders and the mechanism of action of antidepressant medications [2, 46 ]. theories of depression also acknowledge that other factors may be involved ; the antidepressants may act on other neurotransmitters, such as acetylcholine and gamma - aminobutyric acid (gaba). the monoamines, serotonin and norepinephrine, also influence and are influenced by other processes in the brain. the neurochemical basis of depression is now considered more complex and not the result of any one specific deficit. for example, the function of the hypothalamic pituitary axis and the involvement of stress - related hormones are increasingly believed to play a role in the development of depression. it has been suggested that depression may result from down - regulation of the noradrenergic neuronal system, and antidepressants act to return the system to a state of equilibrium by increasing neurotransmitter availability by a process that involves blocking reuptake in the presynaptic neuron. depression may also be due to a change in receptor function, not neurotransmitter concentration. as a result of preclinical investigation of antidepressant mechanisms of action, the monoamine hypothesis of depression was refined to include alterations in noradrenergic receptor function [1012 ]. it has been suggested that the centrally active 1 and 2 adrenergic agonists produce antidepressant - like effects in several behavioral tests, suggesting that these receptors may be involved in the mediation of the effects of antidepressant drugs. down - regulation of -receptors was proposed as the neuronal target for the effects of some antidepressants. duncan., reported that imipramine, a common antidepressant drug, induces down - regulation of beta adrenergic receptors. also, several studies revealed that -adrenergic receptors may play an early role in the mechanism of depression and in the mechanism of action of antidepressants [1618 ]. the forced swimming test (fst) is a behavioral paradigm predicative of antidepressant activity in rodents. the immobility exhibited by rodents when they are placed in an inescapable cylinder of water reflects the cessation of persistent escape - directed behavior. exposure to the forced swimming test is also known to produce changes in the release of dopamine, norepinephrine, and serotonin in a variety of brain regions, and these effects interact with antidepressant drug treatments [21, 22 ]. experimental work on the antidepressant effect of alprazolam on animal behavior is scanty. to further understand the significance of alprazolam in treating depression, it is essential to characterize the mechanisms underlying its action, as they may relate to the proposed mechanisms of depression. norepinephrine is a candidate in both the pathogenesis of affective disorders and the mechanism of action of antidepressants [23, 24 ]. therefore, the present study was conducted to investigate the effect of sympathetic antagonists on the proposed antidepressant action of alprazolam in mice subjected to the behavior despair method. groups of 7 mice each were kept in separate cages at 2025c with 12 hours dark / light cycles. the drugs were suspended in 1% tween 80 in water because alprazolam is not freely soluble in saline ; they were administered by the intraperitoneal route. imipramine was used as the antidepressant, and the dose of 10 mg / kg was selected on the basis of a pilot test. an experiment was conducted for each antagonist : 5 mg / kg of prazocin [2730 ], 5 mg / kg of atenolol [31, 32 ], and 1 mg / kg of propranolol [27, 30, 31, 33 ]. in each experiment, the mice were divided into six groups (n=7). group 1 (control) received only a single dose of 5 ml / kg of 1% tween 80 (t80). group 2 received a single dose of 5 mg / kg alprazolam and group 3 received a single dose of the antagonist followed by the same dose of alprazolam (5 mg / kg). group 4 was treated with a single dose of imipramine (10 mg / kg) alone and group 5 received a single dose of the antagonist followed by the same dose of imipramine (10 mg / kg) ; group 6 received a single dose of the antagonist alone. a modified behavioral model of immobility, known as behavioral despair [3437 ], was used. in this model, mice are forced to swim in a restricted space from which there is no escape. following an initial period of vigorous activity, the mice adopt a characteristic immobile posture and no longer attempt to escape. they were forced to swim for six minutes in a vertical glass cylinder (height : 27 cm ; diameter : 16.5 cm) containing fresh tap water at 27c and a depth of 15 cm. the onset of immobility was recorded during the last four minutes of the six - minute testing period ; mice were judged immobile when they floated in an upright position and made only small movements to keep their head above water.. the kolmogrov simonov maximum deviation test for goodness of fit was used to determine if the data were normally distributed. treatments were compared by one - way anova if the parameters were parametric and by the mann whitney two samples (non matched) test if they were not. administration of prazocin alone resulted in significantly faster onset of immobility as compared to the control group. by contrast, imipramine alone or alprazolam alone produced significant delay of the onset compared to the control group. treatment with prazocin together with imipramine delayed the onset of immobility compared to imipramine treatment alone (table 1). effect of prazocin on the onset of immobility significantly different from the control t80-treated group at p0.05. a = significantly different from alpr+prz treated group at p0.05 ; b = significantly different from impr+prz treated group at p0.05. administration of atenolol alone significantly delayed the onset of immobility compared to the control group. administration of alprazolam alone caused a substantial delay (3048%) in the onset of immobility compared to the control group (vehicle alone). imipramine administration produced significant delay in the onset of immobility compared to the control group. administration of atenolol combined with imipramine did not significantly change the effect of imipramine (table 2). effect of atenolol on the onset of immobility significantly different from the control t80-treated group at p0.05. a = significantly different from alpr+aten treated group at p0.05 ; b = significantly different from impr+aten treated group at p0.05. propranolol alone resulted in a significantly earlier onset of immobility compared to the control group. alprazolam produced a significant delay in the onset of immobility compared to the control group. the combination of propranolol and alprazolam produced significantly faster onset of immobility than observed in either the alprazolam or the control group. propranolol significantly shortened the time to onset of immobility compared to the imipramine treated group or the control group (table 3). effect of propranolol on the onset of immobility significantly different from the control t80-treated group at p0.05. a = significantly different from alpr+prop treated group at p0.05 ; b = significantly different from impr+prop treated group at p0.05. the antidepressant effect of alprazolam was investigated using the forced swimming test as an acute stress model. although the forced swimming test does not induce in mice symptoms similar to human depression, it was used because it is simple and reliable across laboratories. in addition, the majority of antidepressants have been shown to prolong the time to onset of the immobility, and their effectiveness correlates significantly with clinical potency alprazolam gave a uniform effect as an antidepressant in this animal model of depression. mice treated with alprazolam showed a delay in the onset of immobility compared to the control group. the putative antidepressant effect of alprazolam may be mediated by a gaba - ergic mechanism that is independent of the benzodiazepine receptor. in a previous communication, it was reported that flumazenil (an antagonist at the benzodiazepine receptor), did not alter the antidepressant effect of alprazolam (or imipramine), whereas these effects were blocked by picrotoxin. unlike diazepam, alprazolam may enhance the release of serotonin (5-ht) in the hippocampus, and this may at least partly explain its antidepressant activity. several observations indicate that alprazolam and standard antidepressants have some similar actions, such as the down - regulation of the beta - adrenergic receptor and their anti - anxiety effect. the circulating level of corticotropin - releasing factor (crf) is elevated in major depression and other psychiatric disorders [43, 44 ]. in the forced swimming test, there is a dose - dependent increase of endogenous crf, which may play a role in the behavioral response in this model. crf serves as a neurotransmitter in locus coeruleus, the largest aggregate of noradrenaline - containing cells in the mammalian brain. it is thought to be hypersecreted in depression and upon initiation of the stress response [4648 ]. the inhibition of 5ht reuptake (by sertraline) may serve as a functional antagonist of crf in depression. alprazolam may produce its antidepressant effect by decreasing the release of crf in locus coeruleus, amygdala and several cortical regions ; it may also enhance the release of 5ht in hippocampus, which would serve as a functional antagonist of crf. imipramine, a typical antidepressant, produced a significant delay in the onset of immobility compared to the control group. imipramine inhibits presynaptic reuptake of the biogenic amines, serotonin and noradrenaline to produce antidepressant action [5052 ]. imipramine may produce its antidepressant action through gaba - ergic mechanisms, causing the release of catecholamine [40, 5356 ]. reduction of 1 noradrenergic neurotransmission increases depressive behavior, coupled with the fact that this change can result from elevated corticosteroid secretion. prazocin may decrease the plasma level of interleukin-1 (stress marker) [59, 60 ], which may lead to the potentiation of imipramine action. imipramine may produce antidepressant effects through postsynaptic 2-agonist (clonidine), which will activate a sub - threshold dose of imipramine. in an earlier study, small doses of clonidine potentiated the effects of antidepressants in the mouse in a similar forced swimming test. prazocin treatment with alprazolam has significant synergistic effects on alprazolam antidepressant action, which may occur by decreasing the level of interleukin1. also, whereas alprazolam increases hippocampal 5-ht release, diazepam decreases it. in the ca1 region of the hippocampus, the neurochemical profile of alprazolam was similar to that of the 2-adrenergic agonist, clonidine. enhanced 5-ht release in the hippocampus, exhibited by the atypical benzodiazepine, alprazolam but not by the typical benzodiazepine, diazepam, may be an underlying mechanism for the antidepressant activity of alprazolam. therefore, blocking 1 by prazocin may potentiate alprazolam action through the activation of 2 receptors. atenolol is a selective 1 adrenoceptor antagonist, and by itself it produced significant antidepressant action. at least in some instances, the antidepressant effect of atenolol may be mediated by the down - regulation of 1-adrenoceptor [64, 65 ]. in general, receptor down - regulation is a long - term effect of chronic drug administration and does not occur acutely following the administration of a single dose. also, atenolol is a hydrophilic molecule that does not easily penetrate the blood - brain barrier. therefore, it is safe to rule out 1-adrenoceptor down - regulation as the mechanism for the atenolol effect observed in our study. the dose used in this study was sufficient to partially penetrate the cns and produce the observed effect. atenolol may act peripherally to initiate an unknown mechanism that affects the noradrenergic system centrally. it is also possible that the atenolol observations may constitute a false positive result of a -blocker. blocking a steady - state agonist response to measure the potency of an antagonist might in some cases yield erroneous results and the response most of the authors suggested that atenolol lowers melatonin release via specific inhibition of 1-adrenoreceptors. the decrease in melatonin may contribute to the disturbance in sleep and mood associated with atenolol use [19, 6770 ]. in our study atenolol did not change the effect of imipramine significantly, possibly because the maximal capacity of imipramine to down regulate the -receptor was reached. therefore, atenolol acting by the same mechanism did not change imipramine antidepressant effects significantly. this effect can be explained by the pronounced 2 receptor activity due to the blocking of 1 receptor. this explanation may be accepted if the dose used in this study was enough for atenolol to partially penetrate the cns, or atenolol could be acting through a peripheral mechanism to induce this effect. alprazolam induces release of noradrenaline through a gaba - ergic mechanism [40, 5255 ], which stimulates the sensitive 2-receptor, and as a result atenolol significantly potentiates the antidepressant action of alprazolam. alprazolam did not change the effect of atenolol ; this is observed by comparing the group treated with atenolol alone to the combined treatment with atenolol and alprazolam. this may be due to the maximum effect produced by both drugs on 1 (inhibition) and 2 (stimulation). blocking 1-receptors produces antidepressant action as observed by the atenolol effect, while blocking both 1 and 2 receptors produced depression. in the forced swimming test, it was found that isoprenaline increases the duration of immobility, while salbutamol decreases it. the central 1and 2 receptors may be acting in opposite direction to modify the duration of immobility which means that activation of 1 leads to enhanced behavioral despair while 2 activation reverses this effect. in animals, the psychopharmacological profile of 2 stimulant (salbutamol) is, to a certain extent, very similar to tricyclic antidepressant drugs such as imipramine [73, 74 ]. in endogenous depressive patients, the antidepressant effect of salbutamol is both clear and rapid [73, 75 ]. it was speculated that the antidepressant effect of imipramine is related to the stimulation of central 2 adrenergic receptors. both salbutamol and imipramine prevent or reverse reserpine induced hypothermia while these effects were antagonized by propranolol, suggesting that the stimulation of -adrenergic receptors could be a common mechanism underlying their effects. stimulation of the central 2 adrenergic receptor, particularly those located in the hippocampus, produces antidepressant - like effects on behavior. also, 2 agonist (salbutamol) facilitates 5ht transmission in the rat brain probably via stimulation of central receptors. the reduction in ca2+i that is caused by inhibiting ca2 + influx through voltage - gated channels and by enhancing ca2 + efflux may contribute in part to the antidepressant - like activity shown by salbutamol, as verapamil and nifedipine possessed antidepressant - like properties [79, 80 ]. imipramine may cause down regulation or blockade of 1 receptors, thus the balance between 1 and 2 receptors is disturbed leading to the predominance of 2receptor activity which produces an antidepressant effect. blocking 1 or stimulation of 2 receptors may mediate the mechanism of imipramine antidepressant action. administration of propranolol with imipramine produced significant antagonism of imipramine antidepressant effect, and even produced significant depression. propranolol, a non - selective -blocker, combined with alprazolam abolished alprazolam antidepressant effects and even produced significant depression. alprazolam induces the release of mono - amine transmitters through gaba - ergic system [5356 ]. our observation of the depressant effect of propranolol agrees with previous studies which associate blockers with induction of symptoms of depression as mentioned above. in conclusion, this study demonstrates that alprazolam has a significant antidepressant effect in the rodent forced swimming behavioral model. our data also indicate that this effect may be mediated by the release of noradrenaline, which stimulates 2-adrenoeceptors. imipramine may produce its antidepressant action through the activation of 2 receptors by down regulating or blocking the 1-receptor. | alprazolam is an anti - anxiety drug shown to be effective in the treatment of depression. in this study, the effect of sympathetic receptor antagonists on alprazolam induced antidepressant action was studied using a mouse model of forced swimming behavioral despair. the interaction of three sympathetic receptor antagonists with benzodiazepines, which may impact the clinical use of alprazolam, was also studied. behavioral despair was examined in six groups of albino mice. drugs were administered intraperitoneally. the control group received only a single dose of 1% tween 80. the second group received a single dose of alprazolam, and the third group received an antagonist followed by alprazolam. the fourth group was treated with imipramine, and the fifth group received an antagonist followed by imipramine. the sixth group was treated with a single dose of an antagonist alone (atenolol, a 1-selective adrenoceptor antagonist ; propranolol, a non selective -adrenoceptor antagonist ; and prazocin, an 1-adrenoceptor antagonist). results confirmed the antidepressant action of alprazolam and imipramine. prazocin treatment alone produced depression, but it significantly potentiated the antidepressant actions of imipramine and alprazolam. atenolol alone produced an antidepressant effect and potentiated the antidepressant action of alprazolam. propranolol treatment alone produced depression, and antagonized the effects of alprazolam and imipramine, even producing depression in combined treatments.in conclusion, our results reveal that alprazolam may produce antidepressant effects through the release of noradrenaline, which stimulates 2 receptors to produce an antidepressant action. imipramine may act by activating 2 receptors by blocking or down - regulating 1 receptors. |
diabetic macular edema (dme) results from the exuding and accumulation of extracellular liquid and proteins in the macula13 following structural changes to the endothelium of the retinal blood vessels that lead to the rupture of the hematoretinal barrier and thus to an increase in vascular permeability.4 the pathological neo - angiogenesis at the basis of such alterations is provoked by the increase in cytokines (like interleukin-6 and -8), prostaglandins, and vascular endothelial growth factor (vegf).4,5 laser photocoagulation, considered for a long time as the main treatment option for dme, may lead to paracentral deficits of the visual field and reduced color vision and sensitivity to contrast.1,2 for these reasons, intravitreal therapies with anti - vegf have been considered as an efficient treatment strategy for patients affected by dme,5,7 with drugs such as pegaptanib6,9,10 ranibizumab,8 and bevacizumab11 being principally used. steroids reduce inflammatory mediators through a more widespread action that blocks vegfs, inflammatory cytokines, and prostaglandins.12 our study investigates an intravitreal dexamethasone implant (ozurdex ; allergan inc, irvine, ca, usa) and its efficacy as a treatment for dme.13 this implant was developed to guarantee sustained levels of dexamethasone in the posterior section of the eye for a period of 6 months.14,15 ozurdex has recently been approved by the us food and drug administration and by the european union (eu), and is licensed in all eu countries for the treatment of macular edema (me) following retinal vein occlusion.16,17 nonetheless, there is evidence of its efficacy in multiple clinical applications including dme, me associated to uveitis or irvine - gass syndrome, dme in eyes having undergone vitrectomy,18 noninfectious vitritis, and as an adjuvant therapy for age - related macular degeneration.19 in this study, we evaluate the effects of a single intravitreal injection of ozurdex, through a 6-month follow - up time period, in eyes affected with persistent dme. the study was conducted at the policlinico umberto i hospital of sapienza university of rome. the eligibility criteria were : age 18, a best - corrected visual acuity (bcva) between 5 (corresponding to 1/10, logarithm of the minimum angle of resolution [logmar ] 1.0 or more) and 40 (corresponding to 5/10, logmar 0.3 or less) letters, and macular edema with a thickness of 275 m. the initial bcva before the implant (at t0) was an average of 18.80 11.06 letters (logmar 0.67 0.23), and the mean central macular thickness (cmt) was 518.80 224.75 m. all patients had persistent dme although 13 of the patients recruited had previously undergone treatment with anti - vegf : three with bevacizumab (avastin ; roche, basel, switzerland), two with pegaptanib (macugen ; eyetech pharmaceuticals, inc, new york, ny, usa), and eight with ranibizumab (lucentis ; genentech inc, south san francisco, ca, usa) in the 3 months prior to investigation. the remaining patients presented counter recommendations to intravitreal injections of anti - vegf (such as a certified diagnosis of vascular accidents). patients were excluded if : pregnant, had uncontrolled arterial hypertension, venous occlusions, evolved cataract, glaucoma, an epiretinal membrane visible by optical coherence tomography (oct), age - related macular degeneration, uveitis, a history of vitreal surgery, cataract surgery (in the previous 6 months), yag laser capsulotomy (within 2 months prior to the trial), or had undergone recent panretinal laser photocoagulation or grid laser photocoagulation (in the 3 months prior to investigation). the treatment was applied to only one eye of each participant : the eye selected for treatment was the one that showed inferior visual acuity (va) and a greater macular thickness with respect to the other eye. the treatment protocol established that should the control eye have deteriorated to such an extent as to require intervention, then the treatment used would be applied to that eye also, if necessary. all patients underwent : general preoperative anamnesis, cardiological examination, electrocardiogram, and blood tests that included glycosylated hemoglobin (hba1c). all patients gave their informed consent to the injection treatment after they had been briefed regarding the benefits, risks, and possible complications of the intervention. at baseline, ocular exploration was carried out : fluorescein angiography was performed to evaluate the presence of macular ischemia only at baseline, whilst bcva was assessed through early treatment diabetic retinopathy study (etdrs) tables placed at a distance of 4 m, by slit - lamp biomicroscopy, ocular tonometry (using a goldman applanation tonometer), fundus biomicroscopy, oct (for the measurement of macular thickness and morphology using a spectralis hra - oct produced by heidelberg engineering [heidelberg, germany ] with a volumetric 512 49-scan), fluorescein angiography, and color fundus photography. these exams were carried out at day 3, and month 1, 3, 4, and 6 post - injection. the controls carried out on the day after the injection were the following : examination of the anterior section of the eye using slit lamp, tonometry, and indirect fundus biomicroscopy. primary outcome measures included mean change from baseline in bcva and central retinal thickness at all follow - up visits. we considered the efficacy of the implant as a mean improvement of visus (va) of 10 letters (two lines) equivalent to a mean logmar of 0.2. the outcomes expected were : a reduced mean cmt 250 m, including a structural layer analysis of the retina with oct. the evaluation of the integrity of the external membrane, and the inner and outer segments of the photoreceptor interface, was carried out at baseline and at 6 months after the implant. all implants were performed under sterile operating room conditions by author ep, after preparation of the conjunctiva using 5% povidone iodine solution, topical anesthetic with ropivacaine, and positioning of the blepharostat. a 700 g slow - release intravitreal dexamethasone implant (ozurdex) was placed in the vitreous cavity, behind the crystalline lens.1114 patients were treated with a topical ophthalmic antibiotic for 7 days after the treatment. all patients were monitored for local or systemic adverse effects relative to the implant for the duration of the study. demographic data of the pooled patients, duration of dme, and previous treatments were recorded. wilcoxon tests were carried out to measure mean differences between pre- and post - implant values of all the parameters evaluated (etdrs, logmar, and cmt) and obtained at different temporal follow - up points (at day 3 to month 6). wilcoxon tests were carried out to measure mean differences between pre- and post - implant values of all the parameters evaluated (etdrs, logmar, and cmt) and obtained at different temporal follow - up points (at day 3 to month 6). the appearance of undesired side - effects correlated to the drug, such as inflammation of the anterior chamber, ocular pain, keratitis, or vitreous opacity, was monitored ; those correlated to the surgical intervention itself, such as endophthalmitis, perforation of the eye, conjunctival hemorrhage, and systemic effects related to the drug, were also monitored closely. patients who showed a worsening of their clinical - functional condition at month 4 were recommended for a second treatment cycle. indicators of this worsening were considered as a reduced va (a reduction of logmar scores of at least 0.2 or 10 letters) and an increase of macular thickness (of at least 150 m, as measured with oct). patients who showed a worsening of their clinical - functional condition at month 4 were recommended for a second treatment cycle. indicators of this worsening were considered as a reduced va (a reduction of logmar scores of at least 0.2 or 10 letters) and an increase of macular thickness (of at least 150 m, as measured with oct). seventeen patients were selected (and a total of 20 eyes) : 14 males and three females, mean age 67 8 years and affected with dme for an average 46.30 18.64 months. the response to treatment was evaluated independent of age, sex, and concurrent pathologies. the final analysis of the data allows us to compare va and cmt from baseline to month 6. no patients had a worsening of their cataract during this (brief) period of study. in two patients, the recorded cmt values at month 6 were higher than those recorded at baseline, and they were thus reconsidered for treatment. an increment of intraocular pressure was seen in one patient only, and this happened 2 months after the implant (26 mmhg). the evaluation of the integrity of the external membrane, and the inner and outer segments of the photoreceptor interface, performed at baseline and at 6 months after the implant was kept. at day 3 after the intravitreal injection, the mean va was 18.80 11.06, mean logmar 0.67 0.23, and mean cmt 412.75 176.23. at 1 month follow - up, patients showed a mean etdrs of 26.15 11.03 (p = 0.04), a mean logmar 0.525 0.190 (p = 0.03), and a mean cmt 292.0 140.8 (p < 0.0001). at month 3, (p = 0.0087), mean logmar was 0.52 0.20 (p = 0.034), and mean cmt was 346.95 135.70 (p = 0.0018). at month 4 follow - up, mean etdrs was 25.95 10.74 (p = 0.045), mean logmar was 0.56 0.22 (p = 0.12), and mean cmt was 476.55 163.14 (p = 0.45). the last follow - up visual examination was carried out at month 6, and the evaluation of all parameters showed that mean etdrs was 21.25 11.46 (p = 0.5), mean logmar was 0.67 0.23 (p = 1) and mean cmt was 494.25 182.7 (p = 0.67) (figure 4). regarding the control eyes (14 eyes, because three patients had bilateral treatment at a later stage due to the worsening of dme), mean va expressed as logmar scores at month 4 follow - up with respect to baseline increased from a mean value of 0.35 (0.20.4) to 0.4 (0.20.5), whilst the mean etdrs reduced from 39 (3548 letters) to 36.5 (2844 letters). mean cmt increased from an initial 325.5 m (260347) to 344 m (285440). at month 6 of follow - up, two eyes had received a cycle of treatment due to the reinjection criteria, ie, a worsening of their condition had been established. in the two patients who received reinjection, levels of glucose in the blood were not balanced, in fact hba1c was on average greater than 11%. from the data at 6 months follow - up, we can see that the slow - release intravitreal dexamethasone implant, ozurdex, shows efficacy for the treatment of dme, as both substantial improvements were registered in bcva values, and significant reductions of cmt observed. in accordance with other literature, this significant improvement is seen from day 3 of the intravitreal implant. the peak efficacy of the implant appears to be reached at month 1 through to month 3, and this then slowly decreases from month 4 to 6. this result may be explained either by the reduced release of the drug, or by the worsening of the chronic diabetes. the etdrs, logmar, and cmt values recorded at the end of the study, at month 6, were less than those recorded at baseline in all but two of the patients. in these two, a rebound effect was seen at month 6 after an initial improvement had been registered. however, these patients had not controlled their glycemic levels adequately, as testified by their high hba1c levels (above 6%). in these patients, a second slow - release intravitreal dexamethasone implant was inserted. regarding the second aim of the study, ie, to evaluate the safety profile of the implant, we can say that our study is in accordance with others (haller and kuppermann,17) and that no particular complications resulted from either the implant or the drug itself. in particular, few eyes were evaluated, with a very short follow - up period, and hence, it is difficult to reach robust conclusions. however, this study suggests that the slow - release intravitreal dexamethasone implant (ozurdex) is both efficient and safe for the treatment of secondary macular edema caused by diabetic retinopathy. the results that ozurdex has a beneficial short - term effect on va and retinal thickness are not surprising, and consistent with previous works.1317,2224 perhaps the association of this treatment intervention and other therapeutic strategies may help better the outcomes for this pathology.20,21 similar efficacy and safety studies are certainly needed, with a greater number of patients and for a longer period of time. | backgroundto evaluate the efficacy and safety of an intravitreal dexamethasone implant (ozurdex ; allergan inc, irvine, ca, usa) in patients with persistent diabetic macular edema (dme) over a 6-month follow - up period.methodsseventeen patients (20 eyes) affected by dme were selected. the mean age was 67 + 8 years, and the mean duration of dme was 46.3 + 18.6 months. the eligibility criteria were : age 18, a best - corrected visual acuity between 5 and 40 letters, and macular edema with a thickness of 275 m. thirteen patients had also previously been treated with anti - vascular endothelial growth factor medication.resultsthe mean etdrs (early treatment diabetic retinopathy study) value went from 18.80 + 11.06 (t0) to 26.15 + 11.03 (p = 0.04), 28.15 + 10.29 (p = 0.0087), 25.95 + 10.74 (p = 0.045), 21.25 + 11.46 (p = 0.5) in month 1, 3, 4, and 6, respectively. the mean logmar (logarithm of the minimum angle of resolution) value went from 0.67 + 0.23 (at t0) to 0.525 + 0.190 (p = 0.03), 0.53 + 0.20 (p = 0.034), and 0.56 + 0.22 (p = 0.12) in month 1, 3, and 4, respectively, to finally reach 0.67 + 0.23 in month 6. the mean central macular thickness value improved from 518.80 + 224.75 m (at t0) to 412.75 + 176.23 m, 292.0 + 140.8 m (p < 0.0001), and 346.95 + 135.70 (p = 0.0018) on day 3 and in month 1 and 3, respectively, to then increase to 476.55 + 163.14 m (p = 0.45) and 494.25 + 182.70 m (p = 0.67) in month 4 and 6.conclusionthe slow - release intravitreal dexamethasone implant, ozurdex, produced significant improvements in best - corrected visual acuity and central macular thickness from the third day of implant in dme sufferers, and this improvement was sustained until the third month. |
invasive fungal infections (ifd) are increasingly recognized and represent a primary cause of morbidity and mortality in critically ill patients [1 - 4 ]. a variety of factors, including immunosuppressive agents, broad - spectrum antibiotics, and antineoplastic agents influence the incidence and severity of ifds. transplant and haematopoietic stem cell transplant recipients, intensive care unit and surgical patients display the population at risk [1,4 - 7 ]. the introduction of voriconazole, posaconazole and echinocandins (caspofungin, micafungin and anidulafungin) improved the therapeutic option for treatment of invasive aspergillosis (ia). although the outcome of ia is largely influenced by the state of immunosuppression, factors related to the fungus also play a role. until recently, species identification was sufficient to guide antifungal therapy, but the emergence of acquired resistance limits the use of species identification for predicting activity of antifungal agents. aspergilli, less susceptible to antifungals emerged and acquired resistance to azoles have been found mainly in aspergillus fumigatus ; this has launched a new era in handling aspergillosis. this article reviews the epidemiology and antifungal resistance against azoles and candins with particular emphasis on aspergillus species. in a 4-year - study pagano. showed that 64% of ifds in patients with haematological malignancies were caused by moulds and among them 90% were due to aspergillus species. overall, the incidence of ia varies according to underlying diseases, pathogen [lf ] and geographic location ; rates of up to 7% are reported in europe. mortality rates for ia are high and vary according to patient population, ranging from 38% in patients with acute myelogenous leukaemia, from 50 - 60% in patients with organ transplantation and from 70 - 85% in other immunosuppressed patients [11 - 16 ]. although a. fumigatus still represents the leading cause of ia, species like aspergillus terreus and aspergillus flavus become more frequent. these non - a. fumigatus may be intrinsically resistant to antifungal agents (eg a. ustus) and the clinical presentation and evolution of ia may differ from commonly observed a. fumigatus infections [18 - 23 ]. maximizing the efforts the mic represents the lowest drug concentration that results in a notably reduction or complete lack of fungal growth. in vitro resistance secondary resistance is generated following exposure to an antifungal and may be associated with an altered gene expression. clinical resistance is defined as the failure to eradicate an infection despite the administration of an adequate antifungal. such failures can be attributed to a combination of the host, the pathogen and the drug. using the european committee for antibiotic susceptibility testingg (eucast) methodology breakpoints were recently proposed for a.fumigatus and itraconazole, voriconazole and posaconazole ; for itraconazole and voriconazole, 2 mg / l (resistant) ; for posaconazole, 0.5 mg / l respectively. it is suggested to differentiate between single - azole, pan - azole and multi - azole resistance (see table 1), the majority of infections is associated with clinical failure when treated with the ascertained agent. azole resistance in a. fumigatus mics = minimal inhibitory concentrations yet reports from the netherlands and manchester display an alarmingg increase of azole resistance in a. fumzgatus since 1998. the first published case of itraconazole - resistance in a. fumigatus appeared in 1997 ; in 2000, a survey testing over 900 isolates showed a 2% prevalence of itc resistance in manchester. in 2007 the percentage of patients with an azole - resistant a. fumigatus increased up to 15%. in the netherlands azole resistance increased dramatically from 2.5% in 2000, to 4.9% in 2002, to 6.6% in 2004 and to 10% in 2009. this represents an increasing frequency of 6% per year and is an issue due to the limited number of antifungals. overall, azole - resistance differs from country to country and occurred sporadically in belgium, denmark, france, sweden, spain and norway [33 - 39 ]. in spain, the prevalence is about 2% among clinical a. fumigatus and in austria about 0%. in the usa, species with mics of voriconazole and posaconazole > 2 mg / l remain rare, less than 1%. the clinical presentation and disease evolution may be related to the underlying genotypes in a. fumigatus (table 2). clinical overview of azole resistance in a. fumigatus abpa = allergic bronchopulmonary aspergillosis azole drug resistance in a. fumigatus has been reported both, before and after drug exposure ; acquired resistance appears to develop through treatment of patients or through exposure of isolates to azole fungicides in the environment. these findings have major implications for clinical practice especially as fungal drug resistance is an acute issue due to the limited number of antifungal compounds. experts expect that triazole resistance in this haploid, sparingly sexual worldwide airborne fungus will increase. key elements in the management of patients will be an accurate speciation of aspergillus species and the performance of in vitro susceptibility testing for an approbiate antifungal treatment. presently we do not have exact data on the prevalence of azole - resistance aspergilli in germany, but seems to be rather low than high. where invasive candidiasis was once the predominant type of invasive fungal infections, invasive mould infections have become increasingly important, including those caused by unusual pathogens. aspergillus species are the most frequent mould pathogens, but the number of infections caused by previously rare pathogens, such as the zygomycetes and fusarium species, is increasing. the reasons for the shift in the epidemiology are multifactorial, but are a result, at least in part, of the increased use of extensive voriconazole and echinocandins as prophylaxis / treatment. as a result of growing numbers of immune - suppressed patients with risk factors, the patient populations for ia will expand and will include patients with haematological malignancy, icu intervention, pulmonary disease [e.g.chronic obstructive pulmonary disease (copd) and asthma ], sot recipients and patients with solid tumours. much less is currently known about echinocandin resistance in aspergillus, in part because susceptibility testing is not routinely performed and because the methods suffer from technical difficulties and suboptimal reproducibility ; breakthrough infections with a. fumigatus showing high minimum effective concentrations have been reported sporadically. so far, the selection pressure of candins has risen and the development of resistance is presumed to be inevitable. these antifungals bind to lanosterol 14--demethylase (14--dm, or cyp51p) which is encoded by the erg11 genes. such step leads to depletion of ergosterol and an accumulation of lanosterol and other toxic 14--methylated sterols. several pathomechanisms account for azole resistance in a. fumigatus ; these include a modification of target enzymes, an increased expression of drug efflux mechanisms, an overexpression of target enzymes, an incorporation of exogenous cholesterol, an overexpression of hsp90 and of a sterole - regulatory element binding protein. the resistance phenotype depends on the amino - acid substitution and more than one azole can be affected. azole - resistant isolates have been reported as multidrug resistant, multiazole resistant, azole cross - resistant and multiple - triazole resistant isolates. in most cases azole resistance has been associated with point mutations in cyp51a, which represents the target enzyme of the azoles ; hot spots at codons 54, 98 and 220 are most frequently characterized [26,33,35,48 - 50 ]. interestingly, other mutations have been found in azole susceptible strains and so are unlikely to be associated with resistance. the resistance mechanisms differ between the dutch and british azole - resistant isolates ; in the netherlands, the presence of a single resistance mechanism (denoted by tr / l98h, a point mutation at codon 98 accompanied by a tandem repeat in the promoter region), was found in over 90% of clinical a. fumigatus isolates. by contrast, several cyp51a mutations are present in the uk strains and no prevalence of any one alteration. the reasons for this might be due to differences in the patient population from which the isolates originate. azole - resistance may develop due to exposure of a. fumigatus to azole fungicides for plant protection. howard. suggest that the reasons of the widespread increase of azole - resistance in the uk may be part of long - term azole drug exposures in patients. echinocandins are a unique class which block the -(1,3)-d - glucan synthesis by inhibiting - (1,3)-d - glucan synthase. much less is currently known about echinocandin resistance in aspergillus and only few clinical isolates associated with treatment failures have been investigated. in such isolate mutation in the fks1 target gene was not detected, but expression of the fks1 gene was found to be upregulated. manipulated or laboratory - selected strains with various degrees of caspofungin resistance have been described. some of these strains have been found to have mutations in the ecm33 gene (afuecm33), encoding cell wall proteins. strains with mutations in the fks1 gene encoding a subunit of the -1,3-d - glucan synthase enzyme have been generated. in other resistant aspergillus mutants the glucan synthase exhibited a wild - type affks1 gene sequence, where the function, level, and the enzyme itself were susceptible to caspofungin. for example, aspergillus niger is much more susceptible to echinocandins than other species probably in charge of its different cell - wall composition. aspergillus lentulus isolates are less susceptible to caspofungin, although they maintain susceptibility to anidulafungin and micafungin. the analysis of the a. lentulus fks sequence did not reveal a polymorphism at any of the known hot - spot regions of the gene. cross - resistance patterns are closely linked with the position of the mutation in the cyp51a gene. isolates with mutations at codon 54 remain voriconazole susceptible although cross - resistant to posaconazole. cross - resistance patterns in isolates with m220 alterations appear to be unpredictable, particularly with respect to voriconazole. the risk of cross - resistance between the azole compounds is high, in one report 74% and 65% of itraconazole resistant isolates were cross - resistant to posaconazole and voriconazole respectively. between itraconazole, voriconazole and ravuconazole cross - resistance was demonstrated in 10 clinical isolates of a. fumigatus obtained from patients with long - term exposure to itraconazole or voriconazole. also, broad - spectrum cross - resistance among all the azoles has been shown in a. fumigatus in a patient receiving prolonged itraconazole prophylaxis. overall, there is a limited number of reported cases that help us to understand the clinical impact of azole resistance on clinical outcome. for example, in a small case series of patients with ia and no respond to voriconazole, treatment with posaconazole was successful in 50% of infections. on the other hand in animal model of ia caused by an itraconazole - resistant a. fumigatus strain, the potential frequency of cross - resistance amongst echinocandins in aspergillus species is still unclear and has not been investigated in detail. at present, there is no evidence that the activity and efficacy of other antifungal compounds, such as the polyenes and echinocandins, is attenuated in azole - resistant isolates. | aspergilli, less susceptible to antifungals emerge and resistance to azoles have been found mainly in aspergillus fumigatus ; this has launched a new phase in handling aspergillosis. resistant strains have currently been reported from belgium, canada, china, denmark, france, norway, spain, sweden, the netherlands, uk and the usa. centres in the uk (manchester) and the netherlands (nijmegen) have described particularly high frequencies (15 and 10% respectively), and a significant increase in azole resistance in recent years. the reason of this high incidence may be due to long term azole therapy in patients with chronic aspergillosis in manchester, and due to high use of agricultural azoles in nijmegen. the primary underlying mechanism of resistance is as a result of alterations in the cyp51a target gene, with a variety of mutations found in clinical isolates and one genotype identified in the environmental (lh98). reports on well documented in vitro and in vivo resistance to echinocandins are rare for aspergillus species and resistance may be under - diagnosed as susceptibility testing is less frequently performed due to technical reasons. |
the human species can be considered as relatively infertile (viudes - de - castro and vicente, 1997 ; moce., 2005). the average monthly fecundity rate of about 20% implies that among human couples trying to conceive many exposure months may be needed to achieve their goal (evers, 2002). it has also been long known that with increasing chronologic age, female fecundity the ability to produce offspring decreases. this knowledge is based on studies involving both natural historical (spira, 1988 ; wood, 1989) and contemporary populations (abma., 1997), as well as on studies of age dependent success rates in assisted reproduction technology (art) 2009 ; templeton., 1996 ; centers for disease control and prevention, 2007). the age related female infertility (stephen and chandra, 2006 ; noord - zaadstra., 1991) ovarian reserve can be defined as the number and quality of the remaining follicles and oocytes in both ovaries at a given age. decline in follicle numbers dictates the occurrence of irregular cycles and menopause, while quality decay of the oocytes results in decreasing fertility, defined as the capacity to conceive and give birth to a child (te velde and pearson, 2002) (figure 1). there is substantial individual variation in the onset of menopause, varying roughly between 40 and 60 years, with a mean age of 51 (morabia and costanza, 1998 ; thomas., 2001). along the same pattern, the rate of decline in fertility may vary considerably between women of the same age. this implies that a woman at the age of 35 either may be close to natural sterility or have a fertility comparable to a 25 year old woman (broekmans., 2004 ; te velde and pearson, 2002 ; eijkemans., 2005) (figure 2). the insights into the process of ovarian ageing imply that for ovarian reserve testing prior to art, female age remains the predictor of first choice. the availability of a test capable of providing reliable information regarding a woman s individual ovarian reserve within a certain age category would enable the clinician to provide an individually tailored treatment plan. for instance, in older women the finding of a high ovarian reserve may justify the decision to allow art treatment, while in young women with exhausted reserve either early application, refusal of art or choosing for egg donation could be the consequence. the first notice on ovarian reserve assessment prior to starting ivf was published in 1988. fsh levels in the early follicular phase appeared related to stimulation response and outcome of ivf treatment (muasher., 1988). soon thereafter, the predictive role for basal fsh in ivf treatment was further confirmed by scott. (scott., 1989), who stated that cycle day three fsh levels predicted pregnancy outcome and stimulation characteristics in ivf, and might be useful in counselling patients. in the two decades thereafter, a large body of additional work was published, showing that several other cycle day 3 parameters, such as inhibin b, the antral follicle count (afc) and antimullerian hormone (amh) were capable of predicting ovarian responsiveness, and, to a much lesser extent, the outcome of ivf in terms of pregnancy (seifer., 1997 ; tests that challenged the cohort of fsh sensitive follicles in various ways had equal predictive capacity to predict response and outcome compared to basal tests, and thus failed to achieve wide application (loumaye., 1990 ; ovarian reserve can be considered normal in conditions where stimulation by exogenous gonadotropins results in the retrieval of some 6 - 14 healthy oocytes at follicle puncture (fasouliotis., 2000 ; with such a yield the chances of producing a live birth through ivf are considered optimal (van der gaast., 2006). in addition to the number of recruitable follicles (a reflection of the ovarian reserve status), follicle sensitivity to fsh as well as the pharmacokinetics of fsh determine a woman s ovarian response to stimulation (karlsson., 1998 ; karlsson., 1997). the dose of fsh used may therefore be a factor of importance, although the therapeutic range of this compound seems quite narrow. higher doses of fsh may lead to higher numbers of oocytes retrieved in younger patients (out., 2001), but not in all studies (harrison., 2001). such an approach will certainly fail in older women (yong., 2003) or in women expected to have a poor response to stimulation based on an abnormal ovarian reserve test (klinkert, 2005). with currently applied dose levels of exogenous fsh (150 - 450 iu) stimulation of the ovaries will be maximal in virtually 100% of cases. the preferred outcome of or test prediction studies would be live birth after a series of art cycles in order to express of a couple s fertility potential. other outcome measures (especially oocyte yield or follicle number and pregnancy after one ivf / icsi cycle) are in fact the most common. however, ovarian reserve tests mainly relate to the size of the follicle cohort that is at any time responsive to fsh. the antral follicle count (afc) assessed by transvaginal ultrasonography provides direct visual assessment of the cohort (hendriks., 2005), while the endocrine markers anti mullerian hormone (amh) and inhibin b are released products from the antral follicle pool (broekmans., 2006 ; seifer., 1997 ; basal fsh provides a more indirect marker, as it reflects a reduced feedback from the antral follicle pool as it becomes smaller in size. it goes without saying that the focus on quantity prohibits high expectations on the relation to oocyte quality and pregnancy as outcome. ovarian reserve test evaluation should imply the assessment of predictive accuracy and clinical value of the test. predictive accuracy refers to the degree by which the outcome condition (pregnancy or poor response) is predicted correctly. summary statistics of accuracy include sensitivity (rate of correct identification of cases with e.g. poor response) and specificity (rate of correct identification of cases without poor response) (deeks, 2001 ; grimes and schulz, 2005). using the sensitivity and specificity for a range of cut off levels a receiver operating characteristic curve can be drawn and area under this curve calculated to represent the overall predictive accuracy of the test. assessment of the clinical value is a complex process through which the applicability in daily practice should become clear. the overall accuracy represented by the roc curve, the choice of a cut off for abnormality, the rate of abnormal tests at that cut off, the post test probability of disease (i.e., poor response or non - pregnancy), the valuation of false positive and false negative test results and the consequence for patient management of an abnormal test will all contribute to the process of deciding whether a test is useful or not. an overall estimate of test quality is the positive likelihood ratio, which describes the chance of an abnormal test over a normal test in the case of non pregnancy or poor response. the cost of carrying out the test as a routine measure and the burden to the patient, balanced against the reduction in costs by excluding cases with low pregnancy prospects need also to be incorporated in the decision process. finally, clinical value may also be influenced by valuation from patients and health insurance preference regarding the consequences that should be drawn from abnormal tests (mol., 2006). ovarian reserve tests are mostly used as a diagnostic test, indicating that in case of an abnormal test result the diagnosis diminished ovarian reserve is made (levi. in fact, ovarian reserve tests may better be considered as screening tests, where an abnormal test necessitates confirmation by another test. this other test may for instance be a first ivf attempt where ovarian response to maximal stimulation is the additional test. alternatively, combinations of independently predictive tests or repeating of the initial test could improve the diagnostic performance of the single test (bancsi., 2002 ; in several systematic reviews in the last decade the true value of ovarian reserve tests for clinical practice has been debated (bancsi., 2003 ; broekmans., 2006 ; broer., 2009 ; hendriks., 2007 ; especially, the limited capacity of or tests to discriminate between pregnant and non pregnant women and the lack of knowledge on the added value of or tests upon knowing the female s age have been reason not to advocate or testing as routine test prior to ivf. ovarian reserve research has mainly focussed on the explanation and prediction of poor responses and low pregnancy outcome in assisted reproduction technology (art) treatment. a hyper response to ovarian stimulation cycle cancellation at any stage in hyperresponders is often necessary to eliminate the risk of developing the ovarian hyperstimulation syndrome (ohss), a potentially life threatening condition. also, the interest in milder stimulation protocols, that lead to lower costs, patient burden and complications (heijnen., 2007 ; polinder., 2007), urges for the availability of reliable markers of hyper response. finally, hyper response to ovarian stimulation is more and more considered as a condition in which low quality or immature oocyes are added to a basal number of best quality oocytes (kok., 2006). factors that are classically associated with hyper - response and ohss are lean habitus, young age, presence of multiple antral follicles, and the presence of the polycystic ovary syndrome (pcos). the prediction of hyper response from prior tests like basal fsh, amh or the afc has shown to be quite inaccurate or at least inconsistent (seifer., 2002 ; van rooij. currently, no definite strategies on management in case of a predicted hyper response based on such prior test are known, although fsh dose reduction would be the logical step (olivennes., 2009). prevention of hyper response therefore is based on patient profiles, like very young age and the presence of the pco syndrome, as well as the general use of modest dosing schemes not exceeding 200 iu in first cycles. the findings in a series of systematic reviews of the existing literature (bancsi., 2003 ; broekmans., 2006 ;, 2005 ; hendriks., 2007 ; verhagen., 2008) have demonstrated that several tests have good capacity to predict poor responders to ovarian hyper stimulation for ivf. the areas under the receiver operator characteristic curve (auc - roc) for baseline fsh, and especially the afc and amh, indicate that the overall accuracy is sufficient (figure 3, (auc - roc : > 0.70). for instance, if the prevalence of poor response was set at 20% and the cut off chosen at a positive likelihood ratio (ie. the chance of an abnormal test over a normal test in poor responders) level of at least 6 (indicating an overall good test), an abnormal afc would indicate a post - test probability of poor ovarian response of around 67%. this would make the afc test a clinically valuable test, especially as an abnormal test result would be found in 12% of patients. the same has shown to be true for amh, where comparable levels for post test probability and abnormal test rate were observed as for the afc. the choice for either of these two tests is mainly directed by practical issues, like availability and stability of the amh assay and the possibilities for standardised use of ultrasound based follicle counting (broekmans. in contrast, for fsh, a positive lr of 6 and over would imply a post - test likelihood of poor response of about 67%, but at such high cut off levels that abnormal tests would occur in only 3% of patients. from the reviews, the predictive ability towards pregnancy after one ivf cycle appeared only marginal for all the tests, as the area under the roc curve remained very close to the non discriminative value of 0.50. only with extreme cut offs for an abnormal test some non pregnant cases were predicted correctly, without too many false positives. at such cut off levels recent literature has focused on the added value of ovarian reserve tests to the information of female age, although reports on the univariate prediction from or tests seem quite hard to eliminate (maseelall., 2009). the relation between age and live birth in art programs is strong, although it remains difficult to decide at which age level the prospects for pregnancy have become poor enough to advice against or refuse treatment. adding information from or tests recent work by scott (scott jr., 2008) has attempted to define age specific cut off levels for basal fsh to predict failure to achieve live birth. it appeared that useful cut offs per age class could only be identified at threshold values where the live birth rates were under 2%. at such, high (15 - 18 iu / l), cut off levels the percentage of abnormal test results appeared to be only 1.6%. also, in the range of fsh results under 12 iu / l, the added predictive value upon female age has demonstrated to be only marginal, so that lower than extreme cut offs will make the test useless, in spite of more abnormal tests obtained (henne., 2008 ; sun., 2008). in general, therefore, ovarian reserve testing prior to starting art treatment should be regarded useful only if the occurrence of poor response to ovarian stimulation is to be predicted and with the assumption that this foreseen poor response can be effectively prevented with improvement of pregnancy chances (nelson., 2009). however, even a normalized response to ovarian stimulation may not alter the prognosis regarding the chances of pregnancy (land. several studies have shown that in observed poor responders in a first ivf cycle no clear benefit can be expected from various changes in management like increasing the dosage (hoveyda., 2002), applying co - medication, or changing the approach of the gnrh agonist administration (tarlatzis., 2003 ; klinkert, 2005 ; shanbhag., 2007 ; this implies that a prior prediction of poor response is to be considered useless, unless this prediction would identify cases with a poor response due to fsh under dosing related to obesity or fsh receptor polymorphisms. in cases without signs of ovarian ageing the use of a prediction model for ovarian response to fsh, containing the afc, ovarian volume, power doppler score, female age and smoking habit, was developed for individualization of the fsh dose from the first cycle onwards (popovic - todorovic., 2003b). to test whether this fsh dosage score performs well in predicting ovarian response, a randomized trial compared ovarian response in women assigned either to an individual dose of fsh based on her score, or a women in the individual dose group had a higher proportion of appropriate ovarian response than women in the standard dose group. even ongoing pregnancy rates were higher in the individualized compared to the standard dose group and dose adjustments were less frequently necessary than in the standard dose group. whether the increased occurrence of pregnancies had been obtained from dose reduced or dose increased (predicted poor responder) cases further research on the issue of patient tailored dosing and its possible beneficial effects upon pregnancy rates needs to be awaited to see whether poor responders due to other factors than ovarian ageing indeed will benefit from adapted treatment schedules (olivennes., 2009). testing for ovarian reserve may also be possible by using the quantity of the ovarian response to maximal ovarian stimulation in the first art cycle. the assumption would be that the antral follicles visible at transvaginal ultrasound will all grow into dominance with the use of dosages of exogenous fsh of 150 iu daily or over. support for this comes from studies where the number of oocytes appeared correlated to the number of antral follicles (hansen., 2003 ; hsieh., 2001 a poor response to stimulation, defined as a low number of mature follicles developed or oocytes obtained after a conventional long gnrh agonist suppression protocol, will generally be interpreted as a proof of diminished ovarian reserve and reduced prognosis for pregnancy. cycle cancellation to a standard ivf stimulation will predict a poor response in a subsequent cycle more accurately than classical ovarian reserve tests (penarrubia., 2005). also, poor responders experience an earlier transition into menopause compared to normal responders, confirming the relation between response and subsequent fertility potential. still, a poor response may also be caused by conditions like sub maximal stimulation in obese women, the presence of a fsh receptor polymorphism or simply by chance. in such poor responders, the prospects in the actual and subsequent cycles are not so unfavourable that refusal of treatment is justified (popovic - todorovic. the same seems to be true for poor responders of younger age as has been shown from several studies (lashen., 1999). only if a poor response occurs in cases with an unfavourable additional profile (female age over 38, abnormal ovarian reserve test, repeated poor response) does prognosis for subsequent cycles becomes cumbersome enough for further denial of treatment (vladimirov., 2005 ; baka., 2006 ; zhen., 2008 ; lawson., 2003 ; de boer., 2003 ; klinkert., the occurrence of a poor response to stimulation would then urge for a further classification : can the response be classified as expected or unexpected in view of female age or the result of an ovarian reserve test. expected poor responders could then be counselled for further refraining from treatment and egg donation, unexpected poor responders may still have reasonable prospects in subsequent cycles and benefit from the use of a higher fsh stimulation dosage (popovic - todorovic., 2003a ; popovic - todorovic. (a) predicted probabilities using female age as predictor. taking a 15% cumulative pregnancy rate in three cycles as minimal level of success, such a poor prospect can not be predicted by age for any single woman. (b) predicted probabilities using female age, poor response, and poor response type as predictors. taking a 10% cumulative pregnancy rate in cycles 2 and 3 as minimum success level, only in cycle 1 poor responders, who were expected based on the ovarian reserve test combination (fsh, antral follicle count, inhibin b), pr = cycle 1 poor responder ; nr = cycle 1 normal responder ; exp pr = cycle 1 poor responder with abnormal ovarian reserve testing ; unexp pr = cycle 1 poor responder with normal ovarian reserve testing. the true challenge for ovarian reserve tests lies in the possibility of identifying women with a reduced reproductive lifespan at such a stage in their lives that adequate action can be taken. in such test the preferable outcome variable to judge the test upon is the age at which a woman will become menopausal. the relation between menopausal age and the end of natural fertility has been hypothesized to be fixed (te velde and pearson, 2002). if a test existed that adequately predicts age at menopause, then adequate prevention of at least age related infertility would become reality. from the overview on ovarian reserve testing provided, two main points of attention can be deduced. first, the routine use of orts prior to starting art can not be justified, as clear therapeutic options in cases with anticipated low response are lacking. second, a first ivf attempt poor responder without signs of advanced ovarian ageing does not bear a poor prognosis and may benefit from adapted treatment schedules in subsequent cycles. | age related fertility decline varies considerably among women. therefore, chronological female age, though informative on pregnancy prospects in assisted reproduction, will often not correctly express a woman s reproductive potential. the value of quantitative ovarian reserve tests prior to ivf / icsi treatment is still subject of debate. from a series of systematic reviews it has become clear that the added value of these tests upon knowing female age has not been clearly established. still, several tests, like the afc and amh are considered adequate in predicting the response to ovarian stimulation. this claim seems to be truer for poor response prediction, compared to hyper response. prediction of the outcome pregnancy has repeatedly shown to be cumbersome. as management options for predicted poor or hyper responders are not fully investigated to date, routine ovarian reserve testing is not to be recommended. a first cycle poor response to adequate stimulation in cases with otherwise no signs of advanced ovarian ageing (based on female age and ovarian reserve tests) may offer a tool to identify cases with sufficient prospects for continuation of art treatment. |
it has been well documented that a high prevalence of myopia exists in east asia and the western pacific regions [15 ], and studies have indicated that myopia is an independent risk factor for primary open angle glaucoma (poag) [68 ]. however, making a glaucoma diagnosis can sometimes be particularly difficult in myopes. optic disc changes due to myopia (e.g., tilt or rotation of the optic disc and/or peripapillary chorioretinal atrophy) make the discrimination of a glaucomatous optic nerve head more difficult. recently, retinal never fiber layer (rnfl) thickness has become more widely used for the diagnosis and follow - up of glaucoma. however, studies have consistently shown that myopia affects the rnfl thickness [1014 ], especially in cases assessed by optical coherence tomography (oct) [15, 16 ]. quantifying the relationship between rnfl and visual function in myopia would be important in understanding the sequelae of myopia and in aiding glaucoma diagnosis in myopes. however, such structure - function association studies are seldom performed in nonpathologic myopia. in the current study, we investigate the association between rnfl thickness and visual field indices in a group of otherwise healthy myopic persons. the current study had a cross - sectional design of otherwise healthy nonpathologic myopic patients. the participants were recruited by advertisement in zhongshan hospital. briefly, the inclusion criteria included the following : (1) male or female aged 18 to 55 years old, (2) mild to high myopia with spherical equivalent 10.00 d, (3) best corrected visual acuities 20/20 in both eyes, (4) otherwise healthy, and (5) without a history of ocular trauma or surgery. we excluded those who (1) showed any signs in the fundus consistent with pathologic myopia, such as choroidal neovascularization, macular hemorrhage, fuchs spot, lacquer cracks, disciform degeneration, or chorioretinal atrophy or (2) required the use of ophthalmic drug treatment. the study was approved by the ethics committee of zhongshan hospital, fudan university, and was conducted according to the tenets of the declaration of helsinki. written informed consent was obtained from all participants. the study was conducted in zhongshan hospital affiliated with fudan university, shanghai, china, from october 2010 to march 2011. general ophthalmic examinations were performed by a trained ophthalmologist in the outpatient clinic of zhongshan hospital, fudan university. intraocular pressure was measured by a noncontact pneumotonometer (tx - f, canon, tokyo, japan). corneal curvature, anterior chamber depth, and axial length were measured without contact according to standard operating procedures (iolmaster 1322 - 734, carl zeiss, jena, germany). patients ' refractive corrections were determined by both objective and subjective refractions (feng cl, zhang ch). the general ophthalmic and refractive examinations were followed by fundus photography, oct, and perimetry, sequentially. the fundus was photographed centered at the optic disc in each participant by a nonmydriatic fundus camera (trc - nw100, topcon, tokyo, japan). then, the area and maximum and minimum diameters of the optic disc were delineated and measured manually (yuan yz) via imagenet professional (topcon, tokyo, japan). a spectral - domain hd - oct (ver. 4.5, cirrus hd - oct 4000, dublin, ca, us) was used in the current study. each eye of every participant had its optic disc scanned in a random sequence following standard imaging procedures. specifically, imaging was obtained using a cube scan featuring 200 200 axial horizontal scans (pixels) centered on the optic nerve. the image quality was assessed by the examiner immediately after each scan. only well - focused, well - centered images without any eye movement and with a signal strength of 7/10 or greater were used. the rnfl parameters of the printout retained for analysis were of inferior, superior, nasal, temporal, and average thickness. central visual field tests using the dg1x program and a dynamic strategy were performed in a dark exam room using an automated perimeter (octopus 1 - 2 - 3 ; interzeag, schlieren, switzerland). to perform the exam, each participant was asked to wear his / her glasses or contact lenses, whichever he / she had ; if needed, trial lenses were provided. before the test, the corrected visual acuity, glasses and contact lenses (when applicable) of each participant were examined by the optometrist (zhang ch) for suitability. if the corrected visual acuity was under 20/20 and/or any problem was noted in the glasses or contact lenses, best corrected trial lenses were used. the mean defect (md), mean sensitivity (ms), and loss of variance (lv) were acquired from the readouts. the reliability factor (rf) was used for quality control ; for any test with an rf exceeding 20%, retest was required. the refraction was expressed by spherical equivalents (se), which were the sphere plus 1/2 the cylinder. the average central corneal curvature was expressed by the mean of the 2 principle meridians. the normally distributed continuous variables were expressed as the means and standard deviations (sds), whereas medians and interquartile range were used to describe the nonnormal data. visual field indices were treated as continuous variables, and the rnfl thickness was treated both as a continuous variable and as a categorical variable (below 1%, 15%, 595% and above 95% in the distribution of normal, based on the build - in normative data of oct manufacturer). type c intraclass correlation coefficients (icc) were employed to describe the correlation and consistency between right eyes and left eyes using two - way mixed models. the associations between the rnfl thickness and visual field indices were first analyzed separately for the right and left eyes by general linear models and later by combining the data and examining them with linear mixed models. a scaling factor based on bennett 's formula [17, 18 ] was included in one model to adjust for ocular magnification. the ocular magnification is estimated based on the location of the second principal point and its normal spatial relationship to the nodal point. given the default axial length (al, 24.46 mm) and refraction (0 d) for a magnification of 1 with the sd - oct system, an individual scaling factor can be expressed as (24.46 1.82)/(al1.82). the pearson correlation between rnfl thickness and ms. a sample size of 58 was able to achieve 81% power to detect a difference of 0.36 between the null hypothesis correlation of 0 and the alternative hypothesis correlation of 0.36, using a two - sided hypothesis test with a significance level of 0.05. finally, 57 participants (52 females and 5 males) were included in our data analysis. the mean age of the participants was 28.3 (ranged 1946, sd 5.8) years. the mean se was 4.79 (ranged 1.80 to 8.80, sd 1.66) diopters in the right eyes and 4.59 (ranged 0.50 to 9.00, sd 1.88) diopters in the left eyes. the rnfl thickness, visual field indices, and other ocular biometric measurements are presented in table 1. the icc for the average rnfl thickness was 0.89 (95%ci 0.810.94). for md and ms, the corresponding iccs were identical, at 0.91 (95%ci 0.850.95). comparing to the normative database, the overall and quadrant rnfl thicknesses of each participant fell into 4 categories : below 1%, 15%, 595% and above 95% in the distribution of the reference values. the categories of rnfl thickness, both overall and quadrant - specific, are shown in figure 1. for the average rnfl thickness, 86.0% of the right eyes and 93.0% of the left eyes were categorized into the normal (595%) group. the temporal rnfls were significantly thicker (43.9% of the eyes were above 95% normative distribution), whereas the nasal rnfls were much thinner (17.5% of the right eyes and 26.3% of the left eyes were classified as below 5% normative distribution). as revealed by the linear mixed model, the higher the refractive error, the thinner the rnfl thickness. a one - diopter increase in the refractive error was associated with 1.61 (95%ci 0.722.50) m thinning of the rnfl. the association between optic axis length and rnfl was also statistically significant ; a 1-mm increase in axis length was accompanied by 3.33 (95%ci 1.445.22) m thinning of the rnfl. neither clinically nor statistically significant associations were found between the myopia and global visual field indices (table 2). a 1-diopter change in the refractive error was associated with only a 0.02 (95%ci 0.14, 0.18) db change in the ms and a 0.01 (95%ci 0.16, 0.15) db change in the md. the corresponding values are 0.03 (95%ci 0.30, 0.37) db and 0.06 (95%ci 0.39, 0.27) db for ms and md, respectively, per 1 mm increase in axial length. a significant association was observed between the overall rnfl thickness and global visual field indices. a thicker overall rnfl was significantly associated with a decreased ms and increased md. adjusting for age, sex, refractive error, optic disc area, or ocular scaling factor had no effect on this association (figure 2, tables 3 and 4). however, the morphologic and functional changes of the optic nerve and rnfl in myopic patients can complicate the diagnosis of glaucoma. it is therefore imperative to understand the rnfl parameters and their potential impact on visual field indices in normal myopic patients. we show in our current study a negative correlation between refractive errors and the rnfl thickness. although the overall thickness of rnfl mainly fell (86%~93%) within the normal range, the nasal thickness tended to be thinner, and the temporal region appeared thicker (figure 1), potentially indicating a temporal rotation of rnfl in myopic eyes. our data also showed that the average rnfl thickness was decreasing with the optic axis length or myopic refractive error. leung., rauscher., kim., kang., and wang. found that the longer axial length or the higher refractive error, the thinner the rnfl ; a significantly thicker rnfl in the temporal quadrant in myopes has also been reported [10, 11, 13 ]. the elongation of the globe due to myopia leads to the retinal nerve fibers stretching and thinning ; the nasally located optic disc moves laterally in this process. we postulate that the nerve fibers in the nasal quadrant stretch either superiorly or inferiorly, whereas the superior temporal and inferior temporal nerve fibers move centrally toward the horizontal meridian or macula in a nonlinear fashion. during this process, the nasal quadrant loses nerve fibers, and the temporal quadrant gains, finally causing the rnfl to tend to be thinner nasally and thicker temporally. the superior and inferior regions gained and lost fibers simultaneously ; therefore, the thicknesses vary depending on the balance between gaining and losing (figure 1). the association between myopia and global visual field indices has been debated in the literature. martin - boglind observed that the mean resolution threshold significantly correlated with the degree of myopia in the central 30-degree field. huang showed that a group of nonpathologic high myopic patients had refractive degrees and axial lengths that were significantly positively correlated with total visual field loss. in araie 's study of glaucoma patients, however, myopic power affected the mean deviation either way. in one of his papers, rudnicka and edgar stated that a sensitivity decline of the central field occurred in subjects with axial lengths > 26 mm and > 5 d of myopia. aung. found that the prevalence of visual field defects was surprisingly low in young males with myopia. czepita and chmielewska reported a positive association between refractive error and visual field defects in low and medium myopia. a group of glaucomatous suspects of chinese ancestry who had not presented for a check of their glaucomatous progression for up to 7 years showed no correlation between axial length and mean deviation on visual field testing. in the current study, the subjects ' ages, the degree of concomitant myopia, and the controls varied from study to study. different perimetries and refractive corrections [24, 28 ] were also reported to have influenced the association between refractive error and visual field indices. there are few studies investigating the association between the retinal nerve fiber layer and visual field in nonglaucomatous populations. plotted a linear regression line of ms or md against rnfl thickness, demonstrating a negligible degree of determination in normal (r = 0.0378 and 0.0121, resp.) and preperimetric glaucoma groups (r = 0.0215 and 0.0151, resp.).. reported weak associations of the thickness of rnfl with visual field indices in ocular hypertension (pearson correlation r = 0.303, p 0.1 for md). such mild correlations were also reported in normal and ocular hypertension groups. compared with the findings of taliantzis and ajtony, the current study showed (tables 3 and 4) slightly higher correlations between rnfl thickness and visual field indices (indicated by r) and greater variation in the visual field indices, which can be explained by the thickness of the rnfl (indicated by r). in contrast to previous studies and common sense, the most interesting finding of the current study is the inverse association between rnfl thickness and visual field indices ; that is, the thinner the rnfl, the better the performance on the perimetry test (higher ms and lower md). this inverse association may partly be due to the increased attention caused by myopia. a study on the correlation between myopia and visuospatial attention found that more severe myopia was associated with a better ability to quickly narrow the focus of visual attention to a small region of space. in our study, the negative association between rnfl thickness and the performance of the visual field test persisted even after adjusting the refractive power, implying that the stretching and thinning of rnfl per se may also affect the visual field test. another explanation of this discrepancy includes gender and race differences [33, 34 ]. further large and well - controlled studies are warranted to answer this question. on the other hand, ocular magnification can change the actual location of the measurement circle on the peripapillary retina, thereby affecting the average rnfl thickness measurements. however, nowroozizadeh. used customized measurement circles in normal and glaucomatous eyes and found that a correction with ocular magnification did not improve global or regional structure - function relationships. 's study, the myopic subjects in the current study had longer axial length. according to bennett 's formula, axial length affects the magnification of the ocular optic system by a scaling factor expressed as (24.46 1.82)/(al1.82) [17, 18 ]. although no individualized measurement circle was employed in the current study to correct the ocular magnification of each eye, we constructed two models that adjusted for this consideration. beside age and sex, refraction and the area of the optic disc were adjusted in one model, and the magnification scaling factor based on bennett 's formula was added in the other model. the association between rnfl thickness and the visual field global indices did not change in either model. given the pattern of rnfl redistribution in myopes indicated by the current and previous studies, a sector - by - sector and point - by - point structure - function correlation study will facilitate a better understanding of the physiopathological changes in the myopic eye. the strengths of the current study included its characterization of rnfl and its association with visual function, which were examined in a group of young and mid - aged myopes. none of the patients had comorbidities (especially the chorioretinal atrophy), ocular hypertension, or glaucoma, which allowed us to better investigate the relationship between structure and function in myopia. first, the sample size of the current study was relatively small, and there was little power for undergoing subgroup analysis. second, as it is limited by its cross - sectional design, the current study can not address questions on the temporal relationships of myopia versus changes in the rnfl or of rnfl thickness versus visual field test performance. according to the present knowledge, however, there is little biological plausibility favoring alternative pathways (the changes of rnfl leading to myopia, or the performance in visual field test causing the changed rnfl). finally, as the trial lenses were not compulsory during the visual field tests for practical reasons (especially for those participants with astigmatism), the type of refractive correction could add some variation, constituting a potential confounding source. given the relatively small sample size and the possibility for multiple interactions, the type of refractive correction could not be adjusted in the current study. however, we attempted to carefully reduce the variation caused by defocusing and inappropriate glasses / contact lenses. in conclusion, we found in this relatively young myopic chinese population that the rnfl was thinner in the nasal quadrant and thicker in the temporal quadrant. further, the average rnfl thickness was independently inversely associated with visual function, as measured by visual field indices. | the aim of the current study was to investigate the association between the thickness of the retinal nerve fiber layer (rnfl) and central visual field indices in otherwise healthy myopes. in total, 57 otherwise healthy subjects were cross - sectionally studied. general ophthalmic examinations, refractive measurements, rnfl thickness by spectral domain optical coherence tomography (oct), and central visual fields were examined. linear models were used to assess the associations. in this young and mid - aged population, the mean spherical equivalent was 4.79 (sd 1.66) and 4.59 (sd 1.88) diopters in the right and left eyes, respectively. approximately 7% to 14% of the eyes showed the average rnfl thickness out of the normal range. the temporal rnfl was remarkably thicker, whereas the nasal rnfl was thinner. the higher the refractive error, the thinner the rnfl thickness. a thicker overall rnfl was significantly associated with decreased mean sensitivity and increased mean defect, and further adjustments for age, sex, refractive error, optic disk area, or ocular magnification did not change the association. although nonpathologic myopia does not significantly affect central visual field global indices, its effects on the rnfl may be linked with performance on the central visual field test. |
gaucher disease type 1 (gd1) is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme acid -glucosidase. as a result, glucosylceramide accumulates primarily in lysosomes of tissue macrophages leading to multisystem manifestations, including hepatosplenomegaly, anemia, thrombocytopenia, and bone disease,. two treatment approaches have been used in gd1 to restore the balance between glucosylceramide synthesis and degradation. enzyme replacement therapy (ert) with recombinant acid -glucosidase, the standard of care for more than two decades, augments the patient 's residual enzyme activity to break down accumulated glucosylceramide and can improve or reverse hematologic, visceral, and skeletal manifestations,,. substrate reduction therapy (srt) inhibits glucosylceramide synthase, thereby slowing production of the substrate glucosylceramide and decreasing its accumulation. eliglustat (cerdelga, sanofi genzyme, cambridge, ma, usa) is an oral srt recently approved by the united states (us) food and drug administration and the european medicines agency as a first - line treatment for adults with gd1 who are cyp2d6 extensive, intermediate, or poor metabolizers (> 90% of patients). clinical trials demonstrated that eliglustat reduces spleen and liver volumes and increases hemoglobin levels and platelet counts in treatment - nave adults with gd1, and maintains stability long - term,. the relevant comparison of treatment - nave gd1 patients treated with eliglustat (srt) and imiglucerase (ert) has not been studied. a head - to - head trial comparing eliglustat to ert in treatment - nave patients is not feasible due to the difficulty of enrolling the large number of patients such a trial would require, given the rarity and heterogeneity of gd and the availability of effective intravenous treatments. the post - hoc analysis we describe compares clinical response to eliglustat in treatment - nave patients in the eliglustat clinical trials with clinical response to imiglucerase in selected treatment - nave patients from an observational database. this evaluation was prepared for european regulatory authorities during their assessment of eliglustat, given that a clinical trial comparing eliglustat to imiglucerase in treatment - nave patients was not feasible. we performed a post - hoc analysis of treatment - nave patients comparing the results of eliglustat treatment in two clinical studies (12-month data from the phase 2 open - label, single - arm clinical study [nct00358150 ] and 912-month data from the phase 3 double - blind, placebo - controlled engage trial [nct00891202 ],) with the results of imiglucerase treatment for up to 12 months in the international collaborative gaucher group (icgg) gaucher registry (nct00358943), an ongoing sanofi genzyme - sponsored, international, observational and voluntary program that, since its establishment in 1991, has tracked demographics and clinical outcomes for approximately 6000 gaucher patients in a real - world setting regardless of treatment status. no experimental intervention is given ; patients in the gaucher registry undergo clinical assessments and receive the standard of care as determined appropriate by their treating physicians in accordance with current gd management guidelines. all participants in the eliglustat clinical trials and the gaucher registry provided written informed consent allowing post - hoc analysis of anonymous data. inclusion and exclusion criteria from the two eliglustat clinical studies, were used to select a similar population of imiglucerase - treated patients from the registry for comparison of organ volume, hematologic, and skeletal outcomes with eliglustat - treated patients. these criteria included known date of gd1 diagnosis ; imiglucerase treatment initiation at age 1665 years ; no splenectomy ; baseline hemoglobin 816 g / dl, platelet count 30120 10/l, spleen volume 5.065.0 multiples of normal (mn), and liver volume 0.54.0 mn ; all baseline values for spleen, hemoglobin, liver, and platelets available and any of the 9-month (8 10.5 months after treatment initiation) or any of the 12-month (> 10.513 months after treatment initiation) values available. baseline was defined as the last assessment prior to the first infusion of ert. as the registry is an observational and voluntary database, timing of assessments varies ; thus, and/or conditions were allowed to ensure that the comparator population would be large enough for comparison. all patients had to have started ert before june 25, 2007 and all subsequent data points had to be before june 25, 2009 to ensure that their clinical responses were not affected by dose reductions or treatment interruptions that occurred during the imiglucerase supply constraint from 2009 to 2011. among the evaluated registry cohort of patients, sufficient data on bone (i.e., bone mineral density, bone pain, and bone crises) were not available ; therefore, bone disease parameters were excluded from this analysis. the study population consisted of 46 eliglustat - treated patients (26 from phase 2, 20 from phase 3 engage) and 75 imiglucerase - treated registry patients who met the inclusion criteria and had an imiglucerase dose 15 u / kg/2 weeks (mean : 35, range : 1560). the three groups were similar with respect to percent male (3849%), mean age at diagnosis (2225 years), and mean age at first treatment (3235 years). registry data were mostly complete through 12 months (n = 64/71 for organ volumes, n = 71/75 for hematologic parameters). eliglustat - treated and imiglucerase - treated patients were comparable on baseline hematologic parameters ; baseline spleen and liver volumes were higher in the eliglustat phase 2 study patients than in the engage and registry patients (fig. 1). mean spleen and liver volumes decreased from baseline with eliglustat treatment, with time courses and degrees of improvement similar to the imiglucerase - treated patients from the registry (fig. the rate and extent of increase for platelet counts and hemoglobin level were similar across the eliglustat - treated and imiglucerase - treated cohorts (fig. 1c and 1d). limitations of this analysis include the post - hoc design and comparison of eliglustat - treated patients in clinical studies with imiglucerase - treated patients in a real - world setting. although the imiglucerase - treated patients met the same inclusion and exclusion criteria as the patients in the phase 2 and phase 3 engage studies, baseline spleen and liver volumes differed across the cohorts, and bone data for the registry patients were too limited to allow for a meaningful comparison. furthermore, because adverse event data are not recorded in the icgg registry, it is not possible to make comparisons or draw conclusions about the safety of eliglustat versus imiglucerase. we could not completely control for baseline characteristics or for imiglucerase dose, both of which can influence treatment response. with regard to eliglustat dosing, the dose - titration scheme utilized in the clinical trials to ensure plasma eliglustat steady - state pre - dose concentrations above 5 ng / ml differs from the approved eliglustat dosing in the us and european product labels, which is determined by the patient 's cyp2d6 metabolizer phenotype (i.e., extensive, intermediate, or poor metabolizer). pharmacokinetic analyses from the clinical trials showed that pre - dose concentrations > 5 ng / ml were not required for therapeutic efficacy and that cyp2d6 phenotype was the most significant determinant of eliglustat exposure. although lacking the rigor of a head - to - head trial, the findings of this post - hoc analysis in treatment - nave gd1 patients suggest that, during the initial 912 months of treatment, oral eliglustat therapy results in improved organ volumes and hematologic parameters that are comparable to those observed with imiglucerase infusions. contributions : mjp, ja, and jst designed the analysis ; ja performed the eliglustat statistical analyses and jst the icgg gaucher registry statistical analyses. ji, ja, lu and mjp analyzed and interpreted the results and wrote the manuscript. all authors reviewed early and final drafts of the manuscript and were fully responsible for the content and editorial decisions related to this manuscript. | eliglustat is a recently approved oral therapy in the united states and europe for adults with gaucher disease type 1 who are cyp2d6 extensive, intermediate, or poor metabolizers (> 90% of patients) that has been shown to decrease spleen and liver volume and increase hemoglobin concentrations and platelet counts in untreated adults with gaucher disease type 1 and maintain these parameters in patients previously stabilized on enzyme replacement therapy. in a post - hoc analysis, we compared the results of eliglustat treatment in treatment - nave patients in two clinical studies with the results of imiglucerase treatment among a cohort of treatment - nave patients with comparable baseline hematologic and visceral parameters in the international collaborative gaucher group gaucher registry. organ volumes and hematologic parameters improved from baseline in both treatment groups, with a time course and degree of improvement in eliglustat - treated patients similar to imiglucerase - treated patients. |
tobacco smoke is a complex mixture of more than 4500 chemicals, many of which have toxic and/or carcinogenic activity. some of the components, which could be in the form of gases, vapors, and particulates, include carbon monoxide, hydrogen cyanide, phenols, acrolein, ammonia, formaldehyde, nicotine, nitrosamine, tar, heavy metals, and at least 48 known cancer - producing substances. cigarette smoking is a worldwide social epidemic and it is one of the main causes of preventable death and disability. it is an established risk factor for premature mortality due to cancer, cardiovascular disease, and chronic obstructive pulmonary disease. the increased susceptibility of cigarette smokers to infections reflects multifunctional alteration of their innate and adaptive immune responses. acrolein, a toxic unsaturated aldehyde, affects neutrophil functions and thus increases susceptibility to lung infections. leukocytosis is a well - known effect of cigarette smoking though the function of these cells is greatly reduced. reported that a single cigarette provided enough toxic material that completely inhibited the function of oral salivary neutrophils in situ. reduced phagocytic activity of neutrophils was also reported in smokers, which could be responsible for decreased defence of the gingival against bacterial attack. continued exposure to cigarette smoking has been shown to affect both humoral and cellular immune responses. initially (hours to days), there is an acute depression of the immune response followed by stimulation (weeks to months), and finally depression of the immune system sets in. this causes a decreased response to antigens and reduced serum concentration of igg, igm and iga, and also increased levels of autoantibodies notably ; anti - nuclear rheumatoid factors. although several reports have shown that cigarette smoking significantly reduces serum levels of immunoglobin classes in humans, a recent report by arinola. similarly, conflicting observations have been reported on levels of salivary immunoglobulin classes in smokers. bennet and read reported significantly reduced level of salivary iga in smokers while engstrm and engstrm reported increased levels of salivary iga in smokers. more so, research on salivary levels of immunoglobulin classes in cigarette smokers has been rarely explored. to explore, for the first time among nigerians, the interplay between components of cigarette smoke and salivary levels of immunoglobulin classes, our study estimated salivary immunoglobulin classes (igg, iga, igm, ige) in nigerian smokers to provide oral immunological based reasons for oral diseases in cigarette smokers. forty - five (45) subjects were recruited for this study after obtaining informed consent from each subject and an ethical approval from the university of ibadan / university college hospital (u.i / u.c.h) joint ethics review committee. the test group consisted of 24 active smokers who smoke at least 6 sticks of cigarette per day while the control group comprised 21 sex- and age - matched non - smokers who were apparently healthy. also excluded were individuals with pregnancy, diabetes, and human immunodeficiency virus (hiv) infection. a short structured questionnaire was administered on each subject to obtain information on age, sex, occupation, cigarette smoking, and drug consumption. about 5 ml of unstimulated saliva was collected from each subject using the spitting method into plain sample bottles. the samples were collected between 9 am and 11 am, at least 1 h after eating or washing of mouth. the samples were centrifuged at 3000 g for 5 min and the clear supernatant gently pipetted out into another clean plain bottle and stored at -20c until analysed. immunoglobulin levels were estimated using enzyme - linked immunosorbent assay (elisa) supplied by immunology consultant laboratory, usa. ige kit was supplied by leinco technologies, usa. the assay was carried out following manufacturer 's instructions. student 's t - test (unpaired) was used to determine significant difference between the means. student 's t - test (unpaired) was used to determine significant difference between the means. the mean ages of smokers and non - smokers were 39.9 and 39.5 years, respectively. no significant differences were observed in the mean salivary levels of igg, iga and ige. only igm was significantly low in smokers compared with non - smokers (p = 0.038). the mean level of salivary ige was lower in smokers compared with control. only 1 smoker (4.17%) had a detectable level of salivary ige (0.04) while two non - smokers (9.52%) had detectable levels of ige (0.24). also the proportion of smokers with detectable level of salivary ige was lower compared with controls [table 1 ]. cigarette smoking is among social practices commonly found in some nigerian youth, despite its adverse health consequences. gingivitis, periodontitis, pocket depth, attachment loss, alveolar bone loss, and tooth loss are some of oral pathologies commonly found in cigarette smokers. herr. reported that current or former smoking is associated with reduced levels of human -defensins 2 (hbd2) in pharyngeal washes and sputum of patients with acute pneumonia. of note, smoking is associated with reduced levels of surfactant proteins a and d (sp - a and sp - d). its increase could be due to increased local infection, increased antigenic inflammatory stimulus, increased local synthesis, and local host reaction against the presence of disease. levels of other immunoglobulin classes (igg, igm and ige) have also been reported to decrease in oral diseases as observed in gingival fluid exudates. the mean levels of all the immunoglobulin classes were reduced in the saliva of smokers compared with non - smokers. however, only the salivary igm mean level was significantly reduced in smokers (p = 0.038). this observation contradicts the report of engstrm and engstrm who observed increased salivary iga only. they suggested that their observation could be a reflection of protection of the oral mucosa. earlier report by bennet and read who reported a significantly low salivary level of iga only partially supports our observed low salivary iga level in smokers. in our study, the exclusion of subjects with oral diseases could be responsible for the observed differences in salivary iga as there could be upsurge in oral antibodies production consequent to oral infection or diseases. more so, our observation could be as a result of nicotine contained in cigarette as nicotine affects the exocrine glands by an initial increase in salivary secretions followed by inhibition of the secretions. a study carried out on patients with oral mucosal disease showed higher level of salivary igg. the causative effect was suggested to be increased permeability of oral mucosa which made it easy for the passage of igg from vascular and extra vascular compartment into saliva by passive transmucosal diffusion. experimental studies also showed that mice that were chronically exposed to cigarette smoke were more susceptible to influenza and murine sarcoma viruses. similarly, enhanced replication of influenza virus and legionella pneumophila was observed in the lungs of nicotine - treated animals and cells lines, respectively. based on this observation, therefore, it could be suggested that reduced salivary immunoglobulin levels, especially igm, could play an important role in the pathogenesis of oral diseases in cigarette smokers and thus could have potential benefit in screening smokers at risk of developing oral diseases. although only salivary igm was significantly low in smokers, the observed non - significant reduction in all the classes of salivary immunoglobulin suggests pan - hypogammaglobulin in them. further study is required to provide explanation for the reported blood polyclonal b cells activation and the significantly reduced salivary igm levels observed in these cigarette smokers. small sample size (due to strict inclusion criteria), unsuitable samples (occasioned by stimulation or collected after 11 am), and exclusion of ziehl - neelson positive patients were some of the limitations of this study. this observation suggests that reduced salivary immunoglobulin level of igm might be involved in the pathogenesis of oral diseases in cigarette smokers. | background : cigarette smoking is a worldwide social epidemic and it is one of the main causes of preventable death and disability. gingivitis, periodontitis, pocket depth, attachment loss, alveolar bone loss, and tooth loss are some of oral pathologies commonly found in cigarette smokers. the aim of this study was to explore, for the first time among nigerians, the interplay between components of cigarette smoke and salivary levels of immunoglobulin classes so as to provide oral immunological based reasons for oral diseases in cigarette smokers.materials and methods : in this case - control study, 5 ml of unstimulated saliva was collected in plain sample bottles from 24 active smokers who smoke at least 6 sticks of cigarette per day and 21 sex and age - matched non - smokers who were apparently healthy. the samples were spun and supernatant stored at -20c until assayed. the immunoglobulin levels of the samples were estimated using enzyme - linked immunosorbent assay (elisa). student 's t - test (unpaired) was used to determine significant differences between the two groups. p values less than 0.05 was considered significant.results:no significant differences were observed in the mean salivary levels of igg, iga, and ige. only igm was significantly lower in smokers compared with non - smokers (p = 0.038). the proportion of smokers with detectable level of salivary ige was lower compared with controls.conclusion:our study showed that there is decreased salivary igm in smokers. this observation suggests that reduced salivary immunoglobulin level of igm might be involved in the pathogenesis of oral diseases in cigarette smokers. |
in order to assess the abilities of alveolar macrophages (ams) to phagocytize adsorbent - adsorbate complexes, rat ams were incubated in vitro with two carbon blacks that have 15-fold differences in specific surface areas (astm classification n339 less than black pearls 2000) sorbed with 0.5 and 1.0 monolayer coverages of a polar and semi - polar adsorbate (acrolein and benzofuran, respectively). one - half monolayer coverages of n339 with either adsorbates significantly suppressed the phagocytosis of the carbon black, whereas one monolayer coverage did not. neither adsorbate at either coverages affected the phagocytosis of black pearls 2000. the capacity of macrophages to phagocytize a subsequent particle challenge via the fc - membrane receptor was quantified following treatment of the macrophages with the carbon black - adsorbate complexes. treatment of the macrophages with carbon black n339-adsorbates complexes at both coverages impaired fc - receptor - mediated phagocytosis, whereas no effect was observed when the carbon black was black pearls 2000. the results of this study indicate that the surface properties of the particles, the chemical properties of the chemical pollutants, and the interactions between particles and pollutants play a major role in defining the biological effect of particle - pollutant complexes.imagesfigure 2. |
|
there is a pressing need for obesity interventions to address the growing prevalence of excess weight in the usa, especially among hispanics, one of the fastest growing racial / ethnic groups in america. obesity is a common condition with associated risks of morbidity and mortality. according to data from the texas department of state health, 68.3% of the hispanic population in the el paso region had a body mass index (bmi) of 25 or greater and 33.5% had a bmi of greater than 30. these numbers are greater than both the 2010 national (64.3% and 28.9%, respectively) and texas averages (66.6% and 31.8%, respectively). effective physician - centered obesity interventions could provide the solution to our nation s obesity epidemic and improve people s overall health. physician centered counseling and intervention is associated with improved diet, physical activity, readiness for lifestyle change, and short - term weight loss. studies have shown that even modest reductions in body weight of 5 - 10%, as opposed to achieving ideal weight, are associated with clinically significant improvements in health risk factors [5, 6 ]. while we know that obesity interventions can bring about positive results, many studies have shown physicians often do not provide adequate counsel to obese patients about their weight [7, 8 ]. previous attempts to understand why weight loss interventions are lacking have identified barriers reported by physicians such as lack of time for counseling, lack of resources for referral, limited training and competence, concerns about reimbursement, and beliefs about futility of treatment. nguyen s study revealed that less educated, obese spanish speaking patients without comorbid conditions were less likely to receive weight loss advice. this could indicate other possible barriers of language and time that physicians might face when working with hispanics. while these studies provide some insight into physician perceptions about weight loss counseling, they offer little information about what weight management advice patients actually want or need from their physicians. to date, only a few studies have explored how patients in general perceive physician attempts at obesity intervention [9 - 11 ]. potter found that patients believed that primary care physicians could help them lose weight and wanted more help than they were getting. yet this study focused on the general population as a whole and only included hispanics that could read english. brown s study found that some patients were reluctant to seek medical help due to their own personal sense of responsibility with their weight. this study however was conducted in the uk and did not include any minority groups. the results of their study highlighted the need for physicians to be culturally aware of potentially offensive techniques and terminology used to counsel african americans about obesity. only recently have studies emerged gathering information on what hispanics think and believe when it comes to obesity management. they have shown that hispanic immigrants expressed desire for weight loss programs that incorporate traditional foods, support cultural traditions, and include a family focus [12 - 14 ]. common barriers to making long - term dietary changes were the social and family pressures to eat like others and partake in food - centered celebrations. many of the studies also found a significant lack of information and a great deal of misinformation regarding nutrition and health among hispanics [12 - 16 ]. these studies however did not focus on the health care system and how physicians could educate and facilitate weight loss. to our knowledge, no study has focused specifically on obese hispanic patients perceptions regarding physician - guided obesity management. whether physician - guided weight management treatments are effective and accepted by hispanic patients will depend on whether physicians are culturally compatible with patient needs and preferences. this has been demonstrated in the past few years by studies implementing culturally appropriate lifestyle interventions among hispanics [13, 18 ]. the purpose of this study was to describe attitudes of hispanic patients towards weight loss, understand their prior experiences with their doctors, what they believe their doctor s role should be and exactly how they believe their doctor should help them to lose weight. the assessment will be made through brief semi - structured interviews conducted in various health clinics throughout el paso. once gathered, the information could prove invaluable to primary physicians as they work to effectively address the issue of obesity among their hispanic patients. a cross - sectional study combining qualitative and quantitative methods was conducted to describe hispanic patients attitudes and beliefs about obesity and the role of their physician. ethical approval for the study was obtained from the institutional review board (e13030). participants were hispanic men and women, 18 - 75 years of age, overweight or obese (bmi greater than 25.0 based on self - reported height and weight), who presented for a primary care visit for any reason. a semi - structured interview guide was used to conduct an interview with each participant. this interview guide was based on questions used in prior studies that have been used to identify thoughts, beliefs, and attitudes of patients regarding weight loss [9 - 11, 19 - 21 ]. four questions covered attitudes about weight loss [19, 21 ], six questions covered the role of physicians in weight loss [11, 20, 21 ], three questions covered patients past experiences with a physician [10, 19, 21 ] and two questions elicited what patients wanted regarding weight loss counseling [10, 11 ]. two de novo questions regarding knowledge of and awareness of bmi were developed and included. patients medical history, health status and demographic information were also collected by self - report. participants were recruited by personal invitation from a trained bilingual research assistant during regularly scheduled clinic visits. participants were approached in the waiting area of the clinic and asked if they were interested in participating, eligibility was then determined and if eligible they were taken to a private room where informed consent was obtained and the interview was conducted. the interviewer took contemporaneous short notes and all interviews were recorded, transcribed and then translated into english by two separate translators and cross - checked for accuracy. the free recall listing technique determines participants understanding of the definition and boundaries of a topic or domain of interest. it is an open - ended interviewing technique in which participants, as a group, generate a list of responses in their own words. free - recall lists have some important cognitive properties. in particular, items that are most salient to participants are mentioned at the beginning of individual lists and also occur more often across interviews. items that are not mentioned on the lists are not as salient as items that appear on the lists. interviewing more participants may increase the number of items, but the list itself becomes stable, and the order of items does not change as new items are added by each new person. the interviews generate many items per participant, maximizing the amount of information collected per individual and allowing for a smaller sample size. the interviewer and the observer recorded comments and phrases contemporaneously, in written format using the participant s own words. the free - recall listing technique lends itself particularly well to transcribing verbatim because the responses are in list format and consist of short phrases or brief sentences. we conducted a semantic thematic analysis using a theoretical framework that was informed by prior work in this area [9 - 11, 19 - 21 ]. the coding and data analysis was done by the team on an ongoing basis throughout. emerging themes were identified by separately reviewing responses to each question and listing unique themes mentioned by the participant. data were summarized by tabulating the frequency with which themes were mentioned ; this is a method of presenting data obtained from the free - recall listing technique. ethical approval for the study was obtained from the institutional review board (e13030). participants were hispanic men and women, 18 - 75 years of age, overweight or obese (bmi greater than 25.0 based on self - reported height and weight), who presented for a primary care visit for any reason. a semi - structured interview guide was used to conduct an interview with each participant. this interview guide was based on questions used in prior studies that have been used to identify thoughts, beliefs, and attitudes of patients regarding weight loss [9 - 11, 19 - 21 ]. four questions covered attitudes about weight loss [19, 21 ], six questions covered the role of physicians in weight loss [11, 20, 21 ], three questions covered patients past experiences with a physician [10, 19, 21 ] and two questions elicited what patients wanted regarding weight loss counseling [10, 11 ]. two de novo questions regarding knowledge of and awareness of bmi were developed and included. patients medical history, health status and demographic information were also collected by self - report. participants were recruited by personal invitation from a trained bilingual research assistant during regularly scheduled clinic visits. participants were approached in the waiting area of the clinic and asked if they were interested in participating, eligibility was then determined and if eligible they were taken to a private room where informed consent was obtained and the interview was conducted. the interviewer took contemporaneous short notes and all interviews were recorded, transcribed and then translated into english by two separate translators and cross - checked for accuracy. the free recall listing technique determines participants understanding of the definition and boundaries of a topic or domain of interest. it is an open - ended interviewing technique in which participants, as a group, generate a list of responses in their own words. free - recall lists have some important cognitive properties. in particular, items that are most salient to participants are mentioned at the beginning of individual lists and also occur more often across interviews. items that are not mentioned on the lists are not as salient as items that appear on the lists. interviewing more participants may increase the number of items, but the list itself becomes stable, and the order of items does not change as new items are added by each new person. the interviews generate many items per participant, maximizing the amount of information collected per individual and allowing for a smaller sample size. the interviewer and the observer recorded comments and phrases contemporaneously, in written format using the participant s own words. the free - recall listing technique lends itself particularly well to transcribing verbatim because the responses are in list format and consist of short phrases or brief sentences. we conducted a semantic thematic analysis using a theoretical framework that was informed by prior work in this area [9 - 11, 19 - 21 ]. the coding and data analysis was done by the team on an ongoing basis throughout. emerging themes were identified by separately reviewing responses to each question and listing unique themes mentioned by the participant. data were summarized by tabulating the frequency with which themes were mentioned ; this is a method of presenting data obtained from the free - recall listing technique. the income level was primarily under $ 10,000 a year, with 65% of the group meeting this category (table 1). very few mentioned that it affected medical conditions such as diabetes, hypertension, or cholesterol. thirty percent knew what a bmi was and 95% reported that had never been told their bmi by their doctor. most patients agreed that they thought their doctor should be involved weight loss but with varied roles. some were not sure how the doctor could help since they themselves already knew what they needed to do or thought they knew what their doctor would say or do. most agreed that their doctors did have the necessary knowledge and if they did not, they should. about half of the patients replied that their doctors did not prioritize weight loss or were unsure of the priority because their doctor had never discussed weight loss with them. nearly all patients said they had attempted to lose weight with either diet and/or exercise, and a few had undergone gastric bypass operations. prominent obstacles to losing weight were identified as perceived high costs of a healthy diet, eating a bad diet, using food to cope with emotion or life changes, a lack of mobility, low energy and poor weight loss knowledge. all of the patients were comfortable discussing their weight loss with their doctor and the healthcare team but had never broached the subject with their doctor. a few mentioned that they only wanted their weight addressed if they were asked permission. sixty percent of the patients remarked that their doctor had never discussed their weight with them. participants reported that if their physician did mention weight loss, they gave very general tips such as eating less and being more active. the most common preferences for physician assistance were specific information such as nutritional advice and specific weight loss goals, encouragement to continue progress, and referrals to nutritionists and weight loss experts. common weight loss motivators and facilitators identified were encouragement from the healthcare team as well as having a support group or partners to join them in weight loss. the income level was primarily under $ 10,000 a year, with 65% of the group meeting this category (table 1). very few mentioned that it affected medical conditions such as diabetes, hypertension, or cholesterol. thirty percent knew what a bmi was and 95% reported that had never been told their bmi by their doctor. most patients agreed that they thought their doctor should be involved weight loss but with varied roles. some were not sure how the doctor could help since they themselves already knew what they needed to do or thought they knew what their doctor would say or do. most agreed that their doctors did have the necessary knowledge and if they did not, they should. about half of the patients replied that their doctors did not prioritize weight loss or were unsure of the priority because their doctor had never discussed weight loss with them. nearly all patients said they had attempted to lose weight with either diet and/or exercise, and a few had undergone gastric bypass operations. prominent obstacles to losing weight were identified as perceived high costs of a healthy diet, eating a bad diet, using food to cope with emotion or life changes, a lack of mobility, low energy and poor weight loss knowledge. all of the patients were comfortable discussing their weight loss with their doctor and the healthcare team but had never broached the subject with their doctor. a few mentioned that they only wanted their weight addressed if they were asked permission. sixty percent of the patients remarked that their doctor had never discussed their weight with them. participants reported that if their physician did mention weight loss, they gave very general tips such as eating less and being more active. the most common preferences for physician assistance were specific information such as nutritional advice and specific weight loss goals, encouragement to continue progress, and referrals to nutritionists and weight loss experts. common weight loss motivators and facilitators identified were encouragement from the healthcare team as well as having a support group or partners to join them in weight loss. our data show that hispanic patients in this study want to lose weight and have tried in the past but have been unsuccessful due to a variety of barriers. these findings are similar to those identified in past studies in other groups [9, 12, 14, 23 ]. while most hispanics in our group realized that excess weight played a role in decreased mobility and energy, not many were aware of the implications of excess weight on their current medical problems or overall health. only a few patients realized that their excess weight had a direct impact on their medical issues. regardless, it is worrisome that the majority of participants did not know the implications of weight on their direct health. our findings are consistent with other studies in the overall lack of awareness of the health consequences of obesity in the hispanic population [15, 16, 23 ]. this observation is important, since diabetes, hypertension, and coronary heart disease are highly prevalent among the hispanic population [1, 24 ]. so it is especially important for the physician and healthcare team to discuss weight management with patients with these conditions. many of the patients interviewed had little understanding of what their ideal or healthy weight should be. when asked to define bmi, only a third knew what it was while almost none had been notified about it by their doctor. using bmi as a teaching tool could demonstrate clearly to patients how much excess weight they have. interestingly, throughout our interviews many patients admitted they were heavy or overweight when in fact the majority fit the bmi criteria for being obese. similar findings from another study showed that us - born obese hispanics were more likely to perceive themselves as overweight and desire to weigh less than their foreign - born counterparts. using bmi could provide patients a clear view of where they currently are in their weight status while also providing goal oriented steps to gain a healthy weight. overall, participants felt comfortable talking about their weight with their doctors or any member of the healthcare team. we found that hispanic patients trust their doctors and believed they had the knowledge necessary for the health problems they faced. they believed that if weight did affect health, a doctor has a responsibility to bring it up. however, for 60% of our interviewees, the topic of weight loss had never been discussed by their physicians. this lack of weight loss discussion made about 50% of our interviewees believe that their doctors did not put weight loss as a priority in their health. some studies have shown that when physician interventions are done, they focus only on those morbidly obese or those with comorbidities [8, 9 ]. from our results, we believe that if doctors made weight loss intervention a priority in their exam regardless of the lack of comorbidities, patients would understand the importance of maintaining a healthy weight allowing prevention of further weight gain. in our study, we found that similar to patients from other races / ethnicities, hispanic patients want detailed information such as specific nutritional advice and need specific goals to help them achieve their proper weight. when physicians do not have the time or the knowledge to discuss weight loss intervention, patients want referrals to nutritionists and weight loss experts. we were able to obtain a fair amount of responses to our open - ended surveys to the point that responses became saturated with similar themes. while we are aware that such a small sample size could not possibly represent the variety of views found in the hispanic population, it does provide a good basis for future study. it is possible that the results obtained could very well be one - sided as they represent a specific demographic that frequents the local health clinic. looking forward we will need to administer the next phase of our study to a much larger sample size to adequately asses the attitudes, experiences, and desires of the hispanic population concerning weight loss. the results of our study demonstrate that the attitudes, experiences, and desires of the hispanic population concerning weight loss intervention with physicians, resemble those observed among other groups. our findings highlight the need for physicians to specifically educate and provide resources to their patients not only concerning the effects of excess weight on health but also where their patients currently stand in their bmi. the hispanic patients in our study wanted more help and advice from their doctors in the form of dietician referrals, specific weight loss goals, and encouragement throughout the process. we strongly believe that continued research into understanding how hispanic patients can be helped by their physicians will lead to improved weight loss management and overall healthcare of the hispanic community. the results of our study demonstrate that the attitudes, experiences, and desires of the hispanic population concerning weight loss intervention with physicians, resemble those observed among other groups. our findings highlight the need for physicians to specifically educate and provide resources to their patients not only concerning the effects of excess weight on health but also where their patients currently stand in their bmi. the hispanic patients in our study wanted more help and advice from their doctors in the form of dietician referrals, specific weight loss goals, and encouragement throughout the process. we strongly believe that continued research into understanding how hispanic patients can be helped by their physicians will lead to improved weight loss management and overall healthcare of the hispanic community. | backgroundlittle is known concerning hispanic patients perceptions about the role of the physician in obesity management. this study seeks to describe the perspectives of hispanic patients toward weight loss, and what they believe their doctor s role should be in the management of obesity.methodsa cross - sectional study utilizing semi - structured interviews was conducted in a university - based family medicine clinic. open - ended questions explored beliefs about the relationship between weight and health, previous weight loss experience, perceptions about the role of the physician in weight loss, past experiences with their physician, and preferences for how a physician could help facilitate weight loss. the free recall listing technique was used to elicit responses. common themes were identified by a group coding process.resultspatients were open to discussion from physicians concerning weight loss but many had not been approached. they wanted assistance from their doctors in the form of dietician referrals, specific weight loss goals, and encouragement. patients knowledge about the implications of excess weight on health was lacking.conclusionhispanic patients want more help and advice from their doctors. general knowledge of the health implications of obesity was lacking, indicating a need for more health education by the healthcare team. |
recognition of the large numbers of chemicals in commerce and increased focus on evaluation of these chemicals from the perspective of potential human health risk has become a focus of attention in north america and europe. these efforts are devoted not only to evaluation of new chemicals but also to an examination of existing chemical substances. these efforts include those under the health canada chemicals management plan, the european registration, evaluation, authorisation and restriction of chemicals (reach), the high production volume (hpv) challenge program, and the us environmental protection agency 's (us epa) chemical assessment and management program (champ) initiatives. chemical evaluation is also being discussed as part of potential improvements to the us toxic substances control act. because of the large number of chemicals involved and the need for efficient processes that assure focus on substances which could pose the greatest health concerns, tiered approaches that begin with conservative risk - based screening - level assumptions and proceed to more refined data - intensive approaches have been recommended for these types of efforts [1, 2 ]. chemical risk assessment evaluations consider both exposure and hazard, and a tiered set of approaches employing various levels of data for screening - level assessments is often recommended [1, 3, 4 ]. exposure screening considers chemical uses, identifies potential exposure media or pathways, and invokes conservative assumptions in the estimation of potential daily exposure rates. hazard evaluation includes the identification of established tolerable exposure levels (e.g., reference doses or tolerable daily intakes [rfds or tdis ]). in the absence of such established guidance values, robust no observed adverse effect levels (noaels) or benchmark doses (bmds) can be used as a point of departure (pod) and adjustment factors for extrapolation applied (as necessary), and margins of safety (mos) can then be calculated for risk - based screening. finally, in the absence of robust toxicological data, a generic screening approach such as that developed under the threshold of toxicological concern (ttc) framework [57 ] for setting conservative tolerable intake rates has been widely used. in this paper, we explore approaches for using chemical biomonitoring data in risk assessment evaluation of chemicals. as with external exposure - based assessments, exposure assessments based on biomonitoring data require health- or risk - based benchmarks for evaluation of biomarker data. however, because biomarker data is typically expressed in units of biomarker concentration (e.g., g / l urine) and risk - based benchmarks are typically expressed in units of applied dose (mg / kg - day), direct comparison can not be made. two approaches are possible : (1) the biomarker can be back calculated to an applied dose (reverse dosimetry ; see, e.g.,), or (2) the benchmark can be forward calculated to a corresponding biomarker concentration for use as a screening value (forward dosimetry ; see hays.). dosimetry calculations, whether forward or reverse, require the use of pharmacokinetic data and modeling and assumptions regarding exposure patterns. this paper describes methods for interpreting human biomonitoring data in a risk context, illustrating the use of the forward dosimetry biomonitoring equivalents approach for five scenarios. the first three are applicable to substances for which toxicokinetics are well understood but that have different levels of toxicity data : (1) substances with established government risk assessments, (2) substances with sufficient toxicity datasets but as of yet no government - generated (or -vetted) risk assessment, and (3) substances amenable to the generic screening ttc approach for setting conservative tolerable intake rates. these latter two approaches are needed because, for many chemicals in common use today, there may not be authoritative, government - conducted, or approved chemical - specific risk assessment - based exposure guidance values available. the additional scenarios addressed in this paper include (4) the absence of chemical - specific toxicokinetic data or models, and (5) the absence of both toxicity - based guidance values and toxicokinetic data. the framework of the cases and approaches described here is summarized in figure 1 and discussed in detail below. human biomonitoring, in which chemicals or their metabolites, are measured in biological media such as blood or urine, has become a powerful tool in the assessment of chemical exposures in the general population and in studies of targeted populations [10, 11 ]. human biomonitoring data provide a reflection of integrated exposure from multiple pathways and routes in terms of internal, biologically relevant dose. in situations in which exposures to a chemical potentially occur through multiple or ill - defined exposure routes or pathways, well - designed and conducted human biomonitoring studies can provide robust and reliable exposure data that can complement and refine or replace external exposure estimation based on more indirect approaches and generic assumptions. biomonitoring can be particularly useful in cases where widespread population exposure is possible (e.g., residues of agricultural chemicals, food packaging constituents, consumer product ingredients, etc.). biomonitoring can also be used as an accessory tool in evaluation of exposure to chemical ingredients in consumer products in targeted, controlled exposure studies (see below for example with triclosan). screening criteria for determining the health significance of human biomonitoring results would ideally be based on robust datasets relating potential adverse effects to biomarker concentrations in human populations (see, e.g., the us centers for disease control and prevention (cdc) blood lead level of concern ; see http://www.cdc.gov/nceh/lead/). however, data to support such assessments exist for only a few environmental chemicals because this approach requires establishment of causality in epidemiological studies and a robust understanding of human dose response. thus, in an alternative approach, the concept of biomonitoring equivalents (bes) has been developed, and guidelines for the derivation and communication of these values have been published [9, 12, 13 ]. in conventional risk assessment, concentrations in environmental media are used with specific contact scenarios to derive an estimate of external dose (mg / kg - day), and this is then compared to an external dose health - based guidance value, such as an adi, rfd or tdi (mg / kg - day). in the initial screening - level evaluation, estimated exposure rates are compared to hazard- or risk - based benchmarks to assess whether more refined evaluations are required. when an rfd, or tdi or analogous screening value such as a ttc is available, the screening - level exposure estimate is compared directly to that value to assess whether exposure rates above that value are anticipated. if a noael or other pod is used as the benchmark, then adjustment factors (afs) (synonymous with uncertainty factors or safety factors) are generally used to extrapolate from animal toxicity to humans (default 10x) and to account for human variability (default 10x). depending upon the database and quality of studies, additional afs may be used. if a toxicity database is not robust, use of an additional database uncertainty factor should be considered. once the screening level health - based exposure guidance value has been determined, then a margin of safety (mos) can be calculated by comparing this to the estimated daily dose rate (d) : (1)mos=(pod / afs)d. mos values below 1 indicate that exposures exceed the screening level health - based exposure guidance value. if screening approaches have been used in the exposure or hazard assessment process, further refinement in those assessments may be warranted. such refinements to provide greater certainty of potential hazards and exposures may include generation of product - specific exposure data for chemical uses with higher estimated exposure rates, conducting specific toxicity studies to address database deficiencies, or other exposure or hazard characterization refinements. results of refined assessments can be used to identify the need for, and useful focus of, potential risk management strategies. in the biomonitoring equivalent approach for interpreting biomonitoring exposure data (internal dose concentrations) in a risk assessment context, external dose health - based guidance values are translated to estimates of corresponding steady - state biomarker concentrations. a biomonitoring equivalent (be) is defined as the concentration or range of concentrations of a chemical or its metabolites in a biological medium (blood, urine, or other medium) that is consistent with an existing health - based exposure guidance value such as a reference dose (rfd) or tolerable or acceptable daily intake (tdi or adi). bes are intended to be used as screening tools to provide an assessment of which chemical biomarkers are present at levels well below, near, or at or above concentrations that are consistent with existing risk assessments and exposure guidance values, and thus can provide an evaluation of relative priority for risk assessment followup. bes provide a translational tool allowing application of the foundational risk assessment paradigm to the evaluation of exposure information provided by biomonitoring data. development of be values requires an underlying exposure guidance value (such as an rfd or tdi) as well as sufficient understanding of pharmacokinetics of the chemical in humans or key laboratory species. bes are similar in concept to the hbm - i assessment values derived by the german human biomonitoring council (reviewed in angerer.). for interpreting human biomonitoring data in a risk context, the margin of safety (mos) approach is used (2)mos = be[biomarker ]. when the mos value is 1 or greater, then the exposure to the substance is not likely to be of concern. be values have been derived for approximately 80 chemicals in a variety of chemical classes (see angerer. for review). be derivations have been published for persistent organic compounds including dioxins, hexachlorobenzene, and ddt and metabolites, for approximately 40 volatile organic compounds, for several phthalates and phenols including di-2(ethylhexyl)phthalate, bisphenol a, and triclosan, for selected pyrethroid pesticides, and for selected brominated flame retardant compounds. for many of these chemicals, multiple be values have been derived corresponding to different available risk assessment exposure guidance value (e.g., epa rfds versus tdi values derived by the european food safety authority [efsa ]). for these chemicals, screening level assessments of population biomonitoring data can be made by comparison of the data to the be value corresponding to the risk assessment exposure guidance value deemed most appropriate. establishing comprehensive, risk assessment - based exposure guidance values such as rfds or tdis is a resource - intensive effort that may take several years to complete for substances with extensive datasets. in many cases, substantial toxicological data exist for chemicals, but no formal risk assessment - based exposure guidance values such as an rfd or tdi have yet been established by a government agency. further, some existing risk - assessment based values may now be outdated, based on the availability of newer, more relevant hazard or exposure data. thus, for many chemicals in common use today, there may not be authoritative, government - conducted or -approved chemical - specific risk assessment - based exposure guidance values available. in the absence of such established guidance values, robust no observed adverse effect levels or benchmark doses based on a review of available datasets can be used as a point of departure, and by use of appropriate afs, screening level health - based exposure guidance values can be derived. if appropriate pharmacokinetic data are available, these screening level health - based exposure guidance values can be translated to corresponding internal biomarker concentrations and used to assess human biomonitoring data in a parallel fashion. a mos based on comparison of the biomonitoring data to the biomarker concentration level consistent with the screening level health - based exposure guidance value can then be calculated. an example of this approach has been presented by aylward and hays for the flame retardant hexabromocyclododecane (hbcd). although a substantial database of toxicity data for both standard and endocrine - sensitive endpoints is available, no exposure guidance values have been established. both health canada and the european union have conducted provisional or draft risk assessments in which sensitive pods were identified [17, 18 ]. data were available on measured or estimated lipid - adjusted hbcd concentrations in experimental animals at the identified pod dose levels. substantial data on lipid - adjusted hbcd concentrations in human serum and milk were available and tabulated. comparison of those data to the biomarker concentrations in the animal studies at the pods showed margins of exposure (moes) in excess of 5,000 for general population exposures to hbcd. in this case, a moe comparison was made, which is analogous to the mos approach, except with the moe, afs are not used, and comparison is made directly to the pod. a similar moe approach was incorporated as part of a risk assessment for triclosan conducted by the european commission scientific committee on consumer products (ecsccp). in this case, serum concentrations of triclosan were measured throughout the course of a chronic animal bioassay selected by the ecsccp as the basis for establishment of a tdi. thus, serum concentrations in rats corresponding to the noael dosing regimen were directly available from the toxicological database. in contrast to hbcd, in which general population exposures are incidental and due to trace levels of hbcd released into the environment, triclosan is added intentionally as an antibacterial agent to a variety of directly applied and used personal care products such as toothpaste or soap. the conventional risk assessment approach entails estimation of exposure levels using generic assumptions about each use scenario, contact rates, absorption, and so forth. however, because consumers may experience exposures to multiple products containing triclosan, with potential exposure via more than one route (dermal, ingestion), the conventional exposure assessment process can be cumbersome, requiring assessment of many exposure scenarios and reliance on multiple conservative, potentially compounding, exposure assumptions. in the ecsccp evaluation, in addition to a conventional moe assessment based on estimated external doses from use of multiple products compared to an animal noael, peak serum levels were measured in volunteers using multiple triclosan - containing products (toothpaste, deodorant stick, and hand soap) and compared to the serum levels at the noael in rats in the chronic bioassay. the conventional assessment based on estimated external doses resulted in an moe of approximately 380 compared to the administered dose rates in rats at the noael. the corresponding assessment based on comparison of human serum levels to serum levels measured in the animal bioassay at the noael resulted in an moe of approximately 940. this result confirms (1) that the approach based on estimated external exposures incorporates conservative assumptions and (2) the practical utility of risk - based screening using biomonitoring data. the triclosan example illustrates the value of including measurements of blood biomarker concentrations in toxicological assays, as recommended by barton. and saghir.. biomarker concentrations, and in particular blood or serum concentrations of chemicals, provide a reflection of biologically relevant absorbed dose and tissue concentrations. comparison of biomarker concentrations in humans under real - world product use scenarios to the corresponding biomarker concentrations in laboratory animals under bioassay conditions at the pod potentially reduces uncertainties associated with reliance on estimated external exposure doses in the process of safety assessment of products. interpreting human biomonitoring data in a risk context for substances that lack comprehensive, health - based exposure guidance values is challenging. programs such as health canada 's chemicals management plan, the european union (eu) registration, evaluation, authorisation, and restriction of chemical (reach), and the us toxic substances control act (hpv challenge program and champ), while they may be lacking health - based exposure guidance values, can often provide sufficient data to support this screening - level approach. for example, under the high production volume (hpv) challenge program (http://www.epa.gov/chemrtk/index.htm) which is now substantially complete, toxicity data and other relevant information on approximately 2,200 chemicals produced or imported into the us, in quantities > 1,000,000 lbs./year, has been submitted to epa to enable screening based on the oecd 's sids paradigm. this data, which covers about 9095% by volume of chemicals in commerce in the us, is publicly available and was evaluated by epa, under the chemical assessment and management program (champ) initiative, to derive screening - level hazard characterizations, and then, for a subset of these, a screening - level risk - based prioritization. from its initiation in 2007 to 2009, when it was superseded, epa 's champ developed 786 hazard characterizations and 220 risk - based prioritizations [22, 23 ]. for each of these substances, the hazard characterizations generated by epa provide a concise assessment of the toxicity data and include delineation of loaels and noaels for effects on (1) major organ systems (from both acute and repeated exposures), (2) the developing organism in utero, (3) reproduction, and (4) the fidelity of dna (http://www.epa.gov/champ/). the loaels or noaels (as appropriate) for these substances can be readily accessed from epa 's hpvis online database (http://www.epa.gov/hpv/hpvis/index.html) and used for deriving a pod. afs for toxicodynamics can then be applied to derive a screening level health - based exposure guidance value, which is also in units of applied dose (mg / kg - bw / day). then, by using chemical - specific toxicokinetic data or models (cstk), a biomarker concentration level typically in units of concentration in blood or urine consistent with this screening level health - based exposure guidance values can be developed. biomonitoring results can then be interpreted in a risk context using the mos procedure. when using this approach, it is important to recognize that the typical afs of 10x for extrapolating to animals to humans and 10x to account for human variability each contain both dynamic and kinetic components. thus, to use this method to interpret human biomonitoring data, when deriving the screening level health - based exposure guidance value from a noael or pod based on an oral toxicity lab animal study, it is important to use in the first step only the dynamic components of the afs (typically 2.5x or 3.16x to extrapolate from animals to humans and 3.16x to account for human variability) should be used [24, 25 ]. then, in a second step, the cstk data or model needs to be used to convert the applied dose into a concentration and in doing so, the cstk may allow replacement of the kinetic components of the typical afs. if both the typical 10x for extrapolating from animals to humans and the 10x to account for human variability are applied to the lab animal toxicity noael and the cstk is also applied,, no robust toxicological data on which to base selection of a pod are available for a chemical. in this case, the ttc approach can provide a method for selection of a provisional, conservative tolerable daily dose level based on historical data and distributions of noael values (or cancer potency values) along with an appropriate uncertainty factor (or low dose linear extrapolation factor) for a wide range of compounds [57, 26, 27 ]. the threshold of toxicological concern (ttc) evolved from concepts initially developed by frawley and further refined by the us fda as the threshold of regulation [29, 30 ] and was initially developed based on extrapolated risk data for carcinogens with the assumption that if the carcinogenicity endpoint was protected, all other toxicological endpoints would also be protected. these concepts were considerably expanded to include consideration of chemical structure in conjunction with toxicity data for other toxicological endpoints [5, 27 ]. one of the most important enhancements to the original work was the consideration of chemical structure and the addition of a decision tree linked to exposures that pose little or no health risk. the acceptable exposure levels were derived by an extensive analysis of the existing toxicology data for 730 chemicals tested for carcinogenicity (low dose risk based) and more than 600 chemicals tested for repeat dose toxicity (noael based). chemical characteristics are used to identify a generic, conservative tolerable daily intake rate, the ttc. the ttc approach is based on an analysis of two comprehensive databases of toxicity data : one that is relevant to genotoxic carcinogens and one that is relevant to repeat dose endpoints not predicted on an assumption of potential genotoxic carcinogenicity. these tools are used by first assessing conservatively whether or not the chemical has structural features (alerts) suggestive of the potential for carcinogenicity via a genotoxic mode of action. chemicals with alerts for potential genotoxic carcinogenicity are subject to an exposure limit based on the distribution of potencies of historically tested carcinogens. chemicals without alerts for genotoxicity may move further along the decision tree, and, based on their structures, be categorized into one of three classes that are associated with three different conservative tolerable intake rates, or ttcs. the category - specific recommended ttc levels are considered to be conservative estimates of chronic daily intake rates that are unlikely to result in adverse effects. this is based on the analysis of the distribution of no observed adverse effect levels (noaels) for compounds in the three categories. these values are based on the 5th percentile noaels along with the application of default uncertainty factors [57 ]. applying the ttc approach permits rapid evaluation of exposure levels to chemicals with little or no chemical - specific toxicology data to determine if exposures are sufficient to trigger concern for a potential for health risk. exposures below the ttcs are judged to pose a very low probability of an appreciable risk to human health. although the approach was originally developed to support exposures to indirect food additives and later to dietary exposures, the underlying datasets are broad and, consequently, application of the ttc concept to a broader range of exposure scenarios has been considered [3237 ]. initial development and application of the ttc approach was focused on systemic exposure resulting from oral administration or exposure to compounds. more recently, the ttc approach was extended to consider systemic exposure following topical application of cosmetic products [32, 35 ]. there has also been the suggestion that ttc can be applied to inhalation exposure and risk assessment [33, 3537 ]. it also has been proposed that the ttc can be applied to intentionally added materials found at low concentrations in food [7, 34 ]. although there are broad categories of chemicals that can be evaluated using the ttc, there are certain materials that have insufficient data in the underlying toxicity datasets, have been identified as carcinogens with potencies that fall outside of the distribution, or have concerns related to bioaccumulation for the ttc to be applied. these include metals, organometals, and the polyhalogenated dioxins, furans and biphenyl derivatives. application of the ttc requires a careful evaluation of the chemical(s) under consideration and application of the decision tree to assign the chemical to the appropriate tier of the decision tree. this decision tree is outlined in several publications and has been implemented as part of the oecd qsar toolbox (available at http://www.oecd.org/document/54/0,3746,en_2649_34379_42923638_1_1_1_1,00.html). an additional module is also available for identifying alerts for carcinogenicity that may be used as part of the weight of the evidence on whether or not to consider the chemical as a potential genotoxic carcinogen. use of the decision tree approach offers a way to prioritize which materials need more in - depth evaluation. the ttc methodology was developed to evaluate the potential for risk to low - level exposure to chemicals in the diet and has subsequently been applied to ingredients or contaminants in pharmaceutical and consumer products. since biomonitoring represents a real - world measurement of such low - level exposure application of the ttc principles offers an approach to evaluating the measured exposures in a risk - based context. ttc values are typically expressed as applied doses, as either mg / kg - day or mg / day (for a defined population). to use a ttc value to interpret human biomonitoring data if sufficient chemical - specific toxicokinetic data are available, the ttc could be translated into a corresponding biomarker concentration under the assumption of chronic steady - state exposure at the ttc level. as discussed above, in converting to an internal dose concentration, attention this typically will entail review of the derivation of ttc, removal of the default af used for toxicokinetics, then applying chemical - specific toxicokinetic data or models to obtain an internal biomarker concentration level equivalent to the ttc. this would also provide a way to identify chemicals where additional biomonitoring would add little value. for example, if a chemical was in cramer class 3, which has an assigned ttc of 90 g / day, and the biomonitoring data such as those from national biomonitoring programs such as the us national health and nutrition examination survey (nhanes) or the canadian health measures survey (chms) indicated, through reverse dosimetry estimations, that exposure levels at the 95th percentile were likely orders of magnitude less than 90 g / day, that chemical could be a candidate for removal from the biomonitoring program. in a practical sense, for a chemical with little or no toxicological data for which the ttc approach is used to identify a screening intake level, the chemical - specific toxicokinetic data or measurements required to estimate corresponding biomarker values may not be available. in such cases, generic toxicokinetic approaches may be considered ; these are discussed further below. for many chemicals, risk assessment - based exposure guidance values or robust pod values are available. however, little or no chemical - specific toxicokinetic data may exist because such data have not necessarily been considered to be part of the core toxicological test batteries used to assess chemical safety. for such chemicals, provisional estimates of biomarker concentrations corresponding to key benchmarks may still be possible, albeit with greater uncertainty or built - in conservatism. one approach relies upon a read - across from other chemicals that are structurally similar or that have similar chemical and physical properties. if chemicals are closely related, data for a well - studied chemical may be used and serve as a surrogate for a structurally similar compound with fewer data. recently criteria have been established for structural analog identification and selection, and this process has been validated with a set of case studies. more broadly, chemicals that exhibit similar physical and chemical properties may be evaluated using a generic model applicable to that class. for example, chiu and white demonstrated the derivation and application of steady - state solutions to a generic physiologically based toxicokinetic (pbtk) model for volatile organic compounds (vocs) in route - to - route extrapolation. the steady - state solutions require very limited chemical - specific data to implement, and such data can often be generated in vitro. aylward. collected the required chemical - specific data from the literature as well as current risk assessment - based exposure guidance values for approximately 40 voc compounds. they implemented the steady - state solutions to the generic pbtk model to estimate steady - state blood concentrations predicted to arise from steady - state exposures. the resulting estimated chemical - specific blood concentrations corresponding to exposure guidance values were proposed for use as screening values for evaluation of biomonitoring data for these vocs. across this class of compounds, variation in physical / chemical and metabolic properties resulted in estimated steady - state blood concentrations for a unit inhalation exposure (e.g., 1 mg / m) that varied by approximately one order of magnitude, while those arising from a unit of oral exposure (1 mg / kg - day) varied over approximately 2 orders of magnitude. therefore, if an exposure guidance value is available for a chemical expected to have similar physical, chemical, and metabolic behavior to those included here, a range of likely steady - state blood concentrations potentially consistent with the exposure guidance value could be estimated. other pbtk model structures potentially applicable to a wider range of compounds have been proposed and used in a variety of contexts. rotroff. used in vitro methods to develop estimates of the metabolic clearance and protein binding for a series of chemicals included in the us epa phase i toxcast program. these parameters were used in a generic pbtk model to relate blood or serum concentrations to corresponding steady - state external dose rates using commercially available software. presented initial results of a comprehensive effort to develop a generic pbtk model structure that accommodates varying levels of chemical - specific information and allows prediction of biomarker concentrations (both urinary and blood) associated with a specified exposure guidance value. on the toxicity assessment front, louisse. demonstrated the integration of in vitro toxicity data with toxicokinetic models to assess glycol ethers. these efforts highlight the potential utility of targeted data development including in vitro assessments of metabolism and measured or estimated chemical and physical properties in allowing development of provisional biomarker screening or assessment values based on current risk assessments. if human biomonitoring data approach or exceed these screening values, allocation of resources to development of more detailed, data - driven evaluations of toxicokinetic characteristics may be appropriate. most of the chemicals currently being assessed in the us nhanes and the canadian health measures surveys are well - studied substances. however, even among this group of compounds, there is sometimes a lack of derived toxicity guidance values, and, more commonly, a lack of detailed chemical - specific toxicokinetic data needed to translate external exposure levels into expected corresponding biomarker concentrations as required to support development of bes. as biomonitoring programs are expanded to include less well - studied substances, compounds that lack both comprehensive toxicity datasets, and toxicokinetic data needed for development of full be values are likely to be included. in such cases, provisional screening assessment values may still be derived using combinations of the approaches outlined above. for example, the aylward. evaluation of screening be values for assessment of vocs could be applied to chemicals lacking both exposure guidance values and toxicokinetic data. the screening values estimated by chiu and white incorporate both the toxicokinetic behavior of the chemicals as well as the risk assessment - based tolerable exposure levels based on noncancer endpoints. the cumulative distribution of estimated screening blood concentrations for these vocs is presented in figure 2. the values span more than five orders of magnitude in blood concentration. if a chemical is judged to be similar in general physical, chemical, and toxicological characteristics to those included in the group evaluated by aylward. but lacks the information necessary for a chemical specific be, a lower percentile of blood concentration from the distribution represented here might be selected as an initial screening value for evaluation of blood concentrations of that chemical measured in humans. this approach is conceptually similar to the ttc approach, but conducted on a biomarker concentration basis rather than an intake dose basis. similarly, the ttc approach could be applied to a chemical to estimate a conservative level of tolerable external exposure, and a generic pbtk model such as that developed by bartels. could be used to estimate a corresponding biomarker concentration for use as a screening value. the flowchart is conceptually similar to the tiered screening process described in a 2001 review by the health council of the netherlands, with the added component of extension of the tiered approach to evaluation of biomonitoring data. these approaches should be applied in an iterative framework, with increasing refinement indicated when mos values are judged to be insufficient. use of all - generic approaches to derive provisional screening values clearly results in values that are highly uncertain, requiring the use of health - protective assumptions in the screening process. if chemicals being detected in biomonitoring surveys fall into this category of lacking both toxicological and toxicokinetic data, these chemicals may be candidates for early research to fill selected data gaps in order to refine the assessments for those chemicals. the collection and reporting of human biomonitoring data continues to grow, and the advanced analytical chemistry techniques employed can now accurately quantify substances in reasonable sample volumes of blood or urine from individuals. and while authoritative organizations have cautioned that detection does not equate to illness or injury, the absence of methods to interpret human biomonitoring in a health risk context reduces the value of these data because of the inability to prioritize among the detected chemicals on the basis of potential risk posed by the detected levels. employing tools to interpret biomonitoring data which results in a risk assessment - based context can assist risk managers in addressing concerns about chemical exposures. it also provides a framework for determining whether additional product stewardship and/or regulatory risk management actions may be warranted. the be approach has proven to be useful as a screening tool to provide an assessment of which chemical biomarkers are present at levels well below, near, or at or above concentrations that are consistent with exposure guidance values derived in existing authoritative government risk assessments. as discussed here, the underlying approach developed for the bes can also be used in cases where such authoritative risk assessments are not yet available or where robust toxicokinetic models are n't at hand. both the noael approach and the ttc method discussed here can be used to establish benchmarks that will allow screening - level evaluation of biomonitoring data. although there are uncertainties when using such methods, by employing health protective assumptions, such as additional uncertainty factors to account for database shortcomings, the derived points of departure from the noael and ttc approaches can be used with a reasonable degree of confidence that they are health protective. as with any method used for chemical exposure assessment, the quality and representativeness of the biomonitoring data must be considered in the process of interpreting the data. while a complete discussion of the factors relevant to evaluation of biomonitoring data is outside the scope of this paper, some of these factors include the stability and specificity of the biomarker and the representativeness of the sampling frame used to generate the data. similarly, the robustness and reliability of the toxicokinetic models and data used to translate affect the confidence in the derived biomonitoring equivalents (discussed in hays.). the methods described here represent a range of approaches that can be applied depending on the level of chemical - specific information available. obviously, as the level of chemical - specific data decreases and reliance on generic assumptions increases, the uncertainty associated with the derived screening values increases. if human biomonitoring data approach or exceed these screening values, allocation of resources to development of more detailed, data - driven evaluations may be appropriate in order to inform risk managers. in such cases, an iterative approach to development and application of human biomonitoring assessment values is appropriate. such data may include in vitro assessments of metabolism, measured or estimated chemical and physical properties, or in vivo toxicokinetics and metabolism studies to refine provisional toxicokinetic estimates. | evaluation of a larger number of chemicals in commerce from the perspective of potential human health risk has become a focus of attention in north america and europe. screening - level chemical risk assessment evaluations consider both exposure and hazard. exposures are increasingly being evaluated through biomonitoring studies in humans. interpreting human biomonitoring results requires comparison to toxicity guidance values. however, conventional chemical - specific risk assessments result in identification of toxicity - based exposure guidance values such as tolerable daily intakes (tdis) as applied doses that can not directly be used to evaluate exposure information provided by biomonitoring data in a health risk context. this paper describes a variety of approaches for development of screening - level exposure guidance values with translation from an external dose to a biomarker concentration framework for interpreting biomonitoring data in a risk context. applications of tools and concepts including biomonitoring equivalents (bes), the threshold of toxicologic concern (ttc), and generic toxicokinetic and physiologically based toxicokinetic models are described. these approaches employ varying levels of existing chemical - specific data, chemical class - specific assessments, and generic modeling tools in response to varying levels of available data in order to allow assessment and prioritization of chemical exposures for refined assessment in a risk management context. |
spinal epidural abscesses (seas) are unusual bacterial infections requiring prompt diagnosis and management to prevent devastating neurologic sequelae. they represent about 7% of vertebral infections and usually occur in subjects with predisposing underlying diseases or conditions such as diabetes mellitus, chronic renal failure, cancer, advanced age, immunodeficiency, alcoholism, intravenous drug abuse, cauda equina, holocord syndrome, neurosurgery, spinal anesthesia and acupuncture, mucocutaneous trauma, or by spreading of a known infection localized at other sites. bacteria gain access to the epidural space through contiguous spread (primary sea) or by hematogenous dissemination (secondary sea) ; the source of infection is not identified in 2040% of cases,,,. the most common causative agent is staphylococcus aureus, both methicillin - susceptible (mssa) or methicillin resistant (mrsa), accounting for 5090% of cases, followed by streptococci (817%) and gram negative bacteria (1017%),,. seas have an insidious onset of pain and a progressively worsening clinical picture characterized by fever and elevation of the indices of inflammation. four clinical stages have been described : stage 1 lumbar pain, fever and local tenderness ; stage 2 radicular pain, nuchal rigidity and changes in the reflexes ; stage 3 sensory and motor abnormalities, with motor weakness and bowel and bladder dysfunction ; stage 4 paralysis with permanent sequelae. it is therefore essential to achieve an early diagnosis, start effective antimicrobial therapy and, if required, proceed with a prompt neurosurgical intervention,. several case series of spinal epidural abscesses in adults have been reported in the scientific literature, whereas reports in children are scanty. we describe a spontaneous sea due to mssa in a 15-year - old boy without risk factors, and have performed a review of publications in the scientific literature regarding management of pediatric seas published in the last 14 years. two previous reviews, including reports on patients with risk factors, reported cases up to calendar year 2000,. in the present work, we analyzed reports and reviews from pediatric patients published from 2001 to 2014 which, as in our case, did not present risk factors. a review of the english literature was performed by an exhaustive pubmed search for case reports and reviews, with publication date january 2001december 2014, using the following terms : spinal subdural abscess, spinal epidural abscess, spontaneous subdural abscess, spontaneous epidural abscess, spontaneous spinal epidural empyema. the exclusion criteria were : (i) adult population (age 18 years), (ii) incomplete clinical or age information or undistinguishable data between pediatric and adult patients, (iii) tubercular spinal epidural abscesses, and (iv) any underlying disease in the medical or surgical history. a manual review of the papers found by the above search method was performed to verify inclusion and exclusion criteria and to exclude cases with risk factors for hematogenous spreading (e.g. impetigo in chickenpox, cat scratch, mucocutaneous trauma) and/or underling predisposing diseases (e.g. cauda equina, holocord syndrome, neurosurgery). in june 2013, a 15-year - old male was referred to the emergency department of siena university hospital, tuscany, italy, from another regional hospital with a provisional diagnosis of meningitis. he reported a history of fever, headache and back pain, mainly in the lumbar - sacral region, during the previous 3 days. no previous trauma, nor minor or major surgery was reported by the boy or his parents. his past medical history was unremarkable, but he received a anti group c meningococcal conjugate vaccine dose together with a anti diphtheria - tetanus booster dose 2 weeks before admission ; seven days of myalgias and low grade fever (maximum 37.5 c) with spontaneous resolution were reported to after those immunizations. on admission the patient was conscious complaining of headache and lumbar pain with bilateral leg weakness ; on physical examination his bmi was 17.3 blood pressure 110/70 mmhg, heart rate 92/min, respiratory rate 20/min, body temperature 36.3 c (he had received 1000 mg of acetaminophen one hour before admission) ; he had a stiff neck with a lumbar pain arising while attempting to flex his neck, and presence of a bilateral straight leg raise sign, there was not any focal tenderness on palpation of the patient 's spine. neurological examination did not demonstrate any motor or sensory deficit, but the patient was not able to walk due to the severe pain. laboratory exams showed : leukocytes 11,300 10/l (86.3% neutrophils), c - reactive protein (crp) 10.2 mg / dl (upper normal value, upper limit of normal, uln 0.5), procalcitonin 0.18 ng / ml (uln 0.5), pt 71% (normal range 80120%), inr 1.24. cerebrospinal fluid (csf) examination revealed a clear fluid with pleocytosis (138 leukocytes / mm, 67% polymorphs), glucose concentration 67 mg / dl (blood glucose 61 mg / dl), protein concentration 145.3 mg / dl (normal range 2040 mg / dl). empirical treatment with intravenous (iv) ceftriaxone 2 g bid and iv acyclovir 500 mg tid was prescribed and the patient was transferred to the infectious disease ward. the day after admission no peripheral nerve conduction deficits were detected on electromyography and no immunoglobulins type g in the alkaline region of csf on isoelectrofocusing were revealed. urgent gadolinium - enhanced cerebral and spinal magnetic resonance imaging (mri) disclosed a large epidural purulent collection in the posterior and median - paramedian left spinal canal, extending from t11 to l2, with mass effect on spinal nerve roots (fig., in the evening of the 2nd day from admission, neurosurgeons performed an l2 laminectomy and abscess drainage. antimicrobial treatment was modified with discontinuation of ceftriaxone and acyclovir, and empirical switch to meropenem 2 g tid and vancomycin 1 g bid., choosing a wider spectra coverage due to the aforementioned neurological deterioration and to the large amount of obtained purulent material, histological analysis of the surgical tissue samples showed chronic purulent inflammation, without neoplastic changes. abscess culture revealed mssa ; the strain was also fully susceptible to all tested antimicrobials. on the basis of the above in vitro susceptibility results, the treatment was modified again with iv ceftriaxone 2 g bid and iv clindamycin 900 mg tid negative results were obtained from blood cultures, csf culture and polymerase chain reaction on csf for borrelia spp., mycobacterial and viral genomes ; serum agglutination tests for typhoid and brucellosis were negative ; no congenital or acquired immunodeficiency was revealed. normal values of crp and white blood cell count were obtained respectively on the 7th and 18th day ; remission of the fever was obtained within 72 h after admission and a progressive improvement of the clinical condition was observed. lumbar pain disappeared and the patient underwent rehabilitation with a complete recovery. on the 21st day, the patient was discharged from hospital with the final diagnosis of sea by mssa and with the advice to continue antimicrobial therapy with iv ceftriaxone 2 g daily and oral clindamycin 600 mg tid lumbar spinal mri, performed approximately one month after surgery (fig. 2a, b), showed complete resolution of the abscess and gadolinium - enhancing postoperative reactive changes in the fascial muscular planes. heart and abdomen ultrasound and chest radiography were negative. at monthly follow - up examinations, white blood cells and crp were constantly normal but the patient complained general asthenia and mild occasional lumbar pain and the same antimicrobial regimen with ceftriaxone and clindamycin was prolonged for further 4 weeks. 2c) showed reduction of reactive changes and the patient complained persistent mild lumbar pain during subsequent clinical evaluation ; amoxicillin / clavulanate 1 g tid plus rifampicin 600 mg per daily orally was prescribed during the subsequent 6 months. after total 8 months of antimicrobial treatment, lumbar spinal mri showed further reduction of reactive changes (fig. including our report, we found a total of 12 pediatric sea cases without predisposing factors : 8 were males, average age was 9.6 years [range 16 days17 years ]. the patient described by prasad and de vere, masquerading as an acute abdomen, was excluded from subsequent analysis, due to lack of complete clinical information (paper submitted as visual diagnosis). in the remaining 11 patients, clinical presentation was : fever and back pain, with elevation of csf cell counts (average, 15,196 cells / mm) and crp levels, except in a single case. lumbar puncture was performed in 4/11 cases (36.3%), with an abnormal result of csf 's cell count in 3 cases. responsible microorganisms were identified in 8 cases (72%) : in 1 case a group a beta - hemolytic streptococcus was isolated from a purulent collection, in the remaining 7 cases s. aureus was cultured from purulent collection (n = 4), from blood (n = 2) or from both sites (n = 1). mssa was reported in 4 patients, mrsa in 1 patient, while in the remaining 2 cases drug susceptibility was not reported. in one of these two latter cases, even if not specified in the original paper, a presumptive diagnosis of mssa can be inferred based on response to antimicrobial therapy (cefazolin, cephalexin). all the patients, were initially misdiagnosed as : back pain, meningitis (even in absence of the classical meningeal syndrome), acute myelitis, diskitis, cord compression by a neoplastic mass and septic arthritis ; patients spine was always evaluated by gadolinium - enhanced mri. most abscesses (n = 10/11) were localized at the thoracic and lumbar area, without signs of osteomyelitis. in 8/11 cases, laminectomy and abscess drainage were performed in association with effective medical therapy ; only in two cases, both without an aetiological diagnosis, treatment was successful with antimicrobial therapy only,. a good outcome, defined as a complete recovery, was obtained in all patients, with the exception of the report of rook. who described residual headache and paraspinal pain lasting for 3 years. complete data of the reviewed literature reports are summarized in table 1, table 2. to the best of our knowledge, this case report is one of the rare descriptions of pediatric sea, without underlying risk factors. in a previous literature review covering reports from 1980 to 2000, 12 pediatric patients with sea without any risk factors were found : no complications or associated osteomyelitis were reported and a favorable outcome was obtained after medical (n = 1/12, 8%) or combined surgical plus medical (n = 11/12, 92%) therapy. mssa was the predominant detected pathogen (n = 7/12, 58%). according to our review of recent literature from 2001 to 2014, sea in pediatric age is confirmed to be very rarely reported, especially in the absence of predisposing risk factors : in the last 14 years only 12 cases were described. a combined surgical and medical therapy was usually performed with favorable outcome and mssa was confirmed to be the main aetiological agent. despite this prominent etiology, given the potential for serious sequelae, anti - mrsa therapy should be considered mandatory in the empiric antimicrobial regimen for pediatric sea in areas with high rates of mrsa, awaiting microbiological identification and drug susceptibility results. an interesting matter of discussion is the possible source of staphylococcal bacteremia in pediatric patients without risk factors : as nares and skin are the primary colonization sites, these should be considered the primary sources. in our case report, no previous trauma could be linked to a bacteremia by microorganisms colonizing the skin, not even acupuncture procedures as previously reported for adult patients. the required duration of antimicrobial therapy remains an issue : a mean duration of 6 weeks was reported in the reviewed cases ; the previous most recent review suggested a 46 weeks regimen. in the case reported herein, antimicrobial therapy was indeed prolonged until 8 months, with a successful outcome and without side effects. the adopted regimen and its duration can be matter of discussion : despite rapid fever remission and normalization of crp, clinicians chose this long lasting dual regimen due to unusual presentation in an otherwise healthy adolescent and to long term persistence of mild referred back pain. it has been previously stated that a prolonged medical therapy is advisable in cases without surgical interventions and its duration should depend on the level of immune competence, clinical improvement and response to treatment demonstrated by subsequent reduction of inflammation at mri,. in the only previous report describing residual 3 years of headache and paraspinal pain, the duration of therapy was the it should be noted that mri follow - up is reported to overestimate inflammatory changes, mainly when bone involvement is present : mri follow - up has been usually reported as unnecessary in spondylodiscitis when the clinical and laboratory abnormalities respond to treatment. despite the absence of specific statements in the literature, this should be reasonably valid also for patients with sea. the patient with sea reported herein did not show a bone involvement, however he complained of a long lasting low back pain. mri follow - up showed complete removal of the abscess, followed by regular reduction of normal postoperative reactive findings, and development of thoracolumbar kyphosis together with increased lumbar lordosis. the case described herein, along with the reviewed literature, underline the diagnostic complexity of this condition, particularly at its onset. many clinical features may be non - specific for sea and the classical ones (fever, back pain and neurological deficits) can be incompletely present at the early stages. headache and neck stiffness are rarely reported, but their presence, as described also in our patient, can contribute to misdiagnosis : in 100% of the reviewed cases, a diagnosis other than sea was postulated on admission and a lumbar puncture was therefore frequently performed, with possible dangerous consequences depending on sea location. in conclusion, it seems essential to maintain a high index of suspicion for sea in children : the rarity and the possible differential diagnosis on the basis of the early signs and symptoms can lead clinicians to underestimate its occurrence, but an as prompt as possible diagnosis is an essential prognostic factor, allowing an immediate medical and surgical intervention before the development of non - reversible sequelae. on behalf of all authors, the corresponding author states that there is no conflict of interest. | spinal epidural abscesses (seas) are unusual bacterial infections, with possible devastating neurologic sequelae. despite abundance of case series in adults, reports in children are scanty.we describe a spontaneous sea due to methicillin susceptible staphylococcus aureus (mssa) in a previously healthy 15-year old male, and we perform a literature review regarding management of pediatric seas without risk factors, from 2001 to 2014.we found a total of 12 cases (8 males, average age 9.6 years). clinical presentation was mainly fever, back pain and elevation of inflammation markers. all cases were initially misdiagnosed. lumbar puncture was performed in 36% of patients. etiological diagnosis was obtained in 8 cases. mssa was isolated in 4 patients, methicillin - resistant s. aureus in 1 patient, and s. aureus with unknown susceptibility patterns in 2 cases. the average of therapy duration was 6 weeks. patients spine was always evaluated by gadolinium - enhanced magnetic resonance imaging ; most abscesses were localized at thoracic and lumbar area, without osteomyelitis. in 8 cases, laminectomy and/or abscess drainage were performed in association with medical therapy ; 3 cases were successfully treated with antimicrobial therapy only ; no data were available in one case. a good outcome was obtained in all patients, except a reported residual headache and paraspinal pain lasting for 3 years.the rarity and the possible differential diagnosis can lead to underestimate sea occurrence in children without risk factors. it seems therefore essential to maintain a high attention to pediatric seas. a prompt diagnosis and adequate therapy are essential prognostic factors for remission. |
as glycemic control deteriorates over time, treatment intensification with the addition of multiple oral antihyperglycemic agents is often required in patients inadequately controlled with monotherapy. polypharmacy and complexity of the treatment regimens are associated with poor adherence to treatment, which in turn is associated with inadequate glycemic control [24 ]. on the other hand, the use of a fixed - dose combination of agents with complementary mechanisms of action is associated with improved patient compliance and adherence to treatment, as well as better glycemic control [5, 6 ]. vildagliptin is a potent and selective oral dipeptidyl peptidase-4 inhibitor that improves glycemic control in patients with t2 dm by increasing both the -cell and -cell responsiveness to glucose [7, 8 ]. in numerous clinical trials, combination therapy with vildagliptin and metformin has demonstrated a better efficacy and safety profile with good gastrointestinal tolerability than high - dose metformin monotherapy [9, 10 ]. a single - pill combination of vildagliptin / metformin has been approved in the european union and across many countries in the world for the treatment of patients with t2 dm inadequately controlled with metformin alone. in the present study, we evaluated the differences in the treatment compliance with vildagliptin / metformin fixed - dose combination and vildagliptin (50 mg bid) added to metformin (free - dose combination) therapy in patients with t2 dm in greece. patients aged > 18 years with t2 dm and inadequate glycemic control with metformin monotherapy (850 mg bid) were eligible to participate in the study. patients were enrolled in two cohorts on 1 : 1 ratio, according to everyday clinical practice : those receiving either vildagliptin / metformin fixed - dose combination pill (hereafter referred to as the fixed - dose combination group) or vildagliptin (50 mg bid) added to metformin (850 mg bid) (hereafter referred to as the free - dose combination group). patients with a history of type 1 diabetes, end stage renal disease, undergoing hemodialysis, congestive heart failure, and pregnant or lactating women were excluded from the study. in order to assess the treatment compliance, investigators were asked to complete a compliance questionnaire by interviewing patients both at the baseline (visit 1) and final visit 3 (24 weeks after baseline) (table 1). patients were considered compliant if they did not miss any drug dose or no more than 2 doses per week, received the correct dosage of the medication, and did not interrupt their treatment. treatment compliance was assessed from the compliance questionnaire, and the difference in compliance between the treatment groups was reported. in addition to the questionnaire, investigator collected clinical, demographic, and relevant medical history data including comorbidities and complications. at the baseline visit, each patient was given a diary to record their medication intake on a daily basis. the patient was asked to return this diary to the physician at the final visit. the study was designed and conducted in accordance with the applicable local regulations and with the ethical principles laid down in the declaration of helsinki. a written, informed consent was requested from each patient before enrollment in the study. the primary objective was to compare the percentage of patients compliant with their prescribed therapy. secondary objectives of the study were to assess the changes in the levels of hba1c from the baseline until the end of the study (day 0 to 6 months after) and to assess the safety and tolerability profile of vildagliptin. assuming 60% of the patients on fixed - dose combination therapy were compliant and a difference in the treatment groups of 12%, 320 patients per treatment group were required to provide 90% power at a significance level of 5%. the primary variable, difference in compliance between the two treatment groups, was assessed using a multiple binary logistic regression model and adjusted for age, sex, comorbidities, concomitant medications, duration of t2 dm, whether patients remembered the names of their medications for t2 dm, difficulties in ingestion, and clinical laboratory test results. the odds ratio (or) with 95% confidence intervals (ci) was also calculated from the multiple binary logistic regression analysis. change in hba1c from baseline to end of study was analyzed using an analysis of covariance model (an.co.va.). all adverse events (aes) and serious adverse events (saes) were recorded and monitored, along with their severity and relationship to the study drug. patient demographics and baseline characteristics were generally comparable between the two treatment groups (table 2). of the 659 patients enrolled, 366 (55.5%) were assigned to the fixed - dose combination group and 293 (44.5%) to the free - dose combination group ; data for 3 patients were missing. overall, 54.4% of patients were men, mean age was 61.9 years, mean body mass index (bmi) was 30.1 kg / m, and mean baseline hba1c was 8.0%. about 9% of patients were taking other concomitant medications and 16% of patients had comorbidities, of which 70% of patients had hypertension, 59% had dyslipidemia, and 12% had ischemic heart disease. overall, 92.6% of patients were compliant with their prescribed therapy according to the definition of compliance used in this study. the percentage of patients compliant with treatment in the fixed - dose combination group was 98.9% compared with 84.6% in the free - dose combination group before adjusting for confounding factors (figure 1). the or for compliance in the fixed - dose combination group versus the free - dose combination group was 18.9 (95% ci : 6.2, 57.7 ; p < 0.001) after adjusting for confounding factors. patients who remembered the names of their medications were five times more likely to comply with their treatment than patients who did not remember the names of their medications. patients who experienced difficulty in swallowing their medications were 31.3 times less likely to comply with their treatment compared with patients who did not experience any difficulty in swallowing their medications. the model was also tested for goodness of fit to the data of the study using the hosmer and lemeshow that proved that the model had a good fit to the study data ; p value = 0.619 (table 3). the mean hba1c decreased from a baseline of 8.1% to 6.9% in the fixed - dose combination group and from 7.9% to 6.8% in the free - dose combination group ; the change was statistically significant from baseline to study end in both groups but not between groups (figure 2). management of t2 dm is complex due to multiple factors such as competing comorbidities, resistance to pharmacotherapy, reluctance to increase the dosage and/or the number of medications, low socioeconomic or educational status, and lack of adherence to lifestyle modifications. one practical way to enhance compliance in patients with multiple comorbidities and receiving concomitant medications is to simplify the treatment regimen with fixed - dose combinations. results from meta - analysis of clinical trials showed that fixed - dose combinations reduce the risk of noncompliance and improve compliance with treatment compared with free - dose combination regimens [13, 14 ]. as treatment compliance may influence the overall glycemic control as well as progression of the disease, findings from this study may prove to be useful when assessing treatment strategies for diabetes mellitus. in the present observational study, more number of patients in the fixed - dose combination group were found to be compliant to the treatment (or 18.9, 95% ci : 6.2, 57.7 ; p < 0.001) compared with the free - dose combination group. this is consistent with the findings from a meta - analysis of seven studies that reported 10% to 13% higher treatment adherence with fixed - dose combination of medications than with free - dose combinations. in this study, patients who did not remember the names of their medications and those who experienced difficulty in swallowing their medications were less likely to comply with their treatment, suggesting that simple names for medications and pill size could help in improving the compliance with medication. the mean hba1c decreased from a baseline of 8.1 to 6.9% in the fixed - dose combination group and from 7.9% to 6.8% in the free - dose combination group. the observed hba1c drop in the present study is consistent with the results reported from a large clinical trial (0.9 0.1%) which assessed the efficacy and safety of vildagliptin add - on to metformin. of note, although there were differences with respect to treatment compliance between the fixed - dose and free - dose combinations, these did not result in a difference in efficacy. the results from the present study showed that the combination of two oral antihyperglycemic agents with complementary mechanisms of action offers benefits of consistent glycemic control and helps to improve medication compliance. in addition, there were no new safety signals observed with either fixed - dose or free - dose combinations of vildagliptin and metformin which was generally consistent with the previously reported tolerability profile of vildagliptin as add - on therapy to metformin. the present study has certain limitations that need to be considered while interpreting the results. only a few patients completed the diaries on a daily basis which resulted in inadequate data for additional analysis and, further, the 6-month follow - up period might be considered a short duration for the measurement of compliance and its effect on efficacy. moreover, it should be added that the method assessing compliance (interview) is not as accurate as the pill count method or the microprocessor method. in conclusion, patients on vildagliptin / metformin fixed - dose combination were more compliant with their treatment when compared with patients on free - dose combination. taking into account that t2 dm is a chronic disease, it is important to emphasize that its management should be a part of a health policy plan, and priority should be given to therapies with proven effectiveness and safety as well as fixed - dose combinations that improve patients ' compliance. | objective. to evaluate the differences in treatment compliance with vildagliptin / metformin fixed - dose versus free - dose combination therapy in patients with type 2 diabetes mellitus (t2 dm) in greece. design. adult patients with t2 dm, inadequately controlled with metformin monotherapy, (850 mg bid), participated in this 24-week, multicenter, observational study. patients were enrolled in two cohorts : vildagliptin / metformin fixed - dose combination (group a) and vildagliptin metformin free - dose combination (group b). results. 659 patients were enrolled, 360 were male, with mean bmi 30.1, mean t2 dm duration 59.6 months, and mean hba1c at baseline 8% ; 366 patients were assigned to group a and 293 to group b ; data for 3 patients was missing. in group a, 98.9% of patients were compliant with their treatment compared to 84.6% of group b. the odds ratio for compliance in group a versus b was (or) 18.9 (95% ci : 6.2, 57.7 ; p < 0.001). in group a mean hba1c decreased from 8.1% at baseline to 6.9% (p < 0.001) at the study end and from 7.9% to 6.8% (p < 0.001) in group b. conclusions. patients in group a were more compliant than patients in group b. these results are in accordance with international literature suggesting that fixed - dose combination therapies lead to increased compliance to treatment. |
its severe forms (hemorrhagic fever and shock syndrome) may lead to multiorgan involvement and death. dengue shock syndrome (dss) is characterized by a massive increase in systemic capillary permeability with consequent hypovolemia. the mortality rate in dengue shock syndrome ranges from 6 to 30 percent, most commonly reported in children., sri lanka has experienced a surge of the disease reaching epidemic proportions associated with a probable change in the virus strain to a more virulent form [4, 5 ]. in this context we have also noted a rise in the number of cases with severe forms of the disease needing intensive care. the peradeniya icu is a tertiary referral centre and it attracts a large number of above patients with dengue shock syndrome in the region who do not respond to standard therapy with intravenous fluids, antibiotics, and supportive care including inotropes. we collected demographic data of all patients referred for intensive care between january 2009 and june 2010 (18 months) and analyzed their outcomes in relation to the complications of dss that ensued during their icu stay and the therapies given. the diagnosis of dengue has been established upon clinical grounds (who guidelines 2009) and treated accordingly by the referring physicians before admission to the icu. on admission to icu, all have had fever, vomiting 78%, abdominal pain 21%, cough 5%, and body ache among 3% of cases. eleven patients have had serological tests and dengue igm was found to be positive amongst 72% and igg in 50% of the tested. they all have had intravenous fluid therapies and antibiotics. during the course of therapy in icu, 43.6% of cases received fresh frozen plasma, 21.8% cryoprecipitate, 32.7% blood, and 56.4% platelet transfusions. furthermore, 36.4% of cases were mechanically ventilated for multiple reasons such as severe respiratory distress (fio2 > 60%), rr > 40/min, myocardial failure needing inotropes for persistent hypotension despite adequate filling, that is, cvp above 12 cm of h2o. the decision for peritoneal dialysis was based on low urine output (< 0.5 ml / kg / hour) detected over a period of time in an icu as a trend despite resuscitation with fluids combined with a situation of fluid overload, high cvp, persistent hypotension, or severe ards. neither the plasma creatinine (rather a delayed indicator of aki) nor blood urea (not reliable in the presence of liver impairment) was used as a determining factor in implementing dialysis. persistent hypotension in the presence of high cvp was interpreted as indication of myocardial involvement (with or without relative bradycardia, ecg changes) and was supported with inotropes, usually a combination of dobutamine and nor - adrenaline and with mechanical ventilation if oxygenation was compromised.. thus, our data is based on a group of severely ill patients diagnosed to be suffering from dengue shock syndrome and continuing to deteriorate despite intravenous therapies administered in the wards. liver failure regime, that is, oral / ng metronidazole and lactulose was introduced in patients who were found to have raised transaminases. it should be noted that the number of icu beds available (10) in our hospital was approximately 1.5% of the total beds and this may have delayed the admission of some cases due to rationing. between january 2009 and june 2010, 54 cases (25 male) diagnosed of dengue shock syndrome were admitted for treatment to the peradeniya icu, a tertiary referral center. half of them were aged 20 or below as shown in figure 1. on admission, their mean (sd) pcv was 44.5% (5.5), wbc 7.710/l (4.6), platelet count 22 10/l (17), respectively. of the 54, 16 died (mortality 29.6%). most deaths (88%) occurred within 3 days of admission to the icu (table 1). the survivors needed intensive care for a median of 2 days (range 18) before being discharged to the referring wards for convalescence. it is also noteworthy that 62.5% deaths occurred below the age 20 (see table 2). although we were unable to prove that risk of death was higher in children, a higher incidence of deaths (38% as opposed to 22%) was observed below the arbitrary cutoff age of 20 years. to evaluate the risk of death according to the manifesting complications we performed a bivariate analysis and calculated the odds ratios (ors) and confidence intervals (ci). or and ci provide information on the strength (level of statistical significance) of association between the complications and the occurrence of deaths. whereever the numbers of subjects were too small, the fisher 's exact test was used to calculate the p value (table 3). we found that the complications of dengue, namely, hemorrhage, pleural effusion, myocarditis, liver failure, and renal failure were independently linked with a 711 times higher risk of death compared to those without (table 3). the effect of treatment modality on the outcome (death) was evaluated with chi - square test (see table 4). chi - square test is widely used to evaluate the association between these predictor and outcome variables. the results revealed that the treatment modalities, namely, the use of inotropes, mechanical ventilation, peritoneal dialysis, and the use of blood products were significantly associated with higher occurrence of deaths among these patients (table 4). however, the use of steroids had no association with death. thereafter a discriminant analysis was used to classify the cases according to the values of categorical dichotomous - dependent variables. this analysis assesses the relative importance of the independent variables in classifying the dependent variable. the standardized canonical discriminant function coefficients identified mechanical ventilation and peritoneal dialysis as therapeutic modalities significantly associated with the deaths of dengue patients presenting with dengue shock syndrome (table 5). a similar discriminant analysis was used to assess the relative importance of complications and outcome. renal failure and hemorrhage were identified as complications significantly associated with deaths in dengue shock syndrome (table 6). we have evaluated the mortality risk factors amongst a cross - section of patients in dengue shock syndrome not responding to standard therapies and as a consequence in a clinical scenario confounded by previous therapies before admission to icu. dengue shock syndrome is a dangerous complication of the dengue infection and is associated with high mortality. almost one - third of our study group received blood transfusions to counter their bleeding manifestations. increased vascular permeability, together with myocardial dysfunction and dehydration due to capillary leakage, contribute to the development of shock, with resultant multiorgan failure. the diagnosis is largely clinical and is supported by serology and identification of viral material in the blood. no specific methods are available to predict outcome and progression. as observed by singhi. the choice of fluids, inotropes, and techniques of organ support and careful fluid management is the mainstay of management. we have recorded a 30% mortality risk for this unique group of patients with dengue shock syndrome who had received prior medical therapies and was admitted to intensive care with further deterioration. unfortunately, the fact that 50% of the patients who succumbed did so within the first 24 hours of admission to the icu indicates their moribund state upon referral to the icu. it should be noted that the presenting clinical status of these patients to the icu was confounded by a variety of treatment regimens that were applied before admission to the icu. for example, in a typical patient admitted with respiratory distress and hypoxia, the clinical picture would easily be modified by overzealous hydration with colloids such as dextran or hetastarch prior to the icu referral. in this study we have not been able to assess the influence of prior therapy on outcome due to poor medical records received at the admission to icu. it is however the general impression of the authors, that the influence of prior therapies could be an important determinant of outcome, especially because some patients were noted to have had a cumulative dose of hetastarch exceeding 25 ml / kg suggesting overload contributing to respiratory distress more than the disease itself. this is a very important aspect that can not be overlooked in future studies of this nature. there is a general impression that fatal dengue is commoner in the younger population compared to middle or old age [7, 8 ]. although we recorded 62.5% deaths amongst patients aged 20 or below, we have no statistical evidence to support the notion that mortality is higher amongst children. this is because our age distribution also indicated that 50% of the age cohort admitted for icu care was above the age of 20. peradeniya icu is multidisciplinary and there are no age restrictions in its admission policy and hence we presume that our data represent the population with dengue shock syndrome with no age bias. in our study, from amongst the dead, 88% expired during first three days of icu care and the highest death rate was reported on day 1 (50% of total deaths). a similar study during an epidemic of dengue hemorrhagic fever in easternmost indonesia showed a case fatality rate of 1.2% from a 172 suspected cases. they too observed that, the survivors needed a range of 18 days of icu care similar to the durations we observed. another study from mumbai during a dengue epidemic reported a case fatality rate of 16.6% amongst pediatric patients suffering from dengue shock syndrome. bleeding has been identified as one of the dreaded manifestations of concern that complicates the outcome of dengue. although our canonical discriminant analysis indicated that hemorrhage and renal failure were the dreaded complications associated with death, from amongst its main therapeutic modalities only peritoneal dialysis (pd) this suggested that the use of blood products has effectively mitigated the effects of hemorrhage upon outcome. however, peritoneal dialysis did not show a similar effect suggesting that pd may not be the most appropriate modality of therapy in these moribund patients with multiorgan failure. it is also our clinical observation that pd cycles in dengue patients produced a relatively large fluid retrieval without the use of additional measures such as dextrose in dialysate fluid. it is likely that these patients had ascites fluid that was also removed by each dialysis cycle and this may have simulated the main problem of dengue, the capillary leak. thus, peritoneal dialysis may have aggravated the clinical effects of continuing capillary leak only 21% of patients who received mechanical ventilation in the icu recovered in this study (4 out of 19). since we had no facility for ecmo, it is difficult to comprehend whether mechanical ventilation is the best supportive mode of therapy to maintain oxygenation in dss. there was a significant relationship between dengue, complications, and the modes of therapies and outcome. hemorrhage, pleural effusion, myocarditis, renal failure, and liver failure were all important predictors of the worst outcomes. a study conducted in thailand implied the importance of detection of abnormal high transaminase enzyme among the patients with dengue infection since the consequently developed hepatic encephalopathy could be expected. in our study 9 out of 12 patients who were treated with liver failure regime expired (75%). dengue induced acute kidney injury (aki) comprising creatinine increase, proteinuria, glomerulonephritis, and haemolytic uremic syndrome has been reported [13, 14 ] and also dengue - haemorrhagic - fever-(dhf-) induced aki even in the absence of shock, haemolysis, or rhabdomyolysis. in our study 8 out of 8 (100%) patients who were suspected as having renal failure in the year 2002 reported a dengue outbreak in taiwan and noted that patients with renal failure (rf) carry a high mortality rate, that is, the morality rate rf group versus non - rf group was 28.6% against 1.2% ; p < 0.001. the variable incidences of dengue myocarditis had been postulated to be due to variable immunopathogenesis secondary to variations in serotypes. dengue myocarditis is generally reversible with favorable outcomes if diagnosed and treated early [17, 18 ]. in our study 15 out of 27 patients (55.55%) who were suspected of having dengue myocarditis were treated with inotropes but they died during their icu stay. it has been reported that corticosteroids were no more effective than the placebo or the no treatment protocol for reducing the number of deaths, the need for blood transfusion, or the number of serious complications or in achieving a higher rise of the platelet count in dengue infection. our study indicates that during this dengue outbreak, patients in dss who were not responding to standard therapies and admitted icu had a 30% risk of death. peritoneal dialysis increases this risk to 100%. | dengue shock syndrome is the most severe form of dengue that can be fatal. nonresponders to standard therapy need intensive care. this paper outlines the clinical features, complications, and outcomes of dengue shock syndrome not responding to standard therapies and needing supportive care in a tertiary referral intensive care unit of a developing country. nearly one - third die within 3 days of admission to icu. peritoneal dialysis predicts the worst outcomes. |
this genus comprises of gram - negative, strictly - aerobic, non - fermenting, non - fastidious, non - motile, catalase - positive, oxidase - negative bacteria with dna g + c content of 39.0% to 47.0%. according to euzeby 's list of prokaryotic names with standing in nomenclature (http://www.bacterio.cict.fr/a/acinetobacter.html) acinetobacter guillouiae proposed by nemec. was isolated from sewage - containing gas - work effluent and shares characteristics corresponding to those of the genus acinetobacter. a. guillouiae strain msp 4 - 18, isolated from a mangrove soil sample from parangipettai (1130n, 7947e), tamil nadu, india, was grown on tryptic soya agar medium (tsa ; himedia) at 30 c. genomic dna was extracted from 36 hour old culture using zr fungal / bacterial dna miniprep as per manufacturer 's instructions. amplification and sequencing of 16s rrna was performed as described by mayilraj.. identification was confirmed using 16s rrna sequencing. to determine the phylogenetic relationship of strain msp4 - 18, the 16s rrna sequence consisting of 1502 bp was compared with those of type strains of species of related genera and identification of phylogenetic neighbors and the calculation of pairwise 16s rrna gene sequence similarities were achieved using the eztaxon server and aligned using mega version 5.0. bootstrap analysis was performed to assess the confidence limits of the branching (fig. 1). the genome of a. guillouiae msp 4 - 18 was sequenced using the illumina - hiseq 1000 technology. sequencing resulted in 26,685,818 paired - end reads (insert size of 350 bp) with a length of 101 bp. a total of 26,465,246 high - quality reads with approximately 550 coverage were assembled with clcbio wb6 (word size 40 and bubble size 60) to obtain 94 contigs (n50, 128,068 bp) of 4,848,959 bp and average g + c content of 38.0%. the functional annotation was carried out by rast (rapid annotation using subsystem technology), fig. 2 shows the subsystem distribution of a. guillouiae strain msp 4 - 18, trna was predicted by trnascan - se 1.23 and rrna genes by rnammer 1.2. the genome contains 3 rrna genes (5s-23s-16s) and 69 aminoacyl - trna synthetase genes. a total of 4543 coding regions (2294 genes transcribed from the positive strand and 2249 from the negative strand) were found in the genome, of which 3052 (67%) could be functionally annotated. the genome coding density is 83% with an average gene length of 883 bp. the annotated genome has 106 genes responsible for resistance to antibiotic and toxic compounds including 13 genes for mdr efflux pumps. sixty five genes are involved in oxidative stress, 12 in osmotic stress, 15 for heat shock and several others for response to various other stresses, to make a total of 107 genes responsible for stress response in this organism. the functional comparison of the genome sequences available on the rast server revealed the closest neighbors of a. guillouiae msp 4 - 18 as acinetobacter baumanii ab0057 (score 502) followed by a. baumanii aye (score 500), acinetobacter johnsonii sh046 (score 494) and a. baumanii acicu (score 494). the a. guillouiae msp 4 - 18 whole genome shot gun (wgs) project which has been deposited at ddbj / embl / genbank under the project accession asqg00000000 of the project (01) has the accession number asqg01000000 and consists of sequences asqg01000001asqg01000094. the authors declare that there is no conflict of interest on any work published in this paper. | the genus acinetobacter consists of 31 validly published species ubiquitously distributed in nature and primarily associated with nosocomial infection. we report the 4.8 mb genome of acinetobacter guillouiae msp 4 - 18, isolated from a mangrove soil sample from parangipettai (1130n, 7947e), tamil nadu, india. the draft genome of a. guillouiae msp 4 - 18 has a g + c content of 38.0% and includes 3 rrna genes (5s, 23s, 16s) and 69 aminoacyl - trna synthetase genes. |
epistaxis (bleeding from nose) in children is generally traumatic in origin, the most common site of bleed being little 's area. besides trauma that generally occurs due to finger nails eroding the nasal mucosa, there are several tumors which can present with epistaxis. we report the first case in literature where a child with giant prolactinoma has presented with epistaxis as a primary complaint with normal serum prolactin level. a thirteen - year - old child had presented with bleeding from right nostril about eight months back. he started taking some homoeopathic medicine which decreased the quantity of bleeding but not the frequency. later he was also prescribed some nasal decongestant drops by the treating doctor, which resulted in some control temporarily. patient had no csf rhinorrhoea, had occasional feeling of nasal obstructions more so after the bout of nasal bleed, he had no respiratory problem. on anterior rhinoscopy examination, turbinates were hypertrophied along with deviated nasal septum toward left side. after about three months, he noticed diminution of vision in right eye which progressed to loss of vision in a month 's time. there was no headache, vomiting, blurring of vision or any complaints pertaining to endocrinopathy., there were no clinical features of any endocrine abnormality. in view of visual deterioration, mri of brain was done which revealed sellar - suprasellar, solid - cystic mass. the cystic component was predominantly suprasellar and hyperintense on t1 and t2 with peripheral rim enhancement. the solid component was both sellar - suprasellar and isointense on t1and t2 with contrast enhancement. the mass was encasing both cavernous sinuses, extending up to bilateral optic canal, encasing canalicular segment of both optic nerves. it was posteriorly extending into interpeduncular fossa and anteroinferiorly into nasopharynx, posterior nasal cavity, eroding the basisphenoid, clivus and posterior ethmoid and sphenoid sinuses [figure 1 ]. mri sagital section showing invasive mass in the sellar, suprasellar, and infrasellar regions his routine investigations included hb 10.8 gm%, hct 37.7%, tlc 9000/micl, dlc -p64, l32, e4 m0.,platelets 1.59 bleeding time was 1.25 min, clotting time 5.10 min, prothrombin time 15.2 s(control 16 s). his hormonal profile included ft3 -1.35(2.0 - 4.4 pg / ml.), ft4 - 0.40(0.93 - 1.7 ng / dl.), tsh-7.6(0.27 - 4.2 mic iu / l). serum prolactin was 203.7(86 - 324 mic iu / ml.).growth hormone was 5.4 ngm / ml (< 7 ngm / ml). right frontotemporal craniotomy and decompression of tumor was achieved via inter - optic and carotico - optic spaces. child had some bleeding from nose for about two days which settled in due course of time. histological examination revealed pituitary adenoma which was staining strongly positive for prolactin and negative for tsh, lh, acth and somatostatin [figures 2 and 3 ]. mitosis / necrosis and sinusoidal arrangement is not seen ihc, 10.tumor is moderate to intense diffusely positive for antiprolactin antibodies the case of a 72-year - old lady has been reported in literature presenting with fatal epistaxis due to rupture of aneurysm enclosed in a large prolactinoma. not all invasive tumors present with epistaxis. in most of the published literature [table 1 ], epistaxis was found to be associated with pituitary macroadenoma and in pituitary adenoma with infrasellar extension into nasopharynx. the published series of epistaxis and pituitary adenoma with duration of bleed and hormone status the massive size of the tumor leading to outstripping its blood supply and resulting in hemorrhagic infarction and necrosis of the tumor may also be responsible for epistaxis. the association of epistaxis and apoplexy with pituitary adenoma has also been described which is related to lymphocytic infiltration of pituitary adenoma, suggesting an immune - mediated response. there are other reports of epistaxis due to ectopic pituitary adenoma which develops from ectopic cell rests that are entrapped along the pathway of craniopharyngeal duct during embryonic development which have aberrantly migrated. epistaxis has also been reported to be associated with adenoma in paranasal sinuses and nasopharynx with empty sella. in such cases, however, pituitary stalk is found to be extending up to floor of the sella. epistaxis in a pituitary adenoma should raise the suspicion of a pituitary apoplexy in a giant pituitary adenoma with an infrasellar extension into the nasopharynx. have reported a case where pituitary adenoma remained undiagnosed for three years with history of intermittent epistaxis. it has been reported that epistaxis can occur with prolactinoma, growth hormone secreting adenoma and rarely due to co secreting pituitary acidophilic stem cell adenoma. other reported causes of epistaxis related to pituitary tumors are an aggressive tsh - secreting adenoma, extensive acidophil - stem - cell adenoma. in patients with acromegaly and another case of rupture of an intratumoral aneurysm enclosed in a large prolactinoma resulting in fatal epistaxis. the pituitary adenoma in our case presented with epistaxis and remained undiagnosed for eight months. despite the complaint of visual deterioration, the child never had a ct / mri study of brain. invasive prolactinoma can have very high levels of prolactin upto 1,000 ng / ml and epistaxis. there may be simultaneous mass effect in the form of compressive optic neuropathy, visual field deficits and ophthalmoplegia. falsely low serum prolactin level due to hook effect has been reported in giant prolactinoma. the hook effect occurs when excessive antigen is added to a hand held assay (hha), which then results in false negative. in this assay, the amount of antigen exceeds the finite amount of colloidal gold antibody or the colored labeling material. the excess unlabeled antigen migrates across the membrane more rapidly than the heavier color - labeled antigen, thus saturating or binding all the binding site on the capture antibodies. when the color - labeled antigen arrives, there are no binding sites available and the colored antigen can not create the colored test line that would produce a positive result. this, in turn, presents the user a false negative result (hook effect) even though high level of the antigen is present. the pituitary adenoma in our case presented with epistaxis and remained undiagnosed for eight months. despite the complaint of visual deterioration the pituitary adenoma remained undiagnosed initially and became invasive in about eight month 's time. pituitary adenoma can also present with epistaxis in children and should be considered as the differential diagnosis along with other uncommon causes. it should be further suspected if there is associated visual loss and in normal anterior rhinoscopy. however, posterior rhinoscopic examination in such cases may pick up the lesion at early stage. treatment of epistaxis with pituitary adenoma is largely symptomatic followed by surgical interventions. for large tumors, there may be some palliative role of chemotherapy or hormone therapy particularly in hormone secreting adenomas. it can be used both pre - op to reduce the size of lesion or post - op for residual lesions. | pituitary tumour have a wide way of presentation. epistaxis due to pituitary adenoma has been rarely reported. there is no report of bleeding from nose as clinical first presentation in a child. we report the first case in literature where a child had epistaxis for eight months before deterioration of vision. he was found to be having a invasive prolactinoma with normal prolactin levels. |
the development of nanotechnology and microsystems has relied, in many ways, on the major progresses accomplished in surface science and materials science. in the past, much effort has been devoted to characterizing the optical, electrical, and magnetic characteristics of the resultant structures and devices. the successful fabrication of devices based on semiconductors requires better understanding of the mechanical characteristics in addition to their optical and electrical performances. this is because that the contact loading during processing or packaging can significantly degrade the performance of these devices. therefore, there is a growing demand of investigating the mechanical characteristics of materials, in particular in the nanoscale regime, for device applications. contact loading is a type of mechanical impact that many electronic materials experience during processing or application, there are several issues to be addressed. firstly, the mechanical responses of materials to an applied load might be vastly different from that of the same bulk ones. for this purpose, unfortunately, the traditional methods such as tensile measurements do not scale well into the micrometer- and nanometer - regions. secondly, the role of structural changes under contact loading are largely underestimated owing to the difficulties in probing the structural characterizations of materials affected by the contact interaction directly. in this respect, depth - sensing indentation (nanoindentation) has proven to be a powerful technique in providing information on mechanical properties (hardness and elastic modulus) of materials and, variation of these properties with the penetration depth, based on the analysis of the respective load - displacement curves [1 - 6 ] while also producing contact - induced damage. while diamond anvil cell (dac) experiments are capable of investigating the mechanical and phase transformation in bulk materials under hydrostatic pressure, the materials behavior under nanoindentation is of more relevance to realistic contact loading conditions. in fact, the load - displacement curves obtained during nanoindentation can be viewed as for example, the onset for dislocation slip or twinning event in inp and gaas and, the solid - state phase transformation in si have been associated with the discontinuities during nanoindentation. for gan thin films, bradby. [10 - 12 ] proposed the mechanical deformation behaviors during nanoindentation with the spherical indenter. during the nanoindentation of gan thin films, a discontinuity (so - called pop - in event) in the loading curve was observed, indicating that the main deformation mechanism appears to be the nucleation of slip. nevertheless, the point indenter induced microstructural changes have not received sufficiently attention yet. as a result, locations of the details of single - crystal si(100) and gan thin films microstructure via a nanoindentation with a berkovich diamond indenter have not been explored. herein, in this study, the deformation behaviors of single - crystal si(100) and metal - organic chemical - vapor deposition (mocvd)-deposited gan thin films under contact loading have been investigated using berkovich nanoindentation, followed by analysis using micro - raman spectroscopy and cross - sectional transmission electron microscopy (xtem) techniques, in order to understand the final structures of the indentation - induced transformation zones observed in experiments. two materials of single - crystal si(100) wafer with light boron doping (1 10 atoms / cm) and, gan thin films deposited on (0001)-sapphire substrates by using the metal - organic chemical vapor deposition (mocvd) method with an average thickness of about 2 m were used in our present experiments. the nanoindentation tests were performed on a nanoindenter mts nanoxpsystem (mts cooperation, nano instruments innovation center, tn, usa) with a diamond pyramid - shaped berkovich - type indenter tip (face angle 65.3), whose radius of curvature is 50 nm. for microstructure analyses, a 10 5 indent array with each indent separated by 100 m was produced by holding at the peak load of 200 mn for 30 s with the same loading / unloading rates of 0.5 mn / s and 10 mn / s for single - crystal si(100) and gan thin films, respectively. the materials residual impressions produced at an indentation load of 200 mn were examined by a micro - raman spectrometer (renishaw, uk) with an arlaser (excitation wavelength 514.5 nm). the size of the laser spot is about 1 m, smaller than the dimension of impressions 5 m. in the raman experiments, a low laser power of 2 mw was used to avoid any possible artifacts from the center of the residual impressions as determined by optical microscopy. the cross - sectional transmission electron microscopy (xtem) samples were prepared by means of a fei nova 220 dual - beam workstation focused ion beam (fib)/scanning electron microscopy (sem) system. this technique enabled us to cut through the nanoindentation and locate the specific site of interesting efficiently. in practice, we first milled two crosses alongside the indented area for markers and, then deposited a 1 m - thick pt layer to protect this area of interest from gaion beam damage and implantation. material was removed from both sides of the selected area with an ion current of 5 na, followed by successive thinning steps with decreasing current ranging from 3 na to 300 pa until the lamella was about 1 m - thick. subsequently, the bottom and one side of the lamella were cut free while titling the sample at an angle of 45 to the ion beam. a central area containing the nanoindentation apex of a few micrometers in length was then chosen and thinned further to a thickness of 100 nm, leaving at the sides thicker areas that prevented the lamella from collapsing. finally, a small area of interest was selected and thinned until electron transparency was achieved. the transfer of the lamella from the sample holder to a holey carbon coated tem grid was made ex situ by using a shape glass tip under an optical microscope outside fib station. a jeol-2010 tem operated at an accelerating voltage of 200 kv was used to study the microstructures of xtem lamella. silicon (si - i) is a technologically very important material and is also of considerable scientific interest for its electrical, mechanical structural and, optical characterizations. in the past four decades there have been a significant number of investigations of the structural phase transformations of si when it is subjected to sufficiently high hydrostatic or non - hydrostatic pressures. it is well accepted from dac high pressure studies that si transforms from the cubic diamond phase (si - i) to the metallic -sn phase (si - ii) at increased pressures. during pressure release si - ii further transforms into several metastable phases including amorphous silicon, body - centered - cubic si - iii phase, rhombohedral distortion si - xii phase and, hexagonal diamond phase si - iv. these pressure - induced phase transitions can also be achieved by indentation tests [9,17 - 21 ]. in addition, it has been demonstrated that the microstructures of si after indentation with a spherical indenter depends on the maximum indentation load, loading / unloading rate and, number of applied stress cycles. and, a larger indentation load endorses crystalline phase transformation, while a high loading / unloading indentation rate promotes an amorphous phase. since phase transformations significantly affect the electrical, optical and mechanical characteristics of machined surface, the machining processes also have important implications for the manufacture of si substrates, microelectromechanical systems and, microelectronics devices. nevertheless, the berkovich indenter induced microstructural changes have not received sufficient attention. moreover, the plan - view tem analyses can not distinguish the phase changes inside the deformation region along the vertical direction. consequently, in this section, we will use the micro - raman spectroscopy and cross - sectional view tem techniques to clarify this problem mainly. figure 1 shows a typical indentation load - displacement curve of single - crystal si(100) subjected to a maximum indentation load of 200 mn, corresponding to different phase transformations as suggested by bradby.. the sudden displacement discontinuities, the pop - ins and pop - out phenomena, were observed in the loading and unloading part, respectively. association of pop - in events with the onset of si - i to si - ii phase transformation was reported recently in, which suggested that phase transformation begins at earlier stages of loading and pop - in is simple a manifestation of the sudden extrusion of highly plastic transformed materials from underneath the indenter. and, there is agreement with the previous study that upon unloading, the formation of si - iii and si - xii is evidenced by pop - out event. this is supported by the results of phase characteristics within the residual indents, carried out primarily by using of raman spectroscopy and tem. in addition, it can be found that only one major subsequent pop - in occurs during the indentation loading curve, there are obviously three cracking events along the corner of residual indentation (please see sem micrograph in the insert of fig., the cause of the subsequent pop - in event is attributed to the berkovich indentation induced cracking on si surface. load - displacement data for single - crystal si(100) obtained during nanoindentation with a berkovich indenter showing two pop - in events during loading and, one pop - out, the inset is a sem micrograph showing the indentation at an applied load of 200 mn figure 2 shows the micro - raman spectra obtained from an indentation load of 200 mn presented in fig. 2, the raman spectra from a 200 mn nanoindentation on si clearly reveal the additional bands at 160, 184, 350, 390, 433 and 486 cm, commonly associated with the si - iii and si - xii phases. the formation region of si - iii and si - xii phases in center and corner of indentation is found to be much stronger than that in edge one, suggesting that the magnitude of shear stress in the central part is higher. shear stress produced by indentation also plays a crucial role in determining which phases are formed. as the metastable phases of si - iii and si - xii can be formed only via a metallic si - ii phase, this observation suggests pressure - induced metallization of si during nanoindentation similar to the results of high - pressure cell experiments. raman spectra taken from the berkovich indentation of single - crystal si(100) at the corner, the center and near edge of indent. because of the nanoindentation - induced phase transformations, there are crystalline metastable phases present. (the symbols and are denoted as si - iii and si - xii phases) figure 3 shows a xtem bright - field image of a 200 mn indent in single - crystal si(100). characteristics 2, which is thought to be associated with a phase transformation. an amorphous phase is obvious in the upper part of the zone. nevertheless, crystalline phases are located in the central part at the bottom of the transformation zone. at the tip of the nanoindentation - induced transformed zone a crack which extends below the surface material from the transformed zone appears to have been extruded into the top of the crack, which is consistent with the formation of a ductile metallic phase under loading. in addition, a selected area diffraction (sad) of the region immediately beneath the residual indent (shown as an insert to this figure) shows that the nanoindentation - induced transformed zone is a mixture of amorphous and, some crystal materials (which are consistent with results from the previous study as arising from the metastable phases of si - iii and si - xii). the location of the crystalline phases is different from those formed in the previous work with the spherical indenter. also, the major difference between these two microstructures is the median crack that is formed under the indent made by berkovich indenter whereas no cracking was observed in the indent subject to the spherical indentation. the bright - field xtem image in the vicinity immediately under the berkovich indent applied on single - crystal si(100) with an indentation load of 200 mn in closing, we have made on indentation in single - crystal si(100) to track the transformation of the metastable phases of si - iii and si - xii using micro - raman spectroscopy in combination with xtem techniques. multiple pop - ins and pop - out events on si have been reported ; the cause of the pop - ins is not clear at this time, but the pop - out is ascribed to the reason of phase transformation. micro - raman spectroscopy demonstrated its ability to detect phase changes beneath the si surface, giving different signature at different location surrounding the indentation. the extra raman bands from the metastable phases of si - iii and si - xii are clearly visible in the continuous load - unload cycle, consistent with the xtem observations. gan, a iii v wide - band - gap semiconductor, has received a great deal of attention in the recent years due to its potential for the realization of photonic devices such as laser and light emitting diodes (leds) operating in the ultraviolet portion of the electromagnetic spectrum as well as solar - blind photodetectors. its wide band gap, high breakdown field and, high electron saturation velocity also make it as an attractive candidate for the development of electronic devices operating at high temperature, high power and high frequency relative to other competing materials such as si and gaas. consequently, majority of researches on this compound have been focused on exploring its optoelectronic characteristics. however, due to the ubiquitously existent lattice mismatch - induced stress between gan thin films and the available substrates, the resultant defects have been found to significantly affect the threshold power density in stimulated emission of gan optoelectronic devices. therefore, it is becoming increasingly evident that research on the mechanical characteristics of gan thin films is important to make gan thin films to be a good candidate for electronic devices. in this work, figure 4 displays the typical indentation load - displacement curve of gan thin films subjected to a maximum indentation load of 200 mn. during loading, prominent multiple pop - ins, or sudden displacement excursions it can be found that the first apparent pop - in occurs at an indentation load about 80 mn. subsequently, the multiple pop - ins are randomly distributed on the loading curve. according to the previous studies, we note here that the critical applied indentation load for direct identification of the multiple pop - ins in the load - displacement curve is not only dependent on the type of indenters used, but also even very much dependent on the test systems and the maximum applied indentation loads used. thus, we reasonably deduce that these discrepancies are mainly due to the various indentation methods used. for example, the tip - surface contact configuration and stress distribution for the berkovich indenter tip can be drastically different from that for the spherical tip or vickers - type indenters ;. load - displacement data for gan thin film obtained during nanoindentation with a berkovich indenter showing multiple pop - ins in addition, the inset is a sem micrograph showing the indentation at an applied load of 200 mn in addition, the multiple pop - ins behavior has been observed in materials with hexagonal structures such as sapphire, gan and single - crystal bulk zno, while for materials like inp and gaas with the cubic structure only single pop - in event was observed. nevertheless, the above discussions do suggest that multiple pop - ins indeed are specific features of materials with the hexagonal lattice structure and, the geometry of the indenter tip may play an important role in determining the nanoindentation - induced mechanical responses. thus, in order to identify the deformation mechanisms specific to the berkovich nanoindentation direct microstructure characteristics in the vicinity of the indented area are needed. 4 displays the typical sem micrograph for an indented surface obtained with the maximum applied indentation load of 200 mn. there is no evidence of dislocation activity or crack formation in the area of indented surface. thus, if the dislocation nucleation and subsequent propagation are indeed the primary mechanism for the observed multiple pop - ins, it should prevail underneath the indented surface. it is also interesting to check if there is any pressure - induced phase transformation involved. at the ambient conditions, gan tends to crystallize into the wurtzite structure. however, theoretical studies, which have been confirmed experimentally, have predicted that, upon applying a hydrostatic pressure on the order of about 50 gpa, gan will undergo the pressure - induced phase transformation into the rocksalt structure. these values are significantly higher than the apparent room - temperature hardness of gan thin films and the maximum load employed in this study. as will be presented in the followings, we used the micro - raman spectroscopy and xtem techniques in trying to clarify some of the issues concerning the nanoindentation - induced phase transformation in gan thin films. the micro - raman spectra for berkovich indenter operated at an indentation load of 200 mn are illustrated in fig. one before nanoindentation and the other two taken at different positions (corner and center of indent) after nanoindentation. the characteristic features of and peaks, locating, respectively, at 568 cm and 733 cm are clearly observed in the pristine gan thin film. as is evident from fig. 5, both and modes are shifted to the higher wavenumbers after berkovich nanoindentation. the fact that the peak displacement is largest at the center of the indented area and decreases outward indicates that the compressive stresses might be the dominant factors. we note that puech. also reported the similar shifts in micro - raman results taken from point - indentation with an indentation load of 100 mn and finally, no extra peaks were observed in our micro - raman spectra from nanoindentation, indicating that no phase transition in the material has occurred. also, the sem image of the same indentation area displayed in the inset of fig. we suspect that, in the film - substrate system, the indentation load applied to the film may have been partly absorbed by the substrate and distributed over a much larger area. consequently, the local stress concentration beneath the indenter is significantly reduced to values insufficient for phase transformation to occur. raman spectra of gan thin film taken on the pristine surface and after nanoindentation (at the corner and center of indent). changes in raman spectra after indentation, though displaying the effects of compressive stress, do not show clear evidence of phase transformation. 4 shows the sem micrograph of the same area after the berkovich indentation on gan thin film obtained at an indentation load of 200 mn. and, no cracking is evident to be responsible for the multiple pop - ins observed in the load - displacement curves to further elucidate the nanoindentation - induced deformation, a bright - field xtem image of an indentation load of 200 mn in gan thin films is displayed in fig. the image clearly displays that, within the film, the deformation features underneath the indented spot are primarily manifested by dislocation activities. namely the slip bands are well aligned in parallel with the { 0001 } basal planes all the way down to the film - substrate interface. moreover, the picture clearly displays a typical microstructure of a heavily deformed material, characterized by features of very high density of dislocations. nevertheless, the slip bands (dark thick lines in the photograph) clearly indicate that during the indentation the rapidly increasing dislocations can glide collectively along the easy directions. in the present case, in addition to those aligning parallel to the gan - sapphire interface along the (0001) basal planes, slip bands oriented at 60 to the sample surface can also be found. the 60 slip bands, which are believed to originate from dislocations gliding along the pyramidal planes, however, distribute in much shallower regions near the contacting surface. it is indicative that much higher stress level is needed to activate this slip system as compared to the one along the basal planes. from fig. 6, it can be seen that a more detailed microstructure near the intersections of the two sets of slip bands. the distorted slip bands and the extremely high dislocation densities at the intersections indicate highly strained state of the material. however, even at the submicron scale, no evidence of subsurface cracking and film fragmentation was observed. in addition, the selected area diffraction (not shown here) of the heavily damaged regions did not shown evidence of newly formed phases either. the bright - field xtem image in the vicinity immediately under the berkovich indent applied on gan thin film with an indentation load of 200 mn in closing, it is apparent that, in the berkovich indentation scheme, the primary deformation mechanism for gan films is dislocation nucleation and propagation along easy slip systems, similar to that concluded with spherical indenter. since the multiple pop - ins are usually observed after permanent plastic deformation has occurred (80 mn in the present case) and two of the possible mechanisms, the deformation - induced phase transformation and fracture of thin films were basically ruled out, the most likely mechanism responsible for the multiple pop - ins appears to be associated with the activation of dislocation sources. in this scenario, plastic deformation prior to the pop - in event is associated with the individual movement of a small number of newly nucleated and pre - existing dislocations. as the number of dislocations is increased and entangled to each other, large shear stress is quickly accumulated underneath the indenter tip. when the local stress underneath the tip reaches some threshold level, a burst of collective dislocation movement on the easy slip systems is activated, leading to a large release of local stress and a pop - in event on the load - displacement curve. each of these collective dislocation movements is reflected as a slip band in the indented microstructure displayed in fig. finally, we note that the so - called slip - stick behavior, characterized by material pile - ups caused by interactions between the as - grown defects and the indentation - induced dislocations, is not significant in this study. whether it is due to the insignificant grown - in defect density of our gan films or is related to the specific geometric shape of the indenter tip used is not clear at present and further studies may be required to clarify this issue. silicon (si - i) is a technologically very important material and is also of considerable scientific interest for its electrical, mechanical structural and, optical characterizations. in the past four decades there have been a significant number of investigations of the structural phase transformations of si when it is subjected to sufficiently high hydrostatic or non - hydrostatic pressures. it is well accepted from dac high pressure studies that si transforms from the cubic diamond phase (si - i) to the metallic -sn phase (si - ii) at increased pressures. during pressure release si - ii further transforms into several metastable phases including amorphous silicon, body - centered - cubic si - iii phase, rhombohedral distortion si - xii phase and, hexagonal diamond phase si - iv. these pressure - induced phase transitions can also be achieved by indentation tests [9,17 - 21 ]. in addition, it has been demonstrated that the microstructures of si after indentation with a spherical indenter depends on the maximum indentation load, loading / unloading rate and, number of applied stress cycles. and, a larger indentation load endorses crystalline phase transformation, while a high loading / unloading indentation rate promotes an amorphous phase. since phase transformations significantly affect the electrical, optical and mechanical characteristics of machined surface, the machining processes also have important implications for the manufacture of si substrates, microelectromechanical systems and, microelectronics devices. nevertheless, the berkovich indenter induced microstructural changes have not received sufficient attention. moreover, the plan - view tem analyses can not distinguish the phase changes inside the deformation region along the vertical direction. consequently, in this section, we will use the micro - raman spectroscopy and cross - sectional view tem techniques to clarify this problem mainly. figure 1 shows a typical indentation load - displacement curve of single - crystal si(100) subjected to a maximum indentation load of 200 mn, corresponding to different phase transformations as suggested by bradby.. the sudden displacement discontinuities, the pop - ins and pop - out phenomena, were observed in the loading and unloading part, respectively. association of pop - in events with the onset of si - i to si - ii phase transformation was reported recently in, which suggested that phase transformation begins at earlier stages of loading and pop - in is simple a manifestation of the sudden extrusion of highly plastic transformed materials from underneath the indenter. and, there is agreement with the previous study that upon unloading, the formation of si - iii and si - xii is evidenced by pop - out event. this is supported by the results of phase characteristics within the residual indents, carried out primarily by using of raman spectroscopy and tem. in addition, it can be found that only one major subsequent pop - in occurs during the indentation loading curve, there are obviously three cracking events along the corner of residual indentation (please see sem micrograph in the insert of fig., the cause of the subsequent pop - in event is attributed to the berkovich indentation induced cracking on si surface. load - displacement data for single - crystal si(100) obtained during nanoindentation with a berkovich indenter showing two pop - in events during loading and, one pop - out, the inset is a sem micrograph showing the indentation at an applied load of 200 mn figure 2 shows the micro - raman spectra obtained from an indentation load of 200 mn presented in fig. 2, the raman spectra from a 200 mn nanoindentation on si clearly reveal the additional bands at 160, 184, 350, 390, 433 and 486 cm, commonly associated with the si - iii and si - xii phases. the formation region of si - iii and si - xii phases in center and corner of indentation is found to be much stronger than that in edge one, suggesting that the magnitude of shear stress in the central part is higher. shear stress produced by indentation also plays a crucial role in determining which phases are formed. as the metastable phases of si - iii and si - xii can be formed only via a metallic si - ii phase, this observation suggests pressure - induced metallization of si during nanoindentation similar to the results of high - pressure cell experiments. raman spectra taken from the berkovich indentation of single - crystal si(100) at the corner, the center and near edge of indent. because of the nanoindentation - induced phase transformations, there are crystalline metastable phases present. (the symbols and are denoted as si - iii and si - xii phases) figure 3 shows a xtem bright - field image of a 200 mn indent in single - crystal si(100). characteristics 2, which is thought to be associated with a phase transformation. an amorphous phase is obvious in the upper part of the zone. nevertheless, crystalline phases are located in the central part at the bottom of the transformation zone. at the tip of the nanoindentation - induced transformed zone a crack which extends below the surface material from the transformed zone appears to have been extruded into the top of the crack, which is consistent with the formation of a ductile metallic phase under loading. in addition, a selected area diffraction (sad) of the region immediately beneath the residual indent (shown as an insert to this figure) shows that the nanoindentation - induced transformed zone is a mixture of amorphous and, some crystal materials (which are consistent with results from the previous study as arising from the metastable phases of si - iii and si - xii). the location of the crystalline phases is different from those formed in the previous work with the spherical indenter. also, the major difference between these two microstructures is the median crack that is formed under the indent made by berkovich indenter whereas no cracking was observed in the indent subject to the spherical indentation. the bright - field xtem image in the vicinity immediately under the berkovich indent applied on single - crystal si(100) with an indentation load of 200 mn in closing, we have made on indentation in single - crystal si(100) to track the transformation of the metastable phases of si - iii and si - xii using micro - raman spectroscopy in combination with xtem techniques. multiple pop - ins and pop - out events on si have been reported ; the cause of the pop - ins is not clear at this time, but the pop - out is ascribed to the reason of phase transformation. micro - raman spectroscopy demonstrated its ability to detect phase changes beneath the si surface, giving different signature at different location surrounding the indentation. the extra raman bands from the metastable phases of si - iii and si - xii are clearly visible in the continuous load - unload cycle, consistent with the xtem observations. gan, a iii v wide - band - gap semiconductor, has received a great deal of attention in the recent years due to its potential for the realization of photonic devices such as laser and light emitting diodes (leds) operating in the ultraviolet portion of the electromagnetic spectrum as well as solar - blind photodetectors. its wide band gap, high breakdown field and, high electron saturation velocity also make it as an attractive candidate for the development of electronic devices operating at high temperature, high power and high frequency relative to other competing materials such as si and gaas. consequently, majority of researches on this compound have been focused on exploring its optoelectronic characteristics. however, due to the ubiquitously existent lattice mismatch - induced stress between gan thin films and the available substrates, the resultant defects have been found to significantly affect the threshold power density in stimulated emission of gan optoelectronic devices. therefore, it is becoming increasingly evident that research on the mechanical characteristics of gan thin films is important to make gan thin films to be a good candidate for electronic devices. in this work, figure 4 displays the typical indentation load - displacement curve of gan thin films subjected to a maximum indentation load of 200 mn. during loading, prominent multiple pop - ins, or sudden displacement excursions it can be found that the first apparent pop - in occurs at an indentation load about 80 mn. subsequently, the multiple pop - ins are randomly distributed on the loading curve. according to the previous studies, we note here that the critical applied indentation load for direct identification of the multiple pop - ins in the load - displacement curve is not only dependent on the type of indenters used, but also even very much dependent on the test systems and the maximum applied indentation loads used. thus, we reasonably deduce that these discrepancies are mainly due to the various indentation methods used. for example, the tip - surface contact configuration and stress distribution for the berkovich indenter tip can be drastically different from that for the spherical tip or vickers - type indenters ;. load - displacement data for gan thin film obtained during nanoindentation with a berkovich indenter showing multiple pop - ins (arrows) during loading. in addition, the inset is a sem micrograph showing the indentation at an applied load of 200 mn in addition, the multiple pop - ins behavior has been observed in materials with hexagonal structures such as sapphire, gan and single - crystal bulk zno, while for materials like inp and gaas with the cubic structure only single pop - in event was observed. nevertheless, the above discussions do suggest that multiple pop - ins indeed are specific features of materials with the hexagonal lattice structure and, the geometry of the indenter tip may play an important role in determining the nanoindentation - induced mechanical responses. thus, in order to identify the deformation mechanisms specific to the berkovich nanoindentation direct microstructure characteristics in the vicinity of the indented area are needed. 4 displays the typical sem micrograph for an indented surface obtained with the maximum applied indentation load of 200 mn. there is no evidence of dislocation activity or crack formation in the area of indented surface. thus, if the dislocation nucleation and subsequent propagation are indeed the primary mechanism for the observed multiple pop - ins, it should prevail underneath the indented surface. it is also interesting to check if there is any pressure - induced phase transformation involved. at the ambient conditions however, theoretical studies, which have been confirmed experimentally, have predicted that, upon applying a hydrostatic pressure on the order of about 50 gpa, gan will undergo the pressure - induced phase transformation into the rocksalt structure. these values are significantly higher than the apparent room - temperature hardness of gan thin films and the maximum load employed in this study. as will be presented in the followings, we used the micro - raman spectroscopy and xtem techniques in trying to clarify some of the issues concerning the nanoindentation - induced phase transformation in gan thin films. the micro - raman spectra for berkovich indenter operated at an indentation load of 200 mn are illustrated in fig. one before nanoindentation and the other two taken at different positions (corner and center of indent) after nanoindentation. the characteristic features of and peaks, locating, respectively, at 568 cm and 733 cm are clearly observed in the pristine gan thin film. as is evident from fig. 5, both and modes are shifted to the higher wavenumbers after berkovich nanoindentation. the fact that the peak displacement is largest at the center of the indented area and decreases outward indicates that the compressive stresses might be the dominant factors. we note that puech. also reported the similar shifts in micro - raman results taken from point - indentation with an indentation load of 100 mn and finally, no extra peaks were observed in our micro - raman spectra from nanoindentation, indicating that no phase transition in the material has occurred. also, the sem image of the same indentation area displayed in the inset of fig. we suspect that, in the film - substrate system, the indentation load applied to the film may have been partly absorbed by the substrate and distributed over a much larger area. consequently, the local stress concentration beneath the indenter is significantly reduced to values insufficient for phase transformation to occur. raman spectra of gan thin film taken on the pristine surface and after nanoindentation (at the corner and center of indent). changes in raman spectra after indentation, though displaying the effects of compressive stress, do not show clear evidence of phase transformation. 4 shows the sem micrograph of the same area after the berkovich indentation on gan thin film obtained at an indentation load of 200 mn. and, no cracking is evident to be responsible for the multiple pop - ins observed in the load - displacement curves to further elucidate the nanoindentation - induced deformation, a bright - field xtem image of an indentation load of 200 mn in gan thin films is displayed in fig. the image clearly displays that, within the film, the deformation features underneath the indented spot are primarily manifested by dislocation activities. namely the slip bands are well aligned in parallel with the { 0001 } basal planes all the way down to the film - substrate interface. moreover, the picture clearly displays a typical microstructure of a heavily deformed material, characterized by features of very high density of dislocations. nevertheless, the slip bands (dark thick lines in the photograph) clearly indicate that during the indentation the rapidly increasing dislocations can glide collectively along the easy directions. in the present case, in addition to those aligning parallel to the gan - sapphire interface along the (0001) basal planes, slip bands oriented at 60 to the sample surface can also be found. the 60 slip bands, which are believed to originate from dislocations gliding along the pyramidal planes, however, distribute in much shallower regions near the contacting surface. it is indicative that much higher stress level is needed to activate this slip system as compared to the one along the basal planes. from fig. 6, it can be seen that a more detailed microstructure near the intersections of the two sets of slip bands. the distorted slip bands and the extremely high dislocation densities at the intersections indicate highly strained state of the material. however, even at the submicron scale, no evidence of subsurface cracking and film fragmentation was observed. in addition, the selected area diffraction (not shown here) of the heavily damaged regions did not shown evidence of newly formed phases either. the bright - field xtem image in the vicinity immediately under the berkovich indent applied on gan thin film with an indentation load of 200 mn in closing, it is apparent that, in the berkovich indentation scheme, the primary deformation mechanism for gan films is dislocation nucleation and propagation along easy slip systems, similar to that concluded with spherical indenter. since the multiple pop - ins are usually observed after permanent plastic deformation has occurred (80 mn in the present case) and two of the possible mechanisms, the deformation - induced phase transformation and fracture of thin films were basically ruled out, the most likely mechanism responsible for the multiple pop - ins appears to be associated with the activation of dislocation sources. in this scenario, plastic deformation prior to the pop - in event is associated with the individual movement of a small number of newly nucleated and pre - existing dislocations. as the number of dislocations is increased and entangled to each other, large shear stress is quickly accumulated underneath the indenter tip. when the local stress underneath the tip reaches some threshold level, a burst of collective dislocation movement on the easy slip systems is activated, leading to a large release of local stress and a pop - in event on the load - displacement curve. each of these collective dislocation movements is reflected as a slip band in the indented microstructure displayed in fig. finally, we note that the so - called slip - stick behavior, characterized by material pile - ups caused by interactions between the as - grown defects and the indentation - induced dislocations, is not significant in this study. whether it is due to the insignificant grown - in defect density of our gan films or is related to the specific geometric shape of the indenter tip used is not clear at present and further studies may be required to clarify this issue. in conclusions, a combination of nanoindentation, micro - raman spectroscopy, fib and tem techniques has been carried out to investigate the contact - induced structural deformation behaviors in single - crystal si(100) and mocvd - deposited gan thin films. the micro - raman analysis, measured from the indented materials which had plastically deformed on loading, showing a phase transformation occurs in si whereas the results for gan thin films do not give sufficient evidence for phase transformations. by using the fib milling to accurately position the cross - section of the indented region, the xtem results demonstrate that the major plastic deformation were taking place through the indentation - induced metastable phases (si - iii and si - xii) and amorphous phase exhibited in si, and the propagation of dislocations displayed in gan thin films. results revealed that the primary indentation - induced deformation mechanism in gan thin films is nucleation and propagation of dislocations, rather than proposed stress - induced phase transformations or crack formations in si via berkovich nanoindentation. this work was partially supported by the national science council of taiwan, under grant no. | details of berkovich nanoindentation - induced mechanical deformation mechanisms of single - crystal si(100) and the metal - organic chemical - vapor deposition (mocvd) derived gan thin films have been systematic investigated by means of micro - raman spectroscopy and cross - sectional transmission electron microscopy (xtem) techniques. the xtem samples were prepared by using focused ion beam (fib) milling to accurately position the cross - section of the nanoindented area. the behaviors of the discontinuities displayed in the loading and unloading segments of the load - displacement curves of si and gan thin films performed with a berkovich diamond indenter tip were explained by the observed microstructure features obtained from xtem analyses. according to the observations of micro - raman and xtem, the nanoindentation - induced mechanical deformation is due primarily to the generation and propagation of dislocations gliding along the pyramidal and basal planes specific to the hexagonal structure of gan thin films rather than by indentation - induced phase transformations displayed in si. |
between june 1998 and december 1999, a total of 247 patients with new diabetic foot ulcers and without previous major amputation presented to the study center. they were included in this prospective study and followed up until 31 may 2011, or until death. seventy - nine patients (32.0%) were treated by the diabetes team of the study center for the whole observation period or until death, while the remaining patients received foot care in various other institutions. patients who were cared for at the study center continuously were invited for control at least every 3 months and treated by the same interdisciplinary team both as inpatients and outpatients when they had any new foot lesion. patients who continued their treatment at external institutions were contacted by the study center at least once yearly. they agreed to be contacted personally or allowed the investigators to obtain information on their outcomes from their relatives or their family physicians. a diabetologist and a diabetic care nurse assessed all patients initially and performed the follow - up visits. patient history included data on demographic characteristics, type and duration of diabetes, microvascular and macrovascular comorbidities, and smoking habits. the physical examination included objective evaluation for peripheral neuropathy and peripheral arterial disease (pad). protective sensation was assessed separately for each leg with the calibrated rydel - seiffer tuning fork and the 5.07 monofilament. loss of protective sensation as a result of neuropathy was presumed in the presence of insensitivity to the 5.07 monofilament or a vibration perception of 4/8 or below. pad was defined by an ankle - brachial pressure index (abi) < 0.9 with additional investigation by means of duplex ultrasonography or angiography. degree of severity of pad was subdivided according to measured abi and classified as mild (abi, 0.70.9), moderate (abi, 0.410.69), or severe (abi, 0.4) (9,10). patients with noncompressible arteries as a result of medial arterial calcification (abi, 1.31) in whom pad had been diagnosed by imaging techniques were analyzed as having not classified pad. ischemic heart disease was defined as the presence of a history of angina pectoris or myocardial infarction, any positive cardiac stress test result, or pathological signs on coronary angiography. history of stroke was assumed to be present with any event of neurologic deficiency, whether persistent or resolved. full definitions and related references have been published elsewhere (11). in accordance with an earlier publication from the same center, we defined chronic renal insufficiency (cri) by a serum creatinine concentration 1.5 mg / dl and dialysis as the continuous need for renal replacement therapy (advanced renal disease) (12). clinical and demographic data, as well as outcome data (healing, amputation, ulcer recurrence, death), were collected continuously according to a preset standardized protocol and sampled in a study database. the reported data concerned first major amputation and mortality rates among the studied individuals. the cause of death was established from clinical findings before death, from death certificates, or by autopsy. baseline variables were described depending on their distributions by means, sds, ranges, or frequency tables. time from study entry to first major limb amputation and time from study entry to death were evaluated separately as censored event times by kaplan - meier curves. patient data were censored at last observation. in case of the outcome first major amputation, death without major amputation potential risk factors or confounders for both outcomes were analyzed by fitting cox multiple regression models. the assumption of proportional hazards was assessed graphically by stratified kaplan - meier curves (not all shown). the following factors were included in the analysis as possible predictors or confounders : sex, age, smoking, living in a nursing home, living alone, diabetes type, diabetes duration, insulin treatment (yes vs. no), neuropathy, charcot foot syndrome, history of coronary heart disease, history of stroke, cri, dialysis, pad, minor amputation before inclusion, and first - ever foot lesion (yes vs. no). at first, with stepwise and backward variable selection (significance level for entry,10% ; significance level for remaining, 15%), the most relevant factors were selected. finally, two resulting main cox multiple regression models were fitted. instead of pad yes or no, the effect of nonselected covariates from above was estimated in some few cases by including this factor additionally in the final model, and interactions between pad and cri were investigated. all statistical tests were performed two - sided at a significance level of 5% if not stated otherwise. statistical analyses were calculated by the sas statistical software package (version 9.3 ; sas institute inc., cary, nc). a diabetologist and a diabetic care nurse assessed all patients initially and performed the follow - up visits. patient history included data on demographic characteristics, type and duration of diabetes, microvascular and macrovascular comorbidities, and smoking habits. the physical examination included objective evaluation for peripheral neuropathy and peripheral arterial disease (pad). protective sensation was assessed separately for each leg with the calibrated rydel - seiffer tuning fork and the 5.07 monofilament. loss of protective sensation as a result of neuropathy was presumed in the presence of insensitivity to the 5.07 monofilament or a vibration perception of 4/8 or below. pad was defined by an ankle - brachial pressure index (abi) < 0.9 with additional investigation by means of duplex ultrasonography or angiography. degree of severity of pad was subdivided according to measured abi and classified as mild (abi, 0.70.9), moderate (abi, 0.410.69), or severe (abi, 0.4) (9,10). patients with noncompressible arteries as a result of medial arterial calcification (abi, 1.31) in whom pad had been diagnosed by imaging techniques were analyzed as having not classified pad. ischemic heart disease was defined as the presence of a history of angina pectoris or myocardial infarction, any positive cardiac stress test result, or pathological signs on coronary angiography. history of stroke was assumed to be present with any event of neurologic deficiency, whether persistent or resolved. full definitions and related references have been published elsewhere (11). in accordance with an earlier publication from the same center, we defined chronic renal insufficiency (cri) by a serum creatinine concentration 1.5 mg / dl and dialysis as the continuous need for renal replacement therapy (advanced renal disease) (12). clinical and demographic data, as well as outcome data (healing, amputation, ulcer recurrence, death), were collected continuously according to a preset standardized protocol and sampled in a study database. the reported data concerned first major amputation and mortality rates among the studied individuals. the cause of death was established from clinical findings before death, from death certificates, or by autopsy. baseline variables were described depending on their distributions by means, sds, ranges, or frequency tables. time from study entry to first major limb amputation and time from study entry to death were evaluated separately as censored event times by kaplan - meier curves. patient data were censored at last observation. in case of the outcome first major amputation, death without major amputation potential risk factors or confounders for both outcomes were analyzed by fitting cox multiple regression models. the assumption of proportional hazards was assessed graphically by stratified kaplan - meier curves (not all shown). the following factors were included in the analysis as possible predictors or confounders : sex, age, smoking, living in a nursing home, living alone, diabetes type, diabetes duration, insulin treatment (yes vs. no), neuropathy, charcot foot syndrome, history of coronary heart disease, history of stroke, cri, dialysis, pad, minor amputation before inclusion, and first - ever foot lesion (yes vs. no). at first, each factor was included as independent variable in an univariate model. with stepwise and backward variable selection (significance level for entry,10% ; significance level for remaining, 15%), the most relevant factors were selected. finally, two resulting main cox multiple regression models were fitted. instead of pad yes or no, the effect of nonselected covariates from above was estimated in some few cases by including this factor additionally in the final model, and interactions between pad and cri were investigated. all statistical tests were performed two - sided at a significance level of 5% if not stated otherwise. statistical analyses were calculated by the sas statistical software package (version 9.3 ; sas institute inc., cary, nc). mean patient age at study inclusion was 68.8 10.9 years (range, 2591 years), mean diabetes duration was 15.7 10.5 years (range, 053 years), 58.7% of the patients were male, and the majority had type 2 diabetes (87.5%). neuropathy and pad were present at study initiation in 86.2% and 55.5% of the patients, respectively. forty - eight patients with pad (37.2%) met the definition for a severe stage of the disease in at least one leg. in 40 patients (16.2%), arteries were noncompressible as a result of medial arterial calcification. in eight patients with medial arterial calcification, pad could not be classified on the basis of abi. in nine patients (3.6%) twenty - nine patients (11.7%) had evidence of active or inactive charcot foot syndrome at the time of inclusion. fifty - eight patients (23.5%) fulfilled the criteria for cri or were receiving renal replacement therapy. fifty - two patients (21.1%) were active smokers (26.9% of the male patients and 12.8% of the female patients), and 94 patients (38.1%) were former smokers (51.7% of the male patients and 18.6% of the female patients). histories of a coronary event or stroke were reported by 51 (20.7%) and 54 (21.9%) of the subjects, respectively, without major differences by sex. demographic data, risk factors, and comorbidities of the study population the mean follow - up period was 5.7 4.4 years (range, 0.00313.2 years), including both survivors and patients who died during the study period. thirty - eight patients had a first major amputation during the follow - up period. the cumulative probabilities of a first major amputation were 8.7% (5.112.4%), 12.5% (8.016.9%), 15.9% (10.721.0%), and 22.3% (15.329.2%) at years 1, 3, 5, and 10, respectively (table 2). all except one of the affected patients had evidence of pad at inclusion in the study, and 51.4% had evidence of severe pad in the concerned extremity. in separate univariate cox regression models, age, dialysis, and pad were significant risk increasing factors, and neuropathy was a significant preventive factor (table 3). after taking pad into the model as the second variable, however, neuropathy no longer remained significant (hazard ratio [hr ], 0.62 ; p = 0.218). thus the seemingly protective effect of neuropathy was almost fully explained by the absence of pad. age, pad, dialysis, and smoking were selected by stepwise and backward selection in cox regression. age (hr per year, 1.05 [95% ci, 1.011.10 ] ; p = 0.023), being on dialysis at baseline (hr, 3.51 [95% ci, 1.0212.07 ] ; p = 0.046) and pad at baseline (35.34 [4.81259.79 ] ; p < 0.001) were the independent predictive variables for a first major limb amputation during follow - up (table 3, model 1). presence of mild pad at baseline increased the risk of a first major amputation 20-fold, moderate pad 34-fold, and severe pad 62-fold (table 3, model 2) relative to no pad. the hr between severe and mild pad was 3.13 (1.118.79 ; p = 0.03), and that between severe and moderate pad was 1.80 (0.843.86 ; p = 0.13). models including an additional interaction variable between pad and dialysis did not yield valid results because of the low number of dialysis cases. kaplan - meier curves corresponding to cumulative probabilities of the first major amputation stratified by pad classes present a visual representation of these results (fig. cumulative probabilities (with 95% ci) of first major limb amputation or death univariate analysis and cox multiple regression models of association between variables and major amputation or death a : relevance of the presence and severity of pad for the cumulative probability of a first major limb amputation. to avoid complexity, pad not classified because of medial arterial calcification is not shown (n = 8 ; only 1 event of first major limb amputation). the highest curve represents no pad, the second curve represents mild pad, the third curve represents moderate pad, and the lowest curve represents severe pad. b : relevance of the presence or absence of pad, advanced renal disease, or both combined for the cumulative probability of death. the highest curve represents no pad and no renal disease, the second curve represents renal disease and no pad, the third curve represents pad and no renal disease, and the lowest curve represents pad and renal disease. there was no statistical difference in the risk of a first major amputation during the follow - up period between patients who had undergone a minor amputation before inclusion in the study when this factor was included as an additional covariate (hr, 1.21 [95% ci, 0.562.59 ] ; p = 0.630) in model 1 of table 3. by 31 may 2011, a total of 174 of the subjects had died, including 83 (47.7%) from cardiac diseases, 17 (9.8%) after stroke, 18 (10.3%) from malignancies, 23 (13.2%) from renal complications, and 24 (13.8%) as a result of septic conditions. septic conditions as cause of death were more prevalent among patients who had cri or were receiving renal replacement therapy than among patients without obvious renal impairment (21.3 vs. 11.0%). the cumulative mortalities for the whole cohort at years 1, 3, 5, and 10 were 15.4% (10.920.0%), 33.1% (27.139.1%), 45.8% (39.452.2%), and 70.4% (64.576.4%), respectively. for patients with pad at baseline, the corresponding numbers were 21.9% (14.89.0%), 44.1% (35.552.7%), 58.8% (50.267.4%), and 81.0 (74.188.0), respectively (table 2). in separate univariate cox regression models, age, living in a nursing home, type 2 diabetes, history of coronary heart disease, history of stroke, cri, dialysis, and pad were significant risk increasing factors, the presence of charcot foot syndrome a significant preventive factor (table 3). age, sex, pad, cri, dialysis, and history of stroke were selected by stepwise and backward selection in cox regression. living in a nursing home was selected only by backward selection, was not significant (p = 0.116), and was not included in the final model. independent predictive variables for death were age (hr per year, 1.08 [95% ci 1.061.10 ] ; p < 0.001), male sex (1.65 [1.182.32 ] ; p = 0.004), cri (1.83 [1.252.66 ] ; p = 0.002), dialysis (6.43 [3.1413.16 ] ; p < 0.001), and pad (1.44 [1.051.98 ] ; p = 0.023) (table 3, model 3). when the classified pad variable was used, severe pad dominated the significant association of pad with the risk of death ; however, the overall p value for the class variable was not significant (p = 0.065). a significant interaction (p = 0.023) between pad (yes or no) and cri was concluded by adding an interaction variable in model 3 of table 3 (changed hrs [95% cis ] of the main variables, pad, 1.72 [1.201.44 ] ; cri, 3.25 [1.805.85 ] ; and interaction of pad and cri, 0.42 [0.200.89 ]). this means higher hrs of the pad - only and cri - only patients but lower hr than the corresponding product hr for patients with pad and cri (reference, no pad and no cri). kaplan - meier survival curves stratified by pad and renal disease are presented in fig. patients who had undergone a minor amputation for a previous foot lesion had no significant increase in probability of death during follow - up (hr, 1.25 [95% ci, 0.851.83 ] ; p = 0.258) (additional covariable in model 3 of table 3). mean patient age at study inclusion was 68.8 10.9 years (range, 2591 years), mean diabetes duration was 15.7 10.5 years (range, 053 years), 58.7% of the patients were male, and the majority had type 2 diabetes (87.5%). neuropathy and pad were present at study initiation in 86.2% and 55.5% of the patients, respectively. forty - eight patients with pad (37.2%) met the definition for a severe stage of the disease in at least one leg. in 40 patients (16.2%), arteries were noncompressible as a result of medial arterial calcification. in eight patients with medial arterial calcification, pad could not be classified on the basis of abi. in nine patients (3.6%) twenty - nine patients (11.7%) had evidence of active or inactive charcot foot syndrome at the time of inclusion. fifty - eight patients (23.5%) fulfilled the criteria for cri or were receiving renal replacement therapy. fifty - two patients (21.1%) were active smokers (26.9% of the male patients and 12.8% of the female patients), and 94 patients (38.1%) were former smokers (51.7% of the male patients and 18.6% of the female patients). histories of a coronary event or stroke were reported by 51 (20.7%) and 54 (21.9%) of the subjects, respectively, without major differences by sex. demographic data, risk factors, and comorbidities of the study population the mean follow - up period was 5.7 4.4 years (range, 0.00313.2 years), including both survivors and patients who died during the study period. thirty - eight patients had a first major amputation during the follow - up period. the cumulative probabilities of a first major amputation were 8.7% (5.112.4%), 12.5% (8.016.9%), 15.9% (10.721.0%), and 22.3% (15.329.2%) at years 1, 3, 5, and 10, respectively (table 2). all except one of the affected patients had evidence of pad at inclusion in the study, and 51.4% had evidence of severe pad in the concerned extremity. in separate univariate cox regression models, age, dialysis, and pad were significant risk increasing factors, and neuropathy was a significant preventive factor (table 3). after taking pad into the model as the second variable, however, neuropathy no longer remained significant (hazard ratio [hr ], 0.62 ; p = 0.218). thus the seemingly protective effect of neuropathy was almost fully explained by the absence of pad. age, pad, dialysis, and smoking were selected by stepwise and backward selection in cox regression. age (hr per year, 1.05 [95% ci, 1.011.10 ] ; p = 0.023), being on dialysis at baseline (hr, 3.51 [95% ci, 1.0212.07 ] ; p = 0.046) and pad at baseline (35.34 [4.81259.79 ] ; p < 0.001) were the independent predictive variables for a first major limb amputation during follow - up (table 3, model 1). presence of mild pad at baseline increased the risk of a first major amputation 20-fold, moderate pad 34-fold, and severe pad 62-fold (table 3, model 2) relative to no pad. the hr between severe and mild pad was 3.13 (1.118.79 ; p = 0.03), and that between severe and moderate pad was 1.80 (0.843.86 ; p = 0.13). models including an additional interaction variable between pad and dialysis did not yield valid results because of the low number of dialysis cases. kaplan - meier curves corresponding to cumulative probabilities of the first major amputation stratified by pad classes present a visual representation of these results (fig. cumulative probabilities (with 95% ci) of first major limb amputation or death univariate analysis and cox multiple regression models of association between variables and major amputation or death a : relevance of the presence and severity of pad for the cumulative probability of a first major limb amputation. to avoid complexity, pad not classified because of medial arterial calcification is not shown (n = 8 ; only 1 event of first major limb amputation). the highest curve represents no pad, the second curve represents mild pad, the third curve represents moderate pad, and the lowest curve represents severe pad. b : relevance of the presence or absence of pad, advanced renal disease, or both combined for the cumulative probability of death. the highest curve represents no pad and no renal disease, the second curve represents renal disease and no pad, the third curve represents pad and no renal disease, and the lowest curve represents pad and renal disease. there was no statistical difference in the risk of a first major amputation during the follow - up period between patients who had undergone a minor amputation before inclusion in the study when this factor was included as an additional covariate (hr, 1.21 [95% ci, 0.562.59 ] ; p = 0.630) in model 1 of table 3. by 31 may 2011, a total of 174 of the subjects had died, including 83 (47.7%) from cardiac diseases, 17 (9.8%) after stroke, 18 (10.3%) from malignancies, 23 (13.2%) from renal complications, and 24 (13.8%) as a result of septic conditions. septic conditions as cause of death were more prevalent among patients who had cri or were receiving renal replacement therapy than among patients without obvious renal impairment (21.3 vs. 11.0%). the cumulative mortalities for the whole cohort at years 1, 3, 5, and 10 were 15.4% (10.920.0%), 33.1% (27.139.1%), 45.8% (39.452.2%), and 70.4% (64.576.4%), respectively. for patients with pad at baseline, the corresponding numbers were 21.9% (14.89.0%), 44.1% (35.552.7%), 58.8% (50.267.4%), and 81.0 (74.188.0), respectively (table 2). in separate univariate cox regression models, age, living in a nursing home, type 2 diabetes, history of coronary heart disease, history of stroke, cri, dialysis, and pad were significant risk increasing factors, the presence of charcot foot syndrome a significant preventive factor (table 3). age, sex, pad, cri, dialysis, and history of stroke were selected by stepwise and backward selection in cox regression. living in a nursing home was selected only by backward selection, was not significant (p = 0.116), and was not included in the final model. independent predictive variables for death were age (hr per year, 1.08 [95% ci 1.061.10 ] ; p < 0.001), male sex (1.65 [1.182.32 ] ; p = 0.004), cri (1.83 [1.252.66 ] ; p = 0.002), dialysis (6.43 [3.1413.16 ] ; p < 0.001), and pad (1.44 [1.051.98 ] ; p = 0.023) (table 3, model 3). when the classified pad variable was used, severe pad dominated the significant association of pad with the risk of death ; however, the overall p value for the class variable was not significant (p = 0.065). a significant interaction (p = 0.023) between pad (yes or no) and cri was concluded by adding an interaction variable in model 3 of table 3 (changed hrs [95% cis ] of the main variables, pad, 1.72 [1.201.44 ] ; cri, 3.25 [1.805.85 ] ; and interaction of pad and cri, 0.42 [0.200.89 ]). this means higher hrs of the pad - only and cri - only patients but lower hr than the corresponding product hr for patients with pad and cri (reference, no pad and no cri). kaplan - meier survival curves stratified by pad and renal disease are presented in fig. patients who had undergone a minor amputation for a previous foot lesion had no significant increase in probability of death during follow - up (hr, 1.25 [95% ci, 0.851.83 ] ; p = 0.258) (additional covariable in model 3 of table 3). the results of this study suggest that although long - term limb salvage in a modern series of diabetic foot patients is favorable, long - term patient survival still appears to be poor, especially among patients with pad, renal insufficiency, or the combination of both. to our knowledge, this is the first report in the medical literature that has examined both risk for major amputation and mortality in a primary data set of patients followed up for more than a decade. our cohort was remarkably similar in patient age and high prevalence of pad to other european cohorts studied for outcomes of diabetic foot disease over shorter (1315) or longer (5 years) (25,16) observation periods. in accordance with other studies, we observed a predominance of male patients in our cohort (58.7%) ; however, it was less pronounced than in some studies (35,13,1618). among the patients in our study, limb loss was observed almost exclusively in patients who had evidence of pad at study initiation. an independent association between pad and amputation has been found in other long - term studies as well (35) ; however, all those studies combined minor and major amputations rather than analyzing major amputation exclusively. in a study investigating a large cohort of diabetic foot patients (n = 1,088) treated at centers of excellence in 10 different european countries, the major amputation rate among patients with pad during a 12-month follow - up was 8%, compared with 2% among patients without pad (p < 0.001) (13). more than 50% of the individuals in our study who had a first major amputation had evidence of severe pad at baseline. the cumulative probability of a first major amputation was strongly linked to the severity of preexisting pad. none of the other three long - term studies provided clear stratification of severity of pad. (5), who followed 84 of 95 hospitalized diabetic foot ulcer patients (95%) for 6.5 years after hospital release, popliteal stenosis as a potential marker for more distal and probably more severe peripheral vascular disease (19) was the only independent predictor of amputation (relative risk, 2.67 [95% ci, 1.3410.07 ] ; p 0.01). in two other recent publications, severe pad (ankle pressure 50 mm hg or toe pressure 30 mm hg) was a predictor of increased major amputation risk in diabetic patients with neuroischemic or ischemic foot ulcers (14,20). being on renal replacement therapy at the time of inclusion (hr, 3.51 [95% ci, 1.0212.07 ] ; p = 0.046) was an independent predictive variable for a first major amputation during follow - up in our study. in the eurodiale (european study group on diabetes and the lower extremity) study, the presence of end - stage renal disease (defined as dependency on hemodialysis or peritoneal dialysis or a previous renal transplant procedure) was an independent predictor of lack of healing (odds ratio, 2.51 [95% ci, 1.414.48 ] ; p = 0.002), and that was true for patients with and without pad (13) ; however, major amputation in that analysis was not a specifically analyzed end point. (14), uremia was significantly associated with major amputation among patients with neuroischemic and ischemic ulcers (2.43 [1.334.45 ] ; p = 0.004) as well as with minor or major amputation among patients with neuropathic ulcers (2.62 [1.394.96 ] ; p = 0.003). (5) among 62 patients without previous amputation after multivariate analysis, only diabetic nephropathy remained as an independent predictor of first amputation (relative risk, 6.00 [95% ci, 1.6222.21 ] ; p < 0.01). authors of the other long - term studies either did not explore renal impairment as an influential factor for major amputation (4) or decided that a possible association with baseline variables would not be significant because of the small numbers of major amputation events in the study (3). excess mortality among patients with diabetic foot disease over observation periods of variable duration has been reported repeatedly during recent decades (25,7,8,1317,20,21). the published results are somewhat difficult to compare, however, because some studies describe death without healing of a distinct diabetic foot lesion as the end point (13,14,18), while others report cumulative mortality 1, 3, or 5 years after patient inclusion in the study (2,4,1618). death before healing of the initial ulcer occurred in 5.8% of our cases, which is almost identical to the results reported from the eurodiale study (6.0%) (13). somewhat higher frequencies were reported from sweden (14) and from the uk (18). (14) included patients who died with unhealed stumps after major amputation, however, while our study and the eurodiale consortium considered the major amputation event to be the definite end point. our 1-year mortality was in the same range as the data reported from nottingham (18) (15.9 and 16.7%, respectively), whereas our 5-year mortality was comparable to the reports of moulik. mortality data during a 10-year follow - up period have only been published in two medical reports so far. izumi. (7) studied 277 patients receiving amputations between 1993 and 1997, following them up for as long as 10 years (through 2003). they reported cerebrovascular, cardiovascular, and end - stage renal diseases as being strongly associated with death in those receiving distal amputations. for those receiving high - level amputations, only coronary artery disease was associated with increased mortality risk. additionally, people with high - level amputations were at greater risk of death than were those receiving low - level amputations. these results confirm the observation of poor survival after major amputation in patients with and without diabetes reported from studies with shorter follow - up periods (2224) ; however, the izumi. study (7) exclusively followed up patients who had received a lower limb amputation, not all patients had the opportunity to complete ten years of follow - up, and 10-year mortality was not stated. (8) used 19951997 data from the nord - trndelag health study (hunt 2) to evaluate mortality among people with diabetes who did or did not have a diabetic foot ulcer. people with a diabetic foot ulcer (n = 155) had a 2.3-fold greater risk for death relative to nondiabetic patients during the 10-year follow - up period (49.0% vs. 35.2%), with age, male sex, and smoking as significant covariate factors. the extent of mortality excess equals that observed after 3 years of follow - up in swedish primary healed diabetic foot ulcer patients (2.35-fold) but is considerably lower than the 3-year value for those with amputation in the same study (3.94-fold) (2). the 10-year cumulative mortality reported from the study of iversen (8) is also substantially lower than the number from our study (70.4%). the difference could be due to a possible underestimation of the mortality risk in hunt 2 explained by its design. it was stated by the authors that diabetic individuals who did not respond to the questionnaire on foot ulcers reported otherwise more advanced disease. in addition, recruitment procedures in that study made it difficult for housebound or institutionalized individuals to participate, which probably led to a reduced number of elderly people with a history of foot ulcers or amputation in that study relative to other studies (2,21). in addition to age (hr per year, 1.08), male sex (hr, 1.65 [95% ci, 1.182.32 ] ; p = 0.004), cri (hr, 1.83), being on dialysis (6.43), and pad (1.44) were independent predictive variables for death in our study. concerning cri, this result is in agreement with those of two other long - term studies that analyzed this variable : ghanassia. (5) found renal impairment (relative risk, 4.57 [95% ci, 1.119.4 ] ; p < 0.05) to be the only independent predictor of mortality in a multivariate analysis (5), and in the work of faglia. (3) renal impairment was a predictor of death in the univariate analysis (hr, 2.57 [95% ci, 1.225.41 ] ; p = 0.013) but was not confirmed in the multivariate analysis. (5) did not report a statistical relevance of pad as a predictor of death, whereas faglia found the independent association of an abi 0.5 (2.29 [1.294.08 ] ; p = 0.005) confirmed by multivariate analysis. this fits nicely with our finding, that severe pad (abi 0.4) dominates the significant association of pad with the risk of death when using the classified pad variables. the increased risk among death of patients with a history of diabetic foot ulcers and its association with a low abi has recently also been described for an asian population (25). in concordance with our own study, male sex was a significant predictor in the 10-year study done by iversen. although three of the other long - term studies did not report significant sex differences concerning mortality (2,4,5), faglia. (3) found female sex to be independently associated with death (hr, 1.96 [95% ci, 1.083.56 ] ; p = 0.027). because the discrepancy in age between male and female patients in that study was comparable to that among our patients (62.0 vs. 67.2 years and 66.5 vs. 72.0 years, respectively), this conflicting age - adjusted observation remains unexplained for the moment. more than half of the deaths in our study were from cardio- and cerebrovascular events. this observation is in full agreement with the information given in other european studies with long - term follow - up (25,8,16). in contrast, studies on mortality of diabetic foot ulcer patients in developing and newly developed countries report a substantially higher proportion of deaths from septic conditions (26). neuropathy in and of itself is known to be highly associated with cardiovascular mortality (2729). this could be explained most plausibly by two factors : 1) nearly all of our patients had clinically significant neuropathy on entry into the cohort, and 2) we did not specifically investigate cardiovascular mortality but rather all - cause mortality. the main strength of the study is the almost complete follow - up over a long observation period, as long as 13.2 years. only 20 patients (8.1%) were unavailable for follow - up before death or the end of the observation period. the comparability with other studies investigating the outcome of diabetic foot disease with regard to relevant demographic variables and risk factors may be a further advantage. a potential negative selection bias always has to be considered when cohorts from specialized diabetic foot care centers are analyzed. it is to be expected that a number of more superficial ulcers of neuropathic origin are treated successfully in primary health care without being seen by a specialized diabetic foot center. therefore our cohort should be typical for high - risk patients in specialized centers but not for the entire diabetic population. in addition, male predominance was less pronounced in our cohort than in some other reported studies. in conclusion, data from this long - term study suggest strongly that limb preservation today is the rule rather than the exception, even in high - risk patients with diabetes. long - term survival remains poor, however, probably because of myriad comorbid conditions for patients whose first presentation to an interdisciplinary clinic is with a diabetic foot ulcer. efforts to assess and capture these patients earlier in such a clinic may ultimately prove beneficial not only in preventing amputation but also in tangentially prolonging life. | objectivethere is a dearth of long - term data regarding patient and limb survival in patients with diabetic foot ulcers (dfus). the purpose of our study was therefore to prospectively investigate the limb and person survival of dfu patients during a follow - up period of more than 10 years.research design and methodstwo hundred forty - seven patients with dfus and without previous major amputation consecutively presenting to a single diabetes center between june 1998 and december 1999 were included in this study and followed up until may 2011. mean patient age was 68.8 10.9 years, 58.7% were male, and 55.5% had peripheral arterial disease (pad). times to first major amputation and to death were analyzed with kaplan - meier curves and cox multiple regression.resultsa first major amputation occurred in 38 patients (15.4%) during follow - up. all but one of these patients had evidence of pad at inclusion in the study, and 51.4% had severe pad [ankle - brachial pressure index 0.4 ]). age (hazard ratio [hr ] per year, 1.05 [95% ci, 1.011.10 ]), being on dialysis (3.51 [1.0212.07 ]), and pad (35.34 [4.81259.79 ]) were significant predictors for first major amputation. cumulative mortalities at years 1, 3, 5, and 10 were 15.4, 33.1, 45.8, and 70.4%, respectively. significant predictors for death were age (hr per year, 1.08 [95% ci, 1.061.10 ]), male sex ([1.182.32 ]), chronic renal insufficiency (1.83 [1.252.66 ]), dialysis (6.43 [3.1413.16 ]), and pad (1.44 [1.051.98]).conclusionsalthough long - term limb salvage in this modern series of diabetic foot patients is favorable, long - term survival remains poor, especially among patients with pad or renal insufficiency. |
taekwondo is a traditional korean martial art currently practiced in over 206 countries around the world1 and has high - profile educational and physical values2. due to its various kicking and efficient attacking techniques3, taekwondo has become a globalized sport and finally obtained full status in the official summer olympic competition program since the 2000 sydney olympics4. as a martial art sport, taekwondo is characterized by its emphasis on dynamic techniques for taking mobile stances, and agility, speed, flexibility, and endurance are required to perform the whole process efficiently5. with respect to fitness, taekwondo can provide participants with health benefits and a sound mind. despite its perceived physical usefulness for all ages, practicing taekwondo may cause various types of injuries because it involves intense full - contact sparring. active participation in sport is generally recognized as positive, but participation in any type of sports always has the possibility of injury5, 6. indeed, it is reported that several taekwondo - related activities cause accidental injuries owing to its attribute of contact7. because of the essential characteristics of martial arts, any collision or injury in taekwondo is considered natural. especially during full - contact sparring in taekwondo, owing to the physical demands and force imposed on the athlete, the risks of injury must be addressed8. according to previous research, some injury risks from practicing martial arts are unnecessary and preventable9, and are seen as critical health problems in western societies10. moreover, some kinds of injuries resulting from competitions can have a negative influence on participants in taekwondo who want to prolong the duration of their participation in the sport. thus, injuries in taekwondo could be considered important issues and should be treated as key factors to help improve conditions for exercising or competing in taekwondo. therefore, examining the types of injuries resulting from performing taekwondo and their locations in detail are useful for deciding how to cope with injuries and prevent participants from unwarranted injuries. also, even though there are some well - documented epidemiology injury profiles on taekwondo around the world11, because taekwondo takes a relatively long time to attain proficiency in various kicking and sparring techniques, few systematic studies have been conducted on the long - term perspective in korea. therefore, this study aimed to provide fundamental information on injuries in taekwondo by investigating the categories of injuries in taekwondo and determining their locations. the survey was conducted from march 5, 2014 to september 24, 2014. questionnaire forms were used to collect the data and were distributed to participants who regularly practiced taekwondo in gyeongnam province. all participants provided consent for data collection. while collecting the data, the purpose of this study was explicitly explained, and participants were asked whether they would like to take part in the research. the fact that any response to the questionnaire items would be confidential in all circumstances participants who had visited a medical institution more than thrice and were diagnosed with injuries resulting from taekwondo activities were included in the study. even though prospective participants experienced medically diagnosed injuries before, participants who visited medical care centers fewer than two times or did not receive a definite diagnosis were excluded. the form included items on age, gender, length of practice, injury diagnosis, and injury locations. on the questionnaire, the length of practice was categorized as follows : 1) less than 1 year since the beginning, 2) 1 through 3 years, and 3) more than 3 years. for the classification of injury diagnosis and locations, the criteria that kazemi. used in 2009 were adopted12. according to these criteria, injury diagnoses were categorized as contusions, sprains, strains, fractures, joint dysfunction, and concussion of the brain, and injury locations were divided into head, foot, thigh, knee, ankle, trunk, wrist, forearm, and other parts of the body. after completing the survey, all of the questionnaires were gathered and examined closely to exclude incorrectly completed forms. after 23 untrustworthy forms were excluded, 512 questionnaires were confirmed as the raw data sources. additionally, by using the multiple response method, the current status and types of injuries that occurred while practicing taekwondo were examined. the characteristics of the subjects are presented in table 1table 1.subject characteristics (n=512)categoryfrequency%age (years)10193516920291142230479gendermale27754female23546length of practice (years)36914. of 512 subjects, there were 277 males and 235 females with injuries recorded using a binary multiple response method. among the participants included, the top five most frequent locations of injury, in order of decreasing frequency, were the foot (n = 93), knee (n = 86), ankle (n = 80), thigh (n = 64), and head (n = 61). other minor locations of injury, such as the leg (n = 48), toes (n = 31), and back (n = 25) were also reported. taking a closer look at the injury diagnoses, the five major injuries were contusions (n = 319), strains (n = 89), sprains (n = 75), fractures (n = 51), and concussions (n = 50). in addition, a few cases of joint dysfunction (n = 47) and lacerations (n = 11) were also reported. the injury locations and diagnoses this study aimed to provide guidance on how to cope with injuries resulting from taekwondo participation by investigating the current status and types of injuries. from the analysis on injury locations, it was found that the vast majority of taekwondo participants experienced podiatric - related injuries. although the term taekwondo is described as the martial art that uses kicks and punches concurrently, and victory at competitions is acquired by getting higher points from the judges and by performing specific techniques including kicks and punches13, foot - related skills were preferred during sparring and demonstrations. however, because kick techniques are more powerful and more effective for scoring points than punches during competitive performances. most of the skills used in taekwondo are dependent on kick techniques to efficiently score points in competitions. in previous studies, it was reported that the vast majority of injuries in participating athletes in taekwondo were found on the lower extremities owing to the exchange of accurate and powerful turning kicks14, 15. furthermore, while conducting kicking techniques during competitions, taekwondo athletes often exchange kicks concurrently, which can result in lower extremity injuries. for athletes who highly use kicking to gain high scores, therefore, it is necessary to decrease the possibility of injury to the lower extremities to extend participation in taekwondo. other research has also cited that head or neck injuries are also prevalent in taekwondo16. therefore, it is necessary to address this issue while practicing taekwondo. the main diagnoses in the present study were contusions, strains, and sprains. taekwondo training generally consists of three sections, which can be divided into poomsae (the form that combines postures of the basic techniques of attack and defense), sparring, and breaking. participants involved in sparring or breaking seem most likely to experience these types of injuries. sparring is performed through fierce and speedy contact with the partner, therefore, contusions and sprains are common. while breaking objects, owing to contact with the object to break, athletes were more likely to experience injuries such as contusions and strains. previous research cites that regardless of experience levels in taekwondo, almost all participants experienced contusions and strains12. in general, these kinds of injuries are expected because martial arts such as taekwondo involve extremely intensive body contact. however, if ignored, these types of injuries could be threats to prolonged participation in taekwondo because the repetitive and sustained activities could worsen the injury. to improve and prevent this situation, establishment of injury surveillance systems focusing on taekwondo is required, and educational programs for taekwondo instructors or coaching staffs to protect athletes should be strengthened. | [purpose ] the present study aims to provide fundamental information on injuries in taekwondo by investigating the categories of injuries that occur in taekwondo and determining the locations of these injuries. [subjects and methods ] the data of 512 taekwondo athletes were collected. the sampling method was convenience sampling along with non - probability sampling extraction methods. questionnaire forms were used to obtain the data. [results ] the foot, knee, ankle, thigh, and head were most frequently injured while practicing taekwondo, and contusions, strains, and sprains were the main injuries diagnosed. [conclusion ] it is desirable to decrease the possibility of injuries to the lower extremities for extending participation in taekwondo. other than the lower extremities, injuries of other specific body parts including the head or neck could be important factors limiting the duration of participation. therefore, it is necessary to cope with these problems before practicing taekwondo. |
ischemia - reperfusion injury (iri) is a phenomenon whereby cellular damage in a hypoxic organ is accentuated following the restoration of oxygen delivery. in the liver, this form of injury is recognized as a clinically important prolonged disorder.1 liver iri occurs as a result of some liver surgeries, liver transplantation, hemorrhagic shock, and prolonged portal triad clamping followed by reperfusion performed as an elective preplanned procedure or as an emergent maneuver to control excessive bleeding from the cut hepatic surface. it may also be said that liver iri is a complex process involving numerous cell types and molecular mediators in various pathophysiological and biochemical ways. the end result is cell death via a combination of apoptosis and necrosis involving a complex web of interactions between the various cellular and humoral contributors to the inflammatory response of kupffer cells, also producing proinflammatory cytokines, tumor necrosis factor alpha (tnf-), interleukin (il)-1, lymphocytes, neutrophils, and hepatocytes.1,2 our knowledge regarding the mitochondria in generating reactive oxygen species (ros), nitrogen species, and ionic disturbances, and in initiating mitochondrial permeability transition with subsequent cellular death in iri is continuously growing. however, the most promising protective strategy against iri explored during the last few years is ischemic preconditioning (ip), which appears capable of increasing the resistance of liver cells to ischemia and reperfusion events. ip refers to brief periods of ischemia - reperfusion (i / r) followed by a prolonged one.3,4 the aim of the present study was to detect some of these mechanisms by subjecting rats to iri. all rats were treated in accordance with the guide for care and use of laboratory animals. the rats were kept at a constant room temperature under air conditioning at 25c for the duration of the study. the rats were divided into three groups (n=10 each) as follows : a sham operated control group (group 1) ; an i / r group in which the liver was rendered ischemic by portal triad occlusion with a small bulldog vascular clamp for 60 minutes followed by reperfusion for 3 hours (group 2) ; and an ip - i / r group in which animals were subjected to three cycles of 10 minutes of ischemia, each followed by 10 minutes of reperfusion prior to prolonged i / r, as in group 2 (group 3). at the end of the experiment, the animals were sacrificed by decapitation, and blood and liver samples were collected for analysis. the plasma was separated, and the liver was excised, de - encapsulated, washed with ice - cold saline, and then homogenized in phosphate - buffered saline. the following parameters were measured in plasma and/or liver homogenate : liver function tests, ie, alanine aminotransferase (alt)5 and aspartate aminotransferase (ast)5 oxidative stress parameters, ie, lipid peroxidation end products, including malondialdehyde (mda) using thiobarbituric acid,6 reduced glutathione (gsh),79 oxidized glutathione (gssg),79 and ratio of nitrite to nitrate in plasma (nox)10,11 antioxidant enzyme activity, ie, superoxide dismutase (sod)12 and glutathione peroxidase (gpx)13 protein concentration in liver homogenate14 proinflammatory cytokines, ie, il-1 and tnf-, by enzyme - linked immunosorbent assays (predicta ; genzyme, san diego, ca, usa) liver glycogen concentration.16 the data are presented as the mean standard error and were analyzed using two - way analysis of variance followed by the least significant difference test using statistical package for the social sciences version 20 software (ibm corporation, armonk, ny, usa). the data are presented as the mean standard error and were analyzed using two - way analysis of variance followed by the least significant difference test using statistical package for the social sciences version 20 software (ibm corporation, armonk, ny, usa). table 1 shows an increase in serum ast, alt, plasma il-1 and tnf-, and tissue il-1 and tnf- in the i / r group as compared with the sham operated control group. however, the ip group showed a decrease in all these parameters compared with the control and i / r groups, but plasma tnf- was increased compared with the control and tissue il-1 was increased when compared with the control group and tissue tnf- was increased when compared with the control and i / r groups. the significant results shown in table 1 show a decrease in ast in the i / r - ip group when compared with the i / r group. alt and plasma il-1 was decreased in the control and i / r groups whereas plasma tnf- was decreased when compared with the i / r - ip group. tissue tnf- was increased compared with all the other groups, ie, control, t / r, and ip. table 2 shows the oxidative stress parameters, including mda, gsh, gssg, and nox. md and nox were increased and gsh and gssg were decreased in the i / r group when compared with controls. mda was decreased in the ip group when compared with the control group and increased when compared with the i / r group. gssg was increased in groups 1 and 2, and nox was decreased compared with the i / r group and increased compared with the control group. in the ip - i / r group, mda and nox were decreased when compared with i / r, and gsh was increased as compared with the ip group and decreased when compared with the control group. table 3 shows that sod was increased significantly in the ip - i / r group when compared with the control, whereas gpx was decreased, but only gpx increased when compared with the i / r group, in which caspase-3 was decreased. ip (group 2) showed significantly increased caspase-3 compared with the control, but a decrease in gpx, while sod showed a significant change in the ip group, the i / r group showed a significant decrease in gpx and an increase in caspase-3 compared to the control. table 4 shows that there was a significant increase in glycogen phosphorylase in the i / r group compared with the control group. there was a significant decrease in glycogen synthase in the i / r group as compared with the control group. glycogen synthase was increased in the i / r - ip group as compared with the i / r group, as shown in table 5. there was a significant decrease in glycogen content in the i / r group compared with the control, and glycogen was significantly increased in the ir / pr group when compared with the i / r group (table 6). although the precise mechanisms by which ip reduces iri are not well understood, several factors have been reported to contribute to ip - mediated tissue protection.17 the protective effects of ip, a phenomenon by which a traumatic or stressful stimulus confers protection against subsequent injury, have been well documented in many organs, including the heart, brain, skeletal muscle, lung, intestine, kidney, retina, and endothelial cells.18 there is increasing evidence that cellular ischemic stressors activate protein kinase via g - protein - coupled receptor binding and membrane phospholipase activation.19 the signal transduction cascade for ip involves activation of protein kinase c, protein tyrosine kinase, and mitogen - activated protein kinase. however, it was found that protein kinase c inhibitors could attenuate the effects of ip induced by one cycle but not repetitive cycles in the heart. these data also suggest that repetitive ip may activate mechanisms other than the antioxidant system. we also examined its effect on the inflammatory response of i / r as exemplified by il-1 determination, in addition to its effects on the oxidant - antioxidant system.1921 increasing evidence has shown that proinflammatory cytokines and ros are both key mediators of liver iri.22 shortly after hepatic iri (16 hours), kupffer cells are activated and release proinflammatory cytokines, such as tnf- and il-1. these cytokines have a dual role : overexpression of tnf- and il-1 can induce more production of cytokines and granulocyte colony - stimulating factor, which enhances activation of kupffer cells and promotes infiltration of neutrophils into the microcirculation of the liver,23 thereby aggravating sterile hepatic inflammation after ischemia and reperfusion. on the other hand, tnf- and il-1 are indispensable for liver regeneration.24 measurement of mda levels is used widely as an indicator of lipid peroxidation. nitric oxide is also recognized as an important mediator of physiological and pathological processes in hepatic and renal iri.25 hepatic i / r can lead to damage and dysfunction (apoptosis) of liver parenchymal and sinusoidal cells. ip is extensively documented to reduce iri in a variety of organs including the liver,2628 and the caspase-3 enzyme is used as an indicator of apoptosis. this parameter was included in the present study to determine if ip can actually protect the liver from cell death. in this study, there was a significant increase in the serum activity of ast and alt in the i / r group when compared with corresponding values in the control group. this result were in agreement with those of wang and jaeschke and lemasters,29 who found that i / r resulted in a significant increase in ast and alt levels when compared with controls. ast and alt concentrations are commonly used as indirect biochemical indices of liver injury.28,29 studies have shown that increased ast and alt levels in iri probably result from cell membrane damage.30,31 on the other hand, the increase in ast and alt activity observed in the i / r group by lipid peroxidation leads to cytolysis, which is caused by oxygen free radicals formed during the reperfusion phase. there was a significant decrease in serum alt activity in the i / r - ip group when compared with corresponding values in the i / r group. these results are in agreement with those of romanque,32 who found that alt was strongly decreased in the ip - i / r group, with net reductions of 57% in serum alt level when compared with the i / r group. at the same time, kupffer cells and hepatocytes also generate ros, leading to direct damage of endothelial cells and hepatocytes. due to their important role, ros levels are tightly regulated through different pathways.33 the major regulators are ros scavengers that include sod, catalase, and glutathione peroxidase (gsh - px). these ros scavengers are responsible for reducing ros inside tissues. another type of oxidative stress regulation is mediated by nitric oxide (no), which is created by endothelial and inducible no synthases in the liver. no can regulate endothelial function, and its levels can affect blood flow in an organ. ros have been considered a major deleterious factor in reperfusion injury.34 in the present study, we found that liver i / r depleted glutathione stores in the liver and plasma. gsh is an important intracellular antioxidant that acts by directly scavenging ros and also by being a cofactor for gpx - catalyzed reactions that degrade hydrogen peroxide.35 depletion of gsh therefore renders cells susceptible to oxidative stress. in accordance with this, we found significant inhibition of gpx in the liver and plasma, resulting in excessive production of ros. this results in local oxidative stress on the liver and also a systemic oxidative state as indicated by increased levels of lipid peroxidation end products (mda) and decreased gssg. in this study, there was a significant increase in the hepatic activity of mda in the i / r group when compared with corresponding values in the control group. this finding is in agreement with that of wang,28 who found that the content of mda, a marker of liver oxidative injury, was significantly increased in the model group when compared with the control group. the results reported by yang show that mda levels in liver tissue were elevated significantly when treated with vehicle after brain i / r in rats, indicating that the function of the liver, although a remote organ, was damaged. lipid peroxidation is known to be responsible for cell membrane damage, and has been implicated in the pathogenesis of iri.22 accumulation of ros may easily overcome endogenous antioxidative systems, such as sod, catalase, gpx, and gsh, and because ros exist in relatively low concentrations in the liver, they have been proposed to be a contributory factor to cellular mechanisms of inflammation and iri.1,4 in the present study, it was found that the presented model of hepatic i / r was accompanied by a 254% increase in protein carbonyl content and a 37% decrease in hepatic gsh, with a net 481 enhanced protein carbonyl / gsh content ratio : sham - operated rats = 0.260.03 (n=23) versus i / r animals = 1.510.27 (n=6 ; p>0.0001). this pro - oxidant state was significantly attenuated by the ip maneuver, as evidenced by total recovery of the protein carbonyl content and an 89% net reduction in gsh depletion, when differences between the i / r and sham - operated groups were compared with those between ip - treated rats with and without i / r (p>0.05). on one hand, no can induce cellular cytotoxicity and tissue injury via the peroxynitrite formation, protein tyrosine nitration, lipid peroxidation, dna damage, and proapoptotic effects included in iri.37 on the other hand, no may have a protective effect in vasodilatation, antiapoptotic action, inhibition of platelet plug formation, and reduction of the inflammatory response.38 thus, the cellular effects of no may depend on its concentration, site of release, and duration of action. the discrepancy in the previously recorded results might be due to different levels of no production associated with the degree or method of iri.39 however, there is evidence that ip decreased no levels significantly after three cycles of i / r.39 in contrast with this finding, group 2 (i / r) showed a slight decrease in this value, which may be attributed to different conditions of induction of i / r. sod catalyzes dysmutation of the superoxide anion to hydrogen peroxide and oxygen, but hydrogen peroxide still produces liver oxidative injury, and gpx further catalyzes the transformation of hydrogen peroxide to form water.40 i / r reduces liver antioxidant capacity, as evidenced by the downregulated activity of gpx. however, our study revealed that i / r treatment did not significantly change sod, implying that i / r with its oxidant metabolites decreased the antioxidant capacity of sod enzyme. previous research also showed that ip increased the expression and activity of antioxidant enzymes in the ischemic kidney and liver,41 which is in accordance with the present study, where ip decreased mda and gssg concentrations and avoided depletion of gsh, sod and gsh - px in the ischemic kidney. ip also decreased mda and increased gsh in the heart, with no significant change in the other parameters in rats exposed to renal i / r. increased gsh and decreased mda and gssg in the plasma of renal i / r rats our data demonstrate that exposure of the rat liver to an i / p maneuver for 3 hours before the i / r protocol significantly diminishes hepatocellular injury, suggesting the development of a second or delayed phase of protection against iri. this ip was characterized by development of transient oxidative stress of a considerably smaller magnitude than that elicited by i / r. the ip mechanisms involved in hepatic iri as investigated in our study suggest that the protective effects of ip against hepatic iri are related to their role in reducing tissue oxidative stress levels. ip may regulate the activity of sod, gsh - px, nos, and ip, and offer additive protection by increasing gsh - px activity. it may also decrease cellular injury and promote cell survival by suppressing release of cytokines. as reported, ip has a similar physiological and cellular protection mechanism : they have the same catalytic substrate adenosine ; they transport bioinformation through the phosphatidylinositol 3-kinase / akt pathway ; and they can decrease cytokines release.35 another contributory factor to parenchymal inflammation in iri is enhanced tnf- generation by activated kupffer cells.42,43 accordingly, a 15.3-fold increase above that of sham operated rats was observed for serum tnf- levels in i / r animals in the present study. our study also showed a high increase in plasma il-1 and an increase in the liver of i / r rats was shown in other studies. our studies also indicated that ip suppressed the tnf- response in plasma and oxidative stress induced by i / r, as evidenced by normalization of i / r - induced gsh depletion and protein oxidation in the ip - i / r group. we also demonstrated liver protection by ip to be related to recovery of cellular signaling functions modified by the i / r protocol. these findings were found by other researchers to help upregulation of nuclear factor kappa beta and downregulation of activator protein 1 (ap-1) dna binding, with significant reduction in the ap-1/nuclear factor kappa beta dna binding ratio, which may afford liver protection by decreasing susceptibility to ros and tnf--induced liver injury. it was suggested that individual application of ip might reach the limit of decreasing cytokines release in rats. recent studies have hypothesized a role for tnf- and il-1 in liver regeneration.44 our data confirm that maintenance of tnf- and il-1 concentrations at a certain level might help recovery of hepatic function in the liver regeneration process. xanthine oxidase is capable of reducing molecular oxygen to both superoxide and hydrogen peroxide, and has been suggested to be the major source of ros metabolites generated during i / r. plasma xanthine oxidase activity was increased dramatically after liver and gut i / r.45 it has been shown that during i / r, sinusoidal cells die only via apoptosis while hepatocytes may die in both ways (necrosis and apoptosis). in sinusoidal cells, apoptosis occurs earlier and at a higher level than in hepatocytes, but the differences in extent of apoptosis between these cells tend to be improved after a prolonged reperfusion period.15 the present results also demonstrate improvement in caspase-3 levels by ip. reduction of iri has been associated with decreased serum aminotransferase, a finding which has also been confirmed in human models, especially in a younger population,27 and it has been shown that ip protects the liver by inhibiting apoptosis of sinusoidal endothelial cells. liver glycogen is the first and immediate source of glucose for the maintenance of blood glucose levels. in the liver, the glucose-6-phosphate generated from degradation of glycogen is hydrolyzed to glucose by glucose 6-phosphatase, an enzyme present only in the liver and kidneys. glycogen degradation thus provides a readily mobilized source of blood glucose.46 the principal enzymes controlling glycogen metabolism, ie, glycogen phosphorylase and glycogen synthase, are regulated by allosteric mechanisms and covalent modifications due to reversible phosphorylation and dephosphorylation of proteins in response to hormone action. in the present study, there was a significant decrease in hepatic glycogen activity in the i / r group as compared with corresponding values in the control group. on the other hand, there was a significant increase in hepatic glycogen activity in the i / r - ip group as compared with corresponding values in the i / r group. these results are in agreement with those of peralta,35 who found that the glycogen concentration decreased as a function of duration of ischemia. however, glycogen levels were always higher in preconditioned livers.47 glycogen concentration decreased as a function of duration of ischemia ; however, glycogen levels were always higher in preconditioned livers.46 in summary, the present study shows that ip can protect sinusoidal endothelial cells as well as hepatocytes during liver iri, and the mechanism partly involves modulation of the imbalance of the endogenous oxidant - antioxidant system in the organism. this may suggest a potential role for antioxidant enzymes in the management of iri, but further studies are needed regarding their injury preventive effects. | ischemia - reperfusion (i / r) injury is a multifactorial process that affects graft function after liver transplantation. an understanding of the mechanisms involved in i / r injury is essential for the design of therapeutic strategies to improve the outcome of liver transplantation. the generation of reactive oxygen species subsequent to reoxygenation inflicts tissue damage and initiates a cascade of deleterious cellular responses, leading to inflammation, cell death, and ultimate organ failure. increasing experimental evidence has suggested that kupffer cells and t - cells mediate activation of neutrophil inflammatory responses. activated neutrophils infiltrate the injured liver in parallel with increased expression of adhesion molecules on endothelial cells. the heme oxygenase system is among the most critical of the cytoprotective mechanisms activated during cellular stress, exerting antioxidant and anti - inflammatory functions, modulating the cell cycle, and maintaining the microcirculation. finally, the activation of toll - like receptors on kupffer cells may play a fundamental role in exploring new therapeutic strategies based on the concept that hepatic i / r injury represents a case for host innate immunity. in the present study, there was a significant decrease in hepatic activity of glycogen in the i / r group as compared with corresponding values in the control group. on the other hand, there was a significant increase in the hepatic activity of glycogen in the i / r - ip (ischemic preconditioning) group as compared with corresponding values in the i / r group. |
crohn 's disease (cd) is a form of inflammatory bowel disease (ibd) that primarily affects the caucasian population [1, 2 ]. therefore, identification of gene risk factors of cd is beneficial for the clinical treatment of patients. interleukin 23 (il-23) plays an important role in the inflammatory response against infection as a regulator of immune cells. il-23r which interacts with il-23 is a protein consisting of an il-121 and an il-23r chain. recently, the mechanisms of il-23r variants have been investigated in different autoimmune diseases [69 ]. studies also have shown that rs7517847, the single nucleotide polymorphisms (snps) of the il23r gene, are associated with cd occurring rate [10, 11 ]. however, the association between il-23r polymorphisms and cd susceptibility are inconclusive and controversial due to small sample size in each of the published studies. to better understand the association of il-23r polymorphisms and cd susceptibility in caucasians, we conducted a meta - analysis of all eligible studies and hope to yield more accurate and robust estimates. available studies for il-23r polymorphism and cd were collected by different combinations of various key words : interleukin-23 receptor, il-23r ; polymorphism, variant, or mutation ; crohn 's disease, cd. languages restriction was not imposed in this research and only published studies with full text were included in this meta - analysis. in the meta - analysis, the following inclusive selection criteria were set : (a) study design evaluating the association between il-23r polymorphism and cd risk ; (b) case control design ; (c) caucasians design. the following exclusive selection criteria were set : (a) no control cases ; (b) duplication of the previous publication ; (c) no available genotype frequency ; for studies with overlapped or repeated data (d) no caucasians. eligible studies were extracted by 2 reviewers (li zhang and yunjie lu) independently according to the predesigned data collection form. the following information was extracted : first author 's name, publication year, country, ethnicity, immune suppressive protocol, number of cases and controls, and genotype distribution in both groups. disagreement was resolved by discussion with a third reviewer (guozhong yao). for each trial, odds ratio (or) with the 95% confidence interval (95% ci) of the survival rate was derived and calculated. increased or decreased risk of cd the pooled ors were estimated for allelic model (t allele versus g allele), homozygote comparison (tt versus gg) and heterozygote comparison (tg versus gg), dominant models (tt / tg versus gg), and recessive model (tt versus tg+gg). z test was performed to assess the significance of the pooled or. between - study the random effects model was conducted if the q test exhibited a p 50%. the begg 's funnel plot and egger 's linear regression test were conducted, and p < 0.05 was considered significant. a fixed - effect model (based on mantel - haenszel method) was utilized to pool the data from different studies if the between - study heterogeneity was absent, or a random - effect model (based on dersimonian and laird method) was applied. the statistical analysis was performed by stata 10.0 (stata corp lp, college station, tx, usa). a total of 133 studies were identified by our first research ; a number of 41 were preliminarily yielded out after excluding inappropriate studies and screening abstract - screening, full - text assessment. in these 41 studies, 30 were excluded, 11 articles containing rs7517847 in caucasians were recruited for detailed analysis (table 1), and these data built table 1 [1323 ]. thus, a total of 3279 cd cases and 4136 healthy controls were included in our meta - analysis. all of them were caucasian and the diagnosis of cd was based on clinical manifestations and laboratory examinations and further biopsy. after pooling all the eligible studies in table 2, we found that the risk of cd was significantly associated with rs7517847 in dominant models (tt / tg versus gg : or = 1.652, 95% ci 1.277, 2.137), allelic model (t allele versus g allele : or = 1.327, 95% ci 1.198, 1.469), homozygote comparison (tt versus gg : or = 1.890, 95% ci 1.465, 2.437, figure 2), heterozygote comparison (tg versus gg : or = 1.509, 95% ci 1.161, 1.960), and recessive model (tt versus tg / gg : or = 1.409, 95% ci 1.279, 1.552). these data demonstrate that rs7517847 increases the risk of cd among caucasians with hospital - based studies. begg 's funnel plot and egger 's test were both performed to assess the publication bias of this meta - analysis. the shape of the funnel plots for homozygote comparison models seemed symmetrical (figure 3). then, the egger 's test was used to provide statistical evidence of funnel plot symmetry. cd is associated with jak2 signaling pathway which is activated by il-23 and il-23r receptor. previous studies suggested that the interruption of il-23r snps might lead to the dysregulation of intestinal inflammation. il-23r gene variants also play an essential role in the development of many autoimmune diseases such as ankylosing spondylitis (as), inflammatory bowel disease (ibd), and systemic lupus erythematosus (sle) [7, 26, 27 ]. therefore, researchers are focusing on observing the relationship between il-23r gene polymorphisms and the risk of cd. however, the results are conflicting and controversial due to the different races and insufficient sample size. after pooling data for 11 studies in this meta - analysis, our results firstly demonstrate that t allele of rs7517847 was highly susceptible to cd in caucasians. one previous study showed that rs7517847 is a protective factor in rheumatoid arthritis (ra) in european population. interestingly, ra is a systemic autoinflammatory disease which is associated with ptpn22/c1858 t, while the organ - specific autoimmune disease cd is not [28, 29 ]. thus, the mechanism of this genetic variant may not play a common role in different autoimmune diseases. secondly, publication bias might occur even if there is no significance in statistical test due to extracting published studies. ultimately, owing to methodological limitations, this meta - analysis is retrospective. two independent authors performed the process of study selection and data extraction and a third author resolved the discrepancy to minimize the bias. in conclusion, our meta - analysis suggests that il-23r rs7517847 confers susceptibility to cd in the caucasians. | the association between interleukin-23r gene polymorphism and crohn 's disease (cd) in caucasians is still controversial. thus, a meta - analysis was performed to evaluate the correlation between this gene variant and cd risk. we retrieved the available data from embase and pubmed until may 1, 2014, and evaluated the effect of rs7517847 in caucasians. the significant associations were confirmed between rs7517847 and cd risk in dominant models (tt / tg versus gg : or = 1.652, 95% ci 1.277, 2.137), allelic model (t allele versus g allele : or = 1.327, 95% ci 1.198, 1.469), homozygote comparison (tt versus gg : or = 1.890, 95% ci 1.465, 2.437), heterozygote comparison (tg versus gg : or = 1.509, 95% ci 1.161, 1.960), and recessive model (tt versus tg / gg : or = 1.409, 95% ci 1.279, 1.552). in conclusion, this meta - analysis demonstrates that rs7517847 is associated with the risk of cd in caucasians. these findings show that il-23r genes confer susceptibility to cd in the caucasians. |
development of methods to evaluate certain classes of polycyclic aromatic compounds (pac) detected in complex mixtures to which humans are exposed would greatly improve the diagnostic potential of 32p - postlabeling analysis. identification of dna adduct patterns or specific exposure - related marker adducts would strengthen associations between observed dna adducts and exposures to different environmental pollutants (e.g., kerosene, cigarette smoke, coke oven, and diesel). we have compared diesel - modified dna adduct patterns in various in vitro and in vivo rodent model systems and compared them to dna reactive oxidative and reductive metabolites of 1-nitropyrene. the formation of nitrated polycyclic aromatic hydrocarbon (nitrated pah) dna adducts, derived from the metabolism of diesel extract constituents, was enhanced relative to other pah - derived dna adducts via xanthine oxidase - catalyzed nitroreduction. these adducts were detectable only by the butanol extraction version of the postlabeling analysis. five major dna adducts were detected in human lymphocytes treated in vitro with diesel extract. a major adduct detected in human lymphocytes treated in vitro with diesel extract comigrated with a major adduct detected in lymphocyte dna treated with benzo[a]pyrene (bap) alone. other adducts that co - migrated with the major bap - derived adducts were detected in skin and lung dna isolated from rodents topically treated with (50 mg) diesel extract and the major adduct detected in calf thymus dna treated with rat liver s9 and diesel particle extract. postlabeling of lung dna isolated from rodents exposed via lung inhalation for 24 months to diesel combustion emissions resulted in the formation of a major nuclease - p1-sensitive dna adduct that did not co - migrate with the major bap - diol epoxide adduct.(abstract truncated at 250 words)imagesfigure 1. afigure 1. bfigure 1. cfigure 2.figure 3. |
|
the term of the global budget system started to appear in medical literature since 50 years ago. the global budget system indicates a payment method by which hospitals are allotted a specific amount of money and are then free to distribute the funds according to their individual priorities. although initial purposes of the system were to provide hospitals and clinics a greater flexibility in the distribution of funds received and a financial incentive to operate as efficiently as possible, cost reduction to health care services by putting hospitals at operating risk has become an expected effect. by year 2014, this payment system has been implemented on different scopes of health care services in countries such as canada, germany, switzerland, the united states of america (usa), and taiwan. to the best of our knowledge, the earliest inclusion of dental services in the global budget system commenced in germany and was followed by taiwan. the global budget system applied to dental care services in taiwan was planned and monitored by the ministry of health and welfare (mohw). to implement the system, a budget was allocated to the six regional branches of the national health insurance (nhi) administration, including taipei region, northern taiwan, middle taiwan, southern taiwan, kaohsiung - pingtung region, and eastern taiwan. an expenditure cap was decided according to the expenditure of dental services in the previous year. although a limited annual increase of the expenditure was included in the cap, the reimbursement value of each item would become lower when the amount of dental services used was larger than a previously planned target. the expected outcomes of the global budget system in taiwan included an increase in the quantity of preventive dental care and a well - controlled expenditure cap under peer pressure within dental associations. the government has been satisfied with the effect of the global budget system and expanded the implementation to the scope of traditional chinese medicine and modern medicine years later. however, dental practitioners were concerned with payment inequity in relation to market competition and disparities of socioeconomic status. health care provider supply and competition in the health care market have a reciprocal influence for each other. dentist over - supply is becoming an issue of concern in developed countries such as canada, sweden, united kingdom and the usa, as well as newly industrialized countries including south africa and india. to boost the work capacity of over - supplied dental manpower, some researchers suggested that the government should offer dental insurance to induce demand for dental services. a previous study reported that new dental graduates did not follow traditional models of dentist distribution when entering a service market. indeed, there was no relationship between young dentists selection of practice location and the population size of an area. the density of dentists in an area was enhanced by factors such as the opportunity of dental training programs, residents per capita income, health care expenditures per resident, the proportion of minority in the community, and the insurance penetration rate. the situation in taiwan was different 50 years ago when baker and perlman reported a low reputation, mild income, and insufficient manpower of dental practices. at that time, a low registration rate at dental schools in taiwan was also identified. in the early 70 's, only three - quarters of dental graduates practiced in private clinics, and 12.1% of them left taiwan for purposes such as advanced studies or immigration. dynamics of the dental manpower in taiwan depended on the size of the community population although the density was as low as 6.6 dentists per 100,000 population then. this was different from young american dentists relocation that was motivated by a variety of factors. an uneven distribution of dentists in taiwan has been reported, and approximately 30% of taiwanese towns have never attracted a qualified dentist before 1990. by the end of the 20 century, the highest and lowest density of dentists was reported in taipei city (67.5 dentists/100,000 population) and penghu county (5.2 dentists/100,000 population), respectively. compared to the capital of the usa which had almost 100 dentists/100,000 population at a similar survey time, the capital of taiwan, taipei city, was not severely overwhelmed by over - supply of dental manpower, not to mention rural and remote areas such as penghu county. when searching the medline literature database with a combination of keywords including dentist, dental practice, dental surgeon or dental manpower and global budget, only four papers were identified. excluding a paper reporting pharmaceutical expenditures but not utilization of dental care services, the remaining studies reported controversial outcomes. suggested that the implementation of the global budget system failed to improve the distribution of dental manpower because of a declined utilization of dental care services. on the contrary, two studies demonstrated an increase in utilization following a more intense competition of dental care market resulting from the implementation of the global budget system. although the nhi system in taiwan has established an international reputation for its effectiveness, dynamics of dental manpower in relation to the global budget system remained under - investigated. therefore, this study aimed to investigate into dentistsupply and practice patterns and to assess structural changes of dental manpower following the implementation of the global budget system in taiwan. an additional interest of this study was to report utilization of dental care services in relation to the application of the payment scheme. the hypothesis of the study was that the distribution of dental manpower was influenced by socioeconomic factors and the implementation of the global budget system. this study compared the difference in dental practice patterns with pre- and post - innovation of the global budget system in taiwan. cross - sectional data were applied to assess distribution of dental manpower among practice locations and longitudinal data were used to analyze transition of practice patterns. because nationwide health care data were unattainable, claim data from the bureau of national health insurance (bnhi) from 1995 to 1998 were used. the data used included frequency of dental visits, diagnoses, treatments and the total fee charged. socioeconomic data collected from the annual report of the ministry of internal affair (moi), including levels of education, family revenue and health expenditures, were divided into six regions according to regional branches of the bnhi. demographic data received from the moi were composed of the density and growth rate of the population, as well as the percentages of elderly and young populations. the elderly and young populations were defined as those residents who were 65-years - or older, and those children who were 14-years - or younger, respectively. dental manpower data such as registrations for practice locations were received from the mohw. to take the effect of market competition on dental manpower into account, the herfindahl - hirschman index (hhi) was applied as estimate the level of competition within a domestic dental market. it was formulated as : where x was the number of dental hospitals / clinics located in a municipality (a city or county), ni was the number of dentists registered at a hospital / clinic of the municipality, and n was the total number of dentists registered in a municipality. an hhi value closer to 1 indicates a market closer to a monopoly, while an hhi approaching to 0 represents a highly competitive market. a dentist move - in rate was defined as the quotient having the move - in number divided by the total number of dentists in a municipality. similarly, a dentist move - out rate was calculated with the same division methods. data entry and statistical analysis were carried out with ibm spss statistics (version 19.0, ibm corporation, somers, ny, usa). a multivariate linear regression method with a step - wise approach was used to assess the individual contribution of socioeconomic and demographic factors in relation to dependent variables of dental manpower. the dependent variables included the number of dentists per 100,000 population, dentists move - out rate and dentists move - in rate. dental claims from july 1997 to december 1998 were analyzed as the global budget system has been implemented in taiwan since july 1998. eight categories of treatment carried out as outpatient dental care services and reimbursed under the global budget system were included in the study. these included radiographic examinations, endodontic treatments, amalgam restorations, tooth - colored restorations (composite resin and/or glass ionomer cement restorations), periodontal surgeries, nonsurgical periodontal treatments, oral and maxillofacial surgeries, and pulpotomies for deciduous teeth. utilization of dental services in each treatment category before and after the implementation of the global budget system was examined with a chi - square method. during the 4 year study, the average number of dentists in a municipality of taiwan ranged from 9.6 to 70.5 dentists/100,000 population. taipei city and chiayi county (an agricultural county located in southern taiwan) reported the highest and lowest density of dentists, respectively. the difference in the density between taipei city and chiayi county reduced from 8.4 times in 1995 to 6.7 times in 1998. on the other hand, the highest move - out rate was seen in penghu county, while the lowest was identified in tainan county, which was another agricultural county located in southern taiwan. the move - in rate among dentists ranged from 0.084 to 0.170, with the lowest and highest value in taipei city and penghu county, respectively. of further note, the range of the hhi calculated was from 0.002 to 0.117. taipei county (a metropolis surrounding taipei city ; with the name changed to new taipei city in year 2010) and penghu county had the lowest and highest values of hhi, respectively. a municipality that reported a higher percentage of tertiary educated population (t = 3.718, p < 0.001), that had a higher per capita income (t = 6.172, p < 0.001), that showed a higher population density (t = 6.172, p < 0.001), that displayed a lower percentage of elderly population (t = 2.506, p = 0.014), or that was located at middle taiwan (t = 4.234, p < 0.001) was more likely to have a higher number of dentists per 100,000 population [table 1 ]. implementation of the global budget system, per capita health care expenditure, the percentage of young population, the hhi value, geographic locations other than middle taiwan, and the years were not related to the density of dentists (p 0.097). individual contribution of socioeconomic and demographic factors to dentists number per 100,000 population in addition, a municipality that reported a higher hhi value (t = 2.880, p = 0.005) was more likely to show a higher move - out rate among dentists [table 2 ]. the rate was lower after the implementation of the global budget system (t = 2.436, p = 0.018) and in southern taiwan (t = -2.949, p = 0.004). other factors were not associated with dentists move - out rate (p 0.258). individual contribution of socioeconomic and demographic factors to dentists move - out rate moreover, a municipality that had a higher percentage of elderly population (t = 3.628, p < 0.001), that reported a lower percentage of young population (t = 2.138, p = 0.035), or that showed a higher rate of population growth (t = 4.412, p < 0.001) was more likely to display a higher move - in rate among dentists [table 3 ]. year 1996 also showed a higher move - in rate compared to year 1995 (t = 3.385, p = 0.001). none of other factors was in connection with the rate (p 0.243). individual contribution of socioeconomic and demographic factors to dentists move - in rate on the other hand, the number of dental management items claimed to the nhi in the years 1997 and 1998 was 11,190,099 and 11,310,202, respectively [table 4 ]. the percentage of amalgam restorations reduced from 19.82% in the year 1997 to 17.94% in the year 1998, while the percentage of tooth - colored material restorations, increased from 25.46% to 28.79%. other categories of dental management items showed a negligible range of changes in the percentages of claims. figures 1 and 2 show a decreasing trend in the percentage of amalgam restorations among total claims, in relation to an increasing trend in tooth - colored material restorations. all other management items displayed a negligible range of changes according to the monthly data. percentages of utilisation of dental services number of amalgam and tooth - coloured material restorations claimed to national health insurance. this is the first study to suggest a stabilizing effect of the global budget system on dynamics of dental manpower. however, this study did not find a relationship between the global budget system and the number of dentists per 100,000 population as well as dentists move - in rate. thus, a lower move - out rate reported by this study indicated that the implementation of the payment system contributed to a more stable market. this resembled a previous study demonstrating that distribution of dentists was not improved by application of the global budget system. restricted by the predetermined expenditure cap, any increase in the service capacity would result in a lower reimbursement value for all items claimed by dentists in a region. thus, local dental associations could have raised the threshold for entry, such as a high registration fee, to deter new dentists from moving into the market. this would consequently prevent existing dentists from moving out of a region since the cost of moving into another region could be too high. nevertheless, new dental graduates could only move in but not move out a market. this might explain the reason why only the move - out rate was reduced following the implementation of the global budget system. this study has also reported for the 1 time a positive relationship between hhi and dentists move - out rate. according to literature, a higher hhi value indicated a market closer to a monopoly. because data of hhi and dentists move out rate were collected from the corresponding years, it became difficult to interpret a causal relationship between these two variables. a possibility was that a higher move - out rate reduced the number of dentists in a municipality and consequently led the market toward a status of a monopoly. on the other hand, a monopoly - like market could indicate a municipality that had fewer dentists. these municipalities were generally agricultural or island countries characterized by a smaller population size and lower per capita income. furthermore, an earlier study reported that a higher hhi value contributed to a lower amount of dental care expenditure per person. thus, dentists could have opted to move out from a high hhi municipality due to a less profitable practicing location and the less preferred living environment. since both situations could result in a positive relationship between hhi and dentists move - out rate, further investigations are indicated. in addition, this study demonstrated a positive relationship between the density of dentists and average socioeconomic status of a municipality. we found that a higher number of dentists per 100,000 population was accompanied by a higher percentage of the tertiary educated population and a higher per capita income of a municipality. this agreed with an earlier study reporting that more dentists opted to practice in areas of high socioeconomic status. relevant reasons for the concentration of dentists in high socioeconomic areas included children 's education, family reasons, financial reasons, and professional development. as a higher number of dentists with more years experience opted to practice in wealthier areas, new dental graduates could have consequently moved into low socioeconomic areas such as municipalities showing a higher percentage of the elderly population. this was also confirmed by our study, since a lower density of dentists and a higher move - in rate among dentists were seen in municipalities with a higher percentage of the elderly population. even though young dental graduates showed a higher likelihood to practice dentistry in disadvantaged areas compared to experienced dentists, years later they may still leave these areas for municipalities with a higher average socioeconomic status. although the implementation of the global budget system had a stabilizing effect on dynamics of dental manpower, establishment of professional development programs and an attractive reimbursement value at underserved areas are indicated. on the other hand, this study has identified a complementary relationship between amalgam and tooth - colored material restorations in the aspect of utilization of dental services. an upward trend of utilization of tooth - colored material restorations this connection could have resulted from a higher reimbursement value of composite resin over amalgam restorations. nevertheless, the influence from the implementation of the global budget system was unclear, as the complementary effect has been seen since a year prior to the commencement of the payment system. of further note, a previous study has reported a transition of dental materials used from amalgam toward tooth - colored materials due to patients esthetic considerations. despite the fact that health risks from dental amalgam restorations remained controversial, amalgam has been permanently replaced by other materials for restorative dentistry in countries such as sweden and norway. thus, the international trend could also have contributed to the complementary relationship between amalgam and tooth - colored materials in taiwan. this study has demonstrated a stabilizing effect of the global budget system on dynamics of dental manpower in taiwan. a positive relationship between hhi and dentists move - out rate has been reported by this study. the relationship between the socioeconomic status of a municipality and the density of dentists has also been confirmed. in addition, reduced utilization of amalgam restorations was accompanied by increased utilization of tooth - colored material restorations. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. | background : this study aimed to investigate dentists supply and practice patterns following the implementation of the global budget system in taiwan.materials and methods : data of reimbursement claims, municipal socioeconomic status and dental manpower were collected from the national health insurance administration, the ministry of internal affair, and the ministry of health and welfare, respectively. a multivariate linear regression method was used for data analysis.results:a municipality that reported a higher percentage of tertiary educated population (t = 3.718, p < 0.001), a higher per capita income (t = 6.172, p < 0.001), a higher population density (t = 6.172, p < 0.001), or a lower percentage of elderly population (t = 2.506, p = 0.014) was more likely to have a higher number of dentists per 100,000 population. a municipality that reported a higher herfindahl - hirschman index (hhi) value (t = 2.880, p = 0.005) was more likely to show a higher move - out rate among dentists. the rate was lower after the implementation of the global budget system (t = 2.436, p = 0.018). a municipality that had a higher percentage of elderly population (t = 3.628, p < 0.001), a lower percentage of young population (t = 2.138, p = 0.035), or a higher rate of population growth (t = 4.412, p < 0.001) was more likely to display a higher move - in rate among dentists. the percentage of amalgam restorations in total claims reduced from 19.82% to 17.94%, while the percentage of tooth - colored material restorations increased from 25.46% to 28.79%.conclusion : this study has demonstrated a stabilizing effect of the global budget system on dynamics of dental manpower in taiwan. a relationship between hhi and dentists move - out rate has been found. the relationship between municipal socioeconomic status and the density of dentists has also been confirmed. in addition, reduced utilization of amalgam restorations was accompanied by increased utilization of tooth - colored material restorations. further investigations are indicated. |
the patients rights movement have promoted patient involvement in health care for 50 years (1). patients rights encompass legal and ethical issues in the provider - patient relationship, including a person s right to privacy, the right to quality medical care without prejudice, the right to make informed decisions about care and treatment options, and the right to refuse treatment. the patients rights charter of iran was approved by health policy council with a new and comprehensive viewpoint which aimed to clarify the rights of the health service recipients and ensure observance of moral standards in the treatment and medical fields on november 26, 2009, and on december 1st of the same year, it was corresponded to all relevant centers (2, 3). one of the health services which patients may get involved in is occupational therapy (ot). according to the world federation of occupational therapy (wfot), ot is a client - centered health profession which concerns promoting the health and well - being of patients through their occupation. professional ethics and standards for regulating ethical conduct will reinforce level of confidence between patients and health professionals in ot, which in turn will result in protection of community (4). galheigo reported in 2011 that occupational therapists and scientists need to be attentive of human rights issues (5) and they must provide a supportive environment which facilitates patients efforts in getting involved in their own health care services (6). furthermore, observing patients rights may help to achieve more satisfaction among patients and the medical team, while not observing these rights would lead to distrust, damages and losses in the patients and the medical team. hence, ot professionals must be aware of patients rights and try to observe them. up to the present time, no descriptive study has been reported in iran in regard to the relationship between occupational therapists work experience, their educational level and level of their knowledge about patients rights in all areas of their clinical practice. therefore, this study was conducted to examine the level of occupational therapists knowledge about patients rights in this effective health care professional service. the study samples consisted of 125 occupational therapists that were chosen by convenience sampling strategy from rehabilitation clinics under the supervision of the university of social welfare rehabilitation sciences (uswr) and departments of ot in the school of rehabilitation of uswr, tehran university of medical sciences and shahid baheshti university of medical sciences. the sample size determined for the current study was five participants per variable (7). the study was approved by the ethical committee of uswr, and all participants were informed about study objectives and their written and signed informed consents were obtained. the data collection instrument was a questionnaire designed by the researchers and consisted of four - parts : the first part included demographic information (age, gender, work experience, educational level) ; the second part consisted of five questions about participants self - assessment of their knowledge about deputy police of the medical council medical council of islamic republic of iran, legal rules and regulations, ethics committee of medical council, ot code of ethics and patients rights charter of iran ; the third part consisted of 20 questions based on ten sections of the patients rights charter of iran (8) and the last part included an open question about facilitating factors involved in patients rights. the participants answers to the questions of the third part were quantified based on a four - score scale : high (4 scores), moderate (3 scores), low (2 scores) and none (1 score). the questionnaire was given to 10 faculty members of uswr and their comments were applied accordingly. the reliability was examined using a test - retest method in which the questionnaire was given to 10 eligible participants twice with a 10-day interval, and the respective interclass correlation coefficient (icc) of the first and second answers obtained was 0.82 (95% ci, 0.146 0.951). the questionnaires were completed within three months in target clinics and universities. obtained quantitative data one - way analysis was used for comparing scores of occupational therapists knowledge of patients rights according to educational level, areas of practice in occupational therapy and work experience. all statistical analyses were done using spss 16.0 statistical software (spss inc, chicago, il). demographic data of the participating occupational therapist, including their gender, educational levels, different areas of clinical practice and periods of their work experiences are shown in table 1. as it is illustrated, the majority of these participants were females (59.2%). the participants self - assessment of their knowledge about deputy police of the medical council of islamic republic of iran, legal rules and regulations, ethics committee of medical council and ot code of ethics (second part of the questionnaire) are shown in table 2. furthermore, the sum of the scores based on answers to the questions in the third part of the questionnaire showed 67 (53.6%) of respondents had scores above the median, hence, 67 (53.6%) of occupational therapists had high level of knowledge about sections of patients rights charter of iran. there was no significant difference between male and female participants knowledge of patients rights charter of iran (p>0.05). likewise, we could not find a relationship between occupational therapists knowledge of patients rights charter of iran and their educational level, areas of practice in ot and periods of their work experience (table 3). in the next step, regression analysis was performed to determine the relationship between total scores of occupational therapists knowledge about patients rights and sex, educational level, areas of practice in ot and work experience. the obtained results showed that the sex, educational level, areas of practice in occupational therapy and work experience did not have any significant correlation with occupational therapists knowledge of patients rights (p>0.05). finally, participants answers to open - ended questions about facilitating factors of patients rights were categorized. these factors were classified in three groups of factors related to the organization, therapists and clients (table 4). one of the most important components of patients rights is to provide an ethical and humanistic care. european regional office of the world health organization (who) states that promoting patients rights is a multi categorized topic, and goals should be followed through multilateral efforts. in addition, who has presented solutions to this issue, most of which involve active participation by both the service recipients and service providers in formulating health policy and developing training programs, specifically for service providers and the entire community (9). based on the findings of the present study, more than half of the participants had a high level of knowledge in regard to patients rights, especially concerning issues of preserving patients privacy, the right to receive respectful and prompt care despite cultural and racial differences and also the right to permit the presence of those who are not directly involved in the treatment process. the present results were more consistent with previous studies, which have shown that health care professionals have a high level of knowledge about their patients rights (8, 10, 11). on the other hand, the level of knowledge about patients rights do not correlate directly to their observance (10, 12). lied showed that 84% of professionals know the patients rights, but a mere 64.4% observe them (10). arnetz also showed that physicians, registered nurses, and practical nurses did not differ significantly in their views of patient involvement, but did differ significantly in behavior (12). another study demonstrated, however, that there is a relationship between awareness of the rights of the patient and observing them (13). this may be due to the research methods and the questionnaire in the mentioned study to determine the tendency of nurses to implement their roles to advocate patients rights. despite the high knowledge of occupational therapists, there seems to be a gap between awareness of patients rights and their observation in practice. suggested in 2006 that there are many other requirements that should be recognized and identified in order for patients rights to be observed in practice (8). jolaee. showed in 2008 through a qualitative study the facilitating factors which affect patients rights in clinical practice. issues categorized as barriers to patients rights practice and facilitators of patients rights practice were further classified into three subgroups : awareness, resources and accountability (14). patients rights can be fully implemented only if health services identify the barriers and strategies in employment of such charters of rights (15, 16). in this regard, another finding of the present study was related to facilitating factors of patients rights, which were classified in three groups (factors related to the organization, factors related to therapists, factors related to clients). the most important organizational factor was a need for approval of health insurances by the responsible organizations. in economic models, the primary function of health insurance is to alleviate the financial risk associated with unanticipated adverse health events. in general, people dislike risk and are willing to trade a small amount of money to buy insurance premium for their protection against a potentially large loss of their income (17). health insurance improves the quality of care and introduces user entitlements known as patients rights (18). the next important factor was to test patients knowledge level and identify responsive organizations roles in accomplishing their needs and rights as clients. findings of other studies have shown only few patients knew the rules composed within their charter of rights (19). this indicates the need for extensive education of patients and healthcare professionals in related subjects. other important facilitating factors in the promotion of patients rights are ethics courses designed for undergraduate students, workshops for therapists, patients rights charter installations in occupational therapy clinics and the generation of ethical committees within related organizations. a report by the association of american medical colleges (aamc) argued that medical schools must ensure that before graduation, a student will have demonstrated knowledge of the theories and principles that govern ethical decision making and of the major ethical dilemmas in medicine (20). the aamc s statement reflects a growing consensus among medical educators that ethics education should be a core component of medical schools curricula. at the present time, there is no ethics curriculum for occupational therapists in iran and this study hopes to initiate introduction of such courses in iranian academic centers. our findings revealed that occupational therapists had a high knowledge of patients rights, but they would need to train for their implementation. students of occupational therapy should graduate with a baseline level of knowledge in ethics, as they do in the basic sciences. the current state of education, however, does not ensure a common standard for ethics education in occupational therapy. furthermore, as the client - centered approach is very important in ot, ethics are dependent on the cultural and social characteristics of societies (21), and therefore the view of the iranian patients towards the issue urges the ministry of health and medical education of the islamic republic of iran to accelerate the implementation of the patients rights charter of iran (22). it is suggested that a qualitative research be done to determine the views of occupational therapists and clients about observing the patients rights in iran. one of the limitations of our study was the greater number of bachelor - level participants compared to other educational levels, due to the convenience sampling method. this may affect the results based on analyses of the relationship between the level of knowledge about patients rights and educational level although, the level of occupational therapists knowledge about the patients rights charter are high, it is necessary to provide context for observing patients rights by involving related facilitating factors such as different organizations, therapists, and clients within the field of clinical occupational therapy professionalism. | addressing patients rights issues brings occupational therapists ethical and political responsibilities that involve patients privileges and new facilitating factors which influence their needs. the goal of this study was to determine the level of occupational therapists knowledge about patients rights.the present research was a cross - sectional study which involved 125 occupational therapists chosen by a convenience sampling strategy in tehran during the year of 2012. a four - part questionnaire was used for data collection, and the degree of the subjects self - assessment of their knowledge was measured based on the obtained numbers of correct answers in the third part. the validity and reliability of this questionnaire were assessed prior to its being distributed among participants.the results demonstrated no significant association between the level of occupational therapists knowledge about patients rights and their existing experiences within their areas of occupational therapy (p>0.05). based on the result, 53.6% of the respondents had high level of knowledge about patients rights. facilitating factors which influence the attainment of patients rights were classified into three groups : organizations, therapists and clients. the results of the present research demonstrated that the level of occupational therapists knowledge about patients rights were high. furthermore, this study showed that for optimal result, there is a need to provide milieu for observing the patients rights in clinical occupational therapy services. |
taken into account the spermatozoa, cryopreservation has several applications, such as seminal storage prior to prostate and vasectomy surgeries, before chemotherapy and radiotherapy treatments, prior to fertility treatments and for donor sperm banking (1). however, sperm freezing and thawing processes lead to intracellular ice crystals formation which causes organelles and cell membrane rupture (1), modifies the structure and integrity of plasma membranes (2), and alters mitochondrial membrane potential and release of reactive oxygen species (ros) (3). in addition, cryopreservation has been shown to diminish the antioxidant activity of the spermatozoa making them more susceptible to ros damage (4). spermatozoa and seminal plasma contain antioxidant enzymes such as superoxide dismutase (sod), catalase and glutathione peroxidase (gpx), although its lack of cytoplasm leads to a decrease of antioxidant defense. moreover, spermatozoa are particularly more susceptible to lipid peroxidation because of the high proportion of polyunsaturated fatty acid in their membranes (5, 6). deleterious effects of oxidative stress can result in several structural alterations of spermatozoa, such as protein fragmentation, lipid peroxidation and dna fragmentation (dnaf) (7). increase in dnaf is related to reduced implantation and pregnancy rates and increased recurrent pregnancy loss (8, 9). in order to minimize deleterious effects of cryopreservation, some studies have focused on testing antioxidants action on sperm cryopreservation (10, 11). zhang. observed a protective effect of l - carnitine, leading to a significant improvement in post - thawed sperm parameters, including dnaf levels (10). mata - campuzano. also observed a reduction of lipid peroxidation and dnaf following antioxidant supplementation of spermatozoa during cryopreservation (11). leptin, a peptide hormone mainly secreted by adipose tissue, is widely known by its functions related to obesity, appetite and food intake inhibition and energy expenditure. however, it has been shown to have roles in diverse physiologic systems, including reproductive system (12). although presence of leptin and its receptor has been demonstrated in spermatozoa (1315), its role on spermatogenesis and sperm still needs to be clarified. literature has some controversial results concerning sperm parameters after leptin in vitro incubation (13, 16). lampiao and du plessis (2008) found an increase in total and progressive motility, in acrosome reaction and nitric oxide (no) production after leptin incubation. (2008) demonstrated no significant effects of leptin incubation on motility, and percentage of capacitated and acrosome reacted sperm after leptin incubation (13). additionally, studies have suggested that leptin has a role in oxidative stress (17, 18) which is still controversial. zheng. demonstrated that leptin increased sod activity in cardiomyocytes (19), while yamagishi. observed a leptin - induced lipid oxidation in endothelial cells (20) our present study aimed at demonstrating the effect of cryopreservation on sperm dnaf and investigating the possible effects of sperm capacitation and leptin incubation on frozen - thawed sperm dnaf and oxidative stress. semen samples were collected from 45 normospermic patients aged 2845 years (35.34.8) from november 2014 to june 2015, by masturbation after 2 to 3 days of ejaculatory abstinence attending for male infertility investigation at vida centro de fertilidade da rede dor in rio de janeiro, brazil. samples were collected into sterile vials and were left to be liquefied at 37c for 30 min. semen samples were analyzed according to who criteria (2010) for concentration (1510 sperm / ml) and motility (32% progressive motility or 40% total motility) and according to kruger s criteria for morphology (4% normal morphology). the evaluation of concentration, motility and morphology were performed using neubauer chamber, makler chamber and spermac staining (sermacstain sperm morphology kit, fertipro ; belgium), respectively. the criteria for exclusion from the study were urogenital infection, varicocele, hydrocele, use of drugs and medications and other diseases such as diabetes mellitus, obesity and malnutrition. the study was approved by the ethics committee of university hospital pedro ernesto (cep / hupe 432.202) and all patients provided written informed consent. in order to achieve the goal of this study, an experiment was initially done comparing fresh raw (fr) and frozen - thawed raw (f - t) semen samples from 15 patients to evaluate the effect of cryopreservation on dnaf. based on the results observed, a second experiment was done using frozen - thawed raw (f - t) and capacitated (f - tcap) semen samples from other 15 patients. in order to evaluate if leptin had some additional effect on the dnaf and oxidative stress of capacitated samples, a new experiment was performed using other 15 semen samples of previously capacitated frozen - thawed treated (f - tcapl) or not treated (f - tcap) ones with leptin. samples were placed with 1:2 cryoprotectants (spermfreeze ; vitrolife ; sweden) on cryovials and frozen in vapor liquid nitrogen for 30 min, followed by liquid nitrogen immersion. thawing, cryovials were taken from liquid nitrogen and immediately immersed in water bath at 37c for 5 min. for sperm capacitation, samples were centrifuged at 180 g for 20 min using 2-layered density gradient centrifugation (dgc) solutions of 45% and 90% (spermgrad ; vitrolife ; sweden). the supernatant was then removed and the pellet was washed in 1.5 ml human tubal fluid medium (htf hepes ; irvine scientific ; united states) supplemented with 10% sps (sps ; irvine scientific ; united states) at 180 g for 10 min. the supernatant was discarded and 1 ml of medium was gently layered on the pellet. then, the tube was inclined at an angle of 45 degrees and incubated at 37c for 1 the upper interface was aspirated to obtain the motile fraction, which was divided in aliquots for leptin incubation. capacitated samples were incubated with 10 ng leptin at 37c and 6% co2 overnight, before freezing. the leptin dose was used based on previous studies (13, 15, 16). semen samples were diluted using 1:1 concentration in melt agarose microgel and placed in pretreated slides. sperm were lysed and dispersed a large or medium dna halo in non - fragmented sperm cells and a small or none dna halo in fragmented sperm cells. dnaf was performed to compare fr versus f - t ; f - t versus f - tcap ; f - tcap versus f - tcapl. pro - oxidant mechanisms were evaluated in f - tcap and f - tcapl. - lipid peroxidation was evaluated through measurement of thiobarbituric acid reactive substances (tbars) at 532 nm using a spectrophotometer (22). - protein oxidation : this method is based on reaction of 5,5-dithiobis-2-nitrobenzoic acid (dtnb) with sulfhydryl (sh) group, which was measured at 412 nm using a spectrophotometer (23). - superoxide dismutase (sod) : in this method, adrenaline undergoes oxidation by anion superoxide action, which is inhibited by sod activity. this oxidation generates adrenochrome, which is measured at 480 nm with a spectrophotometer (24). - catalase : catalase activity was measured by h2o2 quantification at 240 nm with a spectrophotometer (240 nm) (25). - glutathione peroxidase (gpx) : gpx activity was determined through decay rate of reduced nicotinamide adenine dinucleotide phosphate (nadph) at 340 nm with a spectrophotometer (26). test was used to compare dnaf between groups before and after freeze - thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. semen samples were collected from 45 normospermic patients aged 2845 years (35.34.8) from november 2014 to june 2015, by masturbation after 2 to 3 days of ejaculatory abstinence attending for male infertility investigation at vida centro de fertilidade da rede dor in rio de janeiro, brazil. samples were collected into sterile vials and were left to be liquefied at 37c for 30 min. semen samples were analyzed according to who criteria (2010) for concentration (1510 sperm / ml) and motility (32% progressive motility or 40% total motility) and according to kruger s criteria for morphology (4% normal morphology). the evaluation of concentration, motility and morphology were performed using neubauer chamber, makler chamber and spermac staining (sermacstain sperm morphology kit, fertipro ; belgium), respectively. the criteria for exclusion from the study were urogenital infection, varicocele, hydrocele, use of drugs and medications and other diseases such as diabetes mellitus, obesity and malnutrition. the study was approved by the ethics committee of university hospital pedro ernesto (cep / hupe 432.202) and all patients provided written informed consent. in order to achieve the goal of this study, an experiment was initially done comparing fresh raw (fr) and frozen - thawed raw (f - t) semen samples from 15 patients to evaluate the effect of cryopreservation on dnaf. based on the results observed, a second experiment was done using frozen - thawed raw (f - t) and capacitated (f - tcap) semen samples from other 15 patients. in order to evaluate if leptin had some additional effect on the dnaf and oxidative stress of capacitated samples, a new experiment was performed using other 15 semen samples of previously capacitated frozen - thawed treated (f - tcapl) or not treated (f - tcap) ones with leptin. samples were placed with 1:2 cryoprotectants (spermfreeze ; vitrolife ; sweden) on cryovials and frozen in vapor liquid nitrogen for 30 min, followed by liquid nitrogen immersion. thawing, cryovials were taken from liquid nitrogen and immediately immersed in water bath at 37c for 5 min. for sperm capacitation, samples were centrifuged at 180 g for 20 min using 2-layered density gradient centrifugation (dgc) solutions of 45% and 90% (spermgrad ; vitrolife ; sweden). the supernatant was then removed and the pellet was washed in 1.5 ml human tubal fluid medium (htf hepes ; irvine scientific ; united states) supplemented with 10% sps (sps ; irvine scientific ; united states) at 180 g for 10 min. the supernatant was discarded and 1 ml of medium was gently layered on the pellet. then, the tube was inclined at an angle of 45 degrees and incubated at 37c for 1 the upper interface was aspirated to obtain the motile fraction, which was divided in aliquots for leptin incubation. capacitated samples were incubated with 10 ng leptin at 37c and 6% co2 overnight, before freezing. the leptin dose was used based on previous studies (13, 15, 16). semen samples were diluted using 1:1 concentration in melt agarose microgel and placed in pretreated slides. sperm were lysed and dispersed a large or medium dna halo in non - fragmented sperm cells and a small or none dna halo in fragmented sperm cells. dnaf was performed to compare fr versus f - t ; f - t versus f - tcap ; f - tcap versus f - tcapl. pro - oxidant mechanisms were evaluated in f - tcap and f - tcapl. - lipid peroxidation was evaluated through measurement of thiobarbituric acid reactive substances (tbars) at 532 nm using a spectrophotometer (22). - protein oxidation : this method is based on reaction of 5,5-dithiobis-2-nitrobenzoic acid (dtnb) with sulfhydryl (sh) group, which was measured at 412 nm using a spectrophotometer (23). - superoxide dismutase (sod) : in this method, adrenaline undergoes oxidation by anion superoxide action, which is inhibited by sod activity. this oxidation generates adrenochrome, which is measured at 480 nm with a spectrophotometer (24). - catalase : catalase activity was measured by h2o2 quantification at 240 nm with a spectrophotometer (240 nm) (25). - glutathione peroxidase (gpx) : gpx activity was determined through decay rate of reduced nicotinamide adenine dinucleotide phosphate (nadph) at 340 nm with a spectrophotometer (26). test was used to compare dnaf between groups before and after freeze - thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. there was a significant increase in sperm dnaf evaluation after freeze - thaw cycle compared to evaluation with the same fresh raw sample (fr=17.61.7, f - t=36.23.5 ; p=0.0003 ; figure 1a). besides that, this increase occurred in such a way that 53.3% of the samples classified as fragmented (above 30%) after freeze - thaw cycle were found to be non - fragmented (under 30%) in the fresh raw evaluation. percentage of sperm dnaf in fresh raw (fr) and frozen - thawed (f - t) samples. horizontal line corresponds to cutoff value of the test ; n=15 (figure 1a). percentage of sperm dna fragmentation in capacitated (f - tcap) and non - capacitated (f - t) samples before freeze - thaw cycle ; n=15 (figure 1b) and capacitated samples with (f - tcapl) or without (f - tcap) leptin addition before freeze - thaw cycle ; n=15 (figure 1c). data are represented as mean and standard error sperm dnaf was significantly reduced when sperm capacitation was performed before freezing samples, when compared to those frozen with no previous capacitation (raw). leptin addition to culture media in capacitated samples, before sperm freezing increased this reduction (f - tcap=19.32.8, f - tcapl=9.52.0 ; p<0.0001 ; figure 1c) in sperm dnaf. leptin addition to capacitated spermatozoa before freezing had no effect on lipid peroxidation (f - tcap=0.040.004, f - tcapl=0.030.002 ; p=0.6 ; table 1) and protein oxidation (f - tcap=4.10.1, f - tcapl=4.00.1 ; p= 0.2 ; table 1). oxidative damage measured by lipid peroxidation (tbars), protein oxidation (sh) in capacitated spermatozoa with (f - tcapl) and without (f - tcap) leptin incubation before freezing. antioxidant activity measured by superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) in capacitated spermatozoa with (f - tcapl) and without (f - tcap) leptin incubation before freezing. data are represented as mean and standard error ; n=15 there was a significant increase in antioxidant activity of sod (f - tcap= 82.46.9, f - tcapl=100.86.2 ; p=0.001 ; table 1) and gpx (88.89.8, f - tcapl=110.78.7 ; p=0.02 ; table 1) when comparing samples with and without leptin addition. however, catalase activity did not differ between groups (f - tcap=0.020.006, f - tcapl=0.020.007 ; p=0.9 ; table 1). there was a significant increase in sperm dnaf evaluation after freeze - thaw cycle compared to evaluation with the same fresh raw sample (fr=17.61.7, f - t=36.23.5 ; p=0.0003 ; figure 1a). besides that, this increase occurred in such a way that 53.3% of the samples classified as fragmented (above 30%) after freeze - thaw cycle were found to be non - fragmented (under 30%) in the fresh raw evaluation. percentage of sperm dnaf in fresh raw (fr) and frozen - thawed (f - t) samples. horizontal line corresponds to cutoff value of the test ; n=15 (figure 1a). percentage of sperm dna fragmentation in capacitated (f - tcap) and non - capacitated (f - t) samples before freeze - thaw cycle ; n=15 (figure 1b) and capacitated samples with (f - tcapl) or without (f - tcap) leptin addition before freeze - thaw cycle ; n=15 (figure 1c). data are represented as mean and standard error sperm dnaf was significantly reduced when sperm capacitation was performed before freezing samples, when compared to those frozen with no previous capacitation (raw). leptin addition to culture media in capacitated samples, before sperm freezing increased this reduction (f - tcap=19.32.8, f - tcapl=9.52.0 ; p<0.0001 ; figure 1c) in sperm dnaf. leptin addition to capacitated spermatozoa before freezing had no effect on lipid peroxidation (f - tcap=0.040.004, f - tcapl=0.030.002 ; p=0.6 ; table 1) and protein oxidation (f - tcap=4.10.1, f - tcapl=4.00.1 ; p= 0.2 ; table 1). oxidative damage measured by lipid peroxidation (tbars), protein oxidation (sh) in capacitated spermatozoa with (f - tcapl) and without (f - tcap) leptin incubation before freezing. antioxidant activity measured by superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) in capacitated spermatozoa with (f - tcapl) and without (f - tcap) leptin incubation before freezing. there was a significant increase in antioxidant activity of sod (f - tcap= 82.46.9, f - tcapl=100.86.2 ; p=0.001 ; table 1) and gpx (88.89.8, f - tcapl=110.78.7 ; p=0.02 ; table 1) when comparing samples with and without leptin addition. however, catalase activity did not differ between groups (f - tcap=0.020.006, f - tcapl=0.020.007 ; p=0.9 ; table 1). while cryo - induced damage to motility, viability, morphology and fertility capacity is already well documented, the possible dna damage following sperm freezing is still not confirmed. there appears to be some contradictions concerning whether or not freezing can affect and the extent of the effect on sperm dnaf (4, 27). some authors reported a significant increase on sperm dna fragmentation caused by cryopreservation (28, 29), while others suggest there is no harm to dna integrity (30, 31). this controversy could be explained by several factors : 1) patients selection ; in the study of ozkavukcu. (2008) (32), 30% of the patients were smokers and it is known that smoking can alter dna fragmentation (33) ; 2) different sperm capacitation techniques before freezing, i.e some studies used raw semen (34), others have isolated sperm by washing (35) or dgc (36) ; 3) different cryoprotectors were used in the studies ; 4) different freezing and thawing protocols ; 5) and the use of different techniques to measure sperm dnaf. in our study, a rise in dnaf after sperm freeze - thaw cycle was observed corroborating some previous studies. in order to decrease the variability in the present study, only patients who had no diseases, or used no medication were evaluated. besides, only normospermic patients according to who criteria were included in the study since sperm of infertile men are less resistant to freezing process, showing poor results concerning sperm dnaf, compared to fertile men (4, 37). in this study, raw semen was used for comparison between before freezing and post - thaw dnaf, taking into account that raw semen has a strong antioxidant defense that could protect sperm against oxidative stress during cryopreservation, which in turn, could induce an increase on dnaf (38, 39). the present results can be explained by previous evidence that the process of freezing and thawing can lead to ice crystals formation, provoking plasma membrane disruption, organelle and nuclear membrane, reaching cell dna (1, 4). the mechanism by which these alterations occur is still unknown. however, a possible reason for this cryo - induced injury could be oxidative stress (40) and the activation of apoptotic cascades (41). some studies observed better dnaf results by addition of cryoprotectors directly to raw semen, supporting the idea that seminal plasma protects spermatozoa during freeze - thaw cycle, once it is rich in antioxidant enzymes (38, 39). other researchers support that seminal plasma needs to be removed before freezing, otherwise it can damage sperm motility and vitality after thawing (42, 43). also, sperm dnaf rates were compared using raw semen for cryopreservation and capacitated samples. our results showed that sperm preparation previous to cryopreservation had reduced sperm dnaf rates. for sperm capacitation the likely reason why better results were obtained concerning sperm dnaf of capacitated sperm before freezing can be raised on the hypothesis that the morphologically abnormal sperm are more susceptible to dna damage during the freezing process than sperm with normal morphology (37). additionally, head abnormalities and irregular chromatin organization may have altered membrane physical properties and thereby have altered tolerance to cold stress (37). by selecting a subpopulation of spermatozoa through sperm capacitation before freezing, a better quality sample can be obtained after thawing. this procedure allows selecting high quality spermatozoa and the removal of seminal plasma which contains germ cells, not viable, apoptotic and other components that can cause oxidative damage and apoptosis during freeze and thaw procedures (43). several studies have been searching for mechanisms to attenuate deleterious effects caused by cryopreservation (10, 11). some of these attempt for a better sperm preparation before freezing, some for an ideal freeze and thaw protocol and others for better cryoprotector agents. as it is known that oxidative stress is one of the most detrimental factors during cryopreservation, recent studies have been reaching for cryoprotective substances which could minimize this damage (44). due to its exclusive structural composition, such as lack of cytoplasm, which leads to decrease of antioxidant defense and a high proportion of polyunsaturated fatty acid in their membranes, making it more susceptible to lipid peroxidation, deleterious effects of oxidative stress can result in several structural alterations of spermatozoa, such as protein fragmentation, lipid peroxidation and membrane and dnaf (7). this was the first study to focus on the possible cryoprotective effect of leptin on spermatozoa. our results showed that leptin incubation with capacitated spermatozoa before cryopreservation could lead to increase in antioxidant enzymes sod and gpx, but not catalase. however, catalase is known to be very effective in high - level oxidative stress and especially important in case of limited glutathione content or reduced gpx activity (45). a likely mechanism by which leptin can act against oxidative stress is by dissipating the excess energy via thermogenic mechanism and thus, preventing the development of excessive mitochondrial membrane potential (18). in another study, zheng. demonstrated that leptin had anti - apoptotic effects on cardiomyocytes by increasing sod activity. leptin signaling could induce sod2 gene expression and directly activate sod2 promoter in cardiomyocytes (19). interestingly, this effect was specific for sod2 which is mitochondria - located, whereas the cytoplasm - located sod1 remained unchanged following leptin treatment. in our study, the reduction of oxidative stress is likely to be due to a rise of antioxidant enzymes activity. controversially, leptin has shown to induce lipid oxidation in endothelial cells, leading to increased mitochondrial potential and consequent elevation of mitochondrial production of ros (20). in the present study, leptin also potentiated dnaf reduction in frozen - thawed capacitated samples in relation to sperm capacitation. it can be speculated that the reduction of dnaf after leptin incubation could be explained by increased antioxidant defense, minimizing deleterious effects of oxidative stress during freezing and thawing processes. in summary, our results show that leptin has a cryoprotective effect and if added to the sample before freezing could reduce deleterious effect of cryopreservation. however, it remains unclear if the addition of this hormone could add value to in vitro fertilization techniques. sperm dnaf increased after freeze - thaw cycle compared to evaluation with the same fresh raw sample. sperm capacitation and leptin addition to culture media showed a cryoprotective effect on spermatozoa by reducing dnaf and increasing antioxidant enzymes activity. the addition of leptin to capacitated sperm samples before freezing could possibly reduce deleterious effects of cryopreservation. | background : leptin and its receptor are present in spermatozoa ; however, the role of leptin in sperm function is still controversial. our present study aimed at demonstrating the effect of cryopreservation on sperm dna fragmentation (dnaf) and investigating the possible effects of sperm capacitation techniques and leptin in vitro incubation on frozen - thawed sperm dnaf and oxidative stress.methods:samples of 45 normospermic men attending for infertility investigation at vida centro de fertilidade, rio de janeiro, brazil, were frozen and thawed with or without capacitation and leptin incubation prior to freezing. sperm dna fragmentation was evaluated by sperm chromatin dispersion assay before and after cryopreservation and oxidative stress parameters were measured by spectrophotometry with and without leptin incubation. statistical analysis was performed using paired t test to compare dnaf between groups before and after freeze - thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. statistical significance was considered when p0.05.results:our results revealed a significant post - thaw rise in sperm dnaf compared with fresh samples (p=0.0003). sperm dnaf was significantly reduced when sperm capacitation was performed before freezing, when compared to those frozen with no previous capacitation (p=0.01). the addition of leptin to capacitated sperm before freezing reduced dnaf (p<0.0001) and enhanced superoxide dismutase (p=0.001) and glutathione peroxidase (p=0.02) antioxidant enzymes activity.conclusion:the addition of leptin to capacitated sperm can improve sperm dna quality following cryopreservation, possibly by inducing the activity of certain antioxidant enzymes. |
studies were performed in 14 lean (bmi 22 1 kg / m), healthy volunteers (7 male and 7 female), aged 1835 years without a personal or family history of hypertension or diabetes and receiving no medications. the studies were not scheduled to correspond to a particular phase of the menstrual cycle in the 7 females because insulin clamp measured insulin sensitivity in lean, healthy young women is not significantly affected by cycle phase (19). the study protocol was approved by the university of virginia institutional review board, and each subject gave written consent. all subjects had normal results of physical examination, liver function tests, fasting glucose, and lipid profile. eight of the 14 subjects performed a treadmill exercise test after an overnight fast, with the standard bruce protocol used to determine vo2max. each participant began walking at an initial velocity of 60 m / min, with velocity increasing by 10 m / min every 3 min (the duration of each stage) until volitional exhaustion. metabolic measures were obtained through standard open - circuit spirometry (viasys vmax 229 ; carefusion, yorba linda, ca). vo2 peak was determined as the highest 1-min oxygen consumption value obtained. for the insulin clamp, each of the 14 subjects was admitted to the general clinical research center the evening before the study. after an overnight 12-h fast, volunteers were studied while supine according to the following protocol : a brachial arterial catheter and a retrograde median antecubital venous catheter were placed for blood sampling. in the contralateral arm, a venous catheter was placed for the infusion of lipid, glucose, insulin and definity microbubbles. an infusion of 20% lipid emulsion (abbott laboratories, chicago, il) was initiated at a rate of 45 ml / h for 1 h and then at 30 ml / h for 4 h. a second venous catheter was placed in the same arm for an infusion of heparin at a rate of 0.2 units / kg / min. both these infusions continued for 5 h. after 150 min of lipid infusion, paired arterial and venous samples were taken every 10 min three times for measurement of plasma glucose, insulin, ffas, and lactate. forearm blood flow was measured after each set of arterial and venous samples by doppler ultrasound. at 175 min of lipid infusion, ceu measurements of forearm muscle mbv were initiated and continued for 40 min as described below. at time 180 min, a primed 3 mu / min / kg insulin infusion was started in the arm contralateral to the arterial catheter. this infusion was decreased by 0.2 mu / min / kg each min during the next 10 min and then maintained at a rate of 1 mu / kg / min for the next 110 min. arterial plasma glucose was maintained at basal levels with a variable rate 20% glucose infusion (euglycemic clamp) (20). whole - body glucose disposal at steady state (80120 min of the clamp) was estimated from the glucose infusion rate (gir) required to keep arterial glucose constant. forearm glucose and ffa balances (net uptake or release) were determined from the arteriovenous concentration difference obtained every 10 min from 150 to 300 min of lipid infusion. to avoid interference with the ceu images, no arterial or venous samples mbv was measured with a sonos 7500 ultrasound system (philips medical systems, bothell, wa) with harmonic imaging during the continuous infusion of perfluorocarbon gas filled lipid microbubbles (definity ; lantheus medical imaging co., billerica, ma), as described previously (2). ceu images were downloaded to an off - line image analysis system (q - laboratory ; philips medical systems, andover, ma). background - subtracted acoustic intensity was measured from a region of interest around the deep forearm flexor muscles, as described previously (12,21). changes in mbv with time during insulin exposure were calculated from the acoustic intensity expressed as mean decibels. brachial artery blood flow was measured at baseline and every 20 min from 40 to 120 min of the insulin clamp with the sonos 7500 ultrasound system with a linear - array transducer and a transmit frequency of 12 mhz. two - dimensional imaging of the brachial artery was performed in the long axis approximately 10 cm proximal to the antecubital fossa. images were triggered to the r wave of the cardiac cycle, and the brachial artery diameter was measured with online video calipers. at the same location, the time average mean blood velocity was measured with pulsed - wave doppler ultrasound. brachial artery mean blood flow was calculated according to the following equation : q = v (d/2), where q is brachial blood flow, v is mean brachial artery blood flow velocity, and d is brachial artery diameter. insulin was measured with a solid - phase two - site chemiluminescent assay (diagnostic products corporation, los angeles, ca). the ffa level was measured with a colorimetric assay (waco diagnostics, richmond, va). glucose and lactate were measured in duplicate with a ysi 2300 analyzer (yellow springs instruments, yellow springs, oh). baseline coagulation parameters, liver function tests, and fasting lipid profile were performed by standard assays in the university of virginia clinical chemistries laboratory. forearm balances for glucose, ffas, and insulin were calculated as follows : balance = ([a ] [v ]) f, where [a ] and [v ] are arterial and venous concentrations and f is forearm blood flow in milliliters per minute per 100 ml forearm volume. a positive balance corresponded to a net uptake, whereas a negative balance signaled a net release of substrate. for calculation of glucose balance, blood flow was used ; for ffa and insulin, we used forearm plasma flow, derived as blood flow (1 hematocrit). the clearance of insulin was calculated as the product of the extraction fraction of insulin, derived as ([a ] [v])/[a ], and forearm plasma flow per 100 ml forearm volume. comparisons were made by paired student t test for the following : between mean baseline (30 to 10 min) and mean steady state (80 to 120 min) values for forearm glucose uptake (fgu), forearm insulin uptake (fiu), ffa balance, insulin clearance and total forearm blood flow ; between baseline and 25 min for ceu acoustic intensity ; between 0 and 30 min for arterial ffa concentration ; and between the highest and lowest tertile of percentage mbv change. pearson product - moment correlation coefficient was computed to determine the relationship between specific variables. for all analyses, p < at time 180 min, a primed 3 mu / min / kg insulin infusion was started in the arm contralateral to the arterial catheter. this infusion was decreased by 0.2 mu / min / kg each min during the next 10 min and then maintained at a rate of 1 mu / kg / min for the next 110 min. arterial plasma glucose was maintained at basal levels with a variable rate 20% glucose infusion (euglycemic clamp) (20). whole - body glucose disposal at steady state (80120 min of the clamp) was estimated from the glucose infusion rate (gir) required to keep arterial glucose constant. forearm glucose and ffa balances (net uptake or release) were determined from the arteriovenous concentration difference obtained every 10 min from 150 to 300 min of lipid infusion. to avoid interference with the ceu images, no arterial or venous samples were collected from 180 to 210 min of lipid infusion. mbv was measured with a sonos 7500 ultrasound system (philips medical systems, bothell, wa) with harmonic imaging during the continuous infusion of perfluorocarbon gas filled lipid microbubbles (definity ; lantheus medical imaging co., billerica, ma), as described previously (2). ceu images were downloaded to an off - line image analysis system (q - laboratory ; philips medical systems, andover, ma). background - subtracted acoustic intensity was measured from a region of interest around the deep forearm flexor muscles, as described previously (12,21). changes in mbv with time during insulin exposure were calculated from the acoustic intensity expressed as mean decibels. brachial artery blood flow was measured at baseline and every 20 min from 40 to 120 min of the insulin clamp with the sonos 7500 ultrasound system with a linear - array transducer and a transmit frequency of 12 mhz. two - dimensional imaging of the brachial artery was performed in the long axis approximately 10 cm proximal to the antecubital fossa. images were triggered to the r wave of the cardiac cycle, and the brachial artery diameter was measured with online video calipers. at the same location, the time average mean blood velocity was measured with pulsed - wave doppler ultrasound. brachial artery mean blood flow was calculated according to the following equation : q = v (d/2), where q is brachial blood flow, v is mean brachial artery blood flow velocity, and d is brachial artery diameter. insulin was measured with a solid - phase two - site chemiluminescent assay (diagnostic products corporation, los angeles, ca). the ffa level was measured with a colorimetric assay (waco diagnostics, richmond, va). glucose and lactate were measured in duplicate with a ysi 2300 analyzer (yellow springs instruments, yellow springs, oh). baseline coagulation parameters, liver function tests, and fasting lipid profile were performed by standard assays in the university of virginia clinical chemistries laboratory. forearm balances for glucose, ffas, and insulin were calculated as follows : balance = ([a ] [v ]) f, where [a ] and [v ] are arterial and venous concentrations and f is forearm blood flow in milliliters per minute per 100 ml forearm volume. a positive balance corresponded to a net uptake, whereas a negative balance signaled a net release of substrate. for calculation of glucose balance, blood flow was used ; for ffa and insulin, we used forearm plasma flow, derived as blood flow (1 hematocrit). the clearance of insulin was calculated as the product of the extraction fraction of insulin, derived as ([a ] [v])/[a ], and forearm plasma flow per 100 ml forearm volume. comparisons were made by paired student t test for the following : between mean baseline (30 to 10 min) and mean steady state (80 to 120 min) values for forearm glucose uptake (fgu), forearm insulin uptake (fiu), ffa balance, insulin clearance and total forearm blood flow ; between baseline and 25 min for ceu acoustic intensity ; between 0 and 30 min for arterial ffa concentration ; and between the highest and lowest tertile of percentage mbv change. pearson product - moment correlation coefficient was computed to determine the relationship between specific variables. for all analyses, p < table 1 gives the clinical characteristics of all 14 subjects studied broken down into the two groups who either did not (group 1) or did (group 2) have vo2max measured. all were normotensive, were nonobese, and had normal values for serum lipids. before beginning the insulin clamp, the 3-h intralipid and heparin infusion had raised the arterial plasma ffa concentration to 2.0 0.2 mmol / l. plasma glucose averaged 5.1 0.1 mmol / l, and forearm blood flow was 6.5 0.4 ml / min/100 ml. the basal forearm glucose and ffa balances averaged 0.65 0.1 and 0.1 0.2 mol / min/100 the basal arterial insulin concentration was 37 5 pmol / l, which significantly (p < 0.001) exceeded that in the forearm venous blood (32 4 pmol / l), resulting in a significant fiu of 17.8 3.0 fmol / min/100 ml. the measured phenotypic characteristics of all subjects studied the intravenous insulin infusion raised arterial insulin concentrations from 36 to 251 11 mmol / l during the baseline period and was maintained within 5% of baseline throughout. arterial plasma ffa concentrations had declined sharply by 30 min of insulin infusion (p < 0.001) and plateaued at 1.1 mmol / l by 80 min. forearm blood flow was unchanged from basal during the insulin infusion (6.5 0.4 vs. 6.7 0.5 ml / min/100 ml). fgu during the last 40 min of the insulin infusion averaged approximately sixfold the basal value (0.65 0.1 vs. 3.8 0.8 mol / min/100ml ; p < 0.01) and ranged across subjects from 0.2 to 9.9 mol / min/100 ml. ffa balance was unchanged (0.1 0.2 vs. 0.1 0.3 mol / min/100 ml). the whole - body gir during the last 40 min of the insulin clamp ranged from 11 to 68 mol / kg / min (average 31.3 4.6 mol / kg / min). there was the expected strong correlation (r = 0.876 ; p < 0.001) between fgu and the gir (fig. 1a), each measured during the last 40 min of the clamp, underscoring the role of skeletal muscle in body glucose disposal under hyperinsulinemic conditions. fiu also rose significantly during hyperinsulinemia (18 3 to 80 12 fmol / min/100ml ; p < 0.01), whereas forearm clearance of insulin trended downward (0.49 0.09 vs. 0.33 0.05 ml / min/100 ml ; p = 0.14). on average, there was no change in the mbv observed between baseline and 30 min of insulin infusion (5.4 1.0 vs. 5.8 1.1 acoustic intensity units), consistent with ffa elevation blocking the vascular effect of insulin to increase mbv. we did, however, observe that the microvascular responses varied considerably across individuals, from a 39% decline to a 69% increase in mbv. furthermore, there was a strong correlation between the increase in fgu and the percentage change in mbv (r = 0.80 ; p < 0.01), consistent with a positive relationship between perfusion volume and the metabolic effect of insulin (fig. 1b). likewise, there was a significant correlation between the gir and percentage change in mbv (fig. there was no significant correlation between forearm blood flow and gir (r = 0.16 ; p = ns). likewise, we found no correlation between percentage change in mbv and the plasma ffa concentration measured during the first hour of the insulin clamp (r = 0.09 ; p = ns). a : the correlation between the gir and fgu, each measured during the last 40 min of the euglycemic insulin clamp. b : the correlation between fgu measured during the last 40 min of the insulin clamp and the percentage change of mbv measured during the first 30 min of insulin infusion. c : the correlation of gir measured during the last 40 min of the insulin clamp with the percentage change in mbv during the initial 30 min of insulin infusion. this study was not powered to address whether there was an effect of sex on this response. we did, however, observe a significant correlation between percentage change in mbv and fgu in both women (r = 0.77 ; p < 0.05) and men (r = 0.88 ; p < 0.01) and a correlation between percentage change in mbv and whole - body gir that was not significant in women (r = 0.60 ; p = ns) although it was nearly significant in men (r = 0.74 ; p = 0.06). this suggests that the relationship between mbv and muscle glucose uptake holds for both sexes. comparing the five subjects in the highest tertile with the five in the lowest tertile of mbv percentage change, we found that fgu was markedly higher (6.5 1.2 vs. 0.7 0.3 mol / min/100 ml ; p < 0.01) in the group that had the more responsive microvasculature (fig. fiu averaged nearly threefold greater in that group(92 29 vs. 32 7 fmol / min/100 ml ; p = 0.08), but this difference was of borderline significance (fig. 2). a : the mean sem of fgu observed in the five individuals who either had no increase in mbv or had an actual decline (lowest tertile) versus that in the five individuals who had the greatest percent increase in mbv (highest tertile). b : the mean sem changes in fiu between baseline and the last 40 min of the insulin clamp in the same two groups. fav, forearm volume. of the 14 subjects, 8 agreed to have vo2 max measured on a separate day from the clamp study. the mean vo2max was 43 4 ml / min / kg and ranged from 29 to 63 ml / min / kg. compared with the other 6 subjects there were no differences in these 8 in bmi, age, fasting insulin or glucose, or the plasma concentrations of ffa (1.05 0.16 vs. 1.14 0.13 mmol / l), insulin (250 15 vs. 252 18 pmol / l), or glucose (4.9 0.1 vs. 5.1 0.1 mmol / l) during the last 40 min of the clamp. we observed that in this subgroup the ranges of responses to insulin of gir (2.012 mg / min / kg), fgu (0.1 to + 8.0 mmol / min/100 ml), and mbv percentage change (40 to + 40) were comparable to those of the group as a whole (fig. there was again the expected correlation (r = 0.823 ; p < 0.02) between fgu and gir (fig. 3c). finally, in this subgroup we again found a significant correlation (r = 0.743 ; p < 0.05) between the percentage change in mbv and fgu (fig., there was no correlation between changes in blood flow (either absolute or percentage change from basal) and gir or vo2max, suggesting that under these experimental conditions regulation of mbv is more closely linked than is total blood flow to insulin s metabolic effect. a : the correlation between whole - body gir and fgu, each measured during the last 40 min of the insulin clamp, in eight individuals in whom fitness was assessed by vo2max. c : the correlation between vo2max and the percentage change in mbv seen during the first 30 min of insulin infusion in the same individuals. d : the correlation between fgu measured during the last 40 min of the insulin clamp and the percentage change of mbv measured during the first 30 min of the insulin infusion in the same individuals. previously, we reported that euglycemic hyperinsulinemia significantly enhanced forearm mbv in healthy humans (3) and that metabolic insulin resistance, such as occurs with obesity (12) and with lipid infusion (16), blunts insulin s action to increase mbv. in those studies we did not directly measure muscle glucose uptake, however, and mbv was measured at baseline and after 2 h of hyperinsulinemia. because insulin s microvascular action in muscle occurs within 1530 min (2) of infusion and because we (22) and others (23,24) have hypothesized that insulin s access to muscle interstitium is rate limiting for insulin s metabolic action in muscle, we wanted to compare early insulin - induced changes in mbv with subsequent muscle glucose metabolism and to do so in the setting of physiological ffa elevation to levels observed in the postprandial state in insulin - resistant individuals. in this study, changes in mbv were measured during the first 30 min of hyperinsulinemia and forearm glucose metabolism between 80 and 120 min. in the current study, by maintaining postprandial plasma ffa concentrations (1.1 mmol / l), we found that both muscle mbv and fgu varied over a wide range in healthy young adults. most intriguingly, there was a strong correlation between insulin s early microvascular action and subsequent metabolic action in muscle, underscoring the physiological importance of microvascular insulin sensitivity to muscle glucose metabolism. beyond that, we noted that the level of fitness appeared to impact both microvascular and metabolic responses to insulin during the lipid infusion. this is of particular interest in light of recent reports that both an acute bout of endurance exercise (17) and overall fitness (18,25) interfere with the ability of lipid infusions to diminish insulin sensitivity. this suggests that muscle microvasculature, like muscle itself, responds to exercise and training to preserve insulin responsiveness. ffas are thought to induce muscle insulin resistance at least in part through the activation of an inflammatory response (9), which itself may result from increased oxidative stress (26). in humans, acutely raising plasma ffa level (as was done here) has been observed to enhance nuclear factor-b activity in circulating mononuclear cells and plasma concentrations of macrophage migration inhibition factor, consistent with an acute inflammatory response. this was accompanied by a decrease in brachial artery flow - mediated dilation consistent with an impact of raised ffa level, with or without inflammation, on endothelial function (10). exercise has repeatedly been shown to increase production of reactive oxygen species ; however this reactive oxygen species production appears to play a synergistic role in activating and regulating antioxidant pathways (27), including manganese superoxide dismutase (28), glutathione peroxidase (29), and heme oxygenase-1 (30). this tightly regulated bidirectional redox signaling appears to occur in part through the nf-b and mitogen - activated protein kinase (27) signal transduction pathways. the observation that fitness mitigates the inhibitory effect of ffas on muscle s microvascular response to insulin suggests that the muscle vasculature of fit volunteers has developed a capacity to protect against oxidative stress induced by ffa infusion. ffas have been shown in rats to impair endothelial cell nitric oxide production acting through the inhibitor of b kinase pathway (31) and to impair insulin - induced nitric oxide production and leg blood flow changes in humans (32). we have shown that insulin s effect to increase mbv is blocked by inhibition of nitric oxide synthase. the greater response of mbv to insulin in fit individuals seen here suggests that fitness may abrogate the effect of ffas to inhibit vascular nitric oxide production. in the current study, we observed a significant uptake of insulin by forearm muscle under both basal and hyperinsulinemic conditions. the basal fiu and clearance of insulin observed here were not different than we reported previously in healthy controls not receiving lipid (2). likewise, insulin uptake by muscle during the clamp was comparable to that which we reported earlier (2). we noted however that there was a wide range of fiu among subjects. as was seen with glucose, there appeared to be greater uptake among persons who responded to insulin by increasing mbv (fig. 1). among the 8 subjects who had vo2max measured, the mean rate of fiu during the last 40 min of the insulin clamp ranged from 22 to 112 fmol / min/100 ml. we divided these 8 subjects into two groups, four with high vo2max and four with low vo2max (average 50 4 vs. 36 3 ml / min / kg) and compared fiu rates. fiu during the clamp was nearly twofold greater in the 4 physically fit individuals (82 16 vs. 46 9 ; p = 0.06). this suggests that enhanced insulin delivery in physically fit individuals may contribute to the increased skeletal muscle insulin sensitivity seen with increasing fitness. as noted in results, fgu was much greater in subjects with good microvascular responses to insulin, as reflected by increases in mbv. in four subjects the mbv actually declined below basal level during insulin infusion. we had previously observed this behavior during intralipid infusion in rat studies and found that the decline could be prevented by coinfusion of bq123, an endothelin a receptor blocker (33). this led us to suggest it may be due to selective inhibition by ffas of endothelial nitric oxide synthase activation with preservation of insulin s action to increase endothelin 1 production in the microvasculature, as has been observed in the zucker (fa / fa) rat (34) and in several in vitro studies (35,36). a similar decrease in microvascular perfusion was reported for human cardiac muscle in response to meal ingestion in diabetic patients but was not seen in healthy volunteers (37). a limitation of the current study is that we do not have measures of mbv before beginning the lipid infusion. this is due to the limitation of the amount of definity that can be infused in humans during a single study. in rats, another limitation relates to whether fitness per se or some other lifestyle difference associated with fitness explains the correlation between vo2max and insulin - induced changes in mbv. in summary, we have observed that during mild, physiological increases in plasma ffa concentrations, both metabolic and vascular insulin sensitivities vary widely in otherwise healthy humans. this is consistent with a role for insulin s microvascular action in modulating insulin s metabolic action in muscle. impaired microvascular responses may also diminish muscle insulin uptake, perhaps accounting in part for the muscle insulin resistance seen. finally, physical fitness appears to blunt the inhibitory effect of raising plasma ffa on insulin - induced muscle microvascular recruitment and glucose. | objectiveto test whether early, insulin - mediated microvascular recruitment in skeletal muscle predicts steady - state glucose metabolism in the setting of physiological elevation of free fatty acid concentrations.research design and methodswe measured insulin s microvascular and metabolic effects in 14 healthy young adults during a 2-h euglycemic insulin clamp. plasma free fatty acid concentrations were raised (intralipid and heparin infusion) for 3 h before the clamp and maintained at postprandial concentrations during the clamp. microvascular blood volume (mbv) was measured by contrast - enhanced ultrasound (ceu) continuously from baseline through the first 30 min of the insulin clamp. muscle glucose and insulin uptake were measured by the forearm balance method.resultsthe glucose infusion rate (gir) necessary to maintain euglycemia during the clamp varied by fivefold across subjects (2.512.5 mg / min / kg). the early mbv responses to insulin, as indicated by ceu video intensity, ranged widely, from a 39% decline to a 69% increase. during the clamp, steady state forearm muscle glucose uptake and gir each correlated significantly with the change in forearm mbv (p < 0.01). to explore the basis for the wide range of vascular and metabolic insulin sensitivity observed, we also measured vo2max in a subset of eight subjects. fitness (vo2max) correlated significantly with the gir, the forearm glucose uptake, and the percentage change in mbv during the insulin clamp (p < 0.05 for each).conclusionsearly microvascular responses to insulin strongly associate with steady state skeletal muscle insulin - mediated glucose uptake. physical fitness predicts both metabolic and vascular insulin responsiveness. |
modern out - of - hospital emergency medical services (ems) systems, as we have come to recognize them today, were established in the 1960s and 1970s when a cadre of intrepid physicians ventured into the streets and later published their successful experiences with lifesaving approaches to managing acute coronary syndromes, trauma care, and cardiopulmonary arrest on - scene [1 - 3 ]. although physician - staffed ambulance services had been in place in many venues worldwide for more than a century, the late 20 century evolution of prehospital care was highlighted by documentation of life - saving outcomes in those first modern ems programs and their use of invasive advanced life support (als) procedures including prehospital endotracheal intubation (eti) and intravascular (i.v.) these life - saving reports helped to propel the widespread adoption of ems systems and the concomitant introduction of specially - trained (non - physician) emergency medical technicians called paramedics [1 - 5 ]. eventually nursing personnel also ventured into the realm of on - scene emergency response, particularly in the arena of air medical services. this evolution in out - of - hospital care was especially remarkable in that the formal training of these non - physician personnel included those advanced care interventions such as eti and i.v. drug administration, interventions traditionally provided in the in - hospital setting by expert physician specialists [1 - 9 ]. paramedic skill portfolios ranged from basic spinal immobilization and extremity splinting to the more advanced skills of electrocardiographic (ekg) interpretation, defibrillation attempts, eti, i.v. catheter placement and even pericardiocentesis and tracheotomies in some communities. the skill of eti had become the definitive airway control for most critically ill and injured patients, be they in the operating room, in the early phases of an intensive care unit (icu) hospitalization, or in the out - of - hospital setting [2 - 9,11 ]. the presumed presence of significant physiological derangements (e.g., hypoxemia, hypercarbia, hypoperfusion) in cardiopulmonary arrest, head injury and hemorrhagic states made eti an intuitive procedure to perform as soon as feasible in the critically ill and injured. in addition, there were other clinical care imperatives (e.g., airway protection, ventilatory control, end - tidal carbon monoxide monitoring, drug administration and airway suctioning) that drove a strong philosophy that ems personnel should provide a definitive airway as soon as possible in the out - of - hospital setting for cardiopulmonary arrest, severe trauma and other life - threatening emergencies [2 - 9,11 ]. nevertheless, although these invasive skills were now being provided by paramedics and nurses, for the most part they were still being delegated under the direction of accountable physician supervisor experts in out - of - hospital care. early studies conducted in ems systems with intensive, expert physician supervision, comprehensive training programs and on - scene supervision of ems personnel reported extremely high rates of successful eti for both children and adults [2 - 8,13 - 15 ]. in most of these studies, success was defined not only by accurate anatomic placement of the endotracheal tube (ett), but also by absence of significant complications [3 - 7 ]. moreover, prehospital eti was soon correlated with positive outcomes particularly in the most dire of circumstances. for the most part, prehospital eti has usually been performed in cardiopulmonary arrest cases and in the most severely injured trauma patients with significant physiological impairment (unconscious) and, generally, no gag reflex. as a result, the procedure can be relatively easy to perform by highly - experienced care providers. however, using unqualified univariate analysis, eti is typically performed in those patients with a high - risk of associated morbidity and mortality and thus can be simplistically correlated with a poor outcome [16 - 20 ]. paradoxically, in some selected ems systems, eti has actually been correlated positively with survival, particularly in cases of post - traumatic circulatory arrest. in turn, this paradoxical finding infers a likely value of eti in these worst - case scenarios. however, despite intuitive biases and impressive inferential studies indicating the positive effects of prehospital eti in certain settings, another evolving body of studies and experiences has unveiled a detrimental effect of prehospital eti or, at least, no significant advantage to providing the procedure [17,20,22 - 29 ]. most notably, a controlled clinical trial conducted in the 1990s in a pediatric population generated significant concern about prehospital eti in that vulnerable population and subsequent studies in adult head injury patients amplified that concern. in the pediatric eti trial, 830 children (age 12 years or younger) were studied over a three - year period. although not statistically significant, survivors with positive neurological outcomes were slightly more frequent (92 of 104 ; 23%) in those managed with bag - valve - mask (bvm) devices (23%), versus 85 of 416 (20%) receiving eti. in a subsequent case control study of severely head - injured patients receiving eti that was facilitated by rapid sequence induction (rsi), outcomes were worse for patients receiving the procedure versus those with similar injuries not receiving it. also, in deference to other studies indicating a survival advantage to eti in post - traumatic circulatory arrest, the on - going univariate association of eti with mortality in recent studies, though predictable, has fueled the debate that eti should no longer be used in the out - of - hospital setting [16 - 18,23 ]. adding to this debate has been the concern over interruptions in well - performed chest compressions during cardiopulmonary resuscitation (cpr), the key factor in restoring return of spontaneous circulation and eventual survival following cardiac resuscitation. it is argued that pausing to intubate could, therefore, be detrimental under these circumstances. in turn along with its lowered prioritization in cardiac arrest management, it has been argued that, overall, there is no strong evidenced - based support for eti in terms of survival advantage. so despite the logical value of performing it in critically ill and injured patients, many have argued that a true value can not be demonstrated, particularly in children. regardless of this evolving sentiment to avoid prehospital eti altogether and even consider it as a deleterious procedure, that evidence - based position may indeed be overly simplistic. in the ensuing discussion, it will be delineated how several under - recognized confounding variables have a major impact on the performance of this skill and even related outcomes. these variables include non - intuitive factors, such as how the ems providers are deployed or how they have been trained to ventilate [32 - 44 ]. these concepts and how they relate to the success of prehospital eti for the critically ill and injured will be addressed in the rest of this article. it is hoped that by being provided these perspectives, one can better delineate the circumstances in which eti should be utilized and those in which it should truly be discouraged. as previously stated, the original ems programs that first published success with paramedic - staffed responses generally reported extremely high rates of success with prehospital eti placement [2 - 10,15 ]. in retrospect, when examining the differences in systems that have or have not had successes in eti, it appears that several factors are actually strong determinants of paramedic and nursing proficiency in the skill of eti. these determinants include : 1) the quality, orientation and types of experiences in the initial training ; 2) the frequency of performance ; and 3) on - scene oversight and supervision of eti performance [3 - 6,12,13,29,32 - 36 ]. in contrast to the typical operating room training experience, the skill of eti performed in the emergency care setting, and particularly in the out - of - hospital environment, is wrought with unique challenges. these challenges range from vomit - flooded airways and ground - level patient positions to ambient lighting and oro - pharyngeal injuries. with full stomachs, relaxed esophageal sphincters and inadvertent gastric insufflation from bvm or mouth - to - mouth ventilation, it is commonplace to approach an airway welled - up with vomit in a circumstance with often less - than - adequate (or delayed) suctioning. in turn, this often requires the ability to intubate almost instantly without adjuncts. unlike the controlled in - hospital environment, in a sunny, bright outdoors setting, the ambient light causes glare and pupillary constriction for the rescuers. tricks of the trade, such as placing a coat or blanket over one s head (and the head of the patient) in order to create a makeshift darkened room akin to an old - time photographer s camera hood. in contrast, even in the dark of night, heavy rain or awkward confined spaces may pose their own barriers to easily visualizing vocal cords. therefore, many of the classical techniques used by other practitioners in more traditional settings would not be as effective in the fast - paced, poorly controlled and mobile prehospital settings where resources and support are limited (figure 1).figure 1 endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. paul pepe). in turn, a key to successful ems intubation in the out - of - hospital setting is the street - wise experience of expert highly - experienced medical trainers and ems medical directors who not only understand these principles, but also are themselves facile in such techniques in the out - of - hospital setting. even if initial training techniques are expert and well - taught, both in the classroom and on - scene, frequency of performance is a critical factor. for example, studies have shown the success rates for eti can be related to the deployment strategy of the ems system. in ems systems using tiered ambulance deployments in which paramedics (als providers) are spared for the most critical calls, many fewer paramedics are needed on the roster and the individual experience of each paramedic, including frequency of eti performance, can be enhanced dramatically. accordingly, this approach has been correlated with improved success rates in terms of eti performance. while eti skills may deteriorate a little with a hiatus from practice, collective experience has demonstrated that most prehospital personnel who have performed eti a hundred times or more in the out - of - hospital setting may still be able to perform the technique quite well despite the hiatus. however, the key issue is getting to that threshold of experience and this prerequisite goal requires high exposure and frequent performance. unfortunately, that level of performance is not always achieved in most ems systems today. as an example, for a five - year veteran paramedic to have achieved a successful eti over 100 times, it would mean successful performance of that procedure at least 20 times a year for five years. so if eti experience were to be shared with a paramedic partner, the implication is that this particular team would need to face 40 eti situations a year on their particular ambulance and shift. in fact, accounting for sick time, vacation time and other factors, it typically takes 5 to 6 fulltime equivalent paramedics to staff one of those two positions and thus 1012 different paramedics will be needed just for that one ambulance around the clock. therefore, that particular response unit would need to face approximately 200 to 250 eti cases a year for each als provider to get 20 opportunities to intubate. considering that cardiac arrest, respiratory distress and major trauma cases requiring eti constitute only 23% of all ems on - scene emergency responses, the ambulance in question would need to experience nearly 10,000 ems incidents a year overall. in most ems system configurations, this level of volume would be a logistical - temporal impossibility for a single ambulance. unless alternate deployment strategies were to be utilized, frequent exposure to eti cases would be clearly limited. specifically, in some communities, paramedics (or other types of als personnel, such as doctors or nurses) are spared from the majority of ems responses. instead of als providers, basic emergency medical technicians (emts) trained to do the non - invasive procedures such as spinal immobilization and splinting are used for most of the responses. under such circumstances, overall staffing could this would permit more frequent exposure to critical illness and injury for the individual paramedics (als providers). the same concept would apply to nurses or apprentice physicians who staff ambulances and air medical units, particularly in some european countries. the fact that air medical units are typically triaged only to the most critical cases means that those als providers staffing the helicopters are part of a deployment strategy that enhances skill use. using this tiered approach, individual paramedics (als personnel), nurses or doctors each get more chances to perform an eti. while there is great variation from one city to another, on average a city with a population of 1 million in the u.s. this volume of cases might predict two or three thousand potential circumstances for eti each year. to optimize individual paramedic exposure, it would be best to limit the number of paramedic (als) ambulances to a maximum of 10 ambulances (250 eti exposures per ambulance per year 10 ambulances covers 2 to 3 thousand cases). in this circumstance, a cadre of 100 to 120 paramedics might be required for the 10 paramedic - staffed units. in a contrast, in a system experiencing 100,000 ems responses a year and using all - paramedic staffing, 35 to 40 ambulances would typically be required minimally and thus 400 to 500 paramedics would be needed. this all - als provider approach decreases individual exposure to eti attempts at least 4 to 5-fold. to make matters worse, in some cities, additional paramedics are also placed on first - responder vehicles such as responding fire engines. in turn, this further compounds the infrequency of exposure for individuals. moreover, some ambulances are situated in lower call volume areas than others, creating even less exposure to eti opportunities. fortunately, the great majority (85 to 95%) of ems incidents do not require an als provider (e. g., authorized physician, nurse, paramedic) and can be managed by basic emts. in turn, using well - established and well - documented dispatch triage protocols, paramedics (als providers) can be spared and basic emts (basic life support [bls ] providers) are deployed directly to manage the cases. in other situations, after an initial paramedic (als) response is made, the basic emt ambulance can be called in to transport the less critical patients thus freeing up paramedics (als providers) for the more critical cases. not only does this type of system configuration permit the need for fewer als personnel, but it also improves response intervals because paramedics are not tied up transporting patients and are thus more available. ironically, by having fewer paramedics, paramedic response can be improved. beyond on - scene procedures and moving the patient from the scene, the time to transport, provide hospital transition, create a record and then return to the primary response territory is the greatest deterrent to the availability of ambulance crews and thus a factor in compromised response times. not surprisingly then, the original ems systems reporting excellent paramedic track records with eti were largely this type of tiered response system with staffing configurations that utilized basic emts for the majority of responses and spared the much smaller cadre of relatively busy paramedics for the more critical calls, therefore creating more opportunities for eti skills usage. furthermore, the paramedics in these systems rapidly achieved experience seeing many dozens of cases per year and they eventually became reliably facile. in turn, as they became exceptionally facile, they deferred eti attempts to new trainees. as a result, in these sophisticated ems systems, the lesser - experienced medics rapidly developed their own skills even faster. veterans also maintained their skills by teaching, supervising and getting to attempt and perform the more difficult intubations when the more novice personnel could not place the tube. and other countries, the majority of ems systems actually utilize all - paramedic (all - als) staffing on their ambulances. in addition, many first - responder crews often supplement ambulance response with additional paramedics (als providers) staffing the first response vehicles as well. therefore, it is no surprise that paramedics may not perform eti as well as their forerunners 40 years ago. despite the described impact of using an all - paramedic system tier in those all - als systems by creating a team of supervisors, field training officers, or expert physician responders who routinely respond to critical calls. depending upon the geography, vertical (high - rise) challenges, and traffic, it would be wise to create a small number of senior personnel who can respond across a designated territory (or even into a fellow senior officer s territory for back - up) as a modified approach to ensure high level skills performance. just as there may be 10 or so battalion fire chiefs in a city of a million residents spread out over a large geographical territory, staffing and responding a similar number of senior ems personnel into high level cases could be another alternative and one that is now being adopted by many progressive ems systems. finally, even with appropriate, tailored initial training and tiered response systems with a high frequency of performance for individual paramedics, if the on - scene medics in training are not properly supervised, they may still develop bad habits in a vacuum. it is critical to reinforce what constitutes a proper technique (e. g., sniffing position in those at low risk of neck injury) and to provide renewed coaching in the actual patient care setting, especially in terms of confirmation of tube placement and proper ventilatory techniques. in most ems systems that provide high rates of eti success, in - field medical directors, highly - experienced ems supervisors and well - coached veteran paramedics are the norm. even if paramedics or other prehospital care providers are expertly trained, highly - skilled, highly - experienced and highly - supervised performers of intubation for both adults and children, their ventilatory techniques may still adversely affect outcome [25,37 - 39 ]. the types of patients most likely to need eti are those with cardiac arrest, chronic lung disease and severe post - traumatic shock conditions. yet these patients are also the most vulnerable to the detrimental cardiovascular effects of the positive pressure breaths that are being delivered through the ett. despite the basic physiological principle that ventilation should match perfusion (blood flow), over the years, in many venues, ems personnel have been trained traditionally to aggressively ventilate patients, usually with the ill - advised rationale that such an approach was the way to ensure oxygenation and offset metabolic acidosis. even with more judicious training, however, emergency workers can still have the tendency to over - zealously ventilate such patients in the heat of the emergency. ironically, while such patients in deep shock actually require infrequent breaths and a lesser minute ventilation, once the ett is placed, they may now receive excessive levels of assisted breathing, not only because of some unsound rote training, but also because of adrenaline - modulated behaviors. accordingly, it is now speculated that low national survival rates for out - of - hospital cardiac arrest and the negative outcomes of several prehospital clinical trials may have been, in part, the result of uncontrolled ventilatory rates using positive pressure breaths. for example, in the study of severe traumatic brain injury (tbi) in which rsi - facilitated eti was associated with worse outcomes, a key correlation with mortality was the finding hyperventilation, defined as an arterial pco2 < 24 mmhg. while one might suspect that these negative outcomes may, therefore, be caused by effects of respiratory alkalosis, such as myocardial depression, cerebral vasoconstriction and a left shift in the hemoglobin dissociation curve, it is most likely that the low arterial pco2 is simply a surrogate variable for overzealous positive pressure ventilation [37 - 39 ]. as aufderheide and colleagues have shown, despite aggressive, targeted re - training on respiratory rates and delivery techniques, paramedics still overzealously ventilate and prolong the duration of positive pressure breaths in the adrenaline - charged environment of a critical emergency. it is likely that this scenario is exaggerated in children, considering that paramedics and other emergency care providers are trained to think that pediatric arrests are mostly the result of hypoxemia and that proscribed respiratory rates are generally higher than those proscribed for adults. also, emotions run even higher in childhood critical emergencies, theoretically compounding any predisposition to overzealously ventilate. therefore, clinical trials that indicated worse outcomes with eti may have been confounded by unrecognized detrimental ventilatory techniques [37 - 39 ]. so, paradoxically, in systems where many paramedics are deployed to all prehospital emergency cases with the rationale of improving response times for als procedures (and thus improved survival chances), worse outcomes might actually be expected, especially with successful eti. in the ems system in which the clinical trial of pediatric intubation was conducted, more than 2000 paramedics were trained to perform what resulted in being less than 150 annual pediatric intubations across the system during the study period. experience - wise, this type of system configuration issue makes it difficult for the individual paramedic to get much exposure, even to adult intubations. clearly, pediatric intubation situations would be uncommon, or even unlikely over his or her entire career. it also means too frequent and too lengthy pauses in chest compressions if the crews are not facile at placing the tube. overall, this scenario provides a clear set - up for under - skilled attempts at eti altogether. coupled with high anxiety when dealing with kids, an ems system that follows typical protocols for ventilation and/or does not control for overzealous ventilation, may likely experience even poorer outcomes. under these circumstances, one can make a strong argument against using eti or attempting eti, especially in children and other vulnerable groups such as spontaneously - breathing head injured patients. nevertheless, it must be kept in mind that there are communities that can safely enjoy high success rates for eti and associated good outcomes for patients, even using certain rsi techniques. but, again, these ems systems are typified by street - wise training, tiered paramedic ambulance response systems, and patient care protocols involving controlled ventilatory techniques for critical cases. places like houston and seattle in the 1980s were delivering only one positive pressure breath every ten seconds to their patients with circulatory arrest and outcomes were exceptional when compared to other sites. therefore, eti should not be discouraged in such appropriate settings. on the other hand, as other researchers have implied, eti and/or rsi should be discouraged in those ems systems that are unable to adapt to those appropriate characteristics that facilitate eti and its proper use. as previously stated, the original ems programs that first published success with paramedic - staffed responses generally reported extremely high rates of success with prehospital eti placement [2 - 10,15 ]. in retrospect, when examining the differences in systems that have or have not had successes in eti, it appears that several factors are actually strong determinants of paramedic and nursing proficiency in the skill of eti. these determinants include : 1) the quality, orientation and types of experiences in the initial training ; 2) the frequency of performance ; and 3) on - scene oversight and supervision of eti performance [3 - 6,12,13,29,32 - 36 ]. in contrast to the typical operating room training experience, the skill of eti performed in the emergency care setting, and particularly in the out - of - hospital environment, is wrought with unique challenges. these challenges range from vomit - flooded airways and ground - level patient positions to ambient lighting and oro - pharyngeal injuries. with full stomachs, relaxed esophageal sphincters and inadvertent gastric insufflation from bvm or mouth - to - mouth ventilation, it is commonplace to approach an airway welled - up with vomit in a circumstance with often less - than - adequate (or delayed) suctioning. in turn, this often requires the ability to intubate almost instantly without adjuncts. unlike the controlled in - hospital environment, in a sunny, bright outdoors setting, tricks of the trade, such as placing a coat or blanket over one s head (and the head of the patient) in order to create a makeshift darkened room akin to an old - time photographer s camera hood. in contrast, even in the dark of night, heavy rain or awkward confined spaces may pose their own barriers to easily visualizing vocal cords. therefore, many of the classical techniques used by other practitioners in more traditional settings would not be as effective in the fast - paced, poorly controlled and mobile prehospital settings where resources and support are limited (figure 1).figure 1 endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. paul pepe). in turn, a key to successful ems intubation in the out - of - hospital setting is the street - wise experience of expert highly - experienced medical trainers and ems medical directors who not only understand these principles, but also are themselves facile in such techniques in the out - of - hospital setting. as previously stated, the original ems programs that first published success with paramedic - staffed responses generally reported extremely high rates of success with prehospital eti placement [2 - 10,15 ]. in retrospect, when examining the differences in systems that have or have not had successes in eti, it appears that several factors are actually strong determinants of paramedic and nursing proficiency in the skill of eti. these determinants include : 1) the quality, orientation and types of experiences in the initial training ; 2) the frequency of performance ; and 3) on - scene oversight and supervision of eti performance [3 - 6,12,13,29,32 - 36 ]. in contrast to the typical operating room training experience, the skill of eti performed in the emergency care setting, and particularly in the out - of - hospital environment, is wrought with unique challenges. these challenges range from vomit - flooded airways and ground - level patient positions to ambient lighting and oro - pharyngeal injuries. with full stomachs, relaxed esophageal sphincters and inadvertent gastric insufflation from bvm or mouth - to - mouth ventilation, it is commonplace to approach an airway welled - up with vomit in a circumstance with often less - than - adequate (or delayed) suctioning. in turn, this often requires the ability to intubate almost instantly without adjuncts. unlike the controlled in - hospital environment, in a sunny, bright outdoors setting, tricks of the trade, such as placing a coat or blanket over one s head (and the head of the patient) in order to create a makeshift darkened room akin to an old - time photographer s camera hood. in contrast, even in the dark of night, heavy rain or awkward confined spaces may pose their own barriers to easily visualizing vocal cords. therefore, many of the classical techniques used by other practitioners in more traditional settings would not be as effective in the fast - paced, poorly controlled and mobile prehospital settings where resources and support are limited (figure 1).figure 1 endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. endotracheal intubation in the out - of - hospital setting. in the early years of out - of - hospital emergency medical services (ems) systems, advanced life support personnel were not only trained in the nuances of how to avoid overzealous ventilation and properly place an endotracheal tube in very challenging circumstances, but they were also well - supervised on - scene by expert physicians who themselves were highly - experienced and exceptionally familiar with those challenges as well as methods to overcome them (photo by dr. paul pepe). in turn, a key to successful ems intubation in the out - of - hospital setting is the street - wise experience of expert highly - experienced medical trainers and ems medical directors who not only understand these principles, but also are themselves facile in such techniques in the out - of - hospital setting. even if initial training techniques are expert and well - taught, both in the classroom and on - scene, frequency of performance is a critical factor. for example, studies have shown the success rates for eti can be related to the deployment strategy of the ems system. in ems systems using tiered ambulance deployments in which paramedics (als providers) are spared for the most critical calls, many fewer paramedics are needed on the roster and the individual experience of each paramedic, including frequency of eti performance, can be enhanced dramatically. accordingly, this approach has been correlated with improved success rates in terms of eti performance. while eti skills may deteriorate a little with a hiatus from practice, collective experience has demonstrated that most prehospital personnel who have performed eti a hundred times or more in the out - of - hospital setting may still be able to perform the technique quite well despite the hiatus. however, the key issue is getting to that threshold of experience and this prerequisite goal requires high exposure and frequent performance. unfortunately, that level of performance is not always achieved in most ems systems today. as an example, for a five - year veteran paramedic to have achieved a successful eti over 100 times, it would mean successful performance of that procedure at least 20 times a year for five years. so if eti experience were to be shared with a paramedic partner, the implication is that this particular team would need to face 40 eti situations a year on their particular ambulance and shift. in fact, accounting for sick time, vacation time and other factors, it typically takes 5 to 6 fulltime equivalent paramedics to staff one of those two positions and thus 1012 different paramedics will be needed just for that one ambulance around the clock. therefore, that particular response unit would need to face approximately 200 to 250 eti cases a year for each als provider to get 20 opportunities to intubate. considering that cardiac arrest, respiratory distress and major trauma cases requiring eti constitute only 23% of all ems on - scene emergency responses, the ambulance in question would need to experience nearly 10,000 ems incidents a year overall. in most ems system configurations, this level of volume would be a logistical - temporal impossibility for a single ambulance. unless alternate deployment strategies were to be utilized, frequent exposure to eti cases would be clearly limited. specifically, in some communities, paramedics (or other types of als personnel, such as doctors or nurses) are spared from the majority of ems responses. instead of als providers, basic emergency medical technicians (emts) trained to do the non - invasive procedures such as spinal immobilization and splinting are used for most of the responses. under such circumstances, overall staffing could this would permit more frequent exposure to critical illness and injury for the individual paramedics (als providers). the same concept would apply to nurses or apprentice physicians who staff ambulances and air medical units, particularly in some european countries. the fact that air medical units are typically triaged only to the most critical cases means that those als providers staffing the helicopters are part of a deployment strategy that enhances skill use. using this so - called tiered approach, individual paramedics (als personnel), nurses or doctors each get more chances to perform an eti. while there is great variation from one city to another, on average a city with a population of 1 million in the u.s. this volume of cases might predict two or three thousand potential circumstances for eti each year. to optimize individual paramedic exposure, it would be best to limit the number of paramedic (als) ambulances to a maximum of 10 ambulances (250 eti exposures per ambulance per year 10 ambulances covers 2 to 3 thousand cases). in this circumstance, a cadre of 100 to 120 paramedics might be required for the 10 paramedic - staffed units. in a contrast, in a system experiencing 100,000 ems responses a year and using all - paramedic staffing, 35 to 40 ambulances would typically be required minimally and thus 400 to 500 paramedics would be needed. this all - als provider approach decreases individual exposure to eti attempts at least 4 to 5-fold. to make matters worse, in some cities, additional paramedics are also placed on first - responder vehicles such as responding fire engines. in turn, this further compounds the infrequency of exposure for individuals. moreover, some ambulances are situated in lower call volume areas than others, creating even less exposure to eti opportunities. fortunately, the great majority (85 to 95%) of ems incidents do not require an als provider (e. g., authorized physician, nurse, paramedic) and can be managed by basic emts. in turn, using well - established and well - documented dispatch triage protocols, paramedics (als providers) can be spared and basic emts (basic life support [bls ] providers) are deployed directly to manage the cases. in other situations, after an initial paramedic (als) response is made, the basic emt ambulance can be called in to transport the less critical patients thus freeing up paramedics (als providers) for the more critical cases. not only does this type of system configuration permit the need for fewer als personnel, but it also improves response intervals because paramedics are not tied up transporting patients and are thus more available. ironically, by having fewer paramedics, paramedic response can be improved. beyond on - scene procedures and moving the patient from the scene, the time to transport, provide hospital transition, create a record and then return to the primary response territory is the greatest deterrent to the availability of ambulance crews and thus a factor in compromised response times. not surprisingly then, the original ems systems reporting excellent paramedic track records with eti were largely this type of tiered response system with staffing configurations that utilized basic emts for the majority of responses and spared the much smaller cadre of relatively busy paramedics for the more critical calls, therefore creating more opportunities for eti skills usage. furthermore, the paramedics in these systems rapidly achieved experience seeing many dozens of cases per year and they eventually became reliably facile. in turn, as they became exceptionally facile, they deferred eti attempts to new trainees. as a result, in these sophisticated ems systems, the lesser - experienced medics rapidly developed their own skills even faster. veterans also maintained their skills by teaching, supervising and getting to attempt and perform the more difficult intubations when the more novice personnel could not place the tube. the majority of ems systems actually utilize all - paramedic (all - als) staffing on their ambulances. in addition, many first - responder crews often supplement ambulance response with additional paramedics (als providers) staffing the first response vehicles as well. therefore, it is no surprise that paramedics may not perform eti as well as their forerunners 40 years ago. despite the described impact of using an all - paramedic system tier in those all - als systems by creating a team of supervisors, field training officers, or expert physician responders who routinely respond to critical calls. depending upon the geography, vertical (high - rise) challenges, and traffic, it would be wise to create a small number of senior personnel who can respond across a designated territory (or even into a fellow senior officer s territory for back - up) as a modified approach to ensure high level skills performance. just as there may be 10 or so battalion fire chiefs in a city of a million residents spread out over a large geographical territory, staffing and responding a similar number of senior ems personnel into high level cases could be another alternative and one that is now being adopted by many progressive ems systems. finally, even with appropriate, tailored initial training and tiered response systems with a high frequency of performance for individual paramedics, if the on - scene medics in training are not properly supervised, they may still develop bad habits in a vacuum. it is critical to reinforce what constitutes a proper technique (e. g., sniffing position in those at low risk of neck injury) and to provide renewed coaching in the actual patient care setting, especially in terms of confirmation of tube placement and proper ventilatory techniques. in most ems systems that provide high rates of eti success, in - field medical directors, highly - experienced ems supervisors and well - coached veteran paramedics even if paramedics or other prehospital care providers are expertly trained, highly - skilled, highly - experienced and highly - supervised performers of intubation for both adults and children, their ventilatory techniques may still adversely affect outcome [25,37 - 39 ]. the types of patients most likely to need eti are those with cardiac arrest, chronic lung disease and severe post - traumatic shock conditions. yet these patients are also the most vulnerable to the detrimental cardiovascular effects of the positive pressure breaths that are being delivered through the ett. despite the basic physiological principle that ventilation should match perfusion (blood flow), over the years, in many venues, ems personnel have been trained traditionally to aggressively ventilate patients, usually with the ill - advised rationale that such an approach was the way to ensure oxygenation and offset metabolic acidosis. even with more judicious training, however, emergency workers can still have the tendency to over - zealously ventilate such patients in the heat of the emergency. ironically, while such patients in deep shock actually require infrequent breaths and a lesser minute ventilation, once the ett is placed, they may now receive excessive levels of assisted breathing, not only because of some unsound rote training, but also because of adrenaline - modulated behaviors. accordingly, it is now speculated that low national survival rates for out - of - hospital cardiac arrest and the negative outcomes of several prehospital clinical trials may have been, in part, the result of uncontrolled ventilatory rates using positive pressure breaths. for example, in the study of severe traumatic brain injury (tbi) in which rsi - facilitated eti was associated with worse outcomes, a key correlation with mortality was the finding hyperventilation, defined as an arterial pco2 < 24 mmhg. while one might suspect that these negative outcomes may, therefore, be caused by effects of respiratory alkalosis, such as myocardial depression, cerebral vasoconstriction and a left shift in the hemoglobin dissociation curve, it is most likely that the low arterial pco2 is simply a surrogate variable for overzealous positive pressure ventilation [37 - 39 ]. as aufderheide and colleagues have shown, despite aggressive, targeted re - training on respiratory rates and delivery techniques, paramedics still overzealously ventilate and prolong the duration of positive pressure breaths in the adrenaline - charged environment of a critical emergency. it is likely that this scenario is exaggerated in children, considering that paramedics and other emergency care providers are trained to think that pediatric arrests are mostly the result of hypoxemia and that proscribed respiratory rates are generally higher than those proscribed for adults. also, emotions run even higher in childhood critical emergencies, theoretically compounding any predisposition to overzealously ventilate. therefore, clinical trials that indicated worse outcomes with eti may have been confounded by unrecognized detrimental ventilatory techniques [37 - 39 ]. so, paradoxically, in systems where many paramedics are deployed to all prehospital emergency cases with the rationale of improving response times for als procedures (and thus improved survival chances), worse outcomes might actually be expected, especially with successful eti. in the ems system in which the clinical trial of pediatric intubation was conducted, more than 2000 paramedics were trained to perform what resulted in being less than 150 annual pediatric intubations across the system during the study period. experience - wise, this type of system configuration issue makes it difficult for the individual paramedic to get much exposure, even to adult intubations. clearly, pediatric intubation situations would be uncommon, or even unlikely over his or her entire career. it also means too frequent and too lengthy pauses in chest compressions if the crews are not facile at placing the tube. overall, this scenario provides a clear set - up for under - skilled attempts at eti altogether. coupled with high anxiety when dealing with kids, an ems system that follows typical protocols for ventilation and/or does not control for overzealous ventilation, may likely experience even poorer outcomes. under these circumstances, one can make a strong argument against using eti or attempting eti, especially in children and other vulnerable groups such as spontaneously - breathing head injured patients. nevertheless, it must be kept in mind that there are communities that can safely enjoy high success rates for eti and associated good outcomes for patients, even using certain rsi techniques. but, again, these ems systems are typified by street - wise training, tiered paramedic ambulance response systems, and patient care protocols involving controlled ventilatory techniques for critical cases. places like houston and seattle in the 1980s were delivering only one positive pressure breath every ten seconds to their patients with circulatory arrest and outcomes were exceptional when compared to other sites. most importantly, these sites also involved intensive on - scene expert medical oversight. therefore, eti should not be discouraged in such appropriate settings. on the other hand, as other researchers have implied, eti and/or rsi should be discouraged in those ems systems that are unable to adapt to those appropriate characteristics that facilitate eti and its proper use. even if paramedics or other prehospital care providers are expertly trained, highly - skilled, highly - experienced and highly - supervised performers of intubation for both adults and children, their ventilatory techniques may still adversely affect outcome [25,37 - 39 ]. the types of patients most likely to need eti are those with cardiac arrest, chronic lung disease and severe post - traumatic shock conditions. yet these patients are also the most vulnerable to the detrimental cardiovascular effects of the positive pressure breaths that are being delivered through the ett. despite the basic physiological principle that ventilation should match perfusion (blood flow), over the years, in many venues, ems personnel have been trained traditionally to aggressively ventilate patients, usually with the ill - advised rationale that such an approach was the way to ensure oxygenation and offset metabolic acidosis. even with more judicious training, however, emergency workers can still have the tendency to over - zealously ventilate such patients in the heat of the emergency. ironically, while such patients in deep shock actually require infrequent breaths and a lesser minute ventilation, once the ett is placed, they may now receive excessive levels of assisted breathing, not only because of some unsound rote training, but also because of adrenaline - modulated behaviors. accordingly, it is now speculated that low national survival rates for out - of - hospital cardiac arrest and the negative outcomes of several prehospital clinical trials may have been, in part, the result of uncontrolled ventilatory rates using positive pressure breaths. for example, in the study of severe traumatic brain injury (tbi) in which rsi - facilitated eti was associated with worse outcomes, a key correlation with mortality was the finding hyperventilation, defined as an arterial pco2 < 24 mmhg. while one might suspect that these negative outcomes may, therefore, be caused by effects of respiratory alkalosis, such as myocardial depression, cerebral vasoconstriction and a left shift in the hemoglobin dissociation curve, it is most likely that the low arterial pco2 is simply a surrogate variable for overzealous positive pressure ventilation [37 - 39 ]. as aufderheide and colleagues have shown, despite aggressive, targeted re - training on respiratory rates and delivery techniques, paramedics still overzealously ventilate and prolong the duration of positive pressure breaths in the adrenaline - charged environment of a critical emergency. it is likely that this scenario is exaggerated in children, considering that paramedics and other emergency care providers are trained to think that pediatric arrests are mostly the result of hypoxemia and that proscribed respiratory rates are generally higher than those proscribed for adults. also, emotions run even higher in childhood critical emergencies, theoretically compounding any predisposition to overzealously ventilate. therefore, clinical trials that indicated worse outcomes with eti may have been confounded by unrecognized detrimental ventilatory techniques [37 - 39 ]. so, paradoxically, in systems where many paramedics are deployed to all prehospital emergency cases with the rationale of improving response times for als procedures (and thus improved survival chances), worse outcomes might actually be expected, especially with successful eti. in the ems system in which the clinical trial of pediatric intubation was conducted, more than 2000 paramedics were trained to perform what resulted in being less than 150 annual pediatric intubations across the system during the study period. experience - wise, this type of system configuration issue makes it difficult for the individual paramedic to get much exposure, even to adult intubations. clearly, pediatric intubation situations would be uncommon, or even unlikely over his or her entire career. it also means too frequent and too lengthy pauses in chest compressions if the crews are not facile at placing the tube. overall, this scenario provides a clear set - up for under - skilled attempts at eti altogether. coupled with high anxiety when dealing with kids, an ems system that follows typical protocols for ventilation and/or does not control for overzealous ventilation, may likely experience even poorer outcomes. under these circumstances, one can make a strong argument against using eti or attempting eti, especially in children and other vulnerable groups such as spontaneously - breathing head injured patients. nevertheless, it must be kept in mind that there are communities that can safely enjoy high success rates for eti and associated good outcomes for patients, even using certain rsi techniques. but, again, these ems systems are typified by street - wise training, tiered paramedic ambulance response systems, and patient care protocols involving controlled ventilatory techniques for critical cases. places like houston and seattle in the 1980s were delivering only one positive pressure breath every ten seconds to their patients with circulatory arrest and outcomes were exceptional when compared to other sites. most importantly, these sites also involved intensive on - scene expert medical oversight. therefore, eti should not be discouraged in such appropriate settings. on the other hand, as other researchers have implied, eti and/or rsi should be discouraged in those ems systems that are unable to adapt to those appropriate characteristics that facilitate eti and its proper use. while eti remains the gold standard for definitive airway management in the emergency care setting, it may beinappropriate in the prehospital setting in the absence of paramedic - sparing deployment systems, controlled ventilatorytechniques and intensive medical oversight that provides street - wise training as well as expert, on - scene supervision of theems personnel providing the eti. while eti may very well be life - saving, particularly in cases of severe trauma withcirculatory arrest successful placement and use of an eti is morelikely to occur in ems systems that provide:street - wise training that is provided by experts in out - of - hospital patient care who themselves are well - experienced in on - scene emergency eti;tiered ems deployment systems that spare a small cadre of highly - skilled (and relatively busy) paramedics from the majority of ems incidents (focusing them on the more critical cases, thus resulting in a very high frequency of eti performance by each individual in the system) ; andintensive, street - wise and expert out - of - hospital medical oversight. street - wise training that is provided by experts in out - of - hospital patient care who themselves are well - experienced in on - scene emergency eti ; tiered ems deployment systems that spare a small cadre of highly - skilled (and relatively busy) paramedics from the majority of ems incidents (focusing them on the more critical cases, thus resulting in a very high frequency of eti performance by each individual in the system) ; and intensive, street - wise and expert out - of - hospital medical oversight. but, even when paramedics (and other als providers) are facile at eti in the unique environmental conditions and challenges of the out - of - hospital setting, inappropriate and overzealous ventilation can still result in detrimental outcomes. in summary, systems unable to adopt the appropriate configurations, protocols, training, monitoring, and all other characteristics that optimize eti may, therefore, need to be discouraged from performing eti or they need to develop alternative mechanisms to better ensure routine success with placement of the tube and its appropriate use. | this article is one of ten reviews selected from the annual update in intensive care and emergency medicine 2015 and co - published as a series in critical care. other articles in the series can be found online at http://ccforum.com/series/annualupdate2015. further information about the annual update in intensive care and emergency medicine is available from http://www.springer.com/series/8901. |
coronary artery perforation (cap) which occurs either during or following percutaneuous coronary intervention (pci) is an infrequent complication, but one of the most disastrous complications of this cardiovascular surgery procedure.1 - 9) it has been related to vessel wall penetration with guidewire, balloon overexpansion or rupture, atheroablative techniques, and stent implantation. some reports have shown that helical platinum microcoil embolization was used for cap.10 - 15) contrasting the usual expectation that an embolized artery will not show any blood flow, this report shows preserved normal blood flow at the embolization site on the 1 year follow - up angiography, following emergent lifesaving microcoil embolization, in a patient with uncontrolled ellis grade 3 guidewire - induced cap, resulting in cardiac tamponade. in the literature, there are no other similar case reports from a follow - up angiography after microcoil embolization, especially in a case such as the one presented herein, where the flow was maintained at the embolized site. a 51-year - old male patient with a 30 pack - year current smoking presented with a recent onset of exertion chest pain. the treadmill test provoked severe chest pain, without st changes on the electrocardiography (ecg), at stage 2 of the modified bruce protocol. the patient underwent elective coronary angiography via the left femoral approach, which revealed a discrete critical narrowing of the middle left anterior descending artery (lad), just distal to the first diagonal branch (d1) (fig. a 7 fr jl4 guide catheter (cordis, miami, fl, usa) was used to engage the left main coronary artery, and a 0.014 " atw guidewire (cordis, miami, fl, usa) was passed into the distal lad, without difficulty. the proposed treatment was a cross - over stenting followed by provisional t stenting at that lesion site. angioplasty was performed twice with the ryujin plus (terumo, tokyo, japan) 3.510 mm balloon at 6-atm (up to 3.5 mm) inflation. a zotarolimus - eluting stent (endeavor resolute, medtronic, minneapolis, mn, usa) 4.015 mm stent was deployed, with 12-atm (up to 4.08 mm) inflation. the repeat angiogram showed adequate deployment of the stent, but a jailed d1 (fig. an attempt was made to pass into d1 using a 0.014 " choicept guidewire (boston scientific, miami, fl, usa) through the instent. after initially inflating a sprinter (medtronic, minneapolis, mn, usa) 3.015 mm semi - compliant balloon, up to 3.18 mm at the d1 ostium, ' kissing ' balloon angioplasty, using a sprinter 3.515 mm for the lad and a sprinter 3.015 mm for d1, was finally performed (fig. three hours after pci, he complained of more severe chest pain, dyspnea, tachypnea and heavy sweating. an emergent echocardiogram was taken at the patient 's bedside, under the suspicion of a cardiac tamponade, due to delayed extravasation after pci. it showed a pericardial effusion of less than 10 mm, as well as the collapse of the right atrium. emergency pericardiocentesis was performed and over 100 ml of bloody pericardial fluid was initially drained. pericardial fluids were drained continuously and blood was transfused through the peripheral vein, with the expectation that it will stop the extravasation spontaneously. considering the drainage rate of the pericardial fluid at over 200 ml / hr, a large perforation of the coronary artery at the stenting site was suspected. however, an emergent angiogram showed massive extravasation (ellis grade 3 coronary perforation) at the far distal area of d1 (fig. an attempt was made to recross d1 and a sprinter 3.015 mm semi - compliant coronary balloon with low pressure was inflated at the middle portion of d1. however, the perforation could not be sealed and continuous leakage occurred after the balloon 's deflation, even after an inflation period of over 60 minutes. we attempted to occlude the d1 branch via a microcoil embolization to repair the perforation site completely. a microferret superselective catheter (cook, sandet, denmark) one 0.018-inch and 14.2-mm - long tapered microcoil (104 mm in diameter ; tornado ; cook, bloomington, in, usa) was quickly released, using the microcatheter 's supporting guidewire (transend 300, boston scientific, miami, fl, usa) as a pusher. a post - coil lad arteriogram demonstrated the total occlusion of d1 and no further extravasation (fig., the post procedure echocardiogram revealed an ejection fraction of 60%, with mild hypokinesia on the apical lateral and apical anterior walls, and no pericardial effusion. the peak infarct size, as measured using serum biomarkers, was a troponin of 2.65 ng / ml and a creatinine phosphokinase - mb of 100.8 ng / ml. the patient was uneventful and discharged 5 days later in a stable condition. the treadmill test provoked chest pain at stage 5, with a 1 mm st depression on the ecg, and the echocardiogram showed no interval change. for 12 months after pci, the patient had several episodes of chest pain and dyspnea on exertion. angiographic findings of d1 showed a normal flow without delay (fig. 2c). cap may range clinically from guidewire - related microperforations, resulting in minimal dye staining without haemodynamic consequences, to vessel rupture followed by active extravasations of blood and dye into the pericardial space, leading to tamponade and sudden haemodynamic collapse. it has been reported at variable rates, depending on the procedures performed, lesions treated and devices used. in the recent registries, perforation has been reported to occur in 0.17 - 1.5% of all patients undergoing pci.9)16 - 18) the incidence of these complications has been increased by the use of debulking devices, such as high - speed rotational atherectomy and directional coronary atherectomy.2)3)5 - 7)17) guidewire - induced perforation seems to be the most frequent cause of cap - accounting for 20 to 68% of cap incidents.2)3)5 - 9)16 - 18) in pci of complex lesions, including chronic total occlusions and bifurcation lesions, the use of both hydrophilic and heavy - weight guidewires has also increased the frequency of this complication.2)4)7)9)17) a choicept wire was used for crossing the d1 branch in this case. we suspected that the hydrophilic guidewire - induced damage occurred with the attempt to treat the jailed branch, using the ' kissing ' balloon technique. this choicept wire was coated with a highly lubricious hydrogel and was moderately stiff, compared with the new - generation guidewires. due to this low coefficient of friction and easy distal migration,10)11)15)19) caution should be exercised in positioning the tip of the guidewire distally. after the perforation diagnosis, treatment strategy varies, according to the clinical situation, i.e., the size of the perforation, the extent of contrast extravasation, and the hemodynamic status of the patients. the initial management involves the inflation of an angioplasty balloon proximal to or at the level of the perforation to seal the leak, and pericardioentesis can be performed if cardiac tamponade is present. depending on the bleeding control, complete management modalities should be considered - from surgery to less invasive percutaneous techniques : covered stents / grafts or thrombus inducing therapies, such as polyvinyl alcohol, autologous blood or intracoronary bead injection. if a cardiac surgeon were unavailable, one might have to choose another option for controlling the patient 's bleeding. because a covered stent is bulky, has a large diameter (of over 3 mm), and is not easy to pass through the side hole of a stent in the parent artery, this device could not be used with the continuous bleeding from the perforation in this case. in the event that the perforation is in a small sized branch, the operator may consider excluding the whole branch by coiling, bead seeding, or implanting other thrombogenic agents directly into the branch. the use of the microcoil was very effective in the patient presented herein. in recent studies, the prevalence of microcoil embolization as a treatment option of cap was 0.1 - 4%.1)8)17) it is not common but it may play an important role when traditional management for cap has failed. microcoils have been used as permanent embolic agents for occlusion of cerebral aneurysms, gastrointestinal hemorrhage, and other peripheral vascular therapeutic maneuvers. however, due to the permanent loss of the vessel lumen beyond the site of the microcoil placement and the subsequent infarction, their use in the coronary arteries should be limited to distal small - vessel perforation in life - threatening circumstances, in which no other options are readily available. the tornade embolization microcoils (cook) have a soft platinum structure with synthetic fibers that maximize thrombogenicity. moreover, their helical configuration maximizes the coil exposure to the cross - section of the lumen for the disruption of the flow. to allow proper adherence to the vessel wall, the coil size should be slightly (25%) larger than the target vessel diameter. this case describes the successful management of cap, with the sacrifice of the d1 via coil embolization. nevertheless, at the follow - up angiography, a patent blood flow was evident at the site where the microcoil was inserted. this case, therefore, is not only the first angiographic result of microcoil embolization presented to date, but also, particularly, the first example of maintenance of the blood flow at the embolized site, which was against our expectations of complete occlusion. the best hypothesis is that, although a very large microcoil was inserted, initially inducing a thrombus, this thrombosis state could not be maintained due to inadequate adherence to the vessel under dual antiplatelet therapy. although some perforations are quite evident in the catheterization laboratory, others remain initially occult, only to become clinically apparent hours after the procedure. cardiac tamponade complications occur in 11 - 46% of patients experiencing cap after pci.1 - 4)7 - 9)16)17) delayed tamponade comprised 20 - 60% of these tamponade complications,3)4)20) and was common in the perforations (e.g., ellis type i) induced by either guidewire or glycoprotein iib / iiia inhibitor use.16)20) thus, this diagnosis should remain high on the list of differential diagnoses of post - pci hypotension. in these cases, the diagnosis of cap should be ruled out, especially if retroperitoneal bleeding is not present. close observation is strongly advised in all pci cases because, if not recognized, bleeding from cap can rapidly lead to death from cardiac tamponade. cap is a rare, but disastrous complication of pci. in selected cases of distal coronary perforations, microcoil embolization may be a reasonable alternative therapeutic approach. however, an obvious drawback of this approach is the permanent loss of the vessel lumen beyond the site of the microcoil placement and the subsequent infarction. therefore, this approach should be limited either to life - threatening circumstances, in which no other options are readily available, or to the treatment of very distal perforations, where the amount of myocardium that is jeopardized is minimal. despite a large, hemodynamically significant perforation, the successful management of cap with the sacrifice of the d1 via coil embolization is described in this case. a notable, unexpected result was evident at the patient 's follow - up one - year angiography, i.e., a patent blood flow existed at the site where the microcoil was inserted. | coronary artery perforation (cap) after percutaneous coronary intervention is a rare, but serious complication. it can cause cardiac tamponade, acute myocardial infarction or death. the treatments of cap involve prolonged balloon inflation, emergent surgery, coil embolization, and implantation of covered stent. we have successfully performed the emergent microcoil embolization in a patient with uncontrolled ellis grade 3 guidewire - induced cap resulting in delayed cardiac tamponade. contrasting our usual expectation, the 1-year follow - up angiography showed a patent flow at the embolized site. |
plants have been considered as sources of medicinal agents for the treatment of various diseases such as malaria, leprotic ulcer, skin infections, high blood pressure. in our earlier work, we reported the effect of rauwolfia vomitoria root bark extract on the activities of cardiac enzymes and how this plant extract affects learning and memory in mice.[24 ] rauwolfia vomitoria root bark extract has also been used extensively by other researchers and is implicated in health problems such as mental depression which may persist for several months, early morning insomnia, and impotence. the mechanism by which rauwolfia vomitoria elicits these health problems is of major research concern. the blood is a major vehicle for the transport of most drugs in the human and animal systems, and as such any alteration in the integrity of blood cells may lead to serious health problems. the vasculature in which blood present surface areas of over 10,000 m permits this system to interact extensively with other systems in the body. therefore, changes in the hematological indices may occur as a result of other systemic disease conditions. reactive oxygen species and free radicals have been implicated in a number of complex biological processes and diseases such as ageing, inflammation and malaria, atherosclerosis, ischemia. the role of vitamin e in preventing or delaying coronary heart disease is well known. vitamin supplementation is known to impart significantly on health and is of special benefits in term of disease prevention and treatment. however, vitamin(s) supplementation during some specific drug therapy may be deleterious to health and hence defeats the very purpose of administration. these effects may be due to nutrient interactions resulting in either alteration in absorption and metabolism of vitamin or increased metabolic clearance of the drug thereby compromising the therapeutic benefit. the aim of this research work was to study the possible interaction of vitamin e with rauwolfia vomitoria root bark extract on some haematological indices of wistar albino rats. rauwolfia vomitoria roots were obtained from a farm land in ekpene obo, nigeria in the month of november. samples of the plant were authenticated by the botanist in the botanical garden of university of calabar. a voucher specimen (no. one hundred gram of root powder was extracted twice with 80% ethanol according to the method of ugochukwu. the filtrate was concentrated using rotary evaporator, and the concentrate was dried in a plus 11 gallenkamp oven at 4550c. one gram of the extract was re - suspended in 10% dmso solution daily whenever required for use. forty - two albino rats of the wistar strain weighing 200 230 g were used in this work. the animals were obtained from the animal house, biochemistry department, university of calabar. they were maintained under standard laboratory conditions with rat chow (guinea feed ltd., all animal experiments were carried out in line with the guidelines of institutional animal ethical committee as approved by the graduate school, university of calabar, nigeria. group 1 animals were the control group ; group 2 was administered with vitamin e (10 iu / kg body weight). groups 3 and 4 were given rauwolfia vomitoria root bark extract (150 and 300 mg mg / kg body weight) respectively. groups 5 and 6 were given vitamin e (10 iu / kg body weight) and r. vomitoria root bark extract (150 and 300 mg / kg body weight) respectively. all experimental animals were anaesthetized using chloroform fumes 24 h after the last administration of the extract. blood samples were collected into ethylenediaminetertraacetic acid (edta) sample bottles for hematological studies. determination of hemoglobin concentration was carried out according to the method described by jain using the cyanomethaemoglobin method. parked cell volume (pcv) red blood cell (rbc) count, white blood cell (wbc) count and count were estimated by visual means using the new improved neubauser counting chamber according to dacie and lewis. rauwolfia vomitoria roots were obtained from a farm land in ekpene obo, nigeria in the month of november. samples of the plant were authenticated by the botanist in the botanical garden of university of calabar. a voucher specimen (no. one hundred gram of root powder was extracted twice with 80% ethanol according to the method of ugochukwu. the filtrate was concentrated using rotary evaporator, and the concentrate was dried in a plus 11 gallenkamp oven at 4550c. one gram of the extract was re - suspended in 10% dmso solution daily whenever required for use. forty - two albino rats of the wistar strain weighing 200 230 g were used in this work. the animals were obtained from the animal house, biochemistry department, university of calabar. they were maintained under standard laboratory conditions with rat chow (guinea feed ltd., all animal experiments were carried out in line with the guidelines of institutional animal ethical committee as approved by the graduate school, university of calabar, nigeria. group 1 animals were the control group ; group 2 was administered with vitamin e (10 iu / kg body weight). groups 3 and 4 were given rauwolfia vomitoria root bark extract (150 and 300 mg mg / kg body weight) respectively. groups 5 and 6 were given vitamin e (10 iu / kg body weight) and r. vomitoria root bark extract (150 and 300 mg / kg body weight) respectively. all experimental animals were anaesthetized using chloroform fumes 24 h after the last administration of the extract. blood samples were collected into ethylenediaminetertraacetic acid (edta) sample bottles for hematological studies. determination of hemoglobin concentration was carried out according to the method described by jain using the cyanomethaemoglobin method. parked cell volume (pcv) red blood cell (rbc) count, white blood cell (wbc) count and count were estimated by visual means using the new improved neubauser counting chamber according to dacie and lewis. determination of hemoglobin concentration was carried out according to the method described by jain using the cyanomethaemoglobin method. parked cell volume (pcv) red blood cell (rbc) count, white blood cell (wbc) count and count were estimated by visual means using the new improved neubauser counting chamber according to dacie and lewis. the effects of vitamin e supplementation during rauwolfia vomitoria (r. vomitoria) root bark extract administration in wistar albino rats were investigated to assess the benefits and possible risk involved. in order to do these hematological indices : hemoglobin, pcv, wbc, rbc platelets were estimated. table 1 shows that hemoglobin levels (gm / dl) increased significantly (p0.005) was observed in other groups when compared with the control. similarly, the result of platelet analysis showed a significant increase when compared with the control. the wbc count was observed to have decreased insignificantly (p>0.005) in all the groups. this observed decreased in total wbc may be due to the toxic effect of r. vomitoria root bark extract on the immune system. literature is replete with the use of plant materials and its derivatives for the prevention and treatment of diseases. the beneficial therapeutic effects of these medicinal herbs are expressed in their scientific implications in health conditions of the users. thus, medicinal herbs such as rauwolfia vomitoria have played a major role in the development of modern medicine and their traditional applications can not be under estimated as some persons in the african societies do not have access to modern medications. generally, there is still need to investigate the potential adverse effects associated with the use of medicinal herbs and the possible way of ameliorating this toxic effects. rauwolfia vomitoria is a natural medicinal herb which has been used for over 2000 years for treatment of diseases such as hypertension and mental disorders. its adverse effects include : decreased heart rate and blood pressure, which is due to dilatation of blood vessels. it also causes low sex drive, increased appetite, weight loss, swellings, stomach upset, hallucinations, poor co - ordination, dizziness, impairment of physical abilities and psychotic depression. however, the possible mechanism by which this plant extract elicits its toxicity is very necessary in order to balance its therapeutic benefits with the associated adverse effects. generation of free radicals by many xenobiotics in biological systems have been implicated in cell membrane damage, depletion of the immune system and many other diseases. vitamin e and other antioxidants protect the cells of the body from the effects of free radicals and the potentially damaging by - products of metabolism. the results of this study showed that rauwolfia vomitoria root bark extract could help to increase some haematological indices like haemoglobin content, packed cell volume, red blood cell count, and total platelet count. its ability to reduce the total white blood cell count in the experimental animals may be a possible mechanism by which its potent active ingredients elicit certain levels of immunological advantage. therefore, the need to administer this extract in health conditions where the immune system is compromised due to microbial and other infections. the effects of vitamin e supplementation in this experiment corroborates its function of boosting the immune system as observed in the group treated with 10 iu / kg body weight of vitamin e over the control group. this result is in agreement with the work of fritsche., who reported that there were significant interactions between vitamin e and (n-3) fatty acids that affect the immune system. we also observed that the effect of r. vomitoria root bark extract at 300mg / kg body weight did not result in any positive effect on the hematological indices. this showed that r. vomitoria activity may be more effective at lower concentrations than a higher one and this may in effect reduce its possible toxicity, thereby favoring its therapeutic activity. in resource - limited environments, people are desperately looking for cure of diseases due to scarcity or high cost of medicine and have resorted to herbal medicinal therapy without adequate knowledge of the possible latent side effects. to this group of persons the beneficial effects of medicinal plants often over shadowed their deleterious effects, hence, the need for concerted efforts in the screening of plants for possible toxicity with the view of advising properly. the simplest method of assessing toxicity in experimental studies is by enzyme assay and hematological indices since these parameters are often affected by introduction of xenobiotics in biological species. also the administration of some vitamins that are capable of quenching free radicals may be of special relevance in ameliorating the toxic effects of medicinal plants. in conclusion, we observed that extract of rauwolfia vomitoria root bark is a useful medication in the treatment of many diseases and the combination of this herbal extract with vitamin e may be of more biochemical and therapeutic significance since the antioxidant vitamin is capable of de - potentiating the adverse effect of this herb. rauwolfia vomitoria with or without vitamin e improved the immunity and enhances the hematological indices of the experimental animals. our findings suggest that interaction of vitamin e with rauwolfia vomitoria root bark extract would be a meaningful approach in medicinal therapeutics of this plant. more work on the interaction of this plant with vitamins is ongoing in our laboratory. | background : vitamin supplementation in rauwolfia vomitoria root bark extract administration may interact and impact significantly on hematology of albino wistar rats.aim:in this investigation we studied vitamin e supplementation with rauwolfia vomitoria root bark extract on the hematology of experimental animals.materials and methods : forty two rats weighing 200 230 g were randomly selected into six groups of seven animals each. group 1 animals serve as controls ; group 2 received vitamin e (10 iu / kg body weight). groups 3 and 4 were given the extract (150 and 300 mg / kg body weight) respectively. groups 5 and 6 were given vitamin e (10 iu / kg body weight), the extract (150 and 300 mg / kg body weight) respectively. the extract and the vitamin were administered daily by oral intubation. blood samples analyzed for hematological indices.results:decrease in white blood cell count (wbc) was observed, indicating improved immunity of animals. extract at 150 and 300 mg / kg body weight with and without vitamin e affected hemoglobin and packed cell volume.conclusion:rauwolfia vomitoria with or without vitamin e improved animal 's immunity and enhances their hematology. interaction of vitamin e with the extract affects medicinal therapeutics of this plant. |
rett syndrome is a childhood neurodevelopmental disorder usually caused by a mutation in the gene encoding mecp2 located on the x chromosome (xq28).1 rett syndrome occurs predominately in females although males have been described with mecp-2 mutations.2 stereotyped movement of the hands such as wringing, washing, hand clapping, and hand - to - mouth movements following the loss of the functional use of the hands is the most characteristic feature of rett syndrome.3 typically, patients with rett syndrome have no verbal skills, and about 50% of them are not ambulatory.3 however, they are reported to respond well to music in comparison with their physical and verbal disabilities.47 synchronized movement to music has been observed in all known human cultures, implying that this ability is universal and perhaps unique to human musical behavior.8 among various musical elements such as pitch, melody, harmony, rhythm, dynamics, timbre, etc., auditory rhythm is known to exhibit a close and fundamental relationship with body movement from early infancy,9,10 and facilitates synchronized body movement not only in healthy subjects of all ages but also in patients with various movement disorders such as parkinson disease,11 huntington s disease,12 stroke,13 and incomplete spinal cord injury.14 improvement in walking speed and stride length by auditory rhythm was demonstrated in patients with parkinson disease.15,16 these findings suggest that auditory rhythm could also modify motor behavior and induce voluntary movement in patients with rett syndrome ; however, little is known about the basic role of auditory rhythm in these patients.17 the motion analysis system is a well established clinical method to examine temporal, spatial, and kinetic movements of various parts of the human body, particularly for the assessment of gait in healthy subjects18 and intervention evaluation in patients with cerebral palsy.19 we utilized this method to investigate the relationship between auditory rhythm and behavioral movement in patients with rett syndrome in order to promote their voluntary hand movement. ten female patients with rett syndrome, aged from three to 17 years, were included in this study. the diagnosis was made by more than two child neurologists according to the diagnostic criteria for rett syndrome established by the rett syndrome diagnostic criteria work group.20 patient profiles are described in table 1. in addition, eight of 10 patients had stereotyped body rocking movement : back and forth, three ; left and right, four ; and both, one (table 1). according to the previous study on motion analysis in a patient with rett syndrome,21 markers were put on the wrists and shoulders in patients to investigate the movement of their hands and upper bodies, respectively. color tapes 20-mm wide were employed as the markers, and their movements were captured using a two - dimensional digital video camera at a sampling frequency of 30 hz. two - dimensional motion analysis software (move - tr/2d ver.7 ; library co., ltd., japan) was utilized to record temporal, spatial, and kinetic changes of movements in response to music in a qualitative manner. when no purposeful movement was observed, music familiar to each patient related to us by caregivers was started with a simple regular rhythm. sometimes, the music was stopped suddenly or changed from a simple regular rhythm to a continuous tone without any rhythm to assess any behavioral changes in movement. this study was approved by the institutional ethics and research board, and informed written consent was obtained from legally authorized representatives of the patients. when music with a simple regular rhythm started, body rocking appeared automatically in a back and forth direction in all four patients who had the same body movement as their stereotyped movement (table 1). through this body rocking induced by music, voluntary hand movement increased gradually and finally became sufficient to beat a tambourine presented in front of the patient (table 1). this voluntary hand movement was observed consistently during and after the study when patients were awake and in a good temper. however, the induction of body rocking by music was not observed in the other six patients who did not have stereotyped body rocking in a back and forth direction (table 1). the motion analysis system demonstrated that body rocking induced by music consisted of primarily repetitive horizontal back and forth movement and little vertical movement (figure 1). it persisted with almost the same regular periodic cycle as the auditory rhythm of music and constant amplitude while the music continued (figure 1). through this regular and cyclic body movement induced by music, voluntary hand movement gradually increased in a vertical direction with the same periodic cycle as the body, and finally allowed the patient to reach the tambourine presented in front of her (figure 1). vertical movement of the hand was much more pronounced than horizontal movement of the body (figures 13). when the music stopped suddenly, voluntary movement of the hand soon disappeared (figure 2). horizontal movement of the body also decreased gradually and stopped after a while (figure 2). horizontal movement of the hand merely reflected the accompanying motion of the body in the same direction. when the music changed from a simple regular rhythm to a continuous tone without an auditory rhythm, movements of both the hand and body became slower and the periodic cycle of the motion prolonged (figure 3). in this study, patients with rett syndrome showing stereotyped body rocking in a back and forth direction were demonstrated to recognize and respond well to changes in the rhythm of music. a simple regular auditory rhythm induced stereotyped body rocking movement in a back and forth direction at first. then, the auditory rhythm as well as induced body rocking movement facilitated voluntary movement of the hands. purposeful hand movement was performed through this regular and cyclic body movement induced by music. this might explain why only stereotyped body rocking in a back and forth direction was induced in this study. stereotyped movement is repetitive, restricted, and nonfunctional motor behavior observed in various neurological and developmental disorders such as rett syndrome,3 autistic disorder,22 and visual or auditory impairment.23 if it is frequent and severe, it may interfere with normal voluntary movement. therefore, treatment and rehabilitation usually focuses on how to suppress it in order to facilitate voluntary movement. however, voluntary movement can be induced more easily through using rather than suppressing stereotyped movement in patients whose voluntary movement is very difficult to promote, such as those with rett syndrome. the reinforcement of rhythm in stereotyped movement by music might be another way of rehabilitation for such patients.17 action induction by music is accompanied by neural impulses in the reticular formation of the brainstem.24 in addition, listening to a simple regular rhythm without any actual movement increases the activity of the basal ganglia and supplementary motor area.9 these brain areas are involved in motor prediction and the timing of future movements.25,26 therefore, listening to music with a simple regular rhythm would prime body movement. this mechanism could be preserved in some patients with rett syndrome, and stimulation with music might be worth attempting for their rehabilitation. actually, patients who responded to music showed some improvement of their hand use in everyday life such as opening the door or holding an object for a while even though rett syndrome progresses with gradual deterioration of the motor system. in this study, the movement of 10 patients was analyzed in a qualitative manner without a control group. further studies involving more subjects including older patients with a control group are necessary to clarify the role of auditory rhythm in patients with rett syndrome. | patients with rett syndrome are known to respond well to music irrespective of their physical and verbal disabilities. therefore, the relationship between auditory rhythm and their behavior was investigated employing a two - dimensional motion analysis system. ten female patients aged from three to 17 years were included. when music with a simple regular rhythm started, body rocking appeared automatically in a back and forth direction in all four patients who showed the same rocking motion as their stereotyped movement. through this body rocking, voluntary movement of the hand increased gradually, and finally became sufficient to beat a tambourine. however, the induction of body rocking by music was not observed in the other six patients who did not show stereotyped body rocking in a back and forth direction. when the music stopped suddenly, voluntary movement of the hand disappeared. when the music changed from a simple regular rhythm to a continuous tone without an auditory rhythm, the periodic movement of both the hand and body prolonged. auditory rhythm shows a close relationship with body movement and facilitates synchronized body movement. this mechanism was demonstrated to be preserved in some patients with rett syndrome, and stimulation with music could be utilized for their rehabilitation. |
pancreatic transplants are performed worldwide for type i diabetes with renal failure and have shown to improve survival and quality of life. most of the reports are from countries with established organ donation programmes, but increasing awareness on organ donation in india could lead to an increase in the use of multi visceral transplants as a treatment modality in the next few years. the types of pancreatic transplants include simultaneous pancreas kidney (spk), pancreas after kidney and pancreas alone transplants. the most commonly performed are the spk transplants usually in young diabetics with renal failure. a 32-year - old male with a history of insulin - dependent diabetes mellitus (iddm) since 19 years of age presented with uncontrolled blood sugars and pedal oedema. he was detected to have diabetic nephropathy for the previous 2 years when he presented with frothing of urine. his glycosylated haemoglobin a1c was 11.8%, and a continuous subcutaneous insulin infusion with a pump had been prescribed as conventional insulin treatment failed to control his blood sugars. he was also hypertensive and hypothyroid for which he was on treatment with a calcium channel blocker and beta blocker and 175 g of tablet thyroxin per day. his renal functions showed a creatinine clearance of 2030ml / min, (class iv chronic kidney disease [ckd ]) and had not been initiated on haemodialysis. in view of his age, difficult blood sugar control, end organ involvement with diabetes and poor quality of life, a multidisciplinary team decision was to list him for a spk transplant. a coronary angiogram was performed in our patient following an inconclusive stress test that revealed mild coronary artery disease. multi systemic examinations including respiratory, central nervous system and liver were also performed to exclude contraindications for the transplant. the challenges anticipated were blood sugar control during the surgery, deterioration of native renal function perioperatively until the function of the new graft picked up, and preparedness of the team at the time of availability of the donor. when a suitable matched donor was identified following brain death, the patient was called in and investigations were performed according to protocols. he was started on infusion of prostaglandin e1 at 0.025 g / kg / h as a vasodilator to optimise vascular flow and 5000 u unfractionated heparin subcutaneously for thromboprophylaxis. his investigations at the time of surgery were haemoglobin 9.9 g / dl, urea 88.8 mg / dl, creatinine 3.79 mg / dl, albumin 2.58 g / dl, sodium 143 meq / l and potassium 4.2 meq / l. immunosuppression was induced with interleukin 2 receptor blocker basiliximab and protocols for maintenance planned with prednisolone, tacrolimus and mycophenolate mofetil. anaesthesia was induced as using fentanyl, midazolam and propofol titrated to the loss of verbal response. atracurium was used to facilitate endotracheal intubation and an infusion at 0.5 mg / kg / h was used during surgery. the radial artery was cannulated under local anaesthesia and the left internal jugular vein was cannulated with a 7.5 fr triple lumen central venous catheter after intubation. a minimally invasive cardiac output monitor (edwards ev-1000) was used to guide fluids and the use of inotropes. the surgical procedure involved implantation of the donor pancreaticoduodenal graft in the right iliac fossa through a long vertical midline incision. vascular inflow anastomosis was made between the graft splenic and superior mesenteric artery through a y - shaped graft to the right common iliac artery. graft duodenum was anastomosed to a roux limb of jejunum after reperfusion to drain the exocrine secretion from the graft [figure 1 ]. insulin infusions were given as per protocols and the target was to maintain blood sugars between 100 and 150 mg / dl. volume rendered technique image of the pancreas and kidney reperfusion of the pancreas is accompanied by a rapid fall in blood sugars needing reduction or cessation of insulin infusions. our patient remained stable perioperatively, sugar levels normalised 2 h after reperfusion while insulin infusions were stopped immediately after reperfusion [table 1 ]. the renal implant was started after the pancreas, the graft being placed extraperitoneally in the left iliac fossa with vascular anastomosis to the external iliac artery and vein. injections of 20% mannitol (100 ml), furosemide 80 mg and methylprednisolone 500 mg were administered at the time of renal graft implant. intraoperative haemodynamics and metabolic profile noradrenaline at 0.08 g / kg / min and vasopressin at 1.8 u / h were used as vasopressors guided by the measurements of systemic vascular resistance. during the surgery, urine output from the native kidney was maintained during surgery (450 ml) and good perfusion of the graft kidney was observed he made an unremarkable recovery with normalisation of blood sugars and improvement of renal functions and was shifted from the intensive care unit by the 6 post - operative day. he was discharged on the 15 post - operative day and has well controlled blood sugars and normal renal functions at 6 months follow - up. the goal in pancreatic transplants is to prevent long - term diabetic complications and ensure normal levels of blood glucose. the progress in pancreatic transplants with improved surgical techniques, better donor and recipient selection and immunosuppression has extended the survival following spk to 14 years. majority of spks are performed for type i iddm with nephropathy in whom islet cells are destroyed by auto antibodies ; however, a small percentage of type ii diabetics also receive spk. the ideal candidates in our country are the young type i diabetics with renal failure necessitating dialysis or impending dialysis. pancreatic transplants may halt the progression of diabetic nephropathy and retinopathy and allow glucose control. a major problem with pancreatic grafts is venous thrombosis of the pancreatic portal vein, and prophylaxis with unfractionated heparin was commenced preoperatively and again at reperfusion of the graft in our patient. the use of epidural has been described to improve the quality of analgesia, but we had not considered an epidural in view of the on - going heparin use. the patient received an infusion of fentanyl at 0.5 g / kg / h with intermittent boluses according to haemodynamic responses. venous thrombi in the legs can form during prolonged surgery, and we had instituted mechanical intermittent pneumatic compression intraoperatively. the maintenance of temperature during the prolonged surgery was facilitated by the use of hemotherm, forced air warming devices and fluid warmers. we monitored the blood sugars hourly during surgery and infused a 5% dextrose in water solution at 50 ml / h and insulin as an infusion targeting blood sugars in the range of 100150 mg / dl until reperfusion and half hourly thereafter. the exocrine pancreatic drainage can be drained into the small bowel [figure 2 ] or the urinary bladder [figure 3 ]. in the bladder drainage, the graft duodenum is anastomosed to the recipient urinary bladder and allows a post - operative evaluation of graft function by a serial estimation of amylase levels. however long - term complications have led to a trend towards enteric drainage as described in our patient where the graft is anastomosed to a bowel loop. simultaneous pancreas kidney transplant with enteric drainage simultaneous pancreas kidney transplant with bladder drainage classically, most spk transplants are done on patients with established renal failure requiring haemodialysis. our patient was in class iv ckd and was maintained without haemodialysis, although an imminent need for haemodialysis had been explained. we had secured a jugular venous dialysis access prior to surgery, but in view of reasonable urine output and acceptable serum electrolytes in particular potassium, we had proceeded with surgery without initiating dialysis. this patient had maintained urine output throughout and careful attention had been given to avoid hypotension during surgery. oliguria or anuria prior to kidney graft reperfusion may have predisposed him to volume overload during surgery or pulmonary oedema. surgical problems anticipated in the immediate post - operative period were bleeding from anastomotic sites, portal venous thrombosis, non - functioning graft, bowel leaks and infections. our patient had an uncomplicated recovery of both renal and pancreatic functions and was put on regular follow - up after discharge from the hospital. spk transplants could offer insulin - free blood glucose control with an improved quality of life and protection against long - term diabetic complications in type i diabetic patients. refinements in techniques and improvements in immunosuppression can prospectively lead to improved survival with fewer complications. we have presented this case to bring out anaesthetic concerns and suggestions for protocols in the management of an uncommon surgical procedure in our country. | pancreatic grafts have been successfully used in patients with diabetes and are combined with kidney transplantation in patients with renal failure. the propagation of awareness in organ donation in india has increased the donor pool of transplantable organs in the last few years making multi visceral transplants feasible in our country. we present the anaesthetic management of a 32-year - old male with diabetes mellitus and end - stage renal failure who was successfully managed with a combined pancreas and kidney transplantation. |
the aim of this study was to evaluate the incidence and etiology of maxillofacial fractures and also to evaluate different treatment modalities. the sample consisted of 1,038 patients, with maxillofacial injuries treated at our center from june 2006 to june 2011. cause, type, site of injury, gender, age and treatment given to them, all these parameter are evaluated. the results of this study exhibit that road traffic accidents is the main reason for maxilla facial injuries followed by fall from height. the miniplate osteosynthesis was the most widespread of the fixation technique but conservative management of the fractured bone also has a significance importance in treatment modalities. hippocrates described an array of facial injuries as long ago as 400 bc. the injuries to the facial regions are clinically highly significant for number of reasons. maxillofacial region is associated with a number of important functions of the daily life sight, smell, eating, breathing, and talking. the number of maxillofacial injuries is continuously increasing due to rise in traffic, and failure to take preventive measures in the traffic leads to road traffic accidents, which is the main etiological factor in maxillofacial fractures. the aim of this study was to find out the incidence and pattern of maxillofacial injuries resulting from various etiological factors and treatment modalities and their complications. the maxillofacial injuries remain serious clinical problems because of its anatomical significance, i.e., important organs are located in this area and digestive and respiratory systems start from this area. due to anatomical proximity together with maxillofacial injuries, the damage to the central nervous system may occur and injuries in this region can result in serious dysfunction. this descriptive analytical study assesses the etiology, type, demographic, and treatment data of maxillofacial fractures managed at our center in the last 5 years. the sample consisted of 1,038 patients, with maxillofacial injuries treated at our center from june 2006 to june 2011. around 350 patients who were not admitted in the department and were treated as the outdoor patients were not included in this study, as it was not possible to obtain their complete data. the diagnosis was made on the basis of history, clinical examinations, and other investigations. radiographs, orthopantomogram, occipitomental view, submentovertex view, posterio - anterior (p.a.) view mandible, lateral oblique view mandible, were the main tools to confirm clinical diagnosis. the parameters assessed included age, sex, etiology, fractured bones, and treatment modalities and complications. different approaches for reduction and fixation of fractures were used according to indications either intra - oral approach or extra - oral approach. the most common site of fracture maxilla was found to be leforte 2 fracture. in our study, gender distribution was 9:1 [table 1 ], but in other studies, it was 2:1. males are more prone for trauma because of outdoor works, rash driving, and alcoholism. the most common involved age group was 21 - 30 (37.66%) years [table 2 ], followed by 31 - 40 years (19.36%). the road traffic accident (97.10%) was the most common etiological factor [table 3 ]. most of the patients had multiple bone fractures including mandible, maxilla, and zygomatic complex fracture (62.42%) [table 4 ]. adeyemo stated that road traffic crashes remain the major cause of maxillofacial injuries, unlike in most developed countries where assaults / interpersonal violence has replaced road traffic crashes as the major cause of the injuries. fracture involving different bones (n=1,038) the most commonly involved site was body of the mandible (51.50%) followed by parasymphysis (45.25%). coronoid fracture was reported to be least common (1.08%) [table 5 ]. among maxillary fractures, the most common fracture was leforte 2 fracture (84.00%) followed by leforte 1 and then leforte 3 [table 6 ]., they found there were (72.9%) mandibular, (13.9%) maxillary, (13.5%) zygomatic, (24.0%) zygomatico - orbital, (2.1%) cranial, (2.1%) nasal, and (1.6%) frontal injuries. car accidents (30.8%), motorcycle accidents (23.2%), altercations (9.7%), sports (6.3%), and warfare (9.7%) caused the maxillofacial injuries. regarding distribution of mandibular fractures, 32% were seen in the condylar region, 29.3% in the symphyseal parasymphyseal regions, 20% in the angle region, 12.5% in the body, 3.1% in the ramus, 1.9% in the dentoalveolar, and 1.2% in the coronoid region. the distribution of maxillary fractures was le fort ii (54.6%), le fort i (24.2%), le fort iii (12.1%), and alveolar (9.1%). of the all mandibular fractures, 56.9% were treated by closed reduction, 39.8% by open reduction, and 3.5% by observation only. of all maxillary fractures, 54.6% were treated using closed reduction, 40.9% using open reduction, and 4.5% with observation only. approximately, 52.1% of the patients were treated under general anesthesia and 47.9% were treated under local anesthesia and sedation. regarding treatment modalities we used, most of the patients were treated by open reduction and fixation (72.83%) and conservative management (22.73%), and 2.50% patients were treated by circum - mandibular wiring mostly in pediatric patients and edentulous patients [table 7 ]. according to ajmal,., open reduction and internal fixation has proven to be the most effective method for treatment of mandibular fractures. in most of the patients, open reduction & internal fixation (orif) was done under general anesthesia, rest of them under local anesthesia and conscious sedation. all the patients of circum - mandibular wiring were treated under general anaesthesia (ga). the close reduction was done under local anesthesia. according to study of back,., most patients were males (76%), the average age was 38 years, and drugs or alcohol were a significant aspect of the history in 30% of the cases. the most common mechanism of injury was assault (47%), followed by falls and sporting injuries. fifty percent of the fractures involved the orbital or orbito - zygomatic complex, and 55% had associated injuries. average follow - up was for 6 weeks (range : 0 - 44 weeks). most patients were managed conservatively based on our current criteria of un - displaced / minimally displaced fracture (57%) or minimal / no symptoms (24%). at final review, the other reasons for conservative management included patient non - compliance (11%) and medical contraindications (8%). being a developing country, the socioeconomic status of the majority is low and the patients coming to our center are from remote areas of the state and from neighboring states with the poor background, so choice of plating systems are limited. miniplates (stainless steel or titanium), 3d plates, locking plates, reconstruction plates, lag screws, and biodegradable systems were used. reconstruction of orbital floor was done with autogenous bone graft and in few cases with medpore. in most of the patients, pre - operative photograph of patient pre - operative photograph of patient pre - operative ct scan pre - operative ct scan intra - operative photograph of patient intra - operative photograph of patient post - operative ct scan post - operative ct scan post - operative photograph of patient in patients with only mandibular fractures (497), 21% patients were treated with intermaxillary fixation and 84.78% with open reduction and fixation with different systems [table 8 ]. treatment modalities used for mandible fractures (n=497) danda,. concluded from their study that the results of this study have shown that no significant clinical difference exists between patients undergoing closed treatment and rigid maxillomandibular fixation or open reduction and internal fixation. however, a radiographically better anatomic reduction of the condylar process was seen in the patients treated with open reduction and internal fixation. out of patients who received orif (64.78%), in 25.19% cases plates were removed within 6 months to 2 years because of secondary infection, sinus formation, or pus discharge from the site. there was no single case of delayed union or non - union reported [tables 6 and 9 ]. world health organization has estimated that nearly 25% of all injuries fatalities worldwide are a result of road traffic crashes with 90% of the fatalities occurring in low- and middle - income countries. road traffic accidents have been steadily falling in the developed countries ; they continue to rise with the horrifying speed in the low- and middle - income countries of africa and asia. fatigue is another important factor especially in commercial vehicle drivers who drives very long distances. bad road conditions also play an important role in rta but some studies reported more rtas on well paved and broad roads. the reason for the accidents in our country is due to violation of traffic rules, whereas in developed countries, accidents are most commonly due to alcoholic intoxications. this study shows that the most common cause of facial injuries was road traffic accidents, which is consistent with observation in other studies in india and other countries. mandible fracture was the most common fracture observed in this study because it is the most prominent bone in the face and is often fractured more than strongly supported middle third of face. fractures have been treated by a series of methods including close reductions, internal fixation, and circum - mandibular wiring. coletti stated that the imf self - drilling / tapping screws has been shown to be a useful modality to establish maxillomandibular fixation. it is a safe and time - sparing technique ; however, it is not without limitations or potential consequences which the surgeon must be aware of in order to provide safe and effective treatment. pediatric patients were treated by circum - mandibular wiring and few cases with bioresorbable plates under general anesthesia. pediatric patients benefit from the advantage of bioresorbable plates as it results in faster mobilization and the avoidance of secondary surgery for removal of implants. the old age successful management of these injuries using close reduction technique should be considered. patients with edentulous atrophic mandible were all so treated with circum - mandibular wiring and results were satisfactory. the minimally displaced fractures can be treated with conservative methods like close reduction to avoid hospitalization, cost factor, and significantly low risk of infections. in our study, there was no infection, non - union, mal union, or any functional disability reported in the patients who received inter maxillary fixation for 4 - 6 weeks. temporomandibular joint stiffness was reported during first week of after releasing imf which comes normal after a week with physiotherapy. however despite the professional and commercial interest in open reduction and semi - rigid fixation, we should think about patient 's interest affordability and well - being. sometimes, patients general condition, neurosurgical conditions, spinal injuries, medically compromised patients should be treated with conservative treatment. it is very cost effective, reduces hospital stay, or even no need for hospitalization. only dietary restrictions due to mouth closure and patient compliance are limitations. in few patients like epileptic, we can not use inter maxillary fixation for the management of maxillofacial trauma in minimally displaced fractures. other studies also did not show a clear overall benefits of the open reduction and fixation over conventional maxillo mandibular fixation (mmf) treatment. marker, etal. found non - surgical treatment of fracture of condoyle is non - traumatic, safe, and predictable and also support the conservative management of mandibular fractures. the cases with extensive displacement, associated fractures of mid - face, open reduction and fixation are indicated. according to worsaae and thorn in the study of open versus closed reduction of unilaterally dislocated low subcondylar fractures, they concluded that complications such as malocclusion, mandibular asymmetry, impaired masticatory function, and pain located to the affected joint or masticatory muscles were seen significantly more frequent in patients treated with closed reduction compared with those treated surgically (p = 0.005). neither the degree of dislocation of the proximal fragment, concomitant mandibular fractures nor the absence of posterior occlusal support seemed to influence the results. the results of this study exhibit that road traffic accidents is the main reason for maxilla facial injuries followed by fall from height. the miniplate osteosynthesis was the most widespread of the fixation technique but conservative management of the fractured bone also has a significance importance in treatment modalities. | objectives : the aim of this study was to evaluate the incidence and etiology of maxillofacial fractures and also to evaluate different treatment modalities.study design : the sample consisted of 1,038 patients, with maxillofacial injuries treated at our center from june 2006 to june 2011. cause, type, site of injury, gender, age and treatment given to them, all these parameter are evaluated.conclusion:the results of this study exhibit that road traffic accidents is the main reason for maxilla facial injuries followed by fall from height. maxillofacial injuries are more frequent in male than in female. the mandible was most frequently involved facial bone. the miniplate osteosynthesis was the most widespread of the fixation technique but conservative management of the fractured bone also has a significance importance in treatment modalities. |
most cases of hematuria can be diagnosed by urinalysis, urine culture, urinary cytology, computed tomography, and rigid and flexible cysto - ureteroscopy. we present a rare cause of hematuria that was challenging both to diagnose and to treat. a 50-year - old woman presented with a 1-month history of painless gross hematuria without clots. she was a known diabetic, hypertensive and ischemic heart disease patient and on regular treatment but not on any anticoagulants. on examination, she was hemodynamically stable. urinalysis revealed significant hematuria. bleeding time, clotting time, liver function tests, urine culture and urine cytology were unremarkable. cystoscopy with retrograde pyelogram revealed efflux of blood from the right ureteric orifice and a filling defect in the renal pelvis [figure 1 ]. biopsy of the floating brownish lesion with attachment to the renal pelvis using a semi - rigid ureteroscope was reported as fibrocollagenous material with no evidence of malignancy. however, the semi - rigid ureterorenoscope was unable to definitively rule out malignancy and flexible ureteroscope was planned. the patient continued to have hematuria and received eight units of packed red blood cells and eight units of fresh frozen plasma. however, she developed swelling and ecchymosis at the right thigh (puncture site), which was confirmed to be a pseudoaneurysm in the duplex scan. during anamnesis, it was revealed that the patient had bruises 2 weeks ago following insulin injection. filling defect rgp retrograde pyelogram at this juncture, her activated partial thromboplastin time (aptt) was raised but d dimer, fibrinogen and fibrin degradation product were normal. the bethesda test confirmed acquired factor viii inhibitor syndrome with a value of 1.6 bethesda unit. flexible ureteroscopy showed hyperemic patches and brownish floating material in the right renal pelvis that was completely removed using a dormia basket. the patient was subsequently managed jointly with a hematologist and treated with prednisolone and factor viii inhibitor bypass activity (feiba), and hematuria settled. isolated presentation of hematuria is even rarer as these bleeding disorders are accompanied by bleeding at other sites. acquired factor viii inhibitor syndrome is one such condition with an incidence of about one case per million per year. in this condition, autoantibodies are formed against factor viii. acquired hemophilia is different from the congenital type as it has no genetic inheritance pattern and hemarthroses are seldom present. it could cause significant morbidity with bleeding tendencies, and the mortality rate is 8 - 22%. acquired hemophilia is associated with autoimmune disorders, malignancy (solid, lymphoproliferative), skin diseases (pemphigus, epidermolysisbullosa), infections, drugs and post - partum state, but these are mostly idiopathic. the diagnosis is based on isolated prolongation of activated partial thromboplastin time not corrected by ptt correction study and confirmation by nijmegen modification of the bethesda assay showing reduced factor viii levels with evidence of factor viii inhibitor activity. treatment is aimed at (1) controlling bleeding and its complications and (2) eradication of the inhibitor. fresh frozen plasma and cryoprecipitate will not control bleeding as they do not contain sufficient factor viii to overcome the inhibitor. if the plasma levels of factor viii are raised to 30 - 50% in an acquired hemophilic patient, hemostasis could be generally achieved if the inhibitor assay is less than 5 bu (bethesda unit). 1-deamino-8-d - arginine vasopressin (ddavp) or infusion of factor viii (either human or porcine) is used to achieve the higher levels. however, if high - titer antibodies are present (more than 5 bethesda unit) to obtain hemostasis, bypassing agents like either activated prothrombin complex concentrate (apcc) (feiba) or recombinant activated factor 7(rfvii) (novaseven) is needed. corticosteroids, cytotoxic drugs such as cyclophosamide, azathioprine, vincristine, cyclosporine and rituximab, and high - dose intravenous immunoglobulins are used alone or in combination to eradicate the autoantibodies. the possibility of acquired hemophilia should be considered if elderly individuals present with severe hematuria, isolated aptt elevation, and when all other urological investigations were not contributory. this case is presented in view of the rarity of acquired factor viii inhibitor syndrome, with only hematuria as the main symptom mimicking urological malignancy. | a 50-year - old woman presented with gross hematuria for 1 month. clinical examinations, laboratory investigations, ultrasound and contrast computed tomography were normal, except anemia. cystoscopy revealed bloody efflux from the right side. retrograde pyelogram showed filling defect in the renal pelvis and biopsy was inconclusive. renal angiogram was normal. she developed ecchymosis on the right thigh and arm with elevated activated partial thromboplastin time. the partial thromboplastin time correction study and bethesda study confirmed the presence of acquired factor viii inhibitor (acquired hemophilia). with flexible ureterorenoscopy, the mass in the renal pelvis was removed and its histopathology revealed clotted blood. the patient was subsequently managed with steroids and factor eight inhibitor bypass activity. |
nasal dermoid sinus cysts (ndsc) are the most common congenital midline nasal lesion, accounting for 1% to 3% of all dermoid cysts, and 4% to 12% of head and neck dermoids (1, 2). ndscs may appear as a cystic mass or sinus opening on the midline nasal dorsum between the glabella and the columella at birth, or during early childhood (3). complete excision of ndscs, regardless of extension, is essential to prevent recurrence, nasal deformity, infection, meningitis, and intracranial abscess formation (4, 5). among the surgical approaches used for the treatment of ndscs are : excision and primary closure, midline vertical incision, transverse incision, lateral rhinotomy, inverted - u incision, and external rhinoplasty (6, 7). open rhinoplasty is the preferred approach, since it provides advantages over the standard incisions, including better cosmetic results, wide exposure and more control over osteotomies, and better visualization of the cribriform plate (7). the selection of the appropriate reconstruction technique, after dermoid resection, however, is also important. autologous septal or costal cartilage has been used for the repair of a dorsal defect (8 - 10). among the limitations of autologous graft material in pediatric patients moreover, it may be necessary to reinforce the nasal dorsal skin, which is damaged by the disease itself as well as the resection procedure. thus, the availability of other graft material might help solve some of these complicated problems. here we report the successful management of ndsc using an open rhinoplasty approach and primary reconstruction using crushed septal cartilage and tutoplast - processed fascia lata (tpfl). a 14-yr - old boy presented with pain, redness, and swelling of the nasal dorsum after being hit in the nose. at birth, this patient had a visible pit on the nasal dorsum, and intermittent discharge was present since early childhood. physical examination revealed a pit with discharge on a swollen and reddish dorsum (fig. a preoperative magnetic resonance imaging scan revealed an enhancing soft tissue lesion on the nasal dorsum and a tortuous sinus tract, from the nasal skin to the nasal septum (fig. the patient underwent complete removal of the ndsc by open rhinoplasty. following the transcollumelar and bilateral marginal incisions, both the lower and 3a) and involving the upper lateral cartilage, dorsal septum, and nasal bones. the nasal pit was removed using a small elliptical skin incision, on the nasal dorsum and the lesion, and dissected from the upper lateral cartilages, dorsal septum, and nasal bone. however, the preexisting severe inflammation led to the rupture of the cystic lesion and severe adhesion to the surrounding tissue ; hence, complete en bloc excision could not be achieved. after removal of the lesion, there were significant nasal defects, including an open roof deformity and decreased projection of the nasal dorsum. both upper lateral cartilages were sutured to the nasal septal cartilage using 4 - 0 polydioxanone. the dorsal defect and irregularities were reconstructed using crushed cartilage harvested from the nasal septum and four - layered tpfl strips (3040 mm, tutoplast, tutogen medical gmbh, industriestrasse, germany), as described previously (11). the tpfl was rehydrated in saline solution for more than 5 min before use and cut into long strips approximately 1 cm in width. the cephalic end of each four - layered strip had a stepladder pattern for smooth elevation of the radix area, and the caudal ends of all strips were rounded using iris scissors. this stack of tpfl strips was inserted into the dorsum, and crushed septal cartilage was inserted under the strips. this was followed by intercrural and interdomal sutures and an onlay graft to produce adequate nasal tip projection. the dorsal skin and rhinoplasty incisions were closed with 6 - 0 nylon sutures, and a nasal aqua splint was placed over the nasal dorsum. histologically, the lesion was lined by stratified squamous epithelium with adnexal structures, including hair follicles and sebaceous glands (fig. the patient was satisfied with the cosmetic results, and there was no evidence of recurrence (fig. most repairs of nasal defects after ndsc excision using an open rhinoplasty approach have used autologous septal and costal cartilages as graft materials for reconstruction (8 - 10). although autologous cartilage is the material of choice for dorsal augmentation, the amount of harvested septal or conchal cartilage is often insufficient ; in addition, costal cartilage has been associated with donor site morbidity. although we initially attempted to reconstruct the defect in this patient using only septal cartilage, the amount harvested was only about 1.51.5 cm, an amount insufficient for dorsal augmentation. in the nose of asians, the septal cartilage is relatively small, and younger patients have even smaller amounts of cartilage and require preservation of the large l - strut. moreover, in this patient, some dorsal irregularity persisted, and the skin had been thinned by aggressive removal of the ruptured cystic lesion using a microdebrider. autologous fascia and costal cartilage were also available, but the patient 's parents were reluctant to approve a separate donor site incision for harvest. in addition, synthetic graft material has a considerable risk for infection and was therefore inappropriate in this boy, who had preoperative infection and inflammation. because of our considerable experience using tpfl for dorsal augmentation, we elected to use tpfl together with crushed septal cartilage to repair the dorsal defect of this patient. when tpfl is used for dorsal augmentation in rhinoplasty procedures, it can be easily cut into the desired shapes and multilayered. furthermore, the soft contour of tpfl enables it to be blended well with the overlying skin - soft tissue envelope. tpfl also has the advantage of a low infection rate, the absence of donor site morbidity, and minimal risk for displacement and extrusion. as in our patient, the sinus tract may be ruptured by severe inflammation, making it virtually impossible to completely remove the entire sinus tract. another potential benefit of tpfl is its ability to separate the remnant sinus tract from the dorsal skin, thus preventing recurrent skin infection. unpredictable resorption of tpfl has been observed in 3 (4.3%) out of 69 patients reported in our previous study (11). however, the patient did not show loss of volume at the two - year follow - up. the open rhinoplasty approach using tfpl can be a useful surgical option as illustrated by the patient presented here. | nasal dermoid sinus cysts are the most common congenital midline nasal lesion, accounting for 1% to 3% of all dermoid cysts, and 4% to 12% of all head and neck dermoids. selection of the appropriate reconstruction technique, after dermoid resection, is important for treatment. here we describe the successful management of a case with a nasal dermoid sinus cyst using an open rhinoplasty approach, and primary reconstruction using tutoplast - processed fascia lata and crushed septal cartilage. |
in the last decade repair of mrna by spliceosome - mediated rna trans - splicing has raised interests as a novel therapeutic intervention (for reviews, see refs.). trans - splicing has many attractive features such as preservation of the endogenous regulation, replacement of selected portions of the target gene and, most importantly, corrections of dominant - negative mutations. the 5-, 3- or even internal exons of a target pre - mrna can be replaced by trans - splicing using engineered pre - trans - splicing molecules (ptms). ptms carry the wild - type (wt) sequence, a binding domain complementary to the endogenous target and an appropriate set of splicing elements. after nuclear import, ptms are transcribed and can specifically hybridize the target mutant pre - mrna via their binding domain, giving rise to a repaired mrna molecule (figure 1a). as a positive side effect of trans - splicing, cis - splicing so far, successful trans - splicing between ptms and endogenous targets has been described for different genetic diseases such as hemophilia a, cystic fibrosis, spinal muscular atrophy, hyper - igm - x - linked immunodeficiency, frontotemporal dementia with parkinsonism linked to chromosome 17, epidermolysis bullosa with muscular dystrophy, and huntington 's disease, most of them being 3-trans - splicing approaches. to the best of our knowledge, no study has provided evidence for successful 5-trans - splicing in vivo yet and this promising strategy has not been evaluated for cardiac genetic diseases. the aim of the present study was therefore to investigate this approach in hypertrophic cardiomyopathy (hcm). hcm is a myocardial disease mainly characterized by left ventricular hypertrophy (lvh) and diastolic dysfunction. the clinical outcome of hcm is highly variable and ranges from an asymptomatic benign course to heart failure, atrial fibrillation and sudden cardiac death caused by arrhythmias. hcm is a genetic disease transmitted as an autosomal - dominant trait and caused by mutations in genes encoding sarcomeric proteins. among them, mutations in mybpc3 encoding cardiac myosin - binding protein c (cmybp - c) are the most frequent ones. cmybp - c is a component of the thick filaments of the sarcomere, and plays important structural and functional roles. in the present study the feasibility of 5-trans - splicing to repair hcm - mutant mrna was assessed in isolated cardiac myocytes and in vivo in mybpc3-targeted knock - in (ki) mice that have been generated previously. ki mice carry a g > a transition on the last nucleotide of exon 6, which is associated with a severe phenotype and a poor prognosis in humans and occurs in 13% of all hcm patients in toscany. the g > a transition mutation leads to three different mutant mrnas in homozygous ki mice (supplementary figure s1). mutant-1 contains the mutation (missense), whereas mutant-2 (nonsense) and mutant-3 (deletion / insertion) are due to skipping of exon 6. we generated different ptms encoding exons 16 of wt mybpc3 under the control of a ubiquitous (cytomegalovirus) or cardiac myocyte - specific (tnnt2, human cardiac troponin t) promoter (figure 1b). to specifically detect repaired mybpc3 mrna and protein, an n - terminal flag - tag was introduced in the coding sequence. in addition, an intron was inserted right after the promoter to increase mrna stability and enhance expression of the constructs. the splicing domain included a canonical 5 splice donor site sequence followed by a downstream intronic sequence enhancer element, which has been shown to markedly increase the trans - splicing efficiency. importantly, the binding domain of the ptm is an essential part because it confers specificity to the target pre - mrna. to evaluate the feasibility and efficacy of 5-trans - splicing, we designed several constructs differing only with respect to the length of the binding domain and to the target site in mybpc3 intron 6 (supplementary table s1). the binding domains are complementary to this intron, but leave out its 3 splicing elements. moreover, to maintain the ptm in the nucleus and reduce its translation we deleted the sv40 polyadenylation (polya) signal, which is known to contribute to mrna stability and nuclear export (figure 1b). as negative controls we designed ptms with reversed binding domains (ptm - r), which should not induce 5-trans - splicing events. ptm - driven 5-trans - splicing on the endogenous ki mybpc3 pre - mrna target should produce a full - length repaired mybpc3 mrna, in which the mutation is bypassed, resulting in a flag - tagged wt repaired cmybp - c protein, and simultaneously cis - splicing should be reduced (figure 1c, d). to allow gene transfer in neonatal mouse cardiac myocytes (nmcms), ptms were packaged into self - complementary adeno - associated virus serotype 6 (aav6), a serotype known to efficiently transduce cardiac myocytes in culture. nmcms were isolated from ki mice and transduced with aav6-ptms either with or without polya signal (pa) or aav6-green fluorescent protein (gfp) as a control. after 4 days of transduction (multiplicity of infection (moi) : 3,000) about 80% of cells expressed gfp (supplementary figure s2). using pcr primers that specifically amplify the repaired mybpc3 mrna (supplementary figure s1 and supplementary table s2), we obtained a specific signal in aav6-ptm- and aav6-ptmpa - transduced nmcms, but not in untransduced or ptm - r - transduced nmcms (figure 2a). the absence of 5-trans - splicing in aav6-ptm - r - transduced nmcm excluded the possibility that recombination occurred between the highly homologous sequences of ptms and endogenous mybpc3. the amount of repaired mybpc3 was higher in the absence than in the presence of the polya signal in the ptm. to evaluate whether cis - splicing was reduced, we used mybpc3 primers binding in exons 1 and 9, which amplify total (repaired plus mutant) mybpc3 mrna. although no major difference was detected between samples, reduced signals were observed for certain mybpc3 mrna species in aav6-ptm- and in aav6-ptmpa - transduced nmcms. this suggests a reduction in mybpc3 cis - splicing when 5-trans - splicing occurred (figure 2a). sequencing of repaired mybpc3 mrna amplicons confirmed the presence of the wt guanine (g) at the exon 6exon 7 junction (figure 2b). conversely, sequencing of the upper 896-bp band of total mybpc3 mrna in aav6-ptmpa- and aav6-ptm - r - transduced nmcms showed the presence of the mutant adenine (a) at the same position (figure 2b). to estimate the amount of repaired mybpc3 mrna, we performed two rounds of pcr to amplify either total or only repaired mybpc3 mrna (figure 2c). comparison of amplicon intensities revealed that up to 33% of total mybpc3 transcripts were repaired. to evaluate whether the efficiency of 5-trans - splicing can be improved by increasing the dose of virus, we generated bicistronic recombinant adenovirus (adv) encoding the ptmpa and gfp both under the control of the tnnt2 promoter. ki nmcms were transduced with different moi of adv - ptmpa and analyzed 7 days after. repaired mybpc3 mrna was detected in all transduced samples and its amount increased with increasing moi (figure 2d). the pattern of total mybpc3 mrna did not reveal major difference from one moi to another, except at a moi of 100 at which the intensity of the mutant-3 and mutant-2 mrnas was lower than in untransduced cardiac myocytes (figure 2d). fluorescence analysis of adv - gfp transduced cardiomyocytes confirmed a complete transduction with a moi of 100 (supplementary figure s2). we further determined the efficiency of 5-trans - splicing in several samples with adv - ptmpa at a moi of 100, and estimated by semi - quantitative analysis that 51 7% of total mybpc3 mrna was repaired (figure 2e). we then investigated whether the repaired mybpc3 mrna is translated into protein and whether the repaired cmybp - c is properly incorporated into the sarcomere. the presence of the flag - tag allowed specific detection of repaired cmybp - c. whereas repaired cmybp - c was not detected by standard western blot with the anti - flag antibody, it was detected at the correct molecular weight after flag - immunoprecipitation (figure 3a, b), confirming that 5-trans - splicing occurred in cardiac myocytes. flag - immunoprecipitation of aav6-ptm - r - transduced nmcms did not show any band at 150 kda, while flag - mybpc3 transfected hek293 cells, used as a positive control, did show it (figure 3b). on the other hand, we detected a major flag - positive band around 35 kda in aav6-ptm- and aav6-ptm - r - transduced nmcms, which corresponds to the translated ptm transcripts (figure 3a). this band was barely detected in aav6-ptmpa - transduced nmcms, supporting the view that the absence of the polya signal prevented translation and putative accumulation of toxic ptm proteins in cells. endogenous and/or repaired cmybp - c, but not translated ptms were stained with a specific cmybp - c antibody, which recognizes the mybp - c motif (figures 3a and 1d). to investigate whether the repaired cmybp - c was incorporated into the sarcomere, we performed immunofluorescence analysis of transduced cardiac myocytes. about 9% of cmybp - c - positive cells (= cardiac myocytes) were co - stained with the anti - flag antibody, and the repaired cmybp - c showed the expected doublets in the a - band of the sarcomeres, indicating correct incorporation (figure 3c). in contrast, the 35-kda flag - ptm - r proteins showed a cellular and nuclear diffuse pattern without colocalization with endogenous cmybp - c (figure 3c). we next assessed the feasibility of ptm - driven 5-trans - splicing in ki mice in vivo. the ptmpa and renilla luciferase (rluc) were inserted in the pdsaav transfer vector under the control of the tnnt2 promoter and were packaged in aav serotype 9 (aav9), which has proven efficient cardiac transduction in mice in vivo. aav9 (mean dose 5.2 10 vg / kg of body weight (bw)) was administered systemically into 7-week - old animals. echocardiographic analysis performed during one month after injection did not display major differences in cardiac function between mice that received either aav9 or nacl (supplementary table s3). after 28 days, luciferase expression was evaluated by in vivo bioluminescence imaging and luminescence was recorded only in the heart of the aav9-rluc injected mouse (supplementary figure s3). accordingly, luciferase mrna level was high in the heart and very low in the liver of the mouse that received aav9-rluc (figure 4a), validating efficient and preferential cardiac transduction with aav9. importantly, the repaired mybpc3 mrna was detected in the heart of the mouse that received aav9-ptmpa, but not in the others (figure 4a). semi - quantitative analysis showed that 0.05% of total mybpc3 mrna was repaired (figure 4b). to augment the dose of virus and thus the 5-trans - splicing efficiency, we performed experiments in neonates (figure 5). longitudinal echo analysis in neonatal mice revealed that ki mice developed first systolic dysfunction, as shown by lower fractional area shortening than wt mice at day 2, followed by lvh, as shown by higher left - ventricular - mass - to - bw than wt mice at day 3 (figure 5a). we then systemically administered aav9-ptmpa into 1-day - old ki mice (3.4 10 vg / kg bw). this dose of aav9 resulted in an almost complete transduction of cardiac tissue at postnatal day 7 (supplementary figure s4). although the dose per bw was ~65-fold higher than in the adult mouse, no beneficial effect on left ventricular mass / bw and on fractional area shortening were observed at day 4 and 7 (figure 5b) as well as 7 weeks after injection (supplementary table s3). despite the absence of rescue, we evaluated the 5-trans - splicing efficiency in one mouse 7 weeks after injection. the full - length repaired mybpc3 mrna was detected by reverse transcription - pcr only in the heart of the aav9-ptmpa - injected mouse and represented 0.14% of total mybpc3 transcripts (figure 5c, d and supplementary figure s5), which thus showed 2.8-fold higher 5-trans - splicing event in the newborn than in the adult mouse. in addition, the repaired cmybp - c was detected, although faintly after flag - immunoprecipitation (figure 5e). the g > a transition mutation leads to three different mutant mrnas in homozygous ki mice (supplementary figure s1). mutant-1 contains the mutation (missense), whereas mutant-2 (nonsense) and mutant-3 (deletion / insertion) are due to skipping of exon 6. we generated different ptms encoding exons 16 of wt mybpc3 under the control of a ubiquitous (cytomegalovirus) or cardiac myocyte - specific (tnnt2, human cardiac troponin t) promoter (figure 1b). to specifically detect repaired mybpc3 mrna and protein, an n - terminal flag - tag was introduced in the coding sequence. in addition, an intron was inserted right after the promoter to increase mrna stability and enhance expression of the constructs. the splicing domain included a canonical 5 splice donor site sequence followed by a downstream intronic sequence enhancer element, which has been shown to markedly increase the trans - splicing efficiency. importantly, the binding domain of the ptm is an essential part because it confers specificity to the target pre - mrna. to evaluate the feasibility and efficacy of 5-trans - splicing, we designed several constructs differing only with respect to the length of the binding domain and to the target site in mybpc3 intron 6 (supplementary table s1). the binding domains are complementary to this intron, but leave out its 3 splicing elements. moreover, to maintain the ptm in the nucleus and reduce its translation we deleted the sv40 polyadenylation (polya) signal, which is known to contribute to mrna stability and nuclear export (figure 1b). as negative controls we designed ptms with reversed binding domains (ptm - r), which should not induce 5-trans - splicing events. ptm - driven 5-trans - splicing on the endogenous ki mybpc3 pre - mrna target should produce a full - length repaired mybpc3 mrna, in which the mutation is bypassed, resulting in a flag - tagged wt repaired cmybp - c protein, and simultaneously cis - splicing should be reduced (figure 1c, d). to allow gene transfer in neonatal mouse cardiac myocytes (nmcms), ptms were packaged into self - complementary adeno - associated virus serotype 6 (aav6), a serotype known to efficiently transduce cardiac myocytes in culture. nmcms were isolated from ki mice and transduced with aav6-ptms either with or without polya signal (pa) or aav6-green fluorescent protein (gfp) as a control. after 4 days of transduction (multiplicity of infection (moi) : 3,000) about 80% of cells expressed gfp (supplementary figure s2). using pcr primers that specifically amplify the repaired mybpc3 mrna (supplementary figure s1 and supplementary table s2), we obtained a specific signal in aav6-ptm- and aav6-ptmpa - transduced nmcms, but not in untransduced or ptm - r - transduced nmcms (figure 2a). the absence of 5-trans - splicing in aav6-ptm - r - transduced nmcm excluded the possibility that recombination occurred between the highly homologous sequences of ptms and endogenous mybpc3. the amount of repaired mybpc3 was higher in the absence than in the presence of the polya signal in the ptm. to evaluate whether cis - splicing was reduced, we used mybpc3 primers binding in exons 1 and 9, which amplify total (repaired plus mutant) mybpc3 mrna. although no major difference was detected between samples, reduced signals were observed for certain mybpc3 mrna species in aav6-ptm- and in aav6-ptmpa - transduced nmcms. this suggests a reduction in mybpc3 cis - splicing when 5-trans - splicing occurred (figure 2a). sequencing of repaired mybpc3 mrna amplicons confirmed the presence of the wt guanine (g) at the exon 6exon 7 junction (figure 2b). conversely, sequencing of the upper 896-bp band of total mybpc3 mrna in aav6-ptmpa- and aav6-ptm - r - transduced nmcms showed the presence of the mutant adenine (a) at the same position (figure 2b). to estimate the amount of repaired mybpc3 mrna, we performed two rounds of pcr to amplify either total or only repaired mybpc3 mrna (figure 2c). comparison of amplicon intensities revealed that up to 33% of total mybpc3 transcripts were repaired. to evaluate whether the efficiency of 5-trans - splicing can be improved by increasing the dose of virus, we generated bicistronic recombinant adenovirus (adv) encoding the ptmpa and gfp both under the control of the tnnt2 promoter. ki nmcms were transduced with different moi of adv - ptmpa and analyzed 7 days after. repaired mybpc3 mrna was detected in all transduced samples and its amount increased with increasing moi (figure 2d). the pattern of total mybpc3 mrna did not reveal major difference from one moi to another, except at a moi of 100 at which the intensity of the mutant-3 and mutant-2 mrnas was lower than in untransduced cardiac myocytes (figure 2d). fluorescence analysis of adv - gfp transduced cardiomyocytes confirmed a complete transduction with a moi of 100 (supplementary figure s2). we further determined the efficiency of 5-trans - splicing in several samples with adv - ptmpa at a moi of 100, and estimated by semi - quantitative analysis that 51 7% of total mybpc3 mrna was repaired (figure 2e). we then investigated whether the repaired mybpc3 mrna is translated into protein and whether the repaired cmybp - c is properly incorporated into the sarcomere. the presence of the flag - tag allowed specific detection of repaired cmybp - c. whereas repaired cmybp - c was not detected by standard western blot with the anti - flag antibody, it was detected at the correct molecular weight after flag - immunoprecipitation (figure 3a, b), confirming that 5-trans - splicing occurred in cardiac myocytes. flag - immunoprecipitation of aav6-ptm - r - transduced nmcms did not show any band at 150 kda, while flag - mybpc3 transfected hek293 cells, used as a positive control, did show it (figure 3b). on the other hand, we detected a major flag - positive band around 35 kda in aav6-ptm- and aav6-ptm - r - transduced nmcms, which corresponds to the translated ptm transcripts (figure 3a). this band was barely detected in aav6-ptmpa - transduced nmcms, supporting the view that the absence of the polya signal prevented translation and putative accumulation of toxic ptm proteins in cells. endogenous and/or repaired cmybp - c, but not translated ptms were stained with a specific cmybp - c antibody, which recognizes the mybp - c motif (figures 3a and 1d). to investigate whether the repaired cmybp - c was incorporated into the sarcomere, we performed immunofluorescence analysis of transduced cardiac myocytes. about 9% of cmybp - c - positive cells (= cardiac myocytes) were co - stained with the anti - flag antibody, and the repaired cmybp - c showed the expected doublets in the a - band of the sarcomeres, indicating correct incorporation (figure 3c). in contrast, the 35-kda flag - ptm - r proteins showed a cellular and nuclear diffuse pattern without colocalization with endogenous cmybp - c (figure 3c). we next assessed the feasibility of ptm - driven 5-trans - splicing in ki mice in vivo. the ptmpa and renilla luciferase (rluc) were inserted in the pdsaav transfer vector under the control of the tnnt2 promoter and were packaged in aav serotype 9 (aav9), which has proven efficient cardiac transduction in mice in vivo. aav9 (mean dose 5.2 10 vg / kg of body weight (bw)) was administered systemically into 7-week - old animals. echocardiographic analysis performed during one month after injection did not display major differences in cardiac function between mice that received either aav9 or nacl (supplementary table s3). after 28 days, luciferase expression was evaluated by in vivo bioluminescence imaging and luminescence was recorded only in the heart of the aav9-rluc injected mouse (supplementary figure s3). accordingly, luciferase mrna level was high in the heart and very low in the liver of the mouse that received aav9-rluc (figure 4a), validating efficient and preferential cardiac transduction with aav9. importantly, the repaired mybpc3 mrna was detected in the heart of the mouse that received aav9-ptmpa, but not in the others (figure 4a). semi - quantitative analysis showed that 0.05% of total mybpc3 mrna was repaired (figure 4b). to augment the dose of virus and thus the 5-trans - splicing efficiency, we performed experiments in neonates (figure 5). longitudinal echo analysis in neonatal mice revealed that ki mice developed first systolic dysfunction, as shown by lower fractional area shortening than wt mice at day 2, followed by lvh, as shown by higher left - ventricular - mass - to - bw than wt mice at day 3 (figure 5a). we then systemically administered aav9-ptmpa into 1-day - old ki mice (3.4 10 vg / kg bw). this dose of aav9 resulted in an almost complete transduction of cardiac tissue at postnatal day 7 (supplementary figure s4). although the dose per bw was ~65-fold higher than in the adult mouse, no beneficial effect on left ventricular mass / bw and on fractional area shortening were observed at day 4 and 7 (figure 5b) as well as 7 weeks after injection (supplementary table s3). despite the absence of rescue, we evaluated the 5-trans - splicing efficiency in one mouse 7 weeks after injection. the full - length repaired mybpc3 mrna was detected by reverse transcription - pcr only in the heart of the aav9-ptmpa - injected mouse and represented 0.14% of total mybpc3 transcripts (figure 5c, d and supplementary figure s5), which thus showed 2.8-fold higher 5-trans - splicing event in the newborn than in the adult mouse. in addition, the repaired cmybp - c was detected, although faintly after flag - immunoprecipitation (figure 5e). rna trans - splicing as a potential therapeutic technology has been applied to several diseases both in cell systems and in mouse models (for reviews, see refs.). the present study provides the first evidence of successful 5-trans - splicing both in cardiac myocytes and in the heart in vivo for the most prevalent cardiac genetic disease. the percentage of total mybpc3 mrna that was repaired was estimated to be between 33 and 66% in transduced ki nmcms. this is much higher than what has been reported in previous studies using endogenous targets. however, despite the high efficiency of mybpc3 5-trans - splicing at the mrna level, the amount of repaired cmybp - c protein was rather low. this suggests a low efficiency of translation and underlines that mrna copy number and protein levels do not need to be correlated. on the other hand, the amount of total mybpc3 mrnas is 80% and the level of cmybp - c protein 90% lower in ki than in wt mice. therefore, when 33% of total mybpc3 mrna is repaired, it represents less than 7% of the mybpc3 mrna amount and less than 4% of the cmybp - c protein amount found in wt mice. our study provides additional evidence for removing the polya signal in the ptm construct to prevent translation and therefore accumulation of ptm proteins that could exert a dominant - negative effect on the structure and/or function of cardiac myocytes. recently, in vivo 3-trans - splicing has been shown to improve the phenotype of a mouse model of spinal muscular atrophy. the present study provides the first evidence for successful mrna repair by 5-trans - splicing in vivo. although this resulted in detectable levels of repaired cmybp - c, the amount was still too low to ameliorate the cardiac phenotype. thus, further optimization of the technique is needed to increase the amount of therapeutic protein. among strategies that aim at specifically targeting mutant mrna in dominant genetic disease, such as mutant - specific rna interference, first, it allows the repair of even complex consequences on rna splicing that, as exemplified in the present hcm mouse model, can result from a single point mutation and will be difficult / impossible to target with sirna without affecting wt mrna. second, and in contrast to rna interference therapies targeting a specific mutation, two different ptms would be enough to treat the 4060% of hcm patients who carry a mybpc3 mutation therefore, trans - splicing represents a promising, potentially causal therapy of severe forms of hcm. the investigation conforms to the guidelines for the care and use of laboratory animals published by the national institutes of health (publication no. the experimental procedures were in accordance with the german law for the protection of animals and accepted by the ministry of science and public health of the city state of hamburg, germany (nr. the coding sequence of the ptms was generated by pcr from wt mybpc3 cdna with a forward primer (ptm f) containing an xhoi restriction site, the atg followed by the flag sequence and the first 20 nucleotides of mybpc3 exon 1. the reverse primer (ptm r) contained a bamhi restriction site and the 5 canonical splice donor site sequence followed by a downstream intronic splicing enhancer element / sequence from the rat fibroblast growth factor receptor 2 gene and last 23 nucleotides of mybpc3 exon 6. the binding domains were obtained by pcr on genomic ki dna using a forward primer (bd f) containing a bamhi restriction site, and 21 nucleotides of mybpc3 intron 6. the reverse primer (bd r) contained a noti restriction site and 28 nucleotides complementary to mybpc3 intron 6. the reverse binding domain was amplified in the same way (primers bd - r f and bd - r r) but reverse complemented. pcr products were sequentially cloned into pdsaav6-tnnt2 vector (human tnnt2 promoter) and accuracy of the insertion was verified by dna sequencing analysis. the sv40 polya signal was removed in one of the ptm plasmids by digestion with noti and mva1269i followed by religation of the plasmid. aav6 pseudotyped vectors were generated by cotransfection of hek293-aav cells (biocat, heidelberg, germany) with the pdsaav - tnnt2 transfer plasmid and the aav packaging plasmid pdp6rs, which provides the aav2 rep and aav6 cap genes and adenoviral helper functions. pseudotyped vectors were generated by triple - transfection of pdsaav - tnnt2 transfer plasmid with paav2/9 and phelper encoding adenoviral helper functions (biocat). generation of recombinant aav6 and aav9 particles was carried out as described previously, with some modifications. hek293-aav cells were cultivated in dulbecco 's modified eagle 's medium (high glucose) supplemented with 10% (vol / vol) heat - inactivated fetal calf serum, 0.1 mmol / l mem non - essential amino acids, 2 mmol / l l - glutamine, 100 u / ml penicillin, and 100 g / ml streptomycin. briefly, 1.5 10 hek293-aav cells were seeded on 15-cm plates and transfected with polyethylenimine. after 72 hours, cells were harvested, washed three - times with phosphate - buffered saline (pbs) and resuspended in pbs. after three freeze thaw cycles, benzonase (merck, darmstadt, germany ; final concentration 250 u / ml) was added and the lysates incubated for 1 hour at 37 c. cell debris was pelleted and vector - containing lysates were purified using iodixanol step gradients. the genomic titers of dnase - resistant recombinant aav particles were determined by quantitative pcr using the sybr green qpcr master mix 2 (fermentas, darmstadt, germany) and an abi prism 7900ht cycler (applied biosystems, foster city, ca). a standard curve for quantification was generated by serial dilutions of the respective plasmid dna. the cycling conditions were as follows : 50 c for 2 minutes, 95 c for 10 minutes, followed by 35 cycles of 95 c for 15 seconds and 60 c for 60 seconds. generation of recombinant adv. to generate the adv - ptmpa under the control of tnnt2 promoter, we used the in - fusion kit (clontech, st germain - en - laye, france) to fuse together the two cassettes into pshuttle85706. the pshuttle containing the tnnt2-ptmpa insert as well as the tnnt2-gfp in a bicistronic manner was electroporated into escherichia coli bj5183-d1 (stratagene, darmstadt, germany) to produce adenoviral dna through recombination. this dna was used to transfect hek293 cells and recombinant adv was amplified using standard techniques. aav6-mediated transductions of cardiac myocytes were performed for 30 minutes at 37 c in suspension before plating (4.4 10 cells / well) at a moi of 3,000 (aav6-ptmpa, both rna and protein analysis, aav6-ptm and aav6-ptm - r for protein analysis) or 30,000 (aav6-ptm and aav6-ptm - r for rna analysis). cardiac myocytes were kept in culture for 7 days at 37 c and 10% co2 before harvesting. in vivo seven - week - old ki mice received aav9-ptmpa (1.04 10 vg), aav9-rluc (1.36 10 vg) or nacl via systemic administration into the tail vein with a 29-g needle. intravenous injections of neonatal ki mice (postnatal day 1) with aav9-ptmpa (4.7 10 vg) or pbs were performed into the temporal vein using a 30-g needle. luciferase activity in the mouse heart was non - invasively assessed by in vivo bioluminescence imaging 4 weeks after aav9 injection. thereafter the substrate coelenterazine (biosynth, staad, switzerland) dissolved in methanol and further diluted in sodium phosphate buffer ph 7, was injected intraperitoneally (i.p.) at a dose of 2.5 mg / kg body weight in both mice. the mice were then placed in the chamber of a xenogen in vivo imaging system under continuous anesthesia. the oxidation of coelenterazine by renilla luciferase releases coelentarimide and blue light at 480 nm. this bioluminescence was recorded in a manually - selected region of interest centered over the mouse heart, using 3-minute scans. transthoracic echocardiography was performed using the vevo 2100 system (visualsonics, toronto, ontario, canada). ki mice were anesthetized with isofluorane (12%) and fixed to a warming platform in a supine position. b - mode images were obtained using a ms400 transducer for adult mice and a ms550 transducer for neonatal mice. images were obtained in a parasternal short and long - axis view and dimensions of the left ventricle were measured in a short - axis view in diastole and systole. total rna was isolated from cultured nmcms or ventricular tissue (30 mg) using the sv total rna isolation system kit (promega, madison, wi) according to the manufacturer 's instructions. rna concentration, purity and quality were determined using the nanodrop nd-1000 spectrophotometer (thermo scientific, darmstadt, germany). reverse transcription was performed from 150 to 200 ng rna using oligo - dt primers (superscript - iii kit ; life technologies, darmstadt, germany). as a control for genomic contamination, touchdown pcr amplifications (6560 c) with different primer pairs (supplementary table s2) were performed using amplitaq gold polymerase (applied biosystems) in a total volume of 20 l for 35 cycles. the full - length repaired mybpc3 mrna was amplified by touchdown pcr (6762 c) with phusion hot startii high - fidelity dna polymerase (biozym, hessisch oldendorf, germany) for 31 cycles. for semi - quantitative analysis a touchdown pcr (6560 c) for 25 cycles was used to amplify either total (primers e1-f, e9-r) or repaired (primers flag, e9-r) mybpc3 mrna. pcr products of the first pcr round were purified on a column (qiaquick pcr purification kit ; qiagen, valencia, ca) prior a second touchdown pcr (6560 c, primers e1-f, e2-r) for 35 cycles. crude protein extract from cultured nmcms or hek293 cells were extracted in lysis buffer (30 mmol / l tris base ph 8.8, 5 mmol / l edta, 30 mmol / l naf, 3% sds, 10% glycerol) and protein concentration was determined by bradford protein assay (bio - rad, hercules, ca). total proteins (nmcms 30 g / lane, hek293 2.5 g / lane) were separated on 10% sds - polyacrylamide (29:1) mini - gels (bio - rad) and transferred on polyvinylidene fluoride membranes by electroblotting. membranes were stained overnight with primary antibodies directed against the flag epitope (1:5,000 ; sigma, st louis, mo) in 5% milk in tbs - t or against the mybp - c motif (1:1,000). after incubation with anti - mouse (1:10,000 ; sigma) or anti - rabbit (1:6,000 ; sigma) peroxidase - conjugated secondary antibodies, proteins were visualized using super signal west dura detection reagent (thermo scientific) and signals were detected with the chemigenius bio imaging system. for immunoprecipitation aav6-ptmpa - transduced nmcms or ventricular tissue of aav9-ptmpa - injected mouse were lysed in modified ripa buffer (500 mmol / l nacl, 1 mmol / l edta, 50 mmol / l tris - hcl ph 7.4, 1% triton x-100, protease inhibitors complete mini ; roche, indianapolis, in), sonicated 2 for 30 seconds and centrifuged for 10 minutes at full speed at 4 c. the supernatant containing soluble proteins was diluted in 500 l modified ripa buffer and gently rolled overnight at 4 c with or without 10 g anti - flag antibody (sigma). the immunocomplexes were recovered by incubation with 50 l of protein a / g plus agarose (santa cruz biotechnology, santa cruz, ca) for 4 hours at 4 c. after three washing steps in ripa buffer and one additional wash step in 1 pbs (200 g, 3 minutes, 4 c) the flag - tagged proteins were eluted in 1 laemmli buffer (20 mmol / l tris - hcl ph 6.8, 200 mmol / l dtt, 4% sds, 0.02% bromophenol blue, 20% glycerol) and used for western blot as described above. anti - mouse exacta cruz (1:2,000 ; santa cruz biotechnology), which does not recognize the heavy and light chains of the immunoprecipitation antibody, was used as secondary antibody. the same immunoprecipitation protocol was applied to hek293 cells (40 g) transiently transfected with flag - wt - cmybp - c used as control. immunofluorescence analysis. for immunofluorescence analysis, aav6-transduced cells were rinsed once with ice - cold 1 pbs and fixed 10 minutes at 20 c in methanol / acetone (20/80). after two short washing steps in 1 pbs, cells were permeabilized 1 hour at room temperature in solution a (10% fcs, 1% bsa, 0.5% triton x-100 in 1 pbs). incubation with primary antibodies (anti - flag, 1:800 ; anti - mybp - c motif, 1:500) was done in solution b (1% bsa, 0.5% triton x-100 in 1 pbs) for 1 hour at room temperature. cells were then rinsed twice in solution b and incubated for 1 hour at room temperature with secondary antibodies (anti - mouse igg alexa 488-conjugated, 1:800 and anti - rabbit igg alexa 546-conjugated 1:800 ; molecular probes, darmstadt, germany) diluted in solution b together with draq5 (1:1,000 ; biostatus, leicestershire, uk) for nuclear staining. coverslips were embedded in mowiol and confocal images were acquired with a zeiss lsm 710 system using a zeiss axiovert microscope (zeiss, jena, germany) and a 40-oil objective. efficiency of aav6- and adv - mediated transduction in mybpc3-targeted knock - in nmcms. efficiency of transduction after systemic administration of aav9-gfp in a mybpc3-targeted knock - in mouse. | rna trans - splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. hypertrophic cardiomyopathy (hcm) is an autosomal - dominant disease, characterized by left ventricular hypertrophy (lvh) and diastolic dysfunction. mybpc3, encoding cardiac myosin - binding protein c (cmybp - c) is frequently mutated. we evaluated the 5-trans - splicing strategy in a mouse model of hcm carrying a mybpc3 mutation. 5-trans - splicing was induced between two independently transcribed molecules, the mutant endogenous mypbc3 pre - mrna and an engineered pre - trans - splicing molecule (ptm) carrying a flag - tagged wild - type (wt) mybpc3 cdna sequence. ptms were packaged into adeno - associated virus (aav) for transduction of cultured cardiac myocytes and the heart in vivo. full - length repaired mybpc3 mrna represented up to 66% of total mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. repaired cmybp - c protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. this study provides (i) the first evidence of successful 5-trans - splicing in vivo and (ii) proof - of - concept of mrna repair in the most prevalent cardiac genetic disease. since current therapeutic options for hcm only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease. |
papillary thyroid carcinoma being the most common type of differentiated thyroid malignancy has a predilection for spread through the lymphatic system and thus commonly involves the central and lateral compartmental lymph nodes of the cervical region. soft tissue metastasis is even rarer and amenable to radioiodine treatment similar to metastatic lesions in the lung and bones when they concentrate iodine. the concerned case is unique in the sense that the involvement simultaneously of three soft tissue organs is present with different iodine - concentrating abilities, whereas a review of the literature did not find the simultaneous presence of iodine - concentrating metastasis in one organ, iodine - nonconcentrating metastasis in another organ, and multiorgan soft tissue metastasis in the same patient. the case report also emphasizes possible hematogenous route of metastasis which is rare in papillary thyroid malignancy. a 42-year - old male patient with a history of total thyroidectomy and bilateral cervical nodal dissection positive for papillary carcinoma thyroid was referred to our department for a follow positron emission tomography - computed tomography (pet - ct) scan for evaluation of lung nodules and muscle lesions that he developed five years after primary treatment. during the follow - up, prompted by elevated thyroglobulin levels, an iodine-131 whole body scan and single photon emission tomography - computed tomography (spect - ct) [figure 1 ] showed positive lesions, one in the liver and another in the left gluteal region. a fludeoxyglucose (fdg) pet - ct scan [figure 2 ] was done to find more lesions and revealed metabolically active lesions in liver segment vi [figure 3 ] and the left gluteus and a new paraspinal muscle lesion at the nape of the neck [figure 4 ], whereas the lung nodules were metabolically inactive [figure 5 ]. a post - therapy scan [figure 6 ] at a therapeutic dose of 200 mci of iodine-131 revealed iodine concentration in the liver and a gluteal lesion, whereas there was no concentration in lung nodules and neck lesion. the present fdg pet ct scan [figure 7 ] as a response evaluation six month post treatment revealed a metabolic response in the liver and gluteal lesion [figure 8 ], but an increasing size of the neck lesion [figure 9 ] and lung nodules [figure 10 ]. presently, the patient is put on redifferentiation therapy with sorafenib and suppressive doses of thyroxin. the single photon emission tomography - computed tomography (spect ct) scan shows iodine - concentrating lesion in left gluteal muscle left gluteal lesions were metabolically active in pretherapy positron emission tomography - computed tomography (pet ct) study the new lesion at nape of neck fludeoxyglucose (fdg) activity but not lung nodules the 200 mci post - therapy scan concentration in liver and gluteal lesion but no concentration in neck lesion and lung nodules. the positron emission tomography - computed tomography (pet ct) scan after six months iodine therapy shows treatment response in liver soft tissue deposit in the neck demonstrating increase in size and fdg uptake (disease progression) papillary thyroid carcinoma is the most common type of differentiated thyroid carcinoma accounting for at least 70% of all follicular cell - derived thyroid malignancies and is considered to be a relatively indolent tumor in which distant metastasis and death are rare. the five - year survival rate for papillary thyroid cancer according to the stage is 100% for stage i and ii, 93% for stage iii, and 51% for stage distant metastasis is the principal cause of death in cases of well - differentiated thyroid carcinomas. about 10% of papillary carcinomas develop distant metastasis, with about 50% of patients having such metastasis at the time of diagnosis. the prognosis of these patients is poor, and over 50% of the patients are likely to die within five years, irrespective of the histology of the tumor. distant metastasis, although relatively uncommon, has been known to occur most commonly in the lungs, followed in frequency by bone and brain (0.1 to 5.0%). isolated cases of soft tissue metastasis have been documented in the orbit, skull, skin, muscles, liver, spleen, pancreas, and adrenal gland.[613 ] metastatic lesions have also been documented in silent or occult thyroid neoplasms. radioactive iodine treatment is considered to be the first line of treatment for distant metastasis from thyroid carcinomas that concentrate a significant amount of radioiodine. unlike our patient who developed metastatic disease and related symptoms five years after treatment of primary disease, for patients who develop distant metastatic disease at the time of initial diagnosis, a positive iodine-131 whole body scan post treatment of primary lesions in the neck makes way for successful radioiodine treatment of metastasis. however, solitary metastatic lesions which do not concentrate radioiodine can be dealt with surgical removal and/or external beam radiation. to add to the confusion, our patient showed radioiodine concentration in one of the muscle lesions and liver but no concentration in another muscle lesion and lung nodules. consequently, the muscle lesions responded to radioiodine treatment, whereas the neck lesion and lung nodules showed progression over the period of treatment. the patient has been put on a suppressive dose of thyroxin and redifferentiation therapy. in the past, retinoic acid, thalidomide, and rosiglitazone showed efficacy in redifferentiation therapy of iodine - nonconcentrating thyroid malignancy metastatic lesions ; however, currently, sorafenib (400 mg twice daily) and sunitinib (50 mg daily for 28 days followed by 14 days of no treatment per cycle), approved for other indications, show promise for thyroid cancer. | distant soft tissue metastasis and the simultaneous presence of iodine concentrating and nonconcentrating lesions in papillary thyroid cancer are extremely rare. the concerned patient, a histopathologically proven case of papillary thyroid cancer with nodal metastases treated with total thyroidectomy, bilateral cervical nodal dissection, and radioablation, subsequently developed lung, muscle, and liver metastasis. triggered by increased thyroglobulin, the iodine-131 whole body scan and 200 mci iodine-131 post - therapy scan showed a left gluteus maximus lesion and a liver lesion. fludeoxyglucose (fdg) positron emission tomography - computed tomography (pet - ct) scan intended to find additional lesions revealed iodine and fdg nonconcentrating bilateral pulmonary nodules and a single fdg avid hepatic and two muscle metastases. although fdg concentration in metastatic pulmonary nodules is generally low, the ct characteristics were classical for metastatic lesion. a follow - up fdg pet - ct study six months after 200 mci iodine-131 radioablation showed treatment response in muscle and liver lesions but not lungs. |
magnetic resonance imaging (mri) of the lung has been a challenge due to limitations such as low proton density in the lung and the fast signal decay due to susceptibility artefacts at air - tissue interfaces. thanks to recent technical advances such as parallel imaging, shared echo - technique and rotating phase encoding, lung mri can be recommended in a number of clinical indications. the introduction into clinical routine is facilitated by customising comprehensive mr protocols that apply fast breath - hold acquisition techniques from a buffet of sequences that are optimised for chest imaging. the basic imaging protocol comprises a non - contrast - enhanced protocol based on fast breath hold t1- and t2-weighted sequences to detect lung infiltrates, nodules or masses. additional steady - state free precession sequence (ssfp) imaging can be performed with free breathing and is highly sensitive for detection of central pulmonary embolism, and provides information on respiratory mechanics [2, 3 ]. respiration - triggered t2-weighted sequences are available for uncooperative patients and those with breath - holding difficulties. the sensitivity of this basic protocol for infiltrates and lung nodules is reported to be similar to ct [57 ]. additional contrast - enhanced fat - saturated three - dimensional gradient echo (3d - gre) sequences are warranted for unclear masses, consolidations or pleural effusion detected in the basic protocol. three components are available for the assement of pulmonary vasculature and lung perfusion : an initial free - breathing unenhanced examination followed by dynamic contrast - enhanced perfusion imaging and a high - resolution angiogram. with these customised protocols, lung mri offers alternative solutions to routine diagnostic challenges, in particular for the imaging of the mediastinum. it also provides an alternative radiation - free diagnostic option that is especially relevant to young and pregnant patients, as well as subjects who need to undergo multiple investigations, e.g. for research purposes. the details of the mr physics background as well as the protocol tree and its branches have been addressed in the two preceeding articles (citations 1/3 and 3/3). the aim of this paper is to discuss the advantages and limitations of lung mri for a number of selected clinical applications and to outline current developments and future perspectives. cystic fibrosis (cf) lung disease is caused by mutations in the cftr gene and remains one of the most frequent lethal inherited diseases in the caucasian population. due to the progress in therapy and management of cf lung disease in the past decades, the life expectancy of cf patients has increased substantially, with a current median survival of approximately 40 years and is expected to increase even further [10, 11 ]. it is known that clinical parameters including spirometric pulmonary function testing (pft) suffer from limited sensitivity and provide no regional information. with the advances in imaging in general and the ability to characterise and quantify cf in greater detail, imaging will likely play an increasing role in the improved understanding of the disease process and the progression of disease. however, this also means that it becomes vital to reduce the overall (cumulative) radiation burden in this population, as this could lead to iatrogenic carcinogenesis. magnetic resonance imaging (mri) is reported to be comparable to ct with regard to the detection of morphological changes in the cf lung [1315 ]. at the same time mri is superior to ct when it comes to the assessment of functional changes such as altered pulmonary perfusion. moreover, using the described mr protocols, it is possible to visualise bronchiectasis, bronchial wall thickening, mucus plugging, air fluid levels, consolidation and segmental consolidation and destruction, (fig. 1a, b).fig. the axial t2-weighted (blade ; a) and the volumetric contrast - enhanced 3d - gre (vibe ; b) breath - hold acquisitions show severe bronchiectasis, bronchial wall thickening, mucus plugging, pleural effusion as well as a destructed middle lobe. the perfusion subtraction image (c) shows a severely impaired perfusion pattern with loss of perfusion in several areas. the maximum enhancement (max) and time to peak anhancement (ttp maps) allow for a further characterisation of the perfusion impairment. most areas with impaired perfusion show a reduced (max map) and delaid (ttp map) perfusion. notice the area in the left upper lobe with reduced but not delayed perfusion (arrowhead) a 29-year - old female with cystic fibrosis. the axial t2-weighted (blade ; a) and the volumetric contrast - enhanced 3d - gre (vibe ; b) breath - hold acquisitions show severe bronchiectasis, bronchial wall thickening, mucus plugging, pleural effusion as well as a destructed middle lobe. the perfusion subtraction image (c) shows a severely impaired perfusion pattern with loss of perfusion in several areas. the maximum enhancement (max) and time to peak anhancement (ttp maps) allow for a further characterisation of the perfusion impairment. most areas with impaired perfusion show a reduced (max map) and delaid (ttp map) perfusion. notice the area in the left upper lobe with reduced but not delayed perfusion (arrowhead) the accuracy of mri in detecting bronchiectasis is dependent on a number of factors, including bronchial level and diameter, wall thickness, and the signal from within the bronchial wall and lumen. central bronchi and bronchiectasis (central, peripheral) are well visualised on mri, whereas normal peripheral bronchi starting at the 3rd to 4th generation are poorly visualised. a high signal of the bronchial wall on t2-weighted (t2w) images represents increased fluid, i.e. oedema, possibly caused by active inflammation. enhancement of the thickened bronchial wall on post - contrast, fat - suppressed t1-weighted images is thought to be related to inflammatory activity. it is important to note that compared to mri, ct can only detect wall thickening and is not able to comment on the cause. mucus plugging is well visualised on mri even down to the small airways due to the high t2 signal of its fluid content. it is recognised as a high t2 signal filling of the bronchus along its course with branching in the periphery giving a grape - like or tree - in - bud appearance, respectively. as mucus plugs bronchial air fluid levels are indicative of active infection, occurring in saccular or varicose bronchiectasis, and can be visualised by their high t2 signal. however, discriminating a bronchus with an air fluid level from one with partial mucus plugging or a severely thickened wall can be difficult. when evaluating the signal characteristics on t2- and t1-weighted images with and without contrast enhancement, air fluid levels can usually be differentiated. pulmonary consolidation in cf is mainly caused by alveolar filling with inflammatory material leading to a high signal on t2w images. comparable to ct, mri is able to visualise air bronchograms as low signal areas following the course of the bronchi within the consolidation [17, 18 ]. with progression of the disease, complete destruction of lung segments or lobes can occur with similar appearances on mri and ct. compared to ct, the strength of mri is the additional assessment of function, i.e. perfusion, pulmonary hemodynamics and ventilation. in cf, regional ventilatory defects cause changes in regional lung perfusion due to the hypoxic vasoconstriction response or tissue destruction. using mri, lung perfusion can be assessed by contrast - enhanced lung perfusion imaging. using contrast - enhanced 3d mri, perfusion defects in 11 children with cf were reported to correlate well with the degree of tissue destruction. furthermore it was shown that at the age of 06 years lung perfusion changes were more prominent than morphological changes. however, establishing quantitative assessment tools for lung morphology is challenging for several reasons. first, signal intensities as derived from mri are not calibrated as compared to ct. second, the signal - to - noise ratio (snr) in the lung is low and heterogeneous due to several physical circumstances. moreover, due to the lack of linearity between the mr signal and the concentration of applied contrast media, quantification of pulmonary perfusion using mri is challenging. described the importance of the qualitative assessment of the contrast time course component when analysing contrast - enhanced 3d mri to categorise perfusion changes as normal, delayed, reduced, reduced and delayed as well as perfusion loss. using dedicated post - processing tools in addition to quantitative and qualitative scoring methods, clinical practice relies on visual assessment. it should be feasible to introduce an mr scoring system that is comparable to ct [2426 ]. up to now published studies either used a modified brody or an adapted bhalla / helbich score. functional parameters are important for a comprehensive diagnosis and have to be integrated into a dedicated mr score, also to generate an additional benefit over ct. a recently presented morpho - functional mri score is easily applicable and reproducible for the semi - quantitative morphological and functional evaluation of a large severity spectrum of cf lung disease. based on the current state of affairs, perfusion mri can be applied to monitor therapy and may be capable of differentiating between regions with reversible and irreversible disease. in contrast to ct [2426, 28, 29 ] a dedicated scoring system as well as quantitative readouts for pulmonary mri is lacking and will require development. the current imaging reference technique in evaluation of acute pulmonary embolism is helical computed tomography. its major advantage over ventilation and perfusion scintigraphy and spect are the availability and the comparably short acquisition time of the study with almost immediate delivery of the necessary information for patient care. however, radiation exposure by ct is significant ; therefore an alternative method for young patients and pregnant women would be appreciated. to be competitive with ct, an abbreviated mr protocol focusing on lung vessel imaging and lung perfusion although mr angiography has been demonstrated as an excellent tool in dedicated centres, more recent data from a large multicentre study suggest that the technique in isolation produced unsatisfactory results. therefore, combinations of different available mri techniques for the detection of pulmonary embolism may be of better value. this protocol was further modified and extended into a two - step algorithm. as a first step, a steady - state gre sequence acquired in two or three planes during free breathing would serve for an early detection of large central emboli within the first 5 min of the examination according to the literature with a sensitivity of 90% and a specificity of close to 100% [3335 ]. any patient with a massive, central embolism detected at this point could be directly referred to intensive care and treatment ; the time to diagnosis would be at least as short as with contrast - enhanced helical ct. if this first step of the examination produces a negative or unclear result, the protocol would be continued with the contrast - enhanced steps including first pass perfusion imaging, high spatial resolution contrast - enhanced (ce) mra and a final acquisition with a volumetric interpolated 3d flash sequence in transverse orientation (fig. 2). despite its composition of multiple sequences, the two - step examination could be completed within 15 min in - room time, which makes it feasible as a quick test for daily clinical routine. in many cases, such as in pregnant woman, when administration of contrast material or radiation exposure is contra - indicated, the examination can be limited to the first step, the free breathing or breathhold acquisition of steady - state gre sequences alone. furthermore, since these steps are partially redundant, at least one acquisition would be expected to be diagnostic even in non - compliant patients.fig. breathing (a) and contrast - enhanced coronal 3d flash angiogram acquired in breathhold (b ; embolus inside the right lower lobe artery circled) ; c series of subtracted images from the first pass perfusion study, perfusion deficits marked with open arrows at the image obtained at peak lung enhancement ; 1.5-t mri scanner a 55-year - old patient with acute pulmonary embolism. coronal steady - state free precession images acquired during free breathing (a) and contrast - enhanced coronal 3d flash angiogram acquired in breathhold (b ; embolus inside the right lower lobe artery circled) ; c series of subtracted images from the first pass perfusion study, perfusion deficits marked with open arrows at the image obtained at peak lung enhancement ; 1.5-t mri scanner cystic fibrosis (cf) lung disease is caused by mutations in the cftr gene and remains one of the most frequent lethal inherited diseases in the caucasian population. due to the progress in therapy and management of cf lung disease in the past decades, the life expectancy of cf patients has increased substantially, with a current median survival of approximately 40 years and is expected to increase even further [10, 11 ]. it is known that clinical parameters including spirometric pulmonary function testing (pft) suffer from limited sensitivity and provide no regional information. with the advances in imaging in general and the ability to characterise and quantify cf in greater detail, imaging will likely play an increasing role in the improved understanding of the disease process and the progression of disease. however, this also means that it becomes vital to reduce the overall (cumulative) radiation burden in this population, as this could lead to iatrogenic carcinogenesis. magnetic resonance imaging (mri) is reported to be comparable to ct with regard to the detection of morphological changes in the cf lung [1315 ]. at the same time mri is superior to ct when it comes to the assessment of functional changes such as altered pulmonary perfusion. moreover, using the described mr protocols, it is possible to visualise bronchiectasis, bronchial wall thickening, mucus plugging, air fluid levels, consolidation and segmental consolidation and destruction, (fig. 1a, b).fig. the axial t2-weighted (blade ; a) and the volumetric contrast - enhanced 3d - gre (vibe ; b) breath - hold acquisitions show severe bronchiectasis, bronchial wall thickening, mucus plugging, pleural effusion as well as a destructed middle lobe. the perfusion subtraction image (c) shows a severely impaired perfusion pattern with loss of perfusion in several areas. the maximum enhancement (max) and time to peak anhancement (ttp maps) allow for a further characterisation of the perfusion impairment. most areas with impaired perfusion show a reduced (max map) and delaid (ttp map) perfusion. notice the area in the left upper lobe with reduced but not delayed perfusion (arrowhead) a 29-year - old female with cystic fibrosis. the axial t2-weighted (blade ; a) and the volumetric contrast - enhanced 3d - gre (vibe ; b) breath - hold acquisitions show severe bronchiectasis, bronchial wall thickening, mucus plugging, pleural effusion as well as a destructed middle lobe. the perfusion subtraction image (c) shows a severely impaired perfusion pattern with loss of perfusion in several areas. the maximum enhancement (max) and time to peak anhancement (ttp maps) allow for a further characterisation of the perfusion impairment. most areas with impaired perfusion show a reduced (max map) and delaid (ttp map) perfusion. notice the area in the left upper lobe with reduced but not delayed perfusion (arrowhead) the accuracy of mri in detecting bronchiectasis is dependent on a number of factors, including bronchial level and diameter, wall thickness, and the signal from within the bronchial wall and lumen. central bronchi and bronchiectasis (central, peripheral) are well visualised on mri, whereas normal peripheral bronchi starting at the 3rd to 4th generation are poorly visualised. a high signal of the bronchial wall on t2-weighted (t2w) images represents increased fluid, i.e. oedema, possibly caused by active inflammation. enhancement of the thickened bronchial wall on post - contrast, fat - suppressed t1-weighted images is thought to be related to inflammatory activity. it is important to note that compared to mri, ct can only detect wall thickening and is not able to comment on the cause. mucus plugging is well visualised on mri even down to the small airways due to the high t2 signal of its fluid content. it is recognised as a high t2 signal filling of the bronchus along its course with branching in the periphery giving a grape - like or tree - in - bud appearance, respectively. as mucus plugs bronchial air fluid levels are indicative of active infection, occurring in saccular or varicose bronchiectasis, and can be visualised by their high t2 signal. however, discriminating a bronchus with an air fluid level from one with partial mucus plugging or a severely thickened wall can be difficult. when evaluating the signal characteristics on t2- and t1-weighted images with and without contrast enhancement, air fluid levels can usually be differentiated. pulmonary consolidation in cf is mainly caused by alveolar filling with inflammatory material leading to a high signal on t2w images. comparable to ct, mri is able to visualise air bronchograms as low signal areas following the course of the bronchi within the consolidation [17, 18 ]. with progression of the disease, complete destruction of lung segments or lobes can occur with similar appearances on mri and ct. compared to ct, the strength of mri is the additional assessment of function, i.e. perfusion, pulmonary hemodynamics and ventilation. in cf, regional ventilatory defects cause changes in regional lung perfusion due to the hypoxic vasoconstriction response or tissue destruction. using mri, lung perfusion can be assessed by contrast - enhanced lung perfusion imaging. using contrast - enhanced 3d mri, perfusion defects in 11 children with cf were reported to correlate well with the degree of tissue destruction. furthermore it was shown that at the age of 06 years lung perfusion changes were more prominent than morphological changes. however, establishing quantitative assessment tools for lung morphology is challenging for several reasons. first, signal intensities as derived from mri are not calibrated as compared to ct. second, the signal - to - noise ratio (snr) in the lung is low and heterogeneous due to several physical circumstances. moreover, due to the lack of linearity between the mr signal and the concentration of applied contrast media, quantification of pulmonary perfusion using mri is challenging. described the importance of the qualitative assessment of the contrast time course component when analysing contrast - enhanced 3d mri to categorise perfusion changes as normal, delayed, reduced, reduced and delayed as well as perfusion loss. using dedicated post - processing tools in addition to quantitative and qualitative scoring methods, clinical practice relies on visual assessment. it should be feasible to introduce an mr scoring system that is comparable to ct [2426 ]. up to now published studies either used a modified brody or an adapted bhalla / helbich score. functional parameters are important for a comprehensive diagnosis and have to be integrated into a dedicated mr score, also to generate an additional benefit over ct. a recently presented morpho - functional mri score is easily applicable and reproducible for the semi - quantitative morphological and functional evaluation of a large severity spectrum of cf lung disease. based on the current state of affairs, perfusion mri can be applied to monitor therapy and may be capable of differentiating between regions with reversible and irreversible disease. in contrast to ct [2426, 28, 29 ] a dedicated scoring system as well as quantitative readouts for pulmonary mri is lacking and will require development. the current imaging reference technique in evaluation of acute pulmonary embolism is helical computed tomography. its major advantage over ventilation and perfusion scintigraphy and spect are the availability and the comparably short acquisition time of the study with almost immediate delivery of the necessary information for patient care. however, radiation exposure by ct is significant ; therefore an alternative method for young patients and pregnant women would be appreciated. to be competitive with ct, an abbreviated mr protocol focusing on lung vessel imaging and lung perfusion although mr angiography has been demonstrated as an excellent tool in dedicated centres, more recent data from a large multicentre study suggest that the technique in isolation produced unsatisfactory results. therefore, combinations of different available mri techniques for the detection of pulmonary embolism may be of better value. this protocol was further modified and extended into a two - step algorithm. as a first step, a steady - state gre sequence acquired in two or three planes during free breathing would serve for an early detection of large central emboli within the first 5 min of the examination according to the literature with a sensitivity of 90% and a specificity of close to 100% [3335 ]. any patient with a massive, central embolism detected at this point could be directly referred to intensive care and treatment ; the time to diagnosis would be at least as short as with contrast - enhanced helical ct. if this first step of the examination produces a negative or unclear result, the protocol would be continued with the contrast - enhanced steps including first pass perfusion imaging, high spatial resolution contrast - enhanced (ce) mra and a final acquisition with a volumetric interpolated 3d flash sequence in transverse orientation (fig. 2). despite its composition of multiple sequences, the two - step examination could be completed within 15 min in - room time, which makes it feasible as a quick test for daily clinical routine. in many cases, such as in pregnant woman, when administration of contrast material or radiation exposure is contra - indicated, the examination can be limited to the first step, the free breathing or breathhold acquisition of steady - state gre sequences alone. furthermore, since these steps are partially redundant, at least one acquisition would be expected to be diagnostic even in non - compliant patients.fig. coronal steady - state free precession images acquired during free breathing (a) and contrast - enhanced coronal 3d flash angiogram acquired in breathhold (b ; embolus inside the right lower lobe artery circled) ; c series of subtracted images from the first pass perfusion study, perfusion deficits marked with open arrows at the image obtained at peak lung enhancement ; 1.5-t mri scanner a 55-year - old patient with acute pulmonary embolism. coronal steady - state free precession images acquired during free breathing (a) and contrast - enhanced coronal 3d flash angiogram acquired in breathhold (b ; embolus inside the right lower lobe artery circled) ; c series of subtracted images from the first pass perfusion study, perfusion deficits marked with open arrows at the image obtained at peak lung enhancement ; 1.5-t mri scanner undoubtedly, contrast - enhanced multiple detector row computed tomography is the method of first choice in imaging thoracic malignancies. mri is considered as an alternative method, e.g. when the application of iodinated contrast media is contraindicated. for this purpose, a contrast - enhanced examination can be achieved within 25 min in - room time. intra - pulmonary masses larger than the clinically relevant size of 45 mm in diameter can be easily detected. the extent of mediastinal, hilar and supraclavicular lymph node enlargment can be assessed with excellent soft tissue contrast. metastatic disease involving the liver, the adrenal glands and the skeleton of the thorax are fully covered. the feasibility of extending the examination to whole - body staging with comparable results as achieved by pet / ct has been demonstrated [3740 ]. the only limitation compared to ct is the detection of nodules smaller than the clinically relevant size of 45 mm. beyond being just a surrogate for a ct scan in some cases, mri can offer additional advantages. in large pulmonary masses, the excellent soft tissue contrast of mri allows for the distinction of tumour from atelectasis and pleural effusion, e.g. for image - guided radiotherapy planning. administration of t1-shortening contrast material specifically contributes to detecting tumour necrosis, chest wall or mediastinal invasion, and pleural reaction / carcinomatosis (fig., mri contributes comprehensive functional information on respiratory mechanics, tumour mobility and lung perfusion [42, 43 ]. the clinical value of complementing the purely morphologic staging by imaging of perfusion and tumour motion in specific clinical settings and situations has been demonstrated and is subject to further investigation.fig. the transverse t2-weighted fat - saturated (a) and t1-weighted contrast - enhanced fat - saturated 3d - gre images (b, c) show a large, centrally necrotic mass in the left upper lobe with large peri - hilar lymph node metastases. note the high soft tissue contrast between alelectatic lung (open arrow), small rim of solid tumour (filled arrow) and colliquated central portion of the mass (asterisk) a 56-year - old female patient with small cell lung cancer. the transverse t2-weighted fat - saturated (a) and t1-weighted contrast - enhanced fat - saturated 3d - gre images (b, c) show a large, centrally necrotic mass in the left upper lobe with large peri - hilar lymph node metastases. note the high soft tissue contrast between alelectatic lung (open arrow), small rim of solid tumour (filled arrow) and colliquated central portion of the mass (asterisk) the potential of mri to replace chest radiography, particularly in very young children, was already investigated several years ago [18, 4446 ]. much of this work was conducted on low - field mri (mainly 0.2-t scanners) using steady - state free precession sequences. on average, three thick slices are acquired in coronal orientation with a mean breathhold time of 45 s. however, nowadays, only a few institutions regularly use low - field lung mri in paediatric radiology. nevertheless, the experience from this work may be considered valid for the suggested protocols for 1.5-t scanners since image quality has significantly improved. therefore, t2-weighted fat - suppressed as well as dynamic contrast - enhanced t1-gre sequences are applied with a slice thickness between 5 and 6 mm. disease entities encompassing community - acquired pneumonia, empyema, fungal infections and chronic bronchitis are detectable (fig. 4).fig. 4a 13-year - old girl with suspected organising pneumonia (boop) in both lungs. transverse t2-weighted tse images (a) were acquired with the navigator technique (sample volume placed on the dome of the right liver lobe). the open arrow indicates an oval - shaped consolidation with pleural contact in the lower left lobe and moderate signal intensity. coronal contrast - enhanced fat - saturated t1-weighted gre images (b) were acquired with the breathhold technique. the open arrow indicates the oval - shaped consolidation in the lower left lobe with contrast enhancement ; this is interpreted as an indicator of an active inflammatory process a 13-year - old girl with suspected organising pneumonia (boop) in both lungs. transverse t2-weighted tse images (a) were acquired with the navigator technique (sample volume placed on the dome of the right liver lobe). the open arrow indicates an oval - shaped consolidation with pleural contact in the lower left lobe and moderate signal intensity. coronal contrast - enhanced fat - saturated t1-weighted gre images (b) were acquired with the breathhold technique. the open arrow indicates the oval - shaped consolidation in the lower left lobe with contrast enhancement ; this is interpreted as an indicator of an active inflammatory process recent studies demonstrated the feasibility of chest examinations on 3-t high - field mri. high - field chest mri may allow differentiation between inflammation- and fibrosis - predominant lesions in uip and nsip in adult patients. moreover, a recent comparison between 3-t lung mri and hrct as gold standard showed an excellent correlation with non - cystic fibrosis chronic lung disease in children. breathhold t2- as well as t1-weighted sequences with ecg triggering were acquired. in summary, lung mri may prove to become a valuable tool for detection as well as characterisation of inflammatory lung disease in children. undoubtedly, contrast - enhanced multiple detector row computed tomography is the method of first choice in imaging thoracic malignancies. mri is considered as an alternative method, e.g. when the application of iodinated contrast media is contraindicated. for this purpose, a contrast - enhanced examination can be achieved within 25 min in - room time. intra - pulmonary masses larger than the clinically relevant size of 45 mm in diameter can be easily detected. the extent of mediastinal, hilar and supraclavicular lymph node enlargment can be assessed with excellent soft tissue contrast. metastatic disease involving the liver, the adrenal glands and the skeleton of the thorax are fully covered. the feasibility of extending the examination to whole - body staging with comparable results as achieved by pet / ct has been demonstrated [3740 ]. the only limitation compared to ct is the detection of nodules smaller than the clinically relevant size of 45 mm. beyond being just a surrogate for a ct scan in some cases, mri can offer additional advantages. in large pulmonary masses, the excellent soft tissue contrast of mri allows for the distinction of tumour from atelectasis and pleural effusion, e.g. for image - guided radiotherapy planning. administration of t1-shortening contrast material specifically contributes to detecting tumour necrosis, chest wall or mediastinal invasion, and pleural reaction / carcinomatosis (fig., mri contributes comprehensive functional information on respiratory mechanics, tumour mobility and lung perfusion [42, 43 ]. the clinical value of complementing the purely morphologic staging by imaging of perfusion and tumour motion in specific clinical settings and situations has been demonstrated and is subject to further investigation.fig. the transverse t2-weighted fat - saturated (a) and t1-weighted contrast - enhanced fat - saturated 3d - gre images (b, c) show a large, centrally necrotic mass in the left upper lobe with large peri - hilar lymph node metastases. note the high soft tissue contrast between alelectatic lung (open arrow), small rim of solid tumour (filled arrow) and colliquated central portion of the mass (asterisk) a 56-year - old female patient with small cell lung cancer. the transverse t2-weighted fat - saturated (a) and t1-weighted contrast - enhanced fat - saturated 3d - gre images (b, c) show a large, centrally necrotic mass in the left upper lobe with large peri - hilar lymph node metastases. note the high soft tissue contrast between alelectatic lung (open arrow), small rim of solid tumour (filled arrow) and colliquated central portion of the mass (asterisk) the potential of mri to replace chest radiography, particularly in very young children, was already investigated several years ago [18, 4446 ]. much of this work was conducted on low - field mri (mainly 0.2-t scanners) using steady - state free precession sequences. on average, three thick slices are acquired in coronal orientation with a mean breathhold time of 45 s. however, nowadays, only a few institutions regularly use low - field lung mri in paediatric radiology. nevertheless, the experience from this work may be considered valid for the suggested protocols for 1.5-t scanners since image quality has significantly improved. therefore, t2-weighted fat - suppressed as well as dynamic contrast - enhanced t1-gre sequences are applied with a slice thickness between 5 and 6 mm. disease entities encompassing community - acquired pneumonia, empyema, fungal infections and chronic bronchitis are detectable (fig. 4).fig. 4a 13-year - old girl with suspected organising pneumonia (boop) in both lungs. transverse t2-weighted tse images (a) were acquired with the navigator technique (sample volume placed on the dome of the right liver lobe). the open arrow indicates an oval - shaped consolidation with pleural contact in the lower left lobe and moderate signal intensity. coronal contrast - enhanced fat - saturated t1-weighted gre images (b) were acquired with the breathhold technique. the open arrow indicates the oval - shaped consolidation in the lower left lobe with contrast enhancement ; this is interpreted as an indicator of an active inflammatory process a 13-year - old girl with suspected organising pneumonia (boop) in both lungs. transverse t2-weighted tse images (a) were acquired with the navigator technique (sample volume placed on the dome of the right liver lobe). the open arrow indicates an oval - shaped consolidation with pleural contact in the lower left lobe and moderate signal intensity. coronal contrast - enhanced fat - saturated t1-weighted gre images (b) were acquired with the breathhold technique. the open arrow indicates the oval - shaped consolidation in the lower left lobe with contrast enhancement ; this is interpreted as an indicator of an active inflammatory process recent studies demonstrated the feasibility of chest examinations on 3-t high - field mri. one study used the navigator techniques to reduce breathing artefacts. high - field chest mri may allow differentiation between inflammation- and fibrosis - predominant lesions in uip and nsip in adult patients. moreover, a recent comparison between 3-t lung mri and hrct as gold standard showed an excellent correlation with non - cystic fibrosis chronic lung disease in children., lung mri may prove to become a valuable tool for detection as well as characterisation of inflammatory lung disease in children. chronic obstructive pulmonary disease (copd) is one of the leading causes of morbidity and mortality worldwide. at present it is the fourth most common cause of death among adults, but its prevalence is increasing. copd is characterised by incompletely reversible airflow obstruction due to a mixture of airway obstruction (obstructive bronchiolitis) and parenchymal destruction (emphysema). severity of copd is clinically assessed by lung function tests and diffusion capacity for carbon monoxide. imaging copd with proton mri is a major challenge due to the loss of lung tissue and reduced blood volume due to hypoxic vasoconstriction combined with hyperinflation, all resulting in a marked reduction of lung parenchymal signal. the strength of mri for imaging copd lies with the assessment of functional parameters like perfusion and respiratory dynamics. in copd emphysematous destruction hyperinflation severely affects diaphragmatic geometry with subsequent reduction of the mechanical properties, while the accessory neck and rib muscles become less effective. the common clinical measurements of copd do not provide insights into how structural alterations in the lung lead to dysfunction in the breathing mechanics, although treatments such as lung volume reduction surgery (lvrs) are thought to improve lung function by facilitating breathing mechanics and increasing elastic recoil. in contrast to normal subjects with regular, synchronous diaphragm and chest wall motion, patients with emphysema frequently have reduced, irregular or asynchronous motion, with a significant decrease in the maximum amplitude and the length of apposition of the diaphragm. in some patients the diaphragm movement is not coordinated (e.g. the ventral portion of the hemidiaphragm moves inferiorly while the dorsal part moves cranially), while paradoxical diaphragmatic motion correlated with mild and moderate hyperinflation. one study demonstrated a correlation between the change of parenchymal signal intensity measured by mri at inspiration and expiration and fev1 (r = 0.508) as a predictor of airflow obstruction. several studies have shown that airway obstruction in patients with copd tends to be located in airways smaller than 2 mm internal diameter. these airways are located between the 4th and the 14th generation of the tracheobronchial tree. severe peripheral airflow obstruction also affects the proximal airways from subsegmental bronchi to trachea. for the assessment of tracheal instability, such as seen in tracheobronchial malacia (which may mimick the clinical appearance of small airways disease), mr cine acquisitions during continuous respiration or forced expiration can be recommended. the depiction of airway dimensions and size of the airway walls by mri in physiological condition is limited to the central bronchial tree. for the depiction of bronchiectasis, the previously described 3d volume interpolated gradient echo sequence (vibe) offers a sufficient spatial resolution with a sensitivity of 79% and a specificity of 98% regarding visual depiction of bronchiectasis compared to ct. gas exchange in the lungs is optimally maintained by matching of ventilation and perfusion. in patients with copd, ventilation is impaired due to airway obstruction and parenchymal destruction. in regions with reduced ventilation, hypoxic vasoconstriction occurs [63, 64 ] causing a reduction of local pulmonary blood flow with redistribution to better ventilated lung regions [65, 66 ]. the reduction of the pulmonary vascular bed is related to the severity of parenchymal destruction ; however the distribution of perfusion does not necessarily match parenchymal destruction [67, 68 ]. conventional radionuclide perfusion scintigraphy has been used to assess these abnormalities, but it has substantial limitations with respect to spatial and temporal resolution. mr perfusion allows for a high diagnostic accuracy in detecting perfusion abnormalities of the lung [69, 70 ]. additionally, mr perfusion ratios correlate well with radionuclide perfusion scintigraphy ratios [71, 72 ]. while wedge - shaped perfusion defects occur in embolic obstruction, a generally low degree of inhomogeneous contrast enhancement is found in copd with emphysema. these features allow for easy visual differentiation and compare well with work done using ct perfusion experiments. in patients with copd the quantitative evaluation of 3d perfusion showed that the mean pulmonary blood flow (pbf), pulmonary blood volume (pbv) and mean transit time (mtt) are diffusely decreased and the changes are heterogeneous. calculated mean pbf and pbv are significantly decreased, and mtt is significantly shortened. interstitial lung disease (ild) encompasses numerous pathologic disorders of different etiologies, generally manifesting with an inflammatory reaction known as alveolitis, which may progress towards fibrosis. because the nature of these disorders is highly heterogeneous, imaging findings alone are often insufficient for making the final diagnosis, and integration of morphologic aspects with clinical and functional data is required. in the last 3 decades, computed tomography (ct) has clarified the elementary alterations and morphologic patterns characterising the infiltrative changes of ild. in contrast, mri has only recently overcome many of the technical issues related to lung imaging, providing a standardised image quality, which in many instances is now comparable to ct. this partly explains the relatively limited number of mri studies that have been clinically performed in ild patients. nonetheless, published data suggest at least three possible applications for lung mri in ild : (1) visualisation and recognition of morphological changes and their patterns, (2) assessment of the inflammatory activity of the disease and (3) effects of lung morphologic changes on functional parameters such as contrast enhancement and perfusion. the essential morphologic findings in ild include air - space disease, interstitial abnormalities or a combination of the two. because mr signal increases proportionally to proton density, air - space infiltrates appear on the t2-weighted images as hyperintense areas against the dark background of the normal lung parenchyma. when pulmonary vascular markings are not obscured, these areas can be assimilated to the ground - glass opacities detected by ct [17, 77 ]. similar to consolidations, interstitial abnormalities increase signal intensity presenting with curvilinear bands, nodules and reticulations, which can be associated to a variable degree of parenchymal distortion [50, 79, 80 ]. fibrotic changes that extensively involve both peripheral and perihilar portions of the lung are generally well demonstrated on t2-weighted images, albeit that one needs to consider extracellular interstitial water as a potential differential diagnosis in patients with suspected congestive heart failure. subtle changes in the subpleural regions may become more difficult to visualise, notably when parenchymal distortion is not present, demonstrating the superiority of ct in this respect. t1-weighted vibe images offer higher spatial resolution, and post - contrast acquisition with fat - suppression is recommended to increase the signal of altered subpleural lung tissue against a background represented by chest wall muscles, ribs and normal lung parenchyma. honeycombing, which manifests with reticular changes and irregular cystic transformation of the lung, can also be assessed using this technique (figs. 5, 6, 7 and 8).fig. 5infiltrative disorder of the lung. extensive reticulation and architectural distortion predominant in the subpleural regions of the lung are well demonstrated by the axial (a, b) and coronal (c e) mr images obtained using the half - fourier single - shot fast spin echo (a, c, e) and post - constrast volume interpolated t1-weighted gre (b, e) sequencesfig. the interlobular reticulation (thin arrows) is more evident after contrast administration (c and f). a perfusion defect (arrowhead in e) is associated to the peripheral fibrotic changes at the left lateral costo - phrenic angle (arrowheads in d and f)fig. a) subpleural reticular changes are visualised at the periphery of the lungs (thin arrows). b) after contrast administration the subtle linear enhancement at the pulmonary - chest wall interface indicates abnormal findings related to subpleural fibrosis (thin arrows). a rounded consolidation is present on the left in the lingula suspected for lung tumour in ild (asterisk)fig. node enlargement (arrows) is demonstrated with gradient echo images before (a) and after administration of contrast material (b). coronal perfusion images indicate vascular compression at the right hilum (arrow, c) and a wedge - shaped perfusion defect (asterisk, d) infiltrative disorder of the lung. extensive reticulation and architectural distortion predominant in the subpleural regions of the lung are well demonstrated by the axial (a, b) and coronal (c e) mr images obtained using the half - fourier single - shot fast spin echo (a, c, e) and post - constrast volume interpolated t1-weighted gre (b, e) sequences subtle subpleural reticulation in a patient with fibrotic - predominant nsip. the interlobular reticulation (thin arrows) is more evident after contrast administration (c and f). a perfusion defect (arrowhead in e) is associated to the peripheral fibrotic changes at the left lateral costo - phrenic angle (arrowheads in d and f) fibrosis associated with rounded consolidation. a) subpleural reticular changes are visualised at the periphery of the lungs (thin arrows). b) after contrast administration the subtle linear enhancement at the pulmonary - chest wall interface indicates abnormal findings related to subpleural fibrosis (thin arrows). a rounded consolidation is present on the left in the lingula suspected for lung tumour in ild (asterisk) bilateral hilar and mediastinal adenomegalies in sarcoidosis. node enlargement (arrows) is demonstrated with gradient echo images before (a) and after administration of contrast material (b). coronal perfusion images indicate vascular compression at the right hilum (arrow, c) and a wedge - shaped perfusion defect (asterisk, d) differentiation of active inflammation from fibrosis is of significant clinical importance both for the prediction of therapy response and clinical outcome of ild. both mr signal and contrast - enhancement characteristics of inflammation and fibrosis have been investigated. although initial studies performed on 1.5 t lacked sufficient image quality [8183 ], the feasibility of the assessment of disease activity in ild was demonstrated. only recently, 3.0-t mri has increased sensitivity to changes in proton density. in particular, yi. reported that mr signal of inflammatory and fibrotic lesions on t2-weighted images is hyperintense and isointense, respectively, compared to the signal from chest wall muscle, indicating an increased water content in the areas of inflammation. dynamic mri using iv contrast administration also indicated that early enhancement and washout with discernible peak enhancement at 1 or 3 min after contrast injection was associated with positive and negative prediction values of 82 and 92%, respectively, in predicting disease activity. the earlier enhancement and rapid washout would be in agreement with higher permeability of capillaries in the areas of inflammation compared to those of fibrosis. a different approach to differentiating active inflammation from fibrosis was attempted in a recent study in a bleomycin - induced lung injury model in rats. proton density and t2 relaxation were computed regionally in the injured lungs, and mr - derived parameters were compared to postmortem measures of water and collagen content. the authors concluded that proton density and t2 relaxation data acquired using mri were sensitive to inflammation and fibrotic changes in the lung. although they were able to distinguish diseased lungs as effectively as postmortem measurements, they were unable to differentiate between fibrosis and inflammation. in conclusion, these data are encouraging and support potential future applications of mri in interstitial lung disease both in research and clinical settings. chronic obstructive pulmonary disease (copd) is one of the leading causes of morbidity and mortality worldwide. at present it is the fourth most common cause of death among adults, but its prevalence is increasing. copd is characterised by incompletely reversible airflow obstruction due to a mixture of airway obstruction (obstructive bronchiolitis) and parenchymal destruction (emphysema). severity of copd is clinically assessed by lung function tests and diffusion capacity for carbon monoxide. imaging copd with proton mri is a major challenge due to the loss of lung tissue and reduced blood volume due to hypoxic vasoconstriction combined with hyperinflation, all resulting in a marked reduction of lung parenchymal signal. the strength of mri for imaging copd lies with the assessment of functional parameters like perfusion and respiratory dynamics. in copd emphysematous destruction hyperinflation severely affects diaphragmatic geometry with subsequent reduction of the mechanical properties, while the accessory neck and rib muscles become less effective. the common clinical measurements of copd do not provide insights into how structural alterations in the lung lead to dysfunction in the breathing mechanics, although treatments such as lung volume reduction surgery (lvrs) are thought to improve lung function by facilitating breathing mechanics and increasing elastic recoil. in contrast to normal subjects with regular, synchronous diaphragm and chest wall motion, patients with emphysema frequently have reduced, irregular or asynchronous motion, with a significant decrease in the maximum amplitude and the length of apposition of the diaphragm. in some patients the diaphragm movement is not coordinated (e.g. the ventral portion of the hemidiaphragm moves inferiorly while the dorsal part moves cranially), while paradoxical diaphragmatic motion correlated with mild and moderate hyperinflation. one study demonstrated a correlation between the change of parenchymal signal intensity measured by mri at inspiration and expiration and fev1 (r = 0.508) as a predictor of airflow obstruction. several studies have shown that airway obstruction in patients with copd tends to be located in airways smaller than 2 mm internal diameter. these airways are located between the 4th and the 14th generation of the tracheobronchial tree. severe peripheral airflow obstruction also affects the proximal airways from subsegmental bronchi to trachea. for the assessment of tracheal instability, such as seen in tracheobronchial malacia (which may mimick the clinical appearance of small airways disease), mr cine acquisitions during continuous respiration or forced expiration can be recommended. the depiction of airway dimensions and size of the airway walls by mri in physiological condition is limited to the central bronchial tree. for the depiction of bronchiectasis, the previously described 3d volume interpolated gradient echo sequence (vibe) offers a sufficient spatial resolution with a sensitivity of 79% and a specificity of 98% regarding visual depiction of bronchiectasis compared to ct. gas exchange in the lungs is optimally maintained by matching of ventilation and perfusion. in patients with copd, ventilation is impaired due to airway obstruction and parenchymal destruction. in regions with reduced ventilation, hypoxic vasoconstriction occurs [63, 64 ] causing a reduction of local pulmonary blood flow with redistribution to better ventilated lung regions [65, 66 ]. the reduction of the pulmonary vascular bed is related to the severity of parenchymal destruction ; however the distribution of perfusion does not necessarily match parenchymal destruction [67, 68 ]. conventional radionuclide perfusion scintigraphy has been used to assess these abnormalities, but it has substantial limitations with respect to spatial and temporal resolution. mr perfusion allows for a high diagnostic accuracy in detecting perfusion abnormalities of the lung [69, 70 ]. additionally, mr perfusion ratios correlate well with radionuclide perfusion scintigraphy ratios [71, 72 ]. while wedge - shaped perfusion defects occur in embolic obstruction, a generally low degree of inhomogeneous contrast enhancement is found in copd with emphysema. these features allow for easy visual differentiation and compare well with work done using ct perfusion experiments. in patients with copd the quantitative evaluation of 3d perfusion showed that the mean pulmonary blood flow (pbf), pulmonary blood volume (pbv) and mean transit time (mtt) are diffusely decreased and the changes are heterogeneous. interstitial lung disease (ild) encompasses numerous pathologic disorders of different etiologies, generally manifesting with an inflammatory reaction known as alveolitis, which may progress towards fibrosis. because the nature of these disorders is highly heterogeneous, imaging findings alone are often insufficient for making the final diagnosis, and integration of morphologic aspects with clinical and functional data is required. in the last 3 decades, computed tomography (ct) has clarified the elementary alterations and morphologic patterns characterising the infiltrative changes of ild. in contrast, mri has only recently overcome many of the technical issues related to lung imaging, providing a standardised image quality, which in many instances is now comparable to ct. this partly explains the relatively limited number of mri studies that have been clinically performed in ild patients. nonetheless, published data suggest at least three possible applications for lung mri in ild : (1) visualisation and recognition of morphological changes and their patterns, (2) assessment of the inflammatory activity of the disease and (3) effects of lung morphologic changes on functional parameters such as contrast enhancement and perfusion. the essential morphologic findings in ild include air - space disease, interstitial abnormalities or a combination of the two. because mr signal increases proportionally to proton density, air - space infiltrates appear on the t2-weighted images as hyperintense areas against the dark background of the normal lung parenchyma. when pulmonary vascular markings are not obscured, these areas can be assimilated to the ground - glass opacities detected by ct [17, 77 ]. similar to consolidations, interstitial abnormalities increase signal intensity presenting with curvilinear bands, nodules and reticulations, which can be associated to a variable degree of parenchymal distortion [50, 79, 80 ]. fibrotic changes that extensively involve both peripheral and perihilar portions of the lung are generally well demonstrated on t2-weighted images, albeit that one needs to consider extracellular interstitial water as a potential differential diagnosis in patients with suspected congestive heart failure. subtle changes in the subpleural regions may become more difficult to visualise, notably when parenchymal distortion is not present, demonstrating the superiority of ct in this respect. t1-weighted vibe images offer higher spatial resolution, and post - contrast acquisition with fat - suppression is recommended to increase the signal of altered subpleural lung tissue against a background represented by chest wall muscles, ribs and normal lung parenchyma. honeycombing, which manifests with reticular changes and irregular cystic transformation of the lung, can also be assessed using this technique (figs. 5, 6, 7 and 8).fig. 5infiltrative disorder of the lung. extensive reticulation and architectural distortion predominant in the subpleural regions of the lung are well demonstrated by the axial (a, b) and coronal (c e) mr images obtained using the half - fourier single - shot fast spin echo (a, c, e) and post - constrast volume interpolated t1-weighted gre (b, e) sequencesfig. the interlobular reticulation (thin arrows) is more evident after contrast administration (c and f). a perfusion defect (arrowhead in e) is associated to the peripheral fibrotic changes at the left lateral costo - phrenic angle (arrowheads in d and f)fig. a) subpleural reticular changes are visualised at the periphery of the lungs (thin arrows). b) after contrast administration the subtle linear enhancement at the pulmonary - chest wall interface indicates abnormal findings related to subpleural fibrosis (thin arrows). a rounded consolidation is present on the left in the lingula suspected for lung tumour in ild (asterisk)fig. node enlargement (arrows) is demonstrated with gradient echo images before (a) and after administration of contrast material (b). coronal perfusion images indicate vascular compression at the right hilum (arrow, c) and a wedge - shaped perfusion defect (asterisk, d) infiltrative disorder of the lung. extensive reticulation and architectural distortion predominant in the subpleural regions of the lung are well demonstrated by the axial (a, b) and coronal (c e) mr images obtained using the half - fourier single - shot fast spin echo (a, c, e) and post - constrast volume interpolated t1-weighted gre (b, e) sequences subtle subpleural reticulation in a patient with fibrotic - predominant nsip. the interlobular reticulation (thin arrows) is more evident after contrast administration (c and f). a perfusion defect (arrowhead in e) is associated to the peripheral fibrotic changes at the left lateral costo - phrenic angle (arrowheads in d and f) fibrosis associated with rounded consolidation. a) subpleural reticular changes are visualised at the periphery of the lungs (thin arrows). b) after contrast administration the subtle linear enhancement at the pulmonary - chest wall interface indicates abnormal findings related to subpleural fibrosis (thin arrows). a rounded consolidation is present on the left in the lingula suspected for lung tumour in ild (asterisk) bilateral hilar and mediastinal adenomegalies in sarcoidosis. node enlargement (arrows) is demonstrated with gradient echo images before (a) and after administration of contrast material (b). coronal perfusion images indicate vascular compression at the right hilum (arrow, c) and a wedge - shaped perfusion defect (asterisk, d) differentiation of active inflammation from fibrosis is of significant clinical importance both for the prediction of therapy response and clinical outcome of ild. both mr signal and contrast - enhancement characteristics of inflammation and fibrosis have been investigated. although initial studies performed on 1.5 t lacked sufficient image quality [8183 ], the feasibility of the assessment of disease activity in ild was demonstrated. only recently, 3.0-t mri has increased sensitivity to changes in proton density. in particular, yi. reported that mr signal of inflammatory and fibrotic lesions on t2-weighted images is hyperintense and isointense, respectively, compared to the signal from chest wall muscle, indicating an increased water content in the areas of inflammation. dynamic mri using iv contrast administration also indicated that early enhancement and washout with discernible peak enhancement at 1 or 3 min after contrast injection was associated with positive and negative prediction values of 82 and 92%, respectively, in predicting disease activity. the earlier enhancement and rapid washout would be in agreement with higher permeability of capillaries in the areas of inflammation compared to those of fibrosis. a different approach to differentiating active inflammation from fibrosis was attempted in a recent study in a bleomycin - induced lung injury model in rats. proton density and t2 relaxation were computed regionally in the injured lungs, and mr - derived parameters were compared to postmortem measures of water and collagen content. the authors concluded that proton density and t2 relaxation data acquired using mri were sensitive to inflammation and fibrotic changes in the lung. although they were able to distinguish diseased lungs as effectively as postmortem measurements, they were unable to differentiate between fibrosis and inflammation. in conclusion, these data are encouraging and support potential future applications of mri in interstitial lung disease both in research and clinical settings. significant efforts have been made to further improve robustness and reproducibility of lung mr image quality. compared with x - ray and ct, the quality of lung mri is more dependent on the ability of patients to follow breath hold instructions. advanced acquisition schemes with inherent correction of respiratory motion and cardiac pulsation are therefore important for future developments. for example, a conjoint research group of medical physics and radiology departments supported by the german research foundation (deutsche forschungsgemeinschaft) is currently working on the development of self - navigated sequence designs and radial k - space methods for the assessment of lung morphology on free breathing. similarly, a group of scientists in the usa is working on dedicated mr imaging in copd within the multi ethnic study of atherosclerosis consortium with particular focus on perfusion and dynamic assessment. one way to improve the robustness of lung mri against respiratory motion is to implement self - navigation. the prototype 3d - mri sequence (a self - navigated t1-weighted 3d flash with quasi - random k - space ordering) acquires multiple full lung volumes during free breathing, which results in a set of images with unsharp delineation of structures that are subject to respiratory motion. with each acquisition non - spatially encoded dc signals are acquired at the center of k - space to be used as navigator. this signal contains sufficient information to detect motion and to select image information of the different acquisitions to either produce one motion corrected data set for morphologic imaging without patient compliance or to perform a detailed motion analysis. another approach is based on radial imaging with k - space weighted image contrast (kwic). motion correction is achieved by radial data acquisition with extraction of the signal from k - space centre for the determination of the respiratory cycle. further improvements of image quality are achieved with autofocusing, 3d image correlation, k - space - weighted image contrast (kwic) and principal component analysis [8688 ]. one of the latest developments in the field of proton - based lung mri appears to be a very promising technology for non - contrast - enhanced ventilation and perfusion scanning. this novel approach, known as fourier decomposition mri, utilises a short echo dynamic ssfp acquisition of lung images with subsequent compensation for respiratory motion by using nonrigid image registration. spectral analysis of the image time series allows for identification of peaks at the respiratory and cardiac frequencies. amplitude of these peaks is related to regional proton density change caused by deformation of lung parenchyma (highest signal with lowest pulmonary air content in expiration) and pulmonary blood flow (lowest signal with maximum blood flow in systole). further image post - processing produces ventilation- and perfusion - weighted maps for regional assessment of lung function from a single acquisition series nevertheless, there is a perspective that the method of choice for morphologic and functional assessment of acute pulmonary embolism in the very near future might be a non - contrast - enhanced free breathing mr scan of 1015 min.fig. 9twenty - three year - old female with acute pulmonary embolism at the time point of diagnosis (a, b) and at follow - up study after 6 months (c, d). the initial dynamic contrast enhanced (dce) study (a) as well as the perfusion - weighted fourier - decomposition (fd) image (b) demonstrate multiple perfusion defects (open arrows). in the follow up study, both techniques (dce ; c and fd ; d) demonstrate an almost homogeneous lung perfusion after effective anticoagulation twenty - three year - old female with acute pulmonary embolism at the time point of diagnosis (a, b) and at follow - up study after 6 months (c, d). the initial dynamic contrast enhanced (dce) study (a) as well as the perfusion - weighted fourier - decomposition (fd) image (b) demonstrate multiple perfusion defects (open arrows). in the follow up study, both techniques (dce ; c and fd ; d) demonstrate an almost homogeneous lung perfusion after effective anticoagulation one of the latest developments in the field of proton - based lung mri appears to be a very promising technology for non - contrast - enhanced ventilation and perfusion scanning. this novel approach, known as fourier decomposition mri, utilises a short echo dynamic ssfp acquisition of lung images with subsequent compensation for respiratory motion by using nonrigid image registration. spectral analysis of the image time series allows for identification of peaks at the respiratory and cardiac frequencies. amplitude of these peaks is related to regional proton density change caused by deformation of lung parenchyma (highest signal with lowest pulmonary air content in expiration) and pulmonary blood flow (lowest signal with maximum blood flow in systole). further image post - processing produces ventilation- and perfusion - weighted maps for regional assessment of lung function from a single acquisition series. nevertheless, there is a perspective that the method of choice for morphologic and functional assessment of acute pulmonary embolism in the very near future might be a non - contrast - enhanced free breathing mr scan of 1015 min.fig. 9twenty - three year - old female with acute pulmonary embolism at the time point of diagnosis (a, b) and at follow - up study after 6 months (c, d). the initial dynamic contrast enhanced (dce) study (a) as well as the perfusion - weighted fourier - decomposition (fd) image (b) demonstrate multiple perfusion defects (open arrows). in the follow up study, both techniques (dce ; c and fd ; d) demonstrate an almost homogeneous lung perfusion after effective anticoagulation twenty - three year - old female with acute pulmonary embolism at the time point of diagnosis (a, b) and at follow - up study after 6 months (c, d). the initial dynamic contrast enhanced (dce) study (a) as well as the perfusion - weighted fourier - decomposition (fd) image (b) demonstrate multiple perfusion defects (open arrows). in the follow up study, both techniques (dce ; c and fd ; d) demonstrate an almost homogeneous lung perfusion after effective anticoagulation hybrid pet / mri has recently become available for clinical research. compared to pet / ct, pet / mri may be advantageous due to higher soft tissue contrast, while there is a slight reduction in ionising radiation dose. technically, integration of the two systems has been a significant challenge and required substantial modifications [9496 ]. the size of the pet detectors had to be minimised ; a pet detector that is insensitive to high magnetic fields had to be developed, and the adverse effect of pet detector parts on the homogeneity of the magnet s b0 field must be minimised. moreover, interference between the radiofrequency signals, mri gradients and pet electronic signals must be avoided. some of these problems were overcome by application of optical fibres and advances in gamma ray detector technology, which were initiated mainly by the advent of avalanche photodiodes ; in addition the routine availability of fast scintillation materials resulted in the development of fully magnetic - field - insensitive high - performance pet detectors. although pet / mri may not necessarily replace the role of pet / ct in thoracic oncological imaging, and specific clinical indications remain to be identified, mri may be advantageous when compared to ct in the investigation of consolidating lung lesions, and malignant mediastinal and chest wall invasion. the additional diagnostic value of pet / mri over pet / ct on nodal staging is questionable since both mri and ct nodal staging is size based. mri, however, has been reported to be of higher accuracy than pet / ct when assessing the brain, liver and the bone for distant metastases. oncological research may be another potential area where multiple follow - up functional and anatomical imaging by pet / mri may be advantageous over pet / ct. lastly, with the increasing availability of radiotracers and novel compounds for molecular and physiological assessment, including labelling of target cells and compounds that target particular cell lines or processes, the combination of these powerful modalities may result in significant advances for research (and potentially clinical) purposes. mri is emerging as a valuable lung imaging modality, together with x - ray and ct. it offers a unique combination of morphological and functional information in a single examination without any radiation burden to the patient. however, although push - button protocols facilitate its clinical application, lung mri can be still challenging, being the most comprehensive but also most expensive and least robust of the three modalities. new users are advised to make themselves familiar with the particular advantages and limitations of the technique and its diagnostic scope to appreciate its potential benefits. given this, lung mri will be increasingly used and even further improved by additional recent and future developments, in particular in the fields of motion compensation and functional imaging. | backgroundmri of the lung is recommended in a number of clinical indications. having a non - radiation alternative is particularly attractive in children and young subjects, or pregnant women.methodsprovided there is sufficient expertise, magnetic resonance imaging (mri) may be considered as the preferential modality in specific clinical conditions such as cystic fibrosis and acute pulmonary embolism, since additional functional information on respiratory mechanics and regional lung perfusion is provided. in other cases, such as tumours and pneumonia in children, lung mri may be considered an alternative or adjunct to other modalities with at least similar diagnostic value.resultsin interstitial lung disease, the clinical utility of mri remains to be proven, but it could provide additional information that will be beneficial in research, or at some stage in clinical practice. customised protocols for chest imaging combine fast breath - hold acquisitions from a buffet of sequences. having introduced details of imaging protocols in previous articles, the aim of this manuscript is to discuss the advantages and limitations of lung mri in current clinical practice.conclusionnew developments and future perspectives such as motion - compensated imaging with self - navigated sequences or fast fourier decomposition mri for non - contrast enhanced ventilation- and perfusion - weighted imaging of the lung are discussed.main messages mri evolves as a third lung imaging modality, combining morphological and functional information. it may be considered first choice in cystic fibrosis and pulmonary embolism of young and pregnant patients. in other cases (tumours, pneumonia in children), it is an alternative or adjunct to x - ray and ct. in interstitial lung disease, it serves for research, but the clinical value remains to be proven. new users are advised to make themselves familiar with the particular advantages and limitations. |
thrombolysis with recombinant tissue plasminogen activator (rtpa, alteplase) is the only effective specific treatment for acute ischemic stroke patients coming in window (4.5 h from onset of symptoms). a milestone study of national institute of neurological disorders and stroke in 1995, demonstrated the benefits of rtpa in to patients of acute ischemic stroke (ais) who came within window period. these patients of ais who were thrombolysed were 30% more likely to survive with minimal disability resulting in a 12% absolute increase in the proportion having excellent functional outcomes at 3 months. stroke study iii trail (2008) window period was extended to 4.5 h. for every 15 min reduction in door to needle time (dtnt) there is 5% reduction in odds of in hospital mortality (odd ratio, 0.95 ; 95% confidence interval, 0.92 - 0.98 : p = 0.0007). all patients who presented with stroke to emergency department (er) from january 2011 to december 2013 were included in the study. after initial assessment by causality personnel a medical / neuro - resident evaluated the patient. radiological diagnosis was obtained with noncontrasted brain computed tomography and/or diffusion weighted magnetic resonance imaging (dwi). after neuro physician opinion or after telephonic discussion by neuro - resident with neuro physician (telestroke) the treatment plan was decided. for the patients who presented within window period thrombolysis was planned. severity of stroke was documented by the national institutes of health stroke scale (nihss) contra - indications for thrombolysis were checked and consent of relatives / patient was taken. those patients who presented out of window period (> 4.5 h after onset of symptom / symptom to door time [std ] > 4.5 h), or patients who had hemorrhagic stroke and those who were not willing for giving consent were excluded [figure 1 ]. algorithm for patient presenting with stroke in emergency room (protocol used in the study) patients record files and charts were used to extract retrospective data. the collected data use to evaluate er to needle [door to needle time-(dtnt) ] time and reasons for delay in thrombolysis therapy in acute stroke patients. the following parameters were studied onset of symptoms to er time, assessment by physician / medical chief resident time (door to physician time [dtpt])er to imaging time (door to imaging time [dtit]),er to needle time (dtnt)contraindications for thrombolysis. onset of symptoms to er time, assessment by physician / medical chief resident time (door to physician time [dtpt ]) er to imaging time (door to imaging time [dtit ]), er to needle time (dtnt) contraindications for thrombolysis. the onset of symptom time for patients with wake up was accepted as last time the patient was seen as healthy. the baseline characteristics of patient with acute ischemic stroke brought / admitted to er, clinical features, arrival time (door time) to er, severity of stroke, imaging time, radiological findings, contraindication for thrombolytic treatment, time of starting recombinant tissue plasminogen activator (rt - pa) and thereafter complications were recorded [table 2 ]. the data abstracted were transferred to the spss 17.0 program (spss statistics is a software package used for statistical analysis. it is statistical package for social science and is produced by spss inc.) of the computer for statistical analysis. six hundred and ninety - five patients with symptoms of stroke were presented to our emergency department in the study period. out of these five hundred and forty seven (78.7%) were excluded as they had come out of window period that is, they had arrived 4.5 h after the onset of stroke symptoms. algorithm for patient presenting with stroke in er (protocol used in the study). further after imaging of these one hundred and forty eight patients, one hundred four (70.27%) were excluded. sixty - two (59.6%) had intra cerebral bleed, 1 (0.9%) had hemoglobin - 3.1 g, 13 (13%) of them had transient ischemic attack (neurological symptoms improved) and dwi images of these patient were normal. 6 (5.7) patients were diagnosed to have metabolic de - rrangement (hypoglycemia, hyperglycemia, hyponatremia). other reasons for exclusion in our study were post - ictal status, financial problem, recent thrombolysis, recent surgery, and delay in contacting senior radiologist [table 1 ]. distribution of contraindications for thrombolytic therapy of patients baseline clinical characteristics of thrombolysed patients (n=44) total 44 (29.7%) patients with ais were thrombolysed. thirty - four (79.5%) were male and nine (20.45%) were female. co - morbid illness in the form of hypertension 6 (13.6%), diabetes 2 (4.5%), ischemic heart diseases (ihd) 2 (4.5%), previous stroke cva 3 (6.5%), > 1 co - morbidity (ht / cva / dm / ihd / hypothyriod) 19 (43%), seizure 1 (2%) and alcoholic liver diseases 1 (2%) patients respectively. the mean time for arrival of patients from onset of symptoms to hospital (std) 1.23 h (15 min-3 h). the mean door to neuro - physician time dtpt was 32 min (5 min-2.23 h). the mean dtnt 1.44 h (40 min-3.3 h) [tables 3 and 4 ], [figures 24 ]. our study dtpt, dtit and dtnt compared with aha guideline interval number of patients thrombolysed per hour door to physician time door to imaging time (recommended standard time : 45 min) analysis of our study clearly states that stdt, dtpt, dtit, and dtnt time are significantly more. thus, we had many hurdles in delivering thrombolysis therapy to these 44 patients. only 7 (15%) patients had dtnt 60 min. the problems / barriers in our study were categorized into three factors : mean symptom to door time was 83 min (median : 69). poor recognition of stroke signs, especially in older patients caused delay in arrival time to hospital. public and emergency medical services staff education play important role in shortening the pre hospital period. in our study - door to physician, door to imaging and door to needle time were significantly ore compared to standard recommendations (aha) [table 3 ]. there was lack of handling stroke patients with high priority at each level er, imaging unit, stroke unit. thus lack of triaging stroke patient at all level of intervention was our weakest point. in one of the patients the on call doctor was very busy attending emergency calls so causing increase in dtpt time. the concept of having second on call doctor who takes care only of patients with acute stroke has being recommended by kobayashi. lack of triaging at radiology unit and performing entire sequences of magnetic resonance imaging (mri) scan lead to increase in dtit. thus again dwi has high sensitivity (88 - 100%) and specificity (95 - 100%) for detecting infarcted regions, within minutes of onset of symptoms. study suggested brain attack team mri sequence of < 10 min to confirm acute ischemia stroke and assess candidacy for iv - rtpa. lack of triaging of bed for stroke patients resulted in increase in dtnt time. to prevent these delay we started thrombolysing ais patient in er. to prevent delay due to inavailability of drug, we have started keeping rtpa in our drug stock our dtnt was 104 min (door to needle time median - 100). relatives with geriatric patient (79 years) took longer time to give consent due to age of the patient and secondly due to financial burden. one of our patients had liver diseases so we had to wait for international normal ratio report for prothrombin time (international normalized ratio). transient ischemic attack patients were not thrombolysed, but in latter half of study dwi images helped us to prevent delay. poor recognition of stroke signs, especially in older patients caused delay in arrival time to hospital. public and emergency medical services staff education play important role in shortening the pre hospital period. in our study - door to physician, door to imaging and door to needle time were significantly ore compared to standard recommendations (aha) [table 3 ]. there was lack of handling stroke patients with high priority at each level er, imaging unit, stroke unit. thus lack of triaging stroke patient at all level of intervention was our weakest point. education of emergency medical services of stroke symptoms will help to triage stroke patient. in one of the patients the on call doctor was very busy attending emergency calls so causing increase in dtpt time. the concept of having second on call doctor who takes care only of patients with acute stroke has being recommended by kobayashi. lack of triaging at radiology unit and performing entire sequences of magnetic resonance imaging (mri) scan lead to increase in dtit. thus again dwi has high sensitivity (88 - 100%) and specificity (95 - 100%) for detecting infarcted regions, within minutes of onset of symptoms. study suggested brain attack team mri sequence of < 10 min to confirm acute ischemia stroke and assess candidacy for iv - rtpa. lack of triaging of bed for stroke patients resulted in increase in dtnt time. to prevent these delay we started thrombolysing ais patient in er. to prevent delay due to inavailability of drug, we have started keeping rtpa in our drug stock our dtnt was 104 min (door to needle time median - 100). relatives with geriatric patient (79 years) took longer time to give consent due to age of the patient and secondly due to financial burden. one of our patients had liver diseases so we had to wait for international normal ratio report for prothrombin time (international normalized ratio). transient ischemic attack patients were not thrombolysed, but in latter half of study dwi images helped us to prevent delay. poor recognition of stroke signs, especially in older patients caused delay in arrival time to hospital. public and emergency medical services staff education play important role in shortening the pre hospital period. in our study - door to physician, door to imaging and door to needle time were significantly ore compared to standard recommendations (aha) [table 3 ]. there was lack of handling stroke patients with high priority at each level er, imaging unit, stroke unit. thus lack of triaging stroke patient at all level of intervention was our weakest point. education of emergency medical services of stroke symptoms will help to triage stroke patient. in one of the patients the on call doctor was very busy attending emergency calls so causing increase in dtpt time. the concept of having second on call doctor who takes care only of patients with acute stroke has being recommended by kobayashi. lack of triaging at radiology unit and performing entire sequences of magnetic resonance imaging (mri) scan lead to increase in dtit. thus again dwi has high sensitivity (88 - 100%) and specificity (95 - 100%) for detecting infarcted regions, within minutes of onset of symptoms. study suggested brain attack team mri sequence of < 10 min to confirm acute ischemia stroke and assess candidacy for iv - rtpa. lack of triaging of bed for stroke patients resulted in increase in dtnt time. to prevent these delay we started thrombolysing ais patient in er. to prevent delay due to inavailability of drug our dtnt was 104 min (door to needle time median - 100). patient with raised blood pressure requiring labetalol infusion caused delay. relatives with geriatric patient (79 years) took longer time to give consent due to age of the patient and secondly due to financial burden. one of our patients had liver diseases so we had to wait for international normal ratio report for prothrombin time (international normalized ratio). transient ischemic attack patients were not thrombolysed, but in latter half of study dwi images helped us to prevent delay. the barriers of thrombolysis in our study included : lack of public awareness and inaccessiblity to emergency medical serviceslack of prioritizing triage system at er, radiology unit and stroke unitlack of a multi - disciplinary stroke care team. lack of public awareness and inaccessiblity to emergency medical services lack of prioritizing triage system at er, radiology unit and stroke unit lack of a multi - disciplinary stroke care team. a multi - disciplinary stroke care team consists of well - established emergency medical services, physicians, neurologist, nurses, radiology staff, neuro - radiologist, and pharmacist. forming a one - call comprehensive stroke code will help in co - ordination at all level. time to time audit of quality indicator of stroke code team may help to overcome the factors for delay in dtnt. | aim:(1) to evaluate the number of patients thrombolysed within 1 h of arrival to emergency room (er) (2) to identify reasons for delay in thrombolysis of acute stroke patients.materials and methods : all patients admitted to er with symptoms suggestive of stroke from january 2011 to november 2013 were studied. retrospective data were collected to evaluate er to needle (door to needle time [dtnt ]) time and reasons for delay in thrombolysis. the parameters studied (1) onset of symptoms to er time, (2) er to imaging time (door to imaging time [dtit ]), (4) er to needle time (door to needle) and (5) contraindications for thrombolysis.results:a total of 695 patients with suspected stroke were admitted during study period. 547 (78%) patients were out of window period. 148 patients (21%, m = 104, f = 44) arrived within window period (< 4.5 h.). 104 (70.27%) were contraindicated for thrombolysis. majority were intracerebral bleeds. 44 (29.7%) were eligible for thrombolysis. 7 (15.9%) were thrombolysed within 1 h. the mean time for arrival of patients from onset of symptoms to hospital (symptom to door) 83 min (median - 47). the mean door to neuro - physician time (dtpt) was 32 min (median - 15 min). the mean dtit was 58 min (median - 50 min). the mean dtnt 104 (median - 100 min).conclusion : reasons for delay in thrombolysis are : absence of stroke education program for common people. lack of priority for triage and imaging for stroke patients. |
prehypertension (pht) was first introduced by the seventh joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc-7) in 2003, replacing former categories of high - normal and above - optimal blood pressure (bp) ; and it was defined as systolic bp of 120139 mmhg or diastolic bp of 8090 mmhg based on 2 or more properly measured seated bp readings on each of 2 or more office visits. the 2005 - 2006 national health and nutrition examination survey estimated 28% of united states adults had pht. patients with pht are at increased risk of developing hypertension and other cardiovascular diseases (cvds) compared to normotensives. although a study done by zhu did not find any statistical difference in the parameters of lv structure between normotensive and prehypertensive subjects, some studies have shown a linear correlation between pht and increased left ventricular mass (lvm), with target organ damage found in both prehypertensive youths [6, 7 ] and older population [3, 4 ] when compared to normotensives. in fact, there has been increased risk of mortality associated with the prehypertensive category of bp. the prospective studies collaboration examined relationship between categories of bp and subsequent mortality by following almost 1 million people with no previous vascular disease prospectively for a total of 12.7 million person - years in 61 observational studies. they concluded that there is a continuous increase in mortality from both stroke and ischemic heart disease from bp of 115/75 mmhg, with a twofold increase in cardiovascular death in those with 20 mmhg higher systolic pressure or a 10 mmhg higher diastolic pressure, a level well within the range of pht. studies have shown that brachial and central bps may differ, especially the systolic component [9, 10 ], but debates are still ongoing on which of the two correlates more strongly with left ventricular mass index (lvmi) and other cvd [1113 ]. the aim of this study was (1) to assess the strength of association of brachial and central pressure with lvmi and (2) to compare these variables in prehypertensive and normotensive study participants. cardiac magnetic resonance imaging (mri) was used to diagnose lvm as it is more precise and reliable compared to other diagnostic modalities. we conducted an observational cross - sectional study of healthy volunteers from september 2008 to september 2009 at cardiology division of new york hospital medical center of queens / weill medical college of cornell university. we enrolled healthy volunteers who were at least 40 years old and not on medication. exclusion criteria for the study were previous diagnosis of hypertension (htn), diabetes mellitus (dm), renal disease, cvd, valvular heart disease, and atrial fibrillation. detailed history, physical examination, and brachial and central bp measurement were completed at the first visit. height (m) and weight (kg) were measured and body mass index (bmi) was calculated as weight (kg)/(height (m) height (m)) and unit was recorded as kg / m. the second visit, which was 4 weeks after the first, included the second measurements of brachial and central bp and performing of a cardiac mri. to ensure a more representative bp value for each patient, the average of the bp measurement at the first and second visit was used for analysis. study population was divided into two groups of normotensives and prehypertensives based on their bp measurement, and lvmi was compared in both groups. pht as defined by jnc-7 criteria is systolic blood pressure (sbp) between 120 and 139 mmhg and diastolic blood pressure (dbp) between 80 and 89 mmhg. the study was approved by institutional review board of new york hospital medical center of queens. brachial bp was measured with a bpm-300 noninvasive bp monitor (vsm medtech ltd., vancouver, canada) after the subject had been in a recumbent position for a minimum of 10 minutes. the device took 6 consecutive bp readings, excluded the first measurement, and derived an average. the tonometer, gently pressed against the radial artery pulse, acquires the radial pulse wave and the sphygmocor 's proprietary algorithm derives the pulse wave as it exists in the ascending aorta producing the central bp measurements noninvasively. we acquired ecg - gating and breath - holding during contrast - enhanced segmented k - space inversion - recovery with steady - state free precession imaging. lvmi was calculated using lv measurements in diastole, divided by height, squared, and expressed as g / m. pearson r was used for correlations for each variable of interest (i.e., brachial sbp, central sbp, brachial dbp, central dbp, and lvmi). multivariate linear regression models were constructed using pht as the primary risk factor and effect size was adjusted for typical potential confounders (e.g., age, race, gender, bmi, and cardiovascular risk factors) ; p values 0.05 were considered statistically significant. the study population (n = 65) consists of healthy volunteers (table 1). the average age at the time of enrollment was 54 8 (range from 43 years to 77 years) ; 65% were female ; mean bmi (kg / m) was 27 4 (range of 1942). 29 (45%) of volunteers were prehypertensive and 36 (55%) were normotensive. 58.5% of volunteers were caucasian, 27.7% were african americans, 6.2% were hispanic, and the rest were asians. there was no statistically significant difference between prehypertensive and normotensive group in all of the following parameters : age, gender, race, bmi, augmentation pressure, augmentation pressure index, and central sbp. the main dependent variable we used in our statistical analysis was end diastolic lvmi (using height when calculating the index). prehypertensives had higher lvmi that was statistically significant as compared to normotensives, p < 0.01 (figure 1). simple linear regression analysis showed that central sbp has positive statistically significant association with end diastolic lvmi with p = 0.014 (standardized beta coefficient = 0.314). there was no difference between brachial and central dbp (mean brachial dbp = 76 9 mmhg versus central dbp = 77 9 mmhg) (figure 2) ; however, there was slightly higher brachial sbp (mean 115.8 12 mmhg) compared with central sbp (mean 106 11 mmhg) (figure 3). pearson 's correlation showed statistically significant correlation between central sbp and lvmi (r = 0.318, p = 0.012) (figure 4) and brachial sbp and lvmi (r = 0.460, p < 0.01) (figure 5). the correlation coefficient between brachial dbp and lvmi (r = 0.521, p < 0.01) and central dbp and lvmi (r = 0.523, p < 0.01) was similar. multivariate linear regression analysis, when adjusted for age, gender, race, and bmi, showed positive statistically significant association between pht and end diastolic lvmi. although hypertension is a well - documented independent predictor of elevated lvmi [6, 15, 16 ], few studies have shown the relationship between pht and structural changes in the lv. our study demonstrated a strong relationship between pht and lvmi when compared to normal bp, even after adjustment for age, gender, race, and bmi. another principal new finding in present study was that, in both prehypertensives and normotensives, brachial and central bp correlated positively with lvmi, and central bp was not superior to brachial bp or vice versa for association with lvmi. manios. analyzed the impact of pht on lvm. they found a statistically significant association between prehypertensives and lvm (p = 0.03) compared to normotensive patients after adjustment for baseline characteristics. we were able to establish the importance of pht category to the increased risk of developing future cvd. left ventricular hypertrophy (lvh), measured by lvmi, has been identified as the most powerful risk factor for future cardiovascular events causing morbidity and mortality. in fact, richey. studied the relationship between ambulatory bp and increased lvm in children at risk for hypertension and found that the odds ratio (or) of having elevated lvmi increased by 54% for each incremental increase of standard deviation score (sds) in 24-hour systolic sds after controlling for race and bmi (or = 1.54, unit = 1 sds, ci = 1.1, 2.15, and p = 0.011) and increased by 88% for each increase of 0.1 in bp index (or = 1.88, ci = 1.03, 3.45, and p = 0.04). pht is associated with an increased prevalence of lvh [4, 17 ]. in the bogalusa heart study, toprak., in addition to finding a significantly higher lvmi in prehypertensives compared to normotensives, a finding supported by present study, also found pht was significantly higher among men than women (35% versus 22%) and among blacks than whites (29% versus 27%). we did not find any significant difference between prehypertensive and normotensive groups by gender or race. the difference between both studies on the significance of race on bp category might be due to the smaller sample size (n = 65) of our study compared to the bogalusa heart study (n = 1379). the average age of present study population was 54 8 (4377 years) as compared to the bogalusa heart study with age range of 2044 years. arterial stiffening increases in both genders with age which may explain why gender was not statistically significant in our prehypertensive group but was in the earlier study which was conducted in a much younger population. however, a recent study that analyzed sex differences in arterial stiffness and ventricular - arterial interactions, done in older population (men 67 9 and women 65 10), showed women had greater aortic stiffening as evidenced by higher aortic characteristic impedance (zc) which should translate into a greater increase in bp from increased flow during lv ejection. this motivates further research to determine the impact of gender on zc and its role as a risk for developing pht and future cvd. recent study on 1,940 young participants found higher lvm values in prehypertensives compared to normotensives even after adjustment for covariates [6, 17 ]. methodological differences (such as age of study population, inclusion criteria, and ambulatory blood pressure measurement protocol) between these studies may have accounted for the different results. elevated lvm is a well - defined independent modifiable risk factor for adverse cardiovascular event [7, 15, 22 ] and for developing hypertension. in fact, as reported by urbina., the progression of pht to sustained hypertension was predicted by baseline systolic bp and baseline lvm, with the probability of developing hypertension increasing by 36% for each standard deviation of lvmi. this describes a vicious cycle in which pht causes elevated lvm which in turn accelerates the progression of pht to sustained hypertension increasing future risk of cvds and mortality. our study showed no difference between the diastolic components of both brachial and central bp but a slightly higher brachial sbp than central sbp (figures 2 and 3). this supports the finding of a cardiovascular physiology study which showed that, for the same mean arterial pressure, sbp and pulse pressure (pp) are higher in peripheral (brachial) than in central arteries (thoracic aorta, carotid arteries). the difference, called sbp or pp amplification, is a result of the progressive reduction of the diameter and increase in stiffness from the proximal to the distal arterial vessels and mostly of the modification in the transit of wave reflections [23, 24 ]. however, even though brachial and centrally measured sbp differ, our observation supports previous evidence that both central and brachial sbp positively correlate with lvmi in normotensive and prehypertensive individuals [11, 12, 25 ]. the central bp was measured using sphygmocor monitor which has been shown to have excellent interobserver reproducibility which accords with that reported by other workers using different methodologies. there is an on - going debate on the bp approach that correlates better with lvmi and cvd. roman. reported that lv relative wall thickness and mass index were more strongly related to central than brachial bp, so were the findings in other studies [11, 12 ]. na. concluded that central bp, measured as central pp, was a stronger predictor of lvmi than peripheral pp (coefficient = 0.311, p = 0.001 versus coefficient 0.281, p = 0.003 resp.). pini. also suggested the superior prognostic utility of central bp compared to brachial bp in an unselected geriatric population.. found that brachial bp but not central bp had a better prognostic impact. a new finding in our study is the nonsuperiority of central bp to brachial bp in correlation with lvmi or vice versa. to the best of our knowledge, this is the first study reporting this finding. further prospective studies are required to determine whether central bp may be a better predictor of lvmi and future cvd and mortality. an increased lvm has been shown to be an independent modifiable risk factor for adverse cardiovascular events and progression of pht to sustained hypertension. current guidelines recommend lifestyle modification for the management of pht, but this has had no demonstrable effect on public health to date. the trial of preventing hypertension (trophy) study demonstrated for the first time that pharmacological treatment of prehypertensives was safe and partially reduced the risk of developing incident hypertension ; however, no difference in the occurrence of cardiovascular events was observed between the treatment groups. we recommend further prospective studies to determine whether pharmacological treatment of prehypertensives provides a cost - effective strategy for reducing cvd risks. in addition, both central and brachial bp positively correlate with lvmi in normotensive and prehypertensive patients and central bp was not superior to brachial bp or vice versa for association with lvmi. our study population consists of relatively small sample of 65 volunteers which may limit the generalizability of the results. furthermore, the average age of our study population was 54 8, with older age individuals underrepresented ; however, pht remained an independent predictor of increased lvmi even in multivariable models where age was entered as covariate. bmi was indexed to body surface area (bsa) (kg / m) in our study ; however, there is on - going controversy on the best method to index lvm so as to account for body size. indexing lvm to bsa is said to underestimate lvm in obese and overweight hypertensive patients when compared to height indexed lvm. indexing lvm to bsa in present study showed that pht is associated with lvmi, and this association could have been stronger if lvm was indexed to height as we might have underestimated the prevalence of increased lvm in our study population by using lvm indexed to bsa. | introduction. the purpose of this observational cross - sectional study was to assess left ventricular mass (lvm) in prehypertensive individuals in comparison to normotensives and to determine if central blood pressure (bp) correlates better with lvm index (lvmi) than brachial bp. methods and result. brachial and central bp measurements were completed at first visit and at 4 weeks in 65 healthy volunteers who were at least 40 years old and not on medication. subjects were divided into two groups of normotensives and prehypertensives based on jnc-7 criteria and lvm was obtained using cardiac magnetic resonance imaging. prehypertensives had significantly higher lvmi compared to normotensives (p < 0.01). brachial and central bp also both positively correlate with lvmi (r = 0.460, p < 0.01 ; r = 0.318, p = 0.012, resp.) in both groups and neither method was superior to the other. after multivariate regression analysis and adjusting for cardiovascular risk factors, prehypertension remained an independent determinant of lvm. conclusion. prehypertension is associated with cardiovascular target organ damage, and central bp was not superior to brachial bp or vice versa for association with lvmi. |
respiratory diseases such as asthma are becoming increasingly prevalent, with reduced longevity and quality of life for those affected as well as causing an economic burden upon healthcare systems worldwide. consequently, there is a need to develop more effective therapies to prevent and treat respiratory diseases. developing new therapies requires extensive testing to ensure efficacy and safety, which is both time - consuming and costly. therapies that show promise during the first stage preclinical in vitro tests may be taken forward for further studies. for all new medications, regulatory authorities insist upon acquiring information from animal studies because the effect upon the whole body can be observed. however, the limited biological relevance of animal models to human diseases means that data obtained from such studies could not always be relied on. in vitro models of human tissues that are biomimetic and closely represent the functional properties of their respective tissues could enable better understanding of disease processes, hence providing more physiologically relevant platforms for identification of targets for therapy as well as testing the efficacy and safety of new drug leads. using such in vitro models in drug discovery cycle could in turn substantially reduce the number of drug leads that need to be taken forward to preclinical studies and, therefore, reducing the number of animals required for such experiments. in addition to providing scientific advantages (e.g., identification of more efficacious targets for therapy), using biomimetic in vitro tissue models also conforms with the 3rs principles of refinement, replacement, and reduction of animal experimentations in research wherever possible. the respiratory system is constantly exposed to potentially harmful particles, allergens, and pathogens. to maintain sterility of the lung the respiratory system has a series of defense mechanisms and the capability to respond to environmental challenges. epithelial cells are the predominant cell type in contact with the air and as such the airway epithelium forms the first line of defense against airborne insults. epithelial cells are structurally arranged to form a continuous layer and are joined via protein junctions to create a paracellular barrier to shield interstitial tissue from the airway. as well as a physical barrier, the epithelium forms a chemical barrier via cellular secretions, for example, mucus that entraps infiltrating particles. furthermore, contact with invading pathogens prompts epithelial cells to release lysozymes and phospholipase that destabilize bacterial membranes, defensins that have antimicrobial activity, and surfactants that promote phagocytosis of invading particles. if the epithelial barrier is compromised, the epithelial cells not only change morphologically and functionally but also communicate reciprocally via paracrine or contact - dependent signaling with other cell types, such as underlying stromal and immune cells including macrophages, dcs, lymphocytes, neutrophils, and mast cells. summoning support from underlying cells can assist in restoring the epithelial barrier or initiate an immune response through expression of adhesion molecules and release of mediators including cytokines and chemokines. the synergistic interactions of cells within human lung tissue remains largely understudied ; in particular, few in vitro lung models report the inclusion of immune cells that are essential for sensing cellular and environmental changes as well as exerting a crucial role in the pathogenesis of lung diseases. the tissue engineering of lung models has largely focused toward engineering tracheal replacements due to the simpler nature of this tissue. the robust architecture of the trachea can withstand the decellularization process and subsequent repopulation, whereas it proves difficult to repopulate decellularized tissue from deeper within the lung that has a more complex construction. the specific structural and cellular architecture of complex lung tissue can be retained for experimentation by using ex vivo tissue explants. these biopsy samples are practical for short - term experimentation, though interindividual variability can have an impact upon the results and the availability of such tissue is limited. to allow high - throughput screening of samples and, particularly, longer - term experiments, it is preferable to have a sustainable source of reproducible tissue models. the use of commercially available two - dimensional (2d) platforms upon which epithelial cells can be cultured at the air liquid interface (ali) is widely practiced. although information regarding cellular interaction can be identified using these methods, the 2d platforms fail to represent the cellular arrangement seen in vivo and, therefore, are not amenable to direct cell the use of a 3d tissue equivalent is favorable over 2d cell culture providing more in - vivo - like morphology, function, and intercellular interactions enabling greater resemblance to physiological conditions. encapsulating cells within synthetic or natural hydrogels has been widely used for culturing cells in a 3d environment and provide greater cell cell contact compared to culturing upon a solid 2d substrate. in addition, hydrogels could provide a cellular microenvironment resembling the native extracellular matrix (ecm), hence supporting key functional properties of different cell types. although many cell types seem to thrive within the 3d environment of hydrogels, encapsulating epithelial cells whose primary function is barrier formation could be counterintuitive. therefore, other types of 3d matrix such as porous fiber sheets could be a favorable alternative for culturing epithelial cells, providing closer morphological resemblance to the basement membrane in barrier tissues such as skin and respiratory epithelium. obviously, this does not preclude use of hydrogel based scaffolds with the optimal topography for 3d culture of epithelial cells. methods to create fibrous 3d platforms include phase separation, electrospraying, or electrospinning. we show that the ecm of lung tissue has a randomly arranged network of nanometer - sized fibers, and as such, the electrospinning method proves a suitable choice to create a matrix that mimics this arrangement for culturing lung associated cells. the porous network of polymer fibers that is produced can be tailored in morphology and dimensions to mimic the native ecm of the cells being cultured. although there has been some success in the construction of pure protein electrospun scaffolds (e.g., collagen and fibrinogen), poor structural strength limits their use. synthetic polymers offer the choice of well - defined batches with a greater range of mechanical and chemical properties than those of natural materials. furthermore, synthetic polymers may adsorb ecm proteins in solution or can be surface modified for enhanced cell attachment if necessary. synthetic polymers pla and plga are the more extensively studied, having been explored for both in vitro and in vivo research. however, pla and plga are biodegradable and, in our preliminary experiments, were found to become quite fragile and difficult to handle after few days of cell culture, hence proving unsuitable to support long - term cell cultures for the 3d lung model. thus, use of other nonbiodegradable and biologically nonfouling polymers such as poly(ethylene terephthalate) (pet), which was reported to support cell culture, was considered. subsequently, in this study, we have used electrospun fibers of pet to create a 3d model of airway epithelium, comprising epithelial cells, dendritic cells, and fibroblasts, cultured at ali. the model has been characterized with regards to its barrier function, responses to environmental stimuli, and migratory properties of the immune cells after allergen challenge. this model possesses reasonable cellular and structural representation of the airway epithelium and is amenable to in situ monitoring, and as such, it presents an invaluable tool for academic and pharmaceutical research within the fields of lung biology, disease modeling, and drug discovery and delivery. all materials were purchased from sigma - aldrich, u.k., unless stated otherwise. electrospun scaffolds were produced by dissolving polyethylene terephthalate (pet) in 1:1 trifluoroacetic acid (tfa):dichloromethane (dcm) (fisher chemicals, u.k.) to create a 10% (w / v) solution. the polymer solution was loaded into a syringe (20 ml), and an 18 gauge needle (bd falcon, u.k.) the syringe was securely fitted to a syringe pump - driver (harvard apparatus ltd. the pet solution was delivered at a constant flow rate of 0.5 ml / hour at 14 kv for 4 h. the scaffolds were air - dried in a fumehood for 24 h to allow residual solvent to evaporate. the epithelial (calu-3) and fibroblast (mrc-5) cell lines (lgc standards cell, u.k.) were routinely cultured at 37 c and 5% co2 in dmem - f12 ham or mem media, respectively. both culture media were supplemented with fetal calf serum (fcs) (10% (v / v)), l - glutamine solution (2 mm) (1% (v / v)), and an antibiotic / antimycotic solution (1% v / v) comprised of penicillin (10 000 units / ml), streptomycin sulfate (100 mg / ml), and amphotericin b (25 g / ml). dendritic cells (dc) were generated from peripheral blood monocytes as we have previously described. briefly, peripheral blood mononuclear cells were isolated from human blood buffy coat (national blood transfusion service, u.k.) using histopaque density gradient centrifugation. monocytes were isolated using cd14 + magnetic beads (milteny biotech, u.k.) to the purity of > 98%. purified monocytes were cultured with gm - csf (50 ng / ml) and il-4 (250 iu / ml) (r&d systems) for 6 days to generate immature dcs. dc phenotype was determined by flow cytometry after staining for cell surface markers including cd11c, cd83, cd83, and hla - dr. electrospun scaffolds were cut to a size of 2 cm and sterilized by irradiating with ultraviolet (uv) light at a distance of 8 cm for 15 min each side. the scaffolds were sterilely transferred to a 12 well culture plate and a steel ring was placed on top to secure the scaffold before further sterilization in an antibiotic / antimycotic solution overnight (37 c, 5% co2). the sterilizing solution was removed and the scaffold washed with pbs before submerging the scaffold in the appropriate cell culture media to precondition the scaffold. calu-3 and mrc-5 cells were inoculated inside of the steel ring onto separate pet scaffolds at a density of 3 10 cells / scaffold (1 10 cells / ml in 300 l) and incubated for 72 h (37 c, 5% co2) (figure 1a). steel rings are used to submerge the electrospun scaffolds and define the cell seeding area (a). the use of scaffholders (b e) allow the 3d tissue engineered constructs of lung tissue to be cultured under appropriate conditions where epithelial cells are at the ali (e) and fibroblast cells remain submerged, mimicking in vitro lung conditions. following 72 h culture, single culture scaffolds were transferred from steel rings into a polytetrafluoroethylene (ptfe) platform support (scaffholder), which was designed and fabricated in - house (figure 1b d). the epithelial scaffold monolayer was placed on top of the fibroblast scaffold layer inside the scaffholder to form the coculture model. single culture controls were assembled by combination of either a calu-3 or mrc-5 scaffold with an acellular scaffold (i.e., without cells). to ensure there is no possibility of separation or movement during culture, scaffolds are secured in place within the scaffholder. alignment of cell layers is ensured because the internal diameter of the steel ring matches that of the scaffholder (figure 1). the cells were submerged in cell media for a further 12 h ; the cell media for cocultures comprised a 50:50 mixture of the mrc-5 and calu-3 cell media. media from the apical surface of the scaffholder was removed to culture epithelial cells at the ali (figure 1e). schematic figure showing different steps of fabrication and configuration of the 3d tissue engineered airway epithelium. calu-3 epithelial cells are seeded onto one pet scaffold and mrc-5 fibroblasts are seeded onto a second, separate scaffold (a). following 72 h culture, scaffolds are combined by layering the epithelial scaffold monolayer on top of the fibroblast scaffold layer to form the coculture model. cells are subsequently cultured for 2 weeks at the ali to allow for differentiation of the epithelial cells, including establishment of tight junctions (b). monocyte - derived dcs are seeded onto separate pet scaffolds and then inserted into the coculture model. the upper epithelial scaffold is temporally lifted away from the lower mrc-5 fibroblast layer so that the separate third scaffold containing dendritic cells may be placed on - top of the mrc-5 scaffold layer (c). the calu-3 layer is placed on - top of the dc scaffold layer, resulting in the dc layer sandwiched between the epithelial and fibroblast scaffold layers to form the triculture model (d). fibroblast cocultures were assembled and cultured for 14 days at the ali prior to insertion of the dc layer. the cell culture media composition remained as a 50:50 mixture of calu-3 and mrc-5 cell media. immature dcs (day 6) were prestained with hoescht nuclear stain (5 g / ml) (invitrogen, u.k.) and inoculated onto pet electrospun scaffolds at a density of 2 10 cells / scaffold and incubated for 24 h (37 c, 5% co2). the culture medium was aspirated to remove dcs that had not attached prior to insertion in between calu-3 and mrc-5 layers in an established coculture model to form an immunocompetent triculture model. a schematic describing the layer - on - layer approach to assembling epithelial and fibroblast cocultures and subsequent insertion of the dc layer is described in figure 2. triculture models were stimulated with house dust mite extract (hdm) (10 g / ml) (greer, u.s.a.) and lipopolysaccharide (lps) (100 ng / ml) (sigma - aldrich, u.k.) or pbs control and incubated for 36 h prior to analysis. the triculture models were fixed with 4% (v / v) paraformaldehyde (electron microscopy sciences, u.s.a.) in pbs, and the three scaffold layers were separated and immunostained with pancytokeratin (epithelial cell marker). scaffolds were then examined by confocal microscopy (leica sp2 confocal laser scanning microscope) (leica microsystems ltd., u.k.) with postvisualization performed using volocity software (perkin - elmer, u.k.). trans - epithelial electrical resistance (teer) measurements were performed across the epithelial cell monolayer of cells cultured at the ali. measurements were performed using an evom volt - ohm - meter and stx2 chopstick electrodes (world precision instruments, u.k.). prior to recording teer, chopstick electrodes were sterilized (70% v / v ethanol in distilled water) and cell culture media was added to the upper chamber (500 l) and lower chamber (1.5 ml total volume) and allowed to equilibrate for 30 min (37 c, 5% co2). cellular samples of pet electrospun scaffold were placed onto carbon - coated electron microscope stubs and sputter - coated with gold (5 min, blazers scd 030 blazers union ltd., liechtenstein) under an argon atmosphere (boc, u.k.) prior to analysis. samples were imaged using sem (scanning electron microscopy) analysis (jeol jms-6060 lv microscope, jeol ltd., u.k.) cellular samples were fixed in 3% (v / v) glutaraldehyde overnight at 4 c before dehydration through an ascending series of ethanol concentrations prior to sem imaging. briefly, cellular scaffolds were fixed with 10% buffered formalin, excised, and embedded in paraffin. the paraffin embedded blocks were then sectioned and stained with hematoxylin and eosin (h&e) before imaging. scaffold samples were washed with pbs prior to fixation with paraformaldehyde (4% (w / v)) or methanol (100% (v / v)) for 15 min at room temperature (rt). samples were washed in pbs (3 5 min each) before being permeabilized using triton x-100 (0.5% (v / v)) for 5 min at room temperature. following a further wash in pbs (3 5 min each), nonspecific antibody binding was blocked with goat serum (10% (v / v) in pbs for 5 min at room temperature. primary antibodies used were anti - mucin 5ac [45m1 ] (ab3649, abcam, u.k.), mouse anti - zo1 (invitrogen, u.k.), anti - fibronectin (ab 23750, abcam, u.k.), anti - ki67 (ab15580, abcam, u.k.), anti - collagen (ab34710, abcam, u.k.), and pan - cytokeratin pk110 (santacruz biotech, u.k.). samples were then washed with pbs (3 5 min each) and incubated with species - appropriate fluorescently labeled secondary antibodies (1:100) for 30 min at room temperature. secondary antibodies included goat antimouse igg rhodamine red x (invitrogen, u.k.), goat antirabbit igg fitc (invitrogen, u.k.), and alexa fluor 488 goat antimouse igm (chain) (invitrogen, u.k.). samples were washed in pbs (3 5 min each), incubated with hoechst (5 g / ml) for 5 min at room temperature, and mounted using fluoromount mounting medium. immunostaining was observed using a confocal microscope (leica sp2 confocal laser scanning microscope, images processed with leica confocal software) with postvisualization performed using volocity software. a papain (60u / ml) solution was prepared with l - cysteine (5 mm) to reconstitute the cysteine active site, and an aliquot (300 l) was applied to the apical surface of calu-3 cells. the present study presents a multilayered 3d electrospun pet lung model capable of incorporating multiple cell types each supported upon their own individual electrospun layer. the porous network of electrospun pet fibers can permit cell interaction through both direct cell cell contact and paracrine factors within the 3d model. we report the incorporation of lung associated epithelial cells and fibroblasts and monocyte - derived dcs within the 3d model and determine how culturing these cells together influences their behavior. sem analysis of decellularised human lung tissue (from healthy sections of lung tissue obtained from patients undergoing surgical procedures (nottingham university hospitals nhs trust) after informed consent and ethics approval) revealed a porous network of nanometer - sized fibers (figure 3a). accordingly, we used a nonbiodegradable polymer (namely, pet) to fabricate a nanoscale porous scaffold (figure 3b). the mean fiber diameter of the pet scaffold (240 nm 70) was similar to the mean diameter of lung ecm (245 nm 83) (figure 3c). the average thickness of the pet scaffold, calculated using histological sections, was 60 10 m. owing to its robust physical properties, the pet electrospun scaffold is capable of withstanding repeat handling allowing separation of individual cell layers for assessment following experimentation. comparisons of scanning electron micrographs of decellularised lung tissue (a) with pet electrospun scaffold (b) showing morphological similarities. the fiber diameter of decellularised lung tissue and pet electrospun scaffolds were measured and show comparable dimensions (c). the main structural cell types of the lung, epithelial, and fibroblast cells were each cultured upon separate pet scaffolds. to incorporate an immune component into the model, a triculture system was formed by culturing dcs upon a third pet scaffold and inserting this between the epithelial and fibroblast layers of an established epithelial epithelial and fibroblast cells were each inoculated onto individual pet scaffold layers and cultured submerged in media to allow cells to establish growth upon the scaffold, following which the cell inoculated scaffolds were assembled together within our developed scaffholders allowing physiologically relevant positioning of the construct. epithelial cells were cultured upon the uppermost layer allowing culture at ali where the upper cell surface is in contact with the air. culturing epithelial cells at the ali mimics in vivo conditions by providing apical basal polarity, which leads to full differentiation of epithelial cells and development of a functional barrier. the underlying fibroblasts were positioned directly beneath the epithelial inoculated scaffold and remained submerged in media much as they would in vivo. the production and maintenance of barrier integrity in epithelial cells cultured upon pet electrospun scaffolds and positioned at the ali using scaffholders was assessed using teer measurements and permeability studies. barrier formation was compared when epithelial cells were cultured alone (single culture) or with fibroblasts (coculture). teer is widely used to monitor barrier integrity, where an increase in resistance to flow of current is due to greater integrity of the barrier, attributed to the formation of cellular tight junctions. an acellular control scaffold was monitored in order to report a control value, as it is known that teer values reportedly vary according to the material upon which the epithelial cells are cultured. in addition, a single culture of mrc-5 fibroblasts was monitored as a control, as an increase in teer value was not expected owing to mrc-5 fibroblasts characteristically not forming tight junctions. statistical analysis was performed using two - way anova with sidaks multiple comparison showed that teer measurements of cocultures produced readings that were significantly greater than those of epithelial cells cultured alone (figure 4a). the teer measurements of cocultures are significantly higher than those of epithelial cells cultured alone. statistical analysis was performed using two - way anova with sidaks multiple comparison with the difference between coculture and single calu-3 culture having a p value < 0.0001 (a). sem images of epithelial cells from single and coculture where the upper surface that has been imaged is thought to be predominantly formed from ecm protein presence. the micrographs show that epithelial cells from coculture have a smoother surface than those from single culture (b). the teer measurements show that after 14 days in culture epithelial cells cultured alone, without fibroblasts, attained measurements of 130 cm. the greatest increase was seen for cocultures of epithelial and fibroblast cells attaining measurements of 200 cm. thus, epithelial cells cultured together with fibroblasts appeared to achieve a confluent differentiated state earlier than single cultures of epithelial cells as shown by earlier and greater increases in teer measurements (figure 4 a). the teer values of the controls, an acellular scaffold and mrc-5 single culture, did not increase, maintaining baseline teer measurements of 100 cm. although human lung ex vivo teer results are not available in the literature, ex vivo rabbit airway epithelium has been reported to be 260320 cm for the trachea and 266 cm for the bronchus. thus, the maximal teer recorded in our 3d model are comparable to the data available for animal ex vivo teers. the topography of the confluent epithelial cell layer was observed using sem. the sem micrographs of epithelial cells from single culture, coculture, and fibroblasts from cocultures showed what is thought to be the presence of ecm protein deposition (figure 4b). h&e staining of the epithelial layer from single and cocultures indicated that single cultures formed a single thin layer of cells, whereas the cocultured epithelial cells had a more dense and layered arrangement after 14 days at ali (figure 5). furthermore, epithelial cells within cocultures appeared to produce mucus earlier than single cultures as shown by immunocytochemical staining (figure 5). histological staining shows the presence of epithelial cells on the uppermost surface of the pet scaffold with a greater number of cells found in cocultures compared to single cultures. mucin production (red) was greater from coculture than single culture, where cell nuclei are stained with dapi (blue). immunocytochemical staining of the deposition of ecm proteins indicated that the epithelial cells deposit collagen and fibronectin when cultured upon pet electrospun scaffolds (figure 6). there appeared to be enhanced production of these ecm proteins from cocultured epithelial cells. the expression of the cell proliferation marker ki67 showed that epithelial cells in coculture had lower levels of ki67, possibly indicating their tendency toward an earlier full differentiated state as also evidenced by an accelerated increase in teer readings in cocultures compared with single cultures of epithelial cells (figure 7a). fibroblast cell growth has not been adversely affected when positioned beneath the epithelial cell layer during coculture experimentation ; the fibroblast cells are still present and in a state of active growth as indicated by the production of ki67 and have retained the ability to produce ecm proteins collagen and fibronectin (figure 6). ecm staining following 14 days culture at the ali shows the presence of ecm proteins : collagen and fibronectin, predominantly found in epithelial and fibroblast cells and also ki67, showing that the cells are in active growth. epithelial cells in cocultures appear to express lower levels of ki67 (compared to single cultures), indicating their tendency toward full differentiation. following the 14 days at the ali, the enzyme papain was applied to the epithelial surface of single and cocultures, which was shown to disrupt tight junctions demonstrated by the measurement of teers (a) and by confocal imaging of zo1 protein (b). the disruption to tight junctions from coculture was observed to be less than that of single cultures. teer measurements were then performed for a further 14 days to monitor the healing process. the cocultures were observed to be recovering from the disruption with increase in teers and increase in the presence of zo1. single cultures did not appear to recover so well with teer measurements remaining around the same value as when the cells were subjected to papain. statistical analysis was performed using two - way anova with sidaks multiple comparison, showing that the difference between coculture and single calu-3 culture that had been subjected to papain having a p value < 0.0001. the 3d lung model was cultured at the ali for 14 days before papain was applied to the epithelial surface of single and cocultures. papain is an allergen with cysteine protease activity and is known to disrupt tight junctions in respiratory epithelium using its enzymatic activity, mimicking the action of other airborne allergens such as house dust mite, which also have a cysteine protease activity. the application of papain on day 14 ali was shown to disrupt epithelial tight junctions in both single and coculture as demonstrated by a sharp reduction in teer values (figure 7a) and disintegration of zo1 protein visualized by immunostaining followed by confocal imaging (figure 7b). the disruption to tight junctions from coculture was thought to be less than that of single cultures, where almost no zo1 could be observed post papain. teer measurements were performed for a further 14 days to monitor the epithelial repair process, and further examination of zo1 expression was carried out. the cocultures were observed to be recovering from the disruption with increased teers and increased presence of zo1 (figure 7a and b). however, teer measurements did not appear to recover in single cultures during the same time frame and remained at approximately the same value as when the cells were subjected to papain (figure 7a). the confocal microscopy images on day 15 (post papain) of single culture shows that the cell growth has been disrupted by papain and that tight junctions are not present over a wide area of the sample. the cells from single cultures recovered slightly as evidenced by the presence of nondisrupted nuclei on day 28 ; however, the presence of tight junctions remain absent (figure 7b). in a proof - of - concept experiment, an immunocompetent triculture model comprising of epithelial, dendritic, and fibroblast layers was constructed and dc migration within the model monitored. the triculture model was stimulated with hdm extract (a common airborne allergen) and toll - like 4 receptor agonist lps (abundant in most airborne bacterial pathogens), and subsequently, the migration of dcs was assessed by confocal microscopy. epithelial cells were identified using pancytokeratin and dcs had been prestained using the nuclear stain hoescht. assessment of the uppermost scaffold (bearing epithelial cells) revealed that after 36 h in the triculture, most dcs seemed to have migrated to this layer and were present in close proximity to the basal (in unstimulated samples) or apical side (in stimulated samples) of the epithelial cell layer (figure 8a). the dcs migration was further confirmed by the fact that dcs were found to be largely absent from the scaffold upon which they were originally inoculated (middle scaffold) (figure 8b). typically, not many dcs could be observed in the majority of lower scaffolds (fibroblast layer) in either stimulated or unstimulated samples (figure 8c). confocal microscopy images (10 mag) of dc migration 36 h after allergen stimulation in 3d tricoculture. the upper scaffold contained the calu-3 epithelial layer (a), a middle layer to which dcs were seeded (b), and a lower scaffold containing mrc-5 (c). dcs were prestained with hoescht nuclear stain (blue) and calu-3 cells were poststained with pancytokeratin (green). in single culture, dcs remained on the middle scaffold where they had been inoculated. in the triculture, most dcs migrated from middle scaffold (b) to upper scaffold (a). upon treatment of the triculture with house dust mite extract (hdm) (10 g / ml) and lipopolysaccharide (lps) (100 ng / ml), dcs appeared to primarily migrate to the apical surface of the epithelial layer, whereas in unstimulated samples they appear to be mainly localized in the basal region of the epithelial layer (a). using electrospun porous fibers, we have engineered a 3d triculture of airway epithelial cells, dendritic cells, and fibroblasts at ali creating a modular construct that mimics the cellular orientation and some functional properties of human airway epithelium. the epithelium in vivo provides the first line of defense against environmental insults, and as such, it is capable of rapidly repairing any cellular damage caused. epithelial cells in vivo are thought to respond to insult and injury through a cascade of events of which there are three distinct stages : dedifferentiation, proliferation, and differentiation. the first stage is the dedifferentiation of underlying stromal cells, such as fibroblasts, that are initially exposed to the environment following injury to epithelial cells. the stromal cells are thought to migrate to the wound site to assist repair by dedifferentiation helping to cover the site of injury and assist in restoring barrier integrity through proliferation. the final stage in restoring barrier integrity is the differentiation of cells involved in the repair process, including the restoration of full function to epithelial cells, particularly their ability to form junctional protein to seal the barrier. the exact mechanisms controlling cell fate during epithelium repair are not clear ; however, it is thought that multiple paracrine factors and direct cell cell contact are key parameters required to restore barrier integrity and function. it is, therefore, important to consider these factors when configuring tissue engineering strategies to create in vitro models of epithelial tissue. this study developed a 3d multicellular lung model that is capable of forming a more physiologically relevant representation of lung tissue than culturing cells alone or in 2d systems. our studies corroborate the relevance of culturing multiple cell types found in lung tissue together, as results demonstrate that when epithelial cells were subjected to chemical insult (i.e., an enzymatically active allergen), they recovered earlier in cocultures than single cultures. this supports the relevance of cocultures as it is thought that the cells signal and respond in alliance. this study also assembled a triculture model in a proof - of - concept experiment to examine whether dc migration could occur within the lung model following stimulation. a large number of dcs were found to migrate from the scaffold in which they were initially inoculated (middle layer) through to the uppermost scaffold bearing the epithelial cells. this occurred both in the presence and absence of stimulation and may suggest that dcs were responding to epithelial secretions. however, in samples stimulated with lps and hdm, most dcs were located closer to the apical side of the epithelial barrier, whereas in unstimulated samples they were mainly located close to the basal side. these results prove that the 3d lung model is amenable to cell migration and that cell cell contact is feasible. individual scaffolds are typically 60 10 m thick and the results of the dc migration study demonstrate that scaffold thickness does not seem to impede dc migration. the pattern of migration observed in this experiment substantiates dc migration in vivo, where upon activation, dcs migrate to the epithelium and survey the immune environment before migrating through the subepithelial compartment before traveling to lymph nodes. this enables future studies focused on inhaled drug delivery systems as well as allergen and inhaled drugs uptake by dcs. we would estimate the triculture would be limited to one week due to migration of dcs and their phenotypical / functional changes upon stimulation, which should provide enough time for performing drug uptake / delivery experiments. limitations to in vitro models, particularly those generated from commercially available 2d inserts, are that the surface upon which cells grow is planar, so cell cell interaction is restricted. cell interactions can occur through the pores of the insert ; however, these are thought to be constrained to paracrine interactions because the pore size of the substrate upon which the epithelial cells are commonly cultured to allow barrier formation are too small, thus preventing cell migration. a 3d human skin triculture model comprising of keratinocytes, dcs, and fibroblasts has been reported. this model shares similarities with the model developed in this work, including a physiologically relevant arrangement of the cell layers each supported on separate 3d scaffolds and the establishment of a differentiated epithelial the keratinocytes and fibroblasts were supported on 3d microfiber - scale scaffolds ; however, the dc layer was inserted into the model in an agarose gel rather than on a fibrous scaffold. migration of dcs in the skin model was reported from the agarose gel layer to only the fibroblast layer. furthermore, the degree of migration appeared to be far less than that observed in the 3d model developed in this study. hence, direct seeding of dcs on the porous scaffolds as opposed to encapsulation within a hydrogel seems to facilitate cell migration. epithelial cell lines such as calu-3, the cell line we have used in this study, are widely used as surrogates for primary cells. despite many similarities between calu-3 and primary cells, particularly in features like barrier formation and mucus production, there are also considerable functional differences (e.g., their cytokine profile). therefore, use of cell lines somewhat limits the physiological relevance of the model and future efforts should focus on replacing cell lines with human primary cells. the 3d immunocompetent lung model presented in this study successfully supports the culture of multiple cell types on electrospun pet scaffolds that structurally resemble the native lung ecm. the porous network of electrospun pet fibers can permit cell interaction through both direct cell cell contact and through paracrine factors within the 3d model as shown by the enhanced formation of a differentiated epithelial layer, enhanced epithelial repair, and migration of immune cells incorporated within the model. furthermore, the modular nature of our approach means that other relevant structural (e.g., smooth muscle cells) and immune cells (e.g., mast cells or eosinophils) could be included into the model with relative ease. collectively, we believe this model possesses adequate cellular and structural representation of the airway epithelium and is amenable to in situ monitoring ; as such, it presents an invaluable tool for academic and pharmaceutical research within the fields of lung biology, disease modeling, and drug discovery and delivery with the potential of reducing the need for some animal experimentation in this area. | the development of more complex in vitro models for the assessment of novel drugs and chemicals is needed because of the limited biological relevance of animal models to humans as well as ethical considerations. although some human - cell - based assays exist, they are usually 2d, consist of single cell type, and have limited cellular and functional representation of the native tissue. in this study, we have used biomimetic porous electrospun scaffolds to develop an immunocompetent 3d model of the human respiratory tract comprised of three key cell types present in upper airway epithelium. the three cell types, namely, epithelial cells (providing a physical barrier), fibroblasts (extracellular matrix production), and dendritic cells (immune sensing), were initially grown on individual scaffolds and then assembled into the 3d multicell tissue model. the epithelial layer was cultured at the air liquid interface for up to four weeks, leading to formation of a functional barrier as evidenced by an increase in transepithelial electrical resistance (teer) and tight junction formation. the response of epithelial cells to allergen exposure was monitored by quantifying changes in teer readings and by assessment of cellular tight junctions using immunostaining. it was found that epithelial cells cocultured with fibroblasts formed a functional epithelial barrier at a quicker rate than single cultures of epithelial cells and that the recovery from allergen exposure was also more rapid. also, our data show that dendritic cells within this model remain viable and responsive to external stimulation as evidenced by their migration within the 3d construct in response to allergen challenge. this model provides an easy to assemble and physiologically relevant 3d model of human airway epithelium that can be used for studies aiming at better understanding lung biology, the cross - talk between immune cells, and airborne allergens and pathogens as well as drug delivery. |
a denoid cystic carcinoma (acc) is a malignant neoplasm of the salivary glands. the term adenoid cystic carcinoma was coined in the year 1928 and is in use till date. adenoid cystic carcinomas constitute less than 1% of all head and neck malignancies with 50% of all accs occurring intraorally, commonly in the hard palate. other less common intraoral sites include the lower lip, retromolar / tonsillar pillar region, sublingual gland, buccal mucosa and floor of the mouth. adenoid cystic carcinomas are clinically innocuous lesions usually characterized by small size and slow growth. pain is an important symptom of the condition due to its propensity for perineural spread. thus, accs have a long clinical course and questionable prognosis with minor salivary gland accs having a worse prognosis than those of the major salivary glands. we describe the features of adenoid cystic carcinoma in the buccal mucosa along with a review of the literature. a 45-year - old female reported to the department with complaint of a painful swelling in the left buccal mucosal region. she first noticed the swelling 3 months ago, which had gradually increased in size. on intraoral examination, there was an ill defined swelling in the left posterior buccal mucosa in the molar region [figure 1 ]. palpation revealed a tender, well - defined, freely movable swelling, 1 1 cm in size, which was soft to firm in consistency. clinical intraoral picture showing swelling in the left posterior buccal mucosa (black arrow) panoramic radiography revealed no evidence of bone changes in the maxilla or mandible [figure 2 ]. ultrasonography of the region showed a hypoechoic mass with uniform internal structure and well defined borders. there were no areas of calcification and it appeared unattached to the neighbouring structures [figure 3 ]. histopathological examination revealed loss of cellular architecture and cribriform pattern of tumor cells with many microcytes. since the surgical margins were free of the disease, it was decided not to give any adjuvant therapy. orthopantomograph showing no bony changes ultrasonographic image showing a well defined hypoechoic mass with uniform internal structure (lesion extent marked with x) photomicrograph 10 showing cribriform pattern of tumor cells adenoid cystic carcinoma is a rare epithelial tumor with an indolent but persistent growth pattern. the world health organisation defines acc as a basaloid tumor consisting of epithelial and myoepithelial cells in various morphological configurations, including tubular, cribriform and solid patterns. acc occurs predominantly in fourth to sixth decade of life with a female predilection of 3:2. in our case a 45-year - old female was affected. among salivary gland neoplasms, most articles in the literature describing the incidence of adenoid cystic carcinoma include both the major and minor salivary glands and no article so far has compiled the number of cases of acc of the buccal mucosa alone. our article is the first to present data gleaned from a total of 41 published articles. only those articles which were specific regarding the intraoral site of involvement were included in our review. after compilation of the cases, we found 2,280 cases of acc in a total of 41 articles. out of these cases 1,382 were reported in intraoral sites and 178 were specifically reported in the buccal mucosa[24643table 1 ]. based on the above findings, we concluded that among intraoral minor salivary gland accs, 12.9% occurred in the buccal mucosa. reported cases of adenoid cystic carcinoma of the buccal mucosa the clinical presentation of acc involves a slow growing, firm, unilobular mass. pain is usually a common and important associated symptom, occasionally occurring before clinical evidence of the disease. accs are graded according to the histological pattern into grade i, grade ii and grade iii with grade i being a combination of cribriform and tubular, grade ii a mixture of cribriform, tubular and solid patterns and grade iii having only solid pattern. the present reported case was of the cribriform variant and was classified as grade i. tendency to show perineural invasion is a highly characteristic feature of acc. perineural invasion occurs through spread along the perineural spaces or within the nerve itself. according to who the influence of perineural invasion on survival has been contradictory. it was stated to have no prognostic significance in some studies whereas some authors mention that it is a negative survival predictor because of greater tendency for distant metastasis. various treatment modalities that have been proposed in acc which include surgery, radiotherapy, chemotherapy and combined therapy. many factors influence the prognosis in cases of adenoid cystic carcinoma. these include tumor stage, positive surgical margins, site of primary, perineural invasion, solid histological type and presence of cervical lymph node metastasis at the time of diagnosis. accs typically have a prolonged clinical course with distant metastasis occurring late in the disease despite adequate locoregional control. one study discovered that the median time between diagnosis of the primary lesion and detection of distant metastasis was 60 months with a range of 18 - 120 months. unlike other malignancies, they usually do not lead to death in the short term but have low long term survival rates. adenoid cystic carcinomas are seemingly innocuous lesions, which show slow growth but due to their propensity for perineural spread and distant metastasis, require prolonged follow - up. | adenoid cystic carcinomas are deceptive malignancies that show slow growth and local invasion with recurrences seen many years after diagnosis. upto 50% of these tumors occur in the intraoral minor salivary glands usually in the hard palate. buccal mucosal tumors are relatively rare. we determined the incidence of buccal mucosal adenoid cystic carcinoma by reviewing the number of reported cases in the literature. this is the first article to analyze the occurrence of adenoid cystic carcinomas in the buccal mucosa through a review of 41 articles. our review revealed 178 buccal mucosal adenoid cystic carcinomas among a total of 2,280 reported cases. we present a case of adenoid cystic carcinoma occurring in the left buccal mucosa of a 45-year - old female. |
in adults, damaged cartilage has a very limited capacity for self - healing due to the absence of blood vessels, the low intrinsic density of chondrocytes, and the low turnover of the extracellular matrix. the absence of stem cells and the low capacity of resident chondrocytes to migrate and proliferate also reduce cartilage regeneration capacity. therefore, numerous methods have been proposed to repair cartilage defects in young or osteoarthritis (oa) subjects. intrinsic repair can be stimulated by bleeding induced by drilling or microfracturing the subchondral bone. fibrin clot formation, vascular invasion, and recruitment of stem cells into the defect result in the formation of scar tissue that is inferior biomechanically to normal cartilage and susceptible, therefore, to degeneration. in osteochondral transplantation (or mosaicplasty), osteochondral plugs are transferred from undamaged and relatively non - weight - bearing regions to a debrided site. however, studies have shown that this technique results in donor site morbidity and extensive chondrocyte death in the margins of the osteochondral plugs. one option to treat focal cartilage lesions is autologous chondrocyte implantation (aci), a procedure developed in the late 1980s to treat traumatic and symptomatic cartilage lesions in the knees of young adults. the idea behind the aci procedure is to take a cartilage biopsy from the knee by arthroscopy, to isolate cells and grow them in the lab and, once millions of cells have been grown, to implant them into the area of cartilage damage beneath a periosteal flap or a collagen sheet sutured to the surrounding healthy cartilage rim. more recently, matrix - assisted autologous chondrocyte implantation (maci) has been proposed as an alternative to aci. this technique uses a biomaterial as a chondrocyte carrier that is directly implanted in the lesion [3 - 5 ]. the matrix can be synthetic, including but not limited to poly(lactic acid), poly(glycolic acid), poly(lactic - co - glycolic acid) copolymer, poly(ethylene oxide), or poly(propylene oxide) polymers that all gel at body temperature, ceramic composite and hydrogel - containing polyethylene glycol polymer - based derivatives, or natural substances such as fibrin, collagens, alginate, agarose, chitosan, and hyaluronic acid. these substances have been used to design and produce scaffolds in a rich variety of configurations, including woven and non - woven meshes, sponges, foam, glues, bilayer or trilayer composites and, more recently, small - size magnetic beads. to promote chondrogenesis (differentiation to chondrocyte - like cells) and scaffold integration, many maci systems using growth factors, either attached to the scaffolds or through recombinant expression, have been proposed. several studies have also shown that pulsed electromagnetic field and continuous passive motion may promote chondrogenesis and implant integration to the existing cartilage. until now, aci / maci techniques have been reserved for patients who meet the following criteria : age 15 - 60 years ; body mass index (bmi) 35 ; presence of disabling pain and/or knee locking ; focal articular cartilage defect down to but not through the subchondral bone on a load bearing surface of the femoral condyle (medial, lateral, trochlear) (not in the patella) ; size of defect < 7 mm in depth, < 6 cm in length, and 1.6 - 10 cm in area ; stable knee with intact meniscus and normal joint space on x - ray ; no active inflammatory or other arthritis, clinically and by x - ray ; procedure is not being done for treatment of degenerative arthritis (oa) ; failure of conservative therapy (minimum of 2 months of physical therapy) as well as established surgical interventions (i.e., microfracture, drilling, abrasion) - diagnostic arthroscopy, lavage, or debridement are not considered adequate to meet this criterion ; cooperation with post - operative weight - bearing restrictions and activity restrictions together with a potential for completion of post - operative rehabilitation ; and informed consent with realistic expectations. currently, aci is contraindicated in oa because the risks of complications and failure are high and clinical superiority has not been demonstrated compared with other treatments. in young adults with traumatic chondral lesions, re - operation is a common sequel of aci with an incidence of 15 - 30%. periosteal hypertrophy and delamination, which account for 22.1% and 17.7%, respectively, of adverse effects reported to the us food and drug administration, frequently require aci site debridement. failure of aci occurs in 4 - 22% of patients depending on defect traits and duration of follow - up. a study of single condylar defects reported 5% failure at 2 years, which increased to 22.5% at 5 years. one comparison of aci by patient age demonstrated good to excellent results in 85.7% of patients younger than 20 years compared with 55.9% in those older than 40 years. recently, an extensive case report has shown that maci based on a bioresorbable two - component gel - polymer scaffold is effective for the treatment of focal degenerative cartilage defects of the knee in subjects between 25 and 50 years of age. this product is composed of fibrin and a polymer - based scaffold of polyglycolic / polylactic acid (polyglactin, vicryl) and polydioxanone. in this study, 18 patients with preoperatively radiologically confirmed oa and a kellgren - lawrence score of 2 or more were included and followed up for 4 years. the average age of patients (8 females and 10 males) was 35 years (25 - 50 years), the mean bmi was 25 (ranging from 19 to 24) and mean defect size was 4 cm (2 - 6 cm). clinical improvement has been assessed by different scoring systems, including the lysholm and international cartilage repair society (icrs) scores and the knee injury and osteoarthritis outcome score (koos). after 1 year, mean scores improved between 30% and 50% compared with the pre - operative situation, depending on which score was analyzed. the clinical results 1 year after implantation of the scaffold were good and remained stable for at least 4 years. this indicates a significant decrease in pain and disability as well as a significant increase in quality of life. however, nine patients were subjected to second - look arthroscopy due to symptoms such as persistent grinding, catching pain, or swelling. magnetic resonance imaging performed 4 years after transplantation in 17 patients showed a complete filling of the defect with cartilage repair tissue in 11 patients. in five patients, the defects were filled more than 50% and one patient showed a defect fill of less than 50%. the cartilage signal in 16 out of 17 defects was normal or showed a slight alteration of the signal. strong to moderate subchondral edema was evident in 6 patients, and 11 out of 17 patients showed no or mild edema. no to mild knee joint effusion was evident in 12 out of 17 patients treated at 4-year follow - up. these results are promising but need to be reproduced in older patients with chondrocytes affected by the aging processes. in a retrospective study by minas., 56 patients 45 years of age were treated with aci using periosteal flap. the mean transplant size was 4.7 cm per defect (range, 1 - 15 cm) and 9.8 cm per knee (range, 2.5 - 31.6 cm). there were eight failures (14%), mainly in patients receiving workers compensation, and 24 additional arthroscopic surgical procedures for periosteal - related problems and adhesion. at their latest available follow - up, 72% of patients rated themselves as good or excellent, and 78% felt improved and would again choose aci as a treatment option. these studies, even if they are limited to prospective or retrospective non - controlled trials, suggest that aci / maci systems could be used to prevent or delay total joint replacement in oa. these repair techniques are invasive, associated with some complications (locking of the joint and adhesions, extension deficit, recurrent knee effusion, and so on) and have an approximately 10% risk of failure. aci / maci show great promise as treatments for chondral lesions, with a potential for them to be highly cost effective. but at present this has not been demonstrated and recommendation of this technique for the treatment of cartilage defects in oa joint can not yet be justified. the superiority of aci / maci compared with other oa treatments, such as hyaluronic acid injection or other surgical procedures (debridement, microfractures), needs to be demonstrated. further, these methods require a complex process of tissue harvest, cell culture, scaffold implantation and finally post - operative rehabilitation. what are the clinical benefits that justify this investment in oa ? promising developments are underway with regards to cell - based techniques in combination with scaffolds, growth factors, and gene therapy. unfortunately, this effort has not been followed by appropriate or sufficient clinical studies to assess these new methods and to compare them with existing systems. further research is needed to simplify the implantation procedure and to improve the success level. oa patients are generally older and heavier than the population included in the current studies and we lack data on the incidence of failure in this population. altogether, the use of aci or maci techniques in oa patients remains experimental but constitutes a real opportunity for such patients in the next decade. the main problems still to be resolved are how to produce a hyaline cartilage rather than a fibrocartilage, how to facilitate the integration of the repair within the surrounding tissue, and how to simplify the implementation procedure. | as medical advances lengthen average life expectancy, osteoarthritis (oa) will become a larger public health problem - not only because it is a manifestation of aging but also because it usually takes many years to reach clinical relevance. oa is already one of the ten most disabling diseases in industrialized countries. the huge financial burden emphasizes the acute need for new and more effective treatments for articular cartilage defects, especially since there are few disease modifying drugs or treatments for oa. there is no cure for oa and the management of oa is largely palliative, focusing on the alleviation of symptoms. recent longitudinal non - controlled trials suggest that autologous chondrocyte transplantation techniques, which are indicated for young people with traumatic cartilage defects, could also be used in degenerative defects of elderly people with oa. this report discusses this therapeutic opportunity in view of some recently published data. |
ten different brands of stps were collected from the local retail markets of cochin (kerala) from the months of august to november 2011. the samples were selected on the basis of popularity among the people especially the teenage community, representing a large and uniform sample pool. they were personally collected from the shops due to the absence of a national level manufacturer. these were then labeled with unique identification codes and stored in airtight polythene bags under refrigeration. the samples were thoroughly ground and passed through 5 mesh size before being taken for analysis. for ease of use, the samples have been identified with numbering as samples 1 - 10 : sample 1 - ds madras snuff, sample 2 - shambhu, sample 3 - minar, sample 4 - madhu, sample 5 - cool lip, sample 6 - hans, sample 7 - parag 9000, sample 8 - chaini khaini, sample 9 - bombay 1000, sample 10 - rajanigandha. the sample preparation procedures adopted to analyse trace metals are based on health canada, centres for disease control and the united states federal registers and modified in - house techniques based on the most up to date information from published literature. all the glassware were washed in distilled deionised water and dried in oven prior to use. three grams of the accurately weighed samples were digested using a 3:1 mixture of concentrated nitric acid and hydrochloric acid over a laboratory grade furnace. the samples were then filtered ; the filtrate washed with distilled deionised water and made up to 100 ml in a standard flask. these samples were subsequently analysed by an atomic absorption spectrometer (gbc avanta, 2 - 0 - 2 version from gbc scientific equipments pvt. ltd., a mixture of air - acetylene was used as fuel for all the elements except chromium and cadmium for which nitrous oxide - acetylene mixture was used. the air flow was maintained at 10 l / min while the fuel flow was adjusted to 2 l / min. instrument parameters for trace metal analysis this protocol revolved around the guidelines prescribed by the federal register with slight in - house modifications. the freshly opened samples were extracted with 20 ml distilled water using a mechanical shaker and the clear supernatant was taken for analysis. the samples were analysed in triplicate using a laboratory grade ph meter (digital ph meter m.k 6, systronics, ahmedabad) and the results expressed as mean phsd. the samples were prepared in accordance with cooperation centre for scientific research relative to tobacco (coresta)-recommended method n56 (for karl fischer method) and 57 (for gas chromatography - thermal conductivity detector (gc - tcd) method) with slight in - house modifications in the techniques. moisture content was analysed by karl fischer titrator (mettler toledo model dl18 equipped with mettler ga44 printer and mettler ae200 balance) under conditions specified in coresta. five hundred milligram of the sample was extracted using karl fisher grade anhydrous carbinol and the clear supernatant was used for further analysis. the whole procedure was carried out in sealed flasks to avoid contamination with atmospheric moisture. the gc was equipped with a porapack column of 25 m length with 0.53 mm internal diameter under conditions similar to those specified in coresta. nitrogen at a flow rate of 30 psi was used as the carrier gas with the injection volume and oven temperature being maintained at 2 l and 130, respectively. the carcinogenic potential of the stps was calculated using the method adopted by ayo - yusuf. for which comparable carcinogenic potency data is available in the university of california carcinogenic potency database. the following formulae were used : lifetime cancer risk = adelifetimecpf (1), where adelifetime= lifetime average daily oral exposure (mg / kg bodyweight / d) and cpf = cancer potency factor ((mg / kg bodyweight / d)). now, adelifetime= ade no. of years of snuffing / average lifetime (2), where the ade was calculated assuming the daily consumption of 10 g of stp by an individual for a period of 30 years out of an average lifespan of 70 years. the cpf values for lead and cadmium to be used in formula 1, were obtained from ayo - yusuf. as cadmium : 46.1 ((mg / kg bodyweight / d)) and lead : 0.02 ((mg / kg bodyweight / d)). this method of estimating potential toxicity is based on the assumption that 100% of the toxicant is potentially bioavailable in ideal conditions and can fully contribute to the overall risk of the product. since some literature reports justify the relevance of 6% bioavailability in the context of potential toxicity, this parameter has also been computed. however, all further calculations have been carried out under the assumption of an ideal 100% transfer. the metals for which data was not available in the carcinogenic potency database were compared with their standard permissible daily intake levels in published literature. these reference values for copper, nickel, iron, zinc, and chromium are 50, 20, 250, 300, and 25 g / kg / day, respectively. the tolerable upper intake level of magnesium for 14 - 70 year old individuals as per the national institutes of health is 350 mg / day. the sample preparation procedures adopted to analyse trace metals are based on health canada, centres for disease control and the united states federal registers and modified in - house techniques based on the most up to date information from published literature. all the glassware were washed in distilled deionised water and dried in oven prior to use. three grams of the accurately weighed samples were digested using a 3:1 mixture of concentrated nitric acid and hydrochloric acid over a laboratory grade furnace. the samples were then filtered ; the filtrate washed with distilled deionised water and made up to 100 ml in a standard flask. these samples were subsequently analysed by an atomic absorption spectrometer (gbc avanta, 2 - 0 - 2 version from gbc scientific equipments pvt. ltd., victoria, australia). a mixture of air - acetylene was used as fuel for all the elements except chromium and cadmium for which nitrous oxide - acetylene mixture was used. the air flow was maintained at 10 l / min while the fuel flow was adjusted to 2 l / min. this protocol revolved around the guidelines prescribed by the federal register with slight in - house modifications. the freshly opened samples were extracted with 20 ml distilled water using a mechanical shaker and the clear supernatant was taken for analysis. the samples were analysed in triplicate using a laboratory grade ph meter (digital ph meter m.k 6, systronics, ahmedabad) and the results expressed as mean phsd. the samples were prepared in accordance with cooperation centre for scientific research relative to tobacco (coresta)-recommended method n56 (for karl fischer method) and 57 (for gas chromatography - thermal conductivity detector (gc - tcd) method) with slight in - house modifications in the techniques. moisture content was analysed by karl fischer titrator (mettler toledo model dl18 equipped with mettler ga44 printer and mettler ae200 balance) under conditions specified in coresta. five hundred milligram of the sample was extracted using karl fisher grade anhydrous carbinol and the clear supernatant was used for further analysis. the whole procedure was carried out in sealed flasks to avoid contamination with atmospheric moisture. the gc was equipped with a porapack column of 25 m length with 0.53 mm internal diameter under conditions similar to those specified in coresta. nitrogen at a flow rate of 30 psi was used as the carrier gas with the injection volume and oven temperature being maintained at 2 l and 130, respectively. the carcinogenic potential of the stps was calculated using the method adopted by ayo - yusuf. for which comparable carcinogenic potency data is available in the university of california carcinogenic potency database. the following formulae were used : lifetime cancer risk = adelifetimecpf (1), where adelifetime= lifetime average daily oral exposure (mg / kg bodyweight / d) and cpf = cancer potency factor ((mg / kg bodyweight / d)). now, adelifetime= ade no. of years of snuffing / average lifetime (2), where the ade was calculated assuming the daily consumption of 10 g of stp by an individual for a period of 30 years out of an average lifespan of 70 years. the cpf values for lead and cadmium to be used in formula 1, were obtained from ayo - yusuf. as cadmium : 46.1 ((mg / kg bodyweight / d)) and lead : 0.02 ((mg / kg bodyweight / d)). this method of estimating potential toxicity is based on the assumption that 100% of the toxicant is potentially bioavailable in ideal conditions and can fully contribute to the overall risk of the product. since some literature reports justify the relevance of 6% bioavailability in the context of potential toxicity, this parameter has also been computed. however, all further calculations have been carried out under the assumption of an ideal 100% transfer. the metals for which data was not available in the carcinogenic potency database were compared with their standard permissible daily intake levels in published literature. these reference values for copper, nickel, iron, zinc, and chromium are 50, 20, 250, 300, and 25 g / kg / day, respectively. the tolerable upper intake level of magnesium for 14 - 70 year old individuals as per the national institutes of health is 350 mg / day. the recorded ph of the 10 samples was found to range from 7.180.00 to 10.210.01 with a mean ph of 9.18. sample 5 had the highest value of 10.21 and sample 3 gave the lowest value of 7.18. samples 2, 5, and 8 presented a ph around 10 (table 2). moisture content and ph of stps moisture content of stps is one of the key players influencing the nicotine delivery capacities of the product. in this study a comparison of these results (table 2) clearly indicated some variation in moisture content among the samples. the data from the karl fischer method was taken for further analytical interpretation due to its higher specificity compared to the gc method. the percentage moisture values obtained encompasses a range from 28.81% for sample 9 to 62.90% for sample 2 with a mean of 44.28%. the heavy metal concentrations of the 10 brands of stps expressed in ppm (i.e., g / g) are represented in table 3 as the mean concentration of triplicate readings along with the corresponding standard deviation (sd). cadmium, chromium and nickel represent the group 1 carcinogenic metals analysed in this study. cadmium levels varied between zero in samples 5, 7, 9, and 10 and 1.431.05 ppm in sample 1 with a mean concentration of 0.470.85 ppm. nickel and chromium the iarc has categorized lead and cobalt as group 2 carcinogenic metals with potential threat to humans. the mean concentration of lead across the samples was 3.301.17 ppm with sample 6 exhibiting the highest concentration (5.060.53 ppm). the levels of iron and magnesium in the samples were significantly higher than all the other trace metals estimated, with iron displaying a mean concentration of 684.531.36 ppm and magnesium 6427.641.30 ppm. mineral and heavy metal composition of selected stps by aas the potential toxicity of the 10 stp brands is listed in tables 4 and 5. the average daily oral exposure (ade) and the lifetime cancer risk for lead and cadmium were computed using the mathematical relationships described earlier. while the former was calculated on the basis of the individual consuming 10 g dry weight of the product, the estimation of the latter utilised the underlying assumption that 100% of this consumed product is bioavailable. for metals other than lead and cadmium, the potential toxicity was assessed by comparison with established permissible daily intake values in g / kg / day. assessment of potential toxicity of lead and cadmium in stps assessment of potential toxicity of zinc, nickel, iron, and copper the daily intake of magnesium from the consumption of 10 g of stp per day is represented in table 6. it is pertinent to note here that the dietary intake of these metals is an important consideration in the context of potential toxicity of an individual metal. the contribution from occupational, environmental as well as geographical factors also needs to be quantified and subjected to in - depth analysis with respect to their toxic potential to humans. the total nicotine content is the primary determinant of addiction potential and consumer attractiveness of stps. the relevant component of this net nicotine content is the amount of biologically available unprotonated nicotine which depends on the product ph. a variety of compounds like ammonium bicarbonate, ammonium chloride, ammonium carbonate, sodium bicarbonate, sodium citrate, potassium bicarbonate, calcium bicarbonate, and slaked lime are deliberately added during the curing process of these products to modulate their acidity levels, increase their bioavailable nicotine content and consequently to create a higher addiction potential. unprotonated nicotine is quickly absorbed through the mucosal membranes of the oral cavity and transported to the central nervous system resulting in the instant stimulation that is desired by the habitual snuff dipper. there is a weak correlation between the ph of snuff brands and the extent of oral lesions like leukoplakia, oral tumors, and overall tumor yields as well as the formation of tsnas in the oral cavity. studies indicate that moist snuff shows the highest amount of nicotine while dry snuff, having the least percentage of moisture, also displays the lowest nicotine content. therefore, it can be safely concluded that the samples with higher moisture content will be more addictive in nature due to the presence of a larger amount of orally available free nicotine in them. the region of tobacco cultivation, climatic conditions prevalent therein and soil chemistry as well as ph are all important in determining the extent and rate of metal accumulation in plants. many metal ions, in their capacity as components of metalloenzymes and metalloproteins, play a significant role in regulating the normal physiological functions of the body. the acceptable daily maximal levels of some of these metals are as follows : iron 8 - 18 mg, manganese 1.8 - 2.3 mg, copper 0.9 mg, zinc 8 - 11 mg, and nickel 0.5 mg. lead has been known to increase the rate of hemolysis, suppress cognitive development, cause renal tumors, hypertension, cardiovascular diseases, and negatively impact the male reproductive system. the food and agricultural association / world health organization expert panel on food additives has established a provisional tolerable daily intake value of 3.57 g of lead per kg of body weight. in this context, all the samples with the exception of samples 9 and 10 are found to contain a very high concentration of lead that ranges from about one to seven times the tolerable limit. this data clearly indicates a deleterious effect on the health of the users upon long - term intake of these products. apart from its carcinogenic potential vis - - vis pancreatic cancer, animal studies have shown that cadmium can cause renal failure, placental necrosis, hypertension, anemia, hepatic damage, osteomalacia, testicular tumors, pulmonary edema, emphysema, and induce deficiencies of other essential minerals like iron, zinc, and copper. the reference daily dose of cadmium for chronic oral ingestion leading to proteinuria is 0.5 g / kg. assuming that a 70 kg individual daily consumes 10 g of stp containing 1000 ng cadmium / g of the product, it can result in an overall exposure of 0.6 g / kg bodyweight / d which is effectively higher than the reference dose for causing proteinuria. the food and drug administration (fda) has set 55 g as the tolerable daily intake of cadmium per individual ; however, none of the samples studied exceeded this limit. although iron and copper are essential minerals for humans, a chronic exposure to iron can cause iron oxide deposition in parkinson patients while high levels of copper have been shown to cause liver damage. in this study, none of the samples was found to exceed the recommended daily intake levels of copper and iron. however, when this data is coupled with accumulation from environmental, occupational and/or geographic sources, the net bioavailable concentration of these trace metals will be much above the permissible limits resulting in toxic effects. it also suppresses immune functions and reduces the high density lipoprotein (hdl) levels in the body. a beneficial effect attributed to zinc is its ability to lower bouts of depression by about 15% for every gram consumed per day. zinc concentration in the stp samples analysed was also found to be below the stipulated daily upper intake levels. consideration of the other accumulation factors mentioned above will, however, lead to greater toxicity, especially in high - risk populations. magnesium has been found to have beneficial roles in reducing hypertension as well as in improving the insulin sensitivity and lipid profile in patients at risk of cardiovascular diseases ; however, a chronic exposure can cause hypermagnesemia and other related toxic effects. table 6 clearly indicates that none of the samples have exceeded the permissible limits of magnesium. again it needs to be noted that stp consumption can increase the risk of magnesium overdose and toxicity when the contributions from other environmental and dietary sources are also considered. the lead and cadmium based total lifetime cancer risk calculated as per the united states environment protection agency (usepa) guidelines ranges from 85.17 for sample 8 to 282.38 for sample 1 with a mean value of 93.31. this is about 18 lac (1.8 million) times higher than the minimum usepa - stipulated target of 5.05e5 for potentially hazardous substances. it is also of significant concern that this risk is besides that posed by the ingestion of carcinogenic materials including other metals and tsnas that has already been reported. metallic contamination of drinking water, soil, air, and food (ecosystem products like fish and grains) may be much higher in areas in the vicinities of mining sites, smelters, and hazardous waste sites. consequently, stp users originating from such high - risk geographical regions may be under an elevated threat of accumulated chronic metal toxicity. it has also been confirmed that natural sources including aquifers are a potentially major source of ground water contamination that again increases the risk of cumulative toxicity from stp use. in the event of exposure to a metallic mixture each component of the mixture may then affect a target organ or a particular system in an unpredictable manner that compounds the risk associated with such an exposure and may even lead to complete organ failure. the results of this analysis point to toxic levels of lead and estimate a prolonged cancer risk associated with the stps under consideration. this warrants an in - depth analysis of other brands of stps that are manufactured and marketed in the south indian states. the trace metal concentration can become a health risk when the additive toxicity potential from dietary as well as miscellaneous environmental exposure is taken into consideration. considering all these facts the authors feel that the production of stps should be coupled with thorough quality control protocols as per international guidelines and that their marketing be under stricter government control. furthermore, marketing strategies like advocating stp as an alternative to cigarette smoking, including stps in smoking cessation programs and promoting stps in areas where smoking is prohibited need to be critically reevaluated in the light of the present results. this study may also act as an impetus for further research with a much larger sample size representative of the indian retail market as well as focussing more on the carcinogenic nitrosamines and other identified mutagens. | the characterization and classification of smokeless tobacco products has been a continuously evolving process. this is based on a number of different parameters like nicotine content, moisture content, amount of heavy metals, ph, and in vitro cytotoxicity assays. their contexts often vary between countries, research institutions, and legal requirements. the categorisation of these products is quite challenging due to the diffused sample sizes, diverse array of branded products on offer, and the absence of a centralized manufacturing facility. this study aims at a systematic classification of 10 smokeless tobacco product samples from the retail market based on their potential toxicity upon long - term use. the estimation of potential toxicity follows a well - established method that employs the concentration of toxic metals in the different samples. the potential toxicity as well as heavy metal concentrations of the smokeless tobacco products analysed was found to be much higher than acceptable limits. for instance, the levels of lead, cadmium, copper and zinc of 2.5, 1, 4 and 23 ppm, respectively, are well above their recommended limits. the results from the study indicate that chronic use of smokeless tobacco products is a significant health risk, especially in the vulnerable population. further studies of this nature will help establish a toxicological fingerprint on the diverse class of products that floods the market now. |
the first nobel prize in physiology or medicine, like those in chemistry, physics, literature and peace, was awarded in 1901. indeed, the twentieth century can be regarded as the century of the nobel prize. from the very outset, however, nobel prizes in general, and the one in physiology or medicine in particular, have been plagued by what can be referred to as a basic tension between reality and ideal. by singling out individual scientists as paragons of scientific achievement in their fields, the nobel prize seems to endorse the idea of a solitary researcher making his or her one great discovery or invention, to the benefit of mankind, as it is stated in alfred nobel s will. although from the 1950s onwards nobel prizes in physiology or medicine have usually been awarded to three persons rather than one, as had been the common rule during the first half of the century, these three persons are still usually seen as individual scientific heavy weights working in a more or less independent and researcher - driven fashion, rather than as science workers firmly embedded in extended research networks or consortia. whereas solitude, perseverance, creativity and flashing insights are bound to remain basic ingredients of scientific discovery, the archetypical idea of the scientific hero seems nonetheless increasingly at odds with the way in which research is actually conducted. and although (as will be pointed out below) this basic tension is as old as the nobel prize in physiology or medicine itself, it has become increasingly problematic as the research practices involved continue to expand in terms of pace, complexity and scale. this intricate issue can be addressed from various angles : history of science, sociology of science, science ethics, etc. in this contribution, i intend to address it from a normative perspective, as a quandary of contemporary science ethics, thereby regarding the awarding of nobel prizes first and foremost as a moral issue. the nobel prize is considered by many as the acme of acknowledgement, and, as merton and others have pointed out, acknowledgement (in various formats, ranging from citations, chairs and appointments up to international prizes) is crucially important in science. but as such, it is also likely to be highly controversial, raising a host of normative issues and deliberations in terms of transparency and fairness, both on a general level and with respect to specific cases. moreover, by being awarded a nobel prize, the researchers involved are singled out as models or examples for others, as exemplary scientists setting a standard, not only in terms of the discoveries or inventions they actually made, but also in terms of crucial scientific values they came to embody such as disinterestedness, reliability, honesty, meticulousness and the like. or, to formulate it in a negative vein : should a nobel prize winner be exposed as a dishonest person and a fraud, science as such would be in danger of seeing its credibility diminished. awarding the prize on the basis of a particular achievement, a groundbreaking experiment or publication, conveys the message that this is how science ought to be done. and this explains why the nobel prize in physiology or medicine has stirred such a plethora of normative controversies and why the actual decisions made seem to become increasingly contested over the years : the awarding of such prizes involves more than counting citations or determining an author s h - factor. it entails a normative statement concerning the value and values involved in a highly dynamical and rapidly evolving phenomenon called science. the type of considerations for selecting and evaluating candidates are bound to change as science develops over time. as robert friedman phrases it, success or failure in winning [the prize ] has not depended upon timeless, fixed standards of excellence. rather, the changing priorities and agendas of committee members, as well as their comprehension of scientific accomplishment, have been critical yet, although my paper will basically take a science ethics perspective, sociological and historical analyses are crucially important when it comes to providing the input for a normative assessment. ethical considerations have to build and critically reflect on empirical analyses of how practices of knowledge production are actually evolving and how controversies and dilemmas concerning the nobel prize in physiology or medicine have actually been dealt with. moreover, rather than addressing this issue on a general level, i will focus on a particular case study, namely the human genome project (hgp). should the sequencing of the human genome be awarded by the nobel prize and, if so, who should be the (one, two or three) persons to receive it ? by focussing on this case study i intend to address in a concrete manner the more general question : to what extent can the nobel prize in physiology or medicine still be regarded as a fair and convincing mechanism of reward in an era of anonymity, global networks, multiple authorship, privatepublic partnerships and big science. the hgp will certainly present an interesting but also a difficult case for nobel prize committees to deal with. the sequencing of the human genome is regarded by many as one of the major scientific highlights in recent science history. and it may be seen as highly symbolic perhaps that the human genome sequence was published in 2001, exactly 100 years after the first nobel prizes were awarded (international human genome sequencing consortium [ihgsc ] 2001). moreover, from the very outset, the hgp has generated a plethora of claims concerning the benefits for humankind that are expected to result from it in terms of health, sustainability and empowerment. thus, from the very start, this project, the final conclusion as it were of a long journey that began with the re - discovery of mendel in 1900 and the disclosure of the structure by dna by watson and crick in 1953 (watson and crick 1953), has raised the question whether a nobel prize should be attached to it not a regular one, moreover, but rather a kind of mega - nobel prize, the nobel prize of nobel prizes, and perhaps even more than just one. the history of the hgp is more or less haunted by this question and the likelihood that some of its key protagonists would one day receive a phone call from stockholm has been an issue of speculation and dispute on various occasions. indeed, protagonists such as craig venter consciously tailored their publication policies so as to increase the likelihood of one day earning the biggest prize in science (shreeve 2004). moreover, the hgp is regarded as the flagship project of genomics as an emerging technoscientific field, an endeavour claimed to have irreversibly and fundamentally changed the way in which research in the life sciences is done (collins. another important reason for focussing the discussion on the hgp is that more than any other recent achievement in science, it exemplifies the current transformations that are taking place in the way in which scientific knowledge is produced. the question basically is what these transformations this scientific revolution that is clearly connected with the emergence of big science imply (from a normative perspective) for the scientific individual. the idea of a nobel prize presupposes that a major scientific feat can still be meaningfully attributed to the talents and commitment of one, two or three concrete individuals at most. the era of big science, as exemplified by the hgp, raises the question to what extent this presupposition is still feasible. the big science concept builds on the scientometric observation, put forward by de solla price (1963) and others, that there is a tendency in modern research towards exponential growth, regardless of whether this refers to the number of researchers, author names, publications, journals, journal articles, citations or any other quantitative indicator. as all these indicators display the tendency to double at regular intervals, scientific inquiry has by now evolved into a rather massive phenomenon, and the archetypical image of the solitary researcher increasingly seems to become marginalised as a relic from the past. moreover, the big science concept not only refers to the actual number of researchers working and collaborating within a particular field, but also to the increased dependence of current research on massive, expensive and sophisticated technologies, as exemplified by the particle collider at cern, but also by the automated sequencing machines of genomics research, involving large - scale investments and sophisticated management structures. publications in particle physics in which cern findings are reported may have several hundred authors, and biology is now moving in the same direction with the advent of the industrial - scale work required for sequencing genomes (bishop 2003). as the number of individuals responsible for single breakthroughs in scientific research has gradually increased, so too has the sentiment that a limit of three recipients for each prize may be too restrictive. whereas most discoveries in modern science arise from the efforts of multiple individuals, no more than three individuals can receive the prize in each category. in fact, the current limit of three for each prize is itself a compromise, representing a revision of nobel s original bequest, which speaks of only one recipient per prize. the nobel foundation does not seem inclined to move in this direction (bishop 2003, 24). in what manner does the emergence of big science undermine the credibility of policies of individual recognition as such ? in this paper i will argue that, as we experience a period of increase in scale and pace, of globalisation of scientific effort, individual researchers increasingly tend to operate in the context of massive knowledge networks., this does not mean that the ethical dimension of individual commitment is being erased altogether, quite the contrary. rather, i see it as a challenge for contemporary science ethics to address the novel ethical problems and dilemmas arising in such complex, competitive and large - scale research environments in a convincing way (zwart 2008a). this implies that, besides traditional values such as autonomy and perseverance in the face of adverse external pressures, academic excellence must increasingly involve other virtues and values as well, such as transparency, fairness and a communicative attitude towards the outside world. and the nobel prize, as a highly visible mechanism of acknowledgement in science, should reflect and acknowledge this. but before turning to the present, allow me to briefly browse through the nobel prize archives to see how this dilemma has been dealt with in the past. the responsibility for awarding the nobel prize in physiology or medicine lies with the karolinska institute in stockholm, but the details of nomination, evaluation and selection are a well preserved secret (feldman 2000). the statutes of the nobel foundation provide for strict secrecy and minutes from committee meetings are non - existent, but we may still get to know something every now and then (http://nobelprize.org ; friedman 2001). letters of correspondence and all letters of nomination since 1901 are kept in the committee s archive, while reports from the committee s advisers have been printed in separate internal volumes for each decade. after 50 years the director of the norwegian nobel institute may give access to these archival sources, primarily for the purpose of historical research. in 1976, for instance, the nobel foundation opened its archives to researchers up to the year 1950 (friedman 2001). these intriguing files and sources indicate that the tension outlined above has been haunting nobel prize procedures from the very start, as exemplified by the case of the russian physiologist ivan pavlov. in four successive years (1901, 1902, 1903, 1904) pavlov was nominated for the nobel prize in physiology or medicine, and each time the award committee confronted the same question : to what extent were the products of pavlov s laboratory truly pavlov s ? the nominee had himself pronounced that his most substantial work, lectures on the work of the main digestive glands (1897), was the achievement of his entire laboratory. he had credited his co - workers for conducting the experiments on which it was based. furthermore, he referred readers seeking evidence for his arguments to their publications. did pavlov s major work, on which his nomination was based, represent his own original contributions to science, or was it merely a compilation of the experimental dissertations ? (todes 2002, xiii). apparently endorsing the archetypical image of the scientist as a heroic lone investigator, the nobel prize committee was now confronted with a more or less novel and apparently somewhat aberrant form of scientific knowledge production. pavlov was actually a research manager rather than a solitary researcher, and his laboratory was a factory, producing series of knowledge claims in a systematic fashion, constituting something of a knowledge production line, rather than a small - scale workshop. although pavlov designed most of the trials and presented the research results in books, papers and lectures, the actual experiments were conducted by the praktikanti working in pavlov s research facilities, hoping to complete their medical education in this manner. still, the tension between the somewhat romantic image of the researcher as an individual, about to make his one key discovery, his highly personal contribution to the benefit of mankind, and the way in which scientific knowledge claims came to be produced by academic professionals in the course of the twentieth century, is bound to increase even further with the emergence of big science as exemplified by the hgp. indeed, one century later, in 2000 and 2001, a similar dilemma presented itself, but on an even grander scale. on june 26 2000, president clinton, together with francis collins (director of the international human genome sequencing consortium) and craig venter (his self - proclaimed rival, representing the privately owned celera company), announced at a press conference that the massive effort to sequence the human genome was reaching its completion. in 2001, both teams published their results in a co - ordinated fashion, through milestone articles in nature and science respectively (ihgsc 2001, venter. authors, and venter s publication 285. these very numbers already indicate that big science as a phenomenon had reached the life sciences by now. as indicated above, cern publications in the field of high energy physics already display such tendencies towards multiple authorship, but for the life sciences, where the bulk of academic research still tended to be conducted on the basis of individual research grants, this was something of a novelty. both the press conference and the two key publications significantly fuelled the debate over the question whether this achievement should merit a nobel prize and, more complicated even, who should be the person or persons to receive it ? shortly after the nature publication, at a follow - up press conference in san francisco organised during a meeting of the american association for the advancement of science (aaas) on february 18 2001, francis collins was explicitly asked whether the sequencing of the human genome warranted the nobel prize. in his (now famous) reply he stated that it would have to be given to 3,492 people to properly recognize everyone who had significantly contributed to this common effort (davies 2002, 266). this attitude of humility and collectivism was already conveyed by the opening pages of the nature publication itself, where collins was listed simply as one author among many, allowing his colleague eric lander (who in fact had done most of the actual writing) to be the first name on the list. in the case of venter, he put his own name first and at various occasions explicitly considered the likelihood that some of his highly cited key publications, notably the one on the human genome, might bring him the nobel prize some day. both collins and venter, however, have subsequently published memoirs containing extensive reflections on their human genome years (collins 2006, venter 2007) and both documents make it abundantly clear how problematic it would be to give credit for the human genome sequencing effort to one, two or three individuals only. although at crucial moments individual initiatives, personalities and eureka - like experiences of enlightenment remain undoubtedly important, life science research in the genomics era as such has irrevocably grown into a large - scale, collective endeavour. if a nobel prize is to be awarded for deciphering the human genome, therefore, it is difficult to see how this can be done in a manner that is both meaningful and fair. as robert cook - deegan phrases it : the final truth is that no individual can take full credit (1995, 71). moreover, he argues that nobel selection committees are perpetually unfair in conferring a prize on winners in science ignoring the way science has changed so that most major advances require the efforts of hundreds of researchers, not one or two (ibid.). when it comes to awarding the nobel prize somewhere in the near future, the human genome sequencing effort seems impossible to ignore. on the other hand it seems equally impossible to single out even a limited number of recipients in a convincing way. since merton we are familiar with the idea that scientists are much more interested in symbolic expressions of acknowledgement than in more mundane forms of reimbursement such as money. in order to organize acknowledgement, moreover, a number of mechanisms have been put in place such as citation indices, invited lectures, nature covers and, at the very summit of the acknowledgement pyramid, the nobel prize. yet, some of these acknowledgement mechanisms apparently stem from more or less outdated views on how science works. nobel s bequest, spelled out in a single handwritten paragraph, seems to convey the idea of solitary researchers who, at a certain point during their long journey, have this one grand idea that will not only further science, but will also bring significant benefits to society. and indeed, discoveries of this type have existed and will no doubt continue to exist. the discovery of the structure of dna by watson and crick may to a certain extent be seen in this manner, namely as a key discovery attributable to discrete individuals who embarked on a research effort of their own design. although even in this case the role of co - discoverers incited much controversy, notably the question of whether the role of rosalind franklin had been duly acknowledged, the consensus gradually seems to have emerged that, although the work of chargaff, franklin and others had been pivotally important, watson and crick were nonetheless the ones who, at the crucial moment, choose the right track and made the final decisive steps (maddox 2002). yet, the overall picture seems to be that the tension between the basic image to which the nobel prize still tends to adhere and the actual practices of knowledge production as they currently evolve, continues to increase. this tension causes similar problems of course for other mechanisms of acknowledgement as well, such as citation indices (wouters 1999, cf. can a citation index be regarded as a reliable indicator when it comes to assessing the academic quality and impact of individuals or research groups ? robert merton himself emphasized the lack of fairness in citation practices when he described what he referred to as the matthew effect in science (merton 1988, cf. this concept builds on a famous saying borrowed from the gospels : for unto everyone that hath shall be given, and he shall have in abundance ; but from him that hath not shall be taken away even what he hath (mt 13:12). in contemporary language : those of us who have are bound to receive even more, while the have - nots will become even more deprived. in terms of citation indices this means that most articles published by scientists will be cited only a few times, and then they will be forgotten completely, as if they had never been written. indeed, most authors will be read and cited by only a limited number of readers, and eventually they will be ignored more or less for ever. some articles, however, will be cited more often and the number of citations may even reach a certain critical limit. beyond that point, the number of citations is bound to increase dramatically and exponentially. colleagues will continue to cite them for 20 or 30 years, until the paradigm to which the publication belongs expires. thus, a limited number of authors may publish articles that really allow them to make their name, although the time and effort spent on writing them may not significantly exceed the amount of time and effort spent on publications that are treated less respectfully. if you want to become academically famous, invent a concept, a test, or identify a new disease to which your name may become attached. functionalsimply a convenient way for referring to tests, concepts, illnesses or bodily parts it works. hardly anyone who refers to the stroop test or stroop effect nowadays, will know anything about the individual bearing the surname stroop. hardly anyone when it comes to defining the contribution made by individual scientists to progress in science, a series of trends can be identified. initially, individuality was the focus of attention. historians and other scholars studying science tended to see scientific progress as the achievement of a limited number of heroes of science, a mere handful of great men. an exemplification of this genre is the book groe mnner, published by nobel prize winner wilhelm ostwald in 1909. the author was an outstanding physical chemist who, later in life, became interested in the history of his field. in this book he describes and analyses the life stories of six prominent scientists of the nineteenth century, all of them male. indeed, ostwald explicitly states that, although examples of female researchers such as madame curie do exist, they are the exception to the rule the rule being that, basically, scientific research is the work of a limited number of very great men. another interesting example is paul de kruif s bestseller the microbe hunters published in 1927 and devoted to scientific heroes such as louis pasteur and robert koch the latter received the nobel prize in 1905, whereas the former died in 1895, six years before the nobel prizes began to be awarded. he published on streptococci and worked at the rockefeller institute until he was fired after publishing an anonymous, critical review of contemporary medical research. he was co - author, but not duly acknowledged as such, of sinclair lewis novel arrowsmith, published in 1925, about a research institute clearly modelled after the rockefeller institute. critics sometimes argue that in his narratives de kruif relied too much on his imagination and enthusiasm for science, but two successful hollywood movies and one successful broadway play were based on microbe hunters, his most famous book. de kruif s lively and readable account presents a rather supportive and protagonist - oriented portrayal of scientists as heroes, emphatically emphasizing the dramatic element inherent in experimental inquiry (zwart 2004). alfred nobel s will, although extremely concise compared to the publications by ostwald and de kruif, seems to convey a similar view on progress in science : it is the epoch - making work of outstanding individuals who, because of one decisive feat that actually represents a life of tenacious effort, manage to contribute significantly and exceptionally to human knowledge and wellbeing. from the 1970s and 1980s onwards, after the decline of existentialism so to speak, scepticism concerning the role of individual heroes quickly began to spread. within the domain of science studies (broadly defined) there has been a conscious shift away from studying the work of individual scientists towards analysing networks, discourses and structures (shortland and yeo 1996). science was no longer seen as the achievement of a limited number of great men (lenard 1933). rather, knowledge claims were now regarded as being produced by networks of more or less anonymous actors, so that any desire to focus on prominent individuals tended to be regarded with suspicion. this trend is exemplified by bruno latour s monograph entitled the pasteurisation of france (1984/1988), which has been referred to as a hamlet without hamlet, since the hero whose name is referred to in the title is virtually absent in the book (shortland and yeo 1996). science studies seemed to proclaim what michel foucault referred to as the death of the author - as - an - individual. according to foucault, the history of scientific authorship displays a definite shift away from grand authoritative names (such as aristotle) as indices of genius and truth towards a purely functional form of authorship, where an author s name predominantly serves as, for instance, a search item in the context of information retrieval in pubmed and similar sources. and insofar authorship has become merely functional, various reward mechanisms, even nobel prizes, may perhaps be seen as predominantly functional as well, as techniques employed in the context of performance assessment of research groups or universities. james watson then means that the scientific establishment in place actually promotes a particular interpretation of what the biomedical life sciences are (namely that they should be regarded as more or less identical with molecular biology), at the expense of other possible interpretations. and indeed, nobel laureates such as watson are very powerful figures in science, deploying laureate status and other achievements to assume pivotal roles in processes of agenda - setting, watson for instance in his role as the first director of the hgp (as collins predecessor). although it is important, of course, to be aware of the pitfalls of hero worship in science, and although the social and political dimensions of science (the structures, networks, institutions and power plays involved) are crucially important when it comes to understanding science as a real - life phenomenon, i believe that time has come to reconsider and re - acknowledge the role played by the individual in scientific research. if we want to understand and assess the dynamics of scientific progress, attention should be paid to the micro - level, the level of individual activity as well (zwart 2008b). after focussing on scientists as heroes (at the expense of social context), and after subsequently dismissing the individual dimension from science studies altogether (in favour of a more sociological, structural or science politics approach), i would like to argue that it is important to pay due attention again to the dimension of individuality as well, notably when it comes to addressing issues of normativity. to phrase it in a foucauldian manner : science must be studied not on the level of science politics or epistemic communities only, but also on the level of the self. the individual is the place where transformations of knowledge production become lively and concrete. how do individuals position themselves as responsible agents in the face of major transformations in knowledge production and technoscientific change ? in other words, what i advocate is not a reframing of science ethics in accordance with the archetypical image of the solitary researcher as a hero, whose heroism notably resides in his willingness to stubbornly oppose (rather than interactively endorse) the forces of collectivism. quite the contrary, the emergence of big science has irrevocably reinforced the shift from researcher - driven research, conducted by autonomous, more or less free - floating individuals, to top - down programmatic efforts involving relatively large numbers of (more or less anonymous) science workers. this basic shift, from autonomy to anonymity, challenges and changes the meaning of research in general and of scientific authorship in particular, but does not erase the dimension of the individual self altogether. to further elaborate this issue, i will build on a line of thinking that is often somewhat neglected in mainstream science ethics, namely the type of thinking about research and normativity exemplified by authors such as nietzsche, weber, foucault and sloterdijk. nietzsche, for instance, whose talent for anticipation can hardly be questioned, already discerned that normal modern science is bound to entail the replacement of exceptional heroes by armies of anonymous individuals (1980, 547). according to nietzsche, however, a true scientist will endorse rather than deplore this anonymity as inevitable. for nietzsche, a true scientist is not only someone who is willing to put his theories to the test, remaining susceptible to criticism, continuously on the alert not to deceive himself ; for nietzsche, the most important scientific virtue of all is self - denial. it is not me that counts ! for nietzsche, this phrase articulates the core of the scientific ethos, the quintessence of being in science his view was taken up many years later by michel foucault who articulated his own version of the same idea : quimporte qui parle ? for foucault, the most fundamental ethical principle of contemporary scientific discourse resides in a basic indifference towards the issue of authorship (1994, 789 ; cf. science is, first and foremost, a discursive phenomenon in which author names serve as functional tools, notably in the context of information retrieval as we have seen. in normal science, academic authorship comes very close to anonymity, and there is a certain moral quality in the stoical acceptance of this fact. and indeed, it is in this vein that many nobel laureates have written in retrospect about their prize. j. michael bishop s how to win the nobel prize may serve as an example here, conveying a basic attitude of unobtrusiveness, for instance in the following sentences inserted right at the beginning of his account : i felt less than fully deserving, because the discovery for which harold [varmus ] and i were being honored was only in modest part of my own making etc. yet, this does not delete the dimension of individuality altogether far from it. rather, self - denial or unobtrusiveness are particular styles or modes for positioning oneself as a scientific individual. following nietzsche s lead in this, foucault (1984, 1994), sloterdijk (2010) and others have argued (as nietzsche already did in more or less similar terms) that scientific research may be regarded as a kind of practice of the self, a form of moral self - edification for the individuals involved. through training and intellectual asceticism, scientists gradually transform themselves, not only into highly reliable sources of information, but also into pioneers who, in the folds and margins of established discourse, are able to enter new terrains, to experiment with new techniques and thus to open up novel perspectives. moreover, these authors emphasise that, although the conditions for scientific research and academic authorship have clearly changed, the axis of the self continues to constitute a pivotal dimension of discourse production. in other words, besides self - denial (i.e. the generous affirmation by scientists of their anonymity and fundamental dependence on others), there is another side to seeing scientific research as a practice of the self, namely the inherent strive towards self - improvement, the basic will to challenge established conventions and the readiness to face new dawns. this means that in science, individual excellence is achieved by those who, through constant training and permanent re - education, remain eager and willing to acquire new vocabularies and skills. those who see novel fields as test - beds and experimental settings, not only in the scientific sense, but also for exploring new dilemmas and trying out new ways of addressing normative issues. and besides the willingness to learn to use new tools, such as ict equipment or new computational techniques, it also implies the willingness to become adept in novel practices and fluent in novel professional vocabularies. important challenges facing scientific individuals in the big science era notably emerge in areas of management and communication. they must acquire the skills not only for governance of the self, in order to establish themselves as a reliable and meticulous individual, as was already the case in the era of the lone scientific individual, but also for the governance of increasing numbers of academic others. whereas traditionally science ethics tended to focus on the dilemmas of autonomous decision - making processes by researchers as individuals (micro - ethics), the new era of big science calls for an ethic that addresses social and political dimensions as well (macro - ethics). subsequently, one could argue that, in order to remain in line with these developments, the nobel prize will gradually have to evolve into a mechanism of acknowledgement for novel types of excellence, namely the excellence of scientists who, at a certain point in their career, successfully transform themselves from outstanding individual researchers into visible, accountable and communicative research managers. the question then remains whether those to whom this applies deserve to be singled out and credited, while the great majority of their devoted colleagues are bound to remain anonymous ? does the desire for individual acknowledgement still make sense ? in the movie casablanca there is a famous and intriguing scene. at a certain point humphrey bogart, albeit with a slightly cynical undertone in his voice, complements him with his achievements. the hero, assuming a quasi - humble posture, replies by saying that he simply tries to make a contribution, like so many other people do. although many individuals are more or less committed, some individuals happen to make a contribution that is more decisive than those of others. in various publications and seminars the french psychoanalyst jacques lacan argued that, rather than money, survival, sexual gratification or big automobiles, acknowledgement is what we are really after. acknowledgement is our basic desire, fuelling creativity and perseverance, even under hazardous conditions, and science continues to rely on individuals who are willing to display this type of behaviour. mechanisms of acknowledgement are important elements in what lacan refers to as the symbolic order., the traditional freudian idea had been that research is a kind of sublimation. in the face of societal constraints, individuals at a certain point decide to invest their libido in activities other than sex and reproduction. for freud, however, research remains a detour for individuals on their way to sexual intercourse and parenthood, their ultimate destination. a high citation index is an important gratification, a rewarding source of pleasure in itself. one of the pitfalls for scholars is what lacan would refer to as imaginary recognition : acknowledgement by a limited number of close followers or friends nearby ; the kind of recognition that typifies sectarism. that is why anonymity, globalisation and quantification are so important when it comes to defining performance indicators. it means that we are no longer dependent on the fragile benevolence (usually based on reciprocity rather than true merit) of those individuals who happen to constitute our immediate academic umwelt. thus, recognition is the symbolic bread we as researchers and scholars live by, and this implies that the nobel prize, rather than having become a ritual devoid of meaning or a relic from the past, still has to be taken quite seriously as an acme of symbolic acknowledgement. yet, building on what has been argued above, time has come to reconsider our basic mechanisms of acknowledgement in order to determine whether they still do justice to the way in which top scientists nowadays have to function. every now and then it is important to update and, if necessary, adjust our ways of operationalizing and valuing excellence. typical lab sites are not the only places where creativity becomes decisive and grand ideas flash up. this may also happen at airports and international conferences, or during board meetings and committee gatherings. the big names in contemporary life sciences, the pioneers and agenda - setters, the first authors of landmark publications, are often heavily involved in acquiring large - scale funding and in management of science. the role of the scientific research manager has become a pivotal one, not only in terms of making discoveries and discerning their importance, but also in assessing and addressing the complex ethical, political and strategic issues emerging in contemporary research. besides a track record in laboratory research, scientists in positions of authority must develop new moral virtues besides the traditional ones connected with bench work, such as meticulousness, trustworthiness and selflessness. it is their challenge to become the visionaries of contemporary science : articulating thick views on what is happening in current research and what this may imply for our understanding of ourselves and nature, as well as for societies of the future ; developing seismographic sensitivities towards important trends and promising developments. they are also the ones in a position to ensure that, as alfred nobel once stated it, expensive, large - scale research endeavours contribute sufficiently to the benefit of mankind. in short, science in the big science era it might be compared to playing a variety of different games on different boards simultaneously, with each game having its own standards of excellence, its own morale. is has become impossible to assess scientific performance (or excellence, as the current jargon calls it) on the basis of a single coherent set of criteria. this heterogeneity was already present in nobel s will, where both academic excellence and societal relevance were regarded as important. nobel prize winners of the present and near future are bound to excel on an even broader spectrum. they will have to be virtuosi of heterogeneity, able to perform outstandingly in a broad range of complex and controversial settings. let me now apply these considerations to the case study at hand, the hgp. it is a complicated file no doubt, but also a timely one, given the fact that, ten years after its completion, the subdued debate is now bound to become acute once again. in his one - page testament, alfred nobel stipulated that the funds involved should go to outstanding research achievements (the most important discovery within the domain of physiology or medicine) which during the preceding year had conferred the greatest benefit on mankind. however, the criterion of promptitude explicitly mentioned in the bequest (during the preceding year) has been dropped. as a rule, the prize is nowadays given to a contribution made something like a decade before. by this time thus, 2010 would be an opportune year for a hgp nobel prize to be awarded. moreover, as was argued above, the hgp exemplifies the emergence of big science in the life sciences, with all the ingredients this involves, ranging from high visibility and lofty societal expectations up to multiple authorship and the intricacies of privatepublic funding. thus, the hgp seems to constitute a perfect test case for the nobel prize as a mechanism of acknowledgment in the era of big life science. still, if the nobel prize committee should want to reward the hgp with a nobel prize, in view of its scientific and societal significance, how are they to identify the one, two, or three individuals who deserve to be singled out, who may be credited for this achievement ? the first stipulation to consider, no doubt, is whether the hgp has produced significant benefit for humankind. from the very outset, the hgp has been presented as a milestone in the history of both science and humanity. while the project in its early days was often compared to landing on the moon, the societal prospects it opened up were fleshed out in an increasingly detailed manner as the project continued to evolve. as collins told cnn during a famous interview, it is hard to overstate the importance of reading our own instruction book. yet, countless critics have argued that the societal relevance of the hgp is far from clear as yet. although genomics has produced an avalanche of bioinformation, concrete social benefits are still sparse compared to the grand promises that have been made at various occasions, such as the claim uttered during the hgp press conference (june 26 2000) that it is now conceivable that our children s children will know the term cancer only as a constellation of stars (http://www.genome.gov/10001356). let us, however, for the sake of the argument, give the hgp the benefit of the doubt in this respect, so that we may focus on the key issue of this article : the extent to which scientific achievement in the contemporary biosciences, such as the hgp, can still be meaningfully attributed to individuals, even if, as was already indicated above, nobel prizes in physiology or medicine are now typically given to three researchers rather than one. i will argue that, for a number of reasons, a hgp nobel prize (in 2010 or so) would still make sense. but how to select an acceptable and credible set of candidates ? one source of information concerning track record and practices of the self of outstanding scientists are biographies and autobiographies, notably the latter. in recent years, a stream of autobiographical accounts has been published concerning the history of the human genome sequencing effort, such as collins (2006), crick (1988), hood (2002), sanger (1988), shreeve (2004), sulston and ferry (2002/2003), venter (2007) and watson (2000), but the list will no doubt continue to expand. these are, if anything, moral documents, devoted to self - assessment, self - criticism and self - justification. in virtually all of them, for instance, issues of agency and responsibility are explicitly addressed. to what extent can individuals really be seen as authors of their scientific lives, as autonomous decision - makers ? rather than presenting themselves as heroes of science, in full control of the events, even highly visible scientists such as sulston, collins and venter emphasize (albeit in terms of their own personal vocabularies) how they see themselves as team workers, as products even, rather than as initiators describing in a lively manner how they, notably at crucial moments, had the experience of being pulled, swept or driven by events, the outcomes of which were often impossible to predict from an individual perspective. their autobiographies describe complex processes of interaction, involving both intricate social dynamics and individual initiatives, and we can not say that primacy is given to the latter. the grand efforts these authors were officially heading are described in terms of unpredictability, uncontrollability sheer chaos even rather than as exemplifications of top - down, management - driven planning and control. moreover, after reading the reminiscences of sulston, collins and venter one is bound to realize that, although their role was important, it was limited as well. after reading venter s autobiography, for instance, in combination with shreeve s history (2004) of the companies (tigr and celera) he headed, it is clear that in various respects, researchers like gene myers, mark adams or hamilton smith were at least as important in terms of decisive scientific contributions as was venter himself. thus, eventually, individual contributions must become contextualized again in a more comprehensive view of science as team or network work. still, i want to argue that, eventually notwithstanding the astonishing scale and complexity of contemporary research efforts, notably in the life - sciences individuals can make a difference at times (notably at crucial moments), and that these decisive contributions are meaningfully attributable as well. when francis collins was appointed director of the hgp in 1993, he already had an impressive track record as a gene hunter. he had made a name for himself by developing gene - finding methods such as positional cloning and chromosome jumping, and by discovering the location of three important disease genes, namely those responsible for cystic fibrosis, neurofibromatosis and huntington s disease (collins. thus, assessed in terms of more or less traditional criteria, focusing on single individuals as researchers, a nomination for the nobel prize would already make sense. yet, in subsequent stages of his career, he was willing and able to develop complementary skills as well, and to excel in other fields. after successfully taking the lead in the human genome venture, he was recently appointed as director of the national institutes of health (nih), an acknowledgement of his managerial performance. moreover, he presented series of lectures on the societal aspects of genomics and significantly contributed to debates on this issue through interviews and panel discussions. thus, besides academic research papers, he also published or co - authored a number of influential papers on the prospects of genomics for society (collins 1999)the famous benefits for mankind to which alfred nobel s will refers. or take the case of craig j. venter. much earlier than most of his competitors, he acknowledged and understood the importance of automated sequencing and some of the specific methodologies involved in this, such as the est (expressed sequence tags) technique he invented, in combination with the notorious whole - genome shotgun approach. the string of genome publications in the 1990s in which he was highly involved, such as the ones on the est technique (adams. 2000) made him one of the most highly cited researchers, while the comparatively small teams he marshaled produced staggering amounts of bioinformation. and his publication on the human genome (venter. rather, he subsequently set sail in order to sequence the metagenome of oceanic life forms. as kevin davies (2002) argues, although venter s restless ambition and single - minded opportunism have alienated him from many of his fellow scientists, his trailblazing accomplishments in dna sequencing over the past decade justify a nobel prize. besides that, he was a pioneer in other fields as well, developing new approaches in science management through privatepublic partnerships and valorization. his well - documented experiences in this realm provide ample material for reflection, also on the risks and pitfalls involved in introducing such strategies in science. and finally, like collins, though lectures, interviews and, eventually, his autobiography, he contributed significantly to current debates on the societal meaning of genomics and the hgp and on the implications it has had for our understanding of ourselves as well as of life on earth. of course, he did not do all this single - handedly he had an eye for recruiting talent as well as for acquiring substantial funding. but when it comes to assessing the hgp, the sometimes - decisive interventions and contributions of individuals such as collins and venter are impossible to ignore. if we adopt this line of reasoning, however, the focus is bound to shift from one particular and definite contribution (discovery) to an extended performance, turning the nobel prize into a lifetime award. the nobel prize would then be granted not on the basis of an assessment of a single discovery or publication, but rather on the basis of a track record, a whole career, a curriculum vitae that not only involves laboratory achievements (technoscientific genius), but managerial and communicational talents and achievements as well. the problem then remains whether it is fair and meaningful to single out individuals (rather than, for instance, institutes or teams) in a time of mass production of knowledge claims. before world war ii the nobel prize had almost always been awarded to a single individual. in the more recent past this already indicates that, as a reward mechanism, the nobel prize acknowledges and reflects the increase of scale that has taken place in science. yet, in my view, to further expand this trend for instance, by singling out teams, consortia or institutes rather than individuals as possible laureates would be deplorable as a symptom of anonymisation. as individual effort continues to be a crucial element in the dynamics of science, as was argued above, the nobel prize is one element (a highly prestigious and visible element no doubt) in a complex network of symbolic as the complexities of the knowledge production process continue to increase, the mechanisms involved must no doubt become more adaptive, differentiated and sophisticated as well in order to remain meaningful and effective. this does not imply, however, that acknowledgement of individual achievement as such is something of the past. nonetheless, achievements deserve to be acknowledged, and some achievements more than others. if we agree that the human genome sequence effort still merits an nobel prize, who should be the laureates ? as other candidates such as james watson, john sulston and hamilton smith will have to be dismissed, simply because they have already been awarded the nobel prize for physiology or medicine (and scientists can become a physiology or medicine laureate only once in a lifetime), the idea of a nobel prize for collins and venter preferably in 2010appears a plausible one, on the basis of their measurable and quantifiable performance. a nobel prize for collins and venter would underline that over and above being excellent researchers earlier in their careers, these individuals became outstanding research managers somewhat later in their lives. and these management responsibilities involved not only scientific and managerial skills, but also the ability to address the complex societal issues involved and to effectively deliberate these issues with policy makers, politicians, entrepreneurs, journalists and the public at large. moreover, to the extent that individuals such as collins and venter are acknowledged for their exceptional merits as research managers, their nobel prizes will be indirectly awarded to the teams they represent, thereby acknowledging the less visible geniuses whom they recruited to work behind the scenes. when it comes to awarding the nobel prize to collins and/or venter, another hazardous issue will be to what extent modes of funding should be taken into consideration. officially, while collins headed the publicly funded sequencing effort, venter led a privately owned company with a stock market quotation. as the history of the nobel prize reflects the idea that academic excellence in combination with working for the benefit of society is somehow incompatible with striving for personal financial gain, this would considerably compromise venter s chances. yet, also in this respect, it has become increasingly difficult to interpret the world in terms of convenient moral dichotomies. not only the funding strategies, but also the work ethic and the reward systems of universities and knowledge enterprises have begun to merge. even publicly funded research efforts have become both costly and potentially profitable endeavours. and while venter at various occasions published staggering amount of genomics data for free, watson, collins and most of the other protagonists of publicly funded genomics research privately own patents. thus, it will become an increasingly intricate matter to determine where to convincingly draw the moral line. thus, notwithstanding the various complications and considerations at stake, a nobel prize for individuals such as collins and venter would do justice to the way in which excellent research in the contemporary life - sciences is done. eric lander, first author of the official nature presentation of our genome sequence, would be a convincing third candidate to join them. | the human genome project (hgp) is regarded by many as one of the major scientific achievements in recent science history, a large - scale endeavour that is changing the way in which biomedical research is done and expected, moreover, to yield considerable benefit for society. thus, since the completion of the human genome sequencing effort, a debate has emerged over the question whether this effort merits to be awarded a nobel prize and if so, who should be the one(s) to receive it, as (according to current procedures) no more than three individuals can be selected. in this article, the hgp is taken as a case study to consider the ethical question to what extent it is still possible, in an era of big science, of large - scale consortia and global team work, to acknowledge and reward individual contributions to important breakthroughs in biomedical fields. is it still viable to single out individuals for their decisive contributions in order to reward them in a fair and convincing way ? whereas the concept of the nobel prize as such seems to reflect an archetypical view of scientists as solitary researchers who, at a certain point in their careers, make their one decisive discovery, this vision has proven to be problematic from the very outset. already during the first decade of the nobel era, ivan pavlov was denied the prize several times before finally receiving it, on the basis of the argument that he had been active as a research manager (a designer and supervisor of research projects) rather than as a researcher himself. the question then is whether, in the case of the hgp, a research effort that involved the contributions of hundreds or even thousands of researchers worldwide, it is still possible to individualise the prize ? the hgp nobel prize problem is regarded as an exemplary issue in current research ethics, highlighting a number of quandaries and trends involved in contemporary life science research practices more broadly. |
varicella zoster virus (vzv) is one of the most prevalent viruses which affects the human race (1). primary infection known as chickenpox, mostly occurs in childhood, with mild clinical course in immunocompetent hosts, but it can cause significant morbidity in healthy adults with life threatening forms in immunocompromised persons. vzv establishes latency in sensory ganglia, and can be reactivated years later as herpes zoster. circulating vzv - specific antibody can prevent primary infection but innate and cellular responses are more important in its severity and duration (2). clinical studies suggest that t cell immunity plays a key role in the protection against vzv primary infection. few studies found significant depression of cd4 + t lymphocytes, augmentation of cd8 + t - lymphocytes and changes in cd4/cd8 ratio but none of them were compared to clinical course of illness (3, 4). we performed a prospective clinical study which included 69 immunocompetent persons with confirmed chickenpox in a period july 2014 - january 2016. patients were divided into two groups : group with mild clinical presentation who were treated as outpatients, and group with moderate, severe or life - threatening clinical presentation which were hospitalized. study also included 30 healthy volunteers with age and sex similar to the other groups. we checked general characteristics such as age, sex, complications, clinical course of illness, percentage values of cd4 +, cd8 + t - lymphocytes and cd4/cd8 ratio. values for cd4 +, cd8 + t - lymphocyte percentage and cd4/cd8 ratio was obtained using facs canto, bd biosciences flow cytometer and bd multitest 6-color tbnk immunofluorescent test. values with normal distribution were expressed as meanstandard deviation. to compare mean values for variables without normal distribution chi - square test was used. one - way anova test was used for statistical evaluation of more than three groups. correlation of monitored variables is determined by pearsons test. to determine influence of these variables to a clinical presentation we used logical regression (backward method). p - values less than 0.05 were considered statistically significant. clinical presentation of the illness cd4 + percentage values in the groups cd8 + percentage values for all groups cd4/cd8 ratio values for the groups mean age in outpatient group was 28.47 years with standard deviation (sd) 10.3 while in the other group was 24.57 (sd=14.14), with no statistically significant difference between groups (p>0.05). females were dominant in outpatients group (65%:35%), while males were in higher number in other group (71%:29%). chi - squared test found statistically significant difference between these two groups (p<0.05). the number of complications vary from zero to four per patient, and all were presented in a hospitalized patients. mean value of cd4 + percentage for outpatient group was 44.06 (sd 11.48) and for a group of hospitalised patients it was 28.20 (sd=12.70). one - way anova test found statistically significant difference between group of hospitalised patients and outpatients, as well as hospitalised patients and control group (p<0.0001). values for percentage of cd8 + t - lymphocytes were as follows : outpatients 30.15 (sd=9.46) ; hospitalised patients 38.34 (sd=15.52) ; control group 28.40 (sd=7.34). one - way anova test found statistically significant difference between group of hospitalised patients and outpatients, as well as hospitalised patients and control group (p<0.0001). cd4/cd8 ratio was 1.74 (0.40 - 8.10) in group of outpatients, 1.05 (0.10 - 3.60) in group of hospitalised patients and 1.73 (0.80 - 3.70) in a control group. one - way anova test found statistically significant difference between group of hospitalised patients and outpatients, as well as hospitalised patients and control group (p=0.006). using pearson s correlation factor we have found very strong negative correlation between cd4 + percentage value and clinical presentation (r=0.61, p<0.0001) ; strong positive correlation between cd8 + percentage value and clinical picture and also weak negative correlation between cd4/cd8 ratio and clinical presentation. in addition we performed regression analysis to determine which of mentioned parameters have the strongest influence to clinical presentation. values of cd4 + percentage have a very strong influence as a prognostic factor to clinical presentation and possible severity of clinical picture (beta coefficient = 0.56, p<0.0001). results of our study correlate with other studies regarding age and sex of the patients, where most of the them with complications were males, age 21 - 40, primary because of preexisting medical conditions (5). several studies suggest that cellular immunity has a key role in control of vzv primary infection (6, 7). patients with primary agamagloblinaemia develop mild clinical form, while patients with deficiency of cellular immunity develop severe and life - threatening forms of chickenpox (8). so far, there is no prognostic marker for a severity of clinical picture and outcome of the disease, which can be measured in short time and predict further development of the illness. in our study we tried to investigate if values of cd4 + and cd8 + t - lymphocytes percentage, so as cd4/cd8 ratio, can be considered as relevant prognostic factors for clinical course of chickenpox. we measured values of these parameters from blood samples taken from the patients in acute phase of disease, 24 - 72h after development of the rash, and correlate with presented complications and severity of clinical picture. values of cd4 + t - lymphocytes mostly decrease in acute phase of all viral infection, so as vzv, with recovery during the time. for significant number of diseases it can be strong prognostic factor for possible coinfections and complications in clinical course (3, 9, 10). in our study decrease of cd4 + percentage was very significant, compared to the group with mild clinical form and control group. also cd8 + percentage value increased in acute phase of illness among hospitalized patients with presented complications. early studies suggest similar dynamic of these subsets of t - lymphocytes (11). significant difference between these patients and outpatients, so as control group, was confirmed with anova test. cd4/cd8 ratio is very important in follow - up of some viral diseases and other medical conditions (12, 13). we found significant decrease in values of cd4/cd8 in hospitalized patients, while values in group of outpatients and controls were almost the same. further comparison using regression analysis found values of cd4 + percentage of t - lymphocytes as a strong predictive factor for clinical course of the disease. also values of cd8 + t - lymphocyte percentage were in a strong correlation with clinical pictures. according to the results of our study, we can consider values of cd4 + and cd8 + t - lymphocyte subsets as strong predictive factor for a clinical course of chickenpox in immunocompetent patients. values of cd4 + t - lymphocytes have the strongest influence, while cd4/cd8 ratio stays as a prognostic factor, but not so strong like in other viral diseases, such as hiv. | objective : to investigate possible prognostic values of cd4 +, cd8 + t - lymphocytes, cd4/cd8 ratio to clinical course of chickenpox in immunocompetent hosts.materials and methods : we performed a prospective study which included 69 immunocompetent patients with chickenpox who were addmited to clinic for infectious disease, clinical center university of sarajevo, in a 18 month period. all patients were divided into two groups depending on clinical presentation on admission. patients with mild clinical form were dedicated to outpatient group, and patients with moderate, severe or life - threatening clinical forms were dedicated to hospitalized group. also 30 healthy volunteers are included in study as a control group. we analyzed values of cd4 +, cd8 + percentage, cd4/cd8 ratio with comparison to clinical course of chickenpox. all specimens were taken in acute phase of illness.results:values of cd4 + percentage were significantly declined in a group of hospitalized patients, compared to group of outpatients and control group. values of cd8 + percentage were higher in a group of hospitalized patients, while cd4/cd8 values were lower in comparison to a group of outpatients and control group.conclusion:we found significant correlation between these parameters and clinical course of chickenpox. |
the authors present a case of giant ureteric calculus highlighting the importance of plain film radiograph in the assessment of children with suspected renal tract calculi. ultrasonography demonstrated left hydronephrosis and a 2 cm echogenic area with acoustic shadow in the proximal ureter, consistent with a ureteric calculus. plain radiography astonishingly revealed a large 7 cm radio - opaque shadow in the line of the left ureter extending from the left pelvi - ureteric junction to the mid - ureter, and a second 4 cm opacity in the left distal ureter (fig 1). plain abdominal radiograph demonstrating large opacity extending from the left pelvi - ureteric junction to the mid ureter, and a smaller opacity in the distal ureter. a dmsa scan showed split renal function of 30% on the left side and 70% on the right. the left distal ureter was identified using an extraperitoneal approach through a left iliac fossa incision. the distal calculus was extracted through a longitudinal ureteric incision. as it was not possible to retrieve the proximal stone through this incision the proximal ureteric calculus measured 6.5 x 1.8 cm and weighed 7991 milligrams (fig 2). ureteric calculi removed from the proximal (left) and distal (right) ureter. it was composed of 85% magnesium ammonium phosphate (struvite) and 15% carbonate apatite. the uk incidence of urolithiasis in children has been estimated to be two per million per annum (1). giant. renal tract calculi commonly present with renal colic, haematuria, urinary tract infections or lower urinary tract symptoms. giant ureteric calculi have been reported to present with urinary tract infection and retention of urine (2,3). they can however be asymptomatic and progress silently, as was characterised by our case, where the giant calculus was discovered incidentally. current practice reflects evidence that ultrasonography is more sensitive than plain radiography in detecting renal tract calculi in children (4,5). however, ultrasonography is less sensitive in detecting and characterising ureteric stones than plain radiography. a combination of ultrasonography and plain radiography has therefore been recommended as the standard assessment. ultrasonography does not always accurately characterise ureteric calculi and if a plain radiograph had not been performed, the extent of the calculus would not have been appreciated and a different course of management may have been undertaken. the management of giant ureteric calculi entails removal of the calculus or nephroureterectomy depending on kidney function. the authors wish to emphasise the importance of including a plain abdominal radiography in the pre - operative assessment of ureteric calculus in children. | giant ureteric calculi are extremely rare in children. we present a case of a child who was originally admitted for observation following non - accidental injury and had an episode of painless haematuria as an inpatient. ultrasonography demonstrated left hydronephrosis and a 2 cm echogenic area in the proximal ureter. a plain abdominal radiograph surprisingly revealed two left ureteric calculi, one 7 cm and the other 4 cm in length. stone extraction was achieved using an open left ureterolithotomy and pyelolithotomy. |
interactions between hormonal contraceptives and antiretroviral (arv) medications to treat hiv are of great importance.1 in 2009, approximately 1.2 million people in the united states were living with hiv.2,3 globally, the scope of the problem is more decimating, as hiv / aids is the leading cause of death among women aged 1844 years.4 arv therapy is the standard of care and reduces morbidity and mortality in hiv - infected women.5 arv therapy typically consists of 2 or more medications from the various classes, including entry inhibitors, integrase inhibitors, ccr5 agonists, protease inhibitors (pi), nonnucleoside reverse transcriptase inhibitors, and nucleoside / nucleotide reverse transcriptase inhibitors. use of hormonal contraception is prevalent in hiv - prevalent regions of the world.3 the center for disease control (cdc) and world health organization (who) state that women living with hiv can safely use hormonal contraceptives.6,7 the prevention of unintended pregnancy with safe and effective contraception to improve maternal health and to prevent mother - to - child transmission of hiv are strategies mentioned in the united nation 's millennium development goals for 20102015.8 multiple arv drugs alter drug metabolizing enzyme activity, which may in turn alter the pharmacokinetics of concurrently administered medications.9 ritonavir, atazanavir, indinavir, nelfinavir, and saquinavir are all strong inhibitors of cytochrome p450 (cyp) 3a4.10 ritonavir acts via rapid, reversible, competitive binding.10 this drug is used synergistically with other pis to increase plasma drug concentrations and enhance arv response in patients. ritonavir is the preferred pi given in conjunction with atazanavir or darunavir to arv - naive patients.9 pis also inhibit udp - glucuronosyl transferase and decrease renal p - glycoprotein transport and excretion activity.10 based on these in vitro observations, it would be expected that plasma steroid levels would be increased after the coadministration of hormonal contraception and pi. however, in vitro models do not always correlate with in vivo drug interactions.10,11 complex alterations in pharmacokinetic processes, namely, absorption, distribution, metabolism, and excretion often make in vitro in vivo correlations of drug drug interactions difficult to predict.10 as evidence of this complexity, empiric trials with sample sizes of 510 hiv - negative women have demonstrated that administration of combined oral contraceptives and a pi or a nonnucleoside reverse transcriptase inhibitor produce decreased, and not increased, plasma ethinyl estradiol concentrations.12 decreased ethinyl estradiol concentrations may result in reduced efficacy, with an increased risk of unintended pregnancy. these combined oral contraceptive studies have demonstrated variable changes in serum progestins with arv therapy.5,1214 daily 0.35 mg of norethindrone (net) is administered as a continuous oral contraceptive in us progestin - only pills. the half - life of net is 812 hours, and its peak plasma concentration occurs within 2 hours of oral ingestion.11 hydroxylation of net to its m1 metabolite is predominately due to cyp3a4 catalyzed reactions in the liver and to a lesser degree in the small intestine.11,15 cyp2c19 enzymes may have a minor role.15 additionally, cyp3a4 is subject to a wide degree of interindividual variability, in the order of 11- to 20-fold, but no relevant genetic polymorphisms have been identified. other cyp isoforms do contribute to intersubject variability in arv metabolism, and they include 2d6, 2c9, and 2c19.11,16,17 manufacturer product labels advise patients to use alternative methods of contraception when any pi is coadministered with combined oral contraceptives or progestin - only pills.9,18 the who and cdc list the use of ritonavir - boosted pi and progestin - only pills as category 3 (risks outweigh benefit), thus limiting their use in hiv - infected women.19,20 the who states, as category 3, use of that method is not usually recommended unless other more appropriate methods are not available or acceptable. where resources for clinical judgment are limited.category 3 indicates that a woman is not medically eligible.20 no previous published pharmacokinetic trials have examined progestin - only pills in hiv - infected women taking any pi.12 we studied hiv - infected women to determine if there was a significant interaction between pi and progestin - only pills. this was a 2 arm, open - label, prospective, nonrandomized, steady - state pharmacokinetic trial of drug drug interactions in hiv - infected women treated with oral net and pi. area under the time concentration curve of net in these women was compared with hiv - infected controls taking net and no arv or an arv regimen without a pi, which have demonstrated no significant interaction with net in previous combined oral contraceptive trials.12,14 approval of the university of southern california (usc) institutional review board was obtained. participants, aged 1844 years, were hiv infected, and had no major lifestyle changes or changes in medications in the month before enrollment, no recent exposure to hormonal contraceptives (combined oral contraceptives > 30 days, depot medroxyprogesterone acetate > 180 days), no evidence of immunocompromise, cd4 count of greater than 200 cells per cubic millimeter, no liver or renal disease, normal ovulatory function, body mass index of 30 days postpartum, abstained from grapefruit products (which contain furanocoumarin) or other cyp3a4-interacting substances, and agreed to use nonhormonal contraception. women who were taking any pi as part of their anti - hiv therapy formed the study group, and those who were not taking a pi served as controls. women were recruited from the maternal child adolescent clinic of los angeles county, university of southern california. after screening and informed consent, women received a 28-day blister pack of net (0.35-mg net, jolivette ; watson pharmaceuticals inc., women took a single fixed dose of 0.35-mg net daily for a minimum of 21 days and also adhered to dietary restrictions as per the protocol. on or after day 22, each woman was admitted to the clinical trials unit at the university of southern california, where a clinician observed her final ingestion of net. blood was collected by venous catheter and venipuncture before net ingestion and at 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after taking net. after allowing the blood to stand for approximately 1 hour, the samples were centrifuged, and serum was removed and stored at 20c until analyzed. for the treatment, 0.35-mg net (jolivette) was ordered, stocked, and monitored by the usc research pharmacy. net was measured in serum by radioimmunoassay, as described previously.21,22 before radioimmunoassay, net was extracted with ethyl acetate : hexane (3:2) and then purified by celite column partition chromatography. procedural losses were followed by adding small amounts of high specific activity tritiated internal standard (h - net) to the serum before the extraction step. a highly specific antiserum was used in conjunction with an iodinated radioligand in the radioimmunoassay. separation of unbound from antiserum - bound net was achieved by the use of second antibody. the sensitivity of the net radioimmunoassay was 0.06 ng / ml. intraassay and interassay coefficients of variation range from 4%7% and 9%12%, respectively. the primary study end point was the serum net area under the time concentration curve from 0 to 72 hours, calculated using the linear trapezoidal approximation. secondary end points were maximum net concentration, minimum net concentration, and the half - life that was estimated from the terminal elimination slope for each patient using concentrations sampled at 12 hours and beyond. for area under the curve and half - life, we used the pmetrics package for r.23 the null hypothesis used was that the 90% confidence interval for area under the curve geometric mean ratio would be within the range of 0.61.67, which is a clinically insignificant difference of 40%.10 to reject the null hypothesis, we estimated that 16 women would be required in each arm to detect a > 40% intergroup difference in area under the curve with a 2-tailed alpha of 0.05 and 80% power. our assumptions for the sample size calculation were based on a previously reported mean (standard deviation) net area under the curve of 22.1 (10.9) nghr / ml after an oral dose of 0.3-mg net.24 peer - review literature does not specify minimum net thresholds for contraceptive efficacy. we summarized normally distributed, continuous data with means and standard deviations and compared groups with student t test. we summarized non normally distributed, continuous data ; we summarized them with medians and interquartile ranges and compared them with the wilcoxon rank sum test. log10 transformation was completed for all pharmacokinetic end points, which were compared with student t test. we used sas (version 9.3 ; sas institute, cary, nc) and r (version 3.0.0 ; r project for statistical computing, vienna, austria) for all analyses and plots. this was a 2 arm, open - label, prospective, nonrandomized, steady - state pharmacokinetic trial of drug drug interactions in hiv - infected women treated with oral net and pi. area under the time concentration curve of net in these women was compared with hiv - infected controls taking net and no arv or an arv regimen without a pi, which have demonstrated no significant interaction with net in previous combined oral contraceptive trials.12,14 approval of the university of southern california (usc) institutional review board was obtained. participants, aged 1844 years, were hiv infected, and had no major lifestyle changes or changes in medications in the month before enrollment, no recent exposure to hormonal contraceptives (combined oral contraceptives > 30 days, depot medroxyprogesterone acetate > 180 days), no evidence of immunocompromise, cd4 count of greater than 200 cells per cubic millimeter, no liver or renal disease, normal ovulatory function, body mass index of 30 days postpartum, abstained from grapefruit products (which contain furanocoumarin) or other cyp3a4-interacting substances, and agreed to use nonhormonal contraception. women who were taking any pi as part of their anti - hiv therapy formed the study group, and those who were not taking a pi served as controls. women were recruited from the maternal child adolescent clinic of los angeles county, university of southern california. after screening and informed consent, women received a 28-day blister pack of net (0.35-mg net, jolivette ; watson pharmaceuticals inc., women took a single fixed dose of 0.35-mg net daily for a minimum of 21 days and also adhered to dietary restrictions as per the protocol. on or after day 22, each woman was admitted to the clinical trials unit at the university of southern california, where a clinician observed her final ingestion of net. blood was collected by venous catheter and venipuncture before net ingestion and at 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after taking net. after allowing the blood to stand for approximately 1 hour, the samples were centrifuged, and serum was removed and stored at 20c until analyzed. for the treatment, 0.35-mg net (jolivette) was ordered, stocked, and monitored by the usc research pharmacy. net was measured in serum by radioimmunoassay, as described previously.21,22 before radioimmunoassay, net was extracted with ethyl acetate : hexane (3:2) and then purified by celite column partition chromatography. procedural losses were followed by adding small amounts of high specific activity tritiated internal standard (h - net) to the serum before the extraction step. a highly specific antiserum was used in conjunction with an iodinated radioligand in the radioimmunoassay. separation of unbound from antiserum - bound net was achieved by the use of second antibody. the sensitivity of the net radioimmunoassay was 0.06 ng / ml. intraassay and interassay coefficients of variation range from 4%7% and 9%12%, respectively. the primary study end point was the serum net area under the time concentration curve from 0 to 72 hours, calculated using the linear trapezoidal approximation. secondary end points were maximum net concentration, minimum net concentration, and the half - life that was estimated from the terminal elimination slope for each patient using concentrations sampled at 12 hours and beyond. for area under the curve and half - life, we used the pmetrics package for r.23 the null hypothesis used was that the 90% confidence interval for area under the curve geometric mean ratio would be within the range of 0.61.67, which is a clinically insignificant difference of 40%.10 to reject the null hypothesis, we estimated that 16 women would be required in each arm to detect a > 40% intergroup difference in area under the curve with a 2-tailed alpha of 0.05 and 80% power. our assumptions for the sample size calculation were based on a previously reported mean (standard deviation) net area under the curve of 22.1 (10.9) nghr / ml after an oral dose of 0.3-mg net.24 peer - review literature does not specify minimum net thresholds for contraceptive efficacy. we summarized normally distributed, continuous data with means and standard deviations and compared groups with student t test. we summarized non normally distributed, continuous data ; we summarized them with medians and interquartile ranges and compared them with the wilcoxon rank sum test. log10 transformation was completed for all pharmacokinetic end points, which were compared with student t test. we used sas (version 9.3 ; sas institute, cary, nc) and r (version 3.0.0 ; r project for statistical computing, vienna, austria) for all analyses and plots. of 167 women who were screened, 132 were ineligible based on protocol restrictions or because they declined to participate, as shown in figure 1. one of 17 women in the study group withdrew due to commitments that conflicted with her scheduled admission. therefore, 16 women in the study group and 17 in the control group completed the trial. there were no significant differences between the 2 groups in terms of mean age, parity, cd4 count, history of opportunistic infections, body mass index, smoking status, ethnicity, or language, as shown in table 1. in the control group other control participants were taking combinations of nucleoside reverse transcriptase inhibitors (n = 13), nonnucleoside reverse transcriptase inhibitors (n = 9), and integrase inhibitors (n = 4). baseline characteristics antiretroviral regimens the pharmacokinetic characteristics of net in the study and control groups are shown in table 3. the geometric mean net area under the curve in the pi study group was 37.8 ngh / ml, and in the control group, it was 25.2 ngh / ml (fig. the geometric mean area under the curve ratio of the pi study group to the controls was 1.50, with a 90% confidence interval of 1.21 to 1.86 (p = 0.004). net minimum concentration was higher among women taking a pi, whereas maximum concentration was not significantly different between the study groups (p = 0.11) and it tended to be higher in the pi group. subset analysis was performed with the 11 women taking atazanavir and the 10 women taking atazanavir / norvir (excluding the participant on atazanavir without norvir) ; the results remained significant and were comparable. pharmacokinetic characteristics of serum net after 0.35 mg of oral net ingestion with and without pi violin plot of net area under the curve by group. a traditional box and whisker plot is centered within each violin, where the filled circle is the median, and the lower and upper bounds of the box are the 25th and 75th percentiles (ie, the interquartile range). whiskers indicate the range of the data within 1.5 interquartile range of the box boundaries, and the open circle is an outlier outside this range. exclusion of this outlier did not change the results significantly (data not shown). it is recognized that there is a dearth of clinical data to guide contraceptive recommendations in hiv - infected women taking arv therapy.1 the who and cdc base their progestin - only pill recommendations on studies of arv drugs and combined oral contraceptives.19,20 progestin - only pills are category 3 with ritonavir - boosted pi. as noted in the cdc appendix m, small mostly unpublished studies suggest that some antiretroviral therapies might alter the pharmacokinetics of combined oral contraceptives.19 progestin - only pills have fewer contraindications than estrogen - containing products, allowing greater use by more women. for example, women with hypertension, a history of venous thrombosis, smokers older than 35 years, and women in the postpartum period may all take progestin - only pills and would be discouraged from using ethinyl estradiol containing combined oral contraceptives. furthermore, many hiv - positive women have comorbidities that would prevent them from using combined oral contraceptives. additionally, they have a compelling need for dual contraception with condoms and an alternative method. this present study showed that area under the curve of net is significantly increased by 50% among hiv - infected women taking pi therapy as compared with controls. this ratio met our predefined criteria for a significant interaction, and we rejected the null hypothesis of no interaction. because many pi, particularly ritonavir, are known to be sytsemic inhibitors of cyp3a4,10 and net is a substrate for cyp3a415, we presume that the mechanism of the interaction relates to the activity of this enzyme. in vivo the cyp3a4 inhibition typical of pi resulted in a significantly increased serum net levels by decreasing systemic metabolism ; this finding is supported by the increased area under the curve and increased minimum concentration of net. the net half - life is not significantly different between the 2 groups, which may be due to changes in steroid distribution. it is interesting that administration of combined oral contraceptives and pi have resulted in decreased serum ethinyl estradiol vis - - vis alterations of microsomal enzymes.12 as per the us food and drug administration product insert, ritonavir is known to be an inducer and an inhibitor of cyp3a4, and drug - to - drug interactions are difficult to predict.25 the hispanic and age demographic of our hiv - positive women at our single site in the united states may not reflect the same demographics of other regions. our sample size was based on an a priori power analysis ; however, it was still small. the variability of serum net levels between different participants was extremely large, yet comparable to the range published in previous clinical research.24 there is extremely limited or no published data to guide research on minimum serum levels of exogenous hormones for contraceptive efficacy. other metabolic considerations, such as genetic differences or behaviors that deviated from protocol, may have also contributed to the significant findings. it may be difficult to generalize these results to women who are immunocompromised, who do not have access to clinicians, or who are unable to demonstrate strict adherence to their contraceptive and arv therapy. the regimens of several participants, including the women who were not taking any arv therapy, are not the standard recommendations for most arv - naive hiv - positive women ; they were specifically tailored to these women by their infectious disease clinician. ten of the 16 in the pi group took atazanavir / ritonavir, and the results remained significant when this subset was analyzed. however, only 3 women took darunavir / ritonavir, and 2 took lopinavir / ritonavir. with these small numbers, it is difficult to know if net is increased among all pi regimens. additionally, none of the participants were taking other pi agents, such as fosamprenavir, indinavir, saquinavir, nelfinavir, or tipranavir. however, ritonavir is a potent inhibitor, and there remains biologic plausibility that it would increase net in combination with other pis. the 50% increase in the area of net noted among women taking pi in our study is not concerning for toxicity and does not warrant dose reduction. many progestins are well tolerated, exhibit minimal side effects, and have excellent safety profiles.26 the safety of this steroid and its metabolites has been demonstrated in clinical trials and postmarket surveillance.26 several current combined oral contraceptive products approved and marketed in the united states contain 1.5 mg of net in addition to ethinyl estradiol, which is over 4 times as much progestin as the 0.35 mg of net in the progestin - only pill.2629 the range of net levels noted in the both groups of women were comparable with serum net levels observed in previous clinical trials.24,26 the dose determined for progestin - only pills contraception was a somewhat arbitrary historic assignment based on suspected bioequivalence of 0.5 mg of chlormadinone acetate.3034 in preliminary trials with the progestin net, it was given in doses up to 20 mg, which demonstrates the wide therapeutic index, safety, and minimal toxicity of net.26 compared with combined oral contraceptives, progestin - only pills require less restrictive screening, have wider distribution potential, and can provide an additional safe contraception option for women with hiv. this is the first trial to describe net progestin - only pill pharmacokinetics in hiv - infected women taking pi. net area under the curve is increased by the coadministration of pi. increased serum net levels are a surrogate marker of continued therapeutic contraceptive efficacy. these findings should alter current progestin - only pill medical eligibility recommendations for women taking pi. | objective : pharmacokinetic interactions exist between combined oral contraceptives and protease inhibitors (pi). however, such information is lacking for progestin - only oral contraception. we sought to define the steady - state pharmacokinetic interaction between norethindrone (net) and pi in hiv - infected women.methods and design : we conducted an open - label, prospective, nonrandomized trial to characterize the steady - state pharmacokinetics of serum net in hiv - infected women receiving pi compared with a control group of hiv - infected women receiving other noninteracting drugs. after 21 days of 0.35 mg of net ingestion once daily, serial serum samples were obtained at 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours. the area under the curve between 0 and 72 hours after ingestion was calculated by trapezoidal approximation.results:thirty-five women were enrolled, 2 withdrew. sixteen women in the pi group and 17 controls completed the study. net half - life and maximum concentration were not significantly different between the 2 groups. minimum concentration of net was significantly higher in the pi group (p = 0.01). the ratio of the geometric mean net area under the curve in the pi group compared with controls was 1.5 (90% confidence interval : 1.21 to 1.86). net serum concentrations were significantly higher in hiv - infected women taking a pi compared with controls (p = 0.004).conclusions : coadministration of pi inhibits net metabolism as shown by higher serum net area under the curve levels, a surrogate marker for therapeutic contraceptive efficacy. this study supports the increased utilization of progestin - only pills in hiv - infected women receiving certain pi regimens. |
details of the step study design have been reported previously (1820). in brief, this was a 12-month, cluster - randomized clinical trial designed to assess the use of structured smbg, as part of a comprehensive, collaborative intervention, on glycemic control compared with enhanced usual care in 483 patients with non insulin - treated t2 dm. patients were randomized to the structured smbg group (stg) or to an active control group (acg) for comparison. stg patients received enhanced usual care and used the accu - chek 360 view blood glucose analysis system (roche diagnostics, indianapolis, in) to record and plot a 7-point smbg profile (preprandial / postprandial at each meal and at bedtime) on 3 consecutive days before a study visit. stg patients received training in the use of the analysis system and how to interpret their results and use their findings to make changes to their diet and physical activity. enhanced usual care comprised quarterly clinic visits focusing on diabetes management with office point - of - care glycated hemoglobin measurement. free blood glucose meters and test strips (accu - chek aviva blood glucose meter system, roche diagnostics) were provided to patients in both study arms. patient visits occurred at baseline and at months 1, 3, 6, 9, and 12. the study protocol was approved by the copernicus group (central institutional review board) and is in compliance with the declaration of helsinki (21). written informed consent was obtained from all patients. as reported previously (18), a1c analysis was conducted by a central laboratory (covance, indianapolis, in) using the variant ii and variant ii turbo hemoglobin testing systems (bio - rad laboratories, hercules, ca). measurements of fasting glucose and postprandial excursions were based on stg patient - reported data from the quarterly 7-point glycemic profiles ; accuracy of these data were confirmed using downloaded blood glucose meter data. the 7-point glycemic profiles were also used to assess glycemic variability, which was reported as the mage. measurements of ldl - cholesterol, hdl - cholesterol, triglycerides, and hs - crp were taken at baseline and at months 3, 6, 9, and 12. the hs - crp analysis was also conducted at a central laboratory (covance). cutoff points for cv risk were defined as low (mean hs - crp levels 3.0 mg / l) (22). details of the step study statistical analysis methodologies have been reported previously (18). briefly, a cluster - randomization strategy was chosen, whereby all patients within a given practice were assigned to the same study arm. the analysis of change in a1c and other dependent variables was performed using linear mixed models (lmm) analysis with sas proc mixed (23,24). control variables in all analyses included baseline - dependent variables of patient age, sex, and race (white / nonwhite) as fixed effects ; and practice site and subject as random effects. the primary analysis methods used for this study were similar to those previously reported in the step study. lmm analysis was performed for the natural logarithm of hs - crp at postbaseline visits, with group (acg or stg), baseline loge (hs - crp), age, sex, and race (white / nonwhite) as fixed effects, and patient and site as random effects. the values reported for change from baseline in hs - crp concentration (mg / l) are absolute differences from baseline in geometric means (95% ci, delta method) at postbaseline visits. relationships between change in glycemic control, glycemic variability, and change in hs - crp (log - scale) were examined with a general linear model, with patient demographics, diabetes duration, and bmi as controls. tests of mediated models via lipids were assessed following the recommendations of baron and kenny (26). as reported previously (18), a1c analysis was conducted by a central laboratory (covance, indianapolis, in) using the variant ii and variant ii turbo hemoglobin testing systems (bio - rad laboratories, hercules, ca). measurements of fasting glucose and postprandial excursions were based on stg patient - reported data from the quarterly 7-point glycemic profiles ; accuracy of these data were confirmed using downloaded blood glucose meter data. the 7-point glycemic profiles were also used to assess glycemic variability, which was reported as the mage. measurements of ldl - cholesterol, hdl - cholesterol, triglycerides, and hs - crp were taken at baseline and at months 3, 6, 9, and 12. the hs - crp analysis was also conducted at a central laboratory (covance). cutoff points for cv risk were defined as low (mean hs - crp levels details of the step study statistical analysis methodologies have been reported previously (18). briefly, a cluster - randomization strategy was chosen, whereby all patients within a given practice were assigned to the same study arm. the analysis of change in a1c and other dependent variables was performed using linear mixed models (lmm) analysis with sas proc mixed (23,24). control variables in all analyses included baseline - dependent variables of patient age, sex, and race (white / nonwhite) as fixed effects ; and practice site and subject as random effects. the primary analysis methods used for this study were similar to those previously reported in the step study. lmm analysis was performed for the natural logarithm of hs - crp at postbaseline visits, with group (acg or stg), baseline loge (hs - crp), age, sex, and race (white / nonwhite) as fixed effects, and patient and site as random effects. the geometric mean estimates at postbaseline visits were derived from the lmm. the values reported for change from baseline in hs - crp concentration (mg / l) are absolute differences from baseline in geometric means (95% ci, delta method) at postbaseline visits. relationships between change in glycemic control, glycemic variability, and change in hs - crp (log - scale) were examined with a general linear model, with patient demographics, diabetes duration, and bmi as controls. tests of mediated models via lipids were assessed following the recommendations of baron and kenny (26). as reported previously (18), 13 primary care practices were randomized to the acg and 21 to the stg. patient demographic and disease - related characteristics at baseline between the two study groups differed only by age and ethnicity ; these differences were controlled in all subsequent analyses (table 1). attrition was higher in the stg (n = 81, 28.6%) than in the acg (n = 43, 18.1%) group baseline characteristics of patients with type 2 diabetes by study group geometric mean baseline hs - crp levels were recorded for 481 patients (table 1). at baseline, more than 30% of patients were classified as at an elevated cv risk ; whereas, almost 60% were found to be at high cv risk, according to hs - crp level. patients at high cv risk tended to be younger, more likely to be female, less educated, have higher bmi, shorter diabetes duration, higher diastolic blood pressure, and higher cholesterol levels. the number of study patients taking statin, -blocker, and/or ace - inhibitor medications at baseline was relatively equal among the groups (acg, n = 157 ; stg, n = 182) and remained so throughout the study period. geometric mean (sd) baseline hs - crp values (mg / l) for these patients were 3.19 (2.76) for acg patients and 3.67 (2.93) for stg patients. mean (sd) baseline lipids for the full intent - to - treat cohort were : ldl - cholesterol, 107.1 (42.1) mg / dl ; hdl - cholesterol, 44.4 (11.9) mg / dl ; and triglycerides, 238.6 (183.6) mg / dl, with no significant between - group differences. in both study arms, there was a consistent decrease in geometric mean hs - crp levels over the study duration, which was significantly associated with reductions in a1c observed throughout the study (p 3 although we were unable to identify the underlying mechanism(s) to explain the relationship between reductions in a1c and hs - crp, our analyses did rule out several commonly hypothesized mechanisms. for example, postprandial excursions and overall glycemic variability have been linked with oxidative stress (28) and other markers of vascular disease (e.g., carotid intima - media thickening) (29) ; however, our analyses found no relationship between reduced postprandial glucose excursions or glycemic variability (as measured by mage and the magnitude of postprandial glucose excursions) and changes in hs - crp, nor did hdl - cholesterol, ldl - cholesterol, or triglycerides (individually or combined) mediate the relation between changes in a1c and changes in hs - crp. moreover, because the reductions in hs - crp seen in both study groups were independent of treatment with statins, -blockers, ace - inhibitors, and/or tzds, use of lipid - lowering and/or antihypertensive medications did not appear to be a factor in our findings. however, when one considers the effects of oxidative stress, which is commonly considered to be the link between hyperglycemia and diabetes complications and is believed to be one of the earliest pathophysiologic changes in the inflammatory process that triggers endothelial dysfunction (14), the effects of metabolic memory may partially explain the relationship between changes in a1c and changes in hs - crp. the concept of metabolic memory hypothesizes that diabetic vascular stresses persist after glycemia has been reduced and that early aggressive treatment aiming to normalize metabolic control, as seen in the step trial (20), in combination with the agents that reduce cellular reactive species and glycation, may minimize long - term diabetes complications (30). given that both step study groups experienced significantly greater a1c reductions early in the study compared with acg patients at month 3 and even greater reductions among stg subjects (adherent and nonadherent) at month 6, this early improvement in glycemia possibly conferred a long - term protective effect against oxidative stress that resulted in lower hs - crp levels even though a1c levels in nonadherent stg patients deteriorated to the same level as in acg patients at 12 months. regardless of the mechanism(s) involved, we showed that reductions in a1c are significantly linked with reductions in cv risk (as assessed by hs - crp levels) in non insulin - treated t2 dm. although there are several possible approaches to reducing a1c levels, there is a growing body of evidence demonstrating the effectiveness and practicality of structured smbg - based interventions in lowering a1c and markers of metabolic risk in this population (31,32). long - term follow - up, including assessment of compliance, would determine whether the results found in our analyses translate into clinical benefits such as long - term improvement of cardiovascular outcome. | objectivethe effect of therapeutic strategies on cardiovascular (cv) disease can be evaluated by monitoring changes in cv risk biomarkers. this study investigated the effect of a structured self - monitoring of blood glucose (smbg) protocol and the resulting improvements in glycemic control on changes in high - sensitivity c - reactive protein (hs - crp) in insulin - nave patients with type 2 diabetes.research design and methodsthe structured testing program (step) study was a prospective, cluster - randomized, multicenter trial in which 483 poorly controlled, insulin - nave patients with type 2 diabetes were randomized to active control (acg) or structured testing (stg) that included quarterly structured smbg. changes in a1c, hs - crp, and glycemic variability (stg subjects only) were measured at baseline and quarterly.resultsreductions in geometric mean hs - crp values were significantly greater in the stg group at months 3 (p = 0.005), 6 (p = 0.0003), and 12 (p = 0.04) than in the acg group. stg patients at high cv risk (> 3 mg / l) showed significantly greater reductions in hs - crp levels than acg patients at high cv risk : 3.64 mg / dl (95% ci 4.21 to 3.06) versus 2.18 mg / dl (2.93 to 1.43), respectively (p = 0.002). there was a strong correlation between reductions in hs - crp and a1c in both groups : standardized coefficient () was 0.25 for the entire cohort (p < 0.0001), 0.31 for stg (p < 0.0001), and 0.16 for acg (p = 0.02).conclusionsreductions in hs - crp level are associated with reductions in a1c but not reductions in lipids or glycemic variability. comprehensive structured smbg - based interventions that lower a1c may translate into improvements in cv risk, as evidenced by levels of the biomarker hs - crp. |
impaired upper urinary tract urine flow is often caused by ureter obstruction due to retro - peritoneal metastatic cancer. obstruction generally begins with pain, then with loss of renal function due to long - lasting congestion, acute or chronic renal insufficiency and/or hydronephrosis of the infected kidneys and may end with generalized sepsis. indwelling ureter splints consisting of polyurethane, silicon, or other polymers are placed to restore urine flow from the renal pelvis to the bladder but gradually become incrusted with crystalline deposits, which can ultimately result in renewed flow obstruction. routinely, indwelling ureter splints are monitored sonographically or by x - ray to detect renewed urinary congestion due to stent obstruction by crystalline deposits. however, early crystalline implant deposits can not be detected by x - ray, presumably due to low spatial resolution and radiolucency of the deposited material. forgotten incrusted dj - stents have been described and continuous monitoring of implanted material and removal or exchange as early as possible is recommended. in vascular doppler sonography, the glittering artifact (= twinkling artifact, ta) appears as a color doppler signal at atheromatous plaque, calcified cardiac valves [2, 3 ], and also at other calcified body structures. here, the doppler signal falsely indicates turbulent current, thus limiting the correct assessment of vascular circulation. however, twinkling artifacts were employed in 2013 to detect early incrustation of a nephrostomy catheter by ta and, with minor sonographic parameter modification in the color doppler mode, they can be used to detect minimal ureter splint incrustation. ureter splints are particularly susceptible to crystalline deposits in patients with tumor lysis syndrome (tls). during tls the high filtration rate of uric acid exerts a severe burden on the kidneys and becomes a decisive factor contributing to impaired renal function that can culminate in renal failure. clinically manifested tls is a life - threatening complication of tumor chemotherapy and results in a high dialysis rate. one option of lowering uric acid is to inhibit xanthine oxidase with allopurinol, as far as the nephrotoxicity of allopurinol will allow, at which point the allopurinol dosage must be reduced. another option is to employ rasburicase, a synthetic recombinant urate oxidase, transforming uric acid into the more water - soluble allantoin. in 2001 although treatment with rasburicase significantly reduces renal complications, including the necessity for dialysis, the high therapeutic cost of rasburicase limits preventive application prior to the occurrence of tls. therefore, an easily applied, inexpensive technique to identify stent incrustation in time to prevent complications is important. the present study aimed to employ ta to detect early ureter splint incrustation in oncologic patients and to follow blood and urine parameters, which might correlate with ureter splint incrustation. twenty - six oncologic patients with implanted dj stents (dj - stent ; 7/28 dj, optimed, ettlingen, germany) and receiving chemotherapy or combined radio / chemotherapy and at high risk for developing tumor lysis syndrome (tls) were included in the investigation. the patients were hospitalized due to routine change of their ureter splints or to change their ureter splints because of presenting complications such as flank pain. sonographic examination employing twinkling artifacts took place the day after implantation and then at weekly intervals. serum creatinine and uric acid as well as urine ph were also measured on the day of stent implantation and weekly thereafter. patients with initially evident tls, increased blood levels of uric acid (> 6 mg / dl) or manifest symptoms of tls were excluded from the study. removal of the newly implanted stent was not influenced by the detection of tas, but was initiated according to customary diagnostic procedure. all procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. ultrasonography was carried out with an acuson sequoia 512 ultrasound system with the following transducers : 4v1 (range 4/3/1.75), 4c1 (range 4/3/175), 6c2 (range 5/3.5/2.5 used for very slim patients) (siemens, mnchen, germany) (normal frequency on color doppler in mhz). the following defined parameters and structured procedure ensured targeted, reproducible tas, which could be distinguished from signals stemming from blood flow. depending on the transducer, the nominal frequency was selected at the lowest possible mhz level. a high pulse repetition frequency (prf, cm / sec) was selected on the velocity scale. transmitting focus on a particular site is essential at highest acoustic energy and post - processing with color variance (cdvv) must be turned on to distinguish ta arising from blood vessels. these parameters were not only employed for implanted stents in patients, but also in a water bath for new, unused dj - stents and stents after removal from the urinary tract. infrared spectroscopy (ft / ir 4100, jasco, gro - umstadt, germany) was used to analyze the incrustation material scratched from ureter splints after explantation. the command variable (time for twinkling artifacts to appear after implantation, revealing incrustation) and cause variables (sex, age, ph of urine, uric acid and creatinine) were correlated by spearman correlation and nonparametric biserial correlation (sex). since three censored data existed (time to appearance of incrustation > 12 weeks after implantation) the significance of the five cause variables was calculated by cox - regression including backward elimination (wald - statistics). twenty - six oncologic patients with implanted dj stents (dj - stent ; 7/28 dj, optimed, ettlingen, germany) and receiving chemotherapy or combined radio / chemotherapy and at high risk for developing tumor lysis syndrome (tls) were included in the investigation. the patients were hospitalized due to routine change of their ureter splints or to change their ureter splints because of presenting complications such as flank pain. sonographic examination employing twinkling artifacts took place the day after implantation and then at weekly intervals. serum creatinine and uric acid as well as urine ph were also measured on the day of stent implantation and weekly thereafter. patients with initially evident tls, increased blood levels of uric acid (> 6 mg / dl) or manifest symptoms of tls were excluded from the study. removal of the newly implanted stent was not influenced by the detection of tas, but was initiated according to customary diagnostic procedure. all procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. ultrasonography was carried out with an acuson sequoia 512 ultrasound system with the following transducers : 4v1 (range 4/3/1.75), 4c1 (range 4/3/175), 6c2 (range 5/3.5/2.5 used for very slim patients) (siemens, mnchen, germany) (normal frequency on color doppler in mhz). the following defined parameters and structured procedure ensured targeted, reproducible tas, which could be distinguished from signals stemming from blood flow. depending on the transducer, the nominal frequency was selected at the lowest possible mhz level. a high pulse repetition frequency (prf, cm / sec) was selected on the velocity scale. transmitting focus on a particular site is essential at highest acoustic energy and post - processing with color variance (cdvv) must be turned on to distinguish ta arising from blood vessels. these parameters were not only employed for implanted stents in patients, but also in a water bath for new, unused dj - stents and stents after removal from the urinary tract. infrared spectroscopy (ft / ir 4100, jasco, gro - umstadt, germany) was used to analyze the incrustation material scratched from ureter splints after explantation. the command variable (time for twinkling artifacts to appear after implantation, revealing incrustation) and cause variables (sex, age, ph of urine, uric acid and creatinine) were correlated by spearman correlation and nonparametric biserial correlation (sex). since three censored data existed (time to appearance of incrustation > 12 weeks after implantation) the significance of the five cause variables was calculated by cox - regression including backward elimination (wald - statistics). no twinkling artifacts were detected in new, unused dj - stents investigated in a water bath. after incrusted implanted stents had been removed from the urinary tract and were investigated in a water bath, tas exclusively and reproducibly were visible where crystals had been deposited and did not appear on the stent material itself (figures 1a and 1b). minor stent incrustation at the distal end (urinary bladder) of a dj - stent, removed six weeks after implantation (upper picture). twinkling artifacts (tas) appear as colored spots on the pigtail, but not on the polyurethane stent itself (lower picture). sonography in water bath. b. minimal stent incrustation of intra - renal pigtail (upper picture). incrustation is not apparent. c. distinct ta at the distal intra - vesical pigtail of the ureter splint (urinary bladder) with detectable incrustation in the intramural course of the ureter (extra - luminary). e : x - ray image of the same explanted ureter splint (d) showing no visible incrustation. one to four weeks after implantation minimal ta activity was apparent at the indwelling splints in all patients (figure 1b). appearance of twinkling artifacts (tas) in indwelling ureter splints as well as urine ph and serum levels of uric acid and creatinine rcc condition after kidney transplantation ; ca carcinoma ; leukemias (among others), ml, all, cll, nhl, aml ; cup cancer of unknown primary ; gist gastrointestinal stroma tumor extremely early incrustation of the ureter splint was found in one patient (metastasizing urothelial carcinoma) with uric acid increased to 11.7 mg / dl, slightly acidic urine (ph 6.0) and compensated renal insufficiency of a single kidney with serum creatinine of 2.9 mg / dl. after one week of implantation, the indwelling ureter splint already displayed distinct tas at the distal end of the stent inside the bladder and in the intramural course of the ureter (figure 1c). this ureter splint required change at this time, since renewed obstruction of the upper urinary tract was diagnosed with conventional ultrasonography. incrustations scratched from the ureter splints were predominantly identified as phosphates (struvite, apatite), uric acid and oxalates (whewellite, weddelite). repeated x - ray examination of clearly incrusted stents, which had been explanted, revealed no deposits. incrustation had previously been identified by ta while these splints were indwelling (example : figures 1d, 1e). a significant correlation was apparent between the serum creatinine level and the time it took after stent implantation for tas to appear, indicating incrustation (correlation coefficient of -0.54) (figure 2). all other cause variables had no significant influence on the time it took after stent implantation to identify incrustation by tas. time between stent placement and appearance of twinkling artifacts (tas) as a function of serum creatinine. conventional x - ray imaging can not be used to detect stent incrustation since uric acid crystals are x - ray translucent. conventional sonographic monitoring of the kidneys in patients with an indwelling stent can only differentiate between renewed urinary congestion due to obstruction by stent incrustation or manifest urolithiasis. during conventional color doppler sonography it was noted that colored spots, dubbed twinkling artifacts, appeared on crystalline surfaces such as arterial plaque or kidney stones [5, 10, 11 ]. these artifacts are common enough to cause considerable misinterpretation of flow where atheromatous vascular plaque occurs. however, with specific defined sonographic parameter selection it is possible to utilize twinkling artifacts to locate even minor incrustation in indwelling ureter splints. patients with impaired urine flow of the upper urinary tract and severe tumor load, require ureter splint implantation, and face increased risk of splint incrustation. tumor lysis syndrome (tls), resulting from the breakdown of dying cells induced by successful cancer treatment manifests itself with high phosphate and uric acid levels in both blood and urine. resulting splint incrustation from crystalline deposits can lead to severe urinary tract complications, including lumen obstruction and renewed kidney congestion. incrustation and protein precipitates in the implant can also lead to an infected biomatrix, possibly causing a urinary tract infection culminating in sepsis, particularly in immunocompromised patients. this investigation shows that serum creatinine negatively correlates with the time it takes for tas to become apparent during examination of the ureter stent : the higher the creatinine, the less time it takes for tas revealing incrustation to appear. this implies that patients with normal kidney function have a significantly lower risk for dj - stent incrustation, while patients with renal insufficiency could require more frequent monitoring by means of ta. removing a highly incrusted dj - stent can cause complications including mechanical strain to the urothelium, injuries, consecutive strictures, bleeding, impossible stent removal or ureter avulsion. since tas appear as a result of minute incrustation, but minute incrustation does not obstruct urine flow, identification and establishment of the rate of increase can facilitate decisions about removal, so that complications do not become manifest. this investigation shows that the use of sonographic twinkling artifacts may be especially useful in cases where a high rate of ureter stent incrustation can be expected, such as in patients at risk for tumor lysis syndrome. | introductionureter obstruction caused by a retro - peritoneal tumor is treated by inserting an indwelling ureter splint (dj - stent). indwelling duration is limited by cumulative crystalline deposits into the splint, eventually causing the repeated impairment of urine flow. deciding when a dj - stent must be replaced is important since belated removal can be accompanied by severe complications. x - ray or conventional sonography do not allow satisfactory evaluation of early incrustation, therefore, the use of sonographic twinkling artifacts (ta) to provide accurate stent surveillance was investigated.material and methods26 patients with indwelling ureter splints carrying a high risk of developing tumor lysis syndrome (tls), which is often accompanied by early splint incrustation, were investigated utilizing ta the day after dj - stent implantation and weekly thereafter. serum creatinine, uric acid, and urine ph were measured at all ta exams.resultsearly incrustation of the ureter splint was detected by ta in all patients 14 weeks after implantation. incrustation occurred sooner with increased uric acid levels, and high creatinine or acidic urine accelerated early implant incrustation.conclusionsta can be used to monitor early crystalline deposits in implanted ureter splints, before they can be detected by conventional sonography or x - ray imaging and before complications occur. |
recent data from routine clinical practice shows that once - daily prolonged - release formulations of tacrolimus result in improved graft survival in liver transplant recipients relative to twice - daily immediate - release tacrolimus.based on these data, a model was constructed to estimate life expectancy, numbers needed to treat to avoid graft failure and death, and costs associated with immunosuppressive medications and graft failure over 3 years after transplantation.while model outcomes were sensitive to tacrolimus dosing assumptions, prolonged - release tacrolimus (advagraf) resulted in improved patient and graft survival and reduced costs when compared with branded ir tacrolimus (prograf) in the base case analysis. liver transplantation is a highly effective treatment option for patients with end - stage liver disease, and as of 2014 there were approximately 8300 patients with a functioning liver transplant in the uk transplant registry, up from 7600 in 2009. one - year graft survival rates are now over 80 % and longer - term graft and patient survival have increased dramatically since the first liver transplants were conducted in the 1960s. while these improvements are a result of changes to many aspects of operative and peri - operative treatment implemented since the early transplants, improvements in post - transplant immunosuppression eras based on the availability of new immunosuppressive regimens and induction therapies at the time of transplant. jain. selected the introduction of ciclosporin, muromonab - cd3 (okt3), and tacrolimus as the cut - off points for three eras, corresponding to the periods spanning 198185, 198690, and 199198. their study of 4000 liver transplant recipients showed that survival in the tacrolimus era was significantly improved relative to the previous eras, reporting 10-year survival of 60 % compared with 52 % and 53 % for the okt3 and ciclosporin eras, respectively. the tacrolimus registration trials for liver transplant, published in 1994, showed a significant reduction in the incidence of acute rejection relative to ciclosporin, although no significant differences in mortality or graft loss compared to ciclosporin were observed over 12 months [4, 5 ]. specifically, the registration trials compared ciclosporin with twice - daily, immediate - release (ir) tacrolimus (prograf, astellas tokyo, japan) that is now a cornerstone of immunosuppressive therapy in liver transplant recipients. since the publication of the registration trials, tacrolimus has been reformulated into a once - daily, prolonged - release (pr) formulation (advagraf, astellas tokyo, japan), which received european medicines agency (ema) marketing authorization in 2007. in the european public assessment report accompanying the marketing authorization, regarding advagraf it is expected that it may help to improve compliance with dosing and that the modified - release profile would be expected to improve the variability in the exposure to tacrolimus. several studies in liver transplant recipients have since confirmed that intra - patient variability is indeed reduced with pr tacrolimus relative to ir tacrolimus, and that the majority of patients prefer once - daily dosing over twice - daily dosing and are more adherent to the once - daily regimen [914 ]. one randomized controlled trial (rct) of pr versus ir tacrolimus has been conducted to date, in which pr tacrolimus showed non - inferiority relative to ir tacrolimus in terms of the primary endpoint of biopsy - confirmed rejection at 24 weeks. while the open - label 12-month extension of the rct also showed non - inferiority in secondary endpoints of graft and patient survival, recent retrospective analyses of 3-year follow - up data from the european liver transplant registry (eltr) have been published demonstrating significant improvements in graft survival and numerical but not statistically significant improvements in patient survival with pr tacrolimus relative to ir tacrolimus. given these emerging data, the increasing size of the patient population with a functioning liver graft, and the concomitant increase in healthcare expenditure in these patients, the aim of the present analysis was to use data from the eltr analysis to project treatment costs, patient and graft life expectancy, and numbers needed to treat to avoid graft loss or death with pr tacrolimus relative to ir tacrolimus. a model was constructed in microsoft excel (microsoft corporation, redmond, wa usa) to project cost and effectiveness outcomes in de novo adult liver transplant recipients using pr tacrolimus (advagraf) or branded ir tacrolimus (prograf) as the primary immunosuppressive regimen in the uk setting. patient and graft survival rates were based on a retrospective analysis of data from the eltr. the eltr includes data on liver transplant recipients from 145 european transplant centers, 21 of which prescribed both pr and ir tacrolimus and were included in the analysis. in brief, adam. conducted a retrospective database analysis of primary liver transplant patients 18 years old who underwent their first liver transplant between january 2008 and december 2012 and received pr tacrolimus or ir tacrolimus, with or without concomitant immunosuppressive agents, within the first month after transplantation. the first analysis was of a modified intent - to - treat (mitt) population that excluded all patients with less than 1 month of post - transplant follow - up (to avoid the confounding factors of post - operative complications). the second analysis looked at the same endpoints in a propensity - score matched (psm) population, in which pr and ir tacrolimus patients were paired in a 1:2 ratio based on a propensity score. the propensity score was based on recipient age, recipient human immunodeficiency virus, hepatitis c and hepatocellular carcinoma status, united network for organ sharing (unos) status, creatinine levels, donor age, date of transplantation, total ischemia time, and administration of other immunosuppressive medications early post - transplant [ciclosporin, mycophenolate mofetil (mmf), corticosteroids, daclizumab, and basiliximab ]. the kaplan meier analyses of patient and graft survival in the mitt cohort were used to calculate rates of graft loss and mortality in the base case analysis. retransplantation rates were derived based on the assumption that retransplantation accounts for the entire difference between patient and graft survival. since the eltr analysis did not report tacrolimus dosing, data from the respective summaries of product characteristics (spc) were used to establish the initial doses of ir and pr tacrolimus in the base case, both of which were taken to be the mid - point of the spc - recommended starting dose range of 0.100.20 mg / kg / day. the initial dose was assumed to be maintained for 1 year after which the dose in both treatment arms was switched to match the end - of - study (eos) ir tacrolimus dose (0.58 mg / kg / day) from the truneka. mean patient bodyweight was taken to be 77.2 kg based on the weighted average from truneka.. the model was designed to evaluate the number needed to treat (nnt) to avoid one graft loss or one death with pr tacrolimus relative to ir tacrolimus, the life expectancy with pr relative to ir tacrolimus, and the number of graft years saved with pr relative to ir tacrolimus in addition to the costs associated with retransplantation and primary immunosuppressive therapy with each formulation. for the base case analysis, the per - milligram cost of pr tacrolimus (advagraf) and branded ir tacrolimus (prograf) were taken from the september 2014 british national formulary (bnf ; table 1). the bnf was also used as the source of an alternative per - milligram cost of generic ir tacrolimus (adoport, sandoz international gmbh, holzkirchen, germany) in one - way sensitivity analysis. the mean cost of liver retransplantation was assumed to be 1.84 times more costly than a first transplant based on the overall retransplant cost ratio reported by azoulay. in a single - center study of 1038 first liver transplants and 139 retransplants.table 1unit costs in cost - effectiveness analyses of prolonged - release (pr) tacrolimus versus branded and generic immediate - release (ir) tacrolimus as the primary immunosuppressive agents in renal transplant recipientscost itemcostreferencespr tacrolimus (advagraf)1.43 (per mg)british national formulary 68 ir tacrolimus (prograf)1.61 (per mg)british national formulary 68 ir tacrolimus (adoport), one - way sensitivity analysis only1.11 (per mg)british national formulary 68 liver retransplantation35,164.23 () nhs tariff information 2014 pounds sterling, ir immediate - release, nhs, national health service, pr prolonged - release unit costs in cost - effectiveness analyses of prolonged - release (pr) tacrolimus versus branded and generic immediate - release (ir) tacrolimus as the primary immunosuppressive agents in renal transplant recipients 2014 pounds sterling, ir immediate - release, nhs, national health service, pr prolonged - release the base case analysis was performed over a 3-year time horizon to avoid extrapolation of the underlying graft and patient survival data from the eltr. the model reported all outcomes annually and applied half - cycle correction to eliminate any systematic over- or underestimation of costs and effects. cost and effectiveness outcomes were measured from the perspective of the uk healthcare payer, and future costs and effects were discounted at 3.5 % per annum in the base case. sensitivity analyses were performed with a 1.5 % annual discount rate for both costs and effects in line with guidance from the national institute for health and care excellence. all analyses were run as probabilistic sensitivity analyses, in which uncertainty around patient body weight, the cost of liver retransplantation, and the kaplan standard errors around the kaplan meier curves were estimated based on binomial proportion 95 % confidence intervals around the percentage of patients and grafts surviving at each time point (eq. 1), ensuring that both were monotonically decreasing functions and that patient survival always equaled or exceeded graft survival. equation 1 : assumed standard error around kaplan meier projections of mortality and graft loss.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{se } } = \sqrt { \frac{1}{n } } p(1 - p) $ $ \end{document}se=1np(1-p) patient body weight was sampled using the weighted standard deviation (sd) body weight from the truneka. rct and a confidence interval around the ratio of retransplantation costs to first transplant costs was approximated using fieller s theorem from standard deviations reported in the azoulay. the sensitivity of the model to changes in individual input parameters was explored in a series of one - way sensitivity analyses. specifically, sensitivity analyses were conducted around the base case analysis in which the eltr psm population data were used in place of the mitt population. the sensitivity to dosing assumptions was investigated by using dosing data directly from the truneka. study for the first year of simulation, followed by holding the dose steady at the final dose as reported by truneka. at day 365. a further dosing sensitivity analysis was conducted in which a rational model (i.e., a ratio of a first- and second - order polynomials) was fitted to the truneka dose curves for each arm of the simulation. the correlation coefficients (r) of the rational model to the extracted data sets were 0.985 and 0.982 for pr tacrolimus and ir tacrolimus, respectively, and the models were used to extrapolate out to the full 3-year time horizon. finally, four cost - centric sensitivity analyses were conducted ; one in which the per - milligram cost of ir tacrolimus was set to the same as that for pr tacrolimus, a second in which the per - milligram cost of generic ir tacrolimus (adoport) was used in place of the branded ir tacrolimus (prograf) cost, and two analyses of retransplantation costs ; one in which the cost of retransplantation was set to the same cost as a first transplant and a second in which the cost of retransplantation was abolished. in line with guidance from the international society for pharmacoeonomics and outcomes research, a deterministic threshold analysis was conducted to establish the pr tacrolimus (advagraf) drug cost at which overall costs would be equivalent in the two treatment arms. the threshold analysis was conducted using both the base case cost of branded ir tacrolimus (prograf) and the cost of generic ir tacrolimus (adoport). a model was constructed in microsoft excel (microsoft corporation, redmond, wa usa) to project cost and effectiveness outcomes in de novo adult liver transplant recipients using pr tacrolimus (advagraf) or branded ir tacrolimus (prograf) as the primary immunosuppressive regimen in the uk setting. patient and graft survival rates were based on a retrospective analysis of data from the eltr. the eltr includes data on liver transplant recipients from 145 european transplant centers, 21 of which prescribed both pr and ir tacrolimus and were included in the analysis. in brief, adam. conducted a retrospective database analysis of primary liver transplant patients 18 years old who underwent their first liver transplant between january 2008 and december 2012 and received pr tacrolimus or ir tacrolimus, with or without concomitant immunosuppressive agents, within the first month after transplantation. the first analysis was of a modified intent - to - treat (mitt) population that excluded all patients with less than 1 month of post - transplant follow - up (to avoid the confounding factors of post - operative complications). the second analysis looked at the same endpoints in a propensity - score matched (psm) population, in which pr and ir tacrolimus patients were paired in a 1:2 ratio based on a propensity score. the propensity score was based on recipient age, recipient human immunodeficiency virus, hepatitis c and hepatocellular carcinoma status, united network for organ sharing (unos) status, creatinine levels, donor age, date of transplantation, total ischemia time, and administration of other immunosuppressive medications early post - transplant [ciclosporin, mycophenolate mofetil (mmf), corticosteroids, daclizumab, and basiliximab ]. the kaplan meier analyses of patient and graft survival in the mitt cohort were used to calculate rates of graft loss and mortality in the base case analysis. retransplantation rates were derived based on the assumption that retransplantation accounts for the entire difference between patient and graft survival. since the eltr analysis did not report tacrolimus dosing, data from the respective summaries of product characteristics (spc) were used to establish the initial doses of ir and pr tacrolimus in the base case, both of which were taken to be the mid - point of the spc - recommended starting dose range of 0.100.20 mg / kg / day. the initial dose was assumed to be maintained for 1 year after which the dose in both treatment arms was switched to match the end - of - study (eos) ir tacrolimus dose (0.58 mg / kg / day) from the truneka. mean patient bodyweight was taken to be 77.2 kg based on the weighted average from truneka.. the model was designed to evaluate the number needed to treat (nnt) to avoid one graft loss or one death with pr tacrolimus relative to ir tacrolimus, the life expectancy with pr relative to ir tacrolimus, and the number of graft years saved with pr relative to ir tacrolimus in addition to the costs associated with retransplantation and primary immunosuppressive therapy with each formulation. for the base case analysis, the per - milligram cost of pr tacrolimus (advagraf) and branded ir tacrolimus (prograf) were taken from the september 2014 british national formulary (bnf ; table 1). the bnf was also used as the source of an alternative per - milligram cost of generic ir tacrolimus (adoport, sandoz international gmbh, holzkirchen, germany) in one - way sensitivity analysis. the mean cost of liver retransplantation was assumed to be 1.84 times more costly than a first transplant based on the overall retransplant cost ratio reported by azoulay. in a single - center study of 1038 first liver transplants and 139 retransplants.table 1unit costs in cost - effectiveness analyses of prolonged - release (pr) tacrolimus versus branded and generic immediate - release (ir) tacrolimus as the primary immunosuppressive agents in renal transplant recipientscost itemcostreferencespr tacrolimus (advagraf)1.43 (per mg)british national formulary 68 ir tacrolimus (prograf)1.61 (per mg)british national formulary 68 ir tacrolimus (adoport), one - way sensitivity analysis only1.11 (per mg)british national formulary 68 liver retransplantation35,164.23 () nhs tariff information 2014 pounds sterling, ir immediate - release, nhs, national health service, pr prolonged - release unit costs in cost - effectiveness analyses of prolonged - release (pr) tacrolimus versus branded and generic immediate - release (ir) tacrolimus as the primary immunosuppressive agents in renal transplant recipients 2014 pounds sterling, ir immediate - release, nhs, national health service, pr prolonged - release the base case analysis was performed over a 3-year time horizon to avoid extrapolation of the underlying graft and patient survival data from the eltr. the model reported all outcomes annually and applied half - cycle correction to eliminate any systematic over- or underestimation of costs and effects. cost and effectiveness outcomes were measured from the perspective of the uk healthcare payer, and future costs and effects were discounted at 3.5 % per annum in the base case. sensitivity analyses were performed with a 1.5 % annual discount rate for both costs and effects in line with guidance from the national institute for health and care excellence. all analyses were run as probabilistic sensitivity analyses, in which uncertainty around patient body weight, the cost of liver retransplantation, and the kaplan meier projections of mortality and graft loss were captured. standard errors around the kaplan meier curves were estimated based on binomial proportion 95 % confidence intervals around the percentage of patients and grafts surviving at each time point (eq. 1), ensuring that both were monotonically decreasing functions and that patient survival always equaled or exceeded graft survival. equation 1 : assumed standard error around kaplan meier projections of mortality and graft loss.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{se } } = \sqrt { \frac{1}{n } } p(1 - p) $ $ \end{document}se=1np(1-p) patient body weight was sampled using the weighted standard deviation (sd) body weight from the truneka. rct and a confidence interval around the ratio of retransplantation costs to first transplant costs was approximated using fieller s theorem from standard deviations reported in the azoulay. the sensitivity of the model to changes in individual input parameters was explored in a series of one - way sensitivity analyses. specifically, sensitivity analyses were conducted around the base case analysis in which the eltr psm population data were used in place of the mitt population. the sensitivity to dosing assumptions was investigated by using dosing data directly from the truneka. study for the first year of simulation, followed by holding the dose steady at the final dose as reported by truneka. at day 365. a further dosing sensitivity analysis was conducted in which a rational model (i.e., a ratio of a first- and second - order polynomials) was fitted to the truneka dose curves for each arm of the simulation. the correlation coefficients (r) of the rational model to the extracted data sets were 0.985 and 0.982 for pr tacrolimus and ir tacrolimus, respectively, and the models were used to extrapolate out to the full 3-year time horizon. finally, four cost - centric sensitivity analyses were conducted ; one in which the per - milligram cost of ir tacrolimus was set to the same as that for pr tacrolimus, a second in which the per - milligram cost of generic ir tacrolimus (adoport) was used in place of the branded ir tacrolimus (prograf) cost, and two analyses of retransplantation costs ; one in which the cost of retransplantation was set to the same cost as a first transplant and a second in which the cost of retransplantation was abolished. in line with guidance from the international society for pharmacoeonomics and outcomes research, a deterministic threshold analysis was conducted to establish the pr tacrolimus (advagraf) drug cost at which overall costs would be equivalent in the two treatment arms. the threshold analysis was conducted using both the base case cost of branded ir tacrolimus (prograf) and the cost of generic ir tacrolimus (adoport). in the probabilistic base case analysis, graft and patient survival estimates matched those from the eltr mitt analysis (fig. 1). the mean nnt to avoid one graft loss with pr tacrolimus relative to ir tacrolimus over 3 years was 14 patients, while the corresponding nnt to avoid one death was 18. mean (sd) patient life expectancy over the 3-year time horizon was 31.52 (0.22) months in the pr tacrolimus arm versus 30.62 (0.09) months with ir tacrolimus, representing an increase of 0.89 (0.23) months, while graft survival was 1.07 (0.21) months higher with pr tacrolimus at 31.2 (0.19) months versus 30.2 (0.09) months with ir tacrolimus (table 2).fig. 1patient and graft survival over time based on the propensity - score matched and modified intent - to - treat analyses of the european liver transplant registry data. ir immediate - release, mitt modified intent - to - treat, psm propensity - score matched, pr prolonged - releasetable 2top - line probabilistic results from a 3-year analysis of the cost - effectiveness of prolonged - release (pr) versus immediate - release (ir) tacrolimus in liver transplant recipients in the ukir tacrolimus (prograf)pr tacrolimus (advagraf)differencecost of immunosuppression, 10,405 (2203)9469 (2006)937 (208)cost of retransplantation, 1654 (443)949 (689)705 (820)total cost, 12,062 (2245)10,420 (2130)1642 (885)life expectancy, months30.62 (0.09)31.52 (0.22)+0.89 (0.23)graft life expectancy, months30.16 (0.09)31.23 (0.19)+1.07 (0.21)annualized probability of graft loss0.0640.0390.025nnt to avoid graft loss with pr vs. ir tacrolimus14annualized probability of death0.0580.0390.019nnt to avoid death with pr vs. ir tacrolimus18values are presented as mean (standard deviation) 2014 pounds sterling, ir immediate - release, nnt number needed to treat, pr prolonged - release patient and graft survival over time based on the propensity - score matched and modified intent - to - treat analyses of the european liver transplant registry data. ir immediate - release, mitt modified intent - to - treat, psm propensity - score matched, pr prolonged - release top - line probabilistic results from a 3-year analysis of the cost - effectiveness of prolonged - release (pr) versus immediate - release (ir) tacrolimus in liver transplant recipients in the uk values are presented as mean (standard deviation) 2014 pounds sterling, ir immediate - release, nnt number needed to treat, pr prolonged - release these increases in effectiveness were accompanied by mean (sd) per - patient cost savings with pr tacrolimus (advagraf) of 1642 (885) over 3 years, with pr tacrolimus (advagraf) thereby exhibiting dominance over the branded ir formulation (prograf). pr tacrolimus (advagraf) was less costly and more effective than branded ir tacrolimus (prograf) in 9559 (95.6 %) of 10,000 iterations (fig. 2). pr tacrolimus (advagraf) was more costly and more effective than branded ir tacrolimus (prograf) in 439 analyses, in which the mean incremental cost - effectiveness ratio (icer) was 4282 per life year gained. two analyses (0.02 % of model iterations) showed reduced effectiveness and reduced costs with pr tacrolimus (advagraf) relative to branded ir tacrolimus (prograf).fig. 2cost - effectiveness scatterplot showing incremental per - patient costs and life expectancy from 10,000 model iterations over a 3-year time horizon. ir immediate - release, pr prolonged - release cost - effectiveness scatterplot showing incremental per - patient costs and life expectancy from 10,000 model iterations over a 3-year time horizon. ir immediate - release, pr prolonged - release findings of one - way sensitivity analyses are presented in table 3. the largest effect on the incremental cost outcomes was observed when the dose data for each arm was based on the dose curves reported in the truneka. rct, in which pr tacrolimus (advagraf) was associated with an increase in costs of 1350 per patient over 3 years relative to branded ir tacrolimus (prograf), resulting in an icer of 18,255 per life year gained. switching the per - milligram ir tacrolimus cost to that of generic tacrolimus (adoport) resulted in incremental costs of 1556, yielding an icer of 21,078 per life year gained for pr tacrolimus (advagraf) relative to generic ir tacrolimus (adoport). using the psm outcomes data from the eltr had a large effect on both incremental costs and effects ; incremental life expectancy increased to 2.00 months, while cost savings decreased to 763 as a result of the increased patient and graft survival with pr tacrolimus (advagraf). the rational model fit and extrapolation from the truneka dosing curves also had a relatively large effect on cost, reducing modelled cost savings with pr tacrolimus (advagraf) to 1237 per patient over 3 years.table 3summary of one - way sensitivity analyses around the base case analysislife expectancy (months)costs () icer (per life year gained)ir tacrolimuspr tacrolimusdifferencebranded ir tacrolimus (prograf)pr tacrolimus (advagraf)differencebase case30.62 (0.09)31.52 (0.22)+0.89 (0.23)12,062 (2245)10,420 (2130)1642 (885)pr dominant1.5 % discount rate31.48 (0.09)32.41 (0.22)+0.93 (0.24)12,502 (2290)10,788 (2170)1714 (895)pr dominanttruneka ir dosing in both arms, held at eos dose 30.62 (0.09)31.52 (0.22)+0.89 (0.23)10,102 (1824)8641 (1760)1461 (866)pr dominanttruneka ir and pr dosing, held at eos dose 30.62 (0.09)31.51 (0.22)+0.89 (0.23)10,098 (1837)11,449 (2340)+1350 (964)18,255rational model fit to truneka ir and pr dose curves 30.62 (0.09)31.52 (0.22)+0.89 (0.23)7445 (1298)6208 (1301)1237 (849)pr dominanteltr psm data used in place of mitt30.05 (0.21)32.06 (0.21)+2.00 (0.30)11,557 (2346)10,794 (2221)763(1220)pr dominantir cost equivalence with pr tacrolimus30.62 (0.09)31.52 (0.22)+0.89 (0.23)10,973 (2114)10,420 (2130)553 (855)pr dominantir cost equivalence with generic ir tacrolimus (adoport)30.62 (0.09)31.52 (0.22)+0.89 (0.23)8862 (1578)10,420 (2130)+1556 (981)21,078cost of retransplant same as first transplant30.62 (0.09)31.52 (0.22)+0.89 (0.23)11,341 (2189)10,005 (2025)1336 (513)pr dominantcost of retransplant abolished30.62 (0.09)31.52 (0.22)+0.89 (0.23)10,476 (2186)9533 (1989)943 (207)pr dominantvalues are presented as mean (standard deviation) 2014 pounds sterling, eltr european liver transplant registry, eos end of study, ir immediate - release, mitt modified intent - to - treat, pr prolonged - release, psm propensity - score matched summary of one - way sensitivity analyses around the base case analysis values are presented as mean (standard deviation) 2014 pounds sterling, eltr european liver transplant registry, eos end of study, ir immediate - release, mitt modified intent - to - treat, pr prolonged - release, psm propensity - score matched deterministic threshold analysis showed that the pr tacrolimus (advagraf) breakeven price (the price at which the cost in both model arms is equivalent) would be 1.77 per milligram when branded ir tacrolimus (prograf) was used for the analysis, 0.34 per milligram higher than the current per - milligram cost of pr tacrolimus (advagraf) in the bnf. threshold analysis using the cost of generic ir tacrolimus (adoport) resulted in a breakeven price of 1.28 per milligram, 0.15 per milligram lower than the per - milligram cost of pr tacrolimus (advagraf) in the bnf. the additional 0.15 per milligram for advagraf yielded 0.89 additional months of life over the model time horizon (resulting in the icer of 21,078 per life year gained as reported in one - way sensitivity analysis, table 3). the present study showed that, based on a recent retrospective analysis of data from 4367 patients in the eltr, pr tacrolimus would be expected to be associated with gains in life expectancy and graft survival relative to ir tacrolimus, while reducing costs borne by the healthcare payer (in comparison to branded ir tacrolimus (prograf). as with any modeling analysis, the present study has a number of limitations that should be acknowledged. the largest limitation of the analysis was the use of heterogeneous data sources to model the clinical outcomes and dosing of the pr and ir tacrolimus regimens. dose data were not recorded in the eltr and as such did not form part of the retrospective analysis by adam. that underpinned the clinical aspects of the model. the most important consequence of this data heterogeneity was that clinical effectiveness outcomes were derived from a different dataset from the estimates of pharmacy dosing and hence also pharmacy costs. to establish the effect of dosing assumptions on model outcomes, an extensive series of sensitivity analyses were conducted around the base case analysis, including switching the model to use dosing data from the truneka. study and either holding the projected dose flat at the eos dose or projecting the dose out using a rational model fit to the truneka dose data. the base case analysis used the bnf unit costs for pr tacrolimus (advagraf) and ir tacrolimus (prograf) to reflect the tacrolimus formulations used in the eltr study on which the clinical outcomes were based. other generic formulations of ir tacrolimus are listed in the bnf, including adoport, which is currently the cheapest twice - daily formulation at 1.11 per milligram. sensitivity analysis using the adoport price showed that using pr tacrolimus (advagraf) in place of adoport would result in an icer of 21,078 per life year gained based on an increase in life expectancy of 0.89 months, while threshold analysis showed the pr tacrolimus (advagraf) breakeven price to be 0.15 per milligram (10.5 %) lower than the current pr tacrolimus (advagraf) list price. taken together, the analyses show that the additional 0.15 per milligram spend on pr tacrolimus (advagraf) resulted in an average of 0.89 additional months of life per patient over a 3-year time horizon. the retransplantation cost estimate was based on the nhs tariff for an adult hepatobiliary transplant multiplied by a cost ratio (of second versus first liver transplant) derived from a single - center analysis. while the size of the population analyzed was large enough (n = 1177) to capture a wide range of surgical complications and indications for transplant and retransplant, center - specific practices and protocols may have affected the cost estimates presented and the final cost estimate may not be applicable to other centers. retransplantation was captured in the model as the difference between patient survival and graft survival. given that retransplantation is the only treatment option for liver graft failure, this assumption is clinically realistic but, while the inclusion of retransplantation is also economically important given the high cost associated with the procedure, its role as a driver of incremental costs is challenging. notably, local organ availability and center - specific ethical considerations such as outcomes - based versus urgency - based approaches to retransplant prioritization make the incidence of retransplantation less of a clinical consideration and more of a logistical and ethical issue. to establish the extent to which retransplantation was driving cost outcomes, sensitivity analyses were conducted in which the cost of retransplantation was firstly set to the same cost as a first liver transplant and, in a separate analysis, abolished completely. both analyses yielded cost savings with pr tacrolimus (advagraf), but the magnitude of the savings was reduced relative to the base case analysis. as the main source of clinical data in the present analysis, the eltr study design and its limitations should also be considered when interpreting the findings of the present analysis. an editorial that accompanied the original manuscript noted that the eltr data is subject to reporting bias (in that it is collected on a voluntary basis) and that characteristics of the patients on ir and pr tacrolimus differed in terms of their age, concomitant mediation use, serum creatinine levels, hepatitis delta or hepatocellular carcinoma (hcc) as the primary indication, and donor age. the psm analysis attempted to address these known differences, but extraneous factors such as socioeconomic differences may have persisted and, as noted in the editorial, 49 % of patients on pr tacrolimus remained unmatched in the psm analysis. the lack of randomization may have also resulted in bias arising from assignment of sicker patients to receive the longer established ir tacrolimus regimen and the effect of the choice of included eltr centers should not be ignored. the authors of the eltr data analysis noted that the 21 centers using pr tacrolimus and ir tacrolimus were selected to prevent center bias, but it is conceivable that outcomes with ir tacrolimus in the 21 included centers may differ from those in the remaining 124 centers participating in the eltr using ir tacrolimus exclusively. while such criticisms of observational data are entirely valid, these issues are not unique to data from routine clinical practice, with small - scale rcts suffering from many of the same methodological issues. in the present analysis, the mitt data were used in the base case and a sensitivity analysis was conducted with the psm data to explore the extent to which the mortality and graft loss outcomes affected the analysis. pr tacrolimus (advagraf) remained cost saving in the psm analysis, but the life expectancy benefit increased to 2.00 months over the 3-year time horizon, extending the dominance of pr tacrolimus (advagraf) over branded ir tacrolimus (prograf). certain drivers of costs were intentionally omitted from the present analysis, including surgical complications, new onset diabetes after transplantation, cytomegalovirus infection, and the myriad costs associated with various recurrent indications for liver transplant such as hcc and hcv. while these sequelae and complications contribute to the absolute cost of treating liver transplant recipients, differences in the incidence would not be anticipated to drive incremental cost or effectiveness outcomes between two tacrolimus formulations. cost estimates in the present analysis should not therefore be considered instructive for the purposes of budget impact analysis. based on the emerging data from the eltr in concert with the previously established non - inferiority in terms of biopsy - confirmed acute rejection, we consider the model to be comprehensive in terms of its ability to capture drivers of incremental costs and effects between the two tacrolimus formulations. based on the present analysis, pr tacrolimus would be expected to prevent one graft loss for every 14 patients and one death for every 18 patients initiated on pr tacrolimus rather than ir tacrolimus. furthermore, pr tacrolimus (advagraf) would be likely to reduce costs associated with immunosuppressive treatment and retransplantation by up to 1642 (885) per patient over 3 years versus branded ir tacrolimus (prograf). these findings, combined with the well established patient preference for once - daily over twice - daily dosing [10, 13 ], and the recent publication of clinical data showing a graft survival benefit with once - daily tacrolimus, provide a strong case for the preferential use of pr tacrolimus over ir tacrolimus in adult liver transplant recipients in the uk setting. rfp is a full - time employee of ossian health economics and communications gmbh, which received consultancy fees from astellas pharma emea limited to construct the model and write the manuscript. gm and io are full - time employees of astellas pharma emea limited, a subsidiary of astellas pharma inc., which manufactures prolonged - release and immediate - released tacrolimus. this article does not contain any studies with human participants or animals performed by any of the authors. rfp developed the cost - effectiveness model, ran the analyses, and drafted the manuscript. gm formulated the research question, reviewed the model, and provided critical revisions to the manuscript. | backgroundas of 2014, there were approximately 8300 patients with a functioning liver transplant in the uk transplant registry, with 880 liver transplants performed in 20132014 alone. tacrolimus, typically used in combination with steroids and mycophenolate mofetil, currently represents the cornerstone of post - transplant immunosuppression in liver transplant recipients.objectivesthe objective of the present study was to evaluate the cost - effectiveness of prolonged - release (pr) tacrolimus (advagraf, astellas pharma inc., tokyo, japan) versus branded immediate - release (ir) tacrolimus (prograf, astellas pharma inc., tokyo, japan) in liver transplant recipients in the uk.methodsa model was developed in microsoft excel to estimate costs associated with immunosuppressive medications and retransplantation. three - year patient and graft survival data were taken from a recent retrospective registry analysis and dose data were taken from prescribing information. costs in 2014 pounds sterling were taken from the british national formulary and the national health service national tariff.resultsover a 3-year time horizon, the numbers needed to treat with pr tacrolimus relative to ir tacrolimus were 14 to avoid one graft loss and 18 to avoid one death. the model was sensitive to dosing assumptions, with incremental cost estimates varying between a saving of 1642 (standard deviation 885) per patient, assuming the same per - kilogram dosing of pr tacrolimus (advagraf) and ir tacrolimus (prograf) and an increase of 1350 (964) using rct dose data.conclusiondata from a recent analysis of routine clinical practice data in liver transplant recipients on pr tacrolimus and ir tacrolimus showed significant differences in long - term graft survival in favor of pr tacrolimus. modeling these data in the uk showed that, over a 3-year time horizon, one graft would be saved for every 14 patients treated with pr tacrolimus with minimal impact on costs when compared with branded ir tacrolimus (prograf). |
wrist injuries caused by fatigue at work or improper exercise are very common. for example, carpal tunnel syndrome, cubital tunnel syndrome, stenosing tenosynovitis, stenosing tenosynovitis of the flexor tendons, locked finger, thecal cyst, and other symptoms cause great trouble for patients, because in in addition to receiving medical treatment, they have to undergo regular physical therapy1,2,3,4,5,6. common exercises include wrist stretches, grip strengthening, wrist extension, wrist radial deviation, wrist flexion, etc. these exercises mainly enhance or recover the patients range of motion of the wrist, including wrist radial and ulnar deviation, flexion, and extension movements, as presented in fig. although these simple wrist exercises are helpful for enhancing hand strength and preventing future injuries, traditional rehabilitation exercises are quite boring, so patients find it difficult to regularly perform the exercises, thus delaying full recovery. rehabilitation through physical therapy needs to be progressive and sustainable, and the early success of physical therapy training often influences further training and recovery. chang. stated that recovery of hand function is the most desired outcome for stroke patients, and that wearing a dynamic hand splint for home - use as a supplementary training program in addition to hospital - based rehabilitation can effectively increase the muscle strength of hemiplegic hands7. kim. reported that passive range of motion exercise in the early stage can improve the function of the upper extremities and activities of daily living of patients with acute stroke8. chang. investigated the jebsen - taylor hand function test, and found that task performance with the cock - up splint was slower than without the splint for all items9. in the current age of advancing information advancements and wireless technology, described a training method that combines a blobo bluetooth ball with music in cooperation with hand gestures to control the game s character s motions in order to enhance subjects motivation to participate in the activity. other kinds of music can be added to make the game even more appealing10. lin noted that patients who have suffered strokes are motivated to participate in rehabilitation that involves an audio - visual game controlled by a blobo bluetooth ball. chen. reported that patients intention to participate in physical therapy increased when the traditional words, digits, and diagrams of the rehabilitation system were replaced by a game with a space shuttle passing through obstacles, a 3d animation game integrated with a human - computer interactive interface of the wrist joint motion rehabilitation system12. this study tested a newly developed lively game using a blobo bluetooth ball that can train and evaluate the progress of wrist rehabilitation with the aim of evaluating its efficacy in the recovery of wrist functions. the hardware includes an rfid reader (including tag), blobo bluetooth ball, bluetooth device, etc. the software includes microsoft visual studio c #, microsoft sql server, adobe flash cs4, etc. visual studio c # is the major program executive, while sql server is used to manage the rfid and store the data of the motion of the blobo bluetooth ball. flash files are plugged in under visual studio c#. the blobo bluetooth ball was developed and manufactured by ball - it, finland13. multiple sensors are installed in the sphere of the blobo : a g - sensor (a triaxial accelerometer detecting g force and sphere movement), a pressure sensor (detecting whether the sphere is squeezed), an electronic gyroscope (calculating the inclination of the sphere), etc. for physical therapy, traditional movements such as wrist flexion, wrist extension, wrist radial deviation, and wrist ulnar deviation, are performed by manipulating the blobo bluetooth ball. 3.wrist training holding a blobo bluetooth ball demonstrates the movements of wrist flexion and wrist extension while holding a blobo bluetooth ball ; figure 3 (b) shows the movements of wrist radial deviation and ulnar deviation while holding a blobo bluetooth ball. wrist training holding a blobo bluetooth ball to launch the system, the user has to throw the blobo bluetooth ball up into the air. once the blobo bluetooth ball is wirelessly linked to a computer through bluetooth, it is ready to be used. the ball is held and moved back and forth horizontally, squeezed, and turned (clockwise, counterclockwise) ; the actions are detected by the built - in electronic gyroscope, pressure sensor and g - sensor, and are used to synthesize the motions (swinging horizontally, confirming, and whirling) used within the computer game. the data retrieved from the game is stored in a sql server database for subsequent analysis of the training effect. the blobo bluetooth ball is first activated by throwing it up or shaking it, which initiates the computer link the blobo bluetooth ball. the database for storing the data needs to be created before authentication work can proceed. the administrator can add, delete, and amend data according to the electronic tags and identity management in the database. after authentication and selection of the level of difficulty (easy, medium, or difficult) and the hand to be trained (right or left), the user can play the manual music player game. when the blobo bluetooth ball is rotated or swung, it activates the music playing function in c#. if the user stops the motions, it arrests the music function in c#. depending on the patient s symptoms, the way the patient holds the blobo bluetooth ball will vary for flexion, extension, ulnar deviation, and radial deviation. the subjects swung the blobo bluetooth ball horizontally and rotated it clockwise (counterclockwise) to play the music while squeezing and patting it to turn the volume up or down. the system records and analyzes the length of time taken and the occurrence of pauses for clinical reference. the experiment consisted of two trials separately involving subjects with normal wrist function, and subjects with impaired wrist function. eight subjects with normal wrist function were invited to participate in 10 training sessions and tests for this study to determine whether the same evaluation effect is generated in terms of the dominant hand to validate the stability and repeatability of the system14, 15. eight subjects with impaired wrist function also participate in training and tests twice a week for eight weeks (three rounds / time). they signed the taipei medical university hospital institutional review board - approved informed consent form prior to participating in this study. each subject was initially interviewed to obtain participant s background information, and exercise habits. the assessments of the design of the game and analysis of the subject s behavior were based on the original length of the music (sec), the length of time taken to play the music (sec), and the occurrence of pauses (occurrence). with the cooperation of the joints, muscles, and bones, hands are able to complete a variety of complicated motions. wrists make the following motions : flexion, extension, ulnar deviation, and radial deviation. this study used the range of motion (rom) of these four motions as indicators. prior to the test, the subjects practiced once and then performed the test twice. the outcome measures were examined before the start of the study, at the end of the fourth week, and at the end of the eighth week. the hand rehabilitation training device developed for testing in the study was verified using subjects with normal wrist function and subjects with impaired wrist function. table 1table 1.the basic characteristics of the subjects with normal wrist functionparticipantsr1r2r3r4l1l2l3l4gender (m / f)mfmfmmfmage4235285356483528height (cm)170159180162168183155175weight (kg)8058845072854873dominant siderightleft shows the 10 training sessions and tests performed by the eight subjects with normal wrist function (r1r4 had dominant right hands while l1l4 had dominant left hands). table 1 shows the averages of the 10 training sessions for either hand performed by the subjects with normal wrist function at the easy, medium, and difficult levels of difficulty (the original lengths of the music were 247 sec, 245 sec, and 237 sec, respectively). based on the length of time taken to play the music and the number of pauses, the subjects all performed better with their dominant hand (p<0.05). table 2table 2.the basic characteristics of the subjects with impaired wrist functionparticipants (p1p8)8 (male:5, female:3)age41.357.52height (cm)163.82 6.95weight (kg)62.519.38dominant side3 (left)5 (right)affected side (left)21affected side (right)14 shows the basic information of the eight subjects with impaired wrist function (p1p8). the subjects participated in training sessions and tests twice a week (three sets / session). the subjects were asked to train repeatedly by holding the blobo bluetooth ball and making flexion, extension, ulnar deviation, and radial deviation movements. p1p8 advanced to the next level of difficulty in the eight - week training when the average length of time taken to play the music and the average number of pauses / fell below threshold values determined the performance of the subjects with normal wrist function : an average time within 120% of the original length of the music, and an average number of pauses in easy (medium) of less than 100 (120) times. table 3table 3.performance measures of the subjects with impaired wrist function in the training of process performanceweeksubject & item p1p2p3p4p5p6p7p8wk-2leveleeeeeeeea.u.t.285.611.8289.315.2308.318.6316.620.7318.918.5291.518.5293.621.3321.519.6a.p.78.277.485.893.6102.395.2883101.8skip levelyynnnyynwk-4levelmmeeemmea.u.t.308.715.5312.518.3289.515.7299.320.3309.616.2311.519.3298.517.2313.616.8a.p.87.585.382.987.385.7109.797.696.4skip levelnnynnnnnwk-6levelmmmeemmea.u.t.283.615.8282.320.8315.519.1292.511.5295.312.1302.617.2287.315.4296.115.7a.p.78.772.678.583.778.398.285.388.5skip levelyynyynyywk-8levelddmmmmdma.u.t.301.718.9298.615.5302.320.3318.521.3321.520.3298.516.6293.713.4323.721.3a.p.89.283.677.480.785.692.899.7108.6wrist sidedominant rrrrrlllaffected rrrrlllrlevel : e : easy, m : medium, d : difficult. a.u.t. : r : right, l : left indicates the performance of the subjects with impaired wrist function during the training. level : e : easy, m : medium, d : difficult. a.u.t. : r : right, l : left as presented in table 3, all of the eight subjects advanced to the next level of difficult during the eight - week training. p1, p2, and p7 advanced to the difficulty level in week 8 because their dominant hand was the affected hand. although their dominant hand was the affected hand, because their diagnosis was stenosing tenosynovitis, their ability to hold the blobo bluetooth ball was worse than that of the subjects with carpal tunnel syndrome. therefore, they could not manipulate the blobo bluetooth ball easily and only achieved the medium level of difficulty in week 8. the performance evaluation was measured at the limit when subjects did not feel pain in flexion, extension, ulnar deviation, and radial deviation. table 4table 4.outcome measures testing results of the patientsindicators & subjectsflexion (deg)extension (deg)ulnar deviation (deg)radial deviation (deg)premiddlepostpremiddlepostpremiddlepostpremiddlepostp1 (r, r)404653303440282828161617p2 (r, r)374349273136272828181818p3 (r, r)7172726263651516186911p4 (r, r)707171606163161718579p5 (r, l)333641283135282828161717p6 (l, l)7373736262631516165710p7 (l, l)384551283238272728171818p8 (l, r)343844242831272828171717(r, l) means the dominant hand is the right (r) while the affected hand is the left (l), and vice versa. pre : at the start of the intervention, middle : at the end of week 4, post : at the end of the intervention shows that the rom of the eight subjects with impaired wrist function improved by 813 in flexion ; by 710 in extension ; by 13 in ulnar deviation ; and by 45 in radial deviation. (r, l) means the dominant hand is the right (r) while the affected hand is the left (l), and vice versa. pre : at the start of the intervention, middle : at the end of week 4, post : at the end of the intervention traditional wrist rehabilitation is repetitive, monotonous, and boring, and the training progress can not be automatically stored and analyzed. this study introduced and tested an interactive multimedia training game using a blobo bluetooth ball with the aim of providing a diversified and lively physical therapy method for wrist rehabilitation. the motivation to participate in training can be enhanced through a fun interactive multimedia device, making traditionally tedious and boring training or health enhancement activities more interesting. the introduction of the rfid technique in this study for identification and authentication allows rapid recording, retrieval, and analysis of data acquired by the rehabilitation game, including training dates, length of time, etc. the system developed for this study automatically collects the data regarding the participants performance, and computes increases in levels of difficulty based on the length of time taken to play music during the training. after the training, all the participants wrist rom were performed better than at the beginning of the physical therapy, proving that wrist rehabilitation with the blobo bluetooth ball has positive effects and is worthy of further study. | [purpose ] the introduction of emerging technologies such as the wireless blobo bluetooth ball with multimedia features can enhance wrist physical therapy training, making it more fun and enhancing its effects. [methods ] wrist injuries caused by fatigue at work, improper exercise, and other conditions are very common. therefore, the reconstruction of wrist joint function is an important issue. the efficacy of a newly developed integrated wrist joint rehabilitation game using a blobo bluetooth ball with c # software installed was tested in wrist rehabilitation (flexion, extension, ulnar deviation, radial deviation). [results ] eight subjects with normal wrist function participated in a test of the system s stability and repeatability. after performing the blobo bluetooth ball wrist physical therapy training, eight patients with wrist dysfunction experienced approximately 10 improvements in range of motion (rom) of flexion extension, and ulnar deviation and about 6 rom improvement in radial deviation. the subjects showed progress in important indicators of wrist function. [conclusion ] this study used the blobo bluetooth ball in wrist physical therapy training and the preliminary results were encouraging. in the future, more diverse wrist or limb rehabilitation games should be developed to meet the needs of physical therapy training. |
as the older adult population grows, the number of older drivers also increases. these drivers tend to drastically reduce the amount that they drive, since many are retired and have a limited range of everyday and economic activities. additionally, drivers abilities are affected by their normal aging as well as geriatric diseases, such as declines in visual function, attention and processing speed, physical movement, and visual perception, along with cognitive disabilities, diabetes mellitus and cardiovascular diseases1. therefore, it is necessary to be able to accurately assess driving abilities and to retrain older individuals in order to reduce the risk of motor vehicle collisions. previous studies have introduced various measures for examining and predicting driving performance declines and the accident risk aging adults for use in research and clinical settings2. in gerontology, some researchers have recognized the diversity of older adults and classified them into three sub - groups : young - old (6574), middle - old (7584), and oldest - old (85 and above)3. this study evaluated two self - report questionnaires assessing the driving performance of young - old self - driving adults : the safe driving behavior measure (sdbm) and the driving habits questionnaire (dhq)4, 5. the purpose of this study was to investigate the accuracy of the sdbm and dhq in identifying the history of collisions in community - dwelling young - old self - driving adults. a total of 45 community - dwelling young old self - driving adults, recruited from three senior community centers, participated in this study. this study was carried out in accordance with the international ethical guidelines and the declaration of helsinki and was approved by the local institutional review board of chosun university. the inclusion criteria were as follows : age between 65 to 74 years, valid driver s license, driving at the time of recruitment, cognitive ability to complete the sdbm and the dhq, no missing limbs or major psychiatric diagnosis, and absence of neurological deficits or severe orthopeadic diseases that might have impaired driving skills. table 1table 1.demographic profile and driving history of the participants (n=45)variablesparticipantsgender (male / female)34 (75.6%)/11 (24.4%)age (years)68.36 3.18height (cm)166.76 6.95weight (kg)66.33 7.58accident history (yes / no)26 (57.8%)/19 (42.2%)driving exposure (months)269.80 102.01mini - mental state examination (scores)28.80 1.29mean standard deviation displays the clinical and demographic characteristics of the participants, including gender, age, height, weight, and driving exposure. mean standard deviation all the participants completed the sdbm and the dhq. the sdbm is a self - report questionnaire of safe driving behaviors that assesses the physical and cognitive aspects of driving such as car controls or features, responses to physical and social factors, and responses to environmental factors. the 68 items are divided into three domains : person - vehicle (11 items), person - environment (42 items), and person - vehicle - environment (15 items). responses are made using a 5-point likert scale ranging from 1 (can not do) to 5 (not difficult), resulting in a total possible score of 340 points. previous studies have demonstrated the good validity and reliability of this instrument4, 6. its counterpart, the dhq is interviewer - administered, and consists of 34 items grouped into six domains, of : current driving status and miscellaneous issues (10 items), driving exposure (4 items), dependence on other drivers (2 items), driving difficulty (8 items), self - reported crashes and citations (4 items), and driving space (5 items). options for rating each item range from 1 (i drive) to 3 (this person drives), or from 1 (so difficult i no longer drive in that situation) to 5 (no difficulty) so that the domain is scored on a 100-point scale. like the sdbm this study focused on only three domains of the dhq : driving difficulty, self - reported crashes and citations, and driving space in order to obtain data on the three domains in relation to traffic accidents. descriptive statistics were used to analyze the demographic characteristics and driving habits, such as current driving status and miscellaneous issues, and driving exposure of the participants. to determine the cut - off values for the sdbm and the dhq, this study used the receiver operating characteristic (roc) curve, and the area under the curve (auc). the auc indicates the probability that young - old self - driving adults who have had collisions experience will be correctly identified. multivariate logistic regression analysis was employed to identify the predictors of the history of accident in community - dwelling young - old self - drivers. analysis was performed with the aid of pasw version 18.0 for windows (spss inc., this study examined 45 licensed drivers (34 males and 11 females) with a mean age of 68.36 years, a mean height of 166.76 cm, and a mean body weight of 66.33 kg. the mean duration of driving experience was 269.80 months, and the mean mmse score was 28.8 (table 1). the cut - off value, auc, sensitivity, and specificity of the sdbm and dhq of community - dwelling young - old self - drivers were examined. the sdbm, cut - off values were found for the person - vehicle domain (53.5), person - environment domain (186.5), person - vehicle - environment domain (52.5). the cut - off values the dhq were 84.3 for difficulty, 0.5 for accidents and citations, and 3.5 for driving space. the auc values for the person - vehicle domain, person - environment domain, and person - vehicle - environment domain of the sdbm were 0.501, 0.517, and 0.549 respectively, and 0.593, 0.616, and 0.669 respectively. the sensitivity was 0.538 for all three domains of the sdbm, while for the dhq, the values for difficulty, crash and citations, and driving space were 0.577, 0.423, and 0.615, respectively, for the dhq. the specificities of in the person - vehicle domain, person - environment domain, and person - vehicle - environment domain of the sdbm were 0.474, 0.526, and 0.421, respectively. for the dhq, difficulty, crash and citations, and driving space had values of 0.526, 0.211, and 0.421, respectively (table 2table 2.sensitivity and specificity of the sdbm and dhq of community - dwelling young - old self - drivers (n=45)variablecut - off valuearea under the curvesensitivity (%) specificity (%) sdbmpv53.5000.5010.5380.474pe186.5000.5170.5380.526pve52.5000.5490.5380.421dhqdifficulty84.3250.5930.5770.526crash and citations0.5000.6160.4230.211driving space3.5000.6690.6150.421dhq : driving habits questionnaire ; pv : person - vehicle domain ; pe : person - environment domain, pve : person - vehicle - environment domain ; sdbm : safe driving behavior measure). according to multivariate regression analysis, the sdbm and dhq were not significant predictors of collision history of community - dwelling young - old self - drivers. none of the three domains of the sdbm (person - vehicle, person - environment, and person - vehicle - environment) significantly predicted collision history, and the three domains of the dhq (difficulty, crash and citations, and driving space) were also not significant predictors of collision history (table 3table 3.multiple regression analysis of history of collisions and various driving elements (n=45)variablebsebetatpsdbmpv0.0390.0450.2330.8640.393pe0.0010.0100.0320.0700.945pve0.0050.0140.1160.3850.703dhqdifficulty0.0060.0120.1040.4970.622crash and citations0.1350.0800.2481.6960.098driving space0.1110.0580.3311.9140.063constant1.4741.6780.8780.385r2=0.098dhq : driving habits questionnaire ; pv : person - vehicle domain ; pe : person - environment domain, pve : person - vehicle - environment domain ; sdbm : safe driving behavior measure). dhq : driving habits questionnaire ; pv : person - vehicle domain ; pe : person - environment domain, pve : person - vehicle - environment domain ; sdbm : safe driving behavior measure dhq : driving habits questionnaire ; pv : person - vehicle domain ; pe : person - environment domain, pve : person - vehicle - environment domain ; sdbm : safe driving behavior measure this study investigated the accuracy of the sdbm and dhq in identifying the automobile accident history of community - dwelling young - old self - driving adults. first, the auc was the greatest for the driving space of the dhq, and the lowest for the person - vehicle domain of the sdbm. second, overall the auc was higher for the dhq than for the sdbm. finally, neither the sdbm or dhq were well - explained predictors of collision history. traffic accident - related injuries of the elderly are steadily increasing, and are becoming a major social problem in post - industrial societies as the older population grows. therefore, screening for problematic driving behaviours of risk of older self - driving adults is vital. previous researchers have studied screening tests for risk of traffic collisions in older drivers2, 7. however, it is insufficient to base screening of driving abilities or the risk of traffic collisions on sub - groups of age, as older adults vary in terms of their physical and cognitive abilities. therefore, this study evaluated two major measurement tools, the sdbm and the dhq. previous studies have identified relationships between aging - related conditions (visual impairment, cognitive impairment, or motor impairment) and problematic traffic collisions5, 8, 9. they reported that older drivers with cataract experience a restriction in their driving mobility and a decrease in their safety on the road5. reported that the prospective follow - up of cohort of older drivers, including both prospective collision records and identification of at - fault status, would help to clarify the relationship between vision, cognition and motor or somatosensory skills and fitness to drive8. however, this study only included normal aging young - old self - driving adults. they reported the sdbm has relevance as a self - report instrument for assessing older drivers6. the results of the current study suggest that the sdbm and dhq can predict driving - related collision history based on the auc values. however, the sdbm and dhq were not accurate screening tests for the traffic collision history of young - old self - driving adults. this may be due to the limitation created by the relatively small sample size of the young - old self - driving adults. this study also had a cross - sectional design and only young - old adults were studied. future studies will be needed using larger samples of young - old self - driving adults, as well as investigations of other sub - groups of older adult drivers such as middle - old and the oldest adults. | [purpose ] to evaluate the sensitivity and specificity of the safe driving behavior measure and the driving habits questionnaire in community - dwelling older self - drivers. [subjects and methods ] forty - five older participated in this study, to measure the safe driving behavior measure and the driving habits questionnaire. sensitivity and specificity were calculated along with cut - off values and overall accuracy of each measure as determined by the participants operating characteristic curve and the area under the curve. multivariate logistic regression analysis was employed to identify predictors of driving abilities. [results ] the sensitivities were 0.538 for safe driving behavior measure, and 0.577, 0.423, and 0.615 for the difficulty, crash and citations, and driving space on domains of the driving habits questionnaire, respectively. the specificities of the person - vehicle domain, person - environment domain, and person - vehicle - environment domain of the safe driving behavior measure were 0.474, 0.526, and 0.421, respectively, while the driving habits questionnaire domains, the specificities of difficulty, crash and citations, and driving space were 0.526, 0.211, and 0.421, respectively. [conclusion ] the results of this study suggest that factors related to the accident history of older self - drivers were not well - explained, although the safe driving behavior measure and driving habits questionnaire domains have the potential to determine driving - related accident history. |
Subsets and Splits
No saved queries yet
Save your SQL queries to embed, download, and access them later. Queries will appear here once saved.