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Intentional replantation (intr) is described as the intervention of purposely removing a tooth and following some extra - oral procedures, replacing it into its socket . Despite increasing tendency to be a favored treatment alternative in cases of periodontally hopeless teeth (pht), enhancement of periodontal tissue support and thus, increasing the lifetime and functional quality of replanted teeth is still one of the main concerns . Supportive treatments have been suggested to improve results obtained with intr . Despite the contribution of these to periodontal health, only periodontal pockets and inflammation were evaluated, and no detailed information was given about periodontal and mucogingival state . Because of thin tissue phenotype, pht in mandibular anterior area may frequently subject to gingival recession in case of periodontal destruction . Therefore, mucogingival problem should be potentially considered in performing intr . Although reports involving intr point out outcomes about survival and reduction of inflammation around treated teeth, there is limited data indicating the importance of mucogingival state . In addition, no report represented treatment of pht with a two - step procedure involving intr and free gingival graft (fgg). Therefore, objective of this report is to present 15-month results of treatment including intr and mucogingival surgery . The report also aims to underline the contribution of supporting approaches to intr procedure in terms of periodontal tissue support and amount of keratinized gingiva . A 20-year - old female was referred to kirikkale university periodontology department in june 2011 with mobility and discomfort in mandibular left lateral incisor . Clinically, tooth had malposition, severe periodontitis with 3 mobility, inflammation, and recession [figure 1]. Baseline measurements included plaque index, gingival index, probing depth (from the gingival margin [gm] to base of pocket), attachment level (from cemento - enamel junction [cej] to base of pocket), gingival recession (from cej to gm), and keratinized gingiva (from gm to mucogingival junction) [table 1]. Radiographically, the root had huge radiolucent area demonstrating crater - shaped bony defect starting from the coronal third reaching to apical with completely lost buccal + lingual walls [figure 2]. Pretreatment view of the mandibular left lateral incisor demonstrating significant coronal and lateral displacement with hyperemic and edematous gingiva baseline and follow - up clinical parameters pretreatment radiographic view . Supporting alveolar bone was completely lost after treatment planning, informed consent was signed, and remaining treatments were completed . One week after endodontic therapy, intr was performed using the technique applied by demiralp et al . In brief, the tooth was extracted and put on a sterilized moisturized sponge [figure 3]. After scaling and root planning, 100 mg / ml tetracycline hcl (boehringer, ingelheim am rhein, germany) was applied to the root surface for 5 min . The surface was rinsed with sterile distilled water for 1 min, granulation tissue inside the socket was gently debrided and tooth was replaced into the socket . Tooth was repositioned in most possible ideal position to eliminate traumatic occlusion resulted due to pathologic migration . Tooth stabilization was achieved from the incisal third with composite restorative material (3 m filtektm z250, 3 m espe, st . After intr, doxycycline was prescribed for 7 days and oral hygiene activities were continued . Professional cleaning was made at 3 week, and maintenance visits were scheduled . The tooth was gently extracted and the root surface was scaled and root planed three months after, radiographic view was uneventful, and pocket depths were shallow [table 1]. However, inflammation and mucogingival stress (no keratinized tissue) was detected [figure 4]. As the second step, fgg was utilized in accordance with the technique described by miller [figure 5]. Sutures were removed 10 days following surgery and toothbrushing was discontinued during this time . Recall visits including professional prophylaxis were arranged . At 15 months, splint was removed, and mobility was reevaluated . Three months following replantation procedure . Note the lack of keratinized tissue (a) recipient site prepared for the graft . (c) view following graft suturation wilcoxon test was performed to evaluate the time - dependent change of the clinical parameters and significance level was set at p = 0.05 . Compared with baseline, significant improvement was detected at 6, 9, and 15 months periodontal measurements [table 1]. Although gingival recession did not change at 6 months postsurgical visit, a slight decrease was observed in the subsequent follow - up . Gingiva was healthy with its firm view and no bleeding on probing was detected [figure 6]. Radiographically, the amount of radiolucent area was diminished, and hard tissue formation was detectable around the root apex with no root resorption [figure 7]. Although slight discoloration occurred in attached gingiva and tooth toward the end of follow - up, patient was satisfied with outcomes of treatment . Note that the splint was removed and the gingiva was firm and healthy with a slight color change fifteen months posttreatment periapical radiographic view . In modern periodontology, growing amount of evidence indicates that several therapeutic modalities can successfully result in keeping pht in place . Cortellini et al . Compared outcomes of periodontal treatment with extraction + prosthetic replacement for pht and suggested that last step therapeutic procedures should be considered before extraction of pht . Intr, another last resort therapy, has demonstrated successful results in the treatment of pht . In the present report, significant hard tissue formation was obtained, and replanted tooth was successfully retained during 15 months . Even though fgg was proposed as supportive in mucogingival problems and prosthodontic and orthodontic treatments, a mucogingival problem was detected after intr and it was decided to utilize fgg around the replanted tooth . Following surgery, in addition to the resolution of inflammation, 8 mm keratinized gingiva and minimal root coverage was obtained . Consequently, it can be recommended that mucogingival problems and supportive treatment alternatives should be considered following intr procedure in which time the inflammation dissolves and clear decision can be made to perform mucogingival surgery . Enhancement of periodontal tissue support and thus, increasing the lifetime and functional quality of the replanted teeth is still one of the main concerns . In 2006, tzm et al . Described use of platelet rich plasma (prp) in intr of pht . Eighteen - month follow - up results of their case demonstrated new bone formation around the root apex . Two study groups combined different regenerative options, including enamel matrix derivatives (emd), bone grafts, membranes and prp with intr to improve tissue support and suggested use of these materials with intr in order to have more successful and predictable results . Evaluated results of intr of pht with emd + demineralized freeze - dried bone allograft (dfdba) and showed that emd + dfdba might develop the outcomes of intr . Despite comments of these literatures, only periodontal pocket depth and gingival inflammation parameters were evaluated, and no detailed information was given about mucogingival state . To the best of our knowledge, the present case is the first one demonstrating use of fgg to increase periodontal tissue support of a replanted tooth . Following utilization, inflammation symptoms were reduced, and minimal root coverage was obtained with keratinized gingiva enhancement . In this case - report, 15-month results of mucogingival surgery after intr were presented . Consequently, treated pht survived with healthy gingiva, reduced pocket depth, acceptable mucogingival relationship and new hard tissue formation . From the results of this single case lack of baseline periapical radiograph and standardized radiographic measurements, intr supported with fgg may be speculated as an alternative to keep pht for a period of time.
Protein folding in the endoplasmic reticulum (er) is monitored by stringent quality control mechanisms that prevent release of immature or misfolded er client proteins to travel further along the secretory pathway . Notable examples include the binding - and - release cycles of bip, recognizing exposed hydrophobic stretches at the surface of nonnative conformers, and of calnexin / calreticulin, which exploit glucosylation status of n - linked glycans on er client proteins to exert quality control (ellgaard and helenius, 2003). Er client proteins are rich in intra- and intermolecular disulfide bonds that are generally essential for their function . Numerous oxidoreductases reside in the er to catalyze oxidative protein folding (ellgaard and ruddock, 2005). Also the process of disulfide bond formation is intimately linked to the quality control systems that prevent nonnative conformers to exit from the er (anelli and sitia, 2008). The same mechanisms that exert er quality control on the folding of subunits also monitor certain steps in their oligomeric assembly . For instance, in the case of antibodies, bip binds the first constant domain (ch1) of ig heavy chain (h) and thereby retains it in the er until it is displaced by the ig light chain (l) to assemble into h2l2 structures, called monomers in the immunological jargon (feige et al ., 2009; haas and wabl, 1983; hendershot and sitia, 2005). The assembly process of 2l2 monomers into igm pentamers(2l2)5j and hexamers(2l2)6is favored by ergic-53 (anelli et al ., 2007), which resides preferentially in the er - to - golgi intermediate compartment (ergic) (schindler et al ., 1993). Interaction of 2l2 monomers with ergic-53 occurs upon release by bip (anelli et al ., 2007), suggesting that these assemblies escape from the grasp of er quality control and travel to more distal compartments of the early secretory pathway (cortini and sitia, 2010). This notion is perhaps not so surprising, as the 2l2 monomers, even if not polymerized, are correctly folded and partially assembled and therefore no longer substrate to the er chaperoning machinery . The only sign that betrays their unpolymerized state is the free tail - piece cysteine, which in mature igm polymers is disulfide linked to corresponding cysteine residues in other 2l2 monomers (sitia et al ., members of the pdi family of oxidoreductases associate with free cysteines of orphan assembly subunits and thereby facilitate their so - called thiol - mediated retention (fra et al ., 1993; reddy et al ., 1996; sitia et al ., we previously identified erp44 as a pdi family member that covalently binds ero1 oxidases and facilitates their intracellular localization (anelli et al . Erp44 is special for having a single cysteine (c29) in its conserved active site (crfs), consistent with a dedicated role in thiol - mediated retention not only of ero1 oxidases but also of orphan subunits of otherwise disulfide - linked oligomers, including igm (anelli et al . Moreover, erp44 localizes predominantly to the ergic (anelli et al ., 2007;, 2006), unlike other pdi family members, which reside in the er (ellgaard and ruddock, 2005). Still, the c - terminal rdel motif of erp44 allows its capture by kdel receptors (kdel - r) in distal stations of the early secretory compartment, presumably for retrieval to the er (anelli et al ., 2003). It is yet unclear how erp44 cycles between its predominant localizations in the distal early secretory pathway and the er, and how such cycling would relate to thiol - mediated retention . Here we show that the ph gradient between cisgolgi and er controls association of erp44 both with its clients and with the kdel - r . Our results suggest a model in which the simultaneous unmasking of the client and kdel - r binding interfaces is facilitated by dislocation of the erp44 c - terminal tail (c - tail), which in turn likely involves protonation state of the active site cysteine (c29) at cisgolgi - equivalent ph . As such, both erp44 activity and its shuttling between the er and cisgolgi are ph regulated to drive a quality control cycle dedicated to the surveillance of secretory protein assembly . Erp44 associates with its client proteins via disulfide linkages to residue c29 in the active site and noncovalent interactions with the surrounding hydrophobic patches at the substrate - binding site (anelli et al ., 2003; wang et al ., 2008). In the erp44 crystal structure (wang et al ., 2008), however, the substrate binding site is shielded from the solvent by the c - tail that submerges into a groove delimited by the a and b domains (see also figure 4a). The ph gradient existing in the early secretory pathway (figure 1a, top panel) regulates ergic-53 (appenzeller - herzog et al ., 2004), a glycoprotein transporter lectin that shares several clients with erp44, including igm (anelli et al ., 2007). Ph also regulates the in vitro binding of peptides to kdel - r (wilson et al ., 1993), which prevent erp44 secretion (anelli et al ., 2003 we thus surmised that the erp44 structure, obtained from crystals grown at ph 7.5, most likely corresponds to an off conformation of the protein, found in the ph - neutral er . Arrival into the distal, more acidic stations of the early secretory pathway, namely ergic and cisgolgi, could entail exposure of the substrate binding site (figure 1a). We therefore assessed ph - dependent erp44 c - tail rearrangements in vitro by 1-anilinonaphthalene-8-sulfonate (ans) binding fluorescence spectroscopy (serve et al . Consistent with the exposure of hydrophobic surfaces in erp44, the ans fluorescence peak was blue - shifted and enhanced when the ph was lowered from 7.5 to 6.5 (figure 1b, top panel). This hypsochromic effect was largely abolished for a mutant in which we had engineered a disulfide bond between c29 and the c - tail (t369c, lower panel) to restrict its movements (wang et al ., 2008), unless the engineered disulfide bond was disrupted with the reducing agent dtt (figure 1b). We concluded that accessibility of erp44 s substrate binding site involves ph - dependent dislocation of the c - tail . Next, we monitored reactivity of the active site c29 as a function of ph and found that erp44 bound more polyethylene glycol 2000-modified maleimide (malpeg) as the ph was lowered from 8.0 to 6.0 (figure 1c), with 50% malpeg binding at ph 6.6 (figure 1c, right panel, red dots). Malpeg binding faithfully reported on c29 reactivity, since its substitution with serine almost abolished binding . The residual binding observed in c29s involved residue c63 (figure 1c, right panel, turquoise dots), as indicated by the absence of mobility shifts in the double mutant c29s - c63a (figure 1c). This finding excludes the possibility that the results obtained with erp44 wt reflected ph dependence of malpeg reactivity (makmura et al ., 2001). The t369c mutant did not bind malpeg, reflecting the constitutive engagement of c29 in a disulfide bond with c369 (figure 1c; wang et al ., 2008). Taken together, these results indicate that both c29 and the surrounding hydrophobic substrate binding site become more exposed at ph values similar to those encountered downstream of the er . Although the rdel motif was not resolved in the crystal structure (wang et al ., 2008), we reasoned that its accessibility would be limited by the adjacent domain a when the c - tail is in a closed conformation, in turn hindering its availability to kdel - r . T369c mutant was secreted at levels comparable to a mutant lacking the rdel motif (rdel) (figure 2, upper panels). Insertion of a spacer peptide (flag) immediately upstream of the rdel tetrapeptide to let it protrude from the protein increased retention of the t369c mutant to levels similar to those for erp44 wt with the flag insert (figure 2 lower panels). The finding that flag - tagged erp44 (whether wt or t369c mutant) was less well retained than flag - less erp44 wt may reflect that rdel recognition by kdel - r depends in part on its context . Indeed, in erp44 the sequence upstream of rdel is remarkably conserved in metazoans (data not shown). In all, these data support the notion that an open and flexible conformation of the c - tail increases accessibility of the rdel retrieval motif to its cognate receptors . Having demonstrated in vitro that accessibility of the erp44 active site is ph regulated, we set out to analyze ph dependency of erp44 activity in vivo . To this end, we silenced expression of the golgi ph regulator (gphr) (maeda et al ., 2008), which specifically raised the ph in the cisgolgi by 0.4 units (figure 3a). In line with our in vitro findings, neutralizing the er - cisgolgi ph gradient inhibited erp44 reactivity with its partners / substrates, as is evident from the gphr silencing - induced decrease of erp44 being engaged in disulfide - linked complexes (figure 3b). Consistently, thiol - mediated retention of erp44 client proteins was inhibited upon gphr silencing, as indicated by increased secretion of overexpressed ero1 (figures 3c and 3f and see figure s2 online), adiponectin (apn, figures 3c and 3f), and igm assembly intermediates (figures 3d and 3f). Intracellular retention of ero1 is a task that erp44 shares with pdi, and overexpression of either erp44 or pdi enhances ero1 retention (anelli et al ., 2003; otsu et al ., 2006 gphr silencing strongly subdued this enhanced ero1 retention when erp44 was overexpressed, but much less so in the case of pdi overexpression (figure 3c). Secretion of proteins that are not erp44 clients, like 1-antitrypsin (1at) or a soluble gfp engineered for entry into the secretory pathway (sgfp), was also unaffected by raising the cisgolgi ph (figures 3e and 3f). We concluded that erp44-mediated retention in particular relies on the er - cisgolgi ph gradient . Accordingly, retention of unassembled monomeric ig- chains, which is mediated not by erp44 but by the chaperone bip, was ph insensitive (figures 3d and 3f). Only when the ch1 domain, which is important for bip binding (haas and wabl, 1983; lee et al ., 1999), is deleted (ch1) or engaged in interactions with ig - l chains (as in 22 complexes) does erp44 become the primary retainer of the ig- chains, and hence their retention becomes ph dependent (figures 3d and 3f). Retention of sgfp - rdel was ph insensitive (figure 3e), as expected for a protein that lacks free cysteines and hence is not an erp44 client . This finding implies at the same time that kdel - r function, which is crucial for sgfp - rdel retention, is not corrupted despite the rise in cisgolgi ph . Likewise, erp44 was retrieved by kdel - r despite gphr silencing (figure s2b), whereas this treatment allowed secretion of erp44 client proteins (figure 3f). Perhaps a partial c - tail opening in distal stations of the secretory pathway, which are less affected by gphr silencing (maeda et al ., 2008), is sufficient for kdel - r recognition but not for client binding . Considering that the conformational changes that facilitate c - tail dislocation are induced by lowering the ph as erp44 travels to the cisgolgi, we surmised that a protonation event may lie at the basis of the switch between the on and off conformations of erp44 . To explore this notion, we analyzed the crystal structure of erp44 with the c - tail in the closed conformation using the program propka3, a state - of - the - art semiempirical method that evaluates pka values of ionizable residues starting from protein structures (li et al ., 2005; olsson, 2011; olsson et al ., 2011). The most striking feature that emerged from the propka3 analysis was the downward shift of the pka of c29 . Its predicted value of 7.7 units (table 1) is significantly lower than the reference value for free cysteine in solution (9.0). This shift is mainly ascribed to the propensity of the neighboring side - chain hydroxyls of s32 and t369 and the main - chain amides of residues f31, s32, and t369 (table 1 and figure 4b) to form hydrogen bonds with c29 when in the thiolate form . Likewise, electrostatic interactions with the r98 side chain stabilize the thiolate form of c29 (table 1). Based on this pka value, a significant percentage of c29 would be in the thiolate form (41%) at ph 7.5 (figure 4b and table 1). Conversely, only 6.5% of c29 was predicted to be in the thiolate form at ph 6.5 (table 1). When we removed the 16 strand from the crystal structure in silico, simulating an c - tail conformation of erp44, the calculated pka of c29 raised to 7.8 (table 1). The effect was even more pronounced in sample structures at the end of molecular dynamics simulations of this 16 model . As a consequence of a greater solvent exposure, loss of hydrogen bonding to t369, and an increased distance to the side chains of both s32 and r98, c29 had a calculated pka of 8.7, implying that only a small percentage (6%) of c29 is in the thiolate form at ph 7.5 (table 1 and figure 4c). Based on our in silico analyses, we mutated the residues predicted to affect the pka of c29 in erp44 . Altering the side - chain groups such that they could no longer contribute to the stabilization of the thiolate form of c29 through hydrogen bonds or electrostatic interactions (s32a, r98q, or t369a) indeed rendered erp44 constitutively accessible to malpeg and abolished the ph sensitivity of the reaction (figure 4d). Deletion of strand 16 gave similar results (figure 4d), supporting the notion that the s32a, r98q, and t369a substitutions similarly led erp44 to adopt the we then analyzed whether the enhanced accessibility of the erp44 active site in the various mutants correlated with its affinity toward substrates . In surface plasmon resonance (spr) assays (masui et al ., 2011), erp44 wt showed a ph - dependent reactivity toward ero1, having a higher affinity for its partner at ph 6.5 than at ph 7.5 (figure 4e). All the mutations that resulted in constitutive and ph - independent malpeg binding of erp44 made it also less ph dependent in spr assays and increased its affinity for ero1 (figure 4e). The effect was weakest for the r98q mutant, in agreement with the modest contribution of this arginine in stabilizing the thiolate form of c29 (figure 4c). In summary, our data indicate that c29, s32, r98, and t369 are components of the erp44 ph - sensing mechanisms . The predicted pka of c29 approaches neutral ph due to the interaction of its side chain with the surrounding residues, suggesting that its protonation, when erp44 encounters the lower ph in the cisgolgi, is a key event for c - tail opening, and, hence, exposure of the substrate binding site . We then further substantiated our findings on the mechanisms mediating erp44 ph sensitivity by analyzing the phenotype of the above mutants in vivo . In line with the in vitro results, mutation of the residues expected to affect the pka of c29 (s32, r98, and t369) dramatically increased the tendency of erp44 to form covalent complexes with client and partner proteins, reaching levels comparable to a mutant lacking the whole 16 strand (see figure 5a and figure s3a). This was particularly evident when anti - ha antibodies were used to selectively analyze the behavior of the overexpressed transgenes (figure s3a). We concluded that the s32a, r98q, and t369a mutations increased the accessibility of c29 also in vivo, likely reflecting a more open conformation of the c - tail . In further support of our model, ph sensitivity of erp44 activity was lost in all these mutants, since the fraction of molecules covalently bound to client and partner proteins did not decrease upon gphr silencing in these mutants, as it did instead in erp44 wt (figure 5a). In addition, the activity of these mutants in retaining adiponectin was no longer sensitive to basification of the cisgolgi (figure 5b and figure s3b). Therefore, ph sensitivity is severely reduced when the critical residues s32, r98, and t369 are mutated and, consequently, the predicted c29 pka is raised over neutral values . The ph gradient between er and golgi may serve multiple roles in the cell, such as calibrating activity of lectins and golgi glycan - modifying enzymes (paroutis et al ., 2004). We have shown here that the ph gradient is exploited also for regulating quality control and secretion of a selection of key proteins . Ph - regulated opening of erp44 s tail determines both client capture and kdel - r - mediated retrieval . Thus, erp44 governs a ph - regulated protein quality control cycle shuttling between cisgolgi, where erp44 engages its clients, and the er, where they must be released and where erp44 s c - tail presumably closes to occlude the client binding active site . Our in vitro malpeg binding studies reveal a midpoint transition for erp44 at a ph of approximately 6.6, a value similar to that found in the cisgolgi (figure 3a). In all likelihood, however, additional mechanisms contribute to the regulation of erp44 activity in living cells . For instance, interactions with kdel - r may prevent closing of the c - tail and perhaps provide interaction with additional proteins . Owing to the fact that the opening of the c - tail is ph dependent, the substrate binding site of erp44 becomes more accessible progressively from er to cisgolgi . Client proteins may thus engage erp44 more proximally or distally along the early secretory pathway, according to their affinity toward the substrate binding site at a given ph . Differences in the ph optimum for erp44 association may thus account for differences in the intracellular distribution of ero1, which is enriched at mitochondrial - associated er membranes (anelli et al ., 2012; simmen et al ., 2010) and resides overall more distally in the early secretory pathway, as compared to another erp44 client, sumf1, which localizes to the more proximal stations (fraldi et al .,, the active site c29 is buried at the interface between domain a and the c - tail (wang et al ., 2008). The environment in which c29 resides induces a downward pka shift of this residue toward neutral values, de facto stabilizing the thiolate form of the amino acid . The negative charge of the deprotonated c29 restrains the c - tail movements via hydrogen bonds to s32 and t369 and electrostatic interactions with r98, which also interacts with the tail backbone . Upon entry of erp44 into the cisgolgi compartment this event, in turn, weakens c29 interactions with surrounding residues, as a cysteine with a side - chain thiol is less effective in forming the same hydrogen - bonding and long - range coulombic interactions that the thiolate can maintain . How c29 of erp44 reacts with clients to form mixed disulfides and whether erp44 can act alone or requires assistance for this process deserves further investigation . In line with our model, silencing of erp44 causes more abundant secretion of many proteins with one or more exposed reactive cysteine, including ig- (anelli et al ., 2007; ronzoni et al ., 2010), adiponectin (wang et al ., 2007; qiang et al ., 2007), sumf1 (fraldi et al ., 2008; mariappan et al ., 2008), and peroxiredoxin 4 (t. kakihana, k. araki, s.v ., i. shun - ichiro, m.c ., similarly, upon neutralization of the golgi ph, erp44 substrates like ero1, adiponectin, and igm subunits are secreted more abundantly, presumably because the active site of erp44 remains largely inaccessible . This observation suggests that erp44 could partially open the c - tail, exposing its rdel, and interact with kdel receptors without binding client proteins through an as - yet - unknown mechanism . Erp44 receives substrates from, and associates with, ergic-53 (anelli et al ., 2007), a lectin that also embraces glycoproteins released by the calnexin / calreticulin quality control (ellgaard and helenius, 2003). The erp44-ergic-53 tandem may thus integrate thiol - mediated and glycan - trimming - dependent er quality control cycles . Similarly, we have shown that upon displacement by ig - l, bip no longer warrants retention of orphan ig - h chains but leaves this role to erp44 instead . Erp44 indeed is strongly upregulated in the course of b cell differentiation (van anken et al ., 2003; anelli et al ., 2007) to help avoid release of incompletely assembled igm, which would jeopardize the efficiency of the humoral immune response . The erp44 assembly control cycle thus embodies the last - resort quality control mechanism to prevent the untimely exit from the early secretory compartment of orphan subunits of otherwise disulfide - linked oligomeric secretory proteins . The cdna encoding human erp44 without the signal sequence was amplified by pcr and cloned into pet28 vector (from novagen) at nhei and xhoi sites . The recombinant form of erp44, which was overproduced in e. coli, lacks the rdel motif at the c terminus . Vectors for expression of ha - erp44 in mammalian cells were previously described (anelli et al ., 2002). Mutants were obtained by polymerase chain reaction (pcr) or site - directed mutagenesis (sdm) (table s1). Human erp44-flag - rdel was a kind gift of professor k. nagata (kyoto - sangyo university). Plasmids encoding wt and mutated secretory ig- chains (s and ch1) were described in detail previously (cenci et al ., 2006; mattioli et al ., 2006). The purification of wild - type and mutant erp44 and ero1 was performed as described (inaba et al ., 2010). The association and dissociation rate constants (kon or koff) for the direct binding of erp44 to immobilized ero1 were determined by spr measurements on a biacore2000 system (ge, healthcare), in the presence of 1 mm gsh and 0.25 mm gssg as described (inaba et al ., 2010). Hyperactive ero1 (with cysteines 104, 131, and 166 replaced by ala) was used in spr assays to limit variations caused by the autoregulatory mechanisms controlling ero1 activity (inaba et al ., 2010). Ans fluorescence spectra were recorded in 1 cm cuvettes on a hitachi f-4500 spectrofluorometer . Erp44 and mutants (5 m) were mixed with 100 m ans in 20 mm tris - hcl (ph 7.5) or mes (ph 6.5) containing 150 mm nacl with or without 10 mm dtt and incubated at 293 k for 10 min . Cells were starved for 5 min in cysteine and methionine - free dmem (gibco, invitrogen), pulsed for 10 min with (s) cysteine and methionine (easy tag, perkin - elmer), washed twice, and chased for the indicated periods in complete medium . Cell lysates and supernatants were immunoprecipitated with anti - ha or anti - flag antibodies and resolved by sds - page . Fluorograms or western blot images were acquired with the chemidoc - it imaging system (uvp, upland, ca) or with fla-900 starion (fujifilm life science, usa) and quantified with image quant 5.2 as described (anelli et al ., 2007). Tissue culture, transfection, and silencing were performed as described previously (anelli et al ., 2007). Fetal calf serum and cell culture medium were from gibco, invitrogen . Unless otherwise indicated, chemicals were from sigma . Polyclonal anti - mouse and anti - erp44 (b68) antibodies were purchased from primm srl (milano, italy). Monoclonal anti - gfp (clones 7.1 and 13.1) was purchased from roche (usa) and polyclonal anti-1 antitrypsin (ncl a1ap) from novocastra . Monoclonal anti - erp44 antibody (36c9) was previously described (anelli et al ., 2007). Goat anti - mouse ig and anti - rabbit ig horse - radish peroxidase (hrp) were from jackson immunoresearch laboratories, inc . Mouse monoclonal antibodies specific for myc (9e10), ha (12ca5), and flag (m2) were immobilized by crosslinking to protein g and protein a beads, respectively . Hrp rabbit anti - mouse igg (hl) was from dako cytomation (glostrup, co). Each erp44 derivative (5 m) was incubated on ice for 30 min in various ph buffers containing 100 mm sodium phosphate and 150 mm nacl, followed by incubation with maleimidyl peg2k (300 m) for 10 min at room temperature . The reaction was stopped by the addition of 5% trichloroacetic acid, and the protein pellet was washed with acetone and dissolved in buffer containing 50 mm tris - hcl (ph 7.0) and 1% sds before loading onto a reducing sds gel (10%). Hela cells expressing pme - zeo - gpp130-phluorin (miesenbck et al ., 1998) were cultured on glass - bottomed dishes and analyzed under a tcs sp2 laser scanning confocal (leica confocal) equipped with an ld laser 405 (405 nm) and a multiline ar laser (457, 488, and 515 nm) at room temperature . Data acquisition and analysis of intensity ratios (405 nm; 457 nm) were performed as described (miesenbck et al ., 1998). Statistical significance was calculated, comparing between conditions indicated by brackets using the two - tailed student s t test . Oligonucleotides for rna interference (silencer select predesigned sirna products) were purchased from ambion (sirna i d #s56768). Lipofectamine rnai max was from invitrogen (carlsbad, usa) and used for silencing in hela cells, according to the suppliers instructions . We used the propka 3.0 software (http://propka.ki.ku.dk) to predict the pka value of erp44 c29 and the role of surrounding amino acids starting from the crystal structure of the entire molecule (wang et al ., 2008; pdb i d code 2r2j) and from in silico models of the 16 mutant.
Systemic sclerosis (ssc, scleroderma) is an autoimmune disease characterized early in the process by vasculopathy and subsequently by varying degrees of fibrosis in skin, lungs, and other tissues . The presence of vasculopathy is the hallmark of this condition, represented clinically as raynaud's syndrome which occurs almost universally in both the limited and diffuse cutaneous subsets of this disease . Vasculopathy possibly results from abnormal vasoreactivity, hypoxia, and/or direct damage of vascular and perivascular cells . Perivascular inflammatory infiltrates and neoangiogenesis ensues resulting in varying degrees of fibrosis in the skin and internal organs . This paper describes why details of the vascular microenvironment might determine the degree of end - organ damage that occurs in ssc, with a focus on vascular cell senescence, endothelial progenitor cells (epc) including mesenchymal stem cells (msc), pericytes, and angiogenic monocytes . An explanation of the role of epc, pericytes, and angiogenic monocytes is important to understanding ssc pathogenesis . Ssc is thought to be a genetically complex disease, influenced by multiple genes, with a substantial environmental component . Nonetheless, ssc occurs significantly more frequently in families with scleroderma (1.6%) than in the general population (0.026%). Genome - wide association studies have found a strong association with the hla ii region on chromosome 6, and non - hla candidate genes that regulate interferon production, such as interferon regulatory factor 5 (irf 5) as well as genes that regulate immunological responses, such as signal transducer and activator 4 (stat 4) [5, 6]. As such, systemic sclerosis is an autoimmune disease; however the inherited effects of vasculopathy and fibrosis remain to be determined . Our previous work showed that vasculopathy imparts a greater relative risk to family members than does autoimmune inflammatory conditions or fibrotic lung disease . Irrespective of the subset of ssc, perivesicular inflammatory infiltrates result in endothelial derangement in lesioned as well as perilesional tissue [10, 11]. These perivascular changes precede the excessive accumulation of extracellular matrix components, and fibrosis may represent a default pathway from vascular failure [12, 13]. The histopathological hallmark in ssc is a result of endothelium activation with cell adhesion molecule expression, inflammatory cell recruitment, intimal proliferation, and adventitial fibrosis, which results in apoptosis of endothelial cells [13, 14]. Despite the ensuing severe tissue hypoxia, proper adapted angiogenesis vascular cells normally have a finite lifespan which is determined in part by telomere length and/or telomerase activity . Telomerase is a reverse transcriptase which helps maintain telomere length, thereby preventing cell senescence and protecting chromosomes from aberration . Although telomerase activity is increased in many connective tissue diseases, it is decreased in systemic sclerosis (ssc), perhaps due to gene polymorphism [16, 17]. Artlett and colleagues reported a decrease telomere length in a combined cohort of limited ssc (lssc) and diffuse ssc (dssc) whereas macintyre and colleagues reported increased telomere length and lack of age - related telomere erosion in lssc [18, 19]. In a pilot study, we used a monochrome multiplex quantitative pcr (mmqpcr) method to evaluate the relative telomere lengths (t / s ratios) in dna samples of 6 lssc (1 male; 5 females) and 6 dssc (3 males; 3 females) aging 4060, and 50 healthy controls (hc) aging 3760 . Two factors were statistically associated (p value <.001) to t / s: age and diagnosis (figure 1). Not correcting for age, the average length measure was 1.2 for normals, 1.15 for dssc and 0.96 for lssc patients (figure 2). Telomere length, which is shorter in ssc patients than in normal hc, is possibly a risk factor for vasculopathy . While the appearance of vasculopathy does not vary per subtype of ssc, the effect of telomere length on the fibrocyte or myofibroblast may be different in lssc and dssc, possibly contributing to differences in disease manifestations . The reduced telomere length in the endothelial cell likely results in chronic underperfusion and ischemia in the skin and internal organs in both lssc and dssc . However, if fibrosis is the default pathway of insufficient angiogenic response, the subsequent reduced lifespan of the fibrocyte (determined by telomere length) may be protective in the lssc subtype . The vascular network is a dynamic organ with an estimated surface area of> 1000 m . Neovascularization is a complex process that requires both the mobilization of cells derived from the bone marrow, named endothelial progenitor cells (epcs), and proliferation and differentiation of resident cells, known as pericytes, to migrate to the correct location and assemble into vascular structures . New vessels are produced by a combination of angiogenesis and vasculogenesis . In angiogenesis, fully differentiated endothelial cells arise from pre - existing vessels whereas vasculogenesis describes the formation of new vessels by circulating epc which act to replenish damaged or senescent blood vessels . This process requires dynamic and temporally regulated interactions between endothelial cells, soluble proangiogenic and antiangiogenic growth factors, and extracellular matrix molecules . Primary contact between endothelial cells and mural cells (pericyte and vascular smooth muscle cells) is central to the regulation of vascular formation in angiogenesis . Recently formed endothelial tubes are initially unstable and become stabilized through the formation of a peri - vascular matrix and the connection with pericytes . Pericytes are embedded within the endothelial basement membrane and are found primarily around blood capillaries, precapillary arterioles, postcapillary venules, and collecting venules . They are arranged to facilitate and assimilate cell communication . With particular interest to ssc, pericytes may play a role in ectopic calcification and are able to transdifferentiate into fibroblast - like cells if they escape from the capillary basement membrane . The pericyte role as a perivasicular mesenchymal stem cell with macrophage - like properties has not been welldefined in ssc, but is intriguing . Its ability to perform this function is correlated with marker expression and the microenvironment of the endothelial - pericyte contact . Most likely, specific intercellular signals mediated by ligand - receptor systems are required for endothelial and pericyte vascular stability . Numerous studies demonstrate the critical importance of transforming growth factor- (tgf)-beta signaling for vascular development and function . Tgf - beta has context - dependent effects on endothelial cells; proliferation is mediated by signaling through alk / smad1/5 and differentiation is mediated by alk smad2/3 . Tgf - beta / smad signaling has been suggested to play a key role in the pathogenesis of ssc . In postnatal vasculogenesis identification and quantification of epc population in ssc has been challenging and has resulted in consensus recommendations to help unify epc research . Research by avouac and colleagues, using an accurate, reliable, and reproducible method of epc quantification, supports that ssc is associated with epc mobilization, but in active or severe stages, epc may be recruited to injured sites and thus decrease in the circulation . Multipotential mesenchymal stem cells (mscs) might be a source of epc in vasculogenesis . Mscs show normal functional properties and a normal pattern of biological markers, but the angiogenic potential of these endothelial - like mscs is reduced . Cipriani and colleagues showed that when msc from ssc patients are seeded on matrigel, they have a reduced ability to form capillary - like structures and give rise to incomplete endothelial networks, even after vascular endothelial growth factor (vegf) and stromal - derived factor (sdf-1) stimulation [23, 32]. Vegf is an important angiogenic peptide with specific proliferative, differentiation, and mobilization effects on epcs, and is known to be upregulated in ssc, especially in advanced disease . Vegf gene expression is also regulated by growth factors (such as tgf - beta) and other proinflammatory cytokines . The platelet - derived growth factor (pdgf) family is essential to vascular remodeling and maturation . In a study of 62 ssc patients, epcs were significantly increased in patients with early - stage ssc disease, but not in those with late disease irrespective of diagnosis subtype, and there was no correlation between the number of circulating epcs and vegf . Bone marrow biopsy samples from 14 of these ssc patients (3 early limited ssc, 4 with late limited ssc, 4 with early diffuse ssc, and 3 with late ssc) showed fewer and functionally impaired epcs in all patients . Another study showed that the subset of ssc patients with digital lesions and high severity scores had low epc counts . It is possible that bone marrow from ssc patients cannot satisfy the continuous and prolonged demand for epcs, despite the target organ increase in vegf . The role of target organ microvascular environment (pericytes and endothelial cells), which is producing tgf - beta, vegf, and pdgf, on ssc pathogenesis is less clear . The elevated total number and activated state of circulating endothelial cells (cecs), suggest vascular damage and endothelial activation in ssc patients regardless of subtype correlates to disease activity . It is also known that tgf - beta and pdgf from this microvasculature cooperate in inducing the activation of fibroblasts and their differentiation into myofibroblasts in ssc patients . Thus, understanding the microvascular environment of target organs in ssc is of primary importance . It is suggested that the major contribution of the bone marrow to angiogenic processes may come from progenitors of the periendothelial vascular mural cells . Endothelial differentiation of monocyte - derived multipotential cells (momcs) can occur with angiogenic stimuli and result in the formation of mature endothelial cell tubules in matrigel cultures . Pericytes establish morphologic interactions with transmigrating leukocytes, mainly monocytes (macrophages). During angiogenesis, macrophages contribute to the dissociation and detachment of pericytes from the endothelial cell . Pericytes can act as antigen - presenting cells and can behave as macrophages; they also can show plasticity with potential to become myofibroblasts . Thus, understanding the role of the interaction of circulating angiogenic monocyte and resident pericyte in ssc microvasculature has important implications . It is possible that this interaction is of primary importance for linking the inflammatory aspect of the disease to the vascular abnormalities . Stromal cell - derived factor-1 (sdf-1) and its receptor (cxcr4) system is a component of the microvascular environment which is extremely important for new vessel formation . Sdf-1 released by endothelial cells creates a gradient dictating directional response of endothelial cells expressing cxcr4 . Skin biopsies in early disease of both ssc subtypes show a strong positive pattern of sdf-1 and its receptor cxcr4 in the endothelial cells and pericytes of microvessels, attesting to an attempted reparative process . Of interest, in diffuse ssc, these skin biopsies also showed dense mononuclear cells in the perivascular infiltrate, possibly suggesting a role of the monocytes in a more fibrotic phenotype . The staining for cxcr4 was weak in the late (sclerotic or atrophic) phases in both ssc subsets . Another study of 40 ssc patients demonstrated higher serum levels of vegf, pdgf, and increased concentration of sdf-1, particularly in the diffuse subset . In this same study population circulating cxcr4 + circulating progenitor cells coexpressing monocytic and endothelial cells positively correlated to the severity score, modified rodnan skin score, and pulmonary involvement . Taken in sum, these results suggest that overall disease activity correlates to the markers of activity in the microvascular environment . It has recently been suggested that the actual angiogenic cell type recruited to the site of tissue injury and incorporated into a newly formed vessel is a monocyte . Activated circulating monocytes have also been reported in ssc patients, supporting a potential role of these cells in disease pathogenesis . Gene expression profiling of peripheral blood monocytes from ssc patients suggest that type i interferon may play a key role in the activation of monocytes in this disease . If during the course of the disease, the mechanism of angiogenesis is impaired, the proangiogenic factors in the microvascular environment may serve to recruit proangiogenic monocytes which, with pericytes, result in overactivity of a myofibroblast phenotype . In a preliminary study, there were no significant differences in the expression of circulating monocyte surface molecules involved with cell transformation, function, or migration presumed to give rise to fibrocytes, in 8 patients with limited ssc . It is possible that the role of the angiogenic monocyte may be greater in the diffuse subset of ssc and have prognostic implications . Therapeutics that modulate the phenotype of reparative cells can offer new opportunities for ssc treatment . In particular, multipotential mscs have attracted interest because of low acute toxicity and their availability . The potential of human momcs which can proliferate and differentiate along the endothelial lineage in a specific permissive environment also may represent an autologous transplantable cell source for therapeutic neovasculogenesis . In early ssc, prevention of vascular senescence may be most important . N - acetyl - cysteine (nac), a chemopreventive antiangiogenic and antiapoptotic drug has been suggested to modulate parameters associated with endothelial cell aging . Pilot data suggests that the statin class of medications may be beneficial in treating vascular manifestations of ssc, through an increase in angiogenic factors and reduction of vascular endothelial activation / injury markers (p <.01 for all comparisons). Cyclophosphamide, which remains the current gold standard for treatment of interstitial lung disease, is known to mobilize epc . Nutraceutical - based mobilization of epc is an area of interest in the biomedical field, and has not yet been reported in ssc . For the fibrotic aspect of ssc, the small molecule tyrosine kinase inhibitor imatinib and related drugs, such as dasatinib and nilotinib, which simultaneously target two of the major profibrotic pathways, tgf - beta- and pdgf - signaling are being studied . The effect of these drugs on the microvascular environment, and their efficacy and tolerability in ssc patients are not yet known . Other anti - tgf - beta therapies are also in development and may have a major impact in systemic sclerosis . However, considerable concern regarding safety is needed given its pro- and antiangiogenic effects at different concentrations . Ifn inhibitors are also under investigation for treatment of ssc, though modulation of interferon may be most effective in the diffuse subset, in which there is a higher perivascular monocyte infiltrate . Specifically, therapies that inhibit transdifferentiation of other cell types, such as pericytes and angiogenic monocytes into fibroblasts and myofibroblasts hold promise . A predisposition to vascular senescence is probable in ssc and the pathogenesis may arise from a subsequent defect in vasculogenesis (possibly due to abnormal bone marrow function) and/or angiogenesis (perhaps due to pericyte and angiogenic monocytes) followed by overactivity of activated fibroblasts and myofibroblasts . . Early ssc may be most amenable to treatments that decrease vascular senescence and increase epc mobilization . Surprisingly, diffuse cutaneous ssc may be more responsive to therapeutics, which modulate pericyte and angiogenic monocyte differentiation into activated fibroblasts and myofibroblasts . The difficulty with therapeutics which modulate growth factor and chemokines is that locally varying levels of these substances are necessary for regulation of migration, proliferation, cell - cell interactions, differentiation, and extracellular matrix deposition . Nonetheless, an improved understanding of the principle regulatory mechanisms of angiogenesis in ssc has profound potential therapeutic value . It is exciting to think that through understanding of the microvascular environment in ssc, that subsequent restoration of proper angiogenesis in ssc could limit fibrotic damage.
The atherosclerosis risk in communities (aric) study is a community - based cohort study of 15,792 people aged 4564 years at baseline sampled from four u.s . Communities: forsyth county, north carolina; suburban minneapolis, minnesota; washington county, maryland; and jackson, mississippi (13). The first examination was conducted during 19871989, with three triennial follow - up visits (visit 2 (19901992), visit 3 (19931995), and visit 4 (19961998)). Visit 2 was the only visit at which a1c was measured and was the baseline for the present study . Of these, we excluded participants reporting race other than caucasian or african american (n = 42); missing values of a1c (n = 278); with prevalent heart failure defined as self - reported treatment for heart failure, hospitalization for heart failure between visit 1 and 2, or the gothenburg stage 3, a status with dyspnea due to cardiac causes and under treatment with digitalis or loop diuretics (n = 455) (14,15); or missing information about incident heart failure during follow - up (n = 245). Mmol / l (126 mg / dl), nonfasting glucose of 11.1 mmol / l (200 mg / dl), a1c 6.5% (12), self - reported physician diagnosis of diabetes, or use of glucose - lowering medication (n = 2,174) or missing information for diabetes (n = 97) at either of visit 1 or visit 2, for a final study population size of 11,057 participants . The study was approved by the institutional review boards of all participating institutions, and all participants gave informed consent . Aric study participants provided information on demographic and behavioral variables and medical history to a trained interviewer at each visit . In this study, we used information obtained at visit 2, unless otherwise noted . Certified technicians measured three systolic and diastolic blood pressures with participants in the sitting position after 5 min of rest using a random - zero sphygmomanometer . A1c was measured using a high - performance liquid chromatography instrument (tosoh 2.2 plus in 20032004 and the tosoh g7 in 20072008, tosoh corporation, tokyo, japan) on all participants with available stored whole blood (16). We have previously demonstrated the reliability of measurements from these stored samples (17). Fasting serum glucose was measured by the optimized dart glucose reagent method and cholesterol, triglycerides, and hdl cholesterol were determined using enzymatic methods . Insulin was measured by radioimmunoassay (125insulin kit; cambridge medical diagnosis, bilerica, ma) at visit 1 (19). Estimated glomerular filtration rate was computed by the modification of diet in renal disease study equation (20). Evidence of atherosclerosis of the common carotid arteries (shadowing / plaque on either side or none) was determined by ultrasound examination (13,21). Aric investigators conduct continuous, comprehensive surveillance for all cardiovascular disease - related hospitalizations and deaths in the four communities . Incident heart failure was defined as death from heart failure in any position on the death certificate or as the first heart failure hospitalization with the international classification of diseases code, ninth revision (icd-9) 428 or tenth revision (icd-10) i50 in any position of the hospital discharge list (6). Incident heart failure from visit 2 to january 1, 2006, was analyzed in the present study . We categorized a1c using the following cut - points: <5.0, 5.05.4, 5.55.9, and 6.06.4% . We evaluated the continuous association between a1c and the incidence rates of heart failure using a poisson regression model incorporating linear spline terms for a1c (knots at 5.0, 5.5, and 6.0%) with adjustment for age, sex, and race . Cox proportional hazards models were used to quantify the association between the above categories of a1c and incident heart failure . We tested for interactions using the likelihood - ratio test . For 10,866 participants (98.3%) who provided fasting (8 h) blood samples, we also evaluated the association of fasting glucose levels and incident heart failure by using clinical categories of glucose concentration as follows: <5.0, 5.05.5, 5.66.0, 6.16.9 mmol / l (<90, 9099, 100109, and 110125 mg / dl). We used the most prevalent category within a normal range as a reference group for both a1c (5.05.4%) and fasting glucose (5.05.5 mmol / l) levels . Model 2 was further adjusted for level of education, carotid atherosclerosis, systolic blood pressure, antihypertensive medication, smoking, alcohol intake, bmi, ldl cholesterol, hdl cholesterol, a self - reported history of coronary heart disease (chd) by visit 2, clinical examination, or hospital records and estimated glomerular filtration rate . Model 3 was adjusted for all variables in model 2 plus either baseline fasting glucose or a1c, as appropriate . We also investigated the association of quartiles of a1c or fasting glucose with heart failure risk . First, we examined heart failure occurring in the absence of clinical chd . To accomplish this, we conducted our analysis censoring incident chd cases that occurred prior to the date of heart failure (n = 1,088). Second, we repeated our analyses after excluding participants who had incident diabetes in the first 6 years of follow - up (between visit 2 and visit 4) defined by a fasting glucose of 7.0 mmol / l, nonfasting glucose of 11.1 mmol / l, self - reported physician diagnosis of diabetes, or use of glucose - lowering medication at visit 3 or visit 4 (n = 600). After visit 4, the aric study obtained self - reported information on diabetes diagnosis and medication use by annual telephone calls, for a maximum of 15 years of follow - up . Using this information, we investigated heart failure occurring in the absence of diagnosed diabetes by censoring incident diabetes cases occurring before the heart failure event (n = 1,497). We also examined the association of 1% unit increase in a1c with heart failure risk . To evaluate whether this association was consistent in groups with low-/high - risk profile, we evaluated this association in the subgroup of participants according to the absence / presence of cardiovascular risk factors . All analyses were conducted using stata 10.1 software (stata corp, college station, tx) and a p value of <0.05 was considered statistically significant . Aric study participants provided information on demographic and behavioral variables and medical history to a trained interviewer at each visit . In this study, we used information obtained at visit 2, unless otherwise noted . Certified technicians measured three systolic and diastolic blood pressures with participants in the sitting position after 5 min of rest using a random - zero sphygmomanometer . A1c was measured using a high - performance liquid chromatography instrument (tosoh 2.2 plus in 20032004 and the tosoh g7 in 20072008, tosoh corporation, tokyo, japan) on all participants with available stored whole blood (16). We have previously demonstrated the reliability of measurements from these stored samples (17). Fasting serum glucose was measured by the optimized dart glucose reagent method and cholesterol, triglycerides, and hdl cholesterol were determined using enzymatic methods . Insulin was measured by radioimmunoassay (125insulin kit; cambridge medical diagnosis, bilerica, ma) at visit 1 (19). Estimated glomerular filtration rate was computed by the modification of diet in renal disease study equation (20). Evidence of atherosclerosis of the common carotid arteries (shadowing / plaque on either side or none) was determined by ultrasound examination (13,21). Aric investigators conduct continuous, comprehensive surveillance for all cardiovascular disease - related hospitalizations and deaths in the four communities . Incident heart failure was defined as death from heart failure in any position on the death certificate or as the first heart failure hospitalization with the international classification of diseases code, ninth revision (icd-9) 428 or tenth revision (icd-10) i50 in any position of the hospital discharge list (6). Incident heart failure from visit 2 to january 1, 2006, was analyzed in the present study . We categorized a1c using the following cut - points: <5.0, 5.05.4, 5.55.9, and 6.06.4% . We evaluated the continuous association between a1c and the incidence rates of heart failure using a poisson regression model incorporating linear spline terms for a1c (knots at 5.0, 5.5, and 6.0%) with adjustment for age, sex, and race . Cox proportional hazards models were used to quantify the association between the above categories of a1c and incident heart failure . We tested for interactions using the likelihood - ratio test . For 10,866 participants (98.3%) who provided fasting (8 h) blood samples, we also evaluated the association of fasting glucose levels and incident heart failure by using clinical categories of glucose concentration as follows: <5.0, 5.05.5, 5.66.0, 6.16.9 mmol / l (<90, 9099, 100109, and 110125 mg / dl). We used the most prevalent category within a normal range as a reference group for both a1c (5.05.4%) and fasting glucose (5.05.5 mmol / l) levels . Model 2 was further adjusted for level of education, carotid atherosclerosis, systolic blood pressure, antihypertensive medication, smoking, alcohol intake, bmi, ldl cholesterol, hdl cholesterol, a self - reported history of coronary heart disease (chd) by visit 2, clinical examination, or hospital records and estimated glomerular filtration rate . Model 3 was adjusted for all variables in model 2 plus either baseline fasting glucose or a1c, as appropriate . We also investigated the association of quartiles of a1c or fasting glucose with heart failure risk . First, we examined heart failure occurring in the absence of clinical chd . To accomplish this, we conducted our analysis censoring incident chd cases that occurred prior to the date of heart failure (n = 1,088). Second, we repeated our analyses after excluding participants who had incident diabetes in the first 6 years of follow - up (between visit 2 and visit 4) defined by a fasting glucose of 7.0 mmol / l, nonfasting glucose of 11.1 mmol / l, self - reported physician diagnosis of diabetes, or use of glucose - lowering medication at visit 3 or visit 4 (n = 600). After visit 4, the aric study obtained self - reported information on diabetes diagnosis and medication use by annual telephone calls, for a maximum of 15 years of follow - up . Using this information, we investigated heart failure occurring in the absence of diagnosed diabetes by censoring incident diabetes cases occurring before the heart failure event (n = 1,497). We also examined the association of 1% unit increase in a1c with heart failure risk . To evaluate whether this association was consistent in groups with low-/high - risk profile, we evaluated this association in the subgroup of participants according to the absence / presence of cardiovascular risk factors . All analyses were conducted using stata 10.1 software (stata corp, college station, tx) and a p value of <0.05 was considered statistically significant . Participants with a1c 5.5% were more likely to be older, african american, and smokers but less likely to be current drinkers as compared with the reference group (a1c 5.05.4%). Individuals with a1c 6.06.4% at baseline were also more likely to have higher bmi, higher blood pressure, adverse lipid profile, and higher prevalence of chd and carotid atherosclerosis . A1c and fasting glucose were weakly, but significantly, correlated (r = 0.32, p <0.001). Characteristics of participants according to categories of a1c data are mean sd or percentage . All comparisons were significant at p <0.001, except for sex (p = 0.017). Egfr = estimated glomerular filtration rate . * missing values (number missing): educational level, 13; current smokers, 1; bmi 9, fasting glucose 191; blood pressure, 1; ldl - c, 136: hdl - c, 37; triglycerides, 7; history of chd, 98; carotid atherosclerosis, 211 . During a median follow - up time of 14.1 years the continuous associations of a1c and fasting glucose levels with incidence rate of heart failure with the adjustment for age, sex, and race are shown in fig . The incidence rate of heart failure increased linearly above a1c 5.0% (tests for differences in slopes above a1c 5.0% were not statistically significant; data not shown) and was 2-fold or higher in a range of a1c 6.0% as compared with that of a1c 5.0% . Mmol / l, the slope was much shallower than that for a1c and was flat at the range of 5.05.5 mmol / l . Increased risk of heart failure was observed at the low ranges of a1c (<5.0%) and fasting glucose (<5.0 mmol / l), although 95% cis were wide, reflecting imprecision of the estimate . The graph shows incidence rates (per 1,000 person - years) and 95% cis (shaded area) of heart failure with spline terms of a1c (knots at 5.0, 5.5, and 6.0%) (a) and fasting glucose (knots at 5.0, 5.6, and 6.1 mmol / l [90, 100, and 110 mg / dl]) (b) adjusted for age, sex, and race . The histograms represent the frequency distribution of a1c (4.56.5%) and fasting glucose (4.26.9 mmol / l [75125 mg / dl]) in the study sample . We estimated the hazard ratios and corresponding 95% cis for incident heart failure by categories of a1c using cox proportional hazards models adjusting for multiple covariates (table 2). As compared with participants with a1c 5.05.4, the hazard ratios of heart failure rose progressively from 1.44 (95% ci, 1.241.68) to 2.04 (95% ci, 1.632.54) across categories of a1c 5.5% in the model adjusted for age, sex, and race (model 1). The association among individuals with a1c 6.06.4% remained significant even after adjustment for all traditional cardiovascular risk factors (model 2, hazard ratio 1.38 [95% ci, 1.091.75]), although the association among participants with a1c 5.55.9% was attenuated to borderline significance (hazard ratio 1.16 [0.981.36], p = 0.08). These associations did not change appreciably after further adjustment for use of antihypertensive drugs (i.e., -blockers, ace inhibitors, or diuretics), which might potentially affect both glucose metabolism and risk of heart failure (data not shown). Adjusted hazard ratios (hrs; 95% ci) for incident heart failure (hf) according to a1c categories * model 1: adjusted for age, race, and sex . Model 2: model 1 + level of education, carotid atherosclerosis, systolic blood pressure, antihypertensive medication, smoking, alcohol intake, bmi, ldl - c, hdl - c, a history of chd at baseline, and egfr . Model 3: model 2 + fasting glucose . There was no evidence of effect modification by a history of chd at baseline (p for interaction = 0.83), and similar, but slightly attenuated, associations were observed when we censored participants without prevalent chd at baseline who developed chd prior to heart failure (hazard ratio 1.27 [95% ci, 0.951.70] for a1c 6.06.4% and trend p = 0.095). The exclusion of participants who developed diabetes during the first 6 years or censoring participants who self - reported diagnosed diabetes before heart failure during follow - up did not alter the results (data not shown). Participants with fasting glucose levels of 6.16.9 mmol / l but not 5.66.0 mmol / l had an increased risk of heart failure as compared with those with glucose levels of 5.05.5 mmol / l in model 1 (table 3). However, the association was greatly attenuated after adjustment for multiple covariates (model 2) and no longer significant when a1c was included in the model (model 3, hazard ratio 1.11 [95% ci, 0.901.35]). Mmol / l was associated with higher risk of heart failure as compared with the reference group, even after adjusted for multiple covariates (model 2, hazard ratio 1.51 [1.142.00]). This association remained significant even after adjusting for a1c (model 3) or restricting the sample to participants who contribute more than 5 years follow - up time (hazard ratio 1.55 [1.132.13]). We repeated the analyses using an average fasting glucose level at visit 1 and visit 2 and obtained similar results (data not shown). Adjusted hrs (95% ci) for incident hf according to fasting glucose categories * model 1: adjusted for age, race, and sex . Model 2: model 1 + level of education, carotid atherosclerosis, systolic blood pressure, antihypertensive medication, smoking, alcohol intake, bmi, ldl - c, hdl - c, a history of chd at baseline, and egfr . When we compared the quartiles of a1c and fasting glucose in model 2, the highest quartile of a1c (5.76.4%) but not fasting glucose (6.06.9 mmol / l [108125 mg / dl]) was associated with heart failure risk as compared with the lowest quartile of a1c (<5.2%) and fasting glucose (<5.3 mmol / l [<95 mg / dl]) (hazard ratio 1.42 [1.131.78] and 1.03 [0.841.27], respectively). Similar results were observed when we used the second quartile of fasting glucose (5.35.6 mmol / l [95100 mg / dl]) as the reference group (data not shown). We also examined the joint association of a1c and fasting glucose with heart failure risk (table 4). A1c 6.06.4% compared with 5.05.4% was significantly associated with increased risk for heart failure at fasting glucose levels of 5.05.5 in contrast, the association of elevated fasting glucose with heart failure was not significant at a1c 5.05.4% . Similarly, there was no consistent increase in heart failure risk associated with higher fasting glucose at other a1c categories . Although the relative risk associated with higher a1c tended to be larger among participants with low / normal fasting glucose levels as compared with those with elevated fasting glucose levels, the interaction of a1c and fasting glucose categories on heart failure risk was not significant (p = 0.257). Adjusted * hrs (95% ci) for incident hf according to the combination of a1c and fasting glucose categories * adjusted for age, race, and sex, level of education, carotid atherosclerosis, systolic blood pressure, antihypertensive medication, smoking, alcohol intake, bmi, ldl - c, hdl - c, a history of chd at baseline, and egfr . Finally, we modeled the association of heart failure risk per 1% unit increase in a1c and examined this association in different subgroups (fig . 2). Overall, each 1% unit increase in a1c was associated with 39% (95% ci, 1370%) increased risk of heart failure after adjusted for multiple covariates . These results were largely consistent across the different subpopulations (all ps for interaction> 0.05). Hazard ratios overall and within subgroups adjusted for the same covariates as model 2 in table 2 are shown . In this community - based population, we found that elevated a1c even in the range under 6.5% was associated with heart failure risk independently of traditional cardiovascular risk factors in middle - aged individuals during a median of 14 years of follow - up . The risk conferred by 1% unit increase in a1c in our study was 39% and was slightly higher than what has been reported in populations of people with diabetes (36). This association remained significant even after censoring participants who developed diabetes during median follow - up of 14 years before incident heart failure, suggesting that impaired glucose metabolism even before the development of diabetes is an independent risk factor of heart failure . Given that the association between a1c and heart failure risk was somewhat attenuated by limiting to heart failure cases without preceding chd, elevated a1c apparently confers heart failure risk partially through its association with increased chd risk (22). Several other mechanisms might explain the positive association between a1c and risk of heart failure . People with glucose intolerance may have other comorbidities like hypertension or obesity predisposing to the development of heart failure (23). Hyperinsulinemia may also play a role by stimulating sodium retention and/or activating the sympathetic nervous system (8). Insulin is a known growth factor and may contribute to myocardial dysfunction via increases in cardiac mass (8). However, the association of a1c with risk of heart failure in our study was independent of hypertension, obesity, other traditional cardiovascular risk factors, and insulin concentration, suggesting direct effects of hyperglycemia on the development of heart failure . Increased oxidative stress is associated with cell injury or apoptosis, resulting in decreased cardiac contractile (24). Glucose may also interact with collagen and stimulate the production of advanced glycation end products (25). Advanced glycation end products are hypothesized to induce fibrosis in the heart, leading to myocardial stiffness and diastolic dysfunction (25,26). Whether lowering a1c via lifestyle modification or medication can reduce the risk of heart failure in nondiabetic populations is an important question . To our knowledge some clinical trials (27,28), but not all (29), demonstrated that interventions with lifestyle modification or glucose - lowering medications may reduce cardiovascular risk in individuals with impaired glucose tolerance . The stop - niddm trial showed reduced risk of cardiovascular events by an -glucosidase inhibitor, acarbose, but had few heart failure cases (27). Although an increased risk of heart failure with rosiglitazone treatment was reported in the dream trial (29), the elevated risk of heart failure may be specific to this pharmacologic agent (30). Further studies are needed to evaluate whether early interventions to prevent glucose intolerance and lower a1c levels might reduce subsequent heart failure risk . In contrast to the robust independent association of a1c with increased risk of heart failure, fasting glucose was only weakly associated and no longer significant after adjusting for a1c in our study population . This weak association between fasting glucose and incident heart failure in a nondiabetic population is consistent with the previous literature (7,8) and may result from relatively high variability in glucose measurements (9). Superiority of a1c to fasting glucose for disease prediction has also been observed for chd, stroke, and total mortality (31). Nevertheless, our results suggest that a1c is a better risk marker of heart failure as compared with fasting glucose in a nondiabetic population . Mmol / l had an increased risk of heart failure compared with individuals with fasting glucose 5.05.5 such a j - shaped association has been observed for all - cause or cardiovascular mortality in some studies (3234) but not for heart failure (7,8,35). Given that excluding heart failure cases within 5 years of follow - up did not alter our findings, reverse causation is unlikely . There remains the possibility that comorbid conditions associated with both lower glucose concentration and risk of heart failure, e.g., liver dysfunction (36), might account for the observed higher heart failure risk at low serum glucose concentrations . First, we evaluated the association of a single measurement of a1c and heart failure . A previous study showed that updated average a1c may predict future heart failure better than a single baseline a1c in patients with diabetes (4). However, in nondiabetic populations, a single a1c measurement is highly reliable (37,38). Second, as with any observational study, we cannot rule out the possibility of residual confounding despite adjustment for all major cardiovascular risk factors . Third, identification of heart failure cases relied entirely on icd codes abstracted from hospital records and death certificates (6,39). Reliance on hospital discharge codes may underestimate heart failure incidence (40). Finally, because the aric study consists of a middle - aged, bi - ethnic community - based population of the u.s ., additional studies are needed in younger populations, the elderly, or other ethnicities . In conclusion, elevated a1c (5.56.0%) was more strongly associated with increased risk of heart failure as compared with fasting glucose in a middle - aged bi - ethnic population without diabetes . Our findings suggest that chronic hyperglycemia even before the development of diabetes is an independent risk factor of heart failure and may contribute to the development of heart failure beyond its effect on chd risk.
Diastolic mitral regurgitation has been described in patients with acute aortic regurgitation and those with atrioventricular block or atrial fibrillation with slow ventricular responses . Evidence of diastolic mitral regurgitation in these patients has been demonstrated previously by left ventriculography and recently by pulse doppler echocardiography . The mechanisms of diastolic mitral regurgitation in patients with acute aortic regurgitation have been considered to be the summation of the following pathophysiology: reversed atrio - ventricular pressure gradient due to aortic regurgitation in the non - compliant ventricle, increased mitral annulus due to ventricular relaxation and lack of papillary muscle tension . That of the patients with atrioventricular block or atrial fibrillation has been considered to be lack of atrial factors for mitral valve closure . We documented diastolic mitral regurgitation by color doppler echocardiography in a patient with acute aortic regurgitation . Color doppler flow imaging is highly useful in detecting valvular regurgitation, especially in determining flow direction . Therefore, this technique may provide additional information regarding the mechanism by which diastolic mitral regurgitation is produced . Reversibility of diastolic mitral regurgitation after correction of hemodynamic loading, that was widely believed but not actually demonstrated, was also observed . A 38-year - old japanese man with no previous cardiovascular disease was admitted to our hospital because of paroxysmal nocturnal dyspnea and low grade fever of four weeks duration . He had the history of the resection of subcutaneous abscess in the neck, several days before the onset of symptom . On admission, an early systolic murmur and a diastolic blowing murmur with thrill were detected at the left parasternal border . Increased pulmonary vascular marking was observed in his chest x - ray and the cardio - thoracic ratio was 50% . Electrocardiogram revealed a prolonged p - q interval of 0.28 second, left ventricular hypertrophy and st depression in the left precordial leads . After admission, first - degree of atrioventricular block normalized in 0.18 second of p - q interval . Echocardiography was performed with a tohshiba 65a after normalization of p - q interval, which revealed the dilated and hyperkinetic left ventricle and the slightly dilated left atrium . 1), and the left ventricular end diastolic dimension was 6.4 cm and the end systolic dimension was 4.4 cm . The right coronary cusp and left coronary cusp of the aortic valve were extremely elongated and the left valsalva sinus was dilated . Both aortic cusps were prolapsing and no coaptation was seen . Severe aortic regurgitation was observed on color doppler flow imaging . In late diastole, mitral regurgitation which ran just behind the posterior mitral leaflet and extended to the posterior wall of the left atrium was detected (fig . The diastolic mitral regurgitation began with the atrial systole and disappeared with the ventricular systole (fig . Although several blood cultures were all negative, the diagnosis of acute aortic regurgitation due to infective endocarditis was made from the clinical history and destructive changes of the aortic valve . After intensive antibiotic therapy for three weeks, aortic valve replacement was performed . At surgery, commissural rupture between the right coronary cusp and the left coronary cusp was detected and an abscess was present in the root of the left coronary cusp . Diastolic mitral regurgitation in patients with acute aortic reguritation in patients with acute aortic regurgitation is considered to be produced by hemodynamic abnormalities, not by intrinsic abnormalities of mitral leaflets . However, the proof of this consideration is somewhat obscure . In most reported cases with diastolic mitral regurgitation, the concurrent presence of systolic mitral regurgitation, which indicates the presence of intrinsic abnormalities of mitral leaflets or apparatus, was reported . If the mitral valve is truly normal, systolic mitral regurgitation should be absent and diastolic mitral regurgitation should disappear with resolution of the hemodynamic abnormalities . The reversibility of diastolic mitral regurgitation after aortic valve replacement has never been described . In this case, the mitral regurgitation was observed only in the period of late diastole, disappearing with the ventricular systole . Furthermore, the diastolic mitral regurgitation in this case could not be detected after the successful aortic valve replacement . Our case clearly indicates that acute aortic regurgitation can produce diastolic mitral regurgitation in a normal mitral valve . In this case, the flow of the diastolic mitral regurgitation was directed to the posterior wall of the left atrium through just behind the posterior mitral leaflet . From above mentioned mechanisms of diastolic mitral regurgitation in acute aortic regurgitation, if intrinsic abnormalities of the mitral leaflets are absent, regurgitant flow should be directed to the center of the left atrium . Extreme deviation of mitral regurgitant flow is observed in cases of mitral valve prolapse or those with severely restricted mitral valve leaflets, as is seen in rheumatic mitral disease . Because this case showed no abnormalities in the mitral leaflets under echocardiography, severely restricted movement of the mitral leaflets is unlikely . We speculated either that the aortic regurgitant jet might depress the anterior mitral leaflet toward the left atrium or that acute left ventricular enlargement due to aortic regurgitation might retract the chordae connecting to the posterior mitral leaflet . These processes would result in the dislodgment of the tips of both leaflets, creating extreme deviation of the mitral regurgitant flow similar to mitral prolapse . There are a few reports on the observation of diastolic mitral regurgitation by color doppler echocardiography, but no comments for the flow direction . This case indicated the possibility of additional mechanisms of diastolic mitral regurgitation, namely, depression of the anterior mitral leaflet or retraction of the chordae connecting to the posterior mitral leaflet.
Modified electrodes (mes) coated with a polymeric film containing particles of noble metals dispersed in the film are useful for the accomplishment of electrocatalytic hydrogenation of organic substrates . The polymeric film exchanges ions with the corresponding salts . These ions are then electrochemically reduced, and the obtained modified electrode (me) containing noble metals is used as the working electrode for hydrogenation of organic substrates . By application of an adequate cathodic potential to the cell containing a mineral acid solution, the h ion is reduced to h, which is adsorbed at metallic surfaces like ni, pd, and pt . Organic compounds with double bonds as olefins, carbonylic functions, or aromatics are also adsorbed, and the transference of hydrogen to these species yields saturated compounds . In our laboratories, we have studied the preparation of an electrode, modified with the film poly-(p - allyl ether benzenesulfonic acid) which is able to perform the electrocatalytic hydrogenation of organic substrates [13]. The film was prepared by electrochemical oxidation of the corresponding monomer, leading to initial formation of the radical cation, which in turn is the initiator of a chain reaction . The resulting polymer coats the electrode, giving rise to a film with high chemical and mechanical stability . This film presents low conductivity but recovers its electroactivity when the sulfonic group changes h for ni, pd, or pt . Some cathodic scans are enough to reduce these ions to metals in the fundamental state . Mixed electrodes containing cu, fe, and ni particles have been studied recently [510]. The electrodes were prepared by electroless or galvanic displacement of noble metals like ni, pd, and pt, thereby yielding the modified electrodes me cu / ni, pd, or pt, me fe / ni, pd, or pt, and me ni / ni, pd, or pt [3, 9, 10]. The electroless process consists of the adsorption of atomic hydrogen [1119], provided by sodium hypophosphite, on the surface of fe, cu, ni, pd, or pt particles previously present in the electrode . Then, an adequate potential must be initially applied to the electrode, dipped into the metal salt solution for some minutes, to produce the microcrystals necessary for the electroless process . The atomic hydrogens that are adsorbed on the electrode transfer electrons to the noble metal ions present in the solution, thereby reducing them to metallic particles . The latter particles become dispersed in the film, resulting in the growth of the previous crystals . Interesting results from previous works have shown that such a film is able to catalyze the reduction of the metallic ions by sodium hypophosphite itself, in the absence of metal crystals . Ni, pd, or pt ions are not reduced when their salts are dissolved in electroless solution; however, the reduction of these cations takes place fast and spontaneously when they are associated with the film poly-(p - allyl ether benzenesulfonic acid) with no need for h adsorption . This work describes the catalytic properties of the film poly-(p - allyl ether benzenesulfonic acid) in the reduction of noble metal ions to their corresponding metals by sodium hypophosphite . All reagents and solvents used here were analytical grade and purified when necessary . A potentiostat / galvanostat par model 273 and the software m270 program were employed in the experiments . Cyclic voltammetry experiments were carried out in a cell (15 ml) using glassy carbon as the working electrode (3 mm diameter), an ag / agcl electrode as the reference electrode, and a platinum wire as the auxiliary electrode . Scans were carried out from 0.0 to 1.0 v versus ag / agcl, at 10 or 100 mvs . The surface morphology, microstructure, and elemental composition of the metals deposited on the film were analyzed by scanning electron microcopy (sem) and energy dispersive x - ray spectroscopy (eds), by means of a leica - zeiss leo 440 model sem coupled to an oxford 7060 model analyzer . The preparation of the monomer p - allyl ether benzenesulfonic acid and its polymerization over a glassy carbon electrode (sulfonic acid film) has been described in the literature . The modified electrode prepared by ion exchange / chemical reduction had been previously characterized by sem and eds . A glassy carbon electrode (3 mm diameter) coated with the sulfonic acid film was dipped into 2.5 ml niso4 (0.12 mol l) solution for one hour . The electrode was washed with deionized water and installed versus ag / agcl in a cell containing 10 ml kcl (0.1 mol l) solution . Then, five scans from 0.1 to 0.7 v versus ag / agcl were carried out at 10 mvs . At the end of the procedure, the same procedure was also carried out in the cases of the platinum and palladium salts (0.12 mol l pdcl4 or ptcl4 solutions). The modified electrodes containing dispersed noble metals were arranged in a cell containing 10 ml h2so4 (0.1 mol l) solution . The carbon glassy electrode coated with the sulfonic acid film was dipped into 10 ml niso4 (50 mmol l) and nah2po2 (0.3 mol l) solution (ph adjusted at 2.0 with concentrated hcl) in different time periods . The electrode was gently washed with water and installed as the working electrode in a cell containing 10 ml h2so4 (0.1 mol l) solution . Scans from 0 to 1.0 v were performed to generate hydrogen . In another set of experiments, both ph - adjusted solutions were dipped into the nickel salt solutions for different time periods, varying from 20 s to 25 min ., and hydrogen generation was carried out in the same way . The same procedure was employed in the case of sulfonic acid dipped into a solution of ptso4 or pdso4 (50 mmoll), and 0.3 mmol l nah2po2 during pre - established immersion periods, at ph 2.0 or 8.0 . The strategy employed here to demonstrate the catalytic effect of the sulfonic acid film containing dispersed ni, pd, and pt on the reduction of these ions using nah2po2 was to compare hydrogen generation by the modified electrodes containing ni, pd, and pt particles prepared by two different methods: (a) the conventional approach consisting of ion exchange and electrochemical reduction of the corresponding ions and (b) dipping of the electrode containing the sulfonic acid film into an electroless solution containing the respective ions . Ion exchange occurs followed by chemical reduction of the metallic ion promoted by nah2po2 at ph 2.0 or 8.0 . Sem and eds analyses were carried out to show the presence of the metal particles in the film . Figure 1(a d) depict the currents for hydrogen generation obtained by scans in h2so4 solution, using the mes with ni (b), pd (c), or pt (d) prepared by method a and designated me ni, me pd, and me pt . Experimental conditions such as ph 7.0 and different immersion periods were applied, since these were the best results obtained in previous studies . For both me pd and me pt, the onset of current due to h2 evolution took place at potential values, about 100 to 200 mv lower compared with me ni . Figures 2(a) and 2(b) display the currents obtained with scans in h2so4 solution, using the mes ni, pd, and pt prepared according to method b, at ph 2.0 and 8.0, using the time period necessary for current stabilization (maximum electrode efficiency). It is important to note that there is not anodic peak of reduction of metallic ions present in solution . The currents were similar and have the same order of magnitude than those generated by the mes prepared by method a. the onset of h2 evolution in the case of the mes pd and pt at ph 2 and ph 8 occurred at potentials 350 and 200 mv lower, compared with me ni . When method b and ph 2 are used to prepare the mes, the highest currents are achieved with the mes pt and pd (table 1). When method b and ph 8 are employed, the highest current is obtained for me ni followed by me pt . These currents are higher than those achieved with the mes prepared by method a. at both phs, me pt stabilizes faster than the other mes . The onset of h2 evolution occurs at the same potential me ni, whatever the preparation method is (a or b). Hydrogen evolution started at lower potentials in the case of the mes pd and pt prepared by method b, indicating that a lower energy is necessary . But this process was ph dependent, so one can conclude that this is due to the nature of the employed metals . It is important to highlight that former studies [9, 10] have demonstrated that the electrochemical hydrogenation of organic substrates begins at potentials close to that for initial h2 generation . Substrate hydrogenation occurs with transfer of the radical hydrogen to the unsaturated organic molecule, which are both adsorbed on the surface of the noble metal . The excess of radical hydrogen at more negative potential produces bubbles that damage the integrity of the film, repel the approximation of the substrate during the electrolysis by convection, and are energetically unnecessary for the hydrogenation reaction . So, one can conclude that mes prepared by method b are more efficient and simpler than those prepared by method a, considering the experimental preparation of the electrodes, the current of hydrogen generation, and the potential necessary for the onset of hydrogen radical production . The different times to stabilize the electrodes in the hydrogen generation process are due to macromodifications of the charged polymer dipped in a solution with electrolyte . Data for the elements carbon, oxygen, sulfur, and the metals ni, pd, and pt are listed in table 2 in the mes ni, pd, and pt prepared by method b. figures 3 and 4 show the eds results obtained for all the investigated electrodes prepared by method b. eds gives anomalous results for carbon and oxygen, because the electrode contains carbon . Moreover, analysis reveals several peaks for metal oxides (figure 4). For me pt, the occurrence of sulfur is smaller than the limit of detection ratio of the instrument . But it is interesting to consider the relation between the [m]/[s] ratio for the mes pd and ni . In both cases, it is considered that there is one sulfur atom per monomer unit in the polymer . Again, one can remark here that pt is more abundant in the me compared with pd and ni, thereby explaining the high efficiency of me pt compared with mes pd and ni . Figure 3 corresponds to the sem image of the surface of the polymeric film (a), and polymeric film with metallic ions pt, pd, or ni, (b, c, and d). Eds was carried out to confirm the presence of the metals (figure 4). Figure 3(a) shows the film roughness and evidences that fibers were formed after deposition of the film onto the glassy carbon (b, c, and d). Figure 3(d) reveals the same effect, but one can notice the presence of cracks that increase with the amount of ni metal incorporated into the film . Figures 3(b) and 3(c) demonstrate that the metals are agglomerated in the film with cracks and without cracks . Figure 4(a) confirms the sem results and the presence of carbon as the major constituent of the samples . Although the film is composed by c, o, s, and h, the major presence of c is due to the electrode support material, as can be seen in all the cases . The minor presence of ni in figure 4(b) is also due to the program system, which compares all the elements with c (the major element). The higher quantity of the metals pd and pt is in agreement with the results obtained previously, which showed that these electrodes are more reactive compared with me ni . The mechanistic proposition for metal dispersion into the film is based on two major facts: (a) hypophosphite is a strong reducing agent, which able to supply hydride ions and then reduce the positive bivalent ions ni, pd, and pt to the corresponding m; and (b) the capacity of the sulfonate groups in the film to fix these ions in their valence sphere . Here, it is necessary to indicate that in the film, the polymeric bonds involve the aromatic groups, leaving the sulfonate group free to associate with ions, as earlier demonstrated in the literature for tyramine electropolymerization and illustrated here in figure 5 as a simplified scheme . The products are phosphoric acid (or the corresponding sodium phosphate, depending on the ph) and m, the metal crystals (figure 6). Hypophosphite acid or sodium hypophosphite is attacked by two water molecules, being oxidized and furnishing two hydride ions that reduce the metallic ion . The binding of the metal ion by sulfonate or sulfonic groups is a well - known step in the catalytic process, since it also occurs in enzymatic catalysis . In acid or basic ph, there is probably a strong decrease in the solvation sphere of the ion for all the cations studied here, since the bulky polymer structure facilitates the approximation of the small hydride ion . This proposed mechanism is quite different from the electroless mechanism based on the homolytic cleavage of the p the film poly-(p - allyl ether benzenesulfonic acid) catalyzes the reduction of ni, pd, and pt to the corresponding m noble metals by binding the ions in the valence sphere of the free sulfonate group . Sodium hypophosphite transfers the electrons through the hydride ion when it is oxidized by water to phosphite or phosphoric acid . Sem and eds analyses showed the presence of metal crystals dispersed in the film in an amount higher than the equivalent molecular polymer unit, which confirms the catalytic effect on the reduction reaction . The electrochemical hydrogen generation of two kinds of electrodes, one prepared by electrochemical reduction of the metal ions and the other by this catalytic process, showed that the latter method is more efficient than the former . The me pt is the most efficient in acid ph, whereas me ni is the most efficient in basic ph . When mes are prepared by the catalytic process, less energy is necessary for initiation of the hydrogen generation useful for the catalytic hydrogenation of organic substrates, compared with the process involving the electrochemical preparation of mes.
Brucellosis (malta fever) is an infectious disease with a wide range of manifestations . There are four types of brucella, b. melitensis, b. abortus, b. canis and b. suis . Bacteria are transmitted to humans via the injection of non - pasteurized dairy products, uncooked raw meat or by contact through skin, blood, conjunctiva, gastrointestinal or respiratory tracts.12 although the incidence of brucellosis has declined, it is still remains an important health problem in endemic areas such as the middle east, the mediterranean and asia . Iran is considered an endemic country as are peru, saudi arabia, kuwait and turkey.34 according to the report of the diseases prevention and fight department of iran s health center, published in 2009, the incidence of brucellosis was 25 in 100,000 people . In hamadan, studies show that b. melitensis is the most common and virulent species with a high prevalence in latin america, mediterranean and developing countries . B. abortus is reported mostly in europe and north america . Despite the decrease in total prevalence of the disease a 29-year - old male was referred to infectious disease clinic of hamadan sina hospital in summer 2008 with acute onset of fever, headache, malaise, sweating and low back pain . On examination laboratory test findings were: white blood cell (wbc) count of 13350 (neutrophil: 85%), erythrocyte sedimentation rate (esr) 48m / h, crp, rf, ana, wright s agglutination titer of 1/1280 . Blood cultures were negative and electrocardiogram and chest - x - ray (cxr) were normal . The patient was diagnosed with brucellosis, and treated with rifampin 600 mg / day and doxycycline 100 mg / twice daily . After a week, the patient was referred for an ophthalmology consult with complaints of ocular pain and redness and visual complaints . On ophthalmic examination, the conjunctional injection was a mixture of ciliary injection, episcleritis and conjunctivitis but more severe in the ciliary area . On funduscopy, there was bilateral optic disc swelling along with retinal hyperemia (optic disc hyperemia and vascular tortuosity) and diffuse intraretinal hemorrhage [figure 1]. Intraocular pressure (iop) was 26 mmhg in the right eye and 24 mmhg in the left eye . The patient was hospitalized with a probable diagnosis of ocular brucellosis and was treated with co - trimoxazole adult two tablets, three times a day (tid), rifampin 600 mg / day doxycycline 100 mg / bid and prednisolone 1 mg / kg for 2 months . Computed tomography and magnetic resonance imaging studies of the brain and optic nerve were requested . Fever and headache diminished within 48 hours of the treatment however, ophthalmic complaints lingered . Bilateral optic disc swelling with retinal hyperemia (optic disc hyperemia and vascular tortuosity) and diffuse intraretinal hemorrhage fundoscopic findings 13 months after treatment for brucellosis brucellosis presents with a spectrum of clinical manifestations and diagnosis of this disease is based on clinical signs, positive bacteriological and serological tests . Some ocular manifestations including dacryoadenitis, episcleritis, chronic iridoscleritis, nummular keratitis, cataract, glaucoma, multifocal choroiditis, exudative retinal detachment, maculopathy, and optic neuritis.814 rolando et al.14 showed that the most frequent ocular manifestation is uveitis predominantly posterior uveitis . It seems that optic nerve involvement is secondary to meningeal inflammation and flow change of the optic nerve due to axonal degeneration.14 visual improvement of the patient following corticosteroid administration is proof of ischemic or vasculitic involvement.1410 ophthalmic manifestations of brucellosis are not common and acceptable outcomes following treatment with antibiotics and steroids are low.9101517 cavallarro et al.18 reported a patient with papilledema due to brucellosis that was treated with sole anti - brucellosis treatment without steroid administration . Abd - elrazak19 reported a case of bilateral optic neuritis caused by brucellosis that resolved following anti - brucellosis and steroid administration . Lashay et al.20 from iran reported a case of bilateral optic nerve head swelling following brucellosis, which led to bilateral optic nerve atrophy and visual loss . In our case, antibiotic and steroid administration led to complete visual recovery and in 13 months follow - up after treatment, ophthalmologic examinations were normal . The outcome in our case is likely due to early diagnosis and treatment, lack of a drug resistant strain and better prognosis in males compared to females.142123 in the current case, imaging studies were normal . However, such lesions may be missed on routine imaging studies if magnetic resonance angiography (mra) is not performed . Considering the rapid response to the therapeutic interventions likewise, other possible causes of conjunctival injection associated with increased venous pressure such as cavernous sinus thrombosis or orbital apex syndromes were ruled out due to this quick and appropriate response to treatment . The prevalence of brucellosis has decreased in many developed countries and ophthalmic complications are rare in these regions, but it is suggested that in endemic areas, routine ophthalmic examination for brucellosis be considered, as it seems that early diagnosis and prompt treatment of the disease could decrease vision - threatening complications.23
Thyroid nodules are common in the united states, with reported prevalence between 35% and 65% . Often, these nodules are evaluated by fine - needle aspiration (fna), which is recognized as the gold standard to evaluate thyroid nodules as it is a minimally invasive procedure that provides an approach for management and assists in determining the correct surgical procedure if necessary . For example, parathyroid glands are located in the neck near the thyroid gland and are sometimes in the thyroid (intrathyroid parathyroid). In one autopsy series, the incidence of ectopic parathyroid glands was reported to be 42.8% with 19.6% in the mediastinum / thymus, 12.5% in the thyroid subcapsular space, and 5.4% in the thyroid parenchyma . Three different types of parathyroid epithelial cells may be seen: (1) chief cells, resembling follicular cells of the thyroid, (2) oxyphil cells, resembling hurthle cells of the thyroid, and (3) water clear cells, which have clear cytoplasm . The overlapping cytologic features of chief cells and oxyphil cells with follicular and hurthle cells, respectively, may make it difficult to distinguish parathyroid from thyroid on both cytologic and sometimes histologic specimens . Fnas from thyroid follicular lesions show cells arranged in a microfollicular pattern (612 crowded cells in a ring- or rosette - like configuration), a pattern that may also be observed in fnas of parathyroid tissue . Similar diagnostic challenge can occur during evaluation of frozen sections, especially when (1) there are oncocyte - rich nodules in the parathyroid, which can mimic hurthle cell lesions of the thyroid, (2) there is follicle formation by parathyroid glands, simulating a cellular thyroid nodule, or (3) there is fat present in a thyroid nodule, which can mimic parathyroid stroma . Previous studies have characterized helpful features suggestive of parathyroid, which include the presence of granular or stippled nuclear chromatin, disorganized sheets (which we designate as loose two - dimensional [2d] clusters), three - dimensional (3d) fragments, nuclear overlapping, nuclear molding, anisonucleosis, prominent vascular proliferation with attached epithelial cells, and frequent occurrence of single cells / naked nuclei . However, to the best of our knowledge, no previous studies have examined the features specifically distinguishing parathyroid from bethesda category iv (beth - iv) thyroid lesions, a potential and most likely pitfall given that both are likely to be cellular and have relatively scant the aim of this study was to assess the distinguishing cytomorphological features of parathyroid (including intrathyroidal) and beth - iv thyroid follicular lesions, which may be particularly useful in recognizing unexpected parathyroid tissue and managing patients with a computerized search was performed from 2006 to 2014, to identify consecutive cases of all parathyroid diagnoses in parathyroid and thyroid fnas and beth - iv thyroid fnas (follicular and hurthle cell), all with diagnostic confirmation through surgical pathology, immunocytochemical stains, afirma analysis, and/or clinical correlation . The cytologic slides were prepared with direct smears, using diff - quik and papanicolaou stains, and thinprep technique . Cell blocks were prepared on a minority of cases (3 of 5 parathyroid and 4 of 12 beth - iv thyroid cases). Ancillary studies were performed on cell blocks, and all surgical pathology slides were prepared in a standard fashion from formalin - fixed paraffin - embedded tissue and stained with hematoxylin and eosin stain . All cytologic slides and their corresponding surgical pathology slides were reviewed by two pathologists and scored from 0 to 3 (0 showing the least quantity or quality of the cytomorphologic feature and 3 showing the most) or as present versus absent on the unique cytomorphologic features . The parameters were identified from a literature search and findings from our own observations, which included epithelial cellularity, predominant pattern (flat sheets, 2d, 3d, microfollicles, loose clusters, papillary, single cells), naked nuclei, vascularity, perivascular epithelial cells, cell size, cell shape, nuclear - to - cytoplasmic (n: c) ratio, chromatin quality, nuclear contour, anisonucleosis, presence / size of nucleoli, cytoplasmic quality, cytoplasmic vacuoles, cytoplasmic borders, and presence of histiocytes and/or hemosiderin - laden macrophages . Categorical and continuous variables were assessed using fisher's exact test and student's t - test, respectively, using a p <0.05 for statistical significance . All covariates with p we identified five fna cases with clinical suspicion of parathyroid or thyroid lesion that had an eventual final diagnosis of the parathyroid lesion (all female; age 2069 years) and 12 beth - iv diagnoses (11 female, 1 male; age 1364 years). Of the five parathyroid cases, 3 were clinically designated as thyroid fnas, 1 as parathyroid fna, and 1 as neck nodule fna . Four of the 5 parathyroid cases had suspected fna diagnoses of the parathyroid lesion [table 1]. Two resection specimens confirmed the diagnosis of intrathyroidal parathyroid tissue, including one parathyroid adenoma . The third surgically resected case was initially designated as a neck nodule and had a clinical suspicion of thyroid versus parathyroid tissue; it was proven to represent parathyroid adenoma . Case #1 without surgical pathology follow - up had positive parathyroid hormone (pth) immunocytochemical stain . Case #5 was diagnosed as beth - iii, follicular lesion of undetermined significance on fna; however, afirma analysis suggested a parathyroid lesion . All 12 beth - iv cases had surgical resection follow - up, and the results are summarized in table 2 . Parathyroid / thyroid - designated fine - needle aspiration diagnoses and follow - up bethesda category - iv thyroid diagnoses and the corresponding surgical pathology diagnoses the parathyroid fnas were moderately cellular with cells arranged predominantly as 2d clusters (i.e., sheets of overlapping cells) and naked nuclei but no single intact cells in the background [figure 1a and b]. Other patterns observed were flat sheets, 3d clusters, occasional microfollicles, and some papillary structures . A moderate amount of colloid - like material was observed in 3 of 5 cases . The cells were round with indistinct cytoplasmic borders and scant, delicate cytoplasm . On average, the cells were about 2.4 the size of a red blood cell (rbc). They tended to have high n: c ratio, uniform round nuclei with inconspicuous nucleoli, and no cytological atypia [figure 1c]. The nuclei displayed an even chromatin quality without clumping except one case, which had some stippling . (a and d) the predominant architectural patterns: two - dimensional in (a) parathyroid and microfollicular in (d) bethesda category - iv (diff quik, 200). Many naked nuclei are present in both (b) parathyroid and (e) bethesda category - iv but more single cells are observed in bethesda category - iv (pap, 400). (c and f) a higher nuclear - to - cytoplasmic ratio of (c) parathyroid compared to (f) bethesda category - iv . Nucleoli and nuclear irregularity are evident in bethesda category - iv and absent in parathyroid (pap, 600) the 12 beth - iv fnas were moderately cellular with cells arranged predominantly as microfollicles, single cells, and some naked nuclei [figure 1d and e]. The other patterns observed in parathyroid cases were also identified with similar frequencies in the beth - iv cases . In addition, some loose clusters were also present . Occasional vessels and varying amount of colloid - like material were observed . The cells were round - to - oval and tended to be large, on average about 7 the size of an rbc . They displayed mild epithelial atypia with indistinct cytoplasmic borders and a moderate amount of delicate and sometimes oncocytic cytoplasm . The nuclei showed mild irregularities with stippled chromatin and conspicuous nucleoli [figure 1f]. The following features were associated with parathyroid lesion: 2d pattern (p = 0.001), even chromatin quality (p = 0.028), high n: c ratio (p = 0.007), and smaller cell size (2.4 rbc, p = 0.001). The features associated with beth - iv lesions were: microfollicular pattern (p = 0.001), presence of single cells (p = 0.001), presence of nucleoli (p = 0.001), nuclear irregularities (p = 0.01), stippled chromatin (p = 0.028), and larger cell size (7.25 rbc on average, p = 0.001). The following features were not significantly different in a univariate analysis: naked nuclei, cellularity, cell shape, anisonucleosis, cytoplasmic borders, presence of histiocytes / hemosiderin - laden macrophages, presence of colloid - like material, presence of vessels / vascularity, papillary architecture, 3d fragments, and nuclear molding / overlap . Multiple logistic regression analysis was performed using the seven cytomorphological variables with p <0.05 in the univariate analysis; no statistically significant associations with lesion type were found . Distinguishing parathyroid (including intrathyroidal) tissue from beth - iv thyroid follicular lesions has significant clinical implications since beth - iv lesions carry a 20%30% risk of malignancy and are often followed up with surgical resection . Ultrasound imaging may aid in clinical suspicion, but there are no specific image findings to distinguish parathyroid, thyroid, and lymph nodes, and fna remains the gold - standard for diagnosing thyroid nodules . Some early literature claimed that parathyroid lesions cannot be reliably distinguished from thyroid lesions on fna alone . Serum pth level or hypercalcemia can be used for suspicion of hyperparathyroidism; however, it is not useful in cases of nonfunctioning parathyroid lesions . More recent studies have described the varying appearance of parathyroid on cytology and the common diagnostic pitfalls . While cytological features of parathyroid tissue fna have been reported, the criteria specifically distinguishing parathyroid tissue from beth - iv thyroid lesions have not been previously reported to the best of our knowledge . The most statistically significant distinguishing features were overall pattern, presence of single cells, cell size, nuclear irregularity, nuclear chromatin quality, presence of nucleoli, and n: c ratio . Parathyroid fna specimens typically show intermediate - to - high cellularity with cohesive groups of cells showing 2d or 3d arrangements . Other described patterns include disorganized sheets (another term for 2d arrangements), prominent vascular network with associated epithelial cells, and microfollicles . In contrast, beth - iv thyroid aspirates, which are typically cellular, consist of follicular / hurthle cells arranged as microfollicles, trabeculae, and syncytia . Crowding and overlapping of cell groups are conspicuous, and the follicular cells are usually larger than normal follicular cells . Compared the cytological findings of parathyroid fnas to thyroid lesion fnas, which included papillary carcinoma, follicular adenoma, adenomatoid nodule, and hashimoto's thyroiditis . They found that overlapping 3d fragments were more likely to be found in parathyroid whereas flat honeycomb sheets are more common in thyroid aspirates . Our study showed similar findings, with 2d sheets representing the statistically significant cell arrangement that distinguishes parathyroid from beth - iv (based on the images in absher et al . 's study, the cell groups that we interpret as 2d sheets are quite similar to what they interpreted as 3d groups; for our study, we interpreted 3d architecture as more of a ball of cells as opposed to a cellular sheet with overlapping cells). Similar to the findings of absher et al ., papillary fragments and microfollicular arrangements were present in both our parathyroid and thyroid aspirates, and neither was a statistically significant distinguishing feature . Although naked nuclei have been described to be more prevalent in parathyroid aspirates, this feature was seen in both parathyroid and beth - iv in our study . In addition, we found both background colloid - like material and macrophages in our parathyroid and thyroid aspirates, indicating that have been previously reported . This features was variably present in the beth - iv cases and noted in only 1 of our parathyroid cases; however, we are uncertain whether these rare clusters represented parathyroid versus thyroid . Either way, it was not a statistically significant finding . Parathyroid cells demonstrate round - to - oval nuclei, regular nuclear membranes, and inconspicuous or absent nucleoli, and the chromatin is hyperchromatic, coarsely granular, or stippled . Moderate - to - high n: c ratio, anisonucleosis, and nuclear overlapping are other features that may be observed . The follicular cells of follicular thyroid lesions (beth - iv) are usually larger than normal follicular cells . Nucleoli are generally inconspicuous in parathyroid and infrequent in follicular nodules but may be seen in both . Compared to oncocytic parathyroid adenoma, hurthle cell thyroid neoplasms have larger nuclei, prominent nucleoli, and tend to be more discohesive . Compared to follicular cells, the nuclei of parathyroid cells contain chromatin that is more stippled or hyperchromatic than thyroid follicular cell nuclei, which contain more finely granular chromatin, although cells from follicular nodules (beth - iv) may have coarse chromatin . The presence of stippled chromatin was not a statistically significant finding in distinguishing parathyroid from beth - iv . We found nucleoli to be a significant finding in distinguishing parathyroid from beth - iv cases with all 5 parathyroid fnas lacking nucleoli and 11 of 12 beth - iv cases containing nucleoli though they ranged from inconspicuous to prominent . Anisonucleosis, although present in only a small portion of the cells, is a reported feature of parathyroid fnas but uncommon in thyroid follicular lesions . Anisonucleosis was minimal to nil in our parathyroid cases and ranged from nil to moderate in the beth - iv cases although the differences were not statistically significant . Described nuclear molding to be a feature that was present in most parathyroid aspirates and rarely seen in thyroid lesions . Well - defined cytoplasmic membranes / borders have been described in a minority of cases . The cytoplasm of follicular cells in beth - iv (follicular adenoma) lesions is usually delicately vacuolated or finely granular (follicular lesions) or granular and oncocytic (hurthle cell lesions). Compared to follicular cells, parathyroid cells are usually smaller and contain less cytoplasm, although they may contain oxyphilic cytoplasm, which can lead to misinterpretation as hurthle cells . While a few cells with more abundant cytoplasm were noted in 3 of our parathyroid cases, none demonstrated the abundant oncocytic cytoplasm that is typical of beth - iv hurthle cell lesions . The other 6 contained cells with oncocytic / hurthle cell features, 5 of which were shown to be follicular adenoma with hurthle cell features / hurthle cell adenoma on resection and 1 of which was shown to represent nodular goiter with focal hurthle cell change on resection . Shidham et al . Described the presence of intracytoplasmic fat vacuoles, best seen on romanowsky stain, in imprint smears from parathyroid gland as being useful in confirming parathyroid tissue . All 5 of our parathyroid specimens and all 12 beth - iv cases lacked cytoplasmic vacuoles . The smaller size of parathyroid cells compared to beth - iv was a significant finding in our study . All 5 of our parathyroid cases showed small cells with high n: c ratio which were 23 the size of an rbc compared to the larger cells of beth - iv cases, which contained cells that were on average 6.95 the size of an rbc . Pth assay and/or immunocytochemical studies performed on fna specimens may be useful in classifying a nodule as parathyroid origin . In parathyroid aspirates, the pth level of aspirated fluid (range 248240,075 pg / ml) and the ratio of pth in the aspirated fluid to pth in serum (range 3.67458.3) are markedly elevated, which may guide in diagnosis . However, in our experience, pth analysis is not typically performed on thyroid fnas unless there is prior clinical suspicion for intrathyroidal parathyroid tissue or cytological suspicion of parathyroid at the time of rapid on - site evaluation . Although we occasionally prepare an extra thinprep slide or destain a papanicolaou - stained direct smear for immunocytochemical stain(s), such procedures require sufficient material remaining in the cytolyt (hologic inc ., marlborough, massachusetts, usa) or the presence of sufficient diagnostic material available for destaining / immunostaining, respectively . Immunocytochemical stains in our institution are almost always performed on cell block material, but with the recent implementation of next - generation sequencing (ngs) analysis on many beth - iii and beth - iv thyroid aspirates, cell blocks are typically not prepared on thyroid fnas . In cases where we suspect that immunocytochemical stains might be more useful than ngs to classify an atypical or suspicious thyroid nodule (e.g., metastatic tumor), we forego possible ngs and have a cell block prepared . Suspicion for intrathyroidal parathyroid tissue may be sufficient reason to have a cell block prepared and omit potential ngs . Nonetheless, this correlates with the small percentage (i.e., approximately 5%) of intrathyroidal parathyroids described in the literature . Further, even smaller numbers are likely to represent enlarged parathyroid glands and present clinically or by imaging for fna . Parathyroid fna specimens typically show intermediate - to - high cellularity with cohesive groups of cells showing 2d or 3d arrangements . Other described patterns include disorganized sheets (another term for 2d arrangements), prominent vascular network with associated epithelial cells, and microfollicles . In contrast, beth - iv thyroid aspirates, which are typically cellular, consist of follicular / hurthle cells arranged as microfollicles, trabeculae, and syncytia . Crowding and overlapping of cell groups are conspicuous, and the follicular cells are usually larger than normal follicular cells . Compared the cytological findings of parathyroid fnas to thyroid lesion fnas, which included papillary carcinoma, follicular adenoma, adenomatoid nodule, and hashimoto's thyroiditis . They found that overlapping 3d fragments were more likely to be found in parathyroid whereas flat honeycomb sheets are more common in thyroid aspirates . Our study showed similar findings, with 2d sheets representing the statistically significant cell arrangement that distinguishes parathyroid from beth - iv (based on the images in absher et al . 's study, the cell groups that we interpret as 2d sheets are quite similar to what they interpreted as 3d groups; for our study, we interpreted 3d architecture as more of a ball of cells as opposed to a cellular sheet with overlapping cells). Similar to the findings of absher et al ., papillary fragments and microfollicular arrangements were present in both our parathyroid and thyroid aspirates, and neither was a statistically significant distinguishing feature . Although naked nuclei have been described to be more prevalent in parathyroid aspirates, this feature was seen in both parathyroid and beth - iv in our study . In addition, we found both background colloid - like material and macrophages in our parathyroid and thyroid aspirates, indicating that have been previously reported . This features was variably present in the beth - iv cases and noted in only 1 of our parathyroid cases; however, we are uncertain whether these rare clusters represented parathyroid versus thyroid . Either way, it was not a statistically significant finding . Parathyroid cells demonstrate round - to - oval nuclei, regular nuclear membranes, and inconspicuous or absent nucleoli, and the chromatin is hyperchromatic, coarsely granular, or stippled . Moderate - to - high n: c ratio, anisonucleosis, and nuclear overlapping are other features that may be observed . The follicular cells of follicular thyroid lesions (beth - iv) are usually larger than normal follicular cells . Nucleoli are generally inconspicuous in parathyroid and infrequent in follicular nodules but may be seen in both . Compared to oncocytic parathyroid adenoma, hurthle cell thyroid neoplasms have larger nuclei, prominent nucleoli, and tend to be more discohesive . Compared to follicular cells, the nuclei of parathyroid cells contain chromatin that is more stippled or hyperchromatic than thyroid follicular cell nuclei, which contain more finely granular chromatin, although cells from follicular nodules (beth - iv) may have coarse chromatin . The presence of stippled chromatin was not a statistically significant finding in distinguishing parathyroid from beth - iv . We found nucleoli to be a significant finding in distinguishing parathyroid from beth - iv cases with all 5 parathyroid fnas lacking nucleoli and 11 of 12 beth - iv cases containing nucleoli though they ranged from inconspicuous to prominent . Anisonucleosis, although present in only a small portion of the cells, is a reported feature of parathyroid fnas but uncommon in thyroid follicular lesions . Anisonucleosis was minimal to nil in our parathyroid cases and ranged from nil to moderate in the beth - iv cases although the differences were not statistically significant . Described nuclear molding to be a feature that was present in most parathyroid aspirates and rarely seen in thyroid lesions . Parathyroid cells are small with cytoplasm that is usually pale and finely granular . Cytoplasmic vacuoles or oxyphilia may also be observed . Well - defined cytoplasmic membranes / borders have been described in a minority of cases . The cytoplasm of follicular cells in beth - iv (follicular adenoma) lesions is usually delicately vacuolated or finely granular (follicular lesions) or granular and oncocytic (hurthle cell lesions). Compared to follicular cells, parathyroid cells are usually smaller and contain less cytoplasm, although they may contain oxyphilic cytoplasm, which can lead to misinterpretation as hurthle cells . While a few cells with more abundant cytoplasm were noted in 3 of our parathyroid cases, none demonstrated the abundant oncocytic cytoplasm that is typical of beth - iv hurthle cell lesions . The other 6 contained cells with oncocytic / hurthle cell features, 5 of which were shown to be follicular adenoma with hurthle cell features / hurthle cell adenoma on resection and 1 of which was shown to represent nodular goiter with focal hurthle cell change on resection . Described the presence of intracytoplasmic fat vacuoles, best seen on romanowsky stain, in imprint smears from parathyroid gland as being useful in confirming parathyroid tissue . All 5 of our parathyroid specimens and all 12 beth - iv cases lacked cytoplasmic vacuoles . The smaller size of parathyroid cells compared to beth - iv was a significant finding in our study . All 5 of our parathyroid cases showed small cells with high n: c ratio which were 23 the size of an rbc compared to the larger cells of beth - iv cases, which contained cells that were on average 6.95 the size of an rbc . Pth assay and/or immunocytochemical studies performed on fna specimens may be useful in classifying a nodule as parathyroid origin . In parathyroid aspirates, the pth level of aspirated fluid (range 248240,075 pg / ml) and the ratio of pth in the aspirated fluid to pth in serum (range 3.67458.3) are markedly elevated, which may guide in diagnosis . However, in our experience, pth analysis is not typically performed on thyroid fnas unless there is prior clinical suspicion for intrathyroidal parathyroid tissue or cytological suspicion of parathyroid at the time of rapid on - site evaluation . Although we occasionally prepare an extra thinprep slide or destain a papanicolaou - stained direct smear for immunocytochemical stain(s), such procedures require sufficient material remaining in the cytolyt (hologic inc ., marlborough, massachusetts, usa) or the presence of sufficient diagnostic material available for destaining / immunostaining, respectively . Immunocytochemical stains in our institution are almost always performed on cell block material, but with the recent implementation of next - generation sequencing (ngs) analysis on many beth - iii and beth - iv thyroid aspirates, cell blocks are typically not prepared on thyroid fnas . In cases where we suspect that immunocytochemical stains might be more useful than ngs to classify an atypical or suspicious thyroid nodule (e.g., metastatic tumor), we forego possible ngs and have a cell block prepared . Suspicion for intrathyroidal parathyroid tissue may be sufficient reason to have a cell block prepared and omit potential ngs . Nonetheless, this correlates with the small percentage (i.e., approximately 5%) of intrathyroidal parathyroids described in the literature . Further, even smaller numbers are likely to represent enlarged parathyroid glands and present clinically or by imaging for fna . Parathyroid and beth - iv thyroid fnas share cytomorphological findings and distinguishing them is often difficult on the aspirate material . However, distinguishing cytological features, most notably the lower n: c ratio and more prominent nucleoli in beth - iv lesions, are seen and can be critical in correctly identifying parathyroid tissue, which can be very challenging, especially when the targeted lesion is intrathyroidal . Additional useful cytological findings separating parathyroid from beth - iv include overall less nuclear irregularity, a predominant 2d pattern cell arrangement, the presence of single cells, and smaller cell size in parathyroid . Each author has participated sufficiently in the work and take public responsibility for appropriate portions of the content of this article . This study was conducted with approval from institutional review board (irb) of the institution associated with this study (columbia university medical center). Fna - fine - needle aspiration ngs - next - generation sequencing pth - parathyroid hormone rbc - red blood cell . To ensure the integrity and highest quality of cytojournal publications, the review process of this manuscript was conducted under a double - blind model (authors are blinded for reviewers and vice versa) through automatic online system.
In a recent nih whitepaper (1) the lack of a comprehensive, curated carbohydrate structure database was identified as the largest deficit in glycomics and glycobiology research . The complex carbohydrate structure database (ccsd) (2), initiated in the 1980s, was the largest effort to date to collect carbohydrate structures, mainly through retrospective manual extraction from the literature . The database contained about 50 000 entries when it ceased to be updated in the late 1990s due to a lack of funding . Since then different specialized databases have been developed, which were initially seeded with a subset of the structures contained in the ccsd (3). Subsequently these databases were further extended with carbohydrate structures reflecting the research focus of the group that maintained the database . As a result, different valuable collections of carbohydrate data have emerged over recent years, for example: the bacterial carbohydrate structure database (bcsdb) (4) that collects all published bacterial carbohydrate structures (including their nmr spectra); the database of the consortium for functional glycomics (cfg) that provides access to primary experimental data like that from glycan microarray screens (5); and the kyoto encyclopedia of genes and genomes (kegg) that contains glycan - related biosynthetic pathways (6). Unfortunately each of these databases uses a different sequence format for encoding carbohydrate structures, making it difficult to query across all public databases and analyze or compare their content, or simply to find out whether some additional information on a particular carbohydrate structure is available in any of the databases . In 2005, a new initiative was begun to overcome the isolation of the public carbohydrate structure databases and to create a comprehensive index of all available structures with cross - links back to the original databases . To achieve this goal, structures of the freely available databases were translated to the glycoct sequence format (7), if possible, and stored in a new database, the glycomedb (8). The integration process is performed incrementally on a weekly basis, updating the glycomedb with the newest structures available in the associated databases . A java software application called glycoupdatedb, which is complemented by a postgresql database, is used to download the data from the public databases, reads their sequence notations and translates them to the glycoct encoding format . In addition, the taxonomic annotations are standardized semi - automatically based on curated tables that map the (free - text) annotations used in the source databases to ncbi taxonomy ids [for more details see (8)]. To extract the carbohydrate structures from the protein data bank (pdb) the pdb2linux tool is used (9). During the integration process automated checks are performed; structures that contain errors are reported to the administrators of the original database . A major challenge during the initial integration process was the lack of a controlled vocabulary for carbohydrate and non - carbohydrate residue names . In total 12 253 different residues names were extracted from the sequences stored in the original carbohydrate databases, 5854 of which were identified as non - carbohydrate residues, mainly aglycons, such as amino acids, lipids or other small organic molecules attached to the reducing end of the carbohydrate . In total 5330 residue names could be identified as monosaccharides and were assigned a standardized glycoct encoding . Based on the initial analysis of the namespace used to encode carbohydrate structures in the various databases, a dictionary has been created that contains mappings of the various encoding formats . The dictionary is now used to support the automated update process . If a new residue name appears, this is reported to the database curator who can then check whether the residue name is valid and include the new residue into the dictionary . Finally, a web interface has been developed (www.glycome-db.org) as a single query point for all open access carbohydrate structure databases (10). Glycomedb contains the unified carbohydrate sequences of all publicly accessible databases that contain carbohydrates structures . In total 121 766 original sequences currently (august 2010) there are 35 873 unique carbohydrate sequences with taxonomic annotations if available stored in glycomedb, 11 822 of which are fully determined carbohydrates . Fully determined if all monosaccharide characteristics (base type, anomer, ring size, substituents, modifications, etc) and all linkage positions are known . For polysaccharides an overview of the number of carbohydrate structures contributed by each database is given in table 1 . Table 1.overview of the number of original unique carbohydrate or glycoconjugate sequences contained in the source databases (encoded in the database - specific format, including the aglycon unit) and the number of unique glycoct sequences generated after removing the aglycon and parsing the remaining codeexternal databasenumber of sequences in external databasenumber of unique glycoct sequencesfully determined carbohydrate sequencesurlbcsdb (4)81196536 (4149)1972 (1277)http://www.glyco.ac.ru / bcsdb3/ccsd (2)23 40214 887 (1544)7406 (462)http://www.genome.jp / dbget - bin / www_bfind?carbbankcfg (5)88736285 (4143)397 (110)http://www.functionalglycomics.org / eurocarbdb13 46713 308 (411)8924 (139)http://www.ebi.ac.uk / eurocarb / glycobase(lille) (11)247197 (145)195 (143)http://glycobase.univ - lille1.fr / base / glycosciences.de (12)23 28515 829 (391)9225 (36)http://www.glycosciences.de / kegg (6)10 96910 160 (6128)1610 (179)http://www.genome.jp / kegg / glycan / pdb (13)905733 (0)708 (0)http://www.rcsb.org / pdb / the numbers in brackets denote the number of sequences that are stored exclusively in this database . Currently glycomedb contains 35 873 unique carbohydrate sequences and 11 822 fully determined carbohydrate sequences . See text for the criteria of a fully determined sequence. Overview of the number of original unique carbohydrate or glycoconjugate sequences contained in the source databases (encoded in the database - specific format, including the aglycon unit) and the number of unique glycoct sequences generated after removing the aglycon and parsing the remaining code the numbers in brackets denote the number of sequences that are stored exclusively in this database . Currently glycomedb contains 35 873 unique carbohydrate sequences and 11 822 fully determined carbohydrate sequences . See text for the criteria of a fully determined sequence. Four major structural query options are implemented in glycomedb, namely exact structure search, structural queries can be entered graphically, either using glycanbuilder (14) as the default, or using drawrings, developed by a japanese group at soka university, tokyo (http://rings.t.soka.ac.jp). It is also possible to specify the query structure by using different machine - readable encoding formats, among which are carbbank format (2), linucs (15), linearcode (16), bcsdb encoding (4) and glyde ii (http://glycomics.ccrc.uga.edu/core4/informatics-glyde-ii.html). Next to the exact structure search, which is based on a comparison of ordered glycoct encodings (7), it is possible to generate queries with partially unknown information on the monosaccharide level, i.e. Unknown anomeric center, ring size, or absolute configuration . It is also possible to restrict the search to specific taxonomic sources, as glycomedb applies consistently the ncbi taxonomy for the taxonomic data (17). The various search options can be combined sequentially to a multistep query refinement workflow, which allows very complex queries to be performed . Using the glycomedb information page for individual structures (figure 1), the user can use hyperlinks to navigate to the relevant pages of the external databases, which offer additional information such as literature references, experimental data or 3d structures . Additionally, information about bound aglycons and structural motifs, and a selectable sequence encoding are displayed . For more detailed information about the various aglycons attached to a particular carbohydrate, the user is guided to the original databases by following the link show remote structure evidences. Figure 1.the structure information page of glycomedb . Four major structural query options are implemented in glycomedb, namely exact structure search, structural queries can be entered graphically, either using glycanbuilder (14) as the default, or using drawrings, developed by a japanese group at soka university, tokyo (http://rings.t.soka.ac.jp). It is also possible to specify the query structure by using different machine - readable encoding formats, among which are carbbank format (2), linucs (15), linearcode (16), bcsdb encoding (4) and glyde ii (http://glycomics.ccrc.uga.edu/core4/informatics-glyde-ii.html). Next to the exact structure search, which is based on a comparison of ordered glycoct encodings (7), it is possible to generate queries with partially unknown information on the monosaccharide level, i.e. Unknown anomeric center, ring size, or absolute configuration . It is also possible to restrict the search to specific taxonomic sources, as glycomedb applies consistently the ncbi taxonomy for the taxonomic data (17). The various search options can be combined sequentially to a multistep query refinement workflow, which allows very complex queries to be performed . Using the glycomedb information page for individual structures (figure 1), the user can use hyperlinks to navigate to the relevant pages of the external databases, which offer additional information such as literature references, experimental data or 3d structures . Additionally, information about bound aglycons and structural motifs, and a selectable sequence encoding are displayed . For more detailed information about the various aglycons attached to a particular carbohydrate, the user is guided to the original databases by following the link show remote structure evidences. Figure 1.the structure information page of glycomedb . Glycomedb integrates the structural and taxonomic data of all major public carbohydrate databases, as well as carbohydrates contained in the protein data bank, which renders the database currently the most comprehensive and unified resource for carbohydrate structures worldwide . Hyperlinks to the original source of the data are established, so users can use the glycomedb web - portal to access efficiently relevant additional information, which is only available in the original databases . Glycomedb is a database that integrates knowledge from other existing databases, therefore only carbohydrate structures that are stored in any of these databases will be integrated and cross - linked in glycomedb . Unfortunately, glycomedb cannot provide access to all published structures because, in contrast to proteomics and genomics, in glycomics there is not yet a procedure established that requires deposition of new structures in the context of publication . Therefore it can be assumed that not all published structures are currently available in a database . However, if a public database will be used in the future to deposit systematically new structures, these structures should also be automatically available in glycomedb . In general, the quality of the data depends on the quality of the referenced databases and their curation processes . Nevertheless glycoupdatedb applies additional validation checks during the integration process in order to improve the quality of the data . It can be assumed that the development of annotation tools in ms and nmr that require a library of existing carbohydrate structures as reference data will benefit from the availability of glycomedb . Additionally, the data contained in glycomedb can facilitate statistical analyses of the glycospace of different organisms (18,19). Glycomedb can be accessed using a web - portal (http://www.glycome-db.org/) or the complete database can be downloaded as a compressed zip archive, containing all structures that have been integrated (http://www.glycome-db.org/downloads/). The structures are stored in regular xml files according to the glyde ii specification and can be used by any software that supports this format . Eu (6th research framework program, rids contract number 011952); german research foundation (dfg bib 46 hddkz 01 - 01). Funding for open access charge: german cancer research center (dkfz), heidelberg, germany.
Staghorn calculi are branched calculi that fill the entire or part of the renal pelvis and extend into the renal calices . If untreated or inadequately treated, they may lead to various complications such as deterioration of renal function and risk of developing urosepsis . In addition, chronic irritation, inflammation and infection from these stones can cause squamous metaplasia of the renal pelvis epithelium that may progress to squamous cell carcinoma . Squamous cell carcinoma of the renal pelvis is a rare tumor, with a prevalence of <1% of urinary tract neoplasms . Here we demonstrate an older woman with staghorn calculi presenting with severe hypercalcemia caused by squamous cell carcinoma of the renal pelvis . A 79-year - old female presented with gradual deterioration of mental status for 1 month . For 2 months prior to admission, she had had intermittent right upper abdominal pain, vomiting, constipation, anorexia and significant weight loss . Her medical conditions were hypertension and dyslipidemia, and she was receiving treatment with amlodipine and simvastatin . Kidney, ureter and bladder (kub) x - ray visualized a large right staghorn stone (fig . 1a), and a retrograde pyelogram showed right staghorn renal calculi with partial obstruction and left middle ureter stricture (fig . A diuretic renal scan was interpreted as a nonfunctioning right kidney and fair renal function of the left kidney . Serum blood urea nitrogen and creatinine were 52 mg / dl (reference range 718) and 6.8 mg / dl (reference range 0.671.17), respectively . She was rehydrated with normal saline and then referred to our hospital for relief of left ureteric obstruction . On examination, the patient appeared lethargic and dehydrated . Her blood pressure was 160/87 mm hg and her pulse rate was 93 beats / min . Two lymph nodes, 0.7 and 1 cm in diameter, were palpated in the left supraclavicular area . She had nonpitting edema on both legs that was more pronounced on the right leg with the presence of homan's sign . Urinary examination showed a ph of 5, white blood cells of 12/high - power field and red blood cells of 01 cells / high - power field . Furthermore, blood chemistries revealed serum total calcium of 14.8 mg / dl (reference range 8.510.1), serum phosphorus of 3 mg / dl (reference range 2.54.9) and serum creatinine of 2 mg / dl (reference range 0.671.17). Serum intact parathyroid hormone (pth) was investigated and found to be 11.5 pg / ml (reference range 1565). In the settings of old age, weight loss and deep vein thrombosis, humoral hypercalcemia of malignancy was mostly suspected . Unfortunately, we could not confirm the diagnosis with pth - related protein (pth - rp) due to test unavailability in our country . Renal ultrasound visualized large right staghorn calculi and moderate left hydronephrosis with proximal hydroureter (fig . Doppler ultrasound showed acute extensive deep vein thrombosis of both legs along the external iliac vein, the common femoral vein, the proximal deep femoral vein to the popliteal vein . There was minimal right pleural effusion on chest x - ray, with no abnormal pulmonary nodules . In searching for the primary site of malignancy, histopathology was compatible with metastatic squamous cell carcinoma . Computed tomography of the chest and abdomen showed an infiltrative tumor with extensive involvement of the right kidney, the right pelvocalyceal system, the right adrenal gland, the right lobe of the liver and the adjacent right hemidiaphragm and psoas muscle (fig . There were inferior vena cava invasion and multiple metastases in both hepatic lobes and intra - abdominal lymph nodes . The final diagnosis was advanced - stage squamous cell carcinoma of the right renal pelvis . To rescue left kidney function, a left retrograde pyelogram with double j stent after restoration of volume status, serum creatinine levels decreased from 2.0 to 1.7 mg / dl . Four days later, serum calcium level subsided to 10.16 mg / dl . It was decided that long - term therapy would be the best supportive care . The patient was discharged to a primary care hospital and passed away 1 month later . The tumor generally presents with abrupt onset, severe symptoms and high serum calcium concentration of> 14 mg / dl . Even though various cells in the body have the potential to produce pth - rp, tumors that commonly produce pth - rp include squamous cell carcinomas of the head and neck, esophagus, cervix and lung; adenocarcinomas of the breast and ovary and renal cell carcinoma . The definite diagnosis can be confirmed by detection of high serum pth - rp, but this is not necessary for diagnosis in most situations . Squamous cell carcinoma of the renal pelvis is a very rare tumor, accounting for only 0.58.0% of malignant renal tumors [5, 6]. The coexistence of renal calculi has been reported in 87100% of cases [6, 8, 9]. It has also been associated with tuberculosis, chronic pyelonephritis, radiation therapy, chronic rejection in a transplanted kidney, analgesic abuse with phenacetin, immunosuppression with azathioprine and previous percutaneous nephrolithotomy . Staghorn calculi are usually composed of magnesium ammonium phosphate (struvite) and/or calcium carbonate apatite . Cystine or uric acid mixed with other components can also grow in a staghorn configuration . Infection stones because of the strong relation with urinary tract infection caused by urease - producing gram - negative bacteria, e.g. Proteus, morganella and providencia spp . However, recent data found that 55% of cases with staghorn calculi were from metabolic stones, in particular of calcium phosphate composition . Moreover, the most common component of staghorn calculi in patients in southern thailand was uric acid . Left untreated, staghorn calculi may cause life - threatening sepsis and renal function impairment . Over time, staghorn stones cause squamous metaplasia and dysplasia of the uroepithelium . Therefore, most patients should require treatment of staghorn stones . The treatment options are medical therapy, surgery, percutaneous nephrolithotomy and shock wave lithotripsy . Analysis of the composition of the stone and metabolic evaluation are essential for preventing recurrence [13, 14]. Increased fluid intake to achieve a urine output of> 2.5 l / day will decrease the risk of urinary supersaturation . Early detection of the tumor by ultrasound would be difficult because the tumor appears as a nonspecific finding, such as a solid mass, calcification or hydronephrosis . As a result, a computed tomography scan may be an option for cancer detection, with better yield for diagnosis . Findings of filling defect in the collecting system, delayed appearance of pyelogram or renal parenchymal thickening should be definitely considered as renal tumor . In conclusion, humoral hypercalcemia of malignancy mediated by pth - rp is an uncommon feature of the tumor . Removal of the staghorn stone is necessary to eliminate infection and prevent squamous metaplasia of the renal epithelium . Written informed consent was obtained from the next of kin for publication of this case report and the accompanying images . A copy of the written consent is available for review by the editor of this journal.
Retinal changes associated with the development of age - related macular degeneration (amd) have become even more important than once described . Even prompt therapy of the wet form of amd with antivascular, endothelial, growth factor drugs has been shown to ameliorate vision loss . If diagnosis is established earlier during the dry form of amd, there is a better chance that visual acuity can be maintained . Rheohemapheresis (formally known as rheopheresis) has been investigated over the past decade as a possible method of positively affecting amd in its dry - form stage . Rheohemapheresis is a method of double plasma filtration performed in order to eliminate high - molecular - weight substances especially proteins such as fibrinogen, 2-macroglobulin, immunoglobulin m (igm), thrombomodulin, and low - density (ldl) cholesterol [36]. This method leads to the improvement of rheological parameters (reduction of plasma and whole blood viscosity), as well as the improvement of erythrocyte aggregation and their flexibility [6, 7]. It can also lead to a significant improvement of blood flow in the choroid, which is reduced in patients with amd . When treating our patients suffering from the dry form of amd accompanied by reticular drusen or even drusenoid detachment of the retinal pigment epithelium, we noticed positive morphological changes of the retina after rheohemapheresis treatment, including the preservation of the photoreceptor inner / outer segment (is / os) junction, as well as stabilisation or even improvement of retinal function: best - corrected visual acuity (bcva) and electroretinography results . A description of the above changes and their correlation is the subject of this work . At our department, we have seven years of experience in treating amd using rheohemapheresis . So far, 61 patients with the dry form of amd, soft drusen, confluent soft drusen, and drusenoid pigment epithelium detachment (dped) have been treated . We perform long - term monitoring of morphological and functional changes in the retina [12, 13]. The interrelationship of morphological changes in the photoreceptor inner and outer segment (is / os) junction retinal layer as well as changes in visual acuity and retinal function (electroretinography) was evaluated in the group of 24 patients (40 eyes) who were long - term followed (for 2.5 years or longer). All patients had the dry form of amd with the presence of drusenoid pigment epithelium detachment . Twelve of these patients (22 eyes) with an average age of 64.3 years (range: 6483 years) were treated with rhf . Twelve controls (18 eyes) with an average age of 65.6 years (range: 6483 years) were randomized . Ophthalmologic inclusion criteria were high - risk, preangiogenic form of amd with soft drusen, reticular drusen, confluent soft drusen, and dped in accordance with the eureye study and ability to complete the series of 8 rheohemapheresis procedures within 10 weeks . Exclusion criteria were any retinal or choroidal disorders other than amd, optic nerve disorders, glaucoma, conditions limiting the examination of the fundus, and acute bleeding in the studied eye . General exclusion criteria for rheohemapheresis treatment were the usual exclusion criteria of extracorporeal circulation or therapeutic hemapheresis and the absence of peripheral veins suitable for establishing an extracorporeal circuit . Rheohemapheresis was performed in the treatment group, as described in detail in our previous work [3, 13]. After plasma separation (blood - cell separator, cobe spectra or optia, terumo, lakewood, co, usa), the separated plasma was pumped through the rheofilter (evaflux, kawasumi, tokyo, japan) to remove high - molecular - weight factors . Using this filter, we repeatedly (8 times over the period of 10 weeks) removed a precisely defined spectrum of high - molecular - weight substances, such as fibrinogen, ldl cholesterol, and 2-macroglobulin, from the blood of the patient, thus reducing blood viscosity in an attempt to improve the perfusion of the retina and the choroid . Fundus photography and fluorescein angiography were performed using a digital fundus camera (zeiss ff 450, jena, germany). The dped area was measured in mm by fundus photography using visupac software (zeiss meditec ag, jena, germany), which is acceptable for measuring the area of retinal affections . Spectral domain- (sd-) oct (cirrus hd - oct, zeiss meditec, jena, germany) with an axial resolution of 6 m was used to evaluate central retinal thickness . Thickness in the central point of the 1 mm fixation zone was evaluated using 6 radial scans . Sd - oct enabled us to distinguish between dped and the vascular type of retinal pigment epithelium detachment . A detailed image of the photoreceptor is / os junction was obtained using a 5-line raster scan, which precisely shows the shape, coherence, and defects of the photoreceptor is / os junction as well as the detachment of this layer from the retinal pigment epithelium (rpe) multifocal erg (mferg; reti - port plus mferg system, roland consult gmbh, brandenburg, germany) was performed according to the standards of the international society for clinical electroretinography and vision (iscev) [16, 17]. Mferg traces were recorded from the central 60 of the retina using a resolution of 61-scaled hexagons . We evaluated the amplitudes of the positive peak component from the first - order kernel analysis of the central element, representing the foveal response (01.8) of the four rings centered on the fovea: 1st ring (1.87.0), 2nd ring (513), 3rd ring (1122), and 4th ring (1730). All examinations were performed at baseline and at 2.5-year follow - up . For statistical analysis, we used nonparametric tests (the kruskal - wallis test, mann - whitney u test, and chi - square approximation). The study protocol was approved by the institutional ethics committee and the reported investigations were in accordance with the principles of the current version of the helsinki declaration . Median best - corrected visual acuity (bcva) before rheohemapheresis in the treatment group was 74.0 letters (56.2 to 81.3 letters; 95% ci) and increased to 79.0 letters (57.3 to 83.4 letters; 95% ci), (p = 0.187) after 2.5 years . In the control group, mean bcva decreased from 74.0 letters (25.2 to 82.6 letters; 95% ci) at baseline to 72.5 letters (23.4 to 83.1 letters) after 2.5 years (p = 0.041). While the difference in bcva between the treatment groups was insignificant at baseline (p = 0.457), bcva was significantly higher in the treatment group after 2.5 years (p = 0.021). At baseline, mean dped was 3.68 4.45 mm in treated patients and 4.12 6.64 mm in controls, reaching the central fovea in all cases . After 2.5 years, the mean dped area decreased significantly to 0.71 1.27 mm in the rheohemapheresis group (p <0.001), whereas it increased significantly to 9.19 9.51 mm in the controls (p <0.01). The differences in size of the dped area were only insignificant at baseline (p = 0.605) and significant after 2.5 years (p <0.001). Reduction of the size of the dped area after rheohemapheresis was found in 19/22 rheohemapheresis - treated eyes (86.4%), as opposed to only 3/18 eyes (16.7%) in the control group . Enlargement of the dped area occurred as part of the natural progress of amd over the same period of 2.5 years in 15/18 (83.3%) eyes in the control group, compared to only 3/22 eyes (13.6%) of treated patients . The study and control groups were comparable.in the group of treated patients, the photoreceptor is / os junction was attached to the dped in 6/22 (27.3%) eyes . (dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 16/22 eyes, i.e., 72.7%, without defect in 15/22 eyes and with defect in 1 eye).in the control group, the photoreceptor is / os junction was attached to the dped in 6/18 (33.3%) (dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 12/18 eyes (66.6%), without defect in 4 eyes and with defect in 8 eyes). In the group of treated patients, the photoreceptor is / os junction was attached to the dped in 6/22 (27.3%) eyes . (dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 16/22 eyes, i.e., 72.7%, without defect in 15/22 eyes and with defect in 1 eye). In the control group, the photoreceptor is / os junction was attached to the dped in 6/18 (33.3%) (dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 12/18 eyes (66.6%), without defect in 4 eyes and with defect in 8 eyes). (1) the group of patients . In the group of treated patients, where the photoreceptor is / os junction was intact in 6/22 (27.3%) eyes, it remained without defect even after 2.5 years . However, in 7 cases of the attached is / os junction, there was a certain residual defect . In one additional eye with the is / os junction at baseline, the defect of this layer significantly improved after attachment . Overall, is / os junction defects were demonstrated in 8/22 eyes (31.8%). Defects reached the central foveola in 4 eyes (8.8%), thus negatively affecting photoreceptor function and vision . Integrity of the is / os junction layer was preserved in only 3/18 eyes (16.7%) after 2.5 years and is / os junction defects were diagnosed in 15/18 eyes (83.3%), 12 of which reached the foveolar region and adversely affected vision . It is important to note that none of the treated patients progressed into the wet form during the 2.5-year follow - up . In the control group, 6 eyes with detachment of the is / os junction at baseline developed choroidal neovascularization (cnv) (confirmed by fluorescein angiography). The amplitudes of foveal responses and responses in the most peripheral areas of the retina, as shown on the multifocal erg, changed only slightly during the entire follow - up period in both groups of patients . In the parafoveal and paramacular regions of eccentricity between 1.8 and 13 the differences were statistically nonsignificant between groups of patients in both periods of examination, except for the activity in the region of eccentricity between 1.8 and 7, which was significantly higher in treated patients (p = 0.04) at 2.5-year follow - up . The implicit times of the majority of responses increased after 2.5 years in all patients . At baseline, they were significantly longer in the controls (p values ranging from <0.05 to <0.01), with the exception of the foveal response . At 2.5 years, the foveal response was significantly longer in the control group (p = 0.035). In general, retinal activity remained stable or even improved in treated patients with early decrease or complete disappearance of dped and detachment of the photoreceptor is / os junction, along with preservation of its integrity or development of only small defects in the parafoveal region (figures 2(a), 2(b), 2(c), 3(b), and 3(c)). In contrast, in patients with long - lasting or persistent dped with detachment of the is / os junction and development of its defects or even development of cnv, retinal activity was even reduced . The photoreceptor is / os junction layer is currently receiving attention from many researchers [10, 1822]. According to baba et al ., the state of this thin hyper - reflective layer, which lies above the rpe layer, directly correlates with bcva after successful macular hole repair . Bcva deterioration has proved to be an early indicator of transformation to the wet form of amd . We have verified the direct correlation of the state of the is / os photoreceptor junction and bcva on patients with stargardt disease . Disruption of the is / os junction is associated with poor vision in uveitic macular edema and retinitis pigmentosa [22, 23]. As a result of our findings, we can add the is / os junction as another indicator of the risk of transformation to the wet form of amd . The detachment of the photoreceptor is / os junction (figure 1) is usually located at the top of the area affected by drusenoid retinal pigment epithelium detachment (dped). Sikorski et al . Found that rupture of is / os photoreceptor junction detachment is directly associated with the emergence of submacular neovascularization . Schuman et al . Reported thinning of the is / os photoreceptor layer and the progress of its changes over drusen and dped . In the present study, we found reattachment of the photoreceptor is / os junction layer in the study group . After the 2.5-year follow - up, 8 eyes were reattached without is / os junction defect after rhf; the is / os junction defect was partially reattached in another 8 of the treated eyes . However, in only 4 of these eyes (18.1% of the entire sample), the defect reached the central fovea . The fact that, after rheohemapheresis, patients only rarely developed the photoreceptor is / os junction defect affecting the central foveola and that mean visual acuity actually slightly improved in the majority of these patients is considered an obvious interdependence . In control patients, we did not observe full restoration (reattachment) of a photoreceptor is / os junction layer when detached at the baseline . Drusenoid retinal pigment epithelium detachment usually progresses (which was observed in 7 eyes); otherwise, rupture occurs caused by submacular neovascularization development, as has also been observed by other authors [24, 25]. The photoreceptor is / os junction layer rupture is usually apparent in patients with neovascularization . The remaining fluid as well as the fluid under the detached drusenoid retinal pigment epithelium then spreads into the inner retinal layers and can cause macular edema . In the control group, we observed 6 cases (33.3%) of submacular choroidal neovascularization . The following is one possible mechanism of its development: the existing dped was later accompanied by a detached layer adjoined to it from above (i.e., by detachment of the photoreceptor is / os junction). Dped and is / os layer detachment gradually grew in size and eventually ruptured due to the development of submacular choroidal neovascularization . As evidence of progression to the wet form of amd, fluid inside and underneath the inner retinal layer appeared on sd - oct . These findings are supported by the findings of occult submacular choroidal neovascularization using fluorescein angiography . Patients with preservation of photoreceptor is / os junction integrity or development of only small, paracentral defects by early decrease or complete disappearance of the dped area also exhibited slightly increased retinal activity, which led to significantly higher parafoveal activity in treated patients using mferg (see section 3.4). We found a significantly higher amplitude of parafoveal responses in eccentricity between 1.8 and 7 in treated patients 2.5 years after initiation of the treatment (p = 0.04). In contrast, in patients with development of is / os junction defects or even progression of the disease to its wet form, central retinal activity and visual acuity were even reduced . Development of photoreceptor is / os junction defects in the control group probably contributed to the slight decrease of electrical retinal activity in the parafoveal and paramacular regions of eccentricity between 1.8 and 13 using mferg . This may be due to the long - term accumulation of fluid between the rpe and is / os junctions, which causes stretching of photoreceptor outer segments, thus leading to their malfunction even without the development of associated rpe atrophy . The question of treating the dry form of amd by rheohemapheresis has not yet been fully answered . Case series, two controlled trials and five completed randomized controlled trials, have reported the efficacy of rheohemapheresis in treating dry amd . These studies (inclusive of our two [3, 26]) have shown an improvement in the number of lines that can be read on etdrs charts, improvement in the pepper visual skills for reading tests, a decrease in viscosity parameters, shortening of arteriovenous passage times, and improvement in electroretinograms . The studies have shown improvements shortly after completion of treatment, lasting up to four years [6, 18]. Considerable confusion has been aroused by the preliminary results from the mira 1 study an extensive, sham - controlled, randomized, multicenter trial conducted in the usa . Its encouraging first results were later challenged and a clear positive effect was not proved [27, 28]. However, analysis revealed that 37% of treated patients and 29% of control patients were protocol violators who did not fulfill the trial's inclusion criteria of amd leading to bias in the study's final outcome . Excluding those subjects who had vision loss due to other causes, this trial demonstrated significant improvement with treatment but the trial was underpowered by us fda licensure . Two hundred and seventy - nine patients with dry amd were treated and compared to 55 untreated controls . In the treated group, visual acuity gain greater than or equal to one etdrs line vision loss greater than or equal to one etdrs line was seen in 17% of the treated patients versus 40% of controls . The asfa (american society for apheresis) issues directives (guidelines) for individual procedures in various diseases; according to the latest criteria from 2013, rheohemapheresis is newly classified as a category ib treatment (first line therapy). The second limitation of this study is obviously the small number of patients, despite the relatively long follow - up . The clinical impression of the importance of the is / os layer was established at the beginning of our research 7 years ago, but only patients with follow - up times longer than 2.5 years, that is, in the case of 40 eyes (22 eyes in the study group and 18 control eyes), were randomized for assessment of morphological and functional changes to the is / os layer . Our results will need to be confirmed on a larger number of patients in the future . With the use of rheohemapheresis, preservation of photoreceptor is / os junction layer coherence in the fovea of patients with high - risk dry amd can be achieved, even when drusenoid retinal pigment epithelium detachment is already present . After rheohemapheresis, there was a significant morphological improvement of the damaged is / os layer, as evidenced by reduction of the scope of the defect or reattachment of the detached is / os layer either with or even without the defect of this layer . After 2.5 years of follow - up, better visual acuity, reduced dped size, and improvement of some functional parameters (electroretinography findings) were observed in the study group.
Sodium - glucose cotransporter 2 (sglt2) inhibitors are oral hypoglycemic agents that augment the urinary excretion of glucose, thereby regulating glycemic control and promoting weight loss (1). Sglt2 inhibitors have an insulin - independent mechanism of action, which is being explored in patients with type 1 diabetes (2). The incidence of diabetic ketoacidosis in patients treated with sglt2 inhibitors is rare (4,5). Recently, the us food and drug administration issued a drug safety communication that sglt2 inhibitors may lead to ketoacidosis with mild to moderate glucose elevation (euglycemic diabetic ketoacidosis) (6). However, only a few cases of euglycemic diabetic ketoacidosis with persistent diuresis after the discontinuation of sglt2 inhibitors have been reported . We herein report a case of euglycemic diabetic ketoacidosis with persistent diuresis in a diabetic patient undergoing treatment with an sglt2 inhibitor, canagliflozin . A 27-year - old woman of asian descent from north america was admitted to our hospital for the treatment of euglycemic diabetic ketoacidosis . She had been taking oral antidiabetic drugs (gliclazide 300 mg, metformin 3,000 mg, and sitagliptin 150 mg per day), but her glycemic control was poor . Three months prior to admission, she started taking canagliflozin (300 mg / day). During that time, she lost 3 kg and her urinary frequency increased . Two days before admission, she experienced dizziness, and the following day, she developed upper abdominal pain . On the day of admission, she visited the emergency department with complaints of thirst and malaise . She did not smoke or drink alcohol, and she followed carbohydrate restriction for weight loss . On examination, the patient's blood pressure was 145/103 mmhg, her heart rate was 123 beats / min, and her temperature was 36.2. her weight, height, and body mass index were 63 kg, 150 cm, and 28 kg / m, respectively . The other neurological and general examination results were normal . A urinalysis detected ketone bodies, while an arterial blood gas analysis showed high anion gap metabolic acidosis and a normal lactate level . Her biochemistry results revealed electrolyte disturbance and mild hyperglycemia (240 mg / dl). In addition, her hba1c level was 9.9%, and her fasting serum c - peptide level was <0.1 ng / ml (reference range, 0.61 - 2.09 ng / ml); this finding indicated the severe impairment of insulin secretion . Taken together, these findings were compatible with a diagnosis of type 1 diabetes mellitus and diabetic ketoacidosis without hyperglycemia . Cpr: c - peptide immunoreactivity, gad: glutamic acid decarboxylase, hba1c: glycated hemoglobin, pco2: partial pressure of carbon dioxide, po2: partial pressure of oxygen the patient was initially administered isotonic saline and a continuous insulin infusion with the discontinuation of all oral antidiabetic agents . An hour after the administration of saline and the insulin infusion, her glucose level decreased to <200 mg / dl; thus, an infusion 5% dextrose was initiated . On the following day, her venous blood ph level still showed acidemia, despite a normal glucose level, and her urine output was> 5,000 ml / day ., oral food intake was initiated and the continuous insulin infusion was switched to multiple daily injections of insulin . At this point, however, we prohibited ad libitum water intake . On day 4 of treatment, her venous blood ph level recovered, and her urinary output peaked (figure). We could not measure the urinary output after the fifth day of treatment due to the discontinuation of urinary catheterization and the patient's menstruation . On the eighth day of treatment, she was discharged but the nocturnal urination had not resolved . The course of osmotic diuresis in a patient with euglycemic diabetic ketoacidosis who was treated with canagliflozin . On the second day of treatment, the patient s urine output increased to over 5,000 ml in the absence of hyperglycemia . On the third day of treatment, oral food intake was initiated, and the patient s urine output increased to over 9,000 ml . At this point, cvii: continuous intravenous insulin infusion, egfr: estimated glomerular filtration rate, fpg: fasting plasma glucose, mdi: multiple daily injections of insulin, na: not available, u - glucose: urinary glucose, u - osm: urine osmolality, serum na: serum sodium, u - na: urinary sodium second, euglycemic diabetic ketoacidosis can accompany persistent diuresis after the administration of sglt2 inhibitors is discontinued . To our knowledge, this is the first report of euglycemic diabetic ketoacidosis with persistent diuresis during treatment with an sglt2 inhibitor . Euglycemic diabetic ketoacidosis is defined as a blood glucose level of <300 mg / dl, and a plasma bicarbonate level 10 meq / l (7). In a previous study of patients with type 2 diabetes, the incidence of diabetic ketoacidosis in patients treated with canagliflozin was more than twice as high as that in patients treated with antidiabetic drugs without canagliflozin (8). Only a few case reports have described the characteristics of euglycemic diabetic ketoacidosis due to the administration of sglt2 inhibitors . The possible causative factors for euglycemic diabetic ketoacidosis due to the administration of sglt2 inhibitors include an insulin dose reduction, alcohol intake, and a low insulin secretion capability . Gastroparesis and a low - carbohydrate diet also can trigger euglycemic diabetic ketoacidosis, especially among diabetic patients who do not use insulin (9 - 12). The time from the first dose of an sglt2 inhibitor to the onset of euglycemic diabetic ketoacidosis has been reported to range from 2 to 13 days in diabetic patients who do not use insulin (9,11,12). In the present case, there are several possible reasons for the patient's development of euglycemic diabetic ketoacidosis, including her low adherence to treatment, the relatively acute autoimmune destruction of cells, and her extreme carbohydrate restriction . This patient experienced euglycemic diabetic ketoacidosis with persistent diuresis via glycosuria, even after the discontinuation of the sglt2 inhibitor . The possible mechanisms of this pathology are as follows: (1) her estimated glomerular filtration rate might have been increasing in association with early type 1 diabetes, thereby promoting glycosuria; (2) exogenous insulin may have augmented the effect of sglt2 inhibition on glycosuria (13); and (3) canagliflozin delays the reversibility of sglt2 inhibition in comparison to its short half - life (10 - 13 hours). In a previous case, burr et al . Reported that their patient had persistent glycosuria in the absence of hyperglycemia for 11 days after the discontinuation of an sglt2 inhibitor (11). The correction of hypovolemia is important for the treatment of diabetic ketoacidosis (3). In the present case, the patient received 3,650 ml of fluid in 12 hours of fluid therapy, which was reasonable from the point of view of treating diabetic ketoacidosis . After the second day of admission, fluid therapy was administered according to her urine volume . Thus, the volume of fluid that the patient received was appropriate for her clinical course . However, we did not rule out central diabetes insipidus . Previous studies have reported cases of central diabetes insipidus during diabetic ketoacidosis (14,15). In our case, thus, it remains unclear whether canagliflozin induced the patient's osmotic diuresis or masked central diabetes insipidus . The insulin - independent actions of sglt2 inhibitors are associated with short - term tolerability and the enhancement of urinary glucose excretion in patients with type 1 diabetes (2). On the other hand, off - label use of sglt2 inhibitors in patients with type 1 diabetes sometimes leads to euglycemic diabetic ketoacidosis (6,9). In addition, sglt2 inhibitor - treated type 2 diabetes patients who develop euglycemic diabetic ketoacidosis are often misdiagnosed as having autoimmune diabetes (8). The possible pathophysiologic characteristics of euglycemic diabetic ketoacidosis due to sglt2 inhibitors are as follows: (1) the urinary glucose excretion of patients with euglycemic diabetic ketoacidosis due to the administration of sglt2 inhibitors is more prominent than that in patients with diabetic ketoacidosis (13); (2) sglt2 inhibitors inhibit the transportation of glucose into cells, thereby enhancing the release of glucagon, resulting in ketogenesis; and (3) sglt2 inhibitors may augment the renal tubular reabsorption of ketone bodies (1). In the present case, the patient's intake of canagliflozin, the restriction of carbohydrates, and low insulin secretion capability led to euglycemic diabetic ketoacidosis . Clinicians should therefore be cautious when prescribing sglt2 inhibitors to diabetic patients who restrict their intake of carbohydrates or who display insufficient insulin secretion . In conclusion, the diuresis of the present patient with euglycemic diabetic ketoacidosis persisted, even after the administration of the sglt2 inhibitor was discontinued . The findings of this case emphasize that euglycemic diabetic ketoacidosis should be considered in sglt2 inhibitor - treated patients who develop nausea or fatigue . Furthermore, adequate hydration may enhance persistent diuresis via glycosuria in patients with euglycemic diabetic ketoacidosis who are treated with sglt2 inhibitors.
With the exception of mammals, the function of the members of the atad2 protein family remains mostly undefined in multicellular eukaryotes . The first reported activity of atad2 (atpase family aaa+ domain - containing protein 2) is its ability to act as a co - activator of estrogen and androgen receptors (zou et al . Other studies later defined atad2 as an e2f and mll co - factor stimulating e2f - dependent cell proliferation (revenko et al ., 2010) and also as a myc partner (ciro et al ., 2009). These transcriptional activities of atad2 have been, at least partly, attributed to its two conserved domains (revenko et al . Indeed, a conserved feature of atad2 and of its homologues across species is the presence of a two partite aaa+ atpase (atpases associated with diverse cellular activities) domain that mediates protein multimerization and of a bromodomain that is responsible for its binding to his - tones (boussouar et al ., 2013). This high conservation of the domain organization of the protein in the vast majority of eukaryotes, including unicellular organisms such as yeasts, is also indicative of its conserved functional features . Functional studies of atad2 family members carried out in mammals were mostly in cancer settings and have recently been reviewed (boussouar et al ., 2013). However, in addition to these studies, there is a relatively rich literature on yta7 (yeast tat - binding analog 7), the unique atad2 homologue expressed in the budding yeast saccharomyces cerevisiae . This review considers all the molecular data available on yta7 in the light of our knowledge on atad2 . The data reported highlight the implication of yta7 not only in gene expression, as expected, but also in genome organization, as a possible histone chaperone acting at boundary sites and regulating transcription . 1a (left), illustrates the strong conservation of this protein family within the eukaryotic kingdom, including yeast . These proteins all share a two - partite aaa+ atpase domain, which is found n - terminal to a bromodomain . These include chordata and the fission yeast schizosaccharomyces pombe that possess two atad2-like proteins known as atad2a and b and abo1 and 2, respectively . It is of note that the vast majority of the published functional studies concerns the atad2a (also termed atad2) member in both human and mouse . The high sequence similarity between these paralogs argues in favor of a possible functional redundancy across all species . Other eukaryotes such as plants (arabidopsis thaliana, zea mays), the worm caenorhabditis elegans, and the budding yeast s. cerevisiae possess only one gene encoding an atad2-like protein . Intriguingly, in drosophila melanogaster and tetrahymena thermophila no gene encoding a homologue of atad2 has been identified . In these two organisms, the closest relative proteins belong to the valosin - containing protein / transitional endoplasmic reticulum atpase (vcp / tera family), which is also a conserved protein family that, to a certain extent, shares a similar domain organization with atad2 family members, although the amino - acid sequence aside the aaa+ atpase domain is poorly conserved . Xenopus laevis is quite peculiar, since it possesses a gene expressing a shorter version of atad2-like protein . This protein contains only the first aaa+ atpase domain and the n - terminal part of the second one . The existence of organisms that have lost part or the totality of atad2-like proteins might indicate that a concomitant functional adaptation has occurred to compensate for the lack of atad2 functions in these organisms . This observation also points to the existence of possible redundant functional pathways in various eukaryotic cells that express atad2 family members . Atad2-like proteins have their highest sequence similarity within the two aaa+ atpase domains and the bromodomain (fig . The aaa+ atpase domain is found in all kingdoms of living organisms, in proteins possessing many different cellular functions . Atad2-like proteins contain two aaa+ atpase domains both located in their n - terminal part . Atp binding and atpase activities were demonstrated for atad2 (zou et al ., 2007). Additionally, mutations in the first aaa+ atpase domain that affect atp binding and hydrolysis impact on the property of mouse atad2 and human atad2 to oligomerize, to bind to acetylated histone h4 (caron et al ., 2010) and to co - activate transcription (zou et al ., 2007), indicating that this domain is critical for atad2 functions . Since the amino - acid sequence of the aaa+ atpase domain is highly similar among atad2-like proteins, it is likely that the activity and function of this domain are also conserved in other eukaryotes . In agreement with such a possibility, mutations in the first aaa+ atpase domain of the s. cerevisiae yta7 protein affect many of its functions (kurat et al ., 2011; lombardi et al ., 2011), as described in the next sections . Atad2-like proteins contain also a putative bromodomain, a module known to bind acetylated lysine in histones and other proteins (filippakopoulos et al ., the binding to acetylated histones was demonstrated for mouse atad2 and human atad2, which show a preferential binding to acetylated histones h3 and h4 (caron et al ., 2010; revenko et al ., bromodomains have a three - dimensional structure that consists of four helix bundles (z, a, b and c) with two interhelical za and bc loops, containing several amino - acids necessary to form an hydrophobic pocket for the interaction with acetylated lysines (dhalluin et al ., 1999). The overall conservation of the bromodomain amino - acid sequence in atad2-like proteins suggests that its global architecture and its capacity to bind to histones may be conserved . Nonetheless, alignment of the yta7 bromodomain with other yeast bromodomains revealed that residues critical for binding acetylated histones are missing, pointing out that the specific binding to acetylated histones is probably not true for all atad2-like proteins (jambunathan et al ., 2005). In vitro histone pull - down experiments have indeed shown that yta7 binds histones but in an acetylation - independent manner (gradolatto et al . Interestingly, similar pull - down experiments, using truncated forms of yta7, revealed a second region that binds histones . This region is located in the n - terminal part of yta7, upstream of the aaa+ atpase domains (fig . 1b), and contains a stretch of acidic residues that may be responsible for electrostatic interactions with charged and unmodified lysine and arginine residues in histones (gradolatto et al ., 2009). Remarkably, a patch of acidic residues in the n - terminal part of the protein seems to be a common feature of all atad2-like proteins, suggesting that the function of this negatively charged region in binding histones may be conserved . Mutations changing the nature of this region would be informative both in relation to the importance of this domain in contributing to atad2-like protein functions, as well as to know whether this region influences the capacity and/or specificity of the bromodomain of yta7 to bind histones . Finally, members of the atad2 family also have in common a fourth region of around 60 amino - acids located at the extreme c - terminal part of the protein (fig . This domain, which is highly conserved among atad2-like proteins, does not correspond to any annotated domain and is not found in any other type of proteins . The analysis of this newly identified and conserved domain should establish its function within atad2 proteins . Interestingly, upstream of this c - terminal domain there is another region conserved in atad2b proteins, but not in the atad2a / atad2 paralogs, indicating that this domain may play an important role in attributing a specific function to the atad2b proteins . Here again, the analysis of this newly identified and conserved region, possibly using the powerful yeast genetics, could provide some clues to the function of atad2-like proteins . A genome - wide chromatin localization approach using chromatin immunoprecipitation (chip)-coupled to chip (chip - chip) analysis demonstrated that yta7 binds to all histone genes (gradolatto et al ., 2008). However, the effects of a deletion of the yta7 gene on the level of histone transcripts are not clear yet . (2008) reported that yta7 is a histone gene repressor based on the fact that yta7 cells present a precocious increase in mrnas of all histone genes after -factor block (g1-phase arrest). 2009) proposed that yta7 could be an activator of histone genes transcription, since a deletion of the yta7 gene correlates with a decrease in hta1 transcripts that is concomitant with a decreased recruitment of rna polymerase ii to both the promoter region and the orf of hta1 (kurat et al ., 2011). More recent studies have shown that the importance of yta7 in regulating histone transcription may in fact differ depending on the analyzed histone gene . Indeed, in yta7 cells hta1 transcripts are significantly reduced, while in these same cells hht1, hhf1 or hta2 transcript levels remain unchanged, despite the fact that yta7 binds to all these histone loci (lombardi et al ., 2011; transcription of the histone genes is tightly regulated throughout the cell cycle in order to provide the histone supply required for the replication of chromatin during the s - phase while avoiding inappropriate and toxic accumulation of neosynthesized histones during the other phases of the cell cycle (gunjan and verreault, 2003). The localization of yta7 to the histone genes indicates a possible direct role for this protein in the phase - specific regulation of histone transcription . Interestingly, yta7 binding to the histone gene hta1 is precisely regulated during the cell cycle (kurat et al ., 2011) after the loading of rna polymerase ii to histone genes in g1/s, yta7 is phosphorylated at multiple sites located in its n - terminal part by at least two different kinases cdk1 (cyclin - dependent kinase 1) and ck2 (casein kinase 2). Phosphorylation of yta7 causes its release from hta1 and correlates with an efficient transcription of hta1 and other histone genes (kurat et al ., 2011). The molecular mechanism underlying the yta7-dependent transcriptional gene activation is not completely understood, but it has been proposed that the phosphorylation - mediated removal of yta7 from a histone gene during the s - phase could be an important step to allow efficient transcriptional elongation along the histone gene . In agreement with this hypothesis, in a mutant of yta7 that cannot be phosphorylated, the recruitment of rna polymerase ii is markedly decreased within the orf of hta1 gene but not at the promoter region (kurat et al ., 2011). Transcriptome analyses of yta7 cells revealed that the expression of inducible genes is also deregulated and that yta7 probably acts as a histone chaperone (lombardi et al . Chip experiments have shown that yta7 localizes to the promoters and 5orf of early meiotic genes and galactose genes while they are induced . Moreover, the presence of yta7 limits the nucleosome density in these genic regions, possibly by promoting histone h3/h4 eviction (fig . In agreement with the existence of an yta7-mediated histone eviction activity, the deficiency in inducing gene expression in yta7 cells is rescued by a decrease in the dosage of histones h3 and h4 caused by the deletion of a pair of histone genes (hht1hhf1). Interestingly, the aaa+ atpase domain is required for this potential chaperone activity of yta7, indicating that yta7 atp hydrolysis may be important for the eviction or degradation of histones (lombardi et al ., 2011). In addition, yta7 was co - purified with several subunits of the rna polymerase ii (rpb2, rpb5 and rpb10), as well as with several histone chaperones, the fact complex (spt16 and pob3) and rtt106 protein (kurat et al ., 2011; tackett et al ., 2005). Nonetheless, it should also be pointed out that so far there is no in vitro evidence that yta7 is a bonafide histone chaperone . Moreover, since in vivo experiments do not exclude the possibility that the change in histone density observed in yta7 cells is due to the activity of one of the histone chaperones interacting with yta7, these findings await further experiments to conclude that yta7 and, by extension, other atad2-like proteins are indeed histone chaperones . Yta7 localization to histone genes promoters was also linked to a boundary activity acting at the promoters of all histone genes . Indeed, at these boundaries, yta7 prevents the spreading of the histone chaperone rtt106 from the promoter to their respective coding regions (fillingham et al ., 2009; kurat et al ., 2011; zunder and rine, 2012) (fig . The recruitment of rtt106 to histone gene promoters depends on asf1 and the hir complex and is not regulated through the cell cycle . Hir / asf1 binding to histone genes and the repression of their transcription rely on a specific dna sequence, the negative regulatory element, present in every histone pairs except for hta2-htb2 . Rtt106 was found to bind and act as a repressor of the histone genes targeted by hir and asf1 (hta1-htb1, hht1-hhf1, hht2-hhf2) (fillingham et al ., 2009). Rtt106 is regulating both positively and negatively the expression of histone genes during the cell cycle . Outside s - phase, when histone genes are repressed, rtt106 recruits the atp - dependent remodeling complex rsc . On the contrary, during histone gene activation in s - phase rtt106 recruits the swi / snf complex (ferreira et al ., 2011). In yta7 cells rtt106 spreads within the coding sequence of histone genes together with the rsc complex, except for the hta2-htb2 histone pair that is not bound by hir / asf1 and rtt106 (fillingham et al ., 2009). The spreading of rtt106 through the coding regions of histone genes is associated with a clear decrease of hta1 transcripts, suggesting that rtt106 and rsc spreading may be responsible for the transcriptional repression of hta1 gene . However, since this spreading occurs on all his - tone genes even though it is not always associated to a decrease in histone mrna levels, for instance at hht1 or hhf1 genes, the potential repressive action of rtt106 requires further investigations to be validated (zunder and rine, 2012). In addition, it is also possible that the regulation of histone genes expression presents a certain degree of locus - specificity that would allow rtt106 spreading to repress transcription of some, but not all histone genes . Earlier evidences implicated yta7, together with other proteins, at barriers that demarcate euchromatin from heterochromatin regions (jambunathan et al ., 2005; tackett et al ., s. cerevisiae contains heterochromatin - like regions located at the mating type, telomeres and rrna - encoding dna loci . The implication of yta7 in such barrier function was first found in a genetic screen aimed at identifying new genes that, when mutated, allow inappropriate spreading of silent chromatin from the mating type locus (hmr) over the adjacent trna gene into the neighboring genomic region which included the ade2 reporter gene (jambunathan et al ., 2005). The boundary activity of yta7 was investigated also by using silencing assays testing the sensitivity to 5 fluoroorotic acid (5foa) drug of yeast cells in which the ura3 reporter gene was ectopically incorporated either within the silenced hmr or the transcriptionally competent adjacent region (tackett et al ., 2005). Furthermore, both the bromodomain and the aaa+ atpase domains are required for yta7 barrier function (gradolatto et al ., 2009; lombardi et al ., 2011). In agreement with a direct implication of yta7 in such a barrier function, it was found to co - purify with a complex localizing at the boundaries between euchromatin and heterochromatin - like regions in s. cerevisiae (tackett et al ., 2005). Nonetheless, the exact role of yta7 at these chromatin barriers remains to be determined . For example, it would be interesting to understand if the probable histone chaperone activity of yta7 plays any important role at these barriers . However, although data on yta7 suggest a role for this factor in the organization of the genome and chromatin dynamics, such indications are scarce for atad2 . Indeed, only one experiment that measured histone h2a mobility in a lung cancer cell line showed that a decrease in atad2 content modifies h2a turnover (caron et al ., 2010). Even in this case considering the importance of yta7 in regulating nucleosome density, it is tempting to propose that atad2 can also act as a histone chaperone, evicting histones from chromatin to avoid potentially deleterious effects associated to an increase in his - tone density . Therefore atad2 and yta7 could primarily be chromatin remodelers and the above - mentioned effects on gene expression regulation could be a mere consequence of their actions on chromatin organization . Alternatively, these proteins could be dual factors playing a role both in transcriptional regulation as scaffolds or as co - activators, and in chromatin remodeling as histone chaperones . In agreement with such a possibility, the acidic n - terminal region of atad2 is a histone - interacting module in the case of yta7 (as described previously), but has also been shown to interact with the androgen receptor (zou et al ., 2009) and e2f transcription factor in the case of human atad2 (revenko et al ., 2010), indicating that this domain may play a central role in the potential dual function of atad2-like proteins . It is also tempting to imagine that this domain may be involved in a cellular protective mechanism which would sense an increase in nucleosome density and in response would favor the chromatin remodeler functions of yta7 instead of its transcriptional role . The phosphorylation of atad2/yta7 in its n - terminal region could allow switching towards a chaperone function of the protein by enabling its detachment from chromatin . The histone binding of yta7 is mediated both by its bromodomain and the n - terminal region (gradolatto et al ., 2009), and we can therefore speculate that a functional inactivation or a lack of the n - terminal part of atad2/yta7 could transform the factor into a pure chromatin regulator . In support of this hypothesis, a spliced form of atad2 lacking this n - terminal acidic domain is expressed exclusively in mouse spermatogenic cells (caron et al ., 2010), where one of the most extensive chromatin remodeling takes place (goudarzi et al ., 2014). However, neither in human cells nor in yeast is such n - terminally shortened protein found expressed, suggesting that, if the above hypothesis is right, in the cases of human or yeast cells, a regulatory mechanism should inactivate the transcriptional role of the n - terminal domain to transform the protein into a pure chromatin regulator . This regulation could involve changes, such as yta7 phosphorylation at its n - terminal part, which would lead to the release of the protein from histone genes (kurat et al ., 2011). The data discussed above therefore support the idea that atad2 and yta7 follow similar regulatory rules, since the underlying activity remains conserved (even if their mechanisms of action could be regulated differently due to specific requirements and evolutionary adaptations). Atad2 is one of the most conserved proteins in eukaryotes although paradoxically its function remains obscure . The remarkable conservation of its functional domains which, as shown here, goes beyond the aaa+ atpase and the bromodomain, pleads in favor of conserved activities . An almost systematic up - regulation of atad2 in all cancers (boussouar et al ., 2013 therefore, an intriguing question is why, despite these important features, the atad2-like proteins have not been identified as critical components in the hundreds of functional screens that have been undertaken in the recent years . One explanation is that the function of atad2/yta7 is so diverse and generalist, involved in many cellular activities, that none of them is dramatically affected by the loss of atad2/yta7, but all work more efficiently in the presence of these proteins . Atad2 family members could therefore be important auxiliary factors in many chromatin related activities, including transcription . Finally, as illustrated in this review, the studies done on the yeast s. cerevisiae yta7 protein are complementary to the studies conducted on atad2 in mammals, which are mostly connected to cancer . In this regard, characterizing the function of atad2 proteins in the fission yeast schizosaccharomyces pombe, another yeast model distantly related to budding yeast and often used for studying chromatin biology, could also be informative . This yeast possesses several pathways and his - tones modifications, like rna interference (rnai) and the h3k9me (histone h3 methylated on lysine 9) heterochromatin mark, that are present in larger eukaryotes but absent in s. cerevisiae . (lin-48 expression abnormal protein 1), the atad2 homologue in the worm caenorhabditis elegans, was found in a screen to control the expression of repeated transgenes, suggesting that repeated sequences may also be genomic targets for atad2 proteins (tseng et al ., 2007). Hence, complementary studies on atad2 homologues conducted in the yeast s. cerevisiae or other eukaryotic model organisms shall continue to bring more valuable information about the general and conserved functions of atad2-like proteins, part of which may be deregulated in cancer.
Light - cured composite resins are being more used specially because they are aesthetically advantageous and enable to be polymerized by physical and chemical activators . However, the crucial point to be reached during restorative dentistry procedures with composite resins is to obtain satisfactory restorations with an adequate light activation technique . Composites consist mainly of filler particles and a resin matrix based on different monomers . Currently, the most widely marketed composite resins are based on the polymerization of bisphenol a glycol dimethacrylate (bis - gma) and urethane dimethacrylate (udma). The curing process occurs in the organic matrix where there is a monomer - polymer conversion through an activation mechanism . However, an insufficient degree of conversion directly affects the physical properties and chemical stability of material . Polymers used in the composition of composite resins may absorb water and chemicals from the oral environment (sorption) and may release some of their own components (solubility). Restorations are subject to tension due to chewing when an excessive or continue non - functional load is applied, internal stresses and strain are generated which can cause the material failure . In this context, the adequate polymerization plays an important role on the longevity of composite resin restorations . This procedure requires sufficient light energy intensity and an adequate wavelength in order to activate the photoinitiator within these materials, which will react with the reducer agent to form free radicals and initiate the polymerization process . Variables such as adequate light intensity, correct wavelength and energy density (power density x exposure time) are essential for achieving the proper depth of cure . Different types of light curing units have been proposed for the polymerization of light activated resinous materials including conventional quartz tungsten halogen (qth) lamp, plasma arc, argon laser and light - emitting diodes (led). All these devices emit blue light in the spectrum of the absorption of camphorquinone, which is the photoinitiator generally found in most resinous material . The qth lights can achieve satisfactory results, but they present certain limitations such as shorter durability of the lamp and heat production . Led have been recognized as a promising technology for polymerization of resin - based materials because all the light emitted is within the spectrum of maximum absorption of camphorquinone at 468 nm . Different curing regimes can exert an influence on the resistance of the union, hardness and crosslinking density . When high power density is applied, more photoinitiator molecules are activated at the same time and consequently acceleration occurs immediately upon light exposure, thereby generating inner stresses that are confined into composite, dental structure and interfaces . In the pulse - delay (pd) polymerization technique there is a decrease in shrinkage and stress . In this technique, there is a short period (about 1 - 3 min) between initial exposition and final cure . The interval between both pulses, an initial pulse with low energy density and a final pulse with high light irradiation, increases the composite resin pre - gel phase improving its flowability and relieving the stress generate by composite shrinkage . It has been hypothesized that low power densities, characterized by reduced power density in the initial seconds, may generate a small number of free radicals . Then, a more linear polymeric structure is obtained, with lower crosslinking density, which negatively affects the material's chemical and physical properties . A previous study concluded that marginal integrity of restorations and the composite " plasticizing effect " are negatively affected when a pd technique is used . The aim of this study was to investigate the effect of different light sources, pd curing protocols and storage media on sorption, solubility and biaxial flexural strength (bfs) of a composite resin . The null hypotheses tested were: (1) qth and led curing light units using different energy densities produce no influence on sorption, solubility and bfs; (2) qth and led curing light units using different curing techniques produce no influence on sorption, solubility and bfs; (3) storage procedure in water and ethanol produces no influence on sorption, solubility and bfs . A nanohybrid composite resin (esthet - x, dentsply, rio de janeiro, rj, brazil) (batch #071116), a2 shade, was used to perform the sorption and solubility test and biaxial piston - on - ring flexural strength test . Specimens were made using a stainless steel mold with 2 mm in thick and 8 mm in diameter . Two hundred and forty specimens were divided into 24 groups (n=10) established according to the 4 study factors: 1- light - curing units: qth lamp (variable intensity polymerizer, bisco inc ., shaumburg, il, usa) and led (ultra blue is, dmc, so carlos, sp, brazil), 2- energy densities: 16 j / cm and 20 j / cm, 3- curing modes: conventional mode (cm), pulse - delay using 3 s (pd3s) and pulse - delay using 10 s (pd10s); 4- storage media (for 28 days): deionized water and 75% ethanol (figure 1) the light irradiance for each curing mode was checked with the in - built radiometer prior to use to ensure consistency of light output . A single increment of composite material was placed into a stainless steel mold and confined between two opposing polyester strips (k - dent, quimidrol, joinville, sc, brazil). The specimens were then removed from the mold and excesses were eliminated with a scalpel blade (free - bac, wuxi xinda medical device co. ltd ., the sorption and solubility tests were performed in compliance with iso 4049:2000 standard specifications, except for the specimen dimensions, types of permeant and storage time . The specimen was placed individually into an open glass bottle of 20 mm (verallia; saint - gobain vidros s.a ., so paulo, sp, brazil) inserted into the desiccators (vidrolabor; vidrolabor ind . Com ., so paulo, sp, brazil) containing freshly dried white silica, batch #0506198, (vetec, vetec qumica fina ltda, rio de janeiro, rj, brazil), and maintained for 22 h at 371c in a vacuum oven (model 440 d, lf equipamentos, so paulo, sp, brazil). Thereafter, desiccators containing the specimens were removed from the oven and placed on a bench for 2 h, at a temperature of 231c, completing a cycle of 24 h. specimens were weighed daily on an analytical scale accurate to 0.001 mg (marte ay220, marte balanas e aparelhos de preciso ltda, santa rita do sapuca, mg, brazil). The complete cycle was repeated until a constant mass (m1) was obtained, that is, until the mass loss of each specimen was not more than 0.1 mg per 24 h cycle . Afterwards, the specimens were returned to their respective labeled bottles, and 15 ml of either deionized water or 75% ethanol were added with manual pipettes . The bottles were capped, replaced in the oven and kept at 371c for 28 days . After this period, all bottles were removed from the oven and kept at room temperature, 231c for 2 h. the specimens were removed from the bottles, dried with absorbent paper for 15 s and left in a sterile bucket (duflex, sswhite, rio de janeiro, rj, brazil) for 1 min . Then the specimens were returned to the desiccators until they reached a constant weight (m3) using the same procedure described to obtain m1 . The values for water sorption and solubility were calculated in g / mm using the following equations: wsp=(m2-m3)/v; wsl=(m1-m3)/v, where wsp was the sorption, wsl was the solubility, m1 was a initial mass of the sample in mg, m2 was a mass of the specimen after immersion into storage media in mg, m3 was a final mass of the specimen recorded after to evaporation of the water or ethanol in mg, and v was a volume of the specimen in mm . After sorption and solubility tests, all specimens were submitted to a biaxial piston - on - ring flexural strength test . The specimen was positioned on a circular metallic device with 3 mm in radius that contain 3 symmetrically spaced steel spheres . Each specimen was placed concentrically on the supporting spheres to ensure that the load was applied at the center of the specimen . A steel piston of 2 mm in diameter was attached to a universal testing machine (emic, dl2000, so jos dos pinhais, pr, brazil) and the test was performed at a crosshead speed of 0.5 mm / min using a 50 kgf load cell until specimen failure . The bfs was calculated according to the astm f394 - 78 specifications as follows: s=-0.2387 p(x - y)/d; where s is the flexural strength in mpa, p is the load at failure in n, and d is the specimen thickness in mm of the failure area . X and y were determined as follows: x=(1+v) ln (b / c)+[(1-v)/2] (b / c) and y=(1+v) [1+ln (a / c)]+(1-v) (a / c), where v is poisson's ratio of the composite resin (0.24), a is the radius of the support circle, b is the radius of the tip of the piston, and c is the radius of the specimen . Data obtained from the sorption, solubility and bfs tests were subjected to three - way analysis of variance and tukey's tests for parametric values, and kruskal - wallis and mann - whitney tests for non - parametric values (=0.05). Pearson's correlation was used to verify the correlation among the sorption, solubility and bfs . All tests were performed using the spss 17.0 for windows statistics software (spss inc ., chicago, il, usa). A nanohybrid composite resin (esthet - x, dentsply, rio de janeiro, rj, brazil) (batch #071116), a2 shade, was used to perform the sorption and solubility test and biaxial piston - on - ring flexural strength test . Specimens were made using a stainless steel mold with 2 mm in thick and 8 mm in diameter . Two hundred and forty specimens were divided into 24 groups (n=10) established according to the 4 study factors: 1- light - curing units: qth lamp (variable intensity polymerizer, bisco inc ., shaumburg, il, usa) and led (ultra blue is, dmc, so carlos, sp, brazil), 2- energy densities: 16 j / cm and 20 j / cm, 3- curing modes: conventional mode (cm), pulse - delay using 3 s (pd3s) and pulse - delay using 10 s (pd10s); 4- storage media (for 28 days): deionized water and 75% ethanol (figure 1) the light irradiance for each curing mode was checked with the in - built radiometer prior to use to ensure consistency of light output . A single increment of composite material was placed into a stainless steel mold and confined between two opposing polyester strips (k - dent, quimidrol, joinville, sc, brazil). The specimens were then removed from the mold and excesses were eliminated with a scalpel blade (free - bac, wuxi xinda medical device co. ltd ., wuxi city, jiangsu, china). The sorption and solubility tests were performed in compliance with iso 4049:2000 standard specifications, except for the specimen dimensions, types of permeant and storage time . The specimen was placed individually into an open glass bottle of 20 mm (verallia; saint - gobain vidros s.a ., so paulo, sp, brazil) inserted into the desiccators (vidrolabor; vidrolabor ind . Com ., so paulo, sp, brazil) containing freshly dried white silica, batch #0506198, (vetec, vetec qumica fina ltda, rio de janeiro, rj, brazil), and maintained for 22 h at 371c in a vacuum oven (model 440 d, lf equipamentos, so paulo, sp, brazil). Thereafter, desiccators containing the specimens were removed from the oven and placed on a bench for 2 h, at a temperature of 231c, completing a cycle of 24 h. specimens were weighed daily on an analytical scale accurate to 0.001 mg (marte ay220, marte balanas e aparelhos de preciso ltda, santa rita do sapuca, mg, brazil). The complete cycle was repeated until a constant mass (m1) was obtained, that is, until the mass loss of each specimen was not more than 0.1 mg per 24 h cycle . Afterwards, the specimens were returned to their respective labeled bottles, and 15 ml of either deionized water or 75% ethanol were added with manual pipettes . The bottles were capped, replaced in the oven and kept at 371c for 28 days . After this period, all bottles were removed from the oven and kept at room temperature, 231c for 2 h. the specimens were removed from the bottles, dried with absorbent paper for 15 s and left in a sterile bucket (duflex, sswhite, rio de janeiro, rj, brazil) for 1 min . Specimens were weighed to obtain m2 . Then the specimens were returned to the desiccators until they reached a constant weight (m3) using the same procedure described to obtain m1 . The values for water sorption and solubility were calculated in g / mm using the following equations: wsp=(m2-m3)/v; wsl=(m1-m3)/v, where wsp was the sorption, wsl was the solubility, m1 was a initial mass of the sample in mg, m2 was a mass of the specimen after immersion into storage media in mg, m3 was a final mass of the specimen recorded after to evaporation of the water or ethanol in mg, and v was a volume of the specimen in mm . After sorption and solubility tests, all specimens were submitted to a biaxial piston - on - ring flexural strength test . The specimen was positioned on a circular metallic device with 3 mm in radius that contain 3 symmetrically spaced steel spheres . Each specimen was placed concentrically on the supporting spheres to ensure that the load was applied at the center of the specimen . A steel piston of 2 mm in diameter was attached to a universal testing machine (emic, dl2000, so jos dos pinhais, pr, brazil) and the test was performed at a crosshead speed of 0.5 mm / min using a 50 kgf load cell until specimen failure . The bfs was calculated according to the astm f394 - 78 specifications as follows: s=-0.2387 p(x - y)/d; where s is the flexural strength in mpa, p is the load at failure in n, and d is the specimen thickness in mm of the failure area . X and y were determined as follows: x=(1+v) ln (b / c)+[(1-v)/2] (b / c) and y=(1+v) [1+ln (a / c)]+(1-v) (a / c), where v is poisson's ratio of the composite resin (0.24), a is the radius of the support circle, b is the radius of the tip of the piston, and c is the radius of the specimen . Data obtained from the sorption, solubility and bfs tests were subjected to three - way analysis of variance and tukey's tests for parametric values, and kruskal - wallis and mann - whitney tests for non - parametric values (=0.05). Pearson's correlation was used to verify the correlation among the sorption, solubility and bfs . All tests were performed using the spss 17.0 for windows statistics software (spss inc ., chicago, il, usa). However, to facilitate understanding, the data presented in the tables are the means of the groups with their respective standard deviations (s.d . ). The results for sorption and solubility at energy densities of 16 j / cm and 20 j / cm are shown in tables 1 and 2, respectively . In general, no significant difference was found between the light sources and between the curing modes tested in this study (p>0.05). However, in the interaction between permeant and light source with a density of 16 j / cm, it was seen that the specimens light - cured with the led unit device in pd10s and stored in ethanol led to higher values of sorption and solubility than those light - cured with qth (p<0.05). Ethanol showed higher sorption and solubility than water irrespective of curing unit or curing method (p<0.05). Means, standard deviations (sd) for comparison between light source and permeant for the measurement of sorption and solubility at 16 j / cm values in the column with same superscript lower caser letter denote no statistical differences (p>0.05) means, standard deviations (sd) for comparison between light source and permeant for the measurement of sorption and solubility at 20 j / cm values in the column with same superscript lower caser letter denote no statistical differences (p>0.05) in the bfs there were interactions between light sources, curing modes and permeants at an energy density of 16 j / cm (p<0.05) (table 3), which did not occur at a density of 20 j / cm, where there were significant differences only between the permeants (water and ethanol) (table 4). Means, standard deviations (sd) for biaxial flexural strength at 16 j / cm values with same superscript capital letter (a, b or c) denote no statistical differences (p>0.05) means, standard deviations (sd) for biaxial flexural strength at 20 j / cm values in the same column with same superscript minute letter (a or b) denote no statistical differences (p>0.05) values in the same line with same superscript capital letter (a or b) denote no statistical differences (p>0.05) the correlation among sorption, solubility and bfs was performed by pearson's correlation coefficient test . Otherwise, bfs were negatively correlated with sorption and solubility (table 5). Successful composite resin restoration depends on the association of low rate shrinkage, good flowability, appropriate cure and satisfactory mechanical properties . Led sources seem to be a promising technology for polymerization of dental resin materials, but its application in composite curing remains controversial . In this study, qth lamp was adopted as a control in order to compare its performance with led . Led units produce a narrow band of wavelengths (450 - 490 nm), conveniently situated within the absorption spectrum of camphorquinone, which is the photoinitiator present in the most of light - activated dental materials . In general, the led unit showed similar behavior to the qth unit, suggesting that similar polymerization quality was reached by the equipment, except when led using 16 j / cm in pd10 s intensity produced higher values of sorption and solubility than qth (p<0.05). Led curing unit using cm at 16 j / cm produced lower values of bfs than qth used with the same protocol (p<0.05). This fact could be associated with the greater heat generated by the qth unit, which may speed up the polymer chain induction process in composite, increasing the mobility of molecules during the reaction and allowing more monomers to react before the curing process ends . The polymerization does not stop right after the photoactivation period, and heat contributes for this post - activation polymerization . Although camphorquinone presents its maximum absorbance at 468 nm, it has an absorption band (380 - 510 nm) that is coincident with the light band emitted by qth lamps resulting in more camphorquinone molecules being activated . Consequently, the crosslinked polymer was probably lower in these led subgroups than in the corresponding qth subgroups and more susceptible to softening in solvents . The size of the specimens used in this study differed from the iso 4049:2000, as the standard dictates that they must be 15 mm in diameter . This contrasts with the diameter of the light guide tip of the curing units, which are 8 to 10 mm in diameter, and which would limit the uniform irradiation throughout the circumference of the specimen . Traditionally, the manufacturers have recommended high light intensity or power density to provide a higher degree of monomer conversion into polymer, thereby improving the mechanical properties of composite resin . High power density used for short light exposures led to a lower degree of cure and lower flexural strength and modulus than when the composite was cured with intermediary power densities for longer exposures . In some studies that used 24 j / cm of energy density, the curing modes tested presented similar behavior . It has been speculated that this occurred as a result of the formation of a densely crosslinked polymer network because an adequate energy density had been used . The same finding was seen in this study in the groups that used 20 j / cm, where there were no significant differences between the subgroups studied . The subgroups that used 16 j / cm presented different values of sorption, solubility and bfs storage in ethanol for the qth and led units (p<0.05). The use of low initial intensity followed by high intensity light with an interval between them seems to create a uniform polymerization of the composite resin, providing its best adaptation to cavity walls and possibly the least polymerization contraction stress . In the delay period, little amount of free radicals and double bond conversion are produced and the composite resin has more time to molecular rearrangement and stress relief . In the pd cure, there was reduced gap formation without any mechanical properties being compromised, which can be considered as an indicative that composite has the same quantity of remaining double bonds . The use of slow - cure methods (pulse curing modes) in combination with the interval between two irradiations seem not to interfere with solvent sorption and could be useful for adhesive composite restorations . In this study, there were no significant differences among the curing modes in terms of sorption, solubility and bfs (p>0.05) except for qth comparing the cm and pd3s at 16 j / cm, where pd3s produced lower values of bfs than the cm (p<0.05). However, this result was different from those of other studies, where the pd mode resulted in a linear polymer structure that was less crosslinked, thereby increasing the susceptibility of polymers to softening in ethanol . Some studies have shown a significant reduction in mechanical properties, such as flexural modulus and hardness, when composites were photo - cured by pd mode and submitted to solvent action . However, based on the methodology used in this study and the obtained results, the curing modes presented similar behavior . According to lopes, et al . (2009), this could have occurred because another study used unfilled resins for their tests instead of filled composite resin . Another explanation for the difference between the results of this study and that where the photo - cured composites using the pd mode were more susceptible to softening in solvent could be attributed to the type of tests applied to assess the amount of crosslinking, such as hardness deterioration, degree of conversion and remaining double bonds, which only make an indirect assessment of the quality of the polymer . The effect of the different solvents was clearly significant in all groups; ethanol resulted in more solubility, sorption and lower bfs than water media . 4049:2000 specifications, the storage time is 7 days, but in the present study the storage time of 28 days in the same way as established for other studies in which most of the composites studied reached saturation within 7 - 60 days . The amount of solvent uptake by the polymer is determined by differences in solubility parameters between the polymer and the permeant . Water or solvent uptake into the resin phase of cr causes two opposing processes: the solvent will extract unreacted components, mainly monomer, thereby resulting in shrinkage, loss of weight and reduction in mechanical properties; conversely, solvent uptake leads to a swelling of the composite resin and an increase in weight . These phenomena of sorption and solubility may serve as precursors to a variety of chemical and physical processes which create biological concerns and produce deleterious effects on the structure and function of the polymer material . It is desirable that composites have all monomers converted into polymers during the polymerization reaction and remain stable for long time . It may be assumed that, whereas the increase in the degree of conversion reduced the solubility because the amount of unreacted monomers available for leaching out was lower due to the high percentage of reacted aliphatic c = c bonds from the dimethacrylate monomers . Most dental polymers present ionic functional groups with water affinity, which in turn optimizes their hydrophilicity . The susceptibility for more linear or less crosslinked polymer to softening in solvents may be explained by the solvent - polymer interaction and, consequently, by hoy's solubility parameter for polar forces . Solvents that can form strong secondary bonds with the polymer chains can penetrate and replace the interchange secondary bonds, and thereby pull apart and dissolve linear and branched polymers . Distilled water, a solvent indicated in iso specifications for resin - based filling materials, simulates the wet intraoral environment provided by saliva and water . However, intraoral conditions are clearly more complex than those achieved with distilled water in the laboratory . According to the us food and drug administration guidelines, a 75% ethanol - water solution is a clinically relevant food / oral simulating liquid . The 75% ethanol solvent is the most frequently used to simulate accelerated ageing of restorations as it has a solubility parameter matching that of bisgma when ethanol penetrates the polymer network, it causes the structure swell, thereby allowing for the release of uncured monomers and causing the dissolution of linear polymer chains . This expansion is facilitated when crosslink density is low, based on the fact that more space and pathways are available for solvent molecules to diffuse within the structure . The properties of composite resins are affected by the degree of conversion, but an analysis of the amount of crosslinking in the resins may provide a closer correlation to mechanical properties . Water sorption may deteriorate polymer mechanical properties and because of this the resin specimens were submitted to a mechanical test after the sorption and solubility tests . Tensile strength is generally considered to be the most meaningful property of these brittle materials in assessing the potential for failure of dental restorations . Flexural strength can be measured using a three - point bending flexural test, a four - point bending flexural test or a biaxial flexure test; the drawback of the three - point and four - point flexural tests is the inherent sensitivity to flaws and defect near specimen edges . As ban and anusavice (1990) related, the biaxial flexure test is recognized as a reliable technique since the maximum tensile stress occurs within the central loading area and edge failures are eliminated . In the evaluation of the correlation among the variables (sorption, solubility and bfs), it was possible to see how much higher the sorption and solubility were and how much lower the bfs (p<0.05) was . The degree of conversion and crosslink density can influence the chemical degradation that is usually caused by oxidation and hydrolysis processes, and consequently reduce the physical and mechanical properties . Thus, polymers with lower crosslink densities are expected to undergo more softening in solvents, which results in greater sorption and solubility values but lower bfs values . Within the limitations of this in vitro study methodology, it was concluded that, in general, the light sources (qth and led) and curing modes (cm and pd) did not influence the sorption, solubility or bfs of the tested resin . However, the different solvents (water and ethanol) did influence its sorption, solubility and bfs behavior . The authors would like to thank nbia miranda ferreira (dentsply) for supplying materials and paulo csar freitas santos filho for his assistance at the federal university of uberlndia's laboratory.
Dna double - strand breaks (dsbs) are the most dangerous type of dna damage, as a single unrepaired dsb can trigger apoptosis . Dsbs are generated during physiological replication, and are induced by radiotherapy and chemotherapeutic reagents such as the topoisomerase poison . There are two major dsb repair pathways in eukaryotic cells: homologous recombination (hr) and non - homologous end joining (nhej). The choice of the two pathways in saccharomyces cerevisiae is alternative so that dsb resection can inhibit nhej, because dsbs containing 3 single - strand dna cannot be repaired by canonical nhej (fig . It has been widely believed that the alternative choice model is also relevant to mammalian cells, though the relevance has not yet been demonstrated . The method of examining the precise structure of resected dsb sites is currently available only for meiotic hr in s. cerevisiae, but not for mammalian cells [2, 3]. Although dsb resection would strongly inhibit canonical nhej, quick initiation of dsb resection does not reduce the overall efficiency of dsb repair in s. cerevisiae due to the very small contribution of nhej to dsb repair . 1a) would result in a considerable decrease in the overall efficiency of dsb repair in mammalian cells due to the major role of nhej in dsb repair . The choice of homologous recombination (hr) or non - homologous end - joining (nhej) is determined by the presence or absence of dsb resection, respectively . (b) bidirectional processing of dsbs during dsb resection for meiotic hr in s. cerevisiae . Spo11 protein, a topoisomerase, generates dsbs, where spo11 is covalently associated with the 5 end of dsbs leading to formation of blocked ends . Single - strand break formation on the spo11-associating strand by mre11 initiates dsb resection, enabling resection in a bidirectional manner, where exo1 digests in the 53 direction away from the dsb, and mre11 digests in the 35 direction towards the dsb end . We exposed the indicated genotypes of chicken dt40 cells to doses equivalent to ld50% (the dose that reduces cellular survival to 50%) of -rays in the g2 phase, harvested mitotic cells at 3 h, and counted the number of chromosomal breaks in mitotic chromosome spreads . The x - axis shows the number of chromosome aberrations per 100 mitotic cells and per gray . (d) in contrast with meiotic hr in s. cerevisiae (b), vertebrate mre11 has a very minor role in dsb resection for subsequent hr in somatic vertebrate cells . Dna2 plays the dominant role in dsb resection in chicken dt40 cells among the ctip, dna2, exo1 and mre11 nucleases . The choice of homologous recombination (hr) or non - homologous end - joining (nhej) is determined by the presence or absence of dsb resection, respectively . (b) bidirectional processing of dsbs during dsb resection for meiotic hr in s. cerevisiae . Spo11 protein, a topoisomerase, generates dsbs, where spo11 is covalently associated with the 5 end of dsbs leading to formation of blocked ends . Single - strand break formation on the spo11-associating strand by mre11 initiates dsb resection, enabling resection in a bidirectional manner, where exo1 digests in the 53 direction away from the dsb, and mre11 digests in the 35 direction towards the dsb end . We exposed the indicated genotypes of chicken dt40 cells to doses equivalent to ld50% (the dose that reduces cellular survival to 50%) of -rays in the g2 phase, harvested mitotic cells at 3 h, and counted the number of chromosomal breaks in mitotic chromosome spreads . The x - axis shows the number of chromosome aberrations per 100 mitotic cells and per gray . (d) in contrast with meiotic hr in s. cerevisiae (b), vertebrate mre11 has a very minor role in dsb resection for subsequent hr in somatic vertebrate cells . Dna2 plays the dominant role in dsb resection in chicken dt40 cells among the ctip, dna2, exo1 and mre11 nucleases . A structural study of resected dsbs during meiotic hr in s. cerevisiae revealed that dsb resection is initiated by a single - strand break (ssb) on the strand to be resected up to 300 bases from the 5-terminus of the dsb (fig . This ssb is subjected to subsequent bidirectional resection, both in the 53 direction away from the dsb and in the 35 direction towards the dsb end . The mre11 nuclease forms a complex with rad50 and xrs2 (the yeast ortholog of mammalian nbs1). The resulting mrx complex is responsible for the formation of the ssb, followed by the 53 direction resection in the meiotic hr of s. cerevisiae . It remains elusive whether this bidirectional resection also plays a role in mitotic hr in mammalian cells as well as in yeast . If resection from the ssb is carried out only in the 53 direction away from the dsb, but not in the 35 direction, dsb ends would be maintained as duplex dna (fig . The absence of homologous single - stranded tails at dsb ends does not significantly interfere with homology search in s. cerevisiae, while the presence of duplex dna at dsb ends would ensure efficient repair by canonical nhej . The new model shown in fig . 1d predicts that hr and nhej are able to work in parallel, without interfering with each other . In other words, while hr undergoes homology search with the rad51 recombinase polymerized on resected dsbs, nhej is able to efficiently ligate dsb ends . 1d is supported by the ionizing radiation sensitivity of dt40 cells deficient in both hr and nhej (fig ., we completely inactivated canonical nhej by disrupting the ku70 gene, and partially inactivated hr by disrupting the rad54 gene . Note that the complete inactivation of hr by disrupting the rad51 gene causes cellular lethality associated with severe genome instability, whereas disrupting the rad54 gene allows for normal mouse development . Nonetheless, the loss of rad54 completely inhibits hr - dependent repair of ionizing radiation induced dsbs, as evidenced by data indicating that the loss of rad54 reduces cellular tolerance to ionizing radiation in the s / g2 phases to the tolerance seen in the g1 phase . Rad54 does not affect dsb resection, but facilitates homology search by resected dsbs associated with polymerized rad51 (fig . Hr is preferentially used over canonical nhej for dsb repair in the g2 phase in dt40 cells, as shown by comparable radiosensitivity between wild - type and canonical - nhej 1a) predicts that canonical nhej could not substitute for abortive hr in rad54 cells because the precedent formation of the 3 single - strand tail would inhibit canonical nhej . However, the radiotolerance of rad54 dt40 cells is considerably higher than that of ku70/rad54 dt40 cells (fig . 1c), indicating that canonical nhej can efficiently ligate abortive hr intermediates generated in rad54 cells . We, therefore, propose that the molecular mechanism for dsb resection in s. cerevisiae distinctly differs from that in metazoan cells (compare fig . The new model agrees with the phenotype of rad54 and ku70/rad54 dt40 cells, where canonical nhej is able to efficiently repair dsbs, even after polymerization of rad51 at the dsb sites in rad54 cells . The current model of dsb resection in mammalian cells is based on the findings about hr in s. cerevisiae, since its molecular mechanism for dsb resection is most precisely defined (figs 1b and 2a). Mre11 also plays an important role in dsb resection when hr repairs ionizing radiation induced dsbs, particularly in the g2 phase . Extrapolating these findings of s. cerevisiae, the vast majority of the manuscripts and reviews have suggested that mre11 also plays the major role in dsb resection in mammalian cells . It should be noted that the essential role of mre11 in meiotic hr is irrelevant to some of the mitotic hr in s. cerevisiae . For example, the selective inactivation of nuclease activity in mre11 causes only a modest delay in dsb resection at the dsb site formed by the ho restriction enzyme, which generates thus, other nucleases can effectively substitute for the defective nuclease activity of mre11 in the resection at moreover, hypersensitivity of mre11-nuclease deficient yeast to ionizing radiation suggests that mre11 eliminates chemical modifications at dirty dsb sites induced by ionizing radiation before dsb repair by hr . This possibility will be discussed in a later section entitled the role of mre11 nuclease activity in elimination of chemical adducts from dsb ends. The importance of the nuclease activity in mammalian mre11 has been indicated by buis et al ., who conditionally generated nuclease - deficient mre11 and mre11-null mutant (mre11) mice . Both mutant mice exhibited a very similar phenotype, including the mortality of cells associated with dramatic genomic instability, hypersensitivity to ionizing radiation, and 2- to 3-fold reduction in the efficiency of dsb resection . The data have been interpreted as compelling evidence for the critical role of mre11-nuclease - dependent dsb resection in hr . First, is only 2- to 3-fold reduction in dsb resection solely responsible for a severe defect in hr? Second, does a defect in hr fully explain the dramatic genomic instability? To define the role of mre11 nuclease activity in dsb resection and genome stability, we conditionally generated mre11 and mre11 cells from the chicken dt40 and human tk6 b cell lines . Note that the tk6 cell line is widely used for evaluating the genotoxicity of industrial chemicals by regulators due to its stable karyotype and phenotype . These mre11 mutants retain the capability of performing dsb resection in nearly normal kinetics in both dt40 (fig . The data agree with a recent study directly measuring the length of resected 3 single - strand overhangs; that study shows only about a 50 to 70% decrease in dsb resection upon depletion of mre11 . Collectively, the loss of mre11 causes only up to 2- to 3-fold reduction in the efficiency of dsb resection in vertebrate cells . If dsb resection initiates from a ssb away from a dsb end, the very poor 3 to 5 exonuclease activity of mre11 would allow dsb ends to be maintained as duplex dna (fig . 2.differential roles for mre11 in (a) meiotic hr in saccharomyces cerevisiae, (b) dsb resection for hr in vertebrate cells and (c) the processing of dirty ends containing base damage and covalently associating topoisomerases in s. cerevisiae and vertebrate cells . Saccharomyces cerevisiae mre11 actively digests in the 35 direction towards the dsb end (a). In mammalian cells, mre11 only contributes a small amount to digestion in the 35 direction (b), while mre11 plays an important role in end processing of non - physiological chemical modifications (c). 3. (a) rad51-focus formation of wild - type chicken dt40 cells at the indicated time after exposure to 2 gy ionizing radiation . The average number of rad51 foci per cell was calculated from at least 100 cells . (b) the average number of rad51 foci per cell having the indicated genotypes of dt40 cells . Mre11 and dna2 are deficient in the nuclease activities of dna2 and mre11, while dna2 is deficient in the helicase activity of dna2 . (c) tk6 cell lines carrying the indicated genotypes were exposed to 0.5 gy -rays at time zero . Mre11 null (mre11) and mre11-nuclease - deficient (mre11) mutants show normal kinetics of dsb resection, but delayed resolution of hr intermediates in comparison with heterozygous mutant (mre11) cells, which show the same phenotype as wild - type . (b) dsb resection for hr in vertebrate cells and (c) the processing of dirty ends containing base damage and covalently associating topoisomerases in s. cerevisiae and vertebrate cells . Saccharomyces cerevisiae mre11 actively digests in the 35 direction towards the dsb end (a). In mammalian cells, mre11 only contributes a small amount to digestion in the 35 direction (b), while mre11 plays an important role in end processing of non - physiological chemical modifications (c). (a) rad51-focus formation of wild - type chicken dt40 cells at the indicated time after exposure to 2 gy ionizing radiation . The average number of rad51 foci per cell was calculated from at least 100 cells . (b) the average number of rad51 foci per cell having the indicated genotypes of dt40 cells . Mre11 and dna2 are deficient in the nuclease activities of dna2 and mre11, while dna2 is deficient in the helicase activity of dna2 . (c) tk6 cell lines carrying the indicated genotypes were exposed to 0.5 gy -rays at time zero . Mre11 null (mre11) and mre11-nuclease - deficient (mre11) mutants show normal kinetics of dsb resection, but delayed resolution of hr intermediates in comparison with heterozygous mutant (mre11) cells, which show the same phenotype as wild - type . The prominent question is whether only 2- to 3-fold reduction in dsb resection has an impact on the efficiency of hr - dependent dsb repair . A previous study of our laboratory, as well as a genetic study of yeast, consistently indicate that such a small reduction has virtually no impact on the efficiency of hr - dependent dsb repair . Dsbs, subsequent hr - dependent dsb repair occurs efficiently in s. cerevisiae [3, 10]. We generated the tk6 mutant cells (ctip cells), where the amount of ctip was reduced by five times and the dsb resection efficiency decreased by two times . The resulting mutant is capable of completing hr - dependent dsb repair with normal kinetics (fig . 3c) and is fully proficient in hr between homologous chromosomes induced by i - sce1 restriction enzyme mediated dsbs . Thus, 2- to 3-fold reduction in dsb resection does not have a negative impact on the overall efficiency of hr - dependent dsb repair . In summary, dsb resection by mre11 does not account for the critical role of mre11 in hr - dependent repair of ionizing radiation induced dsbs . If dsb resection by mre11 has a minor role in dsb resection, an important question is which nuclease plays a critical role in dsb resection and hr? Four nucleases (ctip, dna2, exo1 and mre11) contribute to dsb resection in both s. cerevisiae and mammalian cells . Short - range dsb resection, removing up to a few hundred nucleotides from the 5 strand by mre11 and sae2 (the ctip ortholog), is followed by long - range dsb resection, more than 10 kb in length, by dna2 and exo1 in s. cerevisiae [15, 16]. The short - range resection is sufficient for efficient hr . To measure the relative contribution of the four nucleases to dsb resection we assessed dsb resection by measuring rad51 focus formation at 1 h after ionizing radiation (fig . 3a). Only ctip and dna2, but not exo1 or mre11, are required for efficient dsb resection (fig . Nuclease - deficient dna2 as well as null - mutant dna2 cells show a significant defect in dsb resection, indicating dna2 contributes to dsb resection as a nuclease . In contrast, mammalian ctip has the non - catalytic role in dsb resection, and does not work as a nuclease in hr . We showed that the non - catalytic role played by ctip is essential for dsb resection by recruiting dna2 to dsb sites . The modest contribution of exo1 to dsb resection agrees with the normal development of exo1-deficient mice, despite the embryonic mortality of various hr - deficient mice . Collectively, dna2 plays the dominant role in dsb resection required for efficient hr among the four nucleases in the chicken dt40 cell line (fig . 2b). Given the very minor contribution of dna2 to dsb resection for efficient hr in s. cerevisiae, the relative contribution of the four nucleases to dsb resection and hr is very different between s. cerevisiae and the dt40 cells . Although human mre11 as well as mre11 tk6 cells retain the capability of efficiently performing dsb resection, these mutants show delayed resolution of rad51 focus formation after ionizing irradiation (fig . Similarly, although chicken mre11 dt40 cells efficiently perform dsb resection, the cells show a severe defect in repairing ionizing radiation induced dsbs by hr [20, 21]. These observations indicate that mre11 plays important roles in dsb repair other than dsb resection . We hereafter discuss two roles played by mre11: (i) removal of chemical adducts from dsb ends, and (ii) maintenance of holliday junction hr intermediates . Deficient s. cerevisiae shows only a modest delay in dsb resection at the ho restriction enzyme induced dsb sites in asynchronous populations, the mutant yeast is hypersensitive to ionizing radiation [3, 22]. Likewise, in the fission yeast, schizosaccharomyces pombe (s. pombe), the mre11 nuclease plays an important role in the repair of ionizing irradiation induced dsbs, but is not required for resection at an ho - induced dsb . The data suggests that mre11 may eliminate chemical modification induced by ionizing radiation before dsb repair by hr as well as nhej . This idea is further supported by the data that mre11 nuclease activity of s. pombe is involved in the removal of both topoisomerase i (topi) and topii from 3 and 5 dna ends, respectively, in vivo . Topii resolves dna catenanes by catalyzing the transient formation of double - strand dna breakage, enabling intact dna to pass through the dsb and re - ligating the dsb . During such transient dsb formation, top2 becomes covalently bound to the 5-dna end of the breakage, forming topii - dna cleavage complex (topiicc) intermediates . A chemotherapeutic agent, etoposide, strongly stabilizes topiicc, leading to the formation of dsbs . Deficient s. pombe shows a more significant accumulation of topiicc than wild - type cells, following treatment with etoposide . Moreover, mre11 promotes nhej of etoposide - induced dsbs in g1 in mammalian cells . These observations indicate that the incision activity of mre11 eliminates topii adducts from dsb ends (fig . Note that the incision activity is likely to be independent of the role of mre11 in dsb resection (fig . 2b), since the incision can occur in the g1 phase, when dsb resection is inhibited . To directly test whether the nuclease activity of mre11 is involved in elimination of topii from dsb ends, we measured the amount of topiicc (topii covalently associated with genomic dna), following exposure of cells to etoposide (fig . We separated topiicc from free topii by subjecting the cellular extract to cesium - chloride - gradient ultracentrifugation, followed by fractionation and western blot analysis with anti - topii antibody . The heterozygous mutation of the nuclease activity (mre11) significantly increased the amount of topiicc . Moreover, overexpression of tyrosyl - dna - phosphodiesterase 2 (tdp2), an enzyme eliminating covalently bound topii, normalized the amount of topiicc . The prominent phenotype of the heterozygous mutant (mre11) cells suggests the important role of the mre11 nuclease activity in elimination of the topii adduct, as yeast mre11 is essential for removing the spo11 topoisomerase from dsb ends (fig . These observations support the notion that the nuclease activity of mammalian mre11 is required for processing various forms of blocked dsb ends, including those containing damaged nucleotides and topoisomerase adducts, for subsequent dsb repair by nhej as well as hr . We propose that mre11 contributes to cellular tolerance to ionizing radiation through processing of dsb ends (fig . 4.accumulation of topoisomerase ii adduct (topiicc) in human tk6 cells heterozygous for nuclease - deficient mre11 (mre11). (a) schematic of the in vivo topiicc measurement by immunodetection with anti - topii antibody . A 200 g genomic dna sample was subjected to sedimentation by cscl - gradient ultracentrifugation . Genomic dna of wild - type tk6 cells that had been treated with 10 m etoposide for 2 h was included as a control in every ultracentrifugation . The treatment reduced the survival of wild - type cells by 3% relative to untreated cells . Individual fractions were blotted to nylon filters, followed by western blot using anti - topii antibody . The remaining two fractions include free topii (yellow circle), while the other fractions include topii covalently associated with genomic dna . (b) western blot analyses of topii in the indicated tk6 cells that had been treated with 10 m etoposide (+) or dmso () for 2 h. accumulation of topoisomerase ii adduct (topiicc) in human tk6 cells heterozygous for nuclease - deficient mre11 (mre11). (a) schematic of the in vivo topiicc measurement by immunodetection with anti - topii antibody . A 200 g genomic dna sample was subjected to sedimentation by cscl - gradient ultracentrifugation . Genomic dna of wild - type tk6 cells that had been treated with 10 m etoposide for 2 h was included as a control in every ultracentrifugation . The treatment reduced the survival of wild - type cells by 3% relative to untreated cells . Individual fractions were blotted to nylon filters, followed by western blot using anti - topii antibody . The remaining two fractions include free topii (yellow circle), while the other fractions include topii covalently associated with genomic dna . (b) western blot analyses of topii in the indicated tk6 cells that had been treated with 10 m etoposide (+) or dmso () for 2 h. nuclease - dead mre11 mutants have a significantly milder phenotype during mitosis than do null - mre11 mutants in s. cerevisiae [3, 29]. Likewise, nuclease - deficient mre11 mutant dt40 cells are able to slowly proliferate, although the null - mre11 mutant dt40 cells are lethal . However, the non - catalytic functioning of mre11 in hr has not yet been defined . This functioning might depend on the capability of the mrn complex to tether two dna strands, since the complex has a structure similar to that of cohesion and condensin . Although human mre11 as well as mre11 tk6 cells initiate dsb resection with nearly normal kinetics, these mutants showed a significant delay in resolution of ionizing radiation the data suggest a role for mre11 in a late step of hr as well as in processing the ends of there is no reliable phenotypic assay for analyzing hr intermediates in mammalian cells or mitotic yeast . However, phenotypic analyses of meiotic hr in s. cerevisiae most significantly contribute to our understanding of the precise molecular mechanism for resolution of hr intermediates . Hr plays the dominant role in repairing ionizing radiation induced dsbs during the g2 phase in dt40 cells . Thus, we can assess the efficiency of hr - dependent dsb repair by exposing g2-phase cells to -irradiation and measuring the number of chromosome aberrations in the subsequent m phase . Moreover, morphological observation of mitotic chromosomes allows for identifying abortive hr intermediates (fig . 5. (a) model for generation of isochromatid - type breaks following ionizing irradiation at the g2 phase . Formation of hr intermediates such as holliday junctions followed by failure of their resolution could generate isochromatid - type breaks, where two sister chromatids are broken at the same sites due to a defect in local chromosome condensation at hr intermediates . (b) the numbers of the indicated chromosome aberrations in the mitotic chromosome spreads after ionizing irradiation at the g2 phase . Dt40 cells carrying the indicated genotypes were exposed to 0.3 gy -rays and harvested at 3 h for chromosome analysis . (c) the spatial distance between two sister chromatids is much bigger in mammalian cells than in yeast . Following dna replication, accordingly, hr intermediates are constantly pulled in opposite directions, as shown by the arrows . (d) model for the repair of ionizing radiation induced dsbs . Ionizing radiation causes dirty dsbs to associate with chemical modifications at dsb ends . The nuclease activity of mre11 plays the major role in eliminating chemical modifications, particularly from 5 ends . Saccharomyces cerevisiae mre11 coordinates initiation of dsb resection at the two ends of individual dsbs . (a) model for generation of isochromatid - type breaks following ionizing irradiation at the g2 phase . Formation of hr intermediates such as holliday junctions followed by failure of their resolution could generate isochromatid - type breaks, where two sister chromatids are broken at the same sites due to a defect in local chromosome condensation at hr intermediates . (b) the numbers of the indicated chromosome aberrations in the mitotic chromosome spreads after ionizing irradiation at the g2 phase . Dt40 cells carrying the indicated genotypes were exposed to 0.3 gy -rays and harvested at 3 h for chromosome analysis . (c) the spatial distance between two sister chromatids is much bigger in mammalian cells than in yeast . Following dna replication, accordingly, hr intermediates are constantly pulled in opposite directions, as shown by the arrows . (d) model for the repair of ionizing radiation induced dsbs . Ionizing radiation causes dirty dsbs to associate with chemical modifications at dsb ends . The nuclease activity of mre11 plays the major role in eliminating chemical modifications, particularly from 5 ends . Saccharomyces cerevisiae mre11 coordinates initiation of dsb resection at the two ends of individual dsbs . We propose that mre11 maintains hr intermediates for their proper resolution . In rad54 cells, where the efficiency of homology search and strand exchange is reduced, -irradiation of g2-phase cells significantly increased the number of chromatid - type breaks (fig . Remarkably, the loss of mre11 caused marked increases in the number of isochromatid - type breaks as well as chromatid - type breaks . Thus, a significant fraction of the -irradiation induced dsbs in one of the sister chromatids is converted to chromosome breaks in both sister chromatids . These observations indicate that mre11 can contribute to hr - dependent repair of dsbs, even after the formation of hr intermediates containing pairs of sister chromatids . We propose that mre11 is required for the stable maintenance of hr intermediates for their proper resolution . A critical difference in hr between yeast cells and vertebrate cells is a spatial distance between donor sequences in intact sister chromatids and recipient homologous sequences in broken sister chromatids . The distance is much smaller in yeast in comparison with mammalian cells (fig . Moreover, dynamic condensation of sister chromatids may constantly increase their distance after dna replication in mammalian cells . Accordingly, hr intermediates containing two sisters may be pulled in opposite directions, leading to dissociation of the intermediates . Extensive strand exchange between two sister chromatids leads to the formation of stable structure such as the double holliday junction . Moreover, the tethering by mrn may further stabilize hr intermediates and promote the completion of dsb repair by hr . This mrn - dependent mechanism might be relevant to mitotic hr in s. cerevisiae, since yeast mre11 also has a prominent non - catalytic role in genome maintenance [3, 29]. The genetic study of yeast species provides a framework for examining molecular mechanisms underlying hr in mammalian cells . The genetic study of yeast, but not mammalian cells, allows for examining the structure of various hr intermediates . In addition, since hr is the most complex dna repair reaction, involving more than 100 molecules (including histone - modifying enzymes), neither the biochemical study nor genetic study of mammalian cells allows for dissecting the role of individual factors in various hr reactions . Accordingly, the functions of individual mammalian hr factors have been postulated by the function of their yeast ortholog proteins . A large number of researchers of mammalian mre11 seem to have interpreted their own data by extrapolating known functions of s. cerevisiae mre11 . However, although mammalian mre11 does contribute to dsb resection, its limited contribution does not necessarily account for the very severe defect in hr - dependent repair of ionizing radiation induced dsbs [11, 14]. The present data suggest that the severe defect reflects the important role of mre11 in dsb repair aside from dsb resection: (i) processing of dsb ends and (ii) maintenance of hr intermediates . This conclusion is also supported by genetic studies of s. cerevisiae indicating (i) the greater contribution of mre11 to repair of clean dsbs, and (ii) a severer phenotype of the null - mre11 mutant compared with that of the nuclease - deficient mre11 mutant . Future studies will clarify the roles of mammalian mre11 in the processing of dsb ends and the maintaining of hr intermediates (fig . This work was supported by the japan society for the promotion of science (jsps) core - to - core program and a grant - in - aid for scientific research (s) (to s.t .) And grants - in - aid from the ministry of education, science, sport and culture (to h.s . The open access publication charges for this article was provided by kakenhi 23221005 and kakenhi 26740018.
In the last two decades it has become increasingly clear that a diverse range of rnas play numerous critical roles in the regulation of gene expression (13). It has also become apparent that rnas hardly function alone in a cellular environment . Prominent examples include the pre - mrna splicing machinery, the apparatus that assembles ribosomes and mrna storage particles (58). A wealth of data indicate that structure and composition of such ribonucleoprotein complexes (rnps) are not static, and that changes in rnp structure and/or composition need to be accurately timed to ensure correct rnp function (911). One group of enzymes pivotal for facilitating rearrangments of cellular rnps are the dexh / d proteins (11,12). These enzymes are known to manipulate rna structure in an atp - dependent manner (13). Members of the dexh / d protein family are present in all forms of cellular life and also in several viruses (12,14). Dexh / d proteins are highly conserved, sharing at least eight characteristic sequence motifs (12). The characteristic sequence motif ii often takes the form dead, deah or dexh [in single letter amino acid code (15)]. These signatures provide the names for the three subgroups of the protein family (12). The name dexh / d proteins stems from averaging the signatures of the three subgroups . Dexh / d proteins are involved in virtually all aspects of rna metabolism (12). Significant evidence suggests that these proteins act as atp - driven motors or switches at very specific points in processes such as pre - mrna splicing or during ribosome biogenesis (12). Yet, it is unknown where precisely the vast majority of dexh / d proteins bind to their targets and which exact conformational changes these proteins catalyze in their respective substrates . Nonetheless, physiological functions of many dexh / d proteins correlate with the ability of the enzymes to hydrolyze atp in an rna - stimulated manner, and/or to unwind rna duplexes in vitro in an atp - dependent fashion (16). For these reasons, and because of the intuitive connection between the unwinding of rna secondary structure and conformational changes in rnps, it has long been assumed that rna helicase activity is central to the biological function of dexh / d proteins . In fact, dexh / d proteins are frequently referred to as rna helicases . However, because cellular rnas are invariably complexed with proteins, dexh / d proteins are most likely to encounter rna protein complexes, rather than the pure rna duplexes that are commonly used to measure rna helicase activity in vitro (4,16,17). Yet, we are only beginning to understand how dexh / d proteins remodel rnps . Here we review recent results indicating that dexh / d proteins can directly target rna protein interactions both in vivo and in vitro and that rnp remodeling does not necessarily require rna duplex unwinding . Based on the available data, we outline a basic mechanism by which dexh / d proteins may displace proteins from rna . The first specific examples of dexh / d proteins targeting rna protein interactions emerged for prp28p, sub2p, prp5p and dbp5p (1821). Dbp5p is involved in mrna export (22), the other three proteins are essential components of the pre - mrna splicing machinery (17). The dead - box protein prp28p is involved in exchanging u1 snrna with u6 snrna on the 5 splice site and has been implicated specifically in the removal of the u1 snrnp from the 5 splice site (23) (figure 1a). One of these stabilizing proteins is u1cp, and prp28 is thought to counteract the stabilizing effect of u1cp (18,26). If u1cp contains a mutation that reduces its affinity for the rna, the otherwise essential prp28p becomes dispensable, suggesting that prp28p is responsible for the removal of u1cp (18) (figure 1a). A similar bypass suppressor strategy illuminated the involvement of the dead - box protein sub2p in the displacement of the protein mud2p (19). Among other functions, which include a role on rna export (27), sub2p participates in early spliceosome assembly by promoting the exchange of the branch point - binding protein (bbp) with the u2 snrnp at the pre - mrna branch site (28) (figure 1b). The binding of bbpp to the branch site is presumably stabilized by the non - essential protein mud2p (29). Deletion of mud2 obviates the need for the essential sub2p, consistent with a role of sub2p in mud2p displacement (19). Prp5p is required for the stable addition of the u2 snrnp to the branch site, which normally depends on the atpase activity of prp5p (30). However, the interaction of the u2 snrnp with the branch site can also occur with atpase - deficient prp5p, but only if the non - essential protein cus2p is deleted (20). Thus, the essential prp5p cannot be completely bypassed by deletion of cus2p, yet the normally essential atpase activity of prp5p can be made obsolete (20), suggesting that prp5p dislodges cus2p in an atp - dependent manner (figure 1c). Recently, it has been shown that the dead - box protein dbp5p, which functions in late steps of mrna export on the cytoplasmic side of the nuclear pore complex, is also likely to be specifically required for displacement of the protein mex67 from rna (21). It was shown that in dbp5 mutant cells, the mrna export receptor mex67 accumulates on mrna, and that these mex67 bound rnas were enriched at the nuclear rim (21). The accumulation of mex67 bound rnas in dbp5 mutant cells were suppressed by a mex67 mutation, consistent with a scenario where dbp5p removes mex67 from the rna (figure 1d). Although the observations for prp28p, sub2p, prp5p and dbp5p strongly suggested the involvement of these enzymes in the removal of other proteins from rna, the mechanisms by which dexh / d enzymes caused protein displacements remained unclear (17). For example, the data obtained with prp28p, sub2p, prp5p and dbp5 did not illuminate whether dexh / d proteins rely on other cofactors or on a specific context to displace proteins, or whether dexh / d helicases alone are sufficient to dislodge other proteins . It also remained unclear whether dexh / d proteins are able to act directly on the respective rna protein interaction, or whether the enzymes displace proteins only indirectly, possibly through the remodeling of rna secondary structure . To elucidate the basic mechanism(s) of protein displacement by dexh / d proteins, it is critical to quantitatively analyze rnp remodeling reactions . Since it is unknown where precisely the vast majority of dexh / d proteins bind to their targets and which exact conformational changes the enzymes catalyze in their respective substrates (12,15), it has not yet been possible to devise model systems that recapitulate a physiological rnp remodeling reaction that can be analyzed quantitatively as well . For example, complex in vitro systems such as pre - mrna splicing extracts provide invaluable qualitative information about dexh / d protein function, but the limited control over parameters such as concentrations of individual factors precludes the use of these systems for quantitative mechanistic studies . Thus, it has been only possible to obtain quantitative mechanistic information about rnp remodeling by dexh / d proteins with simple, yet non - physiological rnp models (3133). The first rnp used for such model studies consisted of two rna strands which formed a binding site for the prototypical rna - binding protein u1a (31) (figure 2a). U1a is part of the pre - mrna splicing machinery, and it also acts as a feedback inhibitor for its own gene expression (34,35). In the model rnp, u1a forms a homo - dimer on the rna, contacting predominantly the single - stranded loops embedded in the helical regions of the rna (36) (figure 2a). It was tested whether the dexh rna helicase nph - ii could displace u1a from the rna (31). Nph - ii, which is involved in the replication of vaccinia virus (37), was selected for these protein displacement studies because a basic mechanism existed for the rna unwinding activity of this enzyme (38). Nph - ii increased the dissociation rate constant of u1a from the rna by more than three orders of magnitude in an atp - dependent fashion (31). Wait until u1a dissociated spontaneously to then unwind the u1a - binding site; rather, nph - ii actively displaced u1a (31). Further kinetic analysis of the rnp remodeling showed that u1a displacement was in fact faster than rna unwinding by nph - ii . The processivity of nph - ii was decreased, but not completely eliminated by the u1a displacement event, i.e. The enzyme could continue to unwind rna duplexes after dislodging u1a without first leaving the rna (31). These results established that dexh / d proteins could directly and actively displace stably bound proteins from rna in an atp - dependent reaction . However, observations made with this u1a - based model rnp do not preclude the requirement of other factors or a specific context for protein displacement by other dexh / d proteins . While the above results clearly showed active protein displacement by nph - ii, it remained unclear whether protein displacement required the unwinding of rna secondary structure, which in the u1a - based rnp surrounded the protein - binding site (figure 2a). To test whether dexh / d rna helicases could also displace proteins from rna without unwinding any duplexes, two model rnps were devised where rna secondary structure played no role in protein binding (32). One complex was formed between the tryptophan rna - binding attenuation protein (trap) and its specific 53-nt - long cognate rna (figure 2b). Trap binds to this rna in a sequence - specific manner as a 11-unit oligomer, and its affinity can be modulated by tryptophan (39). The second complex was the multi - component exon junction complex (ejc) that is deposited on mrnas as a consequence of splicing (40) (figure 2c). While the exact composition of the ejc is presently unclear, the complex is known to contain at least five distinct proteins that bind tightly in a non - sequence - specific manner 20 nt upstream of exon junctions (40). The ejc plays a variety of roles in postprocessing mrna metabolism, including nonsense - mediated decay and translational efficiency (41). In an atp - dependent fashion, nph - ii accelerated the dissociation rate constants for both the trap and the ejc - based complexes by several orders of magnitude (32). Thus, nph - ii actively displaced protein complexes from unstructured rna, indicating that unwinding of rna duplexes was not required for the removal of proteins from rna (32). However, while nph - ii actively disrupted both rnps, the ejc was displaced at a significantly slower rate than trap, suggesting that the properties of a given rnp might affect the rate by which it can be remodeled by dexh / d proteins (32) (table 1). The ability of a dexh / d protein to perform atp - driven conformational work on single - stranded rna is thought to resemble the movement (tracking) of the enzyme on single - stranded nucleic acids that has been observed for some sf1 dna helicases (42). A notable difference may be the inability of many dexh / d proteins to move with high processivity on the rna . Many dexh / d proteins may only be able to track one or a few steps before dissociating from the rna . The active displacement of u1a, trap and the ejc by nph - ii indicated that dexh / d proteins could efficiently disrupt rna protein interactions . However, many physiological rnps, such as the spliceosomal complexes that contain the small nuclear rnas, bind to their targets through a combination of rna rna and rna protein interactions (11). To illuminate the range of rnps which dexh / d proteins can disassemble, it was tested whether nph - ii could displace the u1snrnp, a complex that bound its target through such a combination of rna rna and multiple rna the u1snrnp is part of the eukaryotic splicing apparatus where it is involved in the recognition of the 5 splice site (43). Nph - ii accelerated the dissociation of purified u1snrnp from a substrate rna (with an authentic 5 splice site) in an atp - dependent fashion, indicating that the enzyme could actively disrupt a more complex rna protein interface (33) (figure 2d). Experiments with four simple model rnps had demonstrated the ability of the dexh / d protein nph - ii to actively displace a diverse range protein complexes from rna (table 1). It was then critical to test whether the efficient rnp remodeling seen with nph - ii was also observed for other dexh / d proteins, especially for enzymes that displayed lower atpase and rna helicase activties than nph - ii (32). Therefore, protein displacement was measured for the dead - box protein ded1 from saccharomyces cerevisiae, which is phylogenetically distant from nph - ii (32,33). Ded1 efficiently unwinds short rna duplexes (45), but the enzyme appears significantly less processive than nph - ii (e. jankowsky, et al . The rna - stimulated atpase activity of ded1 is also considerably lower than that of nph - ii (45). Nevertheless, ded1 actively displaced the ejc and the u1snrnp from their respective rnas, even with an efficiency comparable to that seen with nph - ii (table 1). However, ded1 was unable to accelerate the dissociation of u1a or trap from their target rnas (table 1). In fact, the kinetics for separation of the rna strands of the u1a - based rnp with u1a bound closely matched the kinetics of spontaneous u1a dissociation, indicating that ded1 did not actively displace u1a from the rna (33). Collectively, these data demonstrated that the ability of dexh / d proteins to actively remodel rnps is not restricted to nph - ii . Yet, different enzymes do not necessarily disrupt the same range of rnp substrates in an active fashion . To understand the functional differences between the two helicases, it will be critical to elucidate which features make an rnp susceptible to an active disruption by ded1 . Although no results have yet been reported that specifically address this question, the available data suggest that basic macroscopic physicochemical properties of the rnp such as the affinity for the rna and the dissociation rate constant are unlikely to dictate whether a given rnp complex will be actively disassembled by ded1 (table 1). However, it is worth noting that ded1 actively disrupted the two rnps that bind their target rna through multiple different proteins, whereas the enzyme failed to actively displace the two homo - oligomers u1a and trap . Perhaps the arrangement of individual protein units within an rnp may determine whether ded1 can actively disrupt a given rna to reconcile the observations made during the remodeling of the four tested rnps by both nph - ii and ded1, it is instructive to propose a basic mechanism by which dexh / d proteins actively displace proteins from rna . We emphasize that although the currently available data set is very limited with respect to the diversity of rnps and dexh / d enzymes, a mechanism needs to be consistent with a rather complex set of observations, including (i) the ability of both nph - ii and ded1 to displace proteins without unwinding rna secondary structure, (ii) the ability of nph - ii to actively displace a greater range of proteins than ded1, and (iii) the capacity of ded1 to actively disassemble the stable multi - component rnps but not the stable homo - oligomeric rnps tested (table 1). We propose that the ability of a dexh / d protein to actively disrupt a given rnp is based on the capacity of the enzyme to track on single - stranded rna in an atp - dependent fashion, which explains the ability of nph - ii and ded1 to displace proteins without unwinding rna secondary structure . During the tracking, the dexh / d protein is able to capture nucleotides that are normally part of the rna protein interface of the rnp . This reduction in the number of rna protein contacts increases the propensity of the protein(s) to dissociate from the rna (figure 3a, step 1). Additional advance of the enzyme on the rna and the sequestering of more nucleotides further reduces the number of rna protein contacts in the rnp, eventually leading to the dissociation of the rnp at a rate greater than that of spontaneous rnp dissociation, i.e. The protein is actively displaced from the rna (figure 3a, step 2). If the dexh / d protein dissociates from the rna before capturing the critical number of nucleotides necessary to accelerate dissociation of the rnp, no active protein displacement is observed (figure 3a). Therefore, a dexh / d protein tracking fast and processively (e.g. Nph - ii) should be able to actively disassemble a wider range of rnps than an enzyme that tracks slowly and/or with low (or no) processivity (e.g. Ded1). This prediction is consistent with our observations: the processive nph - ii actively displaces a greater range of proteins than the less processive ded1 . However, active protein displacement is observed even with a less processive enzyme, provided the capture of one (or few) nucleotides from the rna for example, a small decrease in the rna protein interface in a multi - component complex such as the ejc might lead to dissociation of one critical component that in turn unravels the entire rnp (figure 3b). This scenario would provide a basis to explain why ded1 actively disassembles the stable multi - component rnps but not the stable homo - oligomeric rnps tested (table 1). It is unclear how exactly the nucleotides are captured by the dexh / d proteins . Conceivably, the dexh / d helicase could exert force on the other protein to free one or several nucleotides, although it is not known whether dexh / d proteins can produce sufficient force when tracking on rna . Alternatively, the dexh / d helicase could simply sequester transiently fraying nucleotides during the tracking on rna . Helicase on single - stranded nucleic acid and capture of fraying nucleotides is also considered to be important for the unwinding of dna or rna duplexes by these enzymes (46). There, a tracking helicase is proposed to capture fraying nucleotides from the termini of the helix interface (46). It is perhaps not surprising that similar mechanisms may underlie both duplex unwinding and protein displacement by because of the limited amount of data for rnp remodeling by dexh / d proteins, other mechanisms by which these enzymes cause active protein displacement should not be discounted . For example, it may be possible that, instead of capturing one or few nucleotides at a time, dexh / d proteins may simply force the entire protein off the nucleic acid by physical clashes between protein domains . While it is unknown whether dexh / d proteins can exert sufficient force when tracking on rna, as mentioned above, it is well established that dexh / d proteins change their domain orientations upon atp binding / hydrolysis (47). Such large - scale conformational changes could be used to induce a physical clash between the dexh / d enzyme and other proteins . However, this scenario is more difficult to reconcile with the similar rate constants by which ded1 and nph - ii displace the ejc, given the different rates by which both enzymes turn over atp . Using simple rnp models to study protein displacement by dexh / d proteins has provided three major insights . First, dexh / d proteins can directly remodel rnps, i.e. The enzymes do not per se require specific cofactors that turn rna helicases into rnp remodelers . Third, not all dexh / d proteins are able to actively disassemble the same range of rnps . The proposed mechanism by which dexh / d proteins actively displace proteins from rna may present a starting point for more targeted questions about the mechanism(s) of rnp remodeling by dexh / d proteins such as defining conformational changes that dexh / d proteins induce in rna in order to displace proteins . Moreover, it will be critical to investigate the rearrangement of more diverse rnps by a larger range of dexh / d proteins . It also remains of central importance to elucidate how dexh / d proteins remodel rnps in vivo . Finally, it will be illuminating to compare mechanisms of protein displacement by dexh / d proteins with mechanisms that dna helicases use to remove proteins from dna (4850). Dexh / d proteins targeting rna protein interactions: physiological examples . (a) displacement of the u1snrnp from an rna containing a 5 splice site by prp28p . The second step shows the atp - independent binding of u2 snrnp to the branch site of the pre - mrna, which is facilitated by prp5 in an atp - independent fashion . Dbp5p is bound to the nuclear pore complex on the cytoplasmic side of the nuclear rim . As mrnas are transported through the nuclear pore, dbp5p specifically facilitates the release of mex67 . Ovals indicate u1a, lines the rna, and arrows represent the reactions catalyzed by the dexh / d protein . Rna removal of the ejc renders the region protected previously from nuclease digestion by the ejc susceptible to degradation . (d) removal of the u1 snrnp from a radiolabeled (asterisk) rna with a 5 splice site . Removal of the u1snrnp was monitored by immunoprecipitation of u1snrnp and subsequent quantification of radioactivity in supernatant and immunoprecipitate . (a) schematic representations for the displacement of a single (homopolymeric) protein from rna . (b) schematic representations for the displacement of a multi - component protein complex from rna . Remodeling of four different rnps by ded1 and nph - ii kd, equilibrium binding constant of the protein(complex) to the rna substrate . Koff, dissociation rate constant of the protein (complex) from the rna substrate . Binding site size judged from the nmr structure of the u1a rna complex (31). In addition, 30 nt in the mrna substrate are protected from digestion with microccocal nuclease (33), suggesting an u1snrnp - binding site on the mrna substrate of 30 nt . An equilibrium dissociation constant (kd) cannot be determined because the ejc is deposited on the rna during pre - mrna splicing (32). Digestion of the rna with bound ejc using micrococcal nuclease yields two bands of 8 and 10 nt (32).
Chikungunya virus (chikv) is an arthropod - borne virus belonging to the family, togaviridae, and genus, alphavirus . In this group of 29 viruses, six cause arthralgia and arthritis upon infection in humans . These six viruses are chikv, ross river virus (rrv), semliki forest virus (sfv), onyong - nyong virus (onnv), sindbis virus (sinv) and mayaro virus (mayv). The name chikungunya means that which bends up and refers to the contorted posture adopted by infected patients . Symptomatic chikv infections are characterised by fever, rashes and arthralgia leading to arthritis . In the last 50 years there have been frequent outbreaks of chikv disease in asia and africa . In 2005 - 2006 an outbreak that occurred in la runion island resulted in approximately 260,000 cases of chikv associated fever with 237 deaths reported2 . Additionally, in 2006 - 2007 around 1.5 million cases of chikv disease were reported in india3 . Through viral adaptation chikv has broadened its vector competence and therefore increased its potential to cause human disease . A single mutation, a226e in the envelope protein 1 (e1) of chikv, now allows aedes albopictus as well as ae . Currently there are no vaccines or antivirals for the prevention or treatment of chikv infections . Treatment is symptomatic with analgesics, antipyretics and non - steroidal anti - inflammatory agents . Passive immunization with human iggs derived from chikv infected patients has been shown to be protective in mice against chikv challenge5 . This approach may prove effective for the prevention and treatment of neonates who are at the risk of infection from viraemic mothers . However, this is not cost - effective and it is critical to develop antiviral drugs and/or vaccines for the control of chikv infections . Owing to the recent outbreaks of chikv in asia, africa and la runion island there has been a renewed interest in the development of antiviral drugs and vaccines to manage chikv infections . In this article, we report on the advancements made towards the development of anti - inflammatory agents, antivirals and vaccines for the efficient control of chikv infections . Chloroquine phosphate was reported to be effective in the treatment of chronic chikv arthritis6 . However, in a clinical trial of chloroquine in chikv infected patients, no difference was observed between the placebo group and the treatment group7 . Maheshwari et al8 have also reported that in a mouse model, chloroquine enhances alphavirus replication and aggravates disease pathogenesis . Ribavirin, an antiviral agent was used to treat chikv induced arthritis in a small cohort of patients and was found to be beneficial in resolving joint and soft tissue swelling9 . Briolant et al10 have reported a synergistic effect of ribavirin and interferon- in the inhibition of chikv and sfv replication in cell culture . Recently rulli et al11 have reported using bindarit, a small molecule anti - inflammatory drug, in a mouse model to effectively treat chikv induced arthritis . An additional 356 compounds (natural and clinically approved drugs) were initially screened for their antiviral effects using a chikv replicon cell line13 . A recombinant chikv with rluc (renilla luciferase) fused with nonstructural protein 3 (chikv - rluc) was constructed and used to validate the most active compounds from the initial screening studies . Out of 12 selected compounds, coumarin 30 was found to be the most potent in inhibiting the replication of chikv - rluc virus (ic50 value of 6.4 m). Arbidol, licensed for the treatment of influenza and other respiratory infections, was found to be effective in inhibiting chikv replication in vero and mrc-5 cell lines (ic50<10g / ml)14 . Recently, screening of a highly purified natural compound library for inhibition of chikv replication identified 44 inhibitors15 . Among the selected compounds, harringtone, a cephalotoxine alkaloid, was found to be a potent inhibitor of chikv infection with an ec50 value of 0.24 m . Was also found to inhibit sinv replication suggesting the drug may be effective against other alphaviruses . In another study, an optimal homology model of chikv nonstructural protein 2(nsp2) based on the active site of nsp2 model, a virtual screening was performed with commercially available compounds subsequently 26 compounds were evaluated for the anti - chikv activity in a cell culture assay16 . Levitt et al17 described the development of an attenuated strain of chikv (181/25) obtained by serial passage in mrc-5 cells . This vaccine candidate was tested in both mice and non - human primates and was found to protect the animals against challenge with parent virus . However, in phase ii trials the vaccine caused mild transient arthralgia in some of vaccinees18 . Recent attempts to develop a chikv vaccine have used formalin inactivated vaccine19, virus like particles (vlps)20 and dna vaccines2122 . Immunization with vlps and dna vaccines resulted in immunogenicity and protected the mice and non - human primates against subsequent challenge with chikv . Chimaeric viruses containing venezeulean equine encephalitis virus (veev) or eastern equine encephalitis virus (eeev) or sinv non - structural proteins with chikv structural proteins elicited neutralizing antibodies and protected the mice against chikv challenge23 . However, these viruses still had the ability to infect mosquitoes and attenuation was dependent on an intact interferon response in mice . Another chimaeric vaccine using veev strain tc-83 and encepahalomyocarditis virus (emcv) ires (internal ribosome entry site) in the subgenomic promoter was developed to reduce the transmission to mosquitoes . Recently chattopadhyay et al25 developed a chimaeric vaccine using a vesiculostomatitis virus (vsv) backbone and chikv structural proteins (vsvg - chikv). This vsvg - chikv chimaeric virus induced a good neutralizing antibody response and protected mice against chikv infection . Plante et al24 designed a vaccine by introducing the emcv ires sequence into the chikv subgenomic promoter . This reduced the replication of the vaccine strain in mosquitoes and was highly immunogenic in mice . Sirnas and shrnas against envelope protein e1, nsp3, capsid and nsp1 proteins of the chikv have been used to inhibit the replication of chikv in mammalian cells with promising results2627 . Furthermore, in vivo studies have shown that shrna e1 offers a strong and sustained protection against chikv infection in suckling mice . Studies on the role of mannose binding lectin (mbl) in rrv - induced arthritis in mice28 suggest that mbl may be a target for therapeutic intervention in the treatment of alphavirus induced arthritis / myositis . Inhibitors targeting the mbl pathway of complement may be useful in the treatment of alphavirus induced diseases . Additionally, a peptide corresponding to the autophagy inducing peptide, beclin, has been shown to be effective against chikv infections in cell culture and in mice29 . A recent study on gene profiling has revealed a significant overlap of differentially expressed genes involved in the inflammatory processes of both chikv- induced arthritis and rheumatoid arthritis (ra). Thus, new drugs being developed for ra might also be beneficial for the treatment of alphavirus induced arthritis30 . Chikungunya fever is a global health problem and several outbreaks have occurred in the last 50 years in asia and africa . Recent outbreaks in la runion and italy have raised the awareness of the need to develop effective antiviral drugs or vaccines against chikv . Although many antiviral compounds have been shown to be effective in cell culture, very few compounds have been evaluated in animal models . Intensive studies are needed to evaluate the effect of appropriate compounds in animal models and humans.
The incidence rate of cancer is increasing in developing countries, but the 5-year survival rate of cancer is also increasing, since the diagnosis and treatment of cancer has improved enormously worldwide . South korea is no exception . According to an analysis of cancer statistics in korea from 2011, the lifetime cumulative cancer incidence rates are 38.1% for men and 33.8% for women, and 5-year survival rates are 57.6% for men and 75.2% for women, for all cancers . Both incidence and survival rates have been increasing consistently over the past 10 years in korea . However, cancer is regarded as one of the most feared diseases among the general population around the world . A cancer diagnosis imposes a serious psychological burden and there is widespread belief that cancer implies with an unpleasant, painful death . Health literacy is defined as the ability to access, understand, appraise, and communicate information in order to apply health information to promote and maintain good health outcomes . More than 40% of us adults (ages 25 and older) were found to have limited health literacy . Health numeracy, referred to as quantitative literacy, is the degree to which individuals have the capacity to access, process, interpret, communicate, and act on numerical, quantitative, graphical, biostatistical, and probabilistic health information needed to make effective health decisions . Perceived risk is regarded as the subjective judgment, without foundation in objective data, of the degree of risk . Risk perception for cancer, with regard to health literacy, plays an important role in participating in preventive action . The level of health literacy or numeracy and an individual s perceived risk for cancer predict attitudes toward cancer related health behaviors and the likelihood of taking appropriate and timely healthcare action . A fear of cancer is associated with a negative attitude toward early detection and may be a barrier to participation in cancer screening . In seeming paradox, some studies have examined perceived cancer risks and related factors in europe and the united states . However, few studies have been carried out on perceptions of the incidence and survival of different and total cancers among the korean general population . The aim of this study was to determine public perceptions of lifetime cumulative incidence rates and 5-year survival rates for common cancers with disparate prognoses and to compare perceived risk with known cancer incidence and survival rates from epidemiologic data . Furthermore, we explored what factors may influence public perceptions of cancer incidence and prognoses . This study was performed as part of the survey, awareness of the quality of cancer treatment among the general population in korea between november and december 2012 . As the title indicates, this study was conducted to explore the perception of cancer incidence rates and the survivability of cancer among the general population . The nationwide health survey was conducted through face - to - face interviews at participants homes by trained interviewers from november 1, 2012 to december 1, 2012 . The survey applied a stratified probability sampling design from the south korean population using a two - stage systematic sampling method . Inclusion criteria were as follows: (1) general population aged between 40 and 70; (2) general population who had not been diagnosed with any cancer were capable of completing a questionnaire without assistance . Four thousand eight hundred and fifty - one korean adults were contacted and of these survey candidates, 2,000 people completed the survey questions (response rate, 41.2%). Socio - demographic data including age, sex, smoking history, alcohol consumption, educational level, religious status, and marital status were obtained from the questionnaires . Participants were also asked about factors that might be related to public perceptions of cancer, such as cancer worry, having cared for a family member or friend with cancer as a caregiver, and having a disease other than cancer . Cancer worry was assessed by averaging responses to four likert scale items adapted from lerman s cancer worry scale (1, not at all or rarely; 2, sometimes; 3, often; and 4, a lot or all the time). The questions addressed the frequency of cancer worry, the impact of worry on mood, the impact of worry on daily functioning, and the level of cancer concern (alpha, 0.70). The survey questionnaire assessed respondents perceived lifetime incidence rate and 5-year survival rate for cancer in general as well as for eight site - specific cancers (stomach, lung, liver, colorectal, breast [women], uterine cervix [women], thyroid [women], and prostate [men]). To assess perception of the lifetime cancer incidence rates among the general population, we asked the participants the following open - ended question: what would you estimate is the average korean person s likelihood of being diagnosed with cancer in their lifetime? Participants answered the question for each cancer using probability estimates . To assess perceptions of the 5-year cancer survival rate, we asked participants the following open - ended question: what would you estimate is the likelihood of 5-year survival after being diagnosed with cancer? The participants baseline socio - demographics are described using frequency and percentages or median values . Descriptive statistics were used to describe responses to the questions regarding incidence and survival rates . We categorized participants into three groups according to their perceptions of lifetime cancer incidence or 5-year survival rates: the accurate estimation group (participants responses in the 5% range of actual cancer incidence or survival statistics were regarded as accurate), the underestimation group (incidence and survival estimates were lower than the accurate range), or overestimation group (incidence or survival estimates higher than the accurate range). For example, when the actual survival rate of total cancer was 57.6% in men, we regarded the range of accurate estimation as 52.6%-62.6% . Associations between participants perceptions of incidence or survival and potential factors, such as cancer worry or having cared for a family member or friend with cancer as a caregiver, were analyzed using a multinomial regression model . In order to control of type 1 error in the analysis of multiple comparisions, all statistical analyses were conducted using stata ver . 12.0 (stata corp ., college station, tx). The purpose of the study was explained to all of the participants and informed consent was procured . This study was approved by the institutional review board of the national cancer center, korea this study was performed as part of the survey, awareness of the quality of cancer treatment among the general population in korea between november and december 2012 . As the title indicates, this study was conducted to explore the perception of cancer incidence rates and the survivability of cancer among the general population . The nationwide health survey was conducted through face - to - face interviews at participants homes by trained interviewers from november 1, 2012 to december 1, 2012 . The survey applied a stratified probability sampling design from the south korean population using a two - stage systematic sampling method . Inclusion criteria were as follows: (1) general population aged between 40 and 70; (2) general population who had not been diagnosed with any cancer were capable of completing a questionnaire without assistance . Four thousand eight hundred and fifty - one korean adults were contacted and of these survey candidates, 2,000 people completed the survey questions (response rate, 41.2%). Socio - demographic data including age, sex, smoking history, alcohol consumption, educational level, religious status, and marital status were obtained from the questionnaires . Participants were also asked about factors that might be related to public perceptions of cancer, such as cancer worry, having cared for a family member or friend with cancer as a caregiver, and having a disease other than cancer . Cancer worry was assessed by averaging responses to four likert scale items adapted from lerman s cancer worry scale (1, not at all or rarely; 2, sometimes; 3, often; and 4, a lot or all the time). The questions addressed the frequency of cancer worry, the impact of worry on mood, the impact of worry on daily functioning, and the level of cancer concern (alpha, 0.70). The survey questionnaire assessed respondents perceived lifetime incidence rate and 5-year survival rate for cancer in general as well as for eight site - specific cancers (stomach, lung, liver, colorectal, breast [women], uterine cervix [women], thyroid [women], and prostate [men]). To assess perception of the lifetime cancer incidence rates among the general population, we asked the participants the following open - ended question: what would you estimate is the average korean person s likelihood of being diagnosed with cancer in their lifetime? Participants answered the question for each cancer using probability estimates . To assess perceptions of the 5-year cancer survival rate, we asked participants the following open - ended question: what would you estimate is the likelihood of 5-year survival after being diagnosed with cancer? The participants baseline socio - demographics are described using frequency and percentages or median values . Descriptive statistics were used to describe responses to the questions regarding incidence and survival rates . We categorized participants into three groups according to their perceptions of lifetime cancer incidence or 5-year survival rates: the accurate estimation group (participants responses in the 5% range of actual cancer incidence or survival statistics were regarded as accurate), the underestimation group (incidence and survival estimates were lower than the accurate range), or overestimation group (incidence or survival estimates higher than the accurate range). For example, when the actual survival rate of total cancer was 57.6% in men, we regarded the range of accurate estimation as 52.6%-62.6% . Associations between participants perceptions of incidence or survival and potential factors, such as cancer worry or having cared for a family member or friend with cancer as a caregiver, were analyzed using a multinomial regression model . In order to control of type 1 error in the analysis of multiple comparisions, all statistical analyses were conducted using stata ver . 12.0 (stata corp ., college station, tx). The purpose of the study was explained to all of the participants and informed consent was procured . This study was approved by the institutional review board of the national cancer center, korea most of the participants were married, and 84% of women and 79% of men carried private cancer insurance . Approximately 40% of the men and women had a family member or friend with a history of cancer . Table 2 and fig . 1 show the perceived lifetime cumulative incidence rate of total cancer, as well as those of eight site - specific cancers in the korean general population . Widespread discrepancies were observed between the perception of probability and the actual epidemiological data regarding cumulative cancer incidence rates . The actual lifetime cumulative incidence rates of total cancer, based on an analysis of cancer statistics in south korea from 2011, are 38.1% in men and 33.8% in women . Defining accurate estimation rate as in the range of 5% of the actual incidence of total cancers (men, 33.1%-43.1%; women, 28.8%-38.8%), only 19% of men and women came close to this actual incidence rate . Korean men have a lifetime cumulative incidence rate of less than 10% for five specific cancers (stomach, lung, liver, colon, and prostate) (table 2). When we considered the accurate estimation rate in the range of 5% that focused on actual incidence of specific cancers (except 0%), most respondents overestimated lifetime cancer incidence rates (e.g., men: stomach cancer, 80.7%; lung cancer, 70.6%; and prostate cancer, 85.9%; women: stomach cancer, 93.0%; lung cancer, 88.0%; breast cancer, 82.0%; and cervical cancer, 92.8%). Korean women have a lifetime cumulative incidence rate of less than 5% for five specific cancers (stomach, lung, liver, colon, and uterine cervix), 5.3% for breast cancer, and 12.1% for thyroid cancer (table 2). When perceived rates of actual cancer incidence in the range of 5% (except 0%) were regarded as accurate, a significantly low proportion of women respondents (6%-18%) had accurate perceptions for seven specific cancers . Table 3 shows the results of multinomial regression analyses regarding factors that potentially affect public perceptions of lifetime, cancer incidence rates . For both men and women, respondents who are current drinkers were significantly more likely to provide higher estimates on incidence rates for total cancer . For men, a high score of cancer worry respondents who had a high score of cancer worry or had cared for family or a friend as a caregiver were significantly more likely to provide higher estimates of incidence rates for total cancers in women . High scores of cancer worry were associated with higher estimates of all specific cancers in male respondents . For women, the association between cancer worry scores and higher cancer incidence estimates was significant in several specific cancers such as lung cancer, liver cancer, breast cancer, and uterine and cervical cancer . 2 show the perceived 5-year survival rates of total cancer and specific cancers in the korean general population . Widespread discrepancies were observed between perceptions of survival for each cancer and actual 5-year survival rates according to cancer statistics in korea from 2011 . In cancers with high actual survival rates (stomach, colon, breast, uterine cervix, thyroid, and prostate), the majority of respondents underestimated 5-year survival rates in cancer . However about 50% of respondents overestimated 5-year survival rate in cancers with low actual survival rates (lung and liver). There was no consistent association between any single factor and respondents perceptions of 5-year survival rates for each cancer (table 5). Most of the participants were married, and 84% of women and 79% of men carried private cancer insurance . Approximately 40% of the men and women had a family member or friend with a history of cancer . Table 2 and fig . 1 show the perceived lifetime cumulative incidence rate of total cancer, as well as those of eight site - specific cancers in the korean general population . Widespread discrepancies were observed between the perception of probability and the actual epidemiological data regarding cumulative cancer incidence rates . The actual lifetime cumulative incidence rates of total cancer, based on an analysis of cancer statistics in south korea from 2011, are 38.1% in men and 33.8% in women . Defining accurate estimation rate as in the range of 5% of the actual incidence of total cancers (men, 33.1%-43.1%; women, 28.8%-38.8%), only 19% of men and women came close to this actual incidence rate . Korean men have a lifetime cumulative incidence rate of less than 10% for five specific cancers (stomach, lung, liver, colon, and prostate) (table 2). When we considered the accurate estimation rate in the range of 5% that focused on actual incidence of specific cancers (except 0%), most respondents overestimated lifetime cancer incidence rates (e.g., men: stomach cancer, 80.7%; lung cancer, 70.6%; and prostate cancer, 85.9%; women: stomach cancer, 93.0%; lung cancer, 88.0%; breast cancer, 82.0%; and cervical cancer, 92.8%). Korean women have a lifetime cumulative incidence rate of less than 5% for five specific cancers (stomach, lung, liver, colon, and uterine cervix), 5.3% for breast cancer, and 12.1% for thyroid cancer (table 2). When perceived rates of actual cancer incidence in the range of 5% (except 0%) were regarded as accurate, a significantly low proportion of women respondents (6%-18%) had accurate perceptions for seven specific cancers . Table 3 shows the results of multinomial regression analyses regarding factors that potentially affect public perceptions of lifetime, cancer incidence rates . For both men and women, respondents who are current drinkers were significantly more likely to provide higher estimates on incidence rates for total cancer . For men, a high score of cancer worry respondents who had a high score of cancer worry or had cared for family or a friend as a caregiver were significantly more likely to provide higher estimates of incidence rates for total cancers in women . High scores of cancer worry were associated with higher estimates of all specific cancers in male respondents . For women, the association between cancer worry scores and higher cancer incidence estimates was significant in several specific cancers such as lung cancer, liver cancer, breast cancer, and uterine and cervical cancer . Table 4 and fig . 2 show the perceived 5-year survival rates of total cancer and specific cancers in the korean general population . Widespread discrepancies were observed between perceptions of survival for each cancer and actual 5-year survival rates according to cancer statistics in korea from 2011 . In cancers with high actual survival rates (stomach, colon, breast, uterine cervix, thyroid, and prostate), the majority of respondents underestimated 5-year survival rates in cancer . However about 50% of respondents overestimated 5-year survival rate in cancers with low actual survival rates (lung and liver). There was no consistent association between any single factor and respondents perceptions of 5-year survival rates for each cancer (table 5). To our knowledge, this is the first study to assess the perception of incidence and survival rates of eight common cancers and total cancer in the korean general population . The use of numerical ratings of probability for cancer incidence and survival rates has the advantage of making it possible to compare the values with a true rate . In addition, this study investigated perceived risks in relation to common cancer sites, thereby enabling comparisons across the various types of specific cancers . Previous research found that 65% of women overestimated the incidence of breast cancer in australia, and 66% of german women inaccurately estimated breast cancer incidence . Our findings were consistent with these previous studies . Lifetime cumulative incidence rate of total cancer should be the sum of incidence rates of each cancer . However, our results showed that the sum of the perceived incidence rates for each cancer was higher than that of total cancer . Furthermore, there was not a significant disparity between perceived incidence rates of each cancer and that of total cancer . Our results suggested a high prevalence of low health literacy regarding the meaning of each cancer and low numeracy in terms of cancer incidence among the korean population . In our study, high estimates of incidence rates in total cancer, as well as the eight specific cancers, were significantly associated with high scores on cancer worry . Fear of cancer is one of the factors that can lead to delays in seeking medical treatment and health promotion activities . Providing people with accurate information about cancer can help them to accurately assess their cancer risk and reduce excessive worry and anxiety . The majority of both men and women tend to underestimate 5-year survival rates in cancers with relatively high actual survival rates such as cancers of the stomach, colon, breast, uterine cervix, thyroid, and prostate . Half of the respondents overestimated 5-year survival rate in cancers with relatively low actual survival rates, such as lung and liver cancer . A british population - based research study showed a tendency to underestimate breast cancer survival and overestimate lung cancer survival . Perceived survival rates for lung cancer and leukemia, which have low actual survival rates, were overestimated in australian adults . This may be associated with a tendency to avoid extremes on rating scales, and because the general population did not distinguish among cancers in terms of outcomes . In our study, we could not find any factor with a consistent correlation with the respondents perceptions of 5-year survival rates for each cancer . Research from japan showed that older respondents significantly overestimate 5-year survival rates for some cancers (e.g., thyroid, breast, cervix, prostate, colorectal, and stomach). Female respondents overestimate survival rates for other types (e.g., prostate and cervix), and a history of having a family member or friend with cancer was associated with survival rates for still other types (e.g., breast, prostate, cervix, colorectal, and stomach). However, consistent with our study, the pattern of association was not uniform across cancer types . While we also found that several factors are associated with underestimation or overestimation of cancer survival in total and specific cancers (e.g., have cared for family or a friend with cancer as a caregiver or have a high level cancer worry), they were not consistent across cancer types . An often - overlooked problem in all areas of cancer communication is health literacy and numeracy . Patients are frequently assumed to have difficulty understanding health statistics such as numeric estimates of risk . Excessive worry about cancer risks or prognosis can lead to unhealthy behaviors such as avoidance of cancer screenings . Thus, it is important to reiterate that appropriate risk communication and patients education on health information is needed to increase health literacy and reduce cancer worry . This study investigated widespread discrepancies between public perceptions and known cancer risks regarding lifetime incidence and survival rates . It is our belief that the findings of our study are important to physicians, particularly for communicating cancer risk and prognoses to the public and to patients . More efforts in risk communication and education on understanding medical information should be made to reduce these discrepancies . Such efforts would contribute to reasonable decision - making in cancer prevention and treatment and eliminate unnecessary fear of cancer.
Metachromatic leukodystrophy (mld) is a severe neuro - metabolic disorder caused by a deficiency in the lysosomal enzyme arylsulfatase a which catalyzes degradation of 3-o - sulfogalactosyl - ceramide, an essential and abundant component of myelin.1 in mld, the storage of sulfatides in the oligodendrocyte and schwann cells causes progressive demyelination in the central and peripheral nervous systems.2 due to the pervasive nature of this disease, metabolic alterations in mld may go beyond sulfatide accumulation and involve other metabolites . Thus far, there have been limited studies of h - magnetic resonance spectroscopy (mrs) of the brain in mld . Bizzi et al implemented a long echo - time (te) h - mrs in 70 patients in an attempt to classify the patterns of metabolite abnormalities in various types of leukodystrophies.3 kruse et al studied seven cases of mld using short te h - mrs and found a marked decrease in the neuronal marker n - acetyl aspartate (naa), and elevated myo - inositol (mi) levels.4 multi - voxel h - mrs can be used to examine large areas of the brain and therefore is an efficient tool for researching mld, which causes diffuse cerebral changes without any focal lesions . Previous studies have implemented short te h - mrs studies, which can be used to evaluate many different brain metabolites . Although long te studies are limited in the type of spectra that can be acquired, they do improve the spectral resolution for certain metabolites . As such, we studied metabolite abnormalities in mld using multi - voxel long te h - mrs of the brain . Four patients with clinical, laboratory, and genetic confirmation of mld were recruited into a recently published treatment trial.5 all patients had the late infantile form of mld and were advanced in their disease . Long te multi - voxel h - mrs was implemented to measure changes in selected metabolites without contamination by lipid signals . Studies were conducted with a siemens magnetom avanto 1.5 tesla mri system (munich, germany). An area of interest measuring 8 8 1.5 cm was marked in the center of the brain and included the bilateral basal ganglia, thalami, white matter, ventricles, and parts of the cortex (fig . 1). Using a spin - echo (press) sequence with tr / te of 1700/135 msec and a spectral width of 1 khz, 1024 complex data points and 4 averages were acquired . Two - dimensional phase- encoding (16 16 matrix, 16.0 16.0 cm fov) with a slice thickness of 1.5 cm was utilized to produce consecutive voxels measuring 1.5 cm each throughout the area of interest . Spectral peak areas were analyzed using the lcmodel program installed on a linux - based 3.8 ghz intel - xeon image server and analysis workstation.6 multi - voxel h - mrs was used to measure metabolite ratios of naa, mi, choline (cho), and lactate (lac) to creatine (cr) in consecutive voxels in the area of interest . This method does not provide accurate metabolite concentrations, although the metabolites can be reported as ratios to cr . All data were compared to age - matched control subjects who had been previously studied in our lab . Figure 2 is an example illustration of the spectral map acquired for one of the patients . Figure 3 demonstrates a sample of the spectra acquired from subject 1 at the level of the peri - ventricular white matter . The highest lac / cr ratios were noted in the peri - ventricular region as illustrated in figure 4 . In order to better understand the value of this finding, we compared the ratios to those of four age - matched controls previously studied in our lab . These were developmentally normal children who had been evaluated for headaches (control subjects 1 and 2) or seizures (control subjects 3 and 4). Figure 5 demonstrates the mean (calculated in 3 voxels) of the lac / cr ratios in the subjects and the age - matched controls at the level of the peri - ventricular matter . The mean of the lac / cr ratios in mld cases was 0.42 compared to 0.11 in the control subjects . Statistical analysis confirmed a significant difference between the mean values (t - test, p <0.01). Table 2 shows the acquired naa and mi metabolite ratios from selected voxels in the four mld cases and the age - matched control subjects . The naa / cr ratios were significantly reduced in the white matter (t - test, p <0.0001) and the bg (t - test, p <0.0001) in the mld patients compared to controls . In contrast, the mi / cr ratios were markedly elevated in the bg (t - test, p <0.01) and the white matter (t - test, p <0.0001) in our subjects compared to normal controls . We also noted a slight elevation in the cho / cr ratio, with peak levels at the basal ganglia as shown in figure 6 . The energy metabolism in the human brain has not been fully elucidated . In the past, direct absorption of glucose from the blood was considered to be the sole source of energy for the brain . In 1994, however, pellerin and magistretti710 introduced the concept of astrocyte - neuron metabolite trafficking . According to this model, which has been verified by additional studies,11,12 glucose crosses the blood - brain barrier, entering the astrocytes to produce lactate via glycolysis . Subsequently, lactate is transported to the neurons where it enters the krebs cycle in order to generate atp . Glycolysis, and therefore lactate production, is enhanced by synaptic activation, which necessitates the reuptake of glutamate by astrocytes . This phenomenon is attributed to the fact that glutamate reuptake is an energy - dependent process and utilizes atp . This study demonstrates a mild diffuse elevation of the lac / cr ratios documented on multi - voxel h - mrs in mld patients which was statistically significant when compared to the controls . Bizzi et al3 reported elevated lactate levels using single - voxel h - mrs of children with various types of leukodystrophies . They used long tes of 136 msec and successfully produced inverted lactate peaks which were easily distinguished from lipid signals . In their small mld case series, kruse et al also reported elevated lactate levels in seven mld patients using h - mrs with short tes of 20 msec . The major limitation of their study was use the of short tes, which is of limited utility in measuring lactate due to contamination caused by lipid signals . However, long tes are not ideal for measuring other metabolites such as naa and mi . To resolve this issue, we implemented a combination of long tes and lcmodel, enabling measurement of these metabolites . Oligodendrocytes are known to support the axons through multiple mechanisms independent of myelination,13 including their contribution to axonal energy supply . The most abundant lactate transporter, monocarboxylate transporter 1, is known to be highly expressed in oligodendrocytes . Oligodendrocyte injury, which is well - documented in leukodystrophies, limits lactate transport into axons, which contributes to accumulation of this metabolite . In contrast, astrocytic gliosis in mld causes an increase in lactate production as a result of increased cell mass . Conceivably, lactate uptake by the neuronal mitochondria is compromised in mld due to progressive neuronal loss . It can be concluded that the elevation in the cerebral lactate is caused by a combination of increased production due to astrocytic gliosis, decreased transportation due to oligodendrocyte injury, and diminished utilization due to neuronal loss . In addition to mld, elevated mi levels have been described previously in alzheimer s dementia.14 this metabolite is generated by astrocytes and therefore an elevated mi level has been attributed to the histopathological finding of reactive gliosis characteristic of mld and many other neurodegenerative diseases.4 the elevation in the cho / cr ratio reflects a high myelin turnover and has been reported by several studies for mld and other leukodystrophies . The decrease in the naa / cr ratio is related to diffuse neuronal loss and, therefore, is not unexpected in mld.4 dali et al15 recoded h - mrs of brain in 13 cases of late infantile mld using intermediate te of 99 msec . They reported a significant decrease in naa levels, which strongly correlated with motor function, suggesting that naa may be a useful marker for disease progression in mld . The results of this study validate our previous observations on short te single - voxel h - mrs in the same subjects,5 showing low naa / cr and high mi / cr ratios . The major limitation of our study is the small sample size, caused by the scarcity of mld cases . Fortunately, we were able to compare our results to those of four age - matched controls and demonstrated statistical significance . We propose that elevated lac / cr and mi / cr ratios are both attributable to the astrocytic gliosis characteristic of mld . These markers, along with decreased naa / cr, are potentially useful for monitoring disease progression in mld . To further confirm these findings, longitudinal studies of mld patients over 12 years are necessary.
Radiculopathy and myelopathy from degenerative, inflammatory, and traumatic processes have been successfully treated with anterior cervical discectomy and fusion (acdf). Despite clinical success with fusion treatment, there are concerns regarding the long - term effects of fusion on the cervical spine . . Reported on the incidence of radiculopathy and myelopathy at adjacent segment to a cervical fusion and found that 25% of acdf patients will have symptomatic adjacent segment deterioration (asd) within 10 years of acdf at a rate of 2.9% each year . It is believed that although some changes in asd occur naturally, the effects of fusion disturb the biomechanics, most likely increasing the incidence of asd . In vitro studies demonstrated that intradiscal pressures, shear strains, and motion increased in upper and lower segments adjacent to fusion levels [47]. Recently, artificial cervical disc replacement (acdr) has become the alternative to fusion, with the potential to preserve the motion of the instrumented level and to prevent overload of the adjacent levels and subsequent asd . Acdr has been proven to be beneficial in terms of avoiding the deleterious effects of fusion . However, indications for acdr are more stringent and hypermobility of the operative levels may occur, which lead to the limitation of multilevel acdr . Clinical studies regarding hybrid combinations of fusion and non - fusion have been reported, with improved total motion and earlier recovery and return to work [1215]. Biomechanical studies of 2-level hybrid acdf and acdr have demonstrated advantages of the hybrid in reducing compensatory adjacent motion and reduced internal stresses in the construct [4,1621]. In a clinical study, kang el al . Reported that the hybrid construct is a safe and effective alternative for 3-level cervical disk disease . The hypothesis was that the motion response of disc replacement adjacent to fusion was comparable to a stand - alone disc replacement, and the non - operated segments in a hybrid construct would experience significantly less motion than with a fusion . This study is a progression to 3-level from the 1- and 2-level biomechanical studies because 3 levels of fusion are common and hybrid combinations either performed initially or for revision of asd are more common today . The objective of the present study was to compare the 3-level acdf to combinations of acdr and acdf with displacement controlled kinematics at instrumented and adjacent levels . Eighteen fresh adult human cadaveric cervical spines (c2-t1; age range, 5273 years) were used for biomechanical testing . All cervical spines were evaluated for bone mineral density (bmd) using dual - energy x - ray absorptiometry scanning and measured bmd values ranged from 0.53 to 0.72 g / cm . Cervical spines with degenerative diseases or traumatic pathology were excluded by anteroposterior and lateral screening radiographs before biomechanical testing . Once harvested, all cervical specimens were immediately conserved in plastic bags and frozen at 20c . In preparation for biomechanical testing, the required spines were thawed at 4c for 12 hours . At room temperature on the testing day, the proximal vertebra (c2) and distal vertebra (t1) were separately mounted in a cylindrical container using a low - fusion point (72c) alloy . And then, the c2 container was attached to the upper fixture while the t1 container was mounted to the lower testing platform . Markers made of 4 plexiglas motion detectors were fixed to the anterior aspects of each vertebra from c2 to t1 . The 3-dimensional motion range of each vertebra was obtained with an optoelectronics measurement system (optotrak, northern digital inc ., waterloo, ontario, canada) capable of capturing the motion curve of the markers . Biomechanical tests were performed under displacement control by an mts machine (cmt6104; mts systems (china) corp ., shenzhen, china) which can replicate physiologic motion with displacement control (figure 1). The flexion extension axis of each tested spine was placed eccentric to the load axis of the actuator . The bending moments were limited to the upper bound of physiological human bending (4.5 nm). An angular displacement transducer was assembled to measure the global rotation of the cervical spine (c2-t1). A displacement transducer was used to measure the changes in moment arm length between the upper container and the load axis of the mts machine . The spine specimens were tested by application of a 50 n preload in flexion - extension and lateral bending circumstances . During the biomechanical tests, after analysis of the intact spine, each specimen was sequentially reconstructed at c3-c6 3-level) motion segments . The conditions were as follows (figure 2): acdr, acdf, acdr, or 3-level disc plate disc (3dpd); acdf, acdr, acdf or 3-level plate disc plate (3pdp); three - level acdf or 3-level plate (3p). Prior to biomechanical tests, measurements included vertebral motion, applied load, and moment . In the biomechanical tests, the acdr was a titanium - ceramic alloy cervical disc (prestige lp cervical disc, medtronic sofamor danek usa, inc . ). The arthrodesis was performed using an interbody cage (telamon tm, medtronic sofamor danek usa, inc .) Combined with an anterior cervical plating (acp) system (doc cervical plate, depuy spine, inc ., raynham, ma, usa) (figure 3). This biomechanical protocol limited motion to 20 degrees in flexion and extension as well as 15 degrees in lateral bending and axial rotation . The ratio of segmental rom to the total c2-t1 rom was applied to evaluate the operation effect by normalization method . One - way anova (p<0.05) was used to determine the statistical differences in both c3-c6 motion and adjacent level motion under the 3-level anterior plate fusion (3p), the 3pdp, and 3dpd conditions . Eighteen fresh adult human cadaveric cervical spines (c2-t1; age range, 5273 years) were used for biomechanical testing . All cervical spines were evaluated for bone mineral density (bmd) using dual - energy x - ray absorptiometry scanning and measured bmd values ranged from 0.53 to 0.72 g / cm . Cervical spines with degenerative diseases or traumatic pathology were excluded by anteroposterior and lateral screening radiographs before biomechanical testing . Once harvested, all cervical specimens were immediately conserved in plastic bags and frozen at 20c . In preparation for biomechanical testing, the required spines were thawed at 4c for 12 hours . At room temperature on the testing day, the proximal vertebra (c2) and distal vertebra (t1) were separately mounted in a cylindrical container using a low - fusion point (72c) alloy . And then, the c2 container was attached to the upper fixture while the t1 container was mounted to the lower testing platform . Markers made of 4 plexiglas motion detectors were fixed to the anterior aspects of each vertebra from c2 to t1 . The 3-dimensional motion range of each vertebra was obtained with an optoelectronics measurement system (optotrak, northern digital inc ., waterloo, ontario, canada) capable of capturing the motion curve of the markers . Biomechanical tests were performed under displacement control by an mts machine (cmt6104; mts systems (china) corp ., shenzhen, china) which can replicate physiologic motion with displacement control (figure 1). The flexion extension axis of each tested spine was placed eccentric to the load axis of the actuator . The bending moments were limited to the upper bound of physiological human bending (4.5 nm). An angular displacement transducer was assembled to measure the global rotation of the cervical spine (c2-t1). A displacement transducer was used to measure the changes in moment arm length between the upper container and the load axis of the mts machine . The spine specimens were tested by application of a 50 n preload in flexion - extension and lateral bending circumstances . During the biomechanical tests, after analysis of the intact spine, each specimen was sequentially reconstructed at c3-c6 3-level) motion segments . The conditions were as follows (figure 2): acdr, acdf, acdr, or 3-level disc plate disc (3dpd); acdf, acdr, acdf or 3-level plate disc plate (3pdp); three - level acdf or 3-level plate (3p). Prior to biomechanical tests, positioning of implants measurements included vertebral motion, applied load, and moment . In the biomechanical tests, the acdr was a titanium - ceramic alloy cervical disc (prestige lp cervical disc, medtronic sofamor danek usa, inc . ). The arthrodesis was performed using an interbody cage (telamon tm, medtronic sofamor danek usa, inc .) Combined with an anterior cervical plating (acp) system (doc cervical plate, depuy spine, inc ., raynham, ma, usa) (figure 3). This biomechanical protocol limited motion to 20 degrees in flexion and extension as well as 15 degrees in lateral bending and axial rotation . The ratio of segmental rom to the total c2-t1 rom was applied to evaluate the operation effect by normalization method . One - way anova (p<0.05) was used to determine the statistical differences in both c3-c6 motion and adjacent level motion under the 3-level anterior plate fusion (3p), the 3pdp, and 3dpd conditions . As expected, 3-level arthrodesis resulted in significant reduction of rom at the three instrumented levels in all 6 loading conditions (flexion, extension, left bending, right bending, left rotation, and right rotation). Compared to intact spines, almost 80% of motion was successfully restricted at c3-c6 fusion levels in flexion, extension, and lateral bending, as well as 65% in axial rotation . For hybrid constructs, 3dpd construct resulted in slight increase at the 3 instrumented levels in extension, lateral bending, and axial rotation compared to intact (p>0.05; maximal variation of + 7%). However, the 3dpd condition resulted in a slight decrease at c3-c6 in flexion (p>0.05; maximal variation of 9%). As another 3-level hybrid construct, 3pdp construct resulted in significant decrease of rom at the c3-c6 instrumented levels in all 6 loading conditions except for left rotation (mean variation of 21%; maximal variation of 38%). Although 3pdp and 3p conditions produced significant motion decrease at the 3 instrumented levels, there was significant difference within the instrumented levels between 3pdp and 3p conditions in all 6 loading conditions (p<0.05). On the other hand, there were also significant differences within the c3c6 levels between 3dp d and 3pdp conditions in all 6 loading conditions except for flexion and left rotation (p<0.05) (figures 4, 5). For each instrumented level, 3dpd and 3pdp hybrid constructs caused significant reduction of rom in all 6 loading conditions at the arthrodesis level compared to intact (p<0.05) and produced motion increase at the arthroplasty level . For the 3dpd hybrid construct, implantation of upper - level (c3-c4) acdr resulted in significant increase of rom only in right rotation (p<0.05; maximal variation of + 36%), while implantation of lower - level (c5-c6) acdr resulted in significant increase of rom in all 6 loading conditions except for flexion (p<0.05; maximal variation of + 41%). For 3pdp hybrid construct, implantation of middle - level (c3-c4) acdr resulted in significant increase of rom in extension, left rotation, and right rotation compared to intact (p<0.05; maximal variation of + 67%) but produced motion increase in flexion, left bending, and right bending, without significant difference (p>0.05) (figure 6, table 1). As suspected, 3-level arthrodesis resulted in an increased contribution of upper and lower adjacent levels . Significant changes were noted at the lower adjacent level in all 6 loading conditions as well as upper adjacent level in flexion and left bending (p<0.05; maximal variation of + 197%). Concerning 3dpd hybrid construct, significant changes of motion increase were only noted at lower adjacent level in left bending, right bending, and right rotation (p<0.05; maximal variation of + 57%). Importantly, there was a significant decrease toward normal in adjacent segment motion adjacent to an acdr . Concerning 3pdp hybrid construct, significant changes of motion increase were noted at both upper and lower adjacent levels and the largest motion increase was noticed at lower adjacent level in extension (p<0.05; maximal variation of + 79%) (figures 4, 5). As expected, 3-level arthrodesis resulted in significant reduction of rom at the three instrumented levels in all 6 loading conditions (flexion, extension, left bending, right bending, left rotation, and right rotation). Compared to intact spines, almost 80% of motion was successfully restricted at c3-c6 fusion levels in flexion, extension, and lateral bending, as well as 65% in axial rotation . For hybrid constructs, 3dpd construct resulted in slight increase at the 3 instrumented levels in extension, lateral bending, and axial rotation compared to intact (p>0.05; maximal variation of + 7%). However, the 3dpd condition resulted in a slight decrease at c3-c6 in flexion (p>0.05; maximal variation of 9%). As another 3-level hybrid construct, 3pdp construct resulted in significant decrease of rom at the c3-c6 instrumented levels in all 6 loading conditions except for left rotation (mean variation of 21%; maximal variation of 38%). Although 3pdp and 3p conditions produced significant motion decrease at the 3 instrumented levels, there was significant difference within the instrumented levels between 3pdp and 3p conditions in all 6 loading conditions (p<0.05). On the other hand, there were also significant differences within the c3c6 levels between 3dp d and 3pdp conditions in all 6 loading conditions except for flexion and left rotation (p<0.05) (figures 4, 5). For each instrumented level, 3dpd and 3pdp hybrid constructs caused significant reduction of rom in all 6 loading conditions at the arthrodesis level compared to intact (p<0.05) and produced motion increase at the arthroplasty level . For the 3dpd hybrid construct, implantation of upper - level (c3-c4) acdr resulted in significant increase of rom only in right rotation (p<0.05; maximal variation of + 36%), while implantation of lower - level (c5-c6) acdr resulted in significant increase of rom in all 6 loading conditions except for flexion (p<0.05; maximal variation of + 41%). For 3pdp hybrid construct, implantation of middle - level (c3-c4) acdr resulted in significant increase of rom in extension, left rotation, and right rotation compared to intact (p<0.05; maximal variation of + 67%) but produced motion increase in flexion, left bending, and right bending, without significant difference (p>0.05) (figure 6, table 1). As suspected, 3-level arthrodesis resulted in an increased contribution of upper and lower adjacent levels . Significant changes were noted at the lower adjacent level in all 6 loading conditions as well as upper adjacent level in flexion and left bending (p<0.05; maximal variation of + 197%). Concerning 3dpd hybrid construct, significant changes of motion increase were only noted at lower adjacent level in left bending, right bending, and right rotation (p<0.05; maximal variation of + 57%). Importantly, there was a significant decrease toward normal in adjacent segment motion adjacent to an acdr . Concerning 3pdp hybrid construct, significant changes of motion increase were noted at both upper and lower adjacent levels and the largest motion increase was noticed at lower adjacent level in extension (p<0.05; maximal variation of + 79%) (figures 4, 5). This study demonstrated that the motion response of an acdr adjacent to a fusion maintains normal motion at the acdr level and normalizes adjacent segment motion in 3-level hybrid constructs . In longer fusions, the intradiscal pressures and the compensatory hyper - mobility this study confirms the work of lee et al . And others [4,1321] that a longer fusion affects all adjacent levels with hyper - mobility . This study shows that an acdr normalized the adjacent level motion in a 3-level construct . Acdr is the accepted alternative to anterior cervical fusion for single - level disc disease . Favorable outcomes and the prospect of a lower incidence of adjacent - level disease have encouraged surgeons to expand current acdr indications to multilevel disc disease . However, the evidence in multilevel acdr is not as well established as its role in single - level disease, and some levels may be too degenerative for acdr . With that in mind, authors are reporting combinations of fusion and arthroplasty as an alternative to multi - level acdf or acdr . Described 2-level, 3-level, and 4-level hybrid surgery results as safe and reliable without revision . Shin el al . Compared hybrid construct to acdf in 2 levels, with improved ndi, pain, return of motion, and reduced asd . Kang el al . Confirmed these findings in 3-level hybrid treatments, with improvement in recovery of total motion and maintenance of adjacent segment motion . Jia et al ., in a systematic review of 8 biomechanical and 7 clinical papers, found a paucity of quality evidence to support hybrid surgeries and recommended prospective randomized trials . Previous biomechanical studies have investigated the operative- and adjacent - level kinematic properties of 2-level acdf and combined acdr / acdf . Acdf plus acdr had less severe biomechanical effects on adjacent levels when compared to 2-level acdf procedure . Reported that the acdf at c6-c7 and acdr at c5-c6 produced increased segmental motion at the arthroplasty level, particularly in axial rotation and flexion - extension . Barrey et al . Analyzed the biomechanics of acdr placed above acdf and found similar kinematics to single - level acdr and adjacent to a previously implanted acdr . Compared the biomechanics of 2-level acdf and hybrid; the hybrid construct had a better biomechanical performance than the fusion and the hybrid avoided excessive increase of adjacent level motion and loads . . Found that the location of the fusion (cephalad or caudad) did not affect the behavior of the disc replacement . Biomechanical studies investigating the kinematic properties of 3-level hybrid arthroplasty - arthrodesis reconstruction are limited . However, multilevel cervical surgeries, such as 3-level anterior surgeries, are more common in clinical practice . Hanai et al . Reported that there was excellent clinical success and a 100% union rate for 3-level and 4-level cervical corpectomy and autograft strut graft reconstruction . . Revealed that the likelihood of pseudarthrosis was 10% for 1-level surgery, 44% for 2-level surgery, and 54% for 3-level surgery . One study investigated acdr as a promising treatment for symptomatic adjacent level after prior 2-level cervical fusion . Characterized acdr kinematics above 2-level fusion and found that acdr placed adjacent to a 2-level fusion was subjected to a more challenging biomechanical environment as compared to a stand - alone acdr . Two groups reported both 2-level and 3-level hybrid surgeries are comparable to acdf and acdr in terms of safety and feasibility with a minimum follow - up of 2 years . . Stated that hybrid surgeries may be an alternative to acdf for 3-level cervical disease due to the equivalent or improved early clinical outcomes, better overall c2-c7 range of motion, and less impact at adjacent levels . We found that rom in instrumented levels after 3p condition was systematically reduced in all planes . In contrast, the 3dpd condition appeared to preserve motion at adjacent intact levels to the acdr segments . Compared to 3dpd condition, rom in instrumented levels after 3pdp condition was slightly reduced but the difference was not significant . In addition, we did not observe an abnormal increase of motion for 3dpd and 3pdp conditions . At the adjacent levels, 3p condition resulted in increased adjacent segment motion, especially in the caudal adjacent segment, which may potentially result in accelerated adjacent segment degeneration . On the contrary, and as expected, although 3dpd and 3pdp conditions did not fully restore motion within the instrumented levels, they did not demonstrate hyper - mobility, and in fact induced only minimal changes in rom at adjacent levels . This confirmed the finding that the hybrid construct could avoid large motion increase in adjacent levels and clinically may reduce the risk of adjacent segment degeneration . The 3dpd construct did produce a smaller motion change in adjacent segments compared with 3pdp construct . This study has the limitations of any cadaveric biomechanical study of cervical fusion in that it allows only observation of the immediate effects of the intervention . It did not remove all motion in the fusion segments and cannot represent the long - term effect of increasing stiffness as the fusion progresses . These biomechanical findings suggest that while hybrid conditions failed to restore normal motion within the construct, they significantly normalized motion in adjacent segments compared with the 3-level acdf condition . The artificial disc in 3-level constructs has biomechanical advantages compared to fusion in normalizing motion.
Since the first successful electroconvulsive therapy (ect) treatment course in 1938, ect been used for a variety of psychiatric disorders, and its use for the treatment of schizophrenia spread from the 1940s through the 1950s . When antipsychotic medication was introduced in the 1950s, the use of ect for schizophrenia declined.1 however, due to resistance and intolerance to pharmacotherapy in some patients, ect can be beneficial2 and therefore ect for schizophrenia is still being performed worldwide.3,4 several national guidelines suggest the use of ect for schizophrenia in particular circumstances.5 the american psychiatric association (apa)6 suggests that ect is effective for psychotic exacerbations in schizophrenic patients, when illness is of the catatonic type, when the psychotic symptoms are abrupt or recent in onset, or when there is a past history of favorable response to ect and ect is effective for psychotic disorders related to schizophrenia, that is, schizophreniform disorders and schizoaffective disorders . Although ect is an effective acute treatment for patients with severe symptoms, one important problem is the high relapse rate . Even with antipsychotic therapy, relapse rates were> 60% at 1 year after ect in schizophrenic patients.7,8 there are only a few studies reporting the factors that predict a relapse after an initial response to ect in schizophrenia . A high neuroleptic dose before acute ect8 and the observation of self - harming behaviors at baseline7 seem to enhance the risk of relapse . Therefore, additional information on the factors associated with relapse is needed in order to determine the optimal prophylaxis for such cases during the stable phase . We investigated the factors that influence the risk of relapse in schizophrenic patients after demonstrating a response to ect . The inclusion criteria for the study were patients who 1) were diagnosed with schizophrenia based on the international classification of diseases (icd)-10 guideline by at least two trained psychiatrists, 2) received an acute ect course between october 2005 and september 2011, regardless of having received an acute course of ect prior to october 2005, at the national hospital organization (nho) kure medical center, and 3) responded to the acute ect course . The exclusion criteria were patients who 1) were diagnosed with schizoaffective disorders or persistent delusional disorders, 2) did not respond to the acute ect course, and 3) received continuous or maintenance (c / m) ect, as the purpose of this study was to identify risk factors of relapse after acute ect, not after c / m ect . In the current study, we evaluated the responsiveness to acute ect using the clinical global impressions improvement (cgi - i) scale score, as follows: 0= not assessed; 1= very much improved; 2= much improved; 3= minimally improved; 4= no change; 5= minimally worse; 6= much worse; and 7= very much worse . Responders to the acute ect course were defined as patients with a cgi - i scale score 3 after acute ect, and nonresponders were defined as those with a cgi - i score 4 . If two or more acute ect courses were received by the same patient during the study period (20052011), the observation period was defined as the time since completing the first acute ect course . The data used for the study were obtained from medical records . The data collected included demographic variables, clinical variables, and maintenance psychotropic - related variables . The demographic and clinical variables included the age, sex, subtype of schizophrenia, age of onset of illness, number of psychotic episodes (including current episode), number of ect sessions, score on the brief psychiatric rating scale (bprs) before and after ect, and chlorpromazine (cpz) equivalence before ect . In this study, the evaluation of the symptom was carried out by a trained psychiatrist, different from those who diagnosed schizophrenia . The maintenance psychotropic - related variables included the classification of antipsychotics, cpz equivalence, and use of additional medication (mood stabilizers [lithium carbonate, sodium valproate, carbamazepine, and lamotrigine], antidepressants, benzodiazepines, and anticholinergics). The daily doses were calculated by converting the antidepressant, benzodiazepine, and anticholinergic doses to imipramine, diazepam, and biperiden equivalents, respectively . The kind of antipsychotic or the daily doses in some patients were changed because of worsening of their condition, but we calculated the kind and cpz equivalence at the time of discharge, not at the time of relapse, because there were many cases that met the criteria of relapse just after the change of medication . When two or more antipsychotic drugs were used for a single patient, the main antipsychotic drug was defined as the one with the highest cpz - equivalent dose . The classification of antipsychotics into the first - generation antipsychotic (fga) or second - generation antipsychotic (sga) category was conducted according to the literature.9 we adhered to ethical considerations when gathering data so that individuals could not be identified . The ethics committees of our centers approved this retrospective study . Only modified ect with the cooperation of an anesthesiologist was used . Without premedication, patients received intravenous thiamylal sodium (23 mg / kg) and suxamethonium chloride (0.51.0 mg / kg) for anesthesia . The ect device used was the thymatron system iv brief - pulse square - wave apparatus (somatics inc ., lake bluff, il, usa). Only one adequate seizure was required for each session, which was defined as an electroencephalographic seizure lasting> 25 seconds with a high - amplitude, slow - wave and postictal suppression . The initial stimulus dose was determined using the half - age method.10 if an adequate electroencephalographic seizure occurred in one session, the stimulus energy of the next session remained the same . When a missed or an inadequate seizure occurred, the patient was restimulated with 1.52 times the stimulus energy . If any adverse effects (eg, cognitive dysfunction, delirium) occurred, the frequency of the ect schedule was reduced to once or twice per week, or the ect was stopped . On the basis of the apa guidelines, ect was continued until the patient was asymptomatic or when the psychiatrist judged that the patient had benefited as much as possible.6 all patients were treated with antipsychotics during the ect course . After the purpose and procedure of ect was fully explained, written informed consent was obtained from the patients or their family members prior to acute ect . All patients were treated with neuroleptic medication to prevent relapse after the acute ect course . The attending psychiatrist selected the type and dosage of antipsychotics and additional medication on a case - by - case basis . For antipsychotics, sgas were mainly used, while an fga was selected when the effects of sgas were insufficient . In the current study, the time to relapse was defined as the time between ect and the date of an evaluation at which patients had a cgi - i score 6 or if they required psychiatric rehospitalization . The starting point was the last day of the acute ect course during the period, and the end point was the day of relapse . The patients were censored if they moved out of the hospital s recruitment area, died, or had not been readmitted as of september 2012 . Cox regression analyses were used to identify factors associated with relapse via univariate and multivariate analyses . Sex (male, female), the schizophrenia subtype (catatonic, noncatatonic), number of antipsychotic drugs after ect (one, two, or more), main antipsychotics (fga, sga), and additional medication use (no, yes) were analyzed as categorical variables . The age at ect, age at onset of illness, number of psychotic episodes, number of ect sessions, cpz equivalence, and bprs score were analyzed as continuous variables . Univariate analyses were conducted, and factors with a trend toward statistical significance (p<0.10) were subsequently examined by a multivariate analysis . The statistical analyses were carried out on a personal computer using spss version 22.0 for windows (ibm japan corporation, tokyo, japan). The inclusion criteria for the study were patients who 1) were diagnosed with schizophrenia based on the international classification of diseases (icd)-10 guideline by at least two trained psychiatrists, 2) received an acute ect course between october 2005 and september 2011, regardless of having received an acute course of ect prior to october 2005, at the national hospital organization (nho) kure medical center, and 3) responded to the acute ect course . The exclusion criteria were patients who 1) were diagnosed with schizoaffective disorders or persistent delusional disorders, 2) did not respond to the acute ect course, and 3) received continuous or maintenance (c / m) ect, as the purpose of this study was to identify risk factors of relapse after acute ect, not after c / m ect . In the current study, we evaluated the responsiveness to acute ect using the clinical global impressions improvement (cgi - i) scale score, as follows: 0= not assessed; 1= very much improved; 2= much improved; 3= minimally improved; 4= no change; 5= minimally worse; 6= much worse; and 7= very much worse . Responders to the acute ect course were defined as patients with a cgi - i scale score 3 after acute ect, and nonresponders were defined as those with a cgi - i score 4 . If two or more acute ect courses were received by the same patient during the study period (20052011), the observation period was defined as the time since completing the first acute ect course . The data used for the study were obtained from medical records . The data collected included demographic variables, clinical variables, and maintenance psychotropic - related variables . The demographic and clinical variables included the age, sex, subtype of schizophrenia, age of onset of illness, number of psychotic episodes (including current episode), number of ect sessions, score on the brief psychiatric rating scale (bprs) before and after ect, and chlorpromazine (cpz) equivalence before ect . In this study, the evaluation of the symptom was carried out by a trained psychiatrist, different from those who diagnosed schizophrenia . The maintenance psychotropic - related variables included the classification of antipsychotics, cpz equivalence, and use of additional medication (mood stabilizers [lithium carbonate, sodium valproate, carbamazepine, and lamotrigine], antidepressants, benzodiazepines, and anticholinergics). The daily doses were calculated by converting the antidepressant, benzodiazepine, and anticholinergic doses to imipramine, diazepam, and biperiden equivalents, respectively . The kind of antipsychotic or the daily doses in some patients were changed because of worsening of their condition, but we calculated the kind and cpz equivalence at the time of discharge, not at the time of relapse, because there were many cases that met the criteria of relapse just after the change of medication . When two or more antipsychotic drugs were used for a single patient, the main antipsychotic drug was defined as the one with the highest cpz - equivalent dose . The classification of antipsychotics into the first - generation antipsychotic (fga) or second - generation antipsychotic (sga) category was conducted according to the literature.9 we adhered to ethical considerations when gathering data so that individuals could not be identified ., patients received intravenous thiamylal sodium (23 mg / kg) and suxamethonium chloride (0.51.0 mg / kg) for anesthesia . The ect device used was the thymatron system iv brief - pulse square - wave apparatus (somatics inc ., lake bluff, il, usa). Only one adequate seizure was required for each session, which was defined as an electroencephalographic seizure lasting> 25 seconds with a high - amplitude, slow - wave and postictal suppression . The initial stimulus dose was determined using the half - age method.10 if an adequate electroencephalographic seizure occurred in one session, the stimulus energy of the next session remained the same . When a missed or an inadequate seizure occurred, the patient was restimulated with 1.52 times the stimulus energy . If any adverse effects (eg, cognitive dysfunction, delirium) occurred, the frequency of the ect schedule was reduced to once or twice per week, or the ect was stopped . On the basis of the apa guidelines, ect was continued until the patient was asymptomatic or when the psychiatrist judged that the patient had benefited as much as possible.6 all patients were treated with antipsychotics during the ect course . After the purpose and procedure of ect was fully explained, written informed consent was obtained from the patients or their family members prior to acute ect . All patients were treated with neuroleptic medication to prevent relapse after the acute ect course . The attending psychiatrist selected the type and dosage of antipsychotics and additional medication on a case - by - case basis . For antipsychotics, sgas were mainly used, while an fga was selected when the effects of sgas were insufficient . In the current study, the time to relapse was defined as the time between ect and the date of an evaluation at which patients had a cgi - i score 6 or if they required psychiatric rehospitalization . The starting point was the last day of the acute ect course during the period, and the end point was the day of relapse . The patients were censored if they moved out of the hospital s recruitment area, died, or had not been readmitted as of september 2012 . Cox regression analyses were used to identify factors associated with relapse via univariate and multivariate analyses . Sex (male, female), the schizophrenia subtype (catatonic, noncatatonic), number of antipsychotic drugs after ect (one, two, or more), main antipsychotics (fga, sga), and additional medication use (no, yes) were analyzed as categorical variables . The age at ect, age at onset of illness, number of psychotic episodes, number of ect sessions, cpz equivalence, and bprs score were analyzed as continuous variables . Univariate analyses were conducted, and factors with a trend toward statistical significance (p<0.10) were subsequently examined by a multivariate analysis . The statistical analyses were carried out on a personal computer using spss version 22.0 for windows (ibm japan corporation, tokyo, japan). Fifty patients received acute ect during the study period . Of these, 43 were responders and seven were nonresponders to the acute ect course . The seven nonresponders included five patients with the paranoid, one patient with the catatonic, and one patient with the hebephrenic types of schizophrenia . There were significant differences in the response rate among the subtypes (responders / nonresponders: catatonic: 22/1, paranoid: 6/5, hebephrenic: 6/1, undifferentiated: 7/0, and residual: 2/0; p=0.013). However, there were no differences in the number of ect sessions between the responders and nonresponders (p=0.673). There were no patients with a first episode; all of the patients had experienced two or more episodes . The mean follow - up period for all 43 patients was 15.820.3 months . During the follow - up, the relapse - free rates of the 43 patients at 6 months, 1 year, and 2 years were 70.7%, 57.3%, and 48.4%, respectively . The factors that were significantly associated with relapse were the number of psychotic episodes (p=0.021) and the bprs score after ect (p=0.048) (table 2). The number of ect sessions tended to increase the risk of the relapse (p=0.091) (table 2). The sex, subtype, age at ect, age of onset of illness, cpz equivalence before the acute ect course, and the bprs score before the acute ect course were not significantly related to relapse . The use of a mood stabilizer tended to reduce the risk of relapse (p=0.058) (table 2). Mood stabilizers were used mainly for drug - resistant, electroencephalographic abnormalities or for symptoms such as emotional instability . Among the 16 (37.2%) patients who used mood stabilizers, nine patients used lithium carbonate, six used sodium valproate, and one used carbamazepine . The mean dose and blood levels of lithium carbonate were 500.0200.0 mg / day and 0.490.16 the mean dose and blood levels of valproic acid were 833.3196.6 mg / day and 59.018.1 g / ml, respectively . The dose and blood levels of carbamazepine were 400 mg / day and 3.0 g / ml, respectively . However, the patients were not randomized for the mood stabilizer treatment . The patients who received mood stabilizers had high bprs scores before ect (54.411.1 versus 46.99.9; p=0.028) and low bprs scores after ect (24.54.8 versus 30.910.3; p=0.009). The frequency of use of mood stabilizers was not significantly different between the catatonic and noncatatonic subtypes (22/21) (40.9% versus 33.3%; p=0.607). The cpz equivalence after ect, the use of two or more antipsychotic drugs, and the use of sga as the main antipsychotic were not associated with relapse . Fourteen (32.5%) patients received sga monotherapy, three (7.0%) received fga monotherapy, and 26 (60.5%) received multiple antipsychotic therapy . As the main antipsychotic after the acute ect course, 35 (81.4%) patients used sga and 8 (18.6%) patients used fga . These included olanzapine (16; 37.2%), risperidone (13; 30.2%), aripiprazole (4; 9.3%), quetiapine (1; 2.3%), zotepine (1; 2.3%), haloperidol (5; 11.6%), and cpz (3; 7.0%). Olanzapine and risperidone were compared with other antipsychotics, and no significant differences in the relapse rates were noted . The use of an antidepressant, use of benzodiazepine, and the use of an anticholinergic were not associated with relapse (table 2). A total of 35 (81.4%) patients received benzodiazepines, 19 (44.2%) received anticholinergics, and six (14.0%) received antidepressants . As additional analysis, because 51% of patients constituted the catatonic subtype of schizophrenia, we tried to perform a similar analysis only by the type . However, it was not possible to perform the analysis for this small sample size . The four factors with a trend toward statistical significance in the univariate analysis were examined by a multivariate analysis . The multivariate analysis showed that two factors significantly affected relapse: the number of ect sessions and the use of a mood stabilizer after ect (table 3). The number of ect sessions was associated with a significantly increased risk of relapse (hazard ratio = 1.159; p=0.033), and patients who used a mood stabilizer were at a lower risk of relapse than were patients who did not (hazard ratio = 0.257; p=0.047). Fifty patients received acute ect during the study period . Of these, 43 were responders and seven were nonresponders to the acute ect course . The seven nonresponders included five patients with the paranoid, one patient with the catatonic, and one patient with the hebephrenic types of schizophrenia . There were significant differences in the response rate among the subtypes (responders / nonresponders: catatonic: 22/1, paranoid: 6/5, hebephrenic: 6/1, undifferentiated: 7/0, and residual: 2/0; p=0.013). However, there were no differences in the number of ect sessions between the responders and nonresponders (p=0.673). There were no patients with a first episode; all of the patients had experienced two or more episodes . The mean follow - up period for all 43 patients was 15.820.3 months . During the follow - up, the relapse - free rates of the 43 patients at 6 months, 1 year, and 2 years were 70.7%, 57.3%, and 48.4%, respectively . The factors that were significantly associated with relapse were the number of psychotic episodes (p=0.021) and the bprs score after ect (p=0.048) (table 2). The number of ect sessions tended to increase the risk of the relapse (p=0.091) (table 2). The sex, subtype, age at ect, age of onset of illness, cpz equivalence before the acute ect course, and the bprs score before the acute ect course were not significantly related to relapse . The use of a mood stabilizer tended to reduce the risk of relapse (p=0.058) (table 2). Mood stabilizers were used mainly for drug - resistant, electroencephalographic abnormalities or for symptoms such as emotional instability . Among the 16 (37.2%) patients who used mood stabilizers, nine patients used lithium carbonate, six used sodium valproate, and one used carbamazepine . The mean dose and blood levels of lithium carbonate were 500.0200.0 mg / day and 0.490.16 meq / l, respectively . The mean dose and blood levels of valproic acid were 833.3196.6 mg / day and 59.018.1 g / ml, respectively . The dose and blood levels of carbamazepine were 400 mg / day and 3.0 g / ml, respectively . However, the patients were not randomized for the mood stabilizer treatment . The patients who received mood stabilizers had high bprs scores before ect (54.411.1 versus 46.99.9; p=0.028) and low bprs scores after ect (24.54.8 versus 30.910.3; p=0.009). The frequency of use of mood stabilizers was not significantly different between the catatonic and noncatatonic subtypes (22/21) (40.9% versus 33.3%; p=0.607). The cpz equivalence after ect, the use of two or more antipsychotic drugs, and the use of sga as the main antipsychotic were not associated with relapse . Fourteen (32.5%) patients received sga monotherapy, three (7.0%) received fga monotherapy, and 26 (60.5%) received multiple antipsychotic therapy . As the main antipsychotic after the acute ect course, 35 (81.4%) patients used sga and 8 (18.6%) patients used fga . These included olanzapine (16; 37.2%), risperidone (13; 30.2%), aripiprazole (4; 9.3%), quetiapine (1; 2.3%), zotepine (1; 2.3%), haloperidol (5; 11.6%), and cpz (3; 7.0%). Olanzapine and risperidone were compared with other antipsychotics, and no significant differences in the relapse rates were noted . The use of an antidepressant, use of benzodiazepine, and the use of an anticholinergic were not associated with relapse (table 2). A total of 35 (81.4%) patients received benzodiazepines, 19 (44.2%) received anticholinergics, and six (14.0%) received antidepressants . As additional analysis, because 51% of patients constituted the catatonic subtype of schizophrenia, we tried to perform a similar analysis only by the type . However, it was not possible to perform the analysis for this small sample size . The four factors with a trend toward statistical significance in the univariate analysis were examined by a multivariate analysis . The multivariate analysis showed that two factors significantly affected relapse: the number of ect sessions and the use of a mood stabilizer after ect (table 3). The number of ect sessions was associated with a significantly increased risk of relapse (hazard ratio = 1.159; p=0.033), and patients who used a mood stabilizer were at a lower risk of relapse than were patients who did not (hazard ratio = 0.257; p=0.047). Following a response to the acute ect course, the relapse rates at 6 months, 1 year, and 2 years were 29.3%, 42.7%, and 51.6%, respectively . Suzuki et al8 reported that seven (63.6%) of eleven patients with catatonic schizophrenia who responded to acute ect relapsed during the 1-year follow - up, despite continuation of antipsychotics, and that all relapses occurred within 6 months . Hustig and onilov7 reported that 17 (62.9%) of 27 treatment - resistant schizophrenic patients relapsed within 1 year after the application of ect, and all relapses occurred within 8 months . The relapse rate in our study cannot be simply compared with those in past reports because of the differences in each study concerning factors such as patient subtype and definition of relapse . However, our study also showed that the relapses occurred soon after the ect course, similar to the past reports, even if antipsychotic therapy was continued . It therefore seems that an intensive treatment strategy is necessary to prevent relapse, especially during the first year after ect . The first major finding of our current study was that additional mood stabilizer use was significantly related to a lower risk of relapse . There was a 74% risk reduction when adjunctive mood stabilizer therapy was used, compared to when no mood stabilizer was used . To the best of our knowledge, there have so far been no reports addressing the effect of mood stabilizers for schizophrenia after a response to ect . Mood stabilizers may be helpful in specific subpopulations of schizophrenic patients, such as those with catatonic symptoms,11 anxiety depression,12 and hostility.13 ect is also effective for patients with similar features, such as catatonic features, affective symptoms,5 tension, and hostility.14 therefore, following an improvement of these symptoms by acute ect, adjunctive mood stabilizer maintenance therapy may be a useful treatment strategy to support remission . Additionally, in a study of mood disorders,15 a combination of lithium and antidepressants had a marked advantage rather than antidepressants alone in terms of the time to relapse for patients with depression after a response to ect . Mood stabilizers, such as lithium, may have a certain protective effect against relapse of not only depression but also schizophrenia after a response to ect . However, with regard to the treatment strategies employed during the follow - up period, it is difficult to draw conclusions from retrospective studies, because the patients were not randomized, and different factors related to the selection of treatment might have influenced the results . It is possible that clinicians may choose to use mood stabilizers in schizophrenic patients with mood symptoms, who have been reported to tend to have a better outcome.16 therefore, it cannot be denied that subpopulations of schizophrenia defined as having used mood stabilizers was less likely to relapse with or without treatment with ect . We were not able to detect an association between antipsychotic therapy (category, number, or daily use) or the use of antidepressants or benzodiazepine after ect and relapse . In our study, because almost 60% of patients had polypharmacy with antipsychotics, and> 80% of patients were taking additional psychotropic agents other than antipsychotics, the possibility of interactions between these drugs cannot be ruled out . A previous study17 comparing sulpiride, risperidone, and olanzapine in combination with c / m ect revealed that risperidone and olanzapine were superior to sulpiride in terms of the clinical effects . In another study in adolescent subjects diagnosed with schizophrenia spectrum disorders,18 the rate of rehospitalization was lower in patients treated with clozapine compared to that of the nonclozapine group (using other antipsychotics or benzodiazepines). Clozapine is used for intractable schizophrenia worldwide; however, it was not approved in japan until 2009, and no patients received clozapine during the investigation period in the current study . Maintenance pharmacotherapy with clozapine seems to be an alternative strategy that should be examined in future studies . The second finding of the current study was that the number of ect sessions was significantly related to risk of relapse . Generally, the number of ect sessions is not decided in advance of the treatment; rather, it is decided during the course based on the improvement in an individual s symptoms . The general number of ect sessions is 612; however, this differs by individual . According to a chart review of 79 cases, schizophrenic patients received between 2 and 26 sessions of acute ect.1 when ect was ineffective during the early sessions, the number of sessions was necessarily increased during that study, but it was not a control study . When more ect sessions are necessary to improve symptoms, it may indicate that the patient is more intractable to treatment and may be more prone to relapse . Therefore, it is a compatible result that the patients received a greater number of ect sessions and seem to easily relapse . When maintenance pharmacological prophylaxis alone is ineffective or not tolerated in the stable phase, the apa guidelines suggest that c / m ect combined with an antipsychotic may be beneficial in patients who respond to acute ect.19 therefore, the number of acute ect sessions could be one of the indicators for applying c / m ect . The findings of this study must be interpreted within the following limitations of this study: 1) this study was a naturalistic and retrospective study, not a randomized controlled trial; 2) the sample size was relatively small, and subjects were enrolled in one hospital; 3) the severity of the disease varied; 4) subjects may have had refractory properties because all of the patients had experienced two or more episodes of psychosis; 5) various maintenance pharmacotherapy was used because the medication was selected based on the attending physicians choices, rather than in a systematic manner; and 6) there was insufficient evaluation of some factors regarded as factors potentially related to the relapse of general schizophrenia, such as adherence to medicines, insight into the illness, adverse life events, the level of social support, premorbid psychosocial functioning, or expressed emotion because of the incomplete medical records . In addition, our data showed schizophrenia was of five subtypes, with 51% belonging to the catatonic type . In the past decade, catatonia has increasingly been recognized as a unique syndrome . An earlier review reported a great variation in the efficacy of ect depending on the type of treatment setting and type of schizophrenia . The efficacy rate in catatonia is high compared to that in other types.2 it was desirable to separate the catatonia group from the other types and analyze the outcome itself, but it was not possible to perform the analysis due to the small sample size . Another concern is whether catatonia had an effect on the prevention of relapse by mood stabilizers after acute ect in this study . Indeed, the patients who received mood stabilizers had higher bprs scores before ect and lower bprs scores after ect (see treatment factors under results section). However, mood stabilizers were used at approximately the same rate in the catatonia and noncatatonia groups (see treatment factors under results section). Therefore, catatonia may not influence the effects of mood stabilizers after acute courses of ect . A prospective and well - designed study with a large sample size our study on the relapse of schizophrenia after a response to acute ect suggested that the number of ect sessions may be related to the risk of relapse . On the other hand, adjunctive pharmacotherapy with mood stabilizers may be effective in preventing relapse.
A detailed description of the design, subjects, and methods applied in the dps has been reported previously (14). In brief, the dps was a randomized lifestyle intervention study in 522 middle - aged overweight participants with impaired glucose tolerance, aimed at the prevention of type 2 diabetes . In the present study, we included those 486 participants (249 in the intervention and 237 in the control group) who had completed a questionnaire quantifying ltpa at baseline and during yearly follow - up visits (11). A subgroup of 137 participants was taking part in supervised resistance training sessions . The study protocol was approved by the ethics committee of the national public health institute in helsinki, and all subjects gave written informed consent . The participants in the intervention group were given detailed and individualized dietary and exercise counseling as described elsewhere (15). Session for supervised and individually tailored progressive circuit - type resistance training with moderate intensity were recommended twice a week and offered free of charge in three of the five study centers . The participants in the control group were given general information about healthy food choices, physical activity, and weight loss at baseline, but no individualized counseling was offered . The validated kuopio ischemic heart disease risk factor study questionnaire (11,12) was used for the assessment of physical activity . The participants estimated the frequency, average duration, and intensity of different forms of exercise for individual months during the past 12 months . Based on the reported intensity of different activities and their corresponding metabolic equivalent (met) values, the total ltpa was divided to low - intensity and to moderate - to - vigorous intensity ltpa (13). Low - intensity ltpa (<3.5 mets) included activities such as gardening, picking berries, casual walking, and bicycling at recreational intensity . Moderate - to - vigorous ltpa (3.5 mets) included activities such as brisk walking, jogging, skiing, swimming, rowing, forest work, gymnastics, resistance training, ball games, snow shoveling, and heavy housework . The duration of total ltpa and its components were calculated as hours / week from the baseline to the end of the follow - up . The changes were calculated by subtracting averaged follow - up value from the corresponding baseline value (11). The participation in resistance training was recorded electronically when the participants visited the resistance training facilities and was analyzed as sessions / year . Medical history and 3-day food records were collected at baseline and at each annual visit . Average intakes of energy (kcal / day), carbohydrates (e%), total fat (e%), saturated fat (e%), and dietary fiber (g/1,000 kcal) were calculated . The average values from years 13 were used to measure dietary intakes during follow - up (11). Serum total and hdl cholesterol and triglyceride levels were determined by enzymatic methods (boehringer mannheim, germany). For the definition of the mets the data were analyzed using spss statistical software (version 11.5; spss, chicago, il). The baseline values are given as mean sd, as median with 0.250.75 interquartile range, or as percentages . The student two - tailed t test, mann - whitney u test (fasting and 2-h serum insulin, triglycerides, and ltpa), and test were applied to compare the differences at baseline and during the follow - up . For participants who dropped out or developed diabetes during the study, the measurements at the last observation year was used as the end value . The primary outcome measure was the change in the mets status in the combined intervention and control group from baseline to the end, i.e., resolution of the mets from baseline, development of mets, or no change with ltpa changes as explanatory variables . Secondary outcome measures were the changes of the mets components . The change of different ltpa was categorized into thirds . The association with the change in mets status and its components was analyzed with multinominal regression . The models were adjusted for age, sex, intervention group, and dps study years (model 1) with further adjustments for changes in diet (intake of total fat, saturated fat, fiber, and energy) (model 2) and bmi (model 3). The change in low - intensity ltpa was also adjusted for the change in moderate - to - vigorous ltpa and vice versa . The participants in the intervention group were given detailed and individualized dietary and exercise counseling as described elsewhere (15). Session for supervised and individually tailored progressive circuit - type resistance training with moderate intensity were recommended twice a week and offered free of charge in three of the five study centers . The participants in the control group were given general information about healthy food choices, physical activity, and weight loss at baseline, but no individualized counseling was offered . The validated kuopio ischemic heart disease risk factor study questionnaire (11,12) was used for the assessment of physical activity . The participants estimated the frequency, average duration, and intensity of different forms of exercise for individual months during the past 12 months . Based on the reported intensity of different activities and their corresponding metabolic equivalent (met) values, the total ltpa was divided to low - intensity and to moderate - to - vigorous intensity ltpa (13). Low - intensity ltpa (<3.5 mets) included activities such as gardening, picking berries, casual walking, and bicycling at recreational intensity . Moderate - to - vigorous ltpa (3.5 mets) included activities such as brisk walking, jogging, skiing, swimming, rowing, forest work, gymnastics, resistance training, ball games, snow shoveling, and heavy housework . The duration of total ltpa and its components were calculated as hours / week from the baseline to the end of the follow - up . The changes were calculated by subtracting averaged follow - up value from the corresponding baseline value (11). The participation in resistance training was recorded electronically when the participants visited the resistance training facilities and was analyzed as sessions / year . Medical history and 3-day food records were collected at baseline and at each annual visit . Average intakes of energy (kcal / day), carbohydrates (e%), total fat (e%), saturated fat (e%), and dietary fiber (g/1,000 kcal) were calculated . The average values from years 13 were used to measure dietary intakes during follow - up (11). Serum total and hdl cholesterol and triglyceride levels were determined by enzymatic methods (boehringer mannheim, germany). For the definition of the mets the data were analyzed using spss statistical software (version 11.5; spss, chicago, il). The baseline values are given as mean sd, as median with 0.250.75 interquartile range, or as percentages . The student two - tailed t test, mann - whitney u test (fasting and 2-h serum insulin, triglycerides, and ltpa), and test were applied to compare the differences at baseline and during the follow - up . For participants who dropped out or developed diabetes during the study, the measurements at the last observation year was used as the end value . The primary outcome measure was the change in the mets status in the combined intervention and control group from baseline to the end, i.e., resolution of the mets from baseline, development of mets, or no change with ltpa changes as explanatory variables . Secondary outcome measures were the changes of the mets components . The change of different ltpa was categorized into thirds . The association with the change in mets status and its components was analyzed with multinominal regression . The models were adjusted for age, sex, intervention group, and dps study years (model 1) with further adjustments for changes in diet (intake of total fat, saturated fat, fiber, and energy) (model 2) and bmi (model 3). The change in low - intensity ltpa was also adjusted for the change in moderate - to - vigorous ltpa and vice versa . The participants with the mets at baseline had significantly higher bmi, waist circumference, blood pressure, fasting and 2-h glucose, fasting and 2-h insulin, and serum triglyceride levels and lower serum hdl cholesterol levels (table 1). Baseline characteristics of the participants according the absence (mets) or presence (mets) of the mets data are means sd for normally distributed or medians (interquartile ranges) for skewed parameters or percentages . In general, men exercised more than women . Women without the mets reported significantly more hours per week spent on total and low - intensity ltpa during the previous 12 months than women with the mets . The median for total ltpa increased from 7.2 (3.610.8) at baseline to an average of 7.7 (4.811.7) hours per week (p = 0.061) in men and from 5.3 (2.88.6) to 5.8 (3.29.0) hours per week (p = 0.016) in women during the follow - up . The median for moderate to vigorous ltpa increased from 2.3 (0.94.8) to 3.1 (1.84.9) (p 0.001) hours per week in men and from 1.4 (0.33.5) to 2.5 (1.14.1) (p 0.001) hours per week in women . Of the 361 participants meeting the mets criteria at baseline, 20.8% (n = 75; 26.6% in the intervention and 14.7% in the control group; p = 0.005) showed resolution during the follow - up . Of the 126 participants not meeting the mets criteria at baseline, 31.2% (n = 39; 30.8% in the intervention and 31.7% in the control group; p = 0.95) developed mets during the follow - up . The development of the mets was associated with weight gain and less ltpa in both groups . The change in total ltpa was associated with the change in mets status (resolution, no change, development) after adjustment for age, sex, intervention group, and dps study years (model 1, fig . The change in moderate - to - vigorous ltpa was even more strongly associated with the change in mets status in analyses adjusting for the variables in model 1 and changes in low - intensity ltpa . The resolution of the mets was seen in 29.7 versus 19.1% (p = 0.004), and the development of mets was seen in 23.5 versus 44.7% (p = 0.041) in the upper versus lower third of change in moderate - to - vigorous ltpa . The associations remained significant after further adjustments for changes in diet (model 2) and bmi (model 3) (fig . Changes in low - intensity ltpa were not associated with the change in mets status (fig . Incidences (%) for the development (for individuals without mets at baseline, n = 125) () and the resolution (for individuals with mets at baseline, n = 361) () of the mets according to ltpa change tertiles for total ltpa (a), moderate - to - vigorous ltpa (b), and low - intensity ltpa (c). Model 1: adjustments for age, sex, intervention group, and dps study years . The change in low - intensity ltpa was also adjusted for change in moderate - to - vigorous ltpa and vice versa . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . The increase in total ltpa was associated with a decrease in the prevalence of hyperglycemia (p = 0.0200.053), low hdl cholesterol (p = 0.0180.057), and hypertriglyceridemia (p = 0.0020.003) (table 2). Increased moderate - to - vigorous ltpa decreased the prevalence of elevated fasting glucose (p = 0.0030.018), but no association with abdominal obesity (p = 0.0650.181), low hdl cholesterol (p = 0.0980.232), and high blood pressure (p = 0.0680.151) was found . In contrast, an increase in low - intensity ltpa was associated with an improvement in hypertriglyceridemia (p = 0.0060.004), but not any of the other components of the mets . Incidences (%) for development and resolution of the mets components according to ltpa change tertiles for total, low - intensity, and moderate - to - vigorous ltpa during the follow - up * model 1: adjustments for age, sex, intervention group, and dps study years . The change in low - intensity ltpa was also adjusted for change in moderate - to - vigorous ltpa and vice versa . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . Model 3: model 2 and change in bmi . In the subgroup of 137 individuals taking part in supervised resistance training, the median attendance rate was 27.0 (13.442.9) sessions / year during the entire study . Of the mets status components, the resistance training attendance rate was associated, even after adjustment for dietary and bmi changes, with improvements in hyperglycemia (p = 0.1270.029), hypertriglyceridemia (p = 0.0460.081), and low hdl cholesterol (p <0.0010.002), but not with elevated blood pressure or abdominal obesity (table 3). The average resistance training attendance rate per year and the change (development and resolution) in the mets components among a subgroup of 137 participants * model 1: adjustments for age, sex, group, dps study years, averaged low - intensity ltpa, and ltpa other than gymnastics and calisthenics . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . The participants with the mets at baseline had significantly higher bmi, waist circumference, blood pressure, fasting and 2-h glucose, fasting and 2-h insulin, and serum triglyceride levels and lower serum hdl cholesterol levels (table 1). Baseline characteristics of the participants according the absence (mets) or presence (mets) of the mets data are means sd for normally distributed or medians (interquartile ranges) for skewed parameters or percentages . In general, men exercised more than women . Women without the mets reported significantly more hours per week spent on total and low - intensity ltpa during the previous 12 months than women with the mets . The median for total ltpa increased from 7.2 (3.610.8) at baseline to an average of 7.7 (4.811.7) hours per week (p = 0.061) in men and from 5.3 (2.88.6) to 5.8 (3.29.0) hours per week (p = 0.016) in women during the follow - up . The median for moderate to vigorous ltpa increased from 2.3 (0.94.8) to 3.1 (1.84.9) (p 0.001) hours per week in men and from 1.4 (0.33.5) to 2.5 (1.14.1) (p 0.001) hours per week in women . Of the 361 participants meeting the mets criteria at baseline, 20.8% (n = 75; 26.6% in the intervention and 14.7% in the control group; p = 0.005) showed resolution during the follow - up . Of the 126 participants not meeting the mets criteria at baseline, 31.2% (n = 39; 30.8% in the intervention and 31.7% in the control group; p = 0.95) developed mets during the follow - up . The development of the mets was associated with weight gain and less ltpa in both groups . The change in total ltpa was associated with the change in mets status (resolution, no change, development) after adjustment for age, sex, intervention group, and dps study years (model 1, fig . The change in moderate - to - vigorous ltpa was even more strongly associated with the change in mets status in analyses adjusting for the variables in model 1 and changes in low - intensity ltpa . The resolution of the mets was seen in 29.7 versus 19.1% (p = 0.004), and the development of mets was seen in 23.5 versus 44.7% (p = 0.041) in the upper versus lower third of change in moderate - to - vigorous ltpa . The associations remained significant after further adjustments for changes in diet (model 2) and bmi (model 3) (fig . Changes in low - intensity ltpa were not associated with the change in mets status (fig . Incidences (%) for the development (for individuals without mets at baseline, n = 125) () and the resolution (for individuals with mets at baseline, n = 361) () of the mets according to ltpa change tertiles for total ltpa (a), moderate - to - vigorous ltpa (b), and low - intensity ltpa (c). Model 1: adjustments for age, sex, intervention group, and dps study years . The change in low - intensity ltpa was also adjusted for change in moderate - to - vigorous ltpa and vice versa . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . The increase in total ltpa was associated with a decrease in the prevalence of hyperglycemia (p = 0.0200.053), low hdl cholesterol (p = 0.0180.057), and hypertriglyceridemia (p = 0.0020.003) (table 2). Increased moderate - to - vigorous ltpa decreased the prevalence of elevated fasting glucose (p = 0.0030.018), but no association with abdominal obesity (p = 0.0650.181), low hdl cholesterol (p = 0.0980.232), and high blood pressure (p = 0.0680.151) was found . In contrast, an increase in low - intensity ltpa was associated with an improvement in hypertriglyceridemia (p = 0.0060.004), but not any of the other components of the mets . Incidences (%) for development and resolution of the mets components according to ltpa change tertiles for total, low - intensity, and moderate - to - vigorous ltpa during the follow - up * model 1: adjustments for age, sex, intervention group, and dps study years . The change in low - intensity ltpa was also adjusted for change in moderate - to - vigorous ltpa and vice versa . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . In the subgroup of 137 individuals taking part in supervised resistance training, the median attendance rate was 27.0 (13.442.9) sessions / year during the entire study . Of the mets status components, the resistance training attendance rate was associated, even after adjustment for dietary and bmi changes, with improvements in hyperglycemia (p = 0.1270.029), hypertriglyceridemia (p = 0.0460.081), and low hdl cholesterol (p <0.0010.002), but not with elevated blood pressure or abdominal obesity (table 3). The average resistance training attendance rate per year and the change (development and resolution) in the mets components among a subgroup of 137 participants * model 1: adjustments for age, sex, group, dps study years, averaged low - intensity ltpa, and ltpa other than gymnastics and calisthenics . Model 2: model 1 and adjustments for change in dietary intakes of total fat, saturated fat, fiber, and energy . Increased moderate - to - vigorous ltpa during the 4.1-year follow - up increased the likelihood for the mets to resolve and decreased the likelihood for the mets to develop, independently of changes in diet and body weight . Moreover, increased moderate - to - vigorous ltpa decreased the prevalence of hyperglycemia . Improvements in fasting plasma glucose, serum triglycerides, and hdl cholesterol, independently of changes in diet, lifestyle ltpa, and other types of ltpa, were associated with participation in resistance training . Overall, strong and mostly linear dose - response associations of the change in total ltpa with the development and resolution of the mets were seen . When breaking down physical activity into moderate - to - vigorous ltpa and low - intensity ltpa, it seems evident that most of the benefit was from moderate- to vigorous - intensity ltpa . Changes in moderate - to - vigorous ltpa were associated with the change in metabolic status, even independently of the changes in bmi, but the association was not linear across categories . Changes in low - intensity ltpa were not associated with the development or resolution of the mets . Why the dose - response association was not apparent for moderate - to - vigorous ltpa is unclear, but it may be related to the difficulty in the precise assessment of ltpa . Overall, however, our findings support efforts to increase or at least maintain ltpa, especially moderate - to - vigorous ltpa, in the prevention and treatment of the mets . In this analysis of the dps,, there was a significant difference between the groups in the resolution of the mets . However, there was no difference in the development of mets between groups; 30% of those without mets at baseline developed mets in both groups during the follow - up they were individuals at high risk for type 2 diabetes and for the mets . During the follow - up while the development of mets was associated with weight gain and less ltpa, those subjects who developed the mets appeared to not adhere with our intervention . The apparent favorable effects of moderate - to - vigorous ltpa on resolution and development of the mets are consistent with the results of the uncontrolled heritage family study (16) and some prospective cohort studies showing that increased moderate - to - vigorous ltpa was associated with a lower incidence of the mets during follow - up (8,12,17). In the dps cohort, increased moderate - to - vigorous ltpa seemed to protect against developing the mets in both men and women . Higher cardiorespiratory fitness, which partly reflects higher levels of moderate - to - vigorous ltpa, has predicted a lower prevalence of the mets independently of major confounding variables also in women in the aerobics center longitudinal study (18) and in the dose - responses to exercise training study (19). Changes in low - intensity ltpa were not associated with changes in the mets status . These findings are consistent with the results of the kuopio ischemic heart disease study, in which moderate - to - vigorous, but not low - intensity, ltpa was associated with development of the mets (12). When examining specific components of the mets, increased moderate - to - vigorous ltpa had the greatest effect on impaired fasting glucose, whereas the benefit on abdominal obesity, low hdl cholesterol, and high blood pressure was not significant . In contrast, changes in low - intensity ltpa had benefits on hypertriglyceridemia, but not on the other components of the mets, and changes in total ltpa improved impaired fasting glucose and dyslipidemia . In intervention trials, low - intensity ltpa has less consistently improved metabolic outcomes than more intense ltpa (8). However, we have previously reported that increased low - intensity and moderate - to - vigorous ltpa were similarly associated with a lower risk of type 2 diabetes in the finnish dps, suggesting that total energy expenditure on ltpa was more important than intensity (11). In line with that finding, the accumulated daily physical activity as measured with an accelerometer was a major determinant of insulin sensitivity, and time spent on moderate - to - vigorous physical activity did not affect insulin sensitivity independently of total activity in the european relationship between insulin sensitivity and cardiovascular risk study (20). The differences may be explained by differences in study populations and specific metabolic outcomes . More information on the long - term metabolic benefits of low - intensity ltpa in different age - groups and risk groups is nonetheless needed . We found that a higher participation rate in resistance training was associated with benefits on impaired fasting glucose, hypertriglyceridemia, and low hdl cholesterol, but not abdominal obesity or blood pressure . In 3- to 6-month trials, resistance training has variably increased muscle mass, decreased fat mass and abdominal obesity, and improved insulin sensitivity in obese adults, hypertensive patients, older men, and older type 2 diabetic patients (8,21,22). In individuals with type 2 diabetes, resistance training resulted in similar improvements of glycemic control as aerobic exercise (23), although the effect on glucose tolerance in impaired glucose tolerance has been less clear (8). Improvements in insulin sensitivity and metabolic risk factors may be mediated in part by changes in body composition, but strength training may also independently affect steps in insulin signaling and glucose transport (24). Based on meta - analyses of trials, resistance training may decrease blood pressure (25), but effects on dyslipidemia have been variable (8). Our findings suggest that there is a graded benefit in the frequency of resistance training in the prevention or treatment of the mets components, with rather substantial benefits for individuals engaging in resistance training on median once a week compared with individuals engaging in resistance training on median less than once a month . In the above - mentioned studies showing an improvement in insulin sensitivity in individuals at risk for type 2 diabetes and in glycemic control in patients with type 2 diabetes, training frequency was generally two to three times per week . The metabolic benefits of resistance training at a lower frequency may become apparent only after much longer periods of training than in previously published trials, which have usually lasted 36 months . Strengths of the dps include its repeated assessments of ltpa and dietary intake . However, the present analyses are post hoc . Furthermore, the intervention had several components . Detailed assessment of the individual lifestyle components allows statistical disentanglement of their individual effects, but residual confounding is possible . Decreases in ltpa may have been related to factors that themselves may be related to the development of the mets . Adherence to resistance training was on average poor . When this study was conducted in the early 1990s, it was uncommon for middle - aged and overweight individuals to attend resistance training facilities, where most of the clientele were young and fit . Some also encountered difficulties with transportation and time schedules . In conclusion, increased participation in moderate - to - vigorous physical activity and regular long - term participation in resistance training improved the mets status among men and women with impaired glucose tolerance in the finnish dps . Physical activity and resistance training also more specifically had benefits with respect to hyperglycemia and dyslipidemia, but improvements in abdominal obesity were not clearly seen . Resolution or prevention of the mets and related features might contribute to the protective effect of physical activity on type 2 diabetes.
Leptospirosis a common and dreaded zoonosis of global distribution, caused by infection with pathogenic spirochetes of the genus leptospira . The disease is maintained in nature by chronic renal infection of carrier animals, which excrete the organism in their urine, contaminating the environment . Human infection occurs by direct contact with infected urine or tissues or, more commonly by indirect exposure to the organism in damp soil or water . The serovar classification remains useful for epidemiological purposes, as many geographically widespread serovar - reservoir associations exist . According to recent studies on the worldwide incidence of leptospirosis, croatia is 13 in the world and 1 in europe, with an incidence rate of 17.3 cases per million population annually . Leptospirosis is endemic in croatia, and most commonly occurs in the sava and drava river valleys of central and northwestern croatia and in the neretva river valley [812]. Leptospirosis in croatia has been systematically monitored and studied for more than 50 years, as a constant object of investigation of infectious disease specialists, epidemiologists, immunologists, veterinarians and foresters . Throughout this time no changes were observed in the incidence rates of leptospirosis in humans and wild animals; however, changes in the incidence rates have been observed among the most likely infectious serovars . Molecular analysis of leptospira spp . The highest incidence in the last 10 years has been observed among the following serovars: leptospira interrogans serovar australis, leptospira kirschneri serovar grippotyphosa and leptospira interrogans serovar saxkoebing, as opposed to france, italy and germany where leptospira interrogans serovar icterohaemorrhagiae is the most common serovar causing infections in humans [5,1316]. The severity of leptospiral infections ranges from a subclinical illness to 2 clinically recognizable syndromes: a self limited, systemic illness seen in approximately 90% of infections, and a severe potentially fatal illness accompanied by any combination of renal failure, liver failure, pneumonitis with hemorrhagic diathesis and myocarditis . Death, occurring in 1015% of these severe cases, is usually due to pulmonary hemorrhage, renal failure or cardiac failure and arrhythmia secondary to myocarditis . The finding of ecg abnormalities in patients with leptospirosis who show clinical evidence of cardiac involvement is well recognized . Clinical reports make it clear that pericarditis, endocarditis, myocarditis and arrhythmias occur in leptospirosis . A few systematic studies of latent cardiac involvement in leptospirosis have been made [2124]. Experimental studies in animals and autopsies in humans have shown that cardiac involvement in leptospirosis is frequent, even though it may occur without clinical manifestations . The aim of this study was to investigate the incidence and type of ecg changes in patients with leptospirosis regardless of clinical evidence of cardiac involvement . The ethics committee of the university hospital for infectious diseases dr fran mihaljevi, zagreb, croatia (uhid) approved the study . A total of 97 patients with serologically confirmed leptospirosis treated at the university hospital for infectious diseases dr . Fran mihaljevi (uhid) in zagreb, croatia, which is the national reference centre for infectious diseases, were included in this retrospective study from 2000 to 2009 . In the investigated period there were 213 hospitalized patients with clinical and epidemiological diagnosis of leptospirosis, which was serologically confirmed in 105 (1 patient with known heart disease was excluded and 7 medical history charts were unavailable). Epidemiological and clinical data including main clinical characteristics and laboratory findings were obtained from hospital charts . The diagnosis was confirmed by microscopic agglutination test (mat) performed at the laboratory for leptospires, department of microbiology and infectious diseases clinic, faculty of veterinary medicine, university of zagreb (20002008) and leptospira laboratory at the croatian national institute of public health (20082009), with 12 leptospira serovars: leptospira kirschneri serovar grippotyphosa, leptospira interrogans serovar sejroe, leptospira interrogans serovar australis, leptospira interrogans serovar pomona, leptospira interrogans serovar canicola, leptospira interrogans serovar icterohaemorrhagiae, leptospira interrogans serovar tarassovi, leptospira interrogans serovar saxkoebing, leptospira interrogans serovar ballum, leptospira interrogans serovar bataviae, leptospira borgpetersenii serovar poi and leptospira interrogans serovar hardjo . A cut - off value of 1:500 was used for a significant titre in patients in whom those with at least a 4-fold increase in titre were considered positive . In case of coagglutination, the serovar with the most expressed increase in antibody titre was accepted as pathogenic, and if antibody titers were equal, the finding was accepted as undetermined . A 12-lead resting ecg was routinely performed in the first 2 days after hospital admission independent of the clinical manifestations of patients and prior to antibiotic administration . We considered all nonspecific st segment and t wave abnormalities to be non - specific ventricular repolarization disturbances . Thorough past and current medical history was obtained, and careful physical examination and laboratory tests were performed . Exclusion criteria were as follows: prior known alterations in ecg finding, and prior intake of drugs that can influence ecg finding . The variables recorded in each patient included sex, age, duration of disease prior to admission, duration of hospitalization, chest x - ray, white blood cell count, platelets, c - reactive protein, serum creatinine, potassium, alanine aminotransferase, aspartate aminotransferase, bilirubin and urine . We describe our data with medians and interquartile range for continuous variables, and frequencies for categorical variables . The main clinical characteristics and laboratory findings of patients with normal and abnormal ecg were compared by the chi - square or wilcoxon rank - sum test . All tests were performed with the sas software system release 9.1.3 (sas institute, cary, nc). We describe our data with medians and interquartile range for continuous variables, and frequencies for categorical variables . The main clinical characteristics and laboratory findings of patients with normal and abnormal ecg were compared by the chi - square or wilcoxon rank - sum test . All tests were performed with the sas software system release 9.1.3 (sas institute, cary, nc). Abnormal ecg findings were found in 56 of 97 (58%) patients with serologically confirmed leptospirosis hospitalized at the university hospital for infectious diseases in zagreb, croatia from 2000 to 2009 . The majority of patients were hospitalized from july to october (68/97 70%) and the majority had positive epidemiological anamnesis (90/97 93%). There were no differences in age, sex, duration of symptoms prior to admission or hospital stay between the patients with normal and abnormal ecg (table 1). The incidence of certain clinical symptoms did not differ with regards to ecg changes (table 2), nor were there statistical differences in white blood cell count, creatinine, c - reactive protein, and aspartate aminotransferase . Patients with abnormal ecg had significantly elevated values of bilirubin and alanine aminotransferase, lower values of potassium and lower number of platelets, as well as more frequently recorded abnormal chest x - ray . Ecg was recorded in all patients in the first 2 days after hospitalization when patients were febrile . Non - specific ventricular repolarization disturbances were the most common abnormal ecg finding (figure 1). Other recorded ecg abnormalities were sinus tachycardia, right branch conduction disturbances, low voltage of the qrs complex in standard limb leads, supraventricular and ventricular extrasystoles, intraventricular conduction disturbances, atrioventricular block first - degree and atrial fibrillation . Myopericarditis was identified in 4 patients on the basis of physician examination (muffled first heart sound), ecg findings (non - specific st segment and t wave abnormalities, tachycardia, arrhythmias) and echocardiography (table 3). In our patients, serovar australis, leptospira interrogans serovar saxkoebing and leptospira kirschneri serovar grippotyphosa (table 4). Electrocardiographic findings of transient changes found on routine ecg in patients with uncomplicated leptospirosis confirm the predictions of first researchers [1921] that ecg evidence of cardiac involvement may be found in the absence of physical signs . The first systematic comparative research of ecg findings in a total of 43 patients with leptospirosis and malaria hospitalized from 1960 to 1962 in the british military hospital in malaga and in healthy persons exposed to artificial hyperpyrexia showed that transient ecg abnormalities are similar in patients with leptospirosis and malaria and are nonspecific results of a febrile infection . Electrocardiographic alterations were detected in 68% of 157 patients hospitalized with leptospirosis in salvador, brazil from 1998 to 1999 . Atrial fibrillation occurred in 11%, and alterations in ventricular repolarization occurred in 39% of patients . The patients with atrial fibrillation were older, had higher levels of creatinine and aminotransferases, and had evidence of more severe disease . The mechanisms determining the ecg alterations were not specifically assessed in the study, nor was the relation between the presence and type of ecg alteration and the prognosis of leptospirosis . The clinical picture of anicteric leptospirosis can closely mimic that of hemorrhagic fever with renal syndrome (hfrs). Both diseases predominantly affect farmers, trappers, veterinarians and military personnel who may have contact with infected animals or their urine . Both diseases are acute febrile diseases most commonly affecting the kidneys, and cause pulmonary and liver disorders as well as ecg abnormalities . Although there is no curative therapy for hfrs, antibiotic therapy for leptospirosis is generally believed to be beneficial if initiated early in the course of the disease . Therefore, it is important to introduce rapid tests for the detection of both diseases, especially in endemic regions . A total of 1208 patients with serologically confirmed leptospirosis were hospitalized at uhid in the period 19471979 . In all patients ecg was performed; however, no subclinical transitory ecg abnormalities were recorded . Several published case reports of patients with clinically severe forms of leptospirosis hospitalized in uhid also reported no ecg abnormalities [3436]. A total of 256 patients with serologically confirmed leptospirosis hospitalized at uhid from 1973 to 1982 were evaluated retrospectively . The purpose of the study was to estimate the incidence of sporadic clinical manifestations in leptospirosis such as electrocardiographic changes . Nonspecific alterations of the ventricular repolarization were found most often (23 patients), while atrial fibrillation was recorded in 2 patients . In patients with ecg changes, the most frequently observed pathogens were leptospira interrogans serovar icterohaemorrhagiae (20), leptospira kirschneri serovar grippotyphosa (12) and leptospira interrogans serovar sejroe (6). Among 130 retrospectively analyzed patients with serologically confirmed leptospirosis hospitalized at uhid in the period from 1997 to 2007, 9 (7%) patients developed weil s disease and were treated at the intensive care unit . Three patients had acute myopericarditis, and 1 of them developed atrial fibrillation, all with a benign clinical course . We retrospectively analyzed 213 hospitalized patients with clinical and epidemiological diagnosis of leptospirosis which was serologically confirmed in less than half of the patients . Although in endemic areas, the classic history of fever, myalgia and headache in patients with positive epidemiological anamnesis should raise the possibility of leptospirosis, it should be confirmed by serology . This is supported by research from iran, where out of a total of 237 patients with clinically and epidemiologically suspected leptospirosis, the diagnosis was confirmed in 74 . Electrocardiograms in our patients were performed in the first 2 days after hospital admission, independent of the clinical manifestations of the patients and prior to antibiotic administration . All patients were febrile at admission and all developed a moderately severe form of disease requiring symptomatic treatment . Atrial fibrillation occurred in 6 patients, 1 of whom also had right bundle branch conduction disturbances, and 4 also had non - specific ventricular repolarization disturbances . In 36 patients control ecgs were performed during hospitalization which proved normal or improved except in 4 patients with atrial fibrillation which did not regress during hospitalization . These patients probably had cardiac predisposition and clinical cardiac manifestation was incited by leptospirosis . In patients with and without ecg changes, the following serovars were most frequently detected: leptospira interrogans serovar australis, leptospira interrogans serovar saxkoebing and leptospira kirschneri serovar grippotyphosa . Ecg alterations and heart disorders indicate that leptospiras may exhibit tropism for myocardium and contribute directly or indirectly to cardiac disorders during mild, moderate or severe forms of leptospirosis . The presence of transient ecg abnormalities such as sinus tachycardia, non - specific ventricular repolarization disturbances, bundle branch or ventricular conduction disturbances and atrial fibrillation are common at the beginning of disease and possibly are caused by the direct effect of leptospires or febrile infection with a combination of metabolic and electrolyte abnormalities . Further research is required for better understanding of the pathogenesis of leptospiral infections and the mechanism of electrocardiographic alterations in leptospirosis, and to explore the possible relationship between electrocardiographic alterations and the prognosis of leptospirosis.
This retrospective, observational case series was performed at a single center according to the tenets of the declaration of helsinki . The present study included patients from the same cohort and used similar inclusion and exclusion criteria as were used in our previous study . A computerized search for patients who were newly diagnosed with exudative amd from september 2009 to november 2012 at our institution was conducted . Fovea - involving subretinal hemorrhages extending over at least 50% of the lesion area or at least three disc areas were included . Additionally, only patients who exhibited initial visual acuity of 20 / 30 or worse and who were treated with intravitreal ranibizumab were included . Patients who had completed two years or more of follow - up were included in the result analysis . All subjects underwent a comprehensive ophthalmologic examination, including best - corrected visual acuity (bcva) measurement, 90-diopter lens slit - lamp biomicroscopy, fundus photography, fluorescein angiography, and spectral domain optical coherence tomography (either spectral oct / slo, oti ophthalmic technologies, ontario, canada; or spectralis, heidelberg engineering, heidelberg, germany). Indocyanine green angiography was performed using a confocal laser - scanning system (hra-2, heidelberg engineering) at the discretion of each physician . The exclusion criteria included less than six months of follow - up, duration of symptoms longer than six months, severe media opacity, evidence of end - stage amd such as central geographic atrophy or disciform scarring, evidence of a macroaneurysm, proliferative diabetic retinopathy, central retinal vascular occlusion, or history of intraocular surgery other than cataract surgery . Eyes that had undergone pneumatic displacement or photodynamic therapy, as well as eyes that had received intravitreal injections of tissue plasminogen activator or photodynamic therapy during the follow - up period, were also excluded . The number of eyes that underwent vitrectomy or cataract surgery during the follow - up period due to the development of severe vitreous hemorrhage was recorded; however, all data pertaining to these eyes were excluded from the analyses . If a submacular hemorrhage developed in both eyes, the eye that was affected first was included; thus, only one eye was included for each patient . Visual acuities were converted to logarithm of minimal angle of resolution (logmar). As recommended by holla - day, " counting fingers " and " hand - motion " central foveal thickness was defined as the distance between the internal limiting membrane and bruch's membrane at the fovea and was manually measured using the calipers provided by an optical coherence tomography software program . We were concerned about the accuracy of hemorrhage - extent measurements exceeding 20 disc areas and central foveal thickness values exceeding 1,500 m and therefore set these as threshold values . Lesions exceeding these measurements were recorded as 20 discs or 1,500 m thickness, respectively . All extent - of - hemorrhage and central foveal thickness measurements were estimated by a single examiner (jhk). The indocyanine green angiography results were analyzed by two independent examiners (jhk and ysc). Cases of exudative amd were classified as typical exudative amd or polypoidal choroidal vasculopathy (pcv) based on the indocyanine green findings . Cases exhibiting branching vascular networks and/or terminating polypoidal lesions were diagnosed as pcv . In some cases, the presence of late geographic hyperfluorescence on indocyanine green angiography and/or the double - layer sign on optical coherence tomography were observed in association with a branching vascular network on indocyanine green angiography . All other cases were classified as typical exudative amd . For cases in which a definite initial diagnosis was not possible using indocyanine green angiography, indocyanine green angiography images collected within six months after diagnosis were reviewed . Patients were initially treated with either ranibizumab (lucentis; genentech, san francisco, ca, usa) or bevacizumab (avastin, genentech). As an initial treatment, 1 to 3 monthly intravitreal anti - vegf injections were administered . Following initial treatment, patients were scheduled to visit the hospital once every 1 to 4 months based on status . Optical coherence tomography examinations were performed once every 1 to 6 months at the discretion of the clinician . Retreatment with intravitreal anti - vegf usually occurred when intraretinal / subretinal fluid was present after the initial injections or when intraretinal / subretinal fluid or retinal / subretinal hemorrhage recurred and was accompanied by an increase in macular thickness . Some pcv cases were treated with photodynamic therapy at the discretion of the treating physician . Similar to our prior study, we also analyzed values of central foveal thickness, extent of hemorrhage, and bcva up to 12 months of follow - up . The bcva in the period between 12 months of follow - up and the final visit was newly measured in the present study . Baseline bcva, bcva at six months, bcva at 12 months, and bcva at the final visit were compared . Eyes with bcva of 20 / 40 or better, between 20 / 400 and 20 / 40, and 20 / 400 or worse were classified into the fair vision group, moderate vision group, and poor vision group, respectively . The distributions of eyes into the three groups at six months and at the final visit were compared . The associations of bcva at the final visit with baseline bcva, bcva at six months, symptom duration, hemorrhage extent, and central foveal thickness were analyzed . Values for symptom duration, central foveal thickness, hemorrhage extent, and number of anti - vegf injections were compared between the typical exudative amd group and the pcv group . Additionally, baseline bcva, bcva at six months, bcva at 12 months, and bcva at the final visit were compared within each group . The number of eyes that experienced a recurrence of fovea - involving submacular hemorrhage of at least one disc area during the follow - up period was noted . Baseline bcva and bcva at the final visit were compared between eyes that did and did not experience recurrent hemorrhage . Baseline characteristics, including age, diagnosis, central foveal thickness, and hemorrhage extent, were compared between the groups . The proportion of cases for each diagnosis (typical exudative amd vs. pcv vs. unclassified), baseline bcva, duration of symptoms, hemorrhage extent, and central foveal thickness were compared between the included eyes and the excluded eyes . The association between the number of anti - vegf injections received throughout the entire follow - up period in patient with bcva at the final visit and overall change in bcva during the follow - up period was also analyzed . Differences among various time points were analyzed using a repeated - measures analysis of variance, and individual comparisons were performed using bonferroni's method . Differences between groups were analyzed using an independent - samples t - test or a chi - square test . Baseline bcva, bcva at six months, bcva at 12 months, and bcva at the final visit were compared . Eyes with bcva of 20 / 40 or better, between 20 / 400 and 20 / 40, and 20 / 400 or worse were classified into the fair vision group, moderate vision group, and poor vision group, respectively . The distributions of eyes into the three groups at six months and at the final visit were compared . The associations of bcva at the final visit with baseline bcva, bcva at six months, symptom duration, hemorrhage extent, and central foveal thickness were analyzed . Values for symptom duration, central foveal thickness, hemorrhage extent, and number of anti - vegf injections were compared between the typical exudative amd group and the pcv group . Additionally, baseline bcva, bcva at six months, bcva at 12 months, and bcva at the final visit were compared within each group . The number of eyes that experienced a recurrence of fovea - involving submacular hemorrhage of at least one disc area during the follow - up period was noted . Baseline bcva and bcva at the final visit were compared between eyes that did and did not experience recurrent hemorrhage . Baseline characteristics, including age, diagnosis, central foveal thickness, and hemorrhage extent, were compared between the groups . The proportion of cases for each diagnosis (typical exudative amd vs. pcv vs. unclassified), baseline bcva, duration of symptoms, hemorrhage extent, and central foveal thickness were compared between the included eyes and the excluded eyes . The association between the number of anti - vegf injections received throughout the entire follow - up period in patient with bcva at the final visit and overall change in bcva during the follow - up period was also analyzed . Differences among various time points were analyzed using a repeated - measures analysis of variance, and individual comparisons were performed using bonferroni's method . Differences between groups were analyzed using an independent - samples t - test or a chi - square test . Among the 159 patients that initially presented with submacular hemorrhage, 91 satisfied all eligibility criteria and were followed - up for six months or longer . The six - month clinical outcomes of these patients were presented in a previous study . Among the 91 patients, 55 completed two or more years of follow - up . Two of these 55 patients underwent cataract surgery, two underwent vitrectomy, and two underwent photodynamic therapy between the first six months post - therapy and the final visit . Finally, 49 of the 91 enrolled patients (53.8%) were included in the analyses for this manuscript (table 1). Thirty of the patients (61.2%) were men and 19 (38.8%) were women . The mean age was 68.6 8.6 years (range, 51 to 86 years), and the mean symptom duration was 15.5 17.1 days (range, 1 to 90 days). The mean bcva was 1.40 0.52 (snellen equivalent, 20 / 502; range, counting fingers to 20 / 40). The mean hemorrhage extent was 7.7 6.1 disc areas (range, 3 to 20), and the mean central foveal thickness was 601.4 228.9 m (range, 331 to 1,300 m). The mean follow - up period was 32.1 8.5 months from diagnosis . During this period, patients were treated with 5.1 2.2 (range, 1 to 11) intravitreal anti - vegf injections . A mean of 3.7 1.2 injections were administered during the first 12 months, and a mean of 1.4 1.4 injections were administered during the period extending from the 12-month follow - up to the final visit . Thirty - five eyes were treated with ranibizumab only, another 14 eyes were treated with both ranibizumab and bevacizumab, and the single remaining eye was treated with bevacizumab only . All the included eyes received at least one intravitreal anti - vegf injection during the first 12 months . Thirty - one eyes (63.3%) received additional treatment at some point between the 12-month follow - up visit and the final follow - up visit . For the 42 excluded eyes, measurement values were: baseline bcva = 1.37 0.60 (snellen equivalent, 20 / 468; range, hand motion to 20 / 30), symptom duration = 41.8 51.9 days, hemorrhage extent = 7.8 5.2 disc areas, and central foveal thickness = 620.3 274.1 m . The group of excluded eyes had a significantly longer mean symptom duration than the included eyes (p = 0.003). None of the differences in baseline bcva, hemorrhage extent, and central foveal thickness between the two groups were significant (p = 0.744, p = 0.827, and p = 0.909, respectively). 1 shows a representative case of long - term change in the macular microstructure of an eye with submacular hemorrhage . The mean bcva values at baseline, six months post - diagnosis, 12 months post - diagnosis, and at the final visit were 1.40 0.52 (snellen equivalent, 20 / 502), 0.87 0.64 (snellen equivalent, 20 / 148), 0.88 0.68 (snellen equivalent, 20 / 151), and 1.03 0.83 (snellen equivalent, 20 / 214), respectively (fig . 2a). The mean bcva at the final visit showed significant improvement compared to the baseline value (p = 0.012), whereas the differences between the bcva values at six months and at 12 months were not significantly different from bcva at the final visit (p = 0.156 and p = 0.113, respectively). Compared to baseline values, a bcva improvement of three lines or more was noted in 28 eyes (57.1%) at the final visit . A deterioration of three or more lines was noted in nine eyes (18.4%). The remaining 12 eyes (24.5%) exhibited stable bcva throughout the follow - up period . Compared to the six - month values, a bcva improvement of three or more lines was noted in seven eyes (14.3%) at the final visit . A deterioration of three or more lines was noted in 15 eyes (30.6%). The remaining 27 eyes (55.1%) exhibited stable bcva during the follow - up period . The number of anti - vegf injections was not associated with bcva at the final visit (p = 0.470) or the degree of change in bcva during the follow - up period (p = 0.151). At six months, the numbers of eyes included in the fair vision group (bcva 20 / 40 or better), moderate vision group (bcva from 20 / 400 to 20 / 40), and poor vision group (20 / 400 or worse) were 15 (30.6%), 20 (40.8%), and 14 (28.6%), respectively . The mean number of eyes in these groups at the final visit were 15 (30.6%), 16 (32.7%), and 18 (36.7%), respectively . The distribution of eyes among the three groups was not different between the six - month and final visits (p = 0.766). When classified based on bcva at six months, six eyes (40.0%) in the fair vision group required additional treatment at some point between 12 months and the final follow - up . The number of eyes that received additional treatment was 15 (75.0%) in the moderate vision group and 10 (71.4%) in the poor vision group . The proportion of eyes that required additional treatment during the aforementioned period was not significantly different among the three groups (p = 0.065). In the fair vision group, only one eye (6.7%) experienced three lines of deterioration in bcva between the six - month visit and the final follow - up . In the remaining 14 eyes (93.3%), bcva remained stable throughout the entire follow - up period . Indocyanine green angiography results obtained at the time of diagnosis or within six months after diagnosis were available for all included eyes . Among them, 15 (30.6%) and 31 eyes (63.3%) were ultimately diagnosed with typical exudative amd and pcv, respectively . A definite diagnosis was not possible in the remaining three cases (6.1%) because a thick subretinal hemorrhage precluded obtainment of reliable indocyanine green angiography images of the lesion . In the typical exudative amd group, symptom duration, central foveal thickness, hemorrhage extent, and number of anti - vegf injections were 17.8 15.7 days, 543.5 213.4 m, 6.2 5.9 disc areas, and 5.5 2.5, respectively . In the pcv group, the values were 13.9 19.1 days, 637.5 239.9 m, 8.3 5.9 disc areas, and 4.9 2.1, respectively . There were no significant differences between the two groups for these four parameters (p = 0.503, p = 0.209, p = 0.264, and p = 0.406, respectively) (table 2). In the typical exudative amd group, the mean baseline bcva, at six months, at 12 months, and at the final visit were 1.49 0.49, 0.94 0.65, 0.97 0.77, and 1.13 0.87, respectively (fig . The values were 1.36 0.54, 0.80 0.64, 0.77 0.64, and 0.87 0.86, respectively . In unclassified eyes, the values were 1.43 0.56, 0.19 0.67, 1.20 0.62, and 1.28 0.53, respectively . Compared with the baseline value, bcva at six months was significantly improved for both the typical exudative amd (p = 0.031) and pcv (p = 0.001) groups . For the pcv group, the changes in bcva at 12 months (p <0.001) and at final follow - up (p = 0.007) compared to baseline were also statistically significant, but these same comparisons were not significant in the amd group (12 months, p = 0.177; final follow - up, p = 0.145). Both baseline bcva and bcva at six months were significantly associated with bcva at the final visit (p = 0.012 and p <0.001, respectively) (table 3). No other factors, including symptom duration, hemorrhage extent, and central foveal thickness, were associated with bcva at the final visit (p = 0.841, p = 0.083, and p = 0.121, respectively) (table 2). In multivariate analysis, bcva at six months was found to be the factor most strongly associated with bcva at the final visit (p <0.001) (table 2). Fifteen recurrences of fovea - involving submacular hemorrhage occurred in 13 eyes (26.5%) during the follow - up period (fig . Two of these eyes experienced multiple recurrences . On average, hemorrhages reoccurred by 15.0 11.1 months (range, 4 to 42) after diagnosis . Four (30.8%) and six eyes (46.2%) were diagnosed with typical exudative amd and pcv, respectively . The remaining three eyes (23.1%) were unclassified . The results of comparisons between eyes with and without hemorrhage recurrence are summarized in table 4 . There was no difference in baseline bcva (p = 0.304), bcva at the final visit (p = 0.709), diagnosis (p = 0.573), symptom duration (p = 0.361), central foveal thickness (p = 0.970), or hemorrhage extent (p = 0.509) between eyes with or without hemorrhage recurrence . 1 shows a representative case of long - term change in the macular microstructure of an eye with submacular hemorrhage . The mean bcva values at baseline, six months post - diagnosis, 12 months post - diagnosis, and at the final visit were 1.40 0.52 (snellen equivalent, 20 / 502), 0.87 0.64 (snellen equivalent, 20 / 148), 0.88 0.68 (snellen equivalent, 20 / 151), and 1.03 0.83 (snellen equivalent, 20 / 214), respectively (fig . The mean bcva at the final visit showed significant improvement compared to the baseline value (p = 0.012), whereas the differences between the bcva values at six months and at 12 months were not significantly different from bcva at the final visit (p = 0.156 and p = 0.113, respectively). Compared to baseline values, a bcva improvement of three lines or more was noted in 28 eyes (57.1%) at the final visit . A deterioration of three or more lines was noted in nine eyes (18.4%). The remaining 12 eyes (24.5%) exhibited stable bcva throughout the follow - up period . Compared to the six - month values, a bcva improvement of three or more lines was noted in seven eyes (14.3%) at the final visit . A deterioration of three or more lines was noted in 15 eyes (30.6%). The remaining 27 eyes (55.1%) exhibited stable bcva during the follow - up period . The number of anti - vegf injections was not associated with bcva at the final visit (p = 0.470) or the degree of change in bcva during the follow - up period (p = 0.151). At six months, the numbers of eyes included in the fair vision group (bcva 20 / 40 or better), moderate vision group (bcva from 20 / 400 to 20 / 40), and poor vision group (20 / 400 or worse) were 15 (30.6%), 20 (40.8%), and 14 (28.6%), respectively . The mean number of eyes in these groups at the final visit were 15 (30.6%), 16 (32.7%), and 18 (36.7%), respectively . The distribution of eyes among the three groups was not different between the six - month and final visits (p = 0.766). When classified based on bcva at six months, six eyes (40.0%) in the fair vision group required additional treatment at some point between 12 months and the final follow - up . The number of eyes that received additional treatment was 15 (75.0%) in the moderate vision group and 10 (71.4%) in the poor vision group . The proportion of eyes that required additional treatment during the aforementioned period was not significantly different among the three groups (p = 0.065). In the fair vision group, only one eye (6.7%) experienced three lines of deterioration in bcva between the six - month visit and the final follow - up . In the remaining 14 eyes (93.3%), bcva remained stable throughout the entire follow - up period . Indocyanine green angiography results obtained at the time of diagnosis or within six months after diagnosis were available for all included eyes . Among them, 15 (30.6%) and 31 eyes (63.3%) were ultimately diagnosed with typical exudative amd and pcv, respectively . A definite diagnosis was not possible in the remaining three cases (6.1%) because a thick subretinal hemorrhage precluded obtainment of reliable indocyanine green angiography images of the lesion . In the typical exudative amd group, symptom duration, central foveal thickness, hemorrhage extent, and number of anti - vegf injections were 17.8 15.7 days, 543.5 213.4 m, 6.2 5.9 disc areas, and 5.5 2.5, respectively . In the pcv group, the values were 13.9 19.1 days, 637.5 239.9 m, 8.3 5.9 disc areas, and 4.9 2.1, respectively . There were no significant differences between the two groups for these four parameters (p = 0.503, p = 0.209, p = 0.264, and p = 0.406, respectively) (table 2). In the typical exudative amd group, the mean baseline bcva, at six months, at 12 months, and at the final visit were 1.49 0.49, 0.94 0.65, 0.97 0.77, and 1.13 0.87, respectively (fig . The values were 1.36 0.54, 0.80 0.64, 0.77 0.64, and 0.87 0.86, respectively . In unclassified eyes, the values were 1.43 0.56, 0.19 0.67, 1.20 0.62, and 1.28 0.53, respectively . Compared with the baseline value, bcva at six months was significantly improved for both the typical exudative amd (p = 0.031) and pcv (p = 0.001) groups . For the pcv group, the changes in bcva at 12 months (p <0.001) and at final follow - up (p = 0.007) compared to baseline were also statistically significant, but these same comparisons were not significant in the amd group (12 months, p = 0.177; final follow - up, p = 0.145). Both baseline bcva and bcva at six months were significantly associated with bcva at the final visit (p = 0.012 and p <0.001, respectively) (table 3). No other factors, including symptom duration, hemorrhage extent, and central foveal thickness, were associated with bcva at the final visit (p = 0.841, p = 0.083, and p = 0.121, respectively) (table 2). In multivariate analysis, bcva at six months was found to be the factor most strongly associated with bcva at the final visit (p <0.001) (table 2). Fifteen recurrences of fovea - involving submacular hemorrhage occurred in 13 eyes (26.5%) during the follow - up period (fig . 3). Two of these eyes experienced multiple recurrences . On average, hemorrhages reoccurred by 15.0 11.1 months (range, 4 to 42) after diagnosis . Four (30.8%) and six eyes (46.2%) were diagnosed with typical exudative amd and pcv, respectively . The remaining three eyes (23.1%) were unclassified . The results of comparisons between eyes with and without hemorrhage recurrence are summarized in table 4 . There was no difference in baseline bcva (p = 0.304), bcva at the final visit (p = 0.709), diagnosis (p = 0.573), symptom duration (p = 0.361), central foveal thickness (p = 0.970), or hemorrhage extent (p = 0.509) between eyes with or without hemorrhage recurrence . In this study, visual acuity in the majority of eyes that initially presented with submacular hemorrhage had improved or remained relatively stable between six months after diagnosis and the final visit (mean, 32.1 months). As a result, this result suggests that, although the development of submacular hemorrhage induces retinal damage, any remaining retinal function can be maintained long - term through continuous anti - vegf therapy . Notably, the number of injections received between 12 months post - diagnosis and the final visit was markedly lower than the number of injections received during the first 12 months after diagnosis . The mean number of injections after 12 months was far less than that administered during the 12- to 24-month period in prior controlled clinical trials, despite our use of a longer time interval between the 12-month follow - up point and the final visit (20.1 months). The lower injection frequency after 12 months may reflect several factors . To facilitate the early detection and prompt treatment of recurrent exudation in patients with exudative amd, it is generally recommended to perform monthly follow - ups, including monthly oct examinations . In the present study, oct examination was not routinely performed during every visit, and the follow - up period varied from 1 to 4 months at the discretion of the treating physician . Moreover, the re - accumulation of small amounts of intraretinal / subretinal fluid may have been missed . Some patients, particularly those with very low visual acuity, refused additional treatment without a guarantee that their vision would subsequently improve . Similarly, treating physicians decided not to administer additional treatments in some cases when no definite benefit was anticipated . Regardless of these possible explanations, however, the findings in patients with relatively good visual acuity (20 / 40 or better) at six months after diagnosis are noteworthy . In this group, almost all the eyes maintained relatively stable bcva, although only 40% of them required additional treatment . We postulate that relatively large, active vascular lesions, such as major polyps, may contribute to frequent re - accumulation of intraretinal / subretinal fluid that may have ruptured . Although other vascular abnormalities persisted, the activity of the entire exudative amd lesion may have decreased after the rupture of a major vascular lesion and thus reduced the frequency of recurrent exudation . In a study by hwang et al . That evaluated the incidence of recurrent submacular hemorrhage in exudative amd, the recurrence rate was 51.1% during the follow - up period (mean, 36.8 months). That rate is much higher than the rate reported in the present study (26.5%). A direct comparison between this study and that of hwang et al . May not be appropriate because they evaluated various treatment modalities, including photodynamic therapy, intravitreal anti - vegf, pneumatic displacement, and vitrectomy, whereas all the patients in the present study were treated with only intravitreal anti - vegf monotherapy . In hwang et al ., the use of intravitreal anti - vegf was associated with a reduced risk of recurrent hemorrhage . The relatively low recurrence rate found in the present study may also suggest a potential role of anti - vegf monotherapy in preventing recurrent hemorrhage . Most hemorrhage recurrences developed within two years of diagnosis . However, some recurrences occurred after more than three years, suggesting that a lack of recurrence over this relatively long - term period may not guarantee complete stabilization of the lesion . We also found a strong association between bcva at six months and bcva at the final visit . Bcva at six months was more closely associated with bcva at the final visit than with baseline bcva . A subretinal hemorrhage typically resolves or at least decreases markedly in size during the first six months . Because a hemorrhage can block the visual stimulus, bcva at six months rather than baseline bcva may more accurately reflect underlying retinal function . In approximately two - thirds of eyes, visual acuity at six months was relatively unchanged from baseline or even improved over the course of a mean 32.1 months of follow - up . As a result, bcva at the final visit had not significantly decreased compared to the values at six months post - diagnosis . This suggests that the sixmonth visual acuity measurement can be used as a reference value in any discussion with patients regarding long - term visual prognosis . More specifically, a favorable long - term visual outcome was achieved only in the pcv group . Pcv is a distinct entity from typical exudative amd, and the incidence of pcv is generally higher in asian populations than in european populations . While anti - vegf monotherapy has been considered the most effective first - line therapy for typical exudative amd, its efficacy in pcv is still a matter of controversy . Although anti - vegf therapy has been found to be effective in previous studies, its efficacy is generally inferior to that of photodynamic therapy for polyp regression . For this reason, some experts recommend photodynamic therapy rather than anti - vegf monotherapy as first - line therapy in pcv . Despite this controversy, the treatment outcomes of anti - vegf monotherapy in our patients with pcv were encouraging . As discussed above, it is possible that this favorable outcome may be partially in response to the rupture of a major polyp after a hemorrhage . Although marked improvement in bcva was noted during the first six months, deterioration of visual acuity typically occurred thereafter . As a result, bcva values at 12 months and later were not different from those at baseline . Although statistical significance was not reached, symptom duration in the typical exudative amd group was relatively longer than that of the pcv group, suggesting more accumulated damage to the retina in the amd group . Another reason may be a possible difference in the degree of retinal degeneration between typical exudative amd and pcv patients . It is well known that age - related degenerative changes in the retina, including drusen and pseudodrusen, are more markedly present in typical exudative amd cases than in pcv cases . Additionally, the choroid of eyes with typical exudative amd is generally thinner than that of eyes with pcv . We think that retinas with pre - existing degeneration may be more vulnerable to damage resulting from the development of subretinal hemorrhage . Although testing these hypotheses is beyond the scope of this study, the less favorable outcomes associated with typical exudative amd suggest the need for other treatments (e.g., pneumatic displacement). Further studies are needed to establish the appropriate treatment method to improve long - term outcomes of submacular hemorrhages that occur secondary to typical exudative amd . First, it was a retrospective study and included only a small number of patients who exhibited submacular hemorrhage as an initial presentation . Second, there was no common treatment protocol or follow - up / optical coherence tomography examination schedule . Because optical coherence tomography, fluorescein angiography, and indocyanine green angiography were not routinely performed during the follow - up period, the longterm anatomical outcomes were not analyzed . In conclusion, relatively stable long - term visual outcome can be achieved for the majority of eyes exhibiting submacular hemorrhage as a secondary symptom to exudative amd when treated with intravitreal anti - vegf monotherapy, despite recurrence of exudation and/or submacular hemorrhage . Bcva at six months was found to be a useful clinical index predictive of long - term visual prognosis.
These injuries affect a large number of americans: in this country, craniofacial injuries make up more than 10% of all annual emergency room visits1 . Furthermore, in recent conflicts in the middle east, craniofacial injuries account for more than one - quarter of all injuries sustained by us soldiers23 . A number of other situations can also lead to the need for mandibular reconstruction or augmentation; for example, periodontal disease, congenital defects, tumor resection of the mandible, and atrophy of the alveolar ridge due to tooth loss45 . Often, mandibular reconstructions are complex and require multiple procedures6 . To maximize the quality of life for these patients, full restoration of mandible function includes the ability to eat normally; this often relies on the placement of dental implant restorations in proper occlusion . For patients undergoing mandibular reconstruction, one significant challenge faced is that the bone does not heal to pre - injury volume; the height and/or width of the regenerated mandibular bone can be insufficient for implant restorations . Ideally, the implant should be encompassed by at least 1 mm of alveolar bone in order to properly support the prosthesis7 . In addition to bone quantity, bone quality is an essential component contributing to implant success8 . Unfortunately, none of these treatments are reliable enough to be considered the single clinical gold - standard5910 . In order to develop improved therapies for craniofacial bone regeneration, it is important to gain a better understanding of the mechanisms involved in the healing process . To ensure safety and efficacy of novel craniofacial - specific biomaterials prior to clinical trials, promising materials are often tested for anatomically appropriate efficacy in a larger pre - clinical animal model with comparable mandibular size and dentition to humans . Testing therapies in these relevant models allows us to be more confident that findings will be clinically translatable . Furthermore, by standardizing the large animal models used across the field, results from numerous studies can be easily compared . Researchers have historically used dogs, goats, sheep, or pigs as a large animal model to perform pre - clinical craniomaxillofacial (cmf) bone healing trials; with each model having specific advantages and drawbacks11 . Goats121314 and sheep1516171819 have been used in numerous studies to further the understanding of craniofacial bone healing . However, the dentition of these animals is characterized by elongated tooth roots, continuously erupting teeth, and herbivorous chewing pattern, which are quite different from humans . Dogs have been used extensively to study bone healing in mandibular defects, including the use of notch - type defects at the inferior margin202122 . Dogs have similar dentition to humans and are considered a good model to study maxillofacial bone healing23 . Even so, they can be expensive; especially when breed - matched studies are performed . Furthermore, ethical concerns regarding the use of companion animals in medical research have prompted many groups to seek alternative models11 . The pig is an attractive model because its mandible closely resembles the human mandible with regard to anatomy, morphology, healing, bone composition, bone remodeling and dentition2425 . Mandibular defect models in pigs are used to evaluate the efficacy of promising new biomaterials26; however, some inconsistency has been noted in the literature as to the size of the defect that defines a critical - sized defect (csd) in these models . The non - segmental bone notch defect model is one such pig model that is useful in evaluating bone healing due to the fact that it mimics localized edentulous bone atrophy . A number of studies have selected a 5 cm defect as the csd to evaluate bone healing potential of various therapies2728 . However, a subsequent report suggests that a 5 cm defect may not be stringent enough to be defined as a csd . That study found that, with the creation of a resected bone block (~10.1 cm) in the anterior alveolar region with periosteal preservation, the defect showed spontaneous regeneration via a normal physiologic response29 . Ma et al.30 demonstrated that segmental defects as large as 6 cm in length in the presence of the periosteum or 2 cm in length when the periosteum was resected could be considered csds in the pig mandible . These results provide further support for the idea that the periosteum plays a key role in bone regeneration in large defects . There is a significant need to develop bone regenerative therapies to restore mandibular and craniofacial defects in a predictable manner . As such, it is essential that our preclinical models are appropriate and stringent for testing these novel therapies so that we have confidence in our study results . Due to the inconsistencies in the available literature regarding the definition and characterization of csd healing in the miniature pig mandibular defect model, further investigation of a non - healing notch defect, we sought to define healing in a surgically - created mandibular notch defect measuring a minimum of 321 cm (volume=6 cm) with adjacent periosteal stripping in sinclair miniature pigs . We hypothesized that this model would mimic a similar human mandibular injury and would not heal without intervention . Upon defining this model as a csd model, studies can then move forward to test promising bone regenerative therapeutics in the cmf region and justify their translation for use in human clinical studies . To evaluate the healing potential in mandibular bone defects, either with no restoration (negative control) or with autologous bone graft restoration (clinical standard), notch defects 6 cm in volume were created bilaterally in five dentally - mature sinclair miniature pigs (> 1 year of age). Bone healing was monitored for 16 weeks; in vivo radiographic assessments were performed prior to surgery, 4 weeks post - surgery, and 16 weeks post - surgery . This research study was approved by the institutional animal care and use committee at the united states army institute of surgical research (no . A-11 - 017). A schematic representation of the surgical protocol is shown in fig . 1 . A. bilateral mandible defects were created using an extra - oral surgical approach; specifically, the defects were anatomically located anterior to the antegonial notch and posterior to the mental foramen . Approximately 24 hours prior to surgery, a fentanyl transdermal patch (100 g / hr) was applied to ensure appropriate levels of peri- and post - surgery analgesia . Thirty minutes prior to surgery, telazol (4.4 mg / kg intramuscular; zoetis, florham park, nj, usa) was administered as anesthetic induction . Following anesthetic induction, prophylactic antibiotics (cefazolin, 1 g) were administered, and the surgery site was prepared to ensure sterility . A skin incision was made parallel to the inferior border on both sides of the mandible, and the skin was reflected . Using a reciprocating bone saw cooled with copious sterile saline, right and left mandibular osseous defects were created, followed by proximal periosteum dissection. (fig . C) these defects were approximately 6 cm in volume (anterior - posterior=3 cm, buccal - lingual=1 cm, inferior border - height of contour=2 cm). (fig . D - f) to aid in histological analysis, gutta - percha was placed into small holes created in the cortical bone at the defect borders . Bone sectioned from each side was ground in the r. qutin bone - mill (qutin dental - products, leimen, germany) to morselize both the cortical and cancellous bone. (fig . H) the morselized bone from both sides was combined, hydrated with sterile saline, compressed (fig . J) a small amount of morselized bone was reserved for scanning electron microscopy evaluation of particle size . K) all defects were plated using a 2.7 mm reconstruction plate with 2.7 mm self - tapping cortex screws (synthes vet, west chester, pa, usa). (fig . K) prior to reapproximating the tissue layers with resorbable 3 - 0 vicryl sutures (ethicon us llc, somerville, nj, usa), a jackson - pratt drain was placed into the surgical wound site . Next, 64-slice computed tomography (ct) scans (aquilion 64 multislice helical ct scanner; toshiba american medical, tustin, ca, usa) were performed under anesthesia (following the above described anesthesia protocol) immediately prior to surgery and 4 and 16 weeks post - surgery . Following the 16 week final ct scan, the pigs were euthanized via intravenously administered fatal - plus (1 ml/4.5 kg; vortech pharmaceuticals, dearborn, mi, usa). Sixty - four slice ct scan images were acquired at an effective pixel size of 500 m, and three - dimensional rendering of the image sets was performed using a standard " bone mask " in vitrea core (version 6.3; vital, minnetonka, mn, usa). The clinical volumes were then upsampled using tri - linear interpolation at a factor of 5 (imagej version 1.47; national institutes of health, bethesda, md, usa) so that the resolution was 100 m31 . Dataviewer (skyscan, kontich, belgium) was used to re - align the mandibles along the physiological axes, and dentition landmarks were used to ensure that the scans at different time points were all aligned in an identical fashion . The image stack was then imported into ctan software version 1.11 (skyscan), and a region of interest (roi) was created to cover the defect site . The region was defined by the contour of the intact bone from the pre - surgical scan and was fixed within each animal for evaluation at 4 and 16 weeks. (fig . A) the defect site roi spanned an average of 3 cm long, 1 cm deep, and 2 cm wide (full thickness). A threshold of 659 mgha / ml was selected across all samples using the otsu algorithm32 to distinguish mineralized tissue from unmineralized tissue within the defect . A secondary roi was defined over the same length in each pig to evaluate any changes in bone morphology above the notch (intact bone excluding teeth, as outlined with a dashed line in fig . The bone volume fraction and bone mineral density of the mineralized tissue were calculated within each roi for each sample using ctan . After the 16 week harvest, the mandibles were hydrated with formalin, and micro - computed tomography (ct) analysis was performed using a skyscan 1072 scanner (bruker microct, kontich, belgium) at a resolution of 36 m / pixel . The images were reconstructed using nrecon software (bruker microct) to generate grayscale images . Dataviewer was used to re - align the mandible images using dentition landmark registration to the 64-slice ct data for consistency . A uniform threshold for bone the excised mandible sections were prepared for histology by dehydration in ascending concentrations of ethanol, followed by xylene at 4 and then embedding in poly(methyl methacrylate). The specimens were then cut and ground to 30-m thick sections using a diamond saw and microgrinder (exakt technologies, oklahoma city, ok, usa). The sections were mounted on slides, stained with sanderson's rapid bone stain, then counterstained with van gieson's picrofuchsin to stain soft tissue blue and stain bone pink / red . Next, 2.0 magnification histology slide images were acquired on an olympus szx16 research high - class stereo microscope (olympus, center valley, pa, usa) with an olympus dp71 microscope digital camera and compiled using photoshop (version 7.0.1; adobe systems inc ., san jose, ca, usa). High magnification images (40 and 100) were acquired on a nikon eclipse 55i research microscope with a ds - f11 digital camera (nikon instruments inc ., significance in 64-slice ct measures was determined using a two - way anova (across time and experimental group) and tukey's test for post - hoc evaluation when significance was found (sigmaplot version 11.0; systat software inc ., san jose, ca, usa). In the case of the ct data, to evaluate the healing potential in mandibular bone defects, either with no restoration (negative control) or with autologous bone graft restoration (clinical standard), notch defects 6 cm in volume were created bilaterally in five dentally - mature sinclair miniature pigs (> 1 year of age). Bone healing was monitored for 16 weeks; in vivo radiographic assessments were performed prior to surgery, 4 weeks post - surgery, and 16 weeks post - surgery . This research study was approved by the institutional animal care and use committee at the united states army institute of surgical research (no . A-11 - 017). A schematic representation of the surgical protocol is shown in fig . 1 . Bilateral mandible defects were created using an extra - oral surgical approach; specifically, the defects were anatomically located anterior to the antegonial notch and posterior to the mental foramen . Approximately 24 hours prior to surgery, a fentanyl transdermal patch (100 g / hr) was applied to ensure appropriate levels of peri- and post - surgery analgesia . Thirty minutes prior to surgery, telazol (4.4 mg / kg intramuscular; zoetis, florham park, nj, usa) was administered as anesthetic induction . Isoflurane (1.5%-4.0%) and oxygen were then used to maintain anesthesia . Following anesthetic induction, prophylactic antibiotics (cefazolin, 1 g) a skin incision was made parallel to the inferior border on both sides of the mandible, and the skin was reflected . Using a reciprocating bone saw cooled with copious sterile saline, right and left mandibular osseous defects were created, followed by proximal periosteum dissection. (fig . C) these defects were approximately 6 cm in volume (anterior - posterior=3 cm, buccal - lingual=1 cm, inferior border - height of contour=2 cm). (fig . D - f) to aid in histological analysis, gutta - percha was placed into small holes created in the cortical bone at the defect borders . Bone sectioned from each side was ground in the r. qutin bone - mill (qutin dental - products, leimen, germany) to morselize both the cortical and cancellous bone. (fig . H) the morselized bone from both sides was combined, hydrated with sterile saline, compressed (fig . J) a small amount of morselized bone was reserved for scanning electron microscopy evaluation of particle size . K) all defects were plated using a 2.7 mm reconstruction plate with 2.7 mm self - tapping cortex screws (synthes vet, west chester, pa, usa). (fig . K) prior to reapproximating the tissue layers with resorbable 3 - 0 vicryl sutures (ethicon us llc, somerville, nj, usa), a jackson - pratt drain was placed into the surgical wound site . Next, 64-slice computed tomography (ct) scans (aquilion 64 multislice helical ct scanner; toshiba american medical, tustin, ca, usa) were performed under anesthesia (following the above described anesthesia protocol) immediately prior to surgery and 4 and 16 weeks post - surgery . Following the 16 week final ct scan, the pigs were euthanized via intravenously administered fatal - plus (1 ml/4.5 kg; vortech pharmaceuticals, dearborn, mi, usa). Sixty - four slice ct scan images were acquired at an effective pixel size of 500 m, and three - dimensional rendering of the image sets was performed using a standard " bone mask " in vitrea core (version 6.3; vital, minnetonka, mn, usa). The clinical volumes were then upsampled using tri - linear interpolation at a factor of 5 (imagej version 1.47; national institutes of health, bethesda, md, usa) so that the resolution was 100 m31 . Dataviewer (skyscan, kontich, belgium) was used to re - align the mandibles along the physiological axes, and dentition landmarks were used to ensure that the scans at different time points were all aligned in an identical fashion . The image stack was then imported into ctan software version 1.11 (skyscan), and a region of interest (roi) was created to cover the defect site . The region was defined by the contour of the intact bone from the pre - surgical scan and was fixed within each animal for evaluation at 4 and 16 weeks. (fig . A) the defect site roi spanned an average of 3 cm long, 1 cm deep, and 2 cm wide (full thickness). A threshold of 659 mgha / ml was selected across all samples using the otsu algorithm32 to distinguish mineralized tissue from unmineralized tissue within the defect . A secondary roi was defined over the same length in each pig to evaluate any changes in bone morphology above the notch (intact bone excluding teeth, as outlined with a dashed line in fig . The bone volume fraction and bone mineral density of the mineralized tissue were calculated within each roi for each sample using ctan . After the 16 week harvest, the mandibles were hydrated with formalin, and micro - computed tomography (ct) analysis was performed using a skyscan 1072 scanner (bruker microct, kontich, belgium) at a resolution of 36 m / pixel . The images were reconstructed using nrecon software (bruker microct) to generate grayscale images . Dataviewer was used to re - align the mandible images using dentition landmark registration to the 64-slice ct data for consistency . A uniform threshold for bone was determined across all samples using the otsu algorithm and the bone defect rois . The excised mandible sections were prepared for histology by dehydration in ascending concentrations of ethanol, followed by xylene at 4 and then embedding in poly(methyl methacrylate). The specimens were then cut and ground to 30-m thick sections using a diamond saw and microgrinder (exakt technologies, oklahoma city, ok, usa). The sections were mounted on slides, stained with sanderson's rapid bone stain, then counterstained with van gieson's picrofuchsin to stain soft tissue blue and stain bone pink / red . Next, 2.0 magnification histology slide images were acquired on an olympus szx16 research high - class stereo microscope (olympus, center valley, pa, usa) with an olympus dp71 microscope digital camera and compiled using photoshop (version 7.0.1; adobe systems inc . High magnification images (40 and 100) were acquired on a nikon eclipse 55i research microscope with a ds - f11 digital camera (nikon instruments inc ., significance in 64-slice ct measures was determined using a two - way anova (across time and experimental group) and tukey's test for post - hoc evaluation when significance was found (sigmaplot version 11.0; systat software inc ., san jose, ca, usa). In the case of the ct data, a paired t - test analysis was used to determine significant difference . In order to evaluate the healing potential of a defined mandibular bone notch defect, we introduced a 7.5 cm notch (average size as determined by ct analysis) on each side of the mandible in each of 5 dentally mature sinclair miniature pigs . The bone removed during both the right and the left defect creations was morselized, combined, and then placed into the left side defect, serving as autograft . The average size of the morselized particles was found to be 670 m by scanning electron microscopy analysis . Bone healing in the pigs was assessed at 4 weeks post - surgery via ct scans and at 16 weeks post - surgery using ct, ct, and histological techniques . The use of clinical ct scans allowed us to monitor bone healing throughout the course of the experiment and compare bone regeneration at multiple time points . Sixty - four slice ct scans were taken at 3 time points: immediately before the defect creation, at 4 weeks post - surgery, and at 16 weeks post - surgery . The bone volume to tissue volume 2 . A; outlined in dotted line) at 4 weeks post - surgery, measurement of bone volume based on ct scan data revealed an absence of mineralized tissue within both the autograft - treated (180.746.5 mm) and untreated defect sites (265.3111.7 mm) in all pigs . In both defect sites at 4 weeks, measurement of the bv / tv ratio showed significantly less bone volume compared to both the pre - surgery and 16 weeks post - surgery time points (p<0.001). (fig . 2 . B) at 16 weeks post - surgery, bone volume measurements showed substantial bone regeneration in both defects: the mean bone volume measured 2,487.2507.8 mm in the autograft - treated defect sites and 1,665.0335.7 mm in the untreated defect sites . Moreover, in both sites, bone volume was approaching pre - surgical bone volume value (2,114.1 175.9 mm). Irrespective of treatment, no significant intragroup differences in bone volume were found between presurgery and 16 weeks post - surgery time points . However, at 16 weeks, significantly higher bone volume was regenerated in the autograft - treated defects compared with the untreated defects (p = 0.013). (fig . B) these same trends in bone regeneration were observed in three - dimensional reconstructions of each hemi - mandible from a representative animal . 4) for the purpose of obtaining higher resolution data regarding the bone healing in this model, ct imaging was used to analyze bv / tv ratio in the defect space post - mortem . A. in the images of both the autograft - treated and untreated groups, significant bone growth was evident in the defect space . The superior margins of the defect were identifiable by white gutta - percha markers (indicated by the two centermost single white dots). Greater bone regeneration was seen at the posterior interface compared to the anterior interface of the notch in both groups; this result was observed in all 5 pigs . Quantification of the bv / tv ratio indicated that there was no significant difference between the autograft - treated and the untreated groups; however, similar to the ct results, more bone was regenerated in the autograft - treated defect compared to the non - treated defect (p=0.46). (fig . B) furthermore, the results of the ct analysis were consistent with the clinical ct analysis in demonstrating significant bone volume regeneration in both the treated and untreated defect spaces . A high correlation exists between the ct and ct results (r=0.83). In order to detect calcified bone, fibrous tissue formation, and the presence of cellular activity, histological analysis was performed on the regenerated mandibular bone . For both the treated and untreated defects, the histological slides revealed intramembranous trabecular - like healing into the defect from the anterior, posterior, and superior walls. (fig . 6) in general, the autograft - treated defects had a greater extent of thin trabecular spindles penetrating deep into the defect space . The non - treated defects showed less bony invasion into the defect space; however, the trabecular - like in - growth was observed to occur in dense bone fronts. (fig . 6) in all pigs, neither defect spaces contain healed dense bone bridging of the inferior cortex or restoration of the bony anatomy to its pre - surgical condition . During the histological analysis process, we noted significant bone regeneration in the marrow space, superior to the initial defect . This observation led to the further investigation of the bone remodeling adjacent to the defect sites . First, analysis of the 64-slice ct scans was performed using an alternate roi encompassing intact bone. (fig . 3 . A; outlined in dashed lines) compared to pre - surgery values, the roi superior to the surgical site had a significant increase in bone volume fraction at 4 weeks post - surgery (p=0.013). (fig . B) at 16 weeks post - surgery, there was a further significant increase in bone volume in the superior site (p<0.001). However, the increase in bone volume in this superior roi was associated with a decrease in bone mineral density: these values were significantly lower after 16 weeks compared to preoperative levels (p=0.005). (fig . C) together, these results suggest that the bone did in fact remodel to increase load - bearing ability and compensate for the lack of bone in the defect area . Consistent with the ct analysis of the defect - superior roi, the histological slides showed that bone invaded the marrow space superior to all defect sites, regardless of the treatment (apparent in fig . In order to evaluate the healing potential of a defined mandibular bone notch defect, we introduced a 7.5 cm notch (average size as determined by ct analysis) on each side of the mandible in each of 5 dentally mature sinclair miniature pigs . The bone removed during both the right and the left defect creations was morselized, combined, and then placed into the left side defect, serving as autograft . The average size of the morselized particles was found to be 670 m by scanning electron microscopy analysis . Bone healing in the pigs was assessed at 4 weeks post - surgery via ct scans and at 16 weeks post - surgery using ct, ct, and histological techniques . The use of clinical ct scans allowed us to monitor bone healing throughout the course of the experiment and compare bone regeneration at multiple time points . Sixty - four slice ct scans were taken at 3 time points: immediately before the defect creation, at 4 weeks post - surgery, and at 16 weeks post - surgery . The bone volume to tissue volume 2 . A; outlined in dotted line) at 4 weeks post - surgery, measurement of bone volume based on ct scan data revealed an absence of mineralized tissue within both the autograft - treated (180.746.5 mm) and untreated defect sites (265.3111.7 mm) in all pigs . In both defect sites at 4 weeks, measurement of the bv / tv ratio showed significantly less bone volume compared to both the pre - surgery and 16 weeks post - surgery time points (p<0.001). (fig . 2 . B) at 16 weeks post - surgery, bone volume measurements showed substantial bone regeneration in both defects: the mean bone volume measured 2,487.2507.8 mm in the autograft - treated defect sites and 1,665.0335.7 mm in the untreated defect sites . Moreover, in both sites, bone volume was approaching pre - surgical bone volume value (2,114.1 175.9 mm). Irrespective of treatment, no significant intragroup differences in bone volume were found between presurgery and 16 weeks post - surgery time points . However, at 16 weeks, significantly higher bone volume was regenerated in the autograft - treated defects compared with the untreated defects (p = 0.013). (fig . B) these same trends in bone regeneration were observed in three - dimensional reconstructions of each hemi - mandible from a representative animal . For the purpose of obtaining higher resolution data regarding the bone healing in this model, ct imaging was used to analyze bv / tv ratio in the defect space post - mortem . A. in the images of both the autograft - treated and untreated groups, significant bone growth was evident in the defect space . The superior margins of the defect were identifiable by white gutta - percha markers (indicated by the two centermost single white dots). Greater bone regeneration was seen at the posterior interface compared to the anterior interface of the notch in both groups; this result was observed in all 5 pigs . Quantification of the bv / tv ratio indicated that there was no significant difference between the autograft - treated and the untreated groups; however, similar to the ct results, more bone was regenerated in the autograft - treated defect compared to the non - treated defect (p=0.46). (fig . B) furthermore, the results of the ct analysis were consistent with the clinical ct analysis in demonstrating significant bone volume regeneration in both the treated and untreated defect spaces . In order to detect calcified bone, fibrous tissue formation, and the presence of cellular activity, histological analysis was performed on the regenerated mandibular bone . For both the treated and untreated defects, the histological slides revealed intramembranous trabecular - like healing into the defect from the anterior, posterior, and superior walls. (fig . 6) in general, the autograft - treated defects had a greater extent of thin trabecular spindles penetrating deep into the defect space . The non - treated defects showed less bony invasion into the defect space; however, the trabecular - like in - growth was observed to occur in dense bone fronts. (fig . 6) in all pigs, neither defect spaces contain healed dense bone bridging of the inferior cortex or restoration of the bony anatomy to its pre - surgical condition . During the histological analysis process, we noted significant bone regeneration in the marrow space, superior to the initial defect . This observation led to the further investigation of the bone remodeling adjacent to the defect sites . First, analysis of the 64-slice ct scans was performed using an alternate roi encompassing intact bone. (fig . 3 . A; outlined in dashed lines) compared to pre - surgery values, the roi superior to the surgical site had a significant increase in bone volume fraction at 4 weeks post - surgery (p=0.013). (fig . B) at 16 weeks post - surgery, there was a further significant increase in bone volume in the superior site (p<0.001). However, the increase in bone volume in this superior roi was associated with a decrease in bone mineral density: these values were significantly lower after 16 weeks compared to preoperative levels (p=0.005). (fig . C) together, these results suggest that the bone did in fact remodel to increase load - bearing ability and compensate for the lack of bone in the defect area . Consistent with the ct analysis of the defect - superior roi, the histological slides showed that bone invaded the marrow space superior to all defect sites, regardless of the treatment (apparent in fig . To ensure stringent pre - clinical evaluation of the healing capability of a novel bone regenerative biomaterial, ideally, the therapy is tested in a standardized csd model . Due to the increasing reluctance to use companion animals for preclinical research and the stark differences between the masticatory biomechanics of sheep or goats and humans, cmf models in pigs have been favored . Pigs demonstrate similarity to human bone in form, function, bone healing characteristics, and bone mineral density29 . Still, the consensus on defining what constitutes a full thickness notch type model in the pig mandible has been limited . Plug type bone defects ranging (diameterdepth) from 48 mm33 to 94 mm34 or 95 mm35 or notch type defects ranging in size from 2020 mm36 to 2010 mm37 have all been evaluated in the minipig mandible . These observations have been primarily histological in nature with limited quantifiable metrics to demonstrate differences between the healing patterns of autologous grafts (positive controls) and sham treated (negative controls) groups . Spontaneous healing of the mandible post bone resection has also been clinically observed in patients383940 . This underlines the need for better characterization of healing patterns in the pig mandible models so that standardized interpretation of bone healing responses to novel bone graft materials is possible . In the current study, we aimed to validate the premise that a mandibular bone notch at least 6 cm in volume in sinclair miniature pigs can serve as a csd . The presence of considerable bone in the untreated defect (average size in this study was 7.5 cm) at 16 weeks indicates that this model does not meet the criteria to be defined as a csd . However, the absence of a significant healing response in either treatment group after 4 weeks suggests that this notch model could be used to investigate bone graft therapies that aim to accelerate the bone healing response . As a potential strategy to induce better bone regeneration by controlled mechanical stimulation, there is an increasingly greater emphasis on immediate implant placement and early loading41; techniques to accelerate new bone regeneration could prove critical to successful outcomes . In this study, measurement of bone regeneration indicated that both autograft - treated and untreated mandibular defects had very similar bone healing profiles . Minimal mineralization had occurred in both the autograft - treated and the untreated notch defect sites over 4 weeks, followed by significant bone regeneration at 16 weeks . These observations suggest that, during the initial 4 week time period, the majority of the autograft was resorbed . Since histological analyses were not performed at 4 weeks, we cannot confirm if the autograft particles were undergoing fragmented remodeling, complete resorption, or only demineralization, each of which would have rendered them undetectable to clinical ct measurement . However, jensen et al.35 observed similar trends over 4 and 8 weeks using 1,000 to 2,000 m range autologous bone chips to treat three - wall defects in the mandibular angles of gottingen minipigs . The results of this study indicate that it is essential to include long - term evaluations of the healing response to biological bone graft particles in large animals . This may potentially apply to human treatments as well because bone regeneration and resorption of graft particles often proceed at varying rates . An additional known factor that plays a role in graft bone healing success is size of the bone particles used . Pig bone particles in the range of 600 to 1,000 m have been tested clinically as xenografts to treat maxillary sinus defects . At 5 months post - treatment with this particle size xenograft, researchers found retention of graft and significant new bone formation42 . In the current study, we used a bone mill to create autologous bone graft chips that had an average size of 670 m, which is within the range reported as ideal for bone graft preparation procedures43 . While significant bone volume recovery was observed in the untreated defects and autologous graft treated groups after 16 weeks, there was very little structural maintenance of the original mandibular geometry . It has been previously reported that the bone in proximity of the injury site adapts and remodels to compensate for the weakness in the injury area44 . Similar results were observed in this study, where the bone superior to the notch defect increased in volume, bulk, and thickness in 4 weeks and further in 16 weeks. (fig . 3) this observed increase in volume with concurrent decrease in mineral density suggests that the mandible undergoes significant remodeling due to the creation of the notch defect in the mandible body and may be compensating for the loss of load carrying capacity . Similar to the loss of height and thickness in the alveolar ridge after the loss of dentition45, this remodeling response in this model could be utilized when evaluating treatments attempting to maintain the original bone geometry, quality, and quantity necessary for implant restorations . It is critical for bone graft therapies to demonstrate not only on bone volume regeneration, but also maintenance of space and structure specific to the function of the cmf components . A systematic standardized evaluation of a ~7.5 cm mandibular bone notch defect model in the inferior margin of the miniature pig with periosteal resection was performed; autologous bone graft treatments were compared to untreated defects . Both experimental groups showed limited mineralized tissue within the defect site after 4 weeks, indicating that this model could be used to investigate therapies targeting accelerated bone regeneration at this early time point . However, the presence of significant bone within the defect site after 16 weeks in the untreated defect precludes the use of this model as a csd for bone graft evaluation . In order to appropriately investigate bone graft materials, future studies should include appropriate negative controls in their choice of bone defect in the pig mandible to ensure lack of a significant spontaneous regenerative response.
Pancreatic stellate cells (pscs) are interstitial cells of the pancreas, which in vitro are rapidly activated and develop the phenotype of myofibroblasts, characterised by enhanced expression of alpha smooth muscle actin (sma). Activation of psc and differentiation into myofibroblasts in vitro is assumed to be the process underlying activation of psc in vivo, leading to enhanced production of extracellular matrix, and ultimately, towards fibrosis . Pancreatic fibrosis is found in chronic pancreatitis (cp), or as a desmoplastic reaction in pancreatic ductal adenocarcinoma (pdac). In rats, deposition and degradation of extracellular matrix is a highly balanced process that occurs during regeneration of the pancreas after induction of acute pancreatitis . Repeated injury most likely leads to impairment of regulatory mechanisms and thus shifts the balanced process towards matrix deposition and detection of sma - positive interstitial cells or myofibroblasts . A similar deregulation could be induced by persistent exposure to toxic agents, such as organic zinc compounds . Pancreatic fibroblasts, or stellate cells, are major producers of extracellular matrix, and thus activation and deregulation of these cells is most likely the key event in formation of fibrotic deposits . Addressing the process of activation appears to be a means towards the treatment of fibrosis . Thiazolidinediones, antidiabetic drugs, and synthetic ligands of peroxisome proliferator - activated receptor gamma (ppar) have been shown to inhibit the activation of psc in vitro, and in animal models, ppargamma ligands have been shown to ameliorate the development of chronic pancreatitis [7, 8]. Ppar is a nuclear receptor that dimerises with retinoid - x - receptor to bind to dna of target genes . Ppar appears to be a central regulator in lipid metabolism and adipocyte differentiation . While ppar ligands have been shown to inhibit culture activation of psc and maintain a more quiescent state in freshly isolated cells, overexpression of ppar itself in immortalized pscs inhibits proliferation and reduces collagen synthesis . In this study, we describe the ultrastructural morphology of human pancreatic fibroblastoid cells isolated by outgrowth from human pancreatic tissue samples in comparison to rat pancreatic stellate cells . The effect of ppar ligands on human pancreatic fibroblastoid cells in vitro and a potential for adipogenic differentiation were investigated . All chemicals were of highest analytical purity purchased from sigma, deisenhofen, biomol hamburg, or merck, darmstadt, germany . All animal work was carried out according to the procedural and ethical guidelines of the local animal care and use committee . Pscs were isolated from female bn / lew rats, as described before by isopycnic density centrifugation, and were allowed to adhere to culture dishes for 3 days prior to further processing . Human pancreatic tissue samples were derived from different patients who gave informed consent according to institutional ethical procedures . For explantation of human pancreatic tissue, culture dishes were either coated with matrigel (becton dickinson) at a concentration of 0.5 mg / ml, or rat tail collagen, or left untreated . Pancreatic tissue was cut into small cubes of roughly 2 mm and placed into droplets of heterologous human blood plasma containing egta . Coagulation of plasma was started by adding thromborel solution (dade - behring, marburg, germany) and calcium solution (10 mm), keeping the tissue in a plasma clot in place . Finally, dmem (dulbecco's modified eagle's medium) supplemented with 10% heat - inactivated horse serum, 20% fbs (fetal bovine serum (biochrom, berlin, germany)), 10 mm hepes, and antibiotics (amphotericin b, 10 g / ml streptomycin, 10 u / ml penicillin, and 5 g / ml gentamicin) was added, and tissue explants were incubated at 37c in a humid atmosphere under 5% co2 . Outgrowing cells were documented using a zeiss axiovert microscope (carl zeis jena, germany). For isolation and passage of outgrowing cells, the plasma clots with remaining tissue were removed, and the cell layer was trypsinised for 10 min at 37c . Cells were plated with ecm, rtc, or on untreated plates in medium containing 10% fbs . Transformed hpf - t cells were generated by transfection with a plasmid containing sv40 large t antigen . Cells were maintained in culture as described above, using dmem supplemented with 10% fbs and 100 g / ml geneticin . Expression of large t antigen increased lifetime of the cells but did not stably immortalise them . Spontaneously immortalized rat pancreatic fibroblastoid cells were cultured as a stable cell line called pfc1 . Cells were kept in dmem supplemented with 10% fbs as described above . For investigation of proliferation as a direct measure of viability, cells were plated onto 96-well plates at a density of 2500 cells per well . After 24 hours of culture, cells were washed with pbs and switched to serum - free medium for another 24 hours . Subsequently, cells were treated with the indicated agents for the indicated times, and 4 h before the end of treatment, mts reagent (promega, madison, wis) was added, and colour development was monitored every 60 min up to 4 h using a dynatech mr 5000 plate reader . All measurements were carried out in triplicate in at least 3 different experiments (n 3)., cells were plated onto glass coverslips with or without extracellular matrix compounds in 12-well plates (costar, acton, mass). Samples were fixed in 2.5% paraformaldehyde (pfa) in pbs for 20 min at room temperature (rt). Oil red staining was carried out on pfa - fixed material, for 10 min in a saturated solution of oil red (polysciences, warrington, pa) in ethanol / acetone, washed in distilled water, counterstained with hematoxylin (vector laboratories, burlingame, calif), and mounted in glycerol gelatine . For flow cytometry analysis, cells were plated at a density of 200.000 cells per well onto 6-well plates and grown overnight . Cells were then incubated with serum - free medium for 24 h and subsequently treated as indicated . At the end of incubation, cells were washed with pbs containing 0.1% sodium azide, and all supernatants were collected; subsequently cells were detached by incubation with trypsin solution for 10 min, neutralised by addition of growth medium, and collected by centrifugation . Cells resuspended in 1 ml of pbs were incubated for 5 min with nile red solubilised in dmso at a concentration of 1 g / ml in the dark . Analysis was carried out using a galaxy analyser (dako, denmark) and flowmax software (dako, denmark) at 488 nm wavelength for excitation with detection filters for fitc and pe for nile red . For cell cycle analysis, cells resuspended in 0.5 ml pbs - edta were fixed by addition of an equal volume of ethanol and incubated for 30 min at room temperature . After washing with pbs - edta, cells were treated for 30 min with rnase a at a concentration of 2 g / ml for 30 min . After another wash with pbs - edta, propidium iodide (10 mg / ml) was added, and cells were incubated in the dark for 15 min . Rna isolation was carried out using trifast reagent (peqlab, germany), according to the manufacturer's protocol . Rna was solubilised in rnase - free water with rnase inhibitor added and stored at 80c . Reverse transcription of 1 g of rna was carried out with oligo - dt priming and superscript 2 reverse transcriptase (invitrogen) according to the manufacturer's protocol, and the resulting cdna was stored at 20c . For amplification, pcr was carried out with hotstar gold pcr mix (eurogentech, belgium), using the following primer pairs for human rna: gapdh (5-tgaaggtcggagtcaacggatttggt-3, 5-catgtgggccatgaggtc - ca - ccac-3, ppar (5-aactgcggggaaacttgggagattctcc-3, 5-aataataa - ggtggagatgcag - gctcc-3), lpl (5-cttggagatgtggaccgac-3, 5-gtgccatacagagaa - atctc-3), fapb (5-ttgctaccaggcaggtggcc-3, 5-ccagtgtggtctctt - gcccg-3). Products were resolved on 8% polyacrylamide - tbe gels, stained with ethidium bromide, and documented with polaroid film (kodak). For analysis of collagen synthesis, cells were plated in 6-well plates and incubated in 1 ml of serum - free medium containing either ciglitazone at a concentration of 3.3 m, or 15-prostaglandin j2 at 2.5 ng / ml . Incubation was carried out for 2472 h. supernatants were collected, and cells were harvested for determination of protein content . Determination of collagen synthesis (n 3) was carried out using a procollagen type i c - peptide (pip) eia kit according to the manufacturer's protocol (takara bio europe s.a.s ., the assay is specific for human procollagen i propeptide released into the cellular supernatant upon collagen assembly, thus it represents a direct measure of collagen synthesis ., labelling of probes, incubation, and gel analysis was done as described before . Nucleotide probes were consensus sequences for rxr and ppar consensus sequences from santa cruz (san jose, calif) and a sequence of rat acyl - coenzyme a oxidase (5-gggaacgtgacctttgtcctggtccc-3) containing ppar - response elements . Cells were fixed in situ with either 3% glutaraldehyde (ga) in cacodylate buffer at ph 7.2 containing 0.1% malachite green or with 2.5% ga and 2.5% pfa in cacodylate buffer for at least 30 min at 4c . Analysis was carried out using em109 and em 902 (zeiss, oberkochen, germany). Human pancreatic fibroblastoid cells (hpfcs) were isolated by outgrowth from human pancreas tissue explanted into tissue culture plates similar to rat pfcs, as described before . For comparison, pancreatic stellate cells (pscs) were isolated from rat pancreatic tissue by differential centrifugation after tissue digest, as described before . Rat pscs were fixed after 3 days of adherence using glutaraldehyde / malachite green (gam) for lipid preservation, as depicted in figure 1(a), and a standard fixation, seen in figure 1(b). They appeared as mostly round dark, electron - dense structures without a membrane boundary and mostly unstructured in gam fixation (figure 1(a)). Conventional fixation revealed a more structured appearance, with a darker outer rim, but still no membrane boundary (figure 1(b)). Alongside the lipid structures, large oil red staining of pscs after 3 days in culture, as depicted in figure 1(c), showed large, bright red irregular - shaped entities throughout the cytoplasm of the cells . Oil red staining of human pancreatic fibroblastoid cells (hpfcs) after three passages revealed a quite different picture, as seen in figure 1(d), and the staining pattern shows small dots, which are more abundant in some cells, while other cells are almost devoid of oil red - positive particles . Ultrastructural analysis of hpfc did not show prominent lipid droplets, but multiple multilamellar vesicles and small structured nonmembrane bounded entities (arrows in figure 1(e)). Higher magnification showed stacks of lipid bilayers within membrane compartments (arrows in figure 1(f)) and structures with reduced, fuzzy, or right next to regular multivesicular structures, nonmembrane - bounded homogenous lipid structures could be seen (figure 1(g)), which gave a similar appearance with darkened outer rim, as seen in rat pscs . High - resolution analysis as seen in figure 1(h) revealed the amorphous nature of a lipid structure, made up of layers of different electron density, without a defining outer lipid bilayer, which is a characteristic of lipid droplets . As seen in oil red staining and at ultrastructural level, lipid droplets were not uniformly distributed in every cell . In order to quantitate a large number of cells and investigate whether lipid storage was a generalised feature, not limited to single individual cells, we used binding of nile red . Nile red is a dye with high affinity for lipids and allows for flow cytometry evaluation . Isolates of hpfc from individual human pancreatic tissues are inhomogeneous in appearance and often limited in cell numbers, due to a limited lifetime . We employed a stable cell line of rat pancreatic fibroblastoid cells, named pfc1, and transformed human pancreatic fibroblastoid cells, named hpf - t (see section 2) for comparison . Cells were treated with ciglitazone, a member of the thiazolidinedione family of ppar ligands to test for its possible influence on lipid storage . Figure 2(a) shows the results of 72 h treatment with 3.3 m ciglitazone . Pfc1 cells showed a mean content of nile red - positive cells of about 60% which rose significantly by 27% after 3 days of ciglitazone treatment . The transformed human cells, hpf - t, showed a significant increase of lipid content by 36% . Human pfcs revealed a higher amount of nile red binding to begin with and showed an increase by about 15% of lipid content, which was not significant, though not surprising, as the cells were derived from individual isolates . We then tested for effects of ppar ligands on cell proliferation, using a tetrazolium blue - based assay that reflects mitochondrial activity, which is proportional to cell numbers . Figure 3 shows results for different treatments on pfc1, hpfc, and hpc - t cells . Cells were incubated with 3.3 m ciglitazone for 24 to 72 h, and hpfc showed a significant decrease in proliferation (figure 3(a)). To test for reversibility of this effect, whether it was due to growth inhibition or cell death, cells were treated for 48 h with ciglitazone and then washed and incubated without the drug . After 24 h of chase, cell proliferation increased to numbers not significantly different from controls . Spontaneously immortalized rat cells (pfc1, figure 3(b)) incubated with 3.3 m ciglitazone also showed significant reduction of proliferation but did not recover from 48 h ciglitazone treatment after 24 h chase . Further tests were performed on the effect of ppar ligands on pdgf- (platelet - derived growth factor)-induced proliferation (figures 3(c) and 3(d)). We tested not only for the synthetic ligand ciglitazone, but also the natural ligand 15--prostaglandin j2 (pgj2). Treatment of hpfc with 5 ng / ml pdgf for 24 h resulted in significant stimulation of proliferation, and incubation of pdgf- stimulated cells with ciglitazone reduced the stimulating effect . The reduction of pdgf - stimulated proliferation was significantly more pronounced with the natural ligand 15--prostaglandin j2 (figure 3(c)). For the transformed cell line hpf - t, the results were similar, with significant reduction of pdgf - stimulated proliferation by synthetic and natural ppar ligands (figure 3(d)). For further investigation of the antiproliferative effect of ciglitazone, cell cycle analysis was performed over the time course of 24 to 72 hours at 3.3 m concentration, as depicted in figure 4 . Propidium iodide staining for dna content showed a significant increase of g1 phase cells after 24 h and 72 h of treatment in hpfc (figure 4(a)). In transformed hpf - t cells, we found a higher rate of apoptosis during standard culture, which was significantly enhanced by ciglitazone treatment after 48 h and 72 h (data not shown). Pfc1 cells were tested as well (figure 4(b)) and showed a constant rise in g1 phase cells from 24 h to 72 h in control cells and after ciglitazone treatment . As the increase in cell numbers in g1 phase was moderate, vinblastine was employed . After 72 h of vinblastine treatment, g1 phase cells were significantly reduced while cells accumulated in g2/m phase . The percentage of cells in g1 phase under combined treatment with ciglitazone and vinblastine was not significantly lower than after ciglitazone only treatment for 72 h. furthermore, cell numbers in g2/m phase were significantly lower under the combined influence of vinblastine and ciglitazone than when treated with vinblastine only . So the effect of ciglitazone on cell cycle progression was confirmed . In order to confirm the influence of ciglitazone on hpfc, we investigated the gene regulation of proteins involved in lipid metabolism . Figure 5 shows rt - pcr analysis of fatty acid binding protein (fabp), lipoprotein lipase (lpl), and ppar itself in two individual isolates of hpfc . Over the time course of 24 to 72 h, rna levels of fabp rose continually, while lpl varied in rna levels, with a maximum after 72 h. ppar itself was induced after 24 h of ciglitazone treatment and maintained the higher levels . These findings were confirmed, when another isolate was investigated after 72 h of ciglitazone treatment . All analyses on lipid content and induction of lipid metabolism were carried out on cells of the same original isolates . We further tested for the effect of ppar ligands on collagen synthesis, using an immunoassay for procollagen i propeptide . During collagen fiber assembly, the propeptide is cleaved and concomitantly released into the cell culture supernatant and can be assessed by immunological techniques . Figure 6(a) shows that in hpfcs ciglitazone and the natural ligand 15-prostaglandin j2 inhibit collagen synthesis after 24, 48 h, and 72 h of incubation, expressed as percent of controls . Apparently, the natural ligand is more effective than ciglitazone in inhibition of collagen synthesis . As seen before, this analysis revealed that collagen synthesis in individual hpfc isolates showed differences; furthermore, synthesis varied throughout the investigated time course . Figure 6(b) gives an example of collagen synthesis and inhibition by ppar ligands for one individual isolate named hpf71ecm, from a patient treated for pancreatic cancer . Figure 6(c) shows the range of collagen synthesis for all untreated controls of hpfc isolates tested at different times of incubation; in general, collagen synthesis appeared to be maximal 2 d after plating and decreased with ongoing culture, while the amount of collagen synthesis differed . Figure 6(d) shows results for the transformed human cell line hpf - t, expressed as percent of control . Ciglitazone treatment for 24 h significantly induced collagen synthesis, while pgj2 treatment resulted in significant inhibition . Prolonged treatment with ppar action in gene regulation was further investigated by dna binding studies, that is, electrophoretic mobility shift assays (emsas), to confirm ciglitazone - induced activity in the cells investigated . Figure 7 shows binding of ppar to different consensus sequences for the three cell types investigated in this study . Ppar binds to dna as a heterodimer with retinoid x receptor (rxr), thus we used probes with rxr binding sites as well as ppar consensus sequences and performed supershift analyses with antibodies specific for ppar to confirm the presence of the molecule in the heterodimers . Analysis of an individual hpfc isolate revealed that ppar bound in the absence of ligand, which is in accord with a corepressor binding in the absence of ppar ligands, which is released upon ligand binding to the dimer . Incubation with 3.3 m ciglitazone for 1 h induced ppar binding to the rxr consensus sequence (figure 7(a)). For pfc1 cells, we showed a dose - dependent increase in ppar binding to both probes investigated (figure 7(b)). In hpf - t cells ciglitazone as well as the natural ligand pgj2 induced ppar, while rxr binding was enhanced by retinoic acid - derivatives like all - trans retinoic acid (atra) and 9-cis - retinoic acid (9cisra), the former inducing preferentially retinoic acid receptor (rar), while the latter has been shown to induce retinoid x receptor (figure 7(c)). Pancreatic stellate cells, mostly isolated from rat pancreas by tissue digestion and differential centrifugation [1, 2], produce cells which show lipid storage, as detected by oil red staining; these cells are isolated by their buoyant density . On the other hand, cells isolated by outgrowth from tissue pieces in cell culture are per se activated, they must migrate to leave their environment, and they undergo cell division in cell culture prior to being harvested as a passageable cell population with features of fibroblasts . In this study, we investigated human pancreatic fibroblast - like cells isolated by outgrowth, and one of the aims was to analyse whether these cells have the capability of lipid storage . Ultrastructural analysis revealed that at the earliest time point of investigation, there were essentially no lipid droplets resembling those found in rat pancreatic stellate cells by size or number . However, there appeared to be a considerable amount of stacked lipid bilayers concentrated in membrane - bounded vesicles, which might give rise to smaller, secondary lipid storing structures with a distinct layering and without a containing lipid bilayer membrane . As structures like these are barely seen in tumour cells or others of epithelial origin, the potential for lipid storage appears to be inherent in mesenchymal / fibroblast lineages . It is generally believed that the activation process for stellate cells goes along with loss of lipid content and expression of alpha smooth muscle actin as a marker . We show here that isolation of a priori activated cells from different individuals with different etiology of disease, leading ultimately to surgical intervention, yields cells which are heterogeneous in appearance but do respond to pharmaceutical manipulation in a coordinated manner . Thiazolidinediones, used as antidiabetics, were shown to inhibit proliferation and activation of rat psc and immortalized rat psc in vitro . Similar results were found for hepatic stellate cells (hscs) in vitro; most of these experiments were carried out on cells for rat origin . Furthermore animal models for chronic pancreatitis and liver fibrosis did respond to treatment with tzds, ppar ligands by ameliorated fibrotic responses, though there is also a report on a mouse model of hepatic fibrosis that was not responsive towards pioglitazone treatment . So far, little is known about human pancreatic fibroblasts in vitro . Investigation of ppar gene expression in human tissues revealed a maximum of ppar 1 and 2 expression in human fat tissue, and to a lower extent in liver, heart, and muscle . Rats and mice show a similar prevalence in ppar 2 expression in fat tissue, but expression levels are far lower than in human tissues, which is even more evident for ppar 1 . We found ppar mrna in hpfc untreated control cells after prolonged time in culture, readily induced by ciglitazone after 24 h, and in rat hscs, ppar expression went down over time in culture but was inducible by pgj2 . However, the most successful model for induction of adipocyte differentiation is mdi (isobutylmethylxanthine / dexamethasone / insulin) treatment, which usually lasts for 3 days followed by a more or less extended culture period which was initially developed for mouse 3t3 l1 cells (and references therein) and was successfully applied to rat hsc . In hsc, mdi treatment restored a quiescent phenotype by induction of genes regulating lipid metabolism, an effect also seen by overexpression of ppar . We report here the induction of fabp and lpl as representatives of lipid metabolism, as well as ppar mrna induction after ciglitazone treatment . Furthermore, we report on dna binding in heterodimers with retinoid x receptor, thus ensuring direct involvement of ppar in enhanced lipid content . However, 72 h of treatment might not be sufficient for a full - fledged adipogenic differentiation in human or rat pancreatic fibroblastoid cells but clearly points towards activation of signalling pathways leading to reduced proliferation and enhanced lipid content, similar to induction of adipogenic differentiation . In addition, we were able to show a reduction of collagen synthesis by ppar ligands, arguing for reversibility of the activation process . It is tempting, to speculate, that a reversal of activation by synthetic ppar ligands might work in humans by promoting the inherent adipogenic potential of pancreatic interstitial fibroblasts.
Every year, a large number of athletes experience knee trauma during training session and competitions . The anterior cruciate ligament (acl) is the most commonly injured ligament of the knee, which mainly affects patients between 15 and 45 years of age . . However, non - contact condition is more prevalent. [14] the acl rupture can occur not only as a sole event but also in association with the rupture of meniscus and other ligaments. [57] while the reconstruction of an injured acl is considered as one of the most important procedures in sport injury, the optimal timing for the operation remains uncertain . The safety and efficacy of the timing of the operation is controversial . It appears that some consensus has been achieved on this issue with more surgeons waiting for the resolution of the acute hemarthrosis and the restoration of normal gait and range of movement before performing the surgery . It has also been suggested that any undue delay in surgery may lead to an increase in meniscal pathology such as experiencing recurrent episodes of giving ways, which could damage the articular surface . The timing of surgery is of importance in counseling patients regarding the outcome of surgery and could be of particular interest while treating patients within a healthcare system with limited resources . Recent long - term clinical researches have pointed out the increased risk of secondary meniscal damage and chondral lesion in patients with chronic acl deficiency. [1113] the present study, therefore, was designed to evaluate the association between acl deficiency and its impact on meniscal and articular cartilage injury among groups of professional iranian athletes and also to determine whether there is a correlation between the timing of reconstruction and these types of injuries . The present retrospective study was conducted on the medical records of 616 consecutive professional athletes gathered from referral hospitals between 1995 and 2009 . Based on these records, the studied athletes had visited a single orthopedic surgeon (m.r .) Because of knee injury and they were diagnosed with acl tear, which was confirmed by magnetic resonance imaging (mri) and arthroscopy . Athletes who were injured through a non - exercise mechanism such as experiencing a car accident, those experiencing concomitant acl tear and tibia or fibula fracture, and those with a positive history of meniscal tear in the same leg were excluded . The demographic information of the athletes including their age, gender, the injured leg, the sport in which they were involved, the duration between the time of injury and the surgery, and the injured ligaments was recorded in a questionnaire . The sport in which the athletes were involved consisted of soccer, wrestling, ball games (handball, volleyball, and basketball), martial arts, and others (ski, tennis, table tennis, running, badminton, skating, etc . ). The duration between the time of injury and that of surgery was classified as <3 months (group a), 3 - 6 months (group b), 6 - 9 months (group c), 9 - 18 months (group d), 18 - 36 months (group e), and> 36 months (group f). In cases with meniscal tear, the data on individuals with total meniscus injury regardless of their grade were entered and those with partial tears were excluded . Severity of chondral pathology was divided into two groups: low graded groups (type 1 and 2) and high grade groups (type 3 and 4). Chi - square test was used to evaluate the correlation between the presence of the meniscal tear, chondral damage, and the duration between the time of injury and that of surgery . Overall, 564 (91.6%) out of a total of 616 athletes with acl tear enrolled in this study were male . Table 1 outlines the gender distribution and the sports in which the athletes were involved . The frequency of athletes involved in different sports the single acl injury was reported in 103 (16.7%) of the athletes . Medial meniscus injuries were more prevalent than lateral meniscus injuries among our professional athletes . The incidence of medial and lateral meniscus injury in different sports also, 115 (18.7%) of athletes had chondral lesions . Among them, there was no significant association between chondral pathology and sex of the athletes (pv> 0.05). After an interval of 6 months, there was a significant increase in the prevalence of chondral lesions (chi square test, p = 0.008) [table 3]. The incidence and severity of chondral injuries among 6 groups based on what was reported and identification by mri and arthroscopic findings, medial femoral condyl showed more prevalent chondral injuries (58.11%) than lateral side due to acl deficiency . The prevalence of medial and lateral menisci tears within the groups is presented in table 4 . Considering the gathered information, the severity of tears in the medial meniscus increases over time since the injury . The present study was conducted on 616 athletes involved in different sports diagnosed with acl tear based on the findings of clinical examination, mri, and arthroscopy . The first strength of our study lies in the ability of examining a large group of patients by a single surgeon (m.r . ). This is of great importance since the identification of pathology during knee arthroscopy has been shown to be related to the observer's capability . We found an increased incidence of medial meniscal tears if the interval between the injury and reconstruction process was> 3 months . A significant reduction was also found in the number of patients with normal articular surfaces after a delay of 6 months in the time of operation . Prade et al ., reported a relatively higher prevalence (44%) of single acl tear among patients with acute knee injuries accompanied with hemarthrosis over a 90-days period . Similarly, frobell et al ., reported the prevalence of acl rupture in the general population to be about 30%, which was higher than previously described . The difference between the diagnostic methods was considered as the main reason contributing to the discrepancies reported in these studies . In other words, while mri was used to detect acl tear in the abovementioned studies, our patients were diagnosed by clinical examination, mri, and arthroscopic evaluations . The higher accuracy of the diagnostic procedures in our study accounts for the reduced number of single acl tear and increased rate of combined meniscal and/or chondral injuries . The other possibility for having differences of the prevalence of single acl tear in our population is on the basis of late referral of athletes to orthopedic surgeon . It can extend their knee complications and decrease the rate of single acl tear . Based on our findings, there was an association between delayed reconstruction and an increased incidence of medial meniscus tear and chondral pathology after 3 and 6 months, respectively, which is clinically important . To our knowledge, few studies have investigated the relationship between a delay in reconstruction and the incidence of secondary pathologies . In line with the results published by papastergiou et al ., our finding showed the association between acl tear and meniscus injury 3 months after the events . It could be concluded that the rate of secondary meniscus injury increases over time and, therefore, the operation should be performed within 3 months of the injury . While rocha et al ., described similar findings, they noted a higher rate of lateral meniscus injury within the first 3 months of acl tear . They examined the incidence of meniscal tears based on the interval between the injury and operation, but failed to assess the possible changes in the articular surface . They found an increased incidence of both medial and lateral meniscal tears when the operation was postponed beyond 3 months . Due to the fact that most of the elite players are expected to continue their participation in competitions of the league despite of their injury, serious rehabilitation will be achieved for their recovery . Therefore, after rehabilitation process and reducing their pain and effusion, pseudo well - being and self confidence will occur . These athletes believed that the recovery of acl tearing was completed and they could continue their playing without any treatment for their knee . But, unfortunately, this type of estimation will increase the chance of severe injury, especially meniscal tear . The incidence of chondral lesion among our population increased as the interval between injury and reconstruction was> 6 months . Church and keating reviewed 187 patients and found a significant increase in degenerative changes of grade 1 to 4 (31.3% vs 10.7%) and also in the overall incidence of meniscal tears among those who had undergone late reconstruction (after> 12 months of injury). Based on our findings, significant changes in the articular surface of the injured knee starts in less than a year . It has been suggested that patients with acl deficiency dispose to the development of chondral injuries in weight bearing activities due to excessive anterior tibial translation and rotational instability in their injured knee . In a similar study conducted by murell et al ., the injuries to the medial condyl of femur were more prevalent than other parts of condyls . It is believed that the increased shear stresses on the meniscus and the application of greater weight - bearing loads on the articular surface (unconditioned cartilage region), which was not previously functionally loaded, may account for the abnormal translation and rotational motions following acl injury . Moreover, opposite to the constantly changing, contact points of the tibio femoral articulation in a normal knee (rollback), the femur in an acl - deficient knee may remain in contact with a section of the articular surface of tibia, resulting in point - loading with non - physiological loads, leading to degeneration process and chondral damage . In their finite element analysis of the kinematic changes related to acl deficiency, confirmed this hypothesis . Barker et al ., found that the relative internal rotation of the tibia secondary to acl injury results in an increased cartilage loss especially in the medial compartment of the injured knee . This finding is believed to be secondary to a shift of load bearing to the thinner regions of articular cartilage . Despite the increased risk of chondral injuries secondary to a delay of> 6 months in the time of surgery, there was a peak of high - grade chondral lesion among athletes operated in the first 3 months . Acute chondral injuries have been reported in 20% of acl tears; however, many authors believe that the common pattern of bone bruise, known as post - truamatic bone marrow lesions, represent occult damage to the cartilage secondary to the compressive force imposed on the joint surface at the time of the acl tear. [2426] one of our limitations include low number of female athletes in the study . As the number of professional female athletes is lower than in some other countries, there are not many high - level competitions; similarly, international competition is subject to considerable limitation . Therefore, we could not compare our results about women's sports to that in other studies . In conclusion, based on our findings, in order to reduce the chance of the occurrence of instability induced medial meniscal tears and chondral lesions, reconstruction should be performed before 6 months after injury . It is recommended that surgical reconstruction should be performed instantaneously after conduction of intensive rehabilitation program, psychological preparation, and economical support . Due to the fact that the return to play is one of the most imperative and challenging subjects in sports medicine field, identifying it seems that time preparation to improve patients' compliance to surgery takes between 3 weeks to 3 months.
Composite resin restorations should be replaced or repaired in case of failure due to discoloration, recurrent caries beneath the restoration, fractures at the margins, etc . [the repair of composite resin restorations is a conservative and minimally invasive procedure, with advantages such as decrease in costs and time the patients spend on the dental chair, a decrease in the amount of tooth structure lost and prevention of further stimulation of dental pulp [14]. Therefore, it is of utmost importance to increase the bond strength and promote the longevity of these repairs . Several techniques have been proposed to improve the bond strength of composite resins, including air abrasion with aluminum oxide particles measuring 50 m in diameter, etching with hydrofluoric acid and phosphoric acid, use of silanes and roughening of composite resin surfaces with diamond burs [512]. Preparation of the composite resin surface plays an important role in the longevity of repaired composite resin . In order to achieve complete adaptation between the old and the new composite, an intermediate adhesive substance should be used because the composite resin itself cannot completely wet the surface of the old composite resin [4,1315]. The flowability and hydrophobic nature of the intermediate material are important factors involved in selection of such materials . According to papacchini et al, use of a flowable hydrophobic composite resin as an intermediate substance increased the repair bond strength of methacrylate - based composite resins . Hydrophilicity of the intermediate material can compromise the longevity of the repair bond because hydrophilic adhesives absorb more water over time and undergo hydrolytic degradation . One of the major disadvantages of methacrylate - based composite resins is polymerization shrinkage, which results in accumulation of stress within the composite resin and at the composite resin - adhesive interface; one of the possible complications of such a shrinkage is the cuspal deflection and loss of marginal adaptation, resulting in failure of the restorative material, staining of restoration margins and microleakage . To overcome such difficulties, silorane - based composite resins were introduced in 2007, which consist of a new matrix composed of siloxane and oxirane . The aim behind their production was to produce a material with less polymerization shrinkage and subsequently less stress accumulation . In addition, silorane exhibits the greatest stability in presence of visible light, along with optimal mechanical properties compared to methacrylate - based composite resins . Furthermore, the siloxane present in this composite resin makes it highly hydrophobic . In order to make the adhesive system compatible with the silorane - based composite resin, the bonding agents for such composite resins contain hydrophobic dimethacrylate monomers (7080 wt%) and are devoid of hydrophilic hema monomer; therefore they are hydrophobic . Only a limited number of studies have evaluated the effects of different repair techniques on the repair bond strength of silorane - based composite resins . Demonstrated that the repair methods used for methacrylate - based composite resins could be also applied for silorane repair . Surface roughening either with sandblasting or silanization followed by the application of the silorane bonding agent resulted in bond strength values comparable to that of the control group . Bacchi et al . Showed that simultaneous use of an adhesive and sandblasting was successful for the repair of silorane - based composite resins . It has been shown that adding an extra layer of hydrophobic resin can improve the repair bond strength of methacrylate - based composite resins and decrease microleakage in such restorations [2527]. Therefore, the aim of the present study was to assess the effect of adding an additional layer of hydrophobic resin on the repair shear bond strength of a silorane - based composite resin with the use of two silorane - based and methacrylate - based composite resins . Twenty cylindrical composite resin blocks, measuring 2.5 mm in diameter and 6 mm in height, were fabricated using filtek p90 silorane - based composite resin (3 m espe, st . Paul, mn, usa) for evaluation of the cohesive strength of silorane - based composite resin (the positive control group). Also, 60 disc - shaped composite resin blocks were fabricated using a silorane - based composite resin (filtek p90, 3 m espe, st . Paul, mn, usa) by applying unpolymerized composite resin in 1.5 mm layers in cylindrical holes, measuring 5.5 mm in diameter and 3.5 mm in height; each layer of each block was light - cured separately for 30 seconds using a demetron a-2 light - curing unit (kerr corporation, middletown, wi, usa) with a light intensity of 1000 mw / cm . The cylindrical holes were created in auto - polymerized resin (pmma) and the resin itself was surrounded by a plastic cylinder . All the samples underwent an aging procedure after complete polymerization, except for the positive control samples . For the aging process, the samples were immersed in 0.9% nacl solution for 72 hours in a container into which light could not penetrate . The samples were divided into three groups (n=20) based on the surface preparation technique: group 1: surface preparation was carried out with a diamond bur (g & z instrumente gmbh, lustenau, austria) and etching was carried out with 37% phosphoric acid (condac, fgm dental products, joinville - sc, brazil). Then silorane bonding agent (filtek silorane adhesive bond, 3 m espe st . Paul, mn, usa) was applied according to the manufacturer s instructions and light cured for 20 seconds . Group 2: surface preparation was carried out with a diamond bur (g & z instrumente gmbh, lustenau, austria), followed by etching with 37% phosphoric acid (condac, fgm dental products, joinville - sc, brazil). Finally, silorane bonding agent (filtek silorane adhesive bond, 3 m espe st . Paul, mn, usa) was applied according to manufacturer s instructions and light cured for 20 seconds, followed by the application of the hydrophobic resin - the third component of adper scotchbond multi - purpose adhesive system (adper scotchbond multi - purpose adhesive, 3 m espe st . After surface preparations were carried out as explained above, a plastic mold with an internal diameter of 2.5 mm and height of 5 mm was used to place the silorane - based composite resin on the surface of aged samples; the mold was placed at the center of the aged samples and the new composite resin (filtek p90, 3 m espe, st . Paul, mn, usa) was packed and light cured for 20 seconds from each side . Then the samples were removed from the molds and light cured again for 40 seconds using demetron a-2 light - curing unit (kerr corporation, middletown, wi, usa) with a light intensity of 1000 mw / cm . Contrary to groups 1 and 2, the blocks made of a methacrylate - based composite resin (z100, 3 m espe, st . Paul, mn, usa) measuring 2.5 mm in diameter and 5 mm in height were used as the repair composite . For the aging process, first all the samples were immersed in 0.9% nacl solution for 24 hours in a container protected from light, followed by thermocycling which consisted of 1500 cycles at 555c with a dwell time of 20 seconds and transfer time of 10 seconds . In the final stage, the shear bond strength values were measured using a universal testing machine (model h5ks, hounsfield test equipment, surrey, uk) at a strain rate of 1mm / min . The bond strength values were converted to mpa by dividing the maximum force at fracture (n) by the surface area of the repair composite resin (mm). The results of levene s test approved the assumption of homogeneity of variances between groups (levene s statistic=5.63). Thus, the data were analyzed using one - way anova and post - hoc tukey s test (=0.05). In addition, the mode of fracture of the samples, consisting of cohesive in the repair composite resin, cohesive in the repaired composite resin, adhesive and mixed, was evaluated under a stereomicroscope (nikon, smz 800, tokyo, japan) at 20 magnification (fig . Streomicroscope images of fracture patterns (20) which are classified as: a: cohesive in the repairing composite resin, b: cohesive in the repaired composite resin, c: adhesive, and d: mixed table 1 presents the mean repair shear bond strength values, standard deviations, standard errors and distribution of modes of fracture in the study groups . The results of one - way anova showed significant differences in repair shear bond strength values between the different study groups (p<0.001). Pairwise comparisons of the groups with post hoc tukey s test showed significant differences in repair shear bond strength between the positive control group and all the other groups (p<0.001). In addition, there was a significant difference in repair shear bond strength between groups1 and 3 (p<0.001); however, the differences between groups 1 and 2 (p=0.18) and groups 2 and 3 (p=0.20) were not statistically significant (fig . The error bar of means and 95% confidence intervals of means bond strengths (bs) value in the study group . The mean repair shear bond strength values (mpa), standard deviations, standard errors and distribution of fracture modes in the study groups (n=20) different superscripts mean statistically significant differences statistical homogeneous subgroups based on post hoc test the repair of composite resin restorations is a conservative and minimally invasive procedure, with some advantages such as decrease in costs and chair time, decreased loss of tooth structure and prevention of dental pulp injuries [14]. It is of significance to improve the repair bond strength and increase the durability of such repairs . Several studies have shown that use of a hydrophobic resin or a hydrophobic flowable composite resin as an intermediate material for the repair of methacrylate - based composite resins decreases hydrolytic degradation of the bonding layer and increases the repair bond strength . However, the aim of the present study was to evaluate the effect of adding an extra layer of hydrophobic resin on the repair bond strength of a silorane - based composite resin . Due to unavailability of different surface preparation techniques in dental offices and also the results of a study by wiegand et al, who showed that the kind of mechanical treatment (roughening with bur, aluminum oxide sandblasting or silica coating) is of minor significance for silorane composites, in the current study in addition, they showed that silane application was not mandatory when silorane composite along with silorane adhesive was used for repair . The silorane bonding agent is not silorane - based but phosphate - dimethacrylate - based . The acrylate group of the phosphate - methacrylate based bonding agent can react with methacrylate - based systems and the phosphate groups react with the silorane repair composite . Therefore, in the current study only the silorane adhesive system was used without additional silane application . The results of the current study indicated that adding an extra layer of hydrophobic resin resulted in an increase in repair bond strength of silorane - based composite resins . However, this increase in bond strength was only significant in the group in which the repair composite resin was methacrylate - based similar to the hydrophobic resin compared to the group in which the hydrophobic resin was not used . In the group in which an extra layer of hydrophobic resin was used but the repair composite resin was silorane - based, the mean bond strength was higher compared to the group without the hydrophobic resin; however, this increase was not statistically significant . It has been shown that with an increase in the hydrophobicity of the intermediate resin, its water sorption and consequently its hydrolytic degradation decrease . This finding is of high clinical significance because dentists often do not know whether the composite to be repaired is a silorane- or a methacrylate - based composite and may routinely use a methacrylate - based composite for this purpose . In the current study, higher repair bond strength in group 3 might be attributed to the use of silorane bonding agent and scotchbond multi - purpose, both of which are hydrophobic; therefore, the bonding layer is more resistant to hydrolytic degradation . As a result, the bond strength after thermocycling was higher compared to that in the other two groups . In addition, in group 3 both the hydrophobic resin and the repair composite resin were methacrylate - based and chemical bonding might have played a role in this group in addition to micromechanical retention . In contrast, da costa et al . Showed that use of a hydrophobic resin did not influence the immediate repair bond strength and the repair bond strength six months after storage in water in methacrylate - based composite resins . However, some signs of penetration of water and deposition of silver nitrate and early disintegration of the bonding layer were observed in groups in which a more hydrophilic resin was used; that means the degree of hydrophilicity of the intermediate resin did not influence the immediate bond strength and the repair bond strength after six months . In the afore - mentioned two studies, only water storage was performed for six months and thermocycling was not performed for aging . Lack of difference in repair bond strength values in the aforementioned two studies might be attributed to the fact that aging was not sufficient in these two studies to result in differences in repair bond strength . In contrast, in the current study, storage in water and thermocycling were used simultaneously for aging . Another finding of the current study was that the repair bond strength values in the positive control group were significantly higher than those in other groups, i.e. None of the groups achieved the cohesive strength of silorane - based composite resin . Based on the results of previous studies, the repair bond strength of composite resin was 2582% of the cohesive strength of composite resin [3335]. In the current study, the bond strength values were 4451% of the cohesive strength of silorane - based composite resins, consistent with the results of previous studies . Evaluation of the modes of fracture of different groups led to the conclusion that the majority of failures were of adhesive type in the group in which an extra hydrophobic layer had been used and the repair composite resin was silorane - based . Group 3 exhibited the highest frequency of cohesive failures and the lowest number of adhesive failures, which was consistent with the bond strength values, i.e. In the group with the highest repair bond strength the maximum cohesive failures and minimum adhesive failures were observed . In addition, group 3 was the only group in which cohesive failure in the repair composite resin was observed . A higher rate of adhesive failure in group 2 shows that in this group the adhesive layer was the weakest part of the bonding layer, which might be attributed to the fact that there is no chemical affinity between the hydrophobic resin and the silorane bonding agent and therefore, micromechanical retention is the only mechanism for the repair bond strength . This means that although adding an extra layer of hydrophobic resin may increase the longevity of the adhesive layer, it cannot result in a significant difference in bond strength due to the lack of chemical similarity between the hydrophobic resin and the superficial composite resin . One of the limitations of the current study was the hydrophobic nature of the resin used, which was an unfilled resin; it is probable that if a filled resin such as a hydrophobic flowable composite resin is used for the repair of a silorane - based composite resin along with a methacrylate - based composite resin, higher repair bond strength values may be achieved . Due to specific limitations, it is probable that if the thermal cycles are increased or multiple loading is carried out, the effect of the degree of hydrophilicity of the intermediate resin on the repair bond strength will be further elucidated . Considering the limitations of the current study and the results achieved, it can be concluded that use of an extra layer of a methacrylate - based hydrophobic resin does not influence the repair bond strength during repair of a silorane - based composite with a silorane - based composite . However, the use of en extra layer of hydrophobic resin is useful in the repair of silorane - based composite resin with a methacrylate - based composite and can increase the repair bond strength.
Vahlkampfiids, acanthamoebidae and techamoebidae are among the important free - living amoebae (fla) and they could lead to severe disease including amoebic keratitis (ak), granulomatose encephalitis and cutaneous ulcers (13). Free - living amoebae belonging to acanthamoeba genus are distributed in many environmental sources such as soil, clay, dust, fresh waters, mineral springs, sea, water - air interface, spas, jacuzzis and hot springs (4, 5). However, despite the widespread distribution of acanthamoeba in the environmental sources the incidence of disease related to these genera is not high . Thus, it is reasonable to predict that not all people are susceptible to such infections (6, 7). Indeed, the high - risk people can be divided in two categories: contact lens wearers and immunosuppressed patients including hiv positive patients, graft patients, patients undergoing corticosteroid and chemotherapy, pregnant women, diabetes, cirrhosis and lupus patients . Corneal trauma even in microscopic form can lead to acanthamoeba keratitis (8, 9). The mannose level of cornea in healing epithelium is twofold more than normal cornea and thus the capacity of acanthamoeba binding is much higher in this situation (1012). Acanthamoeba trophozoites are flat in shape with large karyo - some and multiple food vacuoles ., various shape of endocyst may be seen in culture including triangular, square, and round forms . To date, acanthamoeba has been divided into 18 different genotypes (t1t18) being most of them the cause of human infections such as severe keratitis (12, 13). The genetic typing is based on 18s rrna gene and the sequencing of diagnostic fragment 3 (df3) of 18s rrna gene (14). Previously only t4, t3 and t2 were introduced as a causal agent of acanthamoeba related infections; however, later it was shown that many genotypes could lead to acanthamoeba keratitis (ak) including t11, t13, t15, etc . Unfortunately, most infections because of acanthamoeba show poor prognosis and the treatment using topical propamidin isethionate (0.1%) and neosporin is still challenging (1, 2, 13). Diagnosis of ak is mainly based on cultivation of corneal scrapes, contact lenses on non - nutrient agar along with heat killed eschershia coli, and molecular based analysis . Confocal microscopy could be non - invasive diagnosis approaches, which could be helpful for ak diagnosis and monitoring, however by using this microscopy - based test, it is not possible to differentiate between various free - living amoebae and therefore culture of corneal specimen could be the gold standard (15). Overall, the present research aimed to review the occurrence of acanthamoeba spp . In addition, their genotypes based on 18s rrna gene in environmental samples and clinical sources such as corneal scrape contact lenses and their paraphernalia . In addition, the present review highlights the increasing trend of amoebic keratitis in the country, which needs improved education regarding this opportunistic free - living protozoan . We conducted a systematic review based on the database sources such as medline, pubmed, scopus and google scholar . We searched all valuable and relevant information considering the occurrence of the acanthamoeba in both environmental and clinical samples . We referred to the information databases of medline, pubmed, scopus and google scholar and the used keywords were the combinations of acanthamoeba and amoebic keratitis, and words associated with environmental sources such as; acanthamoeba and soil, acanthamoeba and water, acanthamoeba and dust sources, acanthamoeba plus treatment . We also referred to some information in books associated with issue of acanthamoeba and health hazard abstracts and full articles that were written in english and relevant to the topic were enrolled in this study and critically studied in detail . There are various researches, which has conducted in iran as following . The first research regarding environmental acanthamoeba distribution these investigators showed the presence of acanthamoeba and naegleria in water samples of kazeroon city using morphological key (15). However, at that time, there was no report regarding various genotypes of acanthamoeba genus and these amoebas classified using morphological based criteria . Later, another morphological - based research was done by rezaeian et al . Interestingly, all of the soil samples (5 out of 5) were positive in the culture . In addition, out of 61 dust samples, 28 of them contained acanthamoeba (16). Consequently, niyyati et al . Performed a sequencing based test for the mentioned environmental sources and the genotypes were belonged to t2, t6, t4 and t11 (17). It is important to mention that all of isolated genotypes have been reported as a cause of ak . Revealed that acanthamoeba present in pond water sources of all districts of tehran, iran . Genotyping of strains showed that they were belonged to t4 and t5 genotype, both genotypes has been identified as corneal pathogens mainly in soft contact lens wearers (18). In another research, niyyati et al . Showed the presence of potentially pathogenic acanthamoeba spp . In recreational river waters of tehran, briefly, 55 water samples from 10 major rivers were analyzed for fla and identified by morphological - based criteria, pcr amplification and sequencing analysis . Acanthamoeba, assigned to the t4 and t15 genotype were then identified using sequencing of df3 region (19). Other researchers showed the presence of thermotolerant acanthamoeba in hot spring of northwester iran (2022). Conducted a survey regarding the pathogenic potential of acanthamoeba t4 type using osmo - tolerance and thermo - tolerance assay . Cysts isolated from water, soil and dust (star shape cysts are shown in clump) (magnification: a: x10; b, c, d: x40) (figures are from pathogenic free living amoebae in human book, rezaeian m and niyyati m, 2009) acanthamoeba spp . Trophozoites isolated from soil and dust (flat shape trophozoites are shown) (magnification: a: x10, b: x40) rezaeian et al . Was the first to report the presence of acanthamoeba in hospital dusts in tehran, iran . This finding could be a health hazard for high - risk people including contact lens wearers and patients with eye surgery (16). The occurrence of fla in immunodeficiency wards of hospitals in tehran, iran were also investigated by lasjerdi et al . Briefly, 70 dust and biofilm samples from immunodeficiency wards of university hospitals were collected and tested for the presence of fla using culturing and molecular approaches presence of the t4 genotype on medical instruments, including an oxygen mask in an isolation room of an immunodeficiency ward, should be of concern for health authorities . Overall, these results reflect a clear need for improved disinfection, especially where high - risk people, such as those who are immune - suppressed or undergo eye surgery such as lasic surgeries, are served . Encephalitis due to acanthamoeba has not been reported yet, mainly due to lack of knowledge regarding acanthamoeba as an agent of central nervous system infections . So far, there are 150 cases of ak in iran, although it seems that these numbers is lower than the true cases . The first cases of ak in iran were reported in a soft contact lens wearer (15). In 2007, interestingly, 44 patients (89.79%) were contact lens wearers for cosmetic purposes or visual corrections . Among them 41 patients (93.18%) wore soft contact lenses and three patients were hard contact lens wearers (25). Among 50 keratitis patient, 13 were positive for acanthamoeba (26). Three species including a. griffin, a. palesinensis and a. castellanii were identified in their samples . Another study revealed acanthamoeba as a causal agent in 15 (30%) of 50 keratitis samples . Among these clinical isolates, 13 (86.7%) belonged to female patients and 2 (13.3%) were male . All positive specimens belonged to soft contact lens wearers and only one belonged to a patient with a history of hard contact lens usage . Regarding genotype identification, 13 (86.7%) of these isolates belonged to t4 genotype . However, it is important to mention there was a mixed genotype belonging to acanthamoebae t4 and t11 genotypes in one patient . Other genotypes identified in the clinical specimens were t11 (13.3%) and t3 (6.7%) (17). Another survey of the 90 asymptomatic contact lens wearers, 9 (10%) were positive for fla outgrowth . Morphological analysis revealed that 3 isolates were belonged to hartmannella genus according to small round cysts and 6 isolates were belonged to acanthamoeba genus based on the star shape of endocysts . Sequencing revealed that acanthamoeba belonged to t4, t3 and t5 genotype (27). Cysts isolated from acanthamoeba keratitis (triangular shape cysts are shown) (magnification: x10) acanthamoeba cysts isolated from acanthamoeba keratitis (triangular shape cysts are shown) (magnification: right: x10, left: x40) so far, other infections due to acanthamoeba spp . Have not been reported in iran . This is due to lack of knowledge regarding these opportunistic protozoan parasites in this region . It should be noted that memari et al . Reported that acanthamoeba belonging to potentially pathogenic t3, t4 and t5 genotypes could be colonize in nasal mucosa of cancer patients which this could be a serious health hazard for developing gae (28). Regarding treatment approaches khojaste et al . Conducted a study targeting the gene profile of acanthamoeba t4 type in trophozoites and cysts . The result revealed that three genes including heat shock protein 70 (hsp70), actin - i and elongation factor-1alpha (ef-1) were differentially expressed during acanthamoeba differentiation and they could be the novel target for treatment (29). A recent research by niyyati et al . Also reported that tap and filtrated waters could be the sources of pathogenic free - living amoebae and this could render contact lens wearers for developing ak (30). There are various researches, which has conducted in iran as following . The first research regarding environmental acanthamoeba distribution these investigators showed the presence of acanthamoeba and naegleria in water samples of kazeroon city using morphological key (15). However, at that time, there was no report regarding various genotypes of acanthamoeba genus and these amoebas classified using morphological based criteria . Interestingly, all of the soil samples (5 out of 5) were positive in the culture . In addition, out of 61 dust samples, 28 of them contained acanthamoeba (16). Consequently, niyyati et al . Performed a sequencing based test for the mentioned environmental sources and the genotypes were belonged to t2, t6, t4 and t11 (17). It is important to mention that all of isolated genotypes have been reported as a cause of ak . Revealed that acanthamoeba present in pond water sources of all districts of tehran, iran . Genotyping of strains showed that they were belonged to t4 and t5 genotype, both genotypes has been identified as corneal pathogens mainly in soft contact lens wearers (18). In another research, niyyati et al . Showed the presence of potentially pathogenic acanthamoeba spp . In recreational river waters of tehran, briefly, 55 water samples from 10 major rivers were analyzed for fla and identified by morphological - based criteria, pcr amplification and sequencing analysis . Acanthamoeba, assigned to the t4 and t15 genotype were then identified using sequencing of df3 region (19). Other researchers showed the presence of thermotolerant acanthamoeba in hot spring of northwester iran (2022). Conducted a survey regarding the pathogenic potential of acanthamoeba t4 type using osmo - tolerance and thermo - tolerance assay . Cysts isolated from water, soil and dust (star shape cysts are shown in clump) (magnification: a: x10; b, c, d: x40) (figures are from pathogenic free living amoebae in human book, rezaeian m and niyyati m, 2009) acanthamoeba spp . Trophozoites isolated from soil and dust (flat shape trophozoites are shown) (magnification: a: x10, b: x40) rezaeian et al . Was the first to report the presence of acanthamoeba in hospital dusts in tehran, iran . This finding could be a health hazard for high - risk people including contact lens wearers and patients with eye surgery (16). The occurrence of fla in immunodeficiency wards of hospitals in tehran, iran were also investigated by lasjerdi et al . Briefly, 70 dust and biofilm samples from immunodeficiency wards of university hospitals were collected and tested for the presence of fla using culturing and molecular approaches . Presence of the t4 genotype on medical instruments, including an oxygen mask in an isolation room of an immunodeficiency ward, should be of concern for health authorities . Overall, these results reflect a clear need for improved disinfection, especially where high - risk people, such as those who are immune - suppressed or undergo eye surgery such as lasic surgeries, are served . . Encephalitis due to acanthamoeba has not been reported yet, mainly due to lack of knowledge regarding acanthamoeba as an agent of central nervous system infections . So far, there are 150 cases of ak in iran, although it seems that these numbers is lower than the true cases . The first cases of ak in iran were reported in a soft contact lens wearer (15). In 2007, interestingly, 44 patients (89.79%) were contact lens wearers for cosmetic purposes or visual corrections . Among them 41 patients (93.18%) wore soft contact lenses and three patients were hard contact lens wearers (25). Among 50 keratitis patient, 13 were positive for acanthamoeba (26). Three species including a. griffin, a. palesinensis and a. castellanii were identified in their samples . Another study revealed acanthamoeba as a causal agent in 15 (30%) of 50 keratitis samples . Among these clinical isolates, 13 (86.7%) belonged to female patients and 2 (13.3%) were male . All positive specimens belonged to soft contact lens wearers and only one belonged to a patient with a history of hard contact lens usage . Regarding genotype identification, 13 (86.7%) of these isolates belonged to t4 genotype . However, it is important to mention there was a mixed genotype belonging to acanthamoebae t4 and t11 genotypes in one patient . Other genotypes identified in the clinical specimens were t11 (13.3%) and t3 (6.7%) (17). Another survey of the 90 asymptomatic contact lens wearers, 9 (10%) were positive for fla outgrowth . Morphological analysis revealed that 3 isolates were belonged to hartmannella genus according to small round cysts and 6 isolates were belonged to acanthamoeba genus based on the star shape of endocysts . Sequencing revealed that acanthamoeba belonged to t4, t3 and t5 genotype (27). Cysts isolated from acanthamoeba keratitis (triangular shape cysts are shown) (magnification: x10) acanthamoeba cysts isolated from acanthamoeba keratitis (triangular shape cysts are shown) (magnification: right: x10, left: x40) so far, other infections due to acanthamoeba spp . Have not been reported in iran . This is due to lack of knowledge regarding these opportunistic protozoan parasites in this region . It should be noted that memari et al . Reported that acanthamoeba belonging to potentially pathogenic t3, t4 and t5 genotypes could be colonize in nasal mucosa of cancer patients which this could be a serious health hazard for developing gae (28). Regarding treatment approaches khojaste et al . Conducted a study targeting the gene profile of acanthamoeba t4 type in trophozoites and cysts . The result revealed that three genes including heat shock protein 70 (hsp70), actin - i and elongation factor-1alpha (ef-1) were differentially expressed during acanthamoeba differentiation and they could be the novel target for treatment (29). A recent research by niyyati et al . Also reported that tap and filtrated waters could be the sources of pathogenic free - living amoebae and this could render contact lens wearers for developing ak (30). The ubiquity of acanthamoeba spp . In environmental sources such as fresh waters, hot springs and tap waters and recreational soil sources and also the occurrence of the amoebae in the dust and biofilm samples of hospitals and clinical settings in iran and around the world could be a health concern specially for those who are within high risk groups such as contact lens wearers and immunosuppressed patients . On the other hand, ak continues to rise in iran mainly in female soft contact lens wearers with a history of poor maintenance of their lenses . There are no reports of acanthamoeba encephalitis in the region yet and thus this point reflects the need for more researches in suspected encephalitis patients . Here, according to our thorough review acanthamoeba belonging to t4 genotype is the most prevalent type strain in environmental and clinical samples in several regions in iran and worldwide, however, there are reports regarding acanthamoeba belonging to other genotypes such as t2 and the mentioned point could leads us to more researches with the goal of presenting the real genotype dominance of acanthamoeba and related disease in this country . Additionally researches regarding new treatment strategies for acanthamoeba - related infections are an utmost priority topic as such infections are manifest with poor prognosis.
Zygomycosis is an uncommon but often fatal opportunistic fungal infection that occurs in the setting of hematologic malignancies, chemotherapy - induced neutropenia, hematopoietic stem cell transplantation, immunosuppressive therapies, diabetes mellitus, and solid organ transplantation [1, 2, 3, 4, 5, 6]. Infection caused by mucorales is called zygomycosis . The members of mucoraceae, which include rhizopus, rhizomucor, mucor, and absidia, cause the vast majority of zygomycosis . Zygomycetes are ubiquitous in nature . Spores are released into the air, rapidly germinate, and gain entry through inoculation of respiratory mucosa, skin, or gastrointestinal (gi) tract, ultimately causing angioinvasion (leading to hematogenous dissemination), thrombosis, infarction, and necrosis [2, 8]. The main manifestations of disease include rhinocerebral, pulmonary, cutaneous / subcutaneous, and disseminated diseases, with the first two being the most common forms of infection [2, 8]. Gi infection is relatively uncommon, but gastric [5, 6], small bowel, ileocecum [4, 10], colon, or liver involvement has been reported . A case of primary gi zygomycosis due to absidia spp . Gi zygomycosis typically presents with nonspecific symptoms, including abdominal pain, nausea, dyspepsia, vomiting, bloody diarrhea, upper gi bleeding, abdominal distension, intestinal obstruction, and perforation peritonitis [5, 6, 10], making early antemortem diagnosis very difficult . As such a high index of suspicion is required for early diagnosis, especially in the presence of predisposing conditions, such as hematologic malignancies, neutropenia, diabetes mellitus, and immunosuppressive therapies [1, 2, 3, 4, 5, 6]. We report a case of disseminated appendiceal zygomycosis in a neutropenic patient who initially presented as acute appendicitis during induction chemotherapy for acute myeloid leukemia (aml) and highlight features that may lead to earlier diagnosis . To our knowledge, appendiceal zygomycosis due to absidia spp . Mimicking acute a 63-year - old woman with relapsed aml and diabetes mellitus was admitted for induction chemotherapy with cytarabine and clofarabine as part of the management plan for allogeneic stem cell transplantation . She was found to have clostridium difficile infection, and oral metronidazole treatment was started . An abdominal and pelvic computer tomography (ct) scan showed a segmental hypoenhancing area in the mid appendix with minimal surrounding fat stranding concerning for appendicitis . Given the patient's high risk of perioperative morbidity and mortality, she was initially treated with broad - spectrum intravenous antibiotics consisting of meropenem . However, her right lower quadrant abdominal pain continued and she developed localized peritoneal signs . A repeat ct scan obtained 3 days later showed stable inflammation of the appendix with a new finding of an adjacent loop of small bowel with thickened wall . Since the patient was not responding to medical therapy, she was taken urgently to the operating room for an appendectomy for treatment of acute appendicitis . Upon inspection of the right lower quadrant, the appendix was found to be completely necrotic down to the base . The necrotic appendix was lying on top of an adjacent loop of terminal ileum that was also segmentally necrotic at the contacting surface (fig . 2). The case was converted to open in order to perform an ileocecectomy with primary stapled anastomosis . The fascia was closed but the skin was left open to heal by secondary intention due to the infected wound classification . The patient was admitted to the intensive care unit postoperatively and extubated on postoperative day (pod) 1 . Despite empiric broad - spectrum antimicrobial therapy with meropenem, linezolid, and fluconazole, a ct scan of the chest showed numerous peripheral cavitary lesions that were either septic emboli or fungal infection . Hematoxylin and eosin (h&e)-stained sections showed ischemic changes, hemorrhage, and thrombosed vessels filled with broad irregular aseptate hyphae (fig . Gomori methenamine silver (gms)-stained sections also demonstrated characteristic wide ribbon - like aseptate hyphae that branch at wide angles, typical of zygomycetes, involving both vessels and adjacent submucosa (fig . 3c) as well as invading through the muscularis propria of the appendix (fig . Antifungal therapy was immediately switched to amphotericin b. on pod6, fungal overgrowth was found in the surgical wound . The patient's clinical condition continued to worsen with severe hypotension that required increasing doses of vasopressors . Given the patient's dismal prognosis of surviving such an overwhelming systemic fungal infection, goals of care were transitioned to comfort and she expired on pod8 . We report an unusual presentation of disseminated gi zygomycosis initially presenting as acute appendicitis in a neutropenic patient with relapsed aml . To our knowledge, appendiceal zygomycosis due to absidia spp . Mimicking acute absidia spp . Is ubiquitous in nature and found in soil, decaying organic matter, cotton, and many different grains, seeds, and nuts . It is a rare pathogen of the order mucorales, accounting for only 23% of all zygomycete infections, and shows almost no pathogenicity in immunocompetent hosts . Infections with absidia spp . Usually occur in immunocompromised hosts, including leukemia or lymphoma patients, especially in the setting of chemotherapy - induced neutropenia and broad - spectrum antibiotics [11, 12]. Although 7% of all zygomycete infections are known to involve the gi tract, zygomycosis due to absidia spp . (absidiomycosis) is extremely rare in these sites, even in patients with leukemia or lymphoma . Several disseminated infections with absidia spp . Have been reported, including a case of brain abscess following bone marrow transplantation as well as a case of metastatic carcinoma with disseminated zygomycosis involving the brain, lungs, and heart . Although it involved a different member of mucoraceae, one case of zygomycosis caused by mucor indicus, presenting as an appendiceal mass with spread to the liver, the diagnosis of zygomycosis requires demonstration of characteristic wide ribbon - like aseptate hyphae that branch at wide angles (4590), especially with fungal specific stains such as gms stain, and should be supported by culture . Vascular invasion resulting in thrombus formation with infarction, necrosis, and hemorrhage is the most characteristic feature of zygomycosis . Disseminated zygomycosis rapidly invades vessels to spread to multiple organs, most frequently involving the lung [2, 3]. Successful treatment of zygomycosis has been attributed to early diagnosis, aggressive surgical intervention with resection of the involved tissue, and antifungal therapy with amphotericin b . Since surgery is most effective in the setting of localized disease without dissemination, early diagnosis is crucial in reducing the high mortality associated with zygomycosis . Pathology of the resected ileocecectomy specimen played a crucial role in diagnosing zygomycosis in this case, leading to the initiation of amphotericin b as the appropriate antifungal therapy . However, the patient developed fatal disease with evidence of dissemination to the lung and abdominal surgical wound at the time of diagnosis . In conclusion, a diagnosis of a fungal infection, including zygomycosis, should be considered in all chemotherapy - induced neutropenic patients who present with symptoms of acute appendicitis . This is further supported by a previous study reporting that immune defects, especially absolute neutrophil count <1,000/l, is a major risk factor for the development of fungal disease . A high index of clinical suspicion with prompt histologic and culture diagnosis of zygomycosis may lead to earlier surgical and/or antifungal treatment that can potentially reduce the high mortality and morbidity associated with gi zygomycosis . Since the patient died, written informed consent was obtained from her next of kin for publication of this case report and any accompanying images.
The majority of patients with cancer undergoing chemotherapy require an efficient venous access for several weeks or months . Central venous port (cvp) catheter is widely used in this setting . The incidence of deep venous thrombosis (dvt) and pulmonary embolism (pe) associated with central venous catheter has been reported between 2% and 67% . The cancer population combines nonspecific thromboembolic risk factors (age, malignancy, hypercoagulability, chemotherapy, infections, and bed rest) and specific risk factors such as catheter material, multiple placement attempts, catheter size and length, number of lumens, and catheter tip localization [35]. Catheter - related vte may be limited to asymptomatic radiological findings but may also lead to significant clinical burden with upper limb postthrombotic syndrome reported in 5 to 28% [6, 7] and respiratory failure in case of pulmonary embolism . Moreover, catheter thrombosis can also lead to catheter occlusion in 14 to 36% and delay chemotherapy . At least eight randomized controlled trials have evaluated antithrombotic therapy versus placebo in the prevention of central venous catheter - associated thrombosis [916]. A small study found that fixed dose of warfarin 1 mg once daily reduced the incidence of upper extremity dvt at the 90th day of venography . However, two subsequent trials failed to confirm any benefit with this regimen [10, 11]. In two large studies, the administration of a prophylactic dose of low molecular weight heparin (lmwh) during at least 6 weeks after the catheter insertion did not reduce significantly the incidence of upper limb dvt compared to placebo [14, 15]. A systematic thromboprophylaxis is therefore not recommended at the time of cvp implantation, and should be considered only for patients with solid tumor and additional risk factors for vte and low bleeding risk . During the last years, khorana and colleagues developed and validated a predictive model for chemotherapy - associated thrombosis [18, 19]. This model allows identification of patients at high risk who may benefit from antithrombotic treatment during chemotherapy . To the best of our knowledge, there is no risk score available to evaluate the risk of vte following cvp insertion and the khorana score has not been validated in this setting . The purpose of this study was to evaluate the incidence of vte closely associated with the insertion and use of cvp catheters and to identify high - risk patients amenable to benefit from a short course of thromboprophylaxis after cvp implantation . From february 2006 to december 2011, all consecutive adult patients suffering from cancer and who were implanted with a cvp in the surgery department of the cantonal hospital, fribourg, switzerland (tertiary care center) were screened for inclusion in this prospective cohort . The operator always attempted to find the cephalic - subclavian junction at the right upper limb and to place a j - curved 0.035 inch guide wire in the superior cava vein . The catheter tip was then placed at the level of the thoracic rib, under fluoroscopy guidance . The chamber was then placed in the pectoral region by tunelisation from the same skin incision . When the cephalic vein was not accessible, the catheter was implanted in the subclavian vein by direct punction . There was no routine sonography or phlebography of the subclavian vein prior to the intervention except in case of known previous dvt, previous central line use or failure to pass the guide wire in a previous attempt . During the study period, patients were regularly followed up clinically during chemotherapy treatment and then every 6 months after chemotherapy completion until complete remission, death of any cause, or loss of follow - up . Patients whose cvp catheter has been removed for lack of use were followed up until 6 weeks after removal . At each visit, the subjects were questioned about any local pain or upper limb swelling at the cvp side . Any other symptom suggesting upper or lower limb dvt or any thoracic symptom suggesting a pe was further investigated by compression venous sonography of the limbs or thoracic ct scan . The main outcome was the 3-month incidence of catheter - related vte, defined as occlusive dvt in the arm along the catheter, with or without pe, or isolated pe of unknown origin . The secondary outcomes were the 12-month incidence of catheter - related vte and the 3- and 12-month incidence of any vte related or not to the catheter, including dvt of the leg, dvt of the other arm and visceral dvt . Asymptomatic dvt or pe observed on ct scan performed for tumoral staging was classified as asymptomatic event . Catheter dysfunction and small, nonoccluding thrombosis along the catheter were not considered as event . Complete occlusion of the vein along the catheter was considered as asymptomatic event if it was not associated with local symptoms . The khorana predictive score assigns 2 points to very high - risk cancer sites (pancreatic, gastric, brain) and 1 point to high risk cancer sites (lung, ovarian, renal, or bladder). In addition, 1 point is assigned for each of the following: platelet count 350 10/l, hemoglobin <10 g / dl, or use of erythropoietin - stimulating agents, leukocyte count 11 10/l, and body mass index 35 kg / m . Incidences of event were expressed as proportions with 95% confidence intervals, calculated by binomial wilson test . Proportions of event were compared using chi test, and continuous variables were compared by the mann - whitney rank sum test according to the normality of their distribution . Statistical significance was considered for <0.05 . The contribution of clinical characteristics (age, sex, weight, body mass index, previous vte, respiratory failure, renal failure, antithrombotic treatment, cancer location, metastatic stage, low performance status, class of chemotherapy agent, major surgery close to cvp implantation or during follow - up, side of cvp implantation, baseline laboratory values, and khorana score) to asymptomatic and symptomatic vte was analysed using multivariable logistic regression analysis . Factors were first analyzed individually in univariate analysis and then selected for multivariable analysis based on a p value <0.2 or known confounding effect . The statistical analysis was performed using stata 9.0 (statacorp, college station, texas, usa). Throughout the 6-year study period, 1243 consecutive patients were candidates for a cvp implantation in our institution and were screened for inclusion . We included 1074 patients with cvp placement at first attempt and 23 patients with failure at first attempt but success at second attempt . We excluded 146 patients (15 patients for previous cvp placement, 129 patients for choice for another intravenous access after cvp placement failure, and 2 patients for failure to place cvp at first and second attempt). The most frequent cancers were lung (21.1%), colo - rectal (18.6%), and oesogastric (13.3%). Table 2 shows the incidence of vte at 3 and 12 months . The incidence of cvp - associated vte was 5.9% of patients (ic95 4.47.3%) at 3 months and 11.3% (ic95 9.413.2%) at 12 months . The incidence of vte at any location was 7.6% of patients (ic95 6.09.3%) at 3 months and 15.3% (ic95 13.117.6%) at 12 months . The multivariate logistic regression analysis identified 2 significant predictors of catheter - related vte at 3 months: a khorana score 3 (odd ratio (or) 3.50, ci95 1.00 to 12.3, and p = 0.05) and lung cancer (or 5.45, ci95 1.87 to 15.87, and p = 0.002). Low performance status was borderline significant (or 4.68, ci95 0.97 to 22.4, and p = 0.054) (table 3). Advanced stage with distant metastases was only borderline significant in the univariate analysis but fell in the multivariable regression . Previous vte, high bmi, age> 70, and platinum - based regimen were not associated with vte at 3 months in this cohort . Most patients had chemotherapy close to the cvp implantation (31% of patients had chemotherapy before cvp implantation, 14.7% of patients had chemotherapy 0 to 3 days after cvp implantation, 20.6% of patients had chemotherapy 4 to 8 days after implantation, and 33.7% of patients> 8 days later). Compared to the 3 other groups, the group of patients receiving chemotherapy within days 0 to 8 after cvp implantation had no additional risk of cvp - related vte at 3 months (or 1.00, 0.67 to 1.57, and p = 0.98). The incidence of vte was particularly high in the 3 subpopulations identified . Among the 102 patients (9.3%) with a baseline khorana score 3, 18.6% (ci95 10.9 to 26.4) had a vte during the first 3 months of follow - up . This incidence was 10.8% (ci95 6.8 to 14.8) among the 232 patients (21.1%) with lung cancer and 10.9% (ci95 7.7 to 14.1) among the 367 patients (33.5%) with metastatic cancer at the time of cvp insertion . The steeper part of the slope is observed during the first 3 months after catheter implantation . However, additional events continue to be observed up to the end of follow - up . Multivariable regression analysis identified the same predictors of vte at 12 months than at 3 months . Khorana score 3 (or 2.67, ci95 1.49 to 4.78, and p = 0.001) and lung cancer (or 1.93, ci95 1.15 to 3.25, p = 0.01) were significantly associated with vte (from any origin) during the 12-month study period . Bevacizumab and platinol based regimen were both borderline significant predictors of 12-month vte events (table 3). This large cohort study, designed to evaluate the incidence of vte closely related to central venous port catheter implantation, shows that 7.6% of the patients will develop dvt or pe during the first 3 months after catheter implantation . This finding confirms the important burden linked to the iv management of chemotherapy and the importance to develop efficient preventive antithrombotic strategies . This study also validates high khorana score and lung cancer as significant predictors of vte during the whole study period . Despite several trials, evaluating different drug regimens to prevent chemotherapy - associated vte, no clear benefit emerged from any specific regimen up to now . One limitation of thromboprophylaxis trials is due to the fact that the benefit from antithrombotic treatment can be overwhelmed by the bleeding risk occuring during chemotherapy . The other difficulty is to identify patients at risk as well as the period at risk . The current practice now is to consider prophylactic antithrombotic treatment for patients with solid tumor and an additional risk factor for vte, such as previous vte, immobilization, and specific anticancer therapy (thalidomide or lenalidomide in association with dexamethasone) [17, 21]. Limiting thromboprophylaxis on a high - risk period could increase efficiency and reduce the bleeding risk inherent to prolonged antithrombotic treatment . The period of cvp implantation concentrates major risk factors for vte (surgery, intravenous foreign material, upper limb immobilization, untreated cancer, and repeated chemotherapy infusions). Our study investigated specifically this period and confirmed the high incidence of vte close to the cvp insertion and its key role in the pathogenesis of cancer - related vte . Our study also identified 2-patient categories prone to develop vte in 10 to 20% at 3 months and who will certainly benefit from thromboprophylaxis, at the time of cvp implantation . Interestingly, identification of these patients based on simple clinical data (lung cancer) or a widely used score based on the type of tumor, bmi, and prechemotherapy laboratory values allows easy identification of patients who could benefit from thromboprophylaxis . In their observational study of 815 cvp implantations, narducci et al . Found that the factor most strongly predictive of complications was a delay shorter than 8 days between cvp implantation and first use . These complications were mostly nonthrombotic (local of systemic infection, port expulsion, catheter dysfunction, or migration). We analyzed the delay between implantation and use in our study and found that a delay> 8 days did not reduce the 3-month vte incidence . One limitation of our study is that the definition of the primary outcome did not include death due to probable or possible vte . At the time of the study design, we were concerned about being unable to exclude formally pe as the cause of death, especially in the setting of palliative care . However, we consider that the conclusions of our study are rather conservative since adding deaths for vte origin could increase the incidence of vte related to cvp insertion . Ultrasound guidance has shown to reduce the risk of infectious and thrombotic complications in all percutaneous venous procedures . Our institution has a surgical team dedicated to cvp placement and during the study period (2006 to 2011) the same senior vascular surgeon was in charge of the team . The subclavian vein was punctured only when cephalic vein was not found . In these cases, the subclavian vein was directly observed and ultrasound would not bring further security . In conclusion, this large cohort study of consecutive patients with first cvp catheter implantation confirms the high incidence of thrombotic events closely associated with the cvp intervention . It confirms khorana score and lung cancer as strong predictors of catheter related vte and vte of any origin at 3 and 12 months . These findings will allow the definition of a risk population in order to assess the best thromboprophylaxis in a randomised trial . Otherwise, we may question whether it is still reasonable to delay efficient thromboprophylaxis for these patient populations.
Posttraumatic stress disorder (ptsd) is a complex mental disorder that can develop in response to a traumatic event such as a motor vehicle accident, rape, combat exposure or natural disaster . The national vietnam veterans readjustment study estimated that 53.4% of us male veterans of the vietnam war developed full or partial ptsd and 15.2% still suffered from the disorder in 1990 1 . The application of conservative procedures for analysis led dohrenwend et al to document that 18.7% of male veterans developed war - related ptsd during their life time and 9.1% were still suffering from ptsd 11 to 12 years after the war 2 . In the general population, the estimated lifetime prevalence of ptsd is 7.8% in adult americans, with women having a 2.3-fold higher prevalence than men 3 . Ptsd symptoms include intrusive thoughts, hyperarousal, nightmares, flashbacks, emotional detachment or numbing of feelings, insomnia, fear, avoidance of reminders, distress when exposed to reminders, irritability, hypervigilance, and heightened startle response 4 . Several lines of evidence support neurochemical, functional and structural alterations in the neuroendocrine system in patients with ptsd . The neurochemical alterations range from adrenergic hyperresponsiveness, increased thyroid activity, increased levels of corticotrophin - releasing factor, to low cortisol levels and increased negative feedback sensitivity of the hypothalamo - pituitary - adrenal - axis after the administration of low - dosage dexamethasone 5 . Functional brain imaging studies in patients with ptsd demonstrate increased function in the amygdala and decreased activity in the hippocampus and prefrontal cortex (pfc) 6;7 . These functional alterations are consistent with the structural findings in animal models of ptsd that neurons in the hippocampus and pfc display atrophy while those in amygdala show growth in response to repeated stress 8 . Dlpfc including ba46 is one of the three regions of pfc, which regulates working memory and execution of fear responses 9 . This brain region has been correlated with structural and functional alterations, and with the treatment response of patients with ptsd . In children with ptsd symptoms, decreased volume of gray matter in the dlpfc is correlated with increased functional impairment 10 . Adult patients with ptsd core symptoms (i.e. Re - experiencing, avoidance) were markedly improved by treatment with 10-hz repetitive transcranial magnetic stimulation over the right dlpfc 11 . Furthermore, the indicator of neuronal viability, n - acetylaspartate, in dlpfc was correlated positively in healthy people with verbal intelligence 12, and negatively in patients with a generalized anxiety disorder following a previous traumatic event 13 . Although the prefrontal cortex, amygdala and hippocampus are the brain regions considered to be related to ptsd 6;7, the underlying molecular mechanisms are unknown . One of the possibilities is that functional and structural changes in the brain may result from mitochondria - centered responses to repeated or chronic harmful stresses 14;15 . Mitochondria provide the cell with energy in the form of atp and play fundamental roles in many metabolic pathways, such as -oxidation, the tricarboxylic acid cycle and urea cycle, the synthesis of steroid hormones and heme, and calcium signaling . In addition, mitochondrial biochemical and molecular pathways are a natural central target of diverse pharmacological agents 16 . However, reactive oxygen species (ros), an inevitable by - product of mitochondrial oxidative phosphorylation, can impair molecules, contributing to various diseases including neurodegeneration 17 . Thus, homeostasis of these mitochondrial functions is critical in human brain because of its high energy demand . Mitochondrial dysfunctions are increasingly recognized as key components in stress related mental disorders 14;15 . Although long or repetitive exposure to stress can induce a neurological change resulting in ptsd, the molecular pathology involving in such a neuronal damage remain elusive . Overexpression of antioxidant enzymes (glyoxalase and glutathione reductase 1) in mouse brain was also associated with increase in anxious behavior 19 . In addition, chronic stress of rats inhibits the activities of mitochondrial respiratory complexes (inhibition of 69% in complex i - iii and of 67% in complex ii - iii) 20 . Cortisol has widespread functions including activation of glucocorticoid receptor which bind directly to mitochondrial membrane and regulate apoptosis 15 . The steroidogenic acute regulatory protein is one of the outer mitochondrial membrane protein required for stress responses 21 . Although all these studies indicate mitochondrial dysfunction, they are all focusing on a specific candidate gene(s). The lack of a holistic study of all the mitochondria - related genes hinders the progress of the ptsd research . To date, methods employing a systems biology approach to identify the mitochondria - focused genes underlying the pathogenesis in ptsd - related brain regions have not been documented, probably due to the lack of human postmortem brain tissue from ptsd patients . The expression signatures in blood cells have been associated with changes in peripheral lymphocytes of subjects with ptsd 22;23, but provide no information for molecular abnormalities in brain tissues . Here we report the identification of expression signatures, canonical pathways, molecular networks and drug targets of dysregulated neuropsychiatric disease - related genes in ba46 of postmortem brain tissue from patients with ptsd using a human mitochondria - focused cdna microarray (hmitchip3) 24 . Our results indicate mitochondrial dysfunction in the dlpfc ba46 and may prove useful for developing methods for diagnosis and treatment of ptsd . Postmortem brain tissue: postmortem brain tissues were collected by the traumatic stress brain study group who diagnosed, collated, planned, coordinated, made decisions on inclusion and exclusion, established procedures of diagnosis and interrogated reliability, and obtained normal brains for controls as well . Medical records were meticulously reviewed and clinical diagnoses were confirmed using dsm - iv criteria 4 . Table 1 lists all available brain tissues employed for this study, with some clinical data including patient's gender and age (4-year difference), brain ph (0.65 difference) and reasons of death . Eight subjects died from myocardial infarction / heart attack, two from suicide by overdose, one from alcoholism complications and another from pulmonary embolism . Tissue (0.4 - 0.8 g) was thawed and homogenized in 10 times the tissue volume of deionized distilled h2o and the homogenate ph was measured with the perphect ph 370 model meter with a compatible perphect sure - flow type probe (ati orion, boston, ma, usa). The ph electrode was washed thoroughly in deionized distilled h2o after each sample and recalibrated at the standard 3 points of ph (4, 7, and 10). Rna preparation: dlpfc ba46 tissues were dissected from postmortem brains and the samples contained primarily gray matter with small but random amount of white matter because the dissections were done from frozen tissue blocks but not from cryostat sections . Carlsbad, ca, usa) and then purified with rneasy kit (qiagen, valencia, ca), following the manufacture's instructions . Gene chip and microarray data analysis: a third - generation human mitochondria - focused cdna microarray (hmitchip3) containing 37 mitochondrial dna - encoded genes, 1,098 nuclear dna - encoded and mitochondria - related genes and 225 controls was printed as described previously 24 . A total of 1135 mitochondria - related genes include 946 genes associated with 645 molecular functions, 930 genes with 612 biological processes, 476 genes with biological chemistry pathways, 227 genes with 23 reactome events, 237 genes with 320 genetic disorders, and 55 genes with 87 drugs targets24 . For examples, 89 genes are related to oxidative phosphorylation, 20 genes to apoptosis, and 14 genes to neurotransmitter (tyrosine, l - dopa and dopamine) metabolism . Five g of rna per sample were used for microarray labeling and hybridization as previously described 24 . Slides were scanned using the scanarray express microarray scanner (perkinelmer, boston, ma, usa) as described previously 24 . Microarray database construction, data filtering and normalization were performed as described previously 24;25 . The normalized data were used to cluster and visualize expression levels of genes in these brain tissue specimens by using eisen's cluster software 26 . Heat map was visualized by using eisen's maple tree software (http://rana.lbl.gov/eisensoftware.htm). Gene information analysis: gene i d, symbols and names were derived from human unigene build 204 (ftp://ftp.ncbi.nih.gov/repository/unigene/) based on human cdna i.m.a.g.e . Ontology, pathways and phenotypes of genes were compiled from entrez (ftp://ftp.ncbi.nlm.nih.gov/gene) and david bioinformatics resources 2008 (http://david.abcc.ncifcrf.gov/). Ingenuity pathway analysis software ipa version 6.0 (ingenuity systems inc ., redwood city, ca, usa) was used to map canonical pathways, diseases, disorders, molecular networks and drug targets and to calculate the percentages and p - values (fisher exact test). Quantitative rt - pcr (qrt - pcr): two g of total rna was reverse - transcribed into cdna by using superscript first - strand synthesis system (invitrogen). 30 ng cdna was used for qpcr reactions with the universal pcr master mix (no amperase ung) on an applied biosystems 7300 real time pcr system (foster city, ca, usa), following the manufacturer's instructions . After 40 cycles, taqman probes and primers for qpcr were purchased from applied biosystems and include those of glyceraldehyde-3-phosphate dehydrogenase (gapdh, 4352934e), dnaj (hsp40) homolog subfamily c member 19 (dnajc19, hs00829488_s1), solute carrier family 1 (high affinity aspartate / glutamate transporter) member 6 (slc1a6, hs00192604_m1), solute carrier family 9 (sodium / hydrogen exchanger) member 6 (slc9a6, hs00234723_m1), amyloid beta (a4) precursor protein (app, hs00169098_m1), and estrogen receptor 2 (er beta) (esr2, hs00230957_m1). Statistics: statistical calculations were performed on triplicate array experiments using xlstat 2006 (xlstat, new york, ny, usa). Differentially expressed genes were identified arbitrarily by 1.25-fold change in the average expression of the background - subtracted mean intensity ratios of a gene between ptsd and control, with p - value <0.05 . Student t - test was used to calculate p - values for gene expression, while fisher exact test in ingenuity pathway analysis software was used to calculate p - values for pathways and diseases . The level of statistical significance was set at a p - value <0.05 . Total rna samples were extracted from gray matter that was dissected from postmortem brain dlpfc ba46 of ptsd brains (n=6) and unaffected controls (n=6) and labeled for triplicate microarray experiments using our recently developed third generation human mitochondria - focused cdna microarray hmitchip3 . Because use of 3 replicates in cdna microarrays analysis of even a single specimen greatly reduces misclassification rates 29, the hmitchip3 genes were all measured 9 times (3 identical probes per microarray and 3 microarray experiments per specimen), which generated reliable expression data for further analysis . The microarray data of 4,080 spots across all 36 gene chips used for 12 rna samples were filtered by uniform statistic and bioinformatic criteria as described previously 24;30, which generated 800 genes with informative expression profiles . The supplemental table 1 reports the expression data of the selected 800 genes in each of the triplicate experiments before and after normalization as the raw and processed data, respectively; while figure 1 shows the box plots of mrna levels of 800 genes before and after the data normalization . The resultant dendrograms for all of these 800 genes and 12 brain dlpfc ba46 specimens classified p1, p2, p3, p5, p6 and n2 in one group and n1, n3, n4 n5, n6 and p4 in another (fig . 2a and supplemental figure 1); these analysis separated the ptsd brain from the control except for n2 and p4 (opposite from their clinical diagnosis). Based on the unsupervised cluster results, we calculated an average expression level of each gene in the ptsd group (p1, p2, p3, p5, p6 and n2) and in the non - ptsd control group (n1, n3, n4 n5, n6 and p4). The ratios and p - values (t - test) were calculated between the ptsd and control specimens, resulting in the identification of 119 genes (p<0.05, 1.25) and 42 genes (p<0.05, 1.60) whose expression were dysregulated in the ptsd dlpfc ba46 as compared to the controls (supplemental table 2). Clustering analysis of these 119 and 42 genes across all 12 brain samples generated double dendrograms and heat maps that clearly distinguish the ptsd dlpfc ba46 specimens from the controls (fig . The identification of these dysregulated genes provides candidates with biological clues for validation of mrna expression and future functional studies . Qrt - pcr confirmation of microarray results . To validate the microarray results, we conducted qrt - pcr analysis on 5 genes including app, dnajc19, esr2, slc1a6 and slc9a6 because of the highest (slc9a6) and lowest (danjc19) expression levels in ptsd ba46 or because of their known relevance to neuropsychiatric disorders (app, est2 and slc1a6). The results showed that the relative mrna levels of these genes measured by qrt - pcr were essentially in agreement with the data detected by microarray experiments (fig . 3). Specifically, 11 of 12 (92%) of expression changes were consistent between these two methods for app, 9 of 12 (75%) for slc9a6, and 8 of 12 (67%) for dnajc19, slc1a6 and esr2 . The discrepant data were randomly distributed among the genes and samples tested (fig . Thus, we subjected all the 119 dysregulated genes to analysis of a systems biology including the pathway, ontology and network . The mitochondrial dysfunction and oxidative phosphorylation pathways contained the highest number of dysregulated genes . To see functional relationships the results showed that out of 94 canonical pathways, 16 (17.0%) contained a significant number of dysregulated genes per pathway, of which 10 were listed in figure 4a . The mitochondrial dysfunction pathway contained 8 (4.8%) dysregulated genes (p=6.61x10) including upregulated app, cat, ndufa10 and ucp2 and downregulated cox8a, ndufb5, ndufs2 and pdha1 . The oxidative phosphorylation pathway had 6 (3.8%) dysregulated genes (p=9.04x10) including upregulated atp5c1 and ndufa10 and downregulated atp5e, cox8a, ndufb5 and ndufs2 . The citrate cycle pathway had 3 (5.1%) dysregulated genes (p=1.71x10) including upregulated idh3b and downregulated cs and suclg1 . The methane metabolism pathway contained 2 (3.1%) dysregulated genes (p=5.38x10) including upregulated cat and downregulated mpo . The ubiquinone biosynthesis pathway had 3 (2.9%) dysregulated genes (p=1.11x10) including upregulated ndufa10 and downregulated ndufb5 and ndufs2 . The erk / mark signaling pathway included upregulated h3f3a, ppp2r1b, prkcd and tln2, and downregulated prkar2b (n=5 [2.8%], p=1.18x10). The propanoate metabolism pathway had upregulated acad8 and acadl and downregulated suclg1 (n=3 [2.4%], p=1.21x10). The xenobiotic metabolism pathway contained upregulated cat, hsp90ab1, ppp2r1b and prkcd and downregulated aldh18a1 and mgst1 (n=6 [2.4%], p=1.28x10). The valine, leucine and isoleucine degradation pathway contained upregulated acad8, acadl and hmgcl (n=3 [2.8%], p=1.38x10). The pyruvate metabolism pathway had downregulated akr1b1, hagh and pdha1 (n=3 [2.1%], p=2.05x10) (fig . 46 dysregulated genes were mapped to neurological diseases and other systemic disorders . To see phenotypes involved, we performed mapping of all 119 dysregulated genes to known human diseases and disorders . The results revealed that 35 dysregulated genes had known roles in a variety of neurological diseases (28 genes) and other systematic disorders including endocrine (14 genes), genetics (11 genes), metabolism (12 genes) and psychology (6 genes). The number of these dysregulated genes were all statistically significant (p<0.05). In addition, 17 (61%) of the 28 genes that were mapped to neurological diseases were known to play a role in survival and apoptosis of a cell . Moreover, a total of 30 dysregulated genes were mapped to the cell survival - apoptosis category (fig . Many of these genes are involved in multiple abnormalities, suggesting that ptsd is a neurological and systemic disorder . Table 2 lists 30 ptsd - dysregulated genes that have been described to play a pathogenic role in various neurological diseases and psychiatric disorders . Upregulated app induces changes in nitric oxide production and mitochondrial activity, leading to apoptosis 31, which have been implicated in amyloidosis 32;33, neurodegeneration 34, alzheimer's disease 35;36 and down syndrome 37 . Considering the role of myeloperoxidase (mpo) in modulating vascular inflammatory responses and transporting molecules as well as its incapability of clearing up amyloid when mutated 38 - 40, mpo downregulation may lead to regional accumulation of amyloid, resulting in mitochondrial dysfunction and cell death . Peptidylprolyl isomerase d (ppid) suppresses apoptosis via a mitochondrial hexokinase ii - dependent mechanism and its decreased expression has been associated with corticobasal degeneration characterized by nerve cell loss and atrophy 41 . Dysregulation of genes with known functions as neurotransmitters in synaptic vesicles (slc1a6 and vamp1) and cell adhesion (pecam1, cdh13 and spg7) are related to diseases in the central nervous system . Dysregulation of these genes might reduce synaptic communication, and jeopardize cell - to - cell interactions and/or cell - to - matrix adhesion . These are but a few examples (table 2). Networks of 54 dysregulated genes involved in neuron function and survival and 7 protein targets for neuropsychiatric drugs: to reveal complex interactions among dysregulated genes and to identify gene targets for the central nervous system drugs, we mapped 54 dysregulated genes to molecular networks . These genes were selected for analysis because of their obvious relevance to neurological diseases, psychological disorders, psychiatric disorders, and stress response in the central nervous system . The results revealed interactive networks with a total of 75 molecular elements that maintain neuronal function and survival . Within these networks, 27 (36%) genes were upregulated; 27 (36%) genes were downregulated; and 21 (28%) elements appear to be unchanged (fig . These data strongly suggest molecular and cellular abnormalities in neuronal function and survival in ptsd dlpfc ba46 . Importantly, 7 gene products within the neuronal function - survival networks were identified as known targets of drugs that are used for neurological diseases, psychological and/or behavioral disorders, including app as a target for bapineuzumab (aab-001), vamp1 for dysport, slc1a6 for riluzole, esr2 for 17-estradiol (agonist) and tamoxifen (antagonist), prkcd for tamoxifen and rottlerin, pp2a for okadaic acid and fostriecin, and ucp2 for mptp (1,2,3,6-methyl - phenyl - tetrahydropyridine). Human brain dlpfc including ba46 is involved in regulation of working memory and preparation and selection of fear responses and has been correlated with structural and functional alterations and treatment response of patients with ptsd 9 - 13 . However, the underlying cellular and molecular mechanisms are unknown . In this study, we applied human mitochondria - focused cdna microarrays (hmitchip3) to ptsd brain samples and have successfully identified expression signatures, canonical pathways, molecular networks and drug targets of neurological disease- and psychological / psychiatric disorder - related genes that are dysregulated in the dlpfc ba46 . Our results indicate mitochondrial dysfunction involved in neuronal function and survival in the dlpfc ba46 and may prove useful for development of methods for diagnosis, prevention and treatment of ptsd . Unsupervised clusters of 12 dlpfc ba46 rna samples based solely on expression similarities of informative 800 mitochondria - focused genes clearly distinguish the ptsd brains from the controls . The identification of 119 (p<0.05, 1.25) and 42 (p<0.05, 1.60) dysregulated genes provides candidates for qrt - pcr validation and for subsequent functional studies . The clustering signatures of these genes may facilitate further development of methods and tools for forensic diagnosis of ptsd . Our approach has generated informative results partially because of our accurate dissection of brain dlpfc ba46 gray matter, and partially because of the utilization of our hypothesis - driven mitochondria - focused cdna microarrays . However, we cannot explain why the samples p4 and n2 had expression profiles different from their clinical classification . Due to a small sample size, p4 and n2 were kept in the groups based on their gene expression rather than a clinical diagnosis because the former might be more objective than the latter . We would like to repeat the study when more ptsd and control specimens become available in order to improve the molecular classification of ptsd . The subsequent experiments should examine both mrna and protein levels in the dlpfc ba46, as well as other ptsd - related brain regions such as medial and ventral pfc, hippocampus, and amygdala 6;7;42 . The mitochondria - mediated cellular and molecular response to stress in the central nervous system of patients with ptsd is the key part of our hypothesis because mitochondria are at the center of a cellular response to stress 14;15 . Study of mitochondrial function is inevitable and our application of the mitochondria - focused cdna microarrays to ptsd is just the beginning . Mitochondrial function in the brain is of particular importance because of its high energy demand . Although human brain represents only 2% of body weight, it receives 15% of the body's cardiac output, and uses 20% of total body oxygen . These high level oxygen and energy requirements are continuous and imply that even brief periods of oxygen or glucose deprivation may impair neuron function and even result in neuron death . Our demonstration that a highly significant number of oxidative phosphorylation genes were dysregulated in the ptsd brain ba46 strongly suggests the presence of at least energy deficiency in this brain region . Our findings support the postulation that mitochondria - focused and stress - responsive genes may play a key role in the pathogenesis of ptsd, although the specific impact of these dysregulated genes and pathways on brain function remains to be determined . The identification and validation of the dysregulated genes enhances our understanding of the cellular and molecular mechanisms underlying the pathophysiology of ptsd . The genes listed in table 2 are critical to the ba46 neuronal functions including survival and apoptosis, carbohydrate and lipid metabolism, atp and ros production, synthesis and transport of neurotransmitter, mitochondrial and nuclear dna expression, and protein phosphorylation and dephosphorylation as well as folding and degradation . Because homeostasis of these mitochondrial functions is vital for neuronal function and survival, the presence of a hundred consistently dysregulated genes in the ptsd dlpfc ba46 implies a pathological role . In addition, more genes were downregulated than those upregulated (63 vs. 56 in supplemental table 1 and 18 vs. 12 in table 2). Moreover, some upregulated genes play a degradative or negative role such as proapoptosis (e.g., app), reduction of atp synthesis (e.g., ucp2) and protein breakdown (e.g., hspa1a). Taken together, functional impairment and atrophy in the ptsd dlpfc ba46 might be a consequence of these dysregulated genes . Thus, the neurons in the ptsd dlpfc ba46 may be unable to regulate working memory and preparation and selection of healthy responses to fear or may even send abnormal signals to their downstream executive neurons such as those in the hippocampus and/or amygdala . Our molecular insight in the ptsd dlpfc ba46 pathogenesis is reminiscent of the neurocircuitry model by bremner 43 and rauch et al 42 that the fear extinction abnormalities in ptsd are characterized by exaggerated amygdala responses and deficiency in ventral / medial pfc and hippocampus . Therefore, our systems biology results appear not only to extend the neurocircuitry model to brain dlpfc ba46, but they also provide genes and proteins that should be further investigated for better understanding of the molecular pathogenesis of ptsd . Seven protein targets for neuropsychiatric drugs are present in the neuronal function - survival networks in human brain dlpfc ba46 . In addition of dysregulation of 72% (54/75) genes in these networks, many dysregulated genes are associated with multiple neuropsychiatric conditions such as alzheimer's disease, parkinson's disease, schizophrenia, and major depressive disorder (mdd), down syndrome, attention disorders and mood disorders (table 2). Obviously, modulations of these genes with drugs would affect neuropsychiatric functions of the neurons in human dlpfc ba46 . For example, the app is a target for the immunotherapeutic drug bapineuzumab that is currently used for the phase 3 clinical trials for the treatment of patients with alzheimer's disease 44 . The esr2 is a target for 17-estradiol (agonist) and tamoxifen (antagonist). Beside the treatment of estrogen - receptor positive breast cancer, tamoxifen is used to treat mania in patients with bipolar disorder, probably by blocking prkcd 45 . The prkcd is also a target for rottlerin used in preclinical study of parkinson's disease 46 . The ucp2 is a target for protection of dopaminergic neurons from oxidative stress caused by 1,2,3,6-methyl - phenyl - tetrahydropyridine (mptp) toxicity 48 . However, whether these drugs and related targets might be useful for prevention and treatment of ptsd is interesting and deserves further studies . Supplemental table 1a: microarray mean data before normalization click here for additional data file . Supplemental table 1b: microarray mean data after normalization click here for additional data file . Click here for additional data file . Traumatic stress brain study group includes david benedek and harry holloway, dept of psychiatry & center for the study of traumatic stress, usuhs, school of medicine; maree j. webster, stanley medical research institute; christopher j. hough, fda; ronald duman, abraham ribicoff research facilities laboratory of molecular psychiatry, yale university school of medicine; matthew friedman, national center for ptsd, us dept of veterans affairs & dartmouth medical school; john krystal, clinical neuroscience division, va national center for ptsd, va connecticut healthcare system, west haven, ct & dept of psychiatry, yale university school of medicine; gregory leskin, national center for ptsd, va palo alto health care system; james meyerhoff, georgetown university school of medicine & division of psychiatry & neurosciences, walter reed army institute of research; elizabeth osuch, dept of psychiatry, university of western ontario, schulich school of medicine and dentistry.
Diabetic retinopathy is the leading cause of blindness in working - age persons around the world.1,2 it can be found in three out of four patients within 15 years of the diagnosis of diabetes.3,4 visual impairment is most commonly a consequence of diabetic macular edema (dme).5,6 while vascular endothelial growth factor inhibitors are usually used first for dme, intravitreal triamcinolone acetonide monotherapy (ivta) has also been proven to be efficacious for refractory dme and vision loss in eyes that fail laser therapy.1,7 it has been shown to improve vision and reduce central macular thickness (cmt), with benefits persisting for up to 5 years.810 moreover, for patients with earlier dme, ivta followed by laser photocoagulation has been shown to double the chance of an improvement in vision by ten or more best - corrected logarithm of minimum angle of resolution letters at 2 years compared to laser alone.11 this result can be contrasted with the diabetic retinopathy clinical research network trial, which found that only the subset of eyes that were pseudophakic at baseline had better visual acuity outcomes after ivta combined with prompt laser treatment.12 the divergent findings may in part be due to differences in study protocol, ie, the shorter period of time between ivta and laser therapy in the diabetic retinopathy clinical research network study (310 days versus 6 weeks) may not have allowed sufficient time for the edema to resolve before application of the laser.11,12 since patients often require more than one injection with ivta, they may be at higher risk of dose - dependent steroid - related adverse events, principally cataract and raised intraocular pressure (iop).1315 this paper examines whether it is possible to predict from baseline characteristics the number of ivta injections patients will require once they embark on a course of combined therapy with laser photocoagulation . This analysis was based on data from the thunderbird (a multicenter randomized clinical trial of laser treatment plus ivta for dme) study, a prospective, randomized, double - masked, placebo - controlled trial.11,16 the thunderbird study tested the hypothesis that there is a synergistic effect of ivta and laser photocoagulation on vision improvement and reduction in macular thickness in eyes with dme . The study was conducted in accordance with the declaration of helsinki and was approved by the research ethics committees of the four participating clinical centers . Patients were included in the thunderbird study if they had focal or diffuse dme involving the central fovea with a cmt of greater than 250 m and best - corrected logarithm of minimum angle of resolution letters read (ie, best - corrected visual acuity [bcva]) in the affected eye(s) of 1770 letters . Time - domain optical coherence tomography was performed (stratus oct; carl zeiss meditec, jena, germany) to determine the average thickness of the central macula (1 mm diameter). Eyes were allocated either to ivta injection or sham - injection 6 weeks prior to laser photocoagulation . Eyes assigned to ivta received an intravitreal injection of 0.1 ml of kenacort 40 (40 mg / ml triamcinolone acetonide; bristol - myers squibb, new york, ny, usa). Eyes assigned to placebo were prepared in the same way but had the barrel of the syringe without a needle pushed firmly against the eye to simulate an injection . Retreatment with injection of study medication followed by laser photocoagulation was considered at the discretion of the chief investigator at each site at each visit, as long as treatments were at least 6 months apart, unless any of the following were present: the investigator considered the macula nearly flat and central optical coherence tomography thickness was <300 m; bcva was 79 letters or better (20/25) or bcva had improved by five letters or more compared with the best bcva after treatment or baseline acuity; or laser treatment was considered by investigator as inappropriate or had no potential for improvement . Only eyes that received ivta in these studies further details on patient enrolment, sample size calculation, treatment assignment, data collection and masking, treatment, and outcomes can be found in previously published reports of this trial.11,16 data are presented as the mean standard deviation when normally distributed or as median (interquartile range) if not . The analysis of the factors predictive of the extent of ivta plus laser treatment was not a prospectively defined outcome of the study . Eyes were analyzed according to the number of treatments they received: one versus two versus three versus four or five . For the purposes of a binary logistic regression analysis, eyes were classified as having either received few (one or two) or many (three or more) treatments . Differences in continuous variables between multiple groups were compared using an analysis of variance or the kruskal - wallis test for normally and not normally distributed data, respectively . In cases of significant analysis of variance results, post hoc testing was performed . If equal variances could be assumed a bonferroni correction was applied . If not the measured baseline characteristics were age, gender, bcva, glycosylated hemoglobin (hba1), phakic status, iop, and cmt to analyze the predictive value of baseline characteristics on the number of treatments received binary logistic regression and multinomial logistic regression models were applied for these, the dependent variable was the number of treatments received classified as described above . Gender (female = reference category) and phakia (pseudophakic = reference category) were defined as categorical variables . Since the a priori risk of retreatment was not known, a cox regression was performed in addition to the logistic regression analyses . All data were analyzed using a commercially available software package (spss 21; ibm corporation, armonk, ny, usa). Data are presented as the mean standard deviation when normally distributed or as median (interquartile range) if not . The analysis of the factors predictive of the extent of ivta plus laser treatment was not a prospectively defined outcome of the study . Eyes were analyzed according to the number of treatments they received: one versus two versus three versus four or five . For the purposes of a binary logistic regression analysis, eyes were classified as having either received few (one or two) or many (three or more) treatments . Differences in continuous variables between multiple groups were compared using an analysis of variance or the kruskal - wallis test for normally and not normally distributed data, respectively . In cases of significant analysis of variance results, post hoc testing was performed . If equal variances could be assumed a bonferroni correction was applied . If not, dunn s test was applied . The measured baseline characteristics were age, gender, bcva, glycosylated hemoglobin (hba1), phakic status, iop, and cmt to analyze the predictive value of baseline characteristics on the number of treatments received binary logistic regression and multinomial logistic regression models were applied . Gender (female = reference category) and phakia (pseudophakic = reference category) were defined as categorical variables . Since the a priori risk of retreatment was not known, a cox regression was performed in addition to the logistic regression analyses . All data were analyzed using a commercially available software package (spss 21; ibm corporation, armonk, ny, usa). Of the 42 eyes treated with ivta plus laser photocoagulation in the thunderbird study, 14 (33%) were pseudophakic at baseline and 16 (38%) patients were female . The mean age at baseline was 66.810.4 years, iop was 16.13.0 mmhg, and cmt was 482123 m . The median baseline bcva was 57 (5370) letters and hba1 was 7.6% (7.2%9.4%). Data for the measured baseline characteristics were complete, except for one eye with no recorded hba1c value . A total of 101 ivta plus laser treatments were given during the 2 years of this study . Only one treatment was required in eleven (26%) eyes, two in ten (24%) eyes, three in 14 (33%) eyes, four in seven (17%) eyes, and five in zero (0%) eyes . Eyes that received more ivta plus laser treatments had significantly thicker maculas at baseline (table 1 and figure 1). There were no other statistically significant differences in measured baseline characteristics between eyes classified on the basis of number of treatments they received . Was found to be a significant predictor of receiving three or more treatments in the binary logistic regression model (odds ratio 5.13, 95% confidence interval 1.7515.04, p=0.003 per 100 m increase in cmt) (table 2). Cox regression analysis produced a result in the same range as the binary logistic regression model . Similarly, a high baseline cmt was significantly associated with an increased risk of receiving three and four, but not two, treatments in the multinomial logistic regression model (table 3). Age, gender, baseline bcva, baseline hba1c, baseline phakia and baseline iop were not predictive of the number of ivta plus laser treatments received . Eyes that received more ivta plus laser treatments had significantly thicker maculas at baseline (table 1 and figure 1). There were no other statistically significant differences in measured baseline characteristics between eyes classified on the basis of number of treatments they received . Was found to be a significant predictor of receiving three or more treatments in the binary logistic regression model (odds ratio 5.13, 95% confidence interval 1.7515.04, p=0.003 per 100 m increase in cmt) (table 2). Cox regression analysis produced a result in the same range as the binary logistic regression model . Similarly, a high baseline cmt was significantly associated with an increased risk of receiving three and four, but not two, treatments in the multinomial logistic regression model (table 3). Age, gender, baseline bcva, baseline hba1c, baseline phakia and baseline iop were not predictive of the number of ivta plus laser treatments received . In this post hoc analysis of data from a prospective, randomized, double - masked, placebo - controlled clinical trial of eyes with dme, increasing baseline cmt was strongly linked with requiring more ivta plus laser treatments.11,16 eyes that eventually received more ivta plus laser during the 2 years of this study had significantly greater mean baseline cmt . In binary and multinomial logistic regression analyses, no relationship was found between other baseline characteristics (bcva, age, gender, hba1c, phakia, and iop) and the number of treatments received . The authors believe that the strong relationship found between a high baseline cmt and the need for ivta retreatment in the patients participating in the thunderbird study is representative of the routine clinical use of this drug . While the data were derived from a randomized controlled trial during which loss to follow up was low, the patients in the study that received a greater number of treatments did so because they met the retreatment criteria more frequently . These criteria were similar to those used in clinical practice, in which treatment is given at the clinician s discretion in light of findings from clinical examination and imaging . If a clinician has a patient similar to those included in this study and applies similar retreatment criteria, then baseline cmt can be used to predict how many treatments will be prescribed . This analysis is not equipped to determine why the eyes in this study with thicker maculas at baseline received more treatments than eyes with less swollen maculas . It can be inferred from the prospectively defined criteria that eyes that received multiple treatments were not responding as well to therapy . In this study, eyes were considered for reinjection plus laser 6 weeks later unless macular edema resolved, bcva improved, or laser photocoagulation was no longer considered appropriate . . It might be that eyes with more fluid require more therapy to dry out the macula, ie, a high cmt is representative of more severe retinal dysfunction which requires more treatments to stabilize.17 alternatively, in contrast to previous reports, it may be that dme is more likely to recur in eyes with a high baseline cmt.18 irrespective of the mechanisms underlying the current findings, the ability to predict which patients will require multiple treatments has important implications for the risk and benefit profile of treatment . The main side effects of ivta treatment are iop elevation and accelerated cataract formation, both of which are dose dependent . The risk of cataract formation and subsequent cataract surgery is strongly dose related.13,1921 similarly, it has been shown that the duration of ocular hypertension is longer with higher doses of ivta.14 the benefits to be gained from treatment in eyes with high baseline cmt should be balanced with the greater risk of developing adverse events . The major weakness of this retrospective analysis is the relatively small number of patients available for analysis . Conversely, the strength of the relationship between cmt and the number of ivta plus laser treatments received should be viewed positively in light of the number of patients analyzed . The need for retreatment was not a prospectively defined outcome and the reasons for a decision to retreat or not were not collected from the investigators . Future trials should assess these factors as the ability to predict the number of intravitreal therapies that will be required before embarking on a course of therapy would be highly beneficial to patient care . This would be equally applicable to vascular endothelial growth factor inhibitors, whose treatment regimen is more intense with less time between injections than that of ivta . In summary, this analysis assessed the predictive value of baseline characteristics of the number of ivta plus laser treatments needed to control dme in the first 2 years of a clinical trial . A high baseline cmt was shown to be strongly associated with receiving more ivta plus laser treatments . The practical implication of the findings of this study is that baseline cmt may be used as a factor when weighing up the risks and benefits of ivta treatment for dme . This may become an important measure in the clinical decision making of the treatment of this disease.
Mucoepidermoid carcinoma (mec) is the most common malignant tumor of salivary glands and comprises approximately 5% of all salivary gland tumors . Laryngeal cases comprise one third of malignant laryngeal salivary - type tumors.1 however, it is a rare entity in the larynx and is frequently misdiagnosed as other laryngeal tumors . Therefore, recognizing its clinical and histological features and distinguishing it from other common laryngeal malignancies are essential . In addition, the most distinctive histological and clinical features of mucoepidermoid carcinoma are clearly stated in tables 1 and 2, respectively . For comparison, histological and clinical features of two major differential diagnoses, squamous cell carcinoma (scc) and adenosquamous carcinoma (asc), are also presented . Laryngeal mucoepidermoid carcinoma develops from the reserve cells in excretory ducts of submucosal glands2 or from squamous cells in the surface epithelium.2 this tumor was first described by arcidiacono and loineo in 1963, as a rare entity.3 the majority of laryngeal cases occur in supraglottis (61%), but they have also been described in glottis (26%) and subglottis (13%) as well as in hypopharynx (fig . 1). This tendency differs from squamous cell carcinoma of larynx, which most frequently affects the glottis.4,5 ho et al argued that this tendency is due to the abundance of laryngeal glands, histologically identical to the minor salivary glands, in the submucosal region of larynx . Mucoepidermoid carcinoma of larynx originates from the intercalated cells which are one part of these glands; therefore mucoepidermoid carcinoma is likely to develop at supraglottis where the laryngeal glands are most frequently distributed.6,7 the infrequency of reports on laryngeal mucoepidermoid carcinoma could be attributable to difficult recognition of this tumor type when it occurs outside the salivary glands . In addition to the possibility of sampling errors, interpretative errors of tumor specimens have been reported.8 laryngeal mecs occur in people of all ages (peak incidence in the 6th decade) and rare cases have been reported in children and adolescents.9,10 this tumor has a definite male predominance and has a wide spectrum of clinical behavior from locally invasive to highly malignant.11,12 nearly half of the cases develop cervical lymph node involvement;10,13,14 and the lungs are the most frequent site for distant metastasis.12 an extremely rare case of metastatic laryngeal mucoepidermoid carcinoma in the temporal bone has also been reported.15 microscopically, these tumors, similar to mucoepidermoid carcinomas in other sites, are composed of varying proportions of mucous, epidermoid, and intermediate - type cells.16 thay are classified as low, intermediate, and high grade (figs ., low - grade tumors are predominantly cystic with few mitotic figures and no cellular anaplasia . Intermediate - grade tumors are usually more solid and have cellular anaplasia; high - grade tumors typically have solid growth, anaplasia, and a high mitotic rate.17 there are pitfalls not only in early detection but also in true histopathologic diagnosis of these lesions . Primary lesions are not detected by laryngoscopy and most patients are diagnosed in the advanced stages . Therefore, progressive hoarseness and dyspnea, which indicate glottic involvement, as well as stridor and airway obstruction, which imply subglottic lesions, should be taken into consideration.18 when a laryngeal tumor is detected, its true histological diagnosis is essential . Scc and mec have similar histopathologic features and presence of intermediate and mucus cells are the only key for differentiation.19 since they have different prognosis and treatment modalities, this differentiation is important . Low - grade mecs have better prognosis in adults than sccs20 whereas high - grade tumors are associated with a lower survival rate, even lower than the survival rates of scc.19 moreover, many early sccs of larynx are best treated by irradiation whereas mecs are successfully treated by surgical excision.21 despite of all these differences, these tumors are vastly misdiagnosed with each other histologically.19 some cases have been reported in the literature that were initially diagnosed as scc but the presence of glandular structures in metastatic lymph nodes and positive mucicarmine stain changed the final diagnosis to mec . For that reason, some authors recommend to apply mucicarmine stain to all mistakable cases except for obvious squamous cell carcinomas arising from the surface mucosa.22 as well, some recent studies have focused on distinctive immunohistochemical expression of muc - type mucin family in salivary gland tumors and head and neck sccs.23 distinguishing high - grade mec and scc by different expressions of cytokeratins (cks), especially ck14, has also been suggested.24 on the other hand, presentation of tumor in metastatic lymph nodes could be helpful in establishing a true diagnosis in high grade tumors . Therefore, careful exploration of metastatic lymph nodes for cystic features and mucus cells in any laryngeal tumor with diagnosis of scc is recommended . A significant number of tumors originally diagnosed as high - grade mec are asc, which has more aggressive behavior and shorter overall survival.25 chenevert et al stated that 22 of 100 alleged mecs in their research study were asc.26 both neoplasms could be of ductal or surface mucosa origin and share some similar cell types . Mucoepidermoid carcinoma does not usually exhibit anaplastic nuclear features and is not associated with carcinoma in situ of the overlying mucosa . Asc, in contrast to mucoepidermoid carcinoma has tendency to demonstrate intercellular bridges, keratin pearl formation and distinct areas of adenocarcinoma.27 the main histological and clinical features of mec, asc and scc are presented in tables 1 and 2 . The prognosis is somehow dependent on the histological features; high - grade tumors have a higher risk of death than low - grade tumors.28 pires et al reviewed the literature and reported that overall 5-year survival rates ranged from 0% to 43% for patients with high - grade mucoepidermoid carcinomas, 62% to 92% for patients with intermediate - grade tumors, and 92% to 100% for patients with low - grade tumors.29 it has been stated that in patients with tracheobronchial mec, the proportion of squamoid cells in tumor histology may be an indicator of tumor malignancy and lymph node metastases.30 the prognosis is not, however, fully dependent on the pathological features . Low to intermediate grade tumors may behave poorly31; even, patients with low - grade mecs have occasionally developed distant metastases at early stages.2,32 the ability to achieve complete surgical resection is one of important prognostic factors and patients who do not achieve complete surgical resection will have a poor prognosis.12 clinical studies of patients with head and neck mucoepidermoid carcinomas have also revealed that patients over 56 years of age are significantly associated with decreased survival rate.2 as a result, considerable note must be taken of the clinical course and both histological classification and clinical behavior, are essential elements to make appropriate therapeutic decisions.2,14 as the main method for therapy, most agree on wide excision; however, radiotherapy or conservative surgery has been used for low grade tumors.33 the extent of excision is comparable to that of squamous cell carcinoma and extensive tumors require total laryngectomy.19 the necessity of radical neck dissection is controversial and is usually performed when lymphadenopathy is present;34 however, it is highly recommended to do elective dissection for all high - grade tumors . Radical surgery followed by radiotherapy has improved local control in salivary gland malignancies . 35 in addition, post - operative irradiation for mec patients with positive surgical margin has been reported to be effective.36 since high grade mecs have a high incidence of local recurrence (up to 50 percent), postoperative radiotherapy is recommended in these cases . Nevertheless, recent studies support the elective neck dissection and postoperative radiotherapy not only for high - grade tumors but also for low - grade histologies with positive margins or extracapsular spread.37 mucoepidermoid carcinoma is a rare entity in the larynx and is frequently misdiagnosed with other laryngeal malignancies especially squamous cell carcinoma and adenosquamous carcinoma . Therefore, recognizing the distinct clinical and histological features of this tumor is essential . Progressive hoarseness and dyspnea without any identifiable lesion in laryngoscopy may implicate a submucosal mass like mucoepidermoid carcinoma . Histological classification and clinical behavior, are both essential elements to make appropriate therapeutic decisions in these tumors . In the case of high - grade histologies, recurrence must be considered and combination of radical surgery, radical neck dissection and postoperative radiotherapy is recommended.
Considerable disparities in cancer survival rates exist between african americans (aas) and white americans (was). Various factors such as socioeconomic status (ses), cancer stage at time of diagnosis, and treatment have accounted for many of these disparities . Other factors include insurance status, social determinants in general and genetics . However, even when analyses of cancer survival data include all known or suspected factors affecting survival, aas still tend to have a lower survival rate than that of was, possibly because of unmodeled factors such as biological differences, and perhaps as a consequence of educational level and access to health care as several authors have noted . Discussions of cancer survival disparities generally overlook the role of vitamin d. for 20012004, aas older than 60 y had a population mean serum 25-hydroxyvitamin d [25(oh)d] concentration of 17 ng / ml compared with 25 ng / ml for was . Prevalence of hypovitaminosis d [(25(oh)d <15 ng / ml] in the south was 45% among blacks and 11% among whites . In patients participating in a randomized controlled trial of chemotherapy, serum 25(oh)d concentrations were lower in black patients than in white patients and patients of other race (median, 10.7 vs 21.1 vs. 19.3 ng / ml, respectively; p <0.001), as well as in females compared with males (median, 18.3 vs 21.7 ng / ml, respectively; p = 0.0005). Solar ultraviolet - b (uvb) irradiance is the primary source of vitamin d for most americans, accounting for 8090% of vitamin d. aas, with darker skin, are less efficient at producing vitamin d from uvb irradiance . In addition, aas are less likely to have as much vitamin d from oral intake . A large body of literature supports a beneficial effect of vitamin d in reducing the risk of cancer incidence and mortality rates . Many ecological studies have supported this hypothesis, as have observational studies of breast and colorectal cancer . Two ecological studies found stronger inverse correlations between solar uvb doses and cancer mortality rates than incidence rates . Several reviews of the uvb - vitamin d - cancer hypothesis have also been published . The dose response relation for vitamin d has been derived from observational studies for breast and colorectal cancer . For the values for cancer incidence are not necessarily the same for cancer survival, but they do suggest the magnitude of the effect . This vitamin d - cancer dose response relation might underestimate the effect of lower serum 25(oh)d concentrations for aas since 20% of the black population is older than 60 y, in contrast to only 6% of whites; also, the risk of cancer increases more rapidly for changes of serum 25(oh)d concentration at lower concentrations . A recent paper addressed vitamin d s role in explaining some of the cancer survival disparities . Data from the third national health and nutrition examination survey (nhanes iii) were used to investigate the role of racial disparity from colorectal cancer; adding vitamin d deficiency to the model attenuated the mortality risk associated with being black by a statistically significant 40% . Grant and peiris investigated vitamin d s role in explaining disease disparities between aas and was in general . This paper surveys the literature on cancer disparities for aas and was as well as the literature on epidemiological studies on vitamin d and cancer to see whether differences in serum 25(oh)d concentrations might explain many of the otherwise - unaccounted - for residual disparities . Table 1 presents the findings regarding cancer survival with respect to serum 25(oh)d concentrations at the time of diagnosis . Significant inverse correlations between 25(oh)d and cancer survival were found for all - cancer, breast, colon, colorectal, lung, prostate cancer, chronic lymphocytic leukemia / chronic lyphocytic lymphoma, hodgkin lymphoma, and non - hodgkin lymphoma . Studies also reported no significant correlation between serum 25(oh)d and survival for bladder, lung and ovarian cancer . Ac, all cause; cs, cancer specific; hr, hazard ratio; nhl, non - hodgkin lymphoma; rr, relative risk . Table 2 presents the multifactor - adjusted hazard ratios for survival for aas vs. was for cancer - specific survival . Inclusion of ses, stage at diagnosis, and treatment in the analyses is indicated . The results for cancer - specific survival rates are a stronger indication of the effects of vitamin d than are all - cause survival rates because some of the all - cause deaths could be due to non - vitamin d - related diseases or to factors such as smoking . Statistically significant disparities emerged for cancer - specific survival rates for 13 types of cancer: bladder, breast, colon, endometrial, lung (non - small cell, stage iii, iv), ovarian (advanced), pancreatic, prostate, rectal, testicular, vaginal cancer, hodgkin lymphoma, stage ii and melanoma . Our analysis also found statistically significant disparities for cancer - specific survival rates for two types of cancer, endometrial and ovarian cancer . There were no statistically significant findings for gastric adenocarcinoma, or head and neck, and oral cancer and leukemia . Aas lower serum 25(oh)d concentrations (mainly from reduced vitamin d photoproduction owing to darker pigmentation) may account for much of the unexplained survival disparity after consideration of such factors as ses, stage at diagnosis, and treatment . All cancers for which a disparity in cancer - specific survival was reported also have evidence for a beneficial role of vitamin d, as do most of those for which we found disparities for all - cause survival . One reason ecological studies are strong include that vitamin d plays an important role in reducing risk of cancer initiation and angiogenesis around tumors and metastases . Since cancer can take years to decades to reach the stage of detection or death, continued high serum 25(oh)d concentrations over much of the lifetime is required for greatest risk reduction . Also, ecological studies include many cases, thereby reducing the uncertainty of the values . Among 45-y - old british citizens, casual solar uvb irradiance in summer increased serum 25(oh)d concentrations by about 15 ng / ml, enough to have an important impact on cancer risk . For example, breast cancer incidence rates are highest in spring and fall . The reasons for the seasonal variations given were increased production of vitamin d in summer and melatonin in winter . Breast cancer has several subtypes, and rate of progression can vary widely, with some being very rapid . For slower growing cancers, serum 25(oh)d concentrations in summer may be sufficient to retard or reverse the growth . Once cancer reaches the point where it can be diagnosed, vitamin d improves cancer - specific survival by several mechanisms, including antiangiogenesis and antimetastases . The disparities for hematopoietic cancers may be weak or nonexistent because angiogenesis and metastases are less important for blood cell - related tumors than for solid tumors . Higher serum 25(oh)d concentrations also affect all - cause mortality rates since vitamin d protects against several major life - threatening conditions for the elderly, including diabetes and cardiovascular disease, influenza and pneumonia, and falls and fractures . Secondary hyperparathyroidism due to osteoblastic metastases and hungry bone syndrome has been described with advanced prostate and breast cancer, it is likely that a vitamin d replete state may minimize such occurrences . Coleman and mccloskey suggest that bisphosphonates may prevent metastases and reduce the risk of disease recurrence . Based on animal data, a vitamin d replete state may be helpful in reducing bisphosphonate induced osteonecrosis of the jaw . Factors other than ses, stage at diagnosis, treatment, and vitamin d status might also explain the cancer survival disparities . For example, the lack of survival disparities for lung cancer may be due to a stronger effect from smoking than from vitamin d. smoking cessation improves lung cancer survival rates associated with early - stage lung cancer . Obesity is significantly correlated with cancer risk for nearly all types of cancer listed in tables 1 and 2 . One reason is that obesity is linked to poverty in the united states because of energy - dense but nutrient - poor foods are cheaper due to subsidies . A second reason is that aas have about twice the prevalence of apolipoprotein e 4 (apoe4) than was . Apoe4 increases production of cholesterol in the liver and of insulin in the pancreas to store excess food as fat for those with sporadic food supplies, such as hunter - gatherers . Interestingly, overweight and obesity rates for white and black men differ little, whereas aa women are much heavier than wa women (http://www.cdc.gov/nchs/data/hestat/obesity_adult_07_08/obesity_adult_07_08.pdf). Thus, obesity does not seem to be a likely explanation for cancer disparities among men but could be for women . On the other hand, serum 25(oh)d concentrations are inversely correlated with body mass index, which has implications for cancer risk . Interestingly, for pancreatic cancer incidence, higher body mass index was significantly associated with risk for aa and wa men and wa women but with only insignificantly reduced risk for aa women . Cancer survival studies with respect to serum 25(oh)d concentrations at time of diagnosis offer strong evidence for a beneficial effect of vitamin d. all cancers with a beneficial effect of vitamin d on survival have been found inversely correlated with solar uvb doses, with the possible exception of chronic lymphocytic leukemia . There are also studies from norway indicating improved survival for those diagnosed with breast, colon, prostate cancer and hodgkin lymphoma in summer compared with winter . The uvb - vitamin d - cancer hypothesis receives its strongest support from ecological studies . Observational studies also provide good support if the various studies are examined carefully and a good reason is found for why many observational studies have not found a beneficial effect of vitamin d in reducing the risk of cancer . Nested case - control studies have a reduced strength since only a single serum 25(oh)d concentration measurement or oral intake assessment is made at time of enrollment, with follow - up periods lasting between 3 and 28 y. as the follow - up time increases beyond about 37 y, the single measurement is less meaningful . Case - control studies, on the other hand, use serum 25(oh)d concentration or vitamin d oral intake values at the time of diagnosis . A review of observational studies of breast and colorectal cancer incidence with respect to serum 25(oh)d concentration found statistically significant inverse correlations for breast cancer out to 3 y and for colorectal cancer out to 12 y of follow - up . Thus, the recently reported results from the vitamin d pooling project study of rarer cancer types (endometrial, esophageal, gastric, ovarian, pancreatic, and renal cancer and non - hodgkin lymphoma) probably failed to find an inverse correlation between incidence of these cancers and prediagnostic serum 25(oh)d concentrations because the mean follow - up period was 6.63 y and because there were so few cases that the 95% confidence intervals were about 50% . The correlation between serum 25(oh)d concentrations measured at different times decreases with time, dropping to a regression coefficient of 0.40 after 14 y. several ways exist to test the uvb - vitamin d - cancer hypothesis as an additional contributing factor for cancer survival disparities . One would be to measure serum 25(oh)d concentrations of newly diagnosed cancer patients and at several intervals during the course of the cancer . Another would be to supplement newly diagnosed cancer patients with sufficient vitamin d to bring serum 25(oh)d concentrations up to 4080 ng / ml and compare results for those not supplemented, perhaps from previous patients in the same practice . A recent publication described the rationale for vitamin d supplementation, which is being done in some cancer treatment centers . Increasing serum 25(oh)d concentrations would also reduce the risk of severe sepsis associated with cancer surgery as well as many other comorbid diseases . We acknowledge that while it appears very likely that vitamin d is an important and often ignored factor in the biology of cancer, the issue of cancer etiology is complex and is clearly multifactorial . Moreover, outcomes studies may have skewed results since aa men are less likely to participate in cancer screening trials . An inverse relationship between physical activity and breast cancer in aa women has been reported . Reported that aa patients are less likely to receive resection in non - metastatic rectal cancer . Demonstrated that aa colorectal cancer survivors are less likely to receive post - treatment colorectal surveillance . These may not necessarily reflect racism in that physicians may make recommendations based on a patient s access to health care, presence of insurance, etc . In addition, poor health literacy in aa women may also impact access to available health care strategies . Cultural differences may also play a role with cultural insensitivity among providers compounding the issue . Margolis et al . Demonstrated significant racial differences in belief prior to lung cancer surgery . Some of these differences result in refusal of surgery on the part of aa patients . Aas have less trust in their health providers and may not accept physicians assertions regarding treatment . Van ness et al . Indicates that lack of religiousness maybe associated with poor cancer survival in aa women . Church attendance may be associated with greater emotional and social support, which is linked to better outcomes in breast cancer . We must also consider the possibility that apart from direct cellular benefits of vitamin d on cancer that vitamin d deficiency has indirect effects which are hard to quantify but may have a significant impact on cancer outcomes . Vitamin d deficiency is also associated with a higher prevalence of depression and neurocognitive symptoms, which makes patients intrinsically less likely to seek medical attention . Treating vitamin d deficiency some risk factors such as diet can be modified and increased consumption of vegetables may decrease the risk of breast cancer in aas, possibly by altering estrogen / progesterone receptor status . Fortunately, it does appear that tumors are not intrinsically more aggressive in aas . In veterans with equal access to health care, dignam reported that black women, diagnosed at comparable disease stage as white women and treated appropriately, tend to experience similar breast cancer prognoses and survival . Some of the residual disparity for prostate cancer may be due to the higher prevalence of the apoe4 allele among aas than was, which is related to increased cholesterol production . Increased low - density lipoprotein concentrations increased the risk of prostate cancer for aas but not was . We acknowledge that while it appears very likely that vitamin d is an important and often ignored factor in the biology of cancer, the issue of cancer etiology is complex and is clearly multifactorial . Moreover, outcomes studies may have skewed results since aa men are less likely to participate in cancer screening trials . An inverse relationship between physical activity and breast cancer in aa women has been reported . Reported that aa patients are less likely to receive resection in non - metastatic rectal cancer . Demonstrated that aa colorectal cancer survivors are less likely to receive post - treatment colorectal surveillance . These may not necessarily reflect racism in that physicians may make recommendations based on a patient s access to health care, presence of insurance, etc . In addition, poor health literacy in aa women may also impact access to available health care strategies . Cultural differences may also play a role with cultural insensitivity among providers compounding the issue . Some of these differences result in refusal of surgery on the part of aa patients . Aas have less trust in their health providers and may not accept physicians assertions regarding treatment . Van ness et al . Indicates that lack of religiousness maybe associated with poor cancer survival in aa women . Church attendance may be associated with greater emotional and social support, which is linked to better outcomes in breast cancer . We must also consider the possibility that apart from direct cellular benefits of vitamin d on cancer that vitamin d deficiency has indirect effects which are hard to quantify but may have a significant impact on cancer outcomes . Vitamin d deficiency is also associated with a higher prevalence of depression and neurocognitive symptoms, which makes patients intrinsically less likely to seek medical attention . Treating vitamin d deficiency some risk factors such as diet can be modified and increased consumption of vegetables may decrease the risk of breast cancer in aas, possibly by altering estrogen / progesterone receptor status . Fortunately, it does appear that tumors are not intrinsically more aggressive in aas . In veterans with equal access to health care, dignam reported that black women, diagnosed at comparable disease stage as white women and treated appropriately, tend to experience similar breast cancer prognoses and survival . Some of the residual disparity for prostate cancer may be due to the higher prevalence of the apoe4 allele among aas than was, which is related to increased cholesterol production . Increased low - density lipoprotein concentrations increased the risk of prostate cancer for aas but not was . Lower serum 25(oh)d concentrations among aas than was may explain many of the cancer survival disparities after consideration of ses, stage at time of diagnosis, and treatment . If substantially correct, programs to increase serum 25(oh)d concentrations among aas could reduce the cancer disparities . This approach would work not only for those of the ages where cancer is more likely but also for those younger . Vitamin d can reduce the risk of cancer at the initiation stage and in the advanced stages, as well as raising serum 25(oh)d concentrations to over 40 ng / ml shortly after cancer diagnosis . Given the biologic plausibility, the currently available evidence of beneficence, and the lack of harm with moderate vitamin d replacement, we recommend oncologists consider a more proactive stance on this issue pending additional studies . Papers cited in this study came from the national library of medicine s pubmed database (http://www.pubmed.gov). As of december 28, 2011, a search for cancer disparity papers (search terms cancer disparities survival, african - american) identified 457 articles . We examined in more detail those reporting hazard ratios for aas vs. was for survival . The tables in this review do not include the component papers of meta - analyses cited . We inspected the results in the papers, preferring those with disparities in survival that included all three factors ses, stage at diagnosis, and treatment, in addition to race over those including fewer or none of these factors . We examined the papers to see whether survival was cancer - specific or all - cause . In addition, papers reporting cancer survival with respect to serum 25(oh)d concentration were also sought.
Three subjects with pi, a clinically healthy implant, and a periodontally diseased tooth were selected . They had not received systemic antibiotics, anti - inflammatory drugs, or oral anti - microbial agents within the last 3 months . The investigation was approved by the ethics committee of the tokyo medical and dental university, and a written informed consent was obtained from all subjects . The following clinical parameters were assessed at six sites per tooth and at six sites per implant (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and distolingual): (1) probing depth (pd), (2) bleeding on probing (bop), (3) suppuration (sup), and (4) gingival index (gi) (27). Intra - oral periapical radiographs (insight dental films, eastman kodak company, sp, japan) were obtained using the parallel technique . Radiographs were analyzed for peri - implant bone loss by the same examiner using the smooth components and threads of the implants as reference points . Based on clinical and radiographic data, a diseased implant, a clinically healthy implant, and a periodontally diseased tooth diseased implants (implants with pi) showed pd5 mm with bop and/or sup and concomitant radiographic bone loss (bone loss more than three threads up to half of the implant length). Healthy implants (h) showed pd<4 mm without bop and sup, and radiographic bone loss . Subgingival plaque samples were obtained from the deepest pockets at the implants with / without pi . In addition, samples from the deepest pockets of the periodontally diseased tooth, not adjacent to the implant were collected . Two weeks before sampling, we performed periodontal examination for all of the residual teeth and implants . Pd, bop, and sup were measured at six points per tooth as pre - examination and together with radiographic evaluation, sampling sites were decided . After 30 s, all paper points were removed and placed in a sterile tube with 1 ml of sterile distilled water . Samples were mixed for 1 min using a vortex mixer . After removing the paper point, the resulting pellet was resuspended in 150 l of lysis buffer from a bacterial dna extraction kit (mora - extract, amr inc ., samples were then incubated for 10 min at 90c and total bacterial genomic dna was isolated using the mora - extract kit . Total bacterial dna was eluted with 200 l of te buffer (amr inc .) And was stored at 20c . 16s rrna gene clone library analysis was performed as described previously (28, 29). Briefly, the primers used for pcr amplification of the 16s rrna gene were 27f (5-agagtttgatcmtggctcag-3) and 1492r (5-tacggytaccttgttacgactt-3). Pcr reaction mixture (100 l) containing 10 l of extracted dna, 2.5 u of takara ex taq (takara bio inc ., otsu, japan), 10 l of 10 ex taq buffer, 8 l of dntp mixture (0.2 mm each), and 50 pmol of each primer . Pcr amplification was performed using a veriti 200 pcr thermal cycler (applied biosystems, foster city, ca, usa) with the following program; 95c for 3 min, followed by 15 cycles of 95c for 30 s, 50c for 30 s, 72c for 1.5 min and a final extension period of 72c for 10 min . Pcr products were purified using a qiaquick pcr purification kit (qiagen, valencia, ca, usa). Purified amplicons were ligated into plasmid vector pcr2.1 and then transformed into one shot invaf competent cells using the original ta cloning kit (invitrogen, san diego, ca, usa). Plasmid dnas were prepared using the templiphi dna amplification kit (ge healthcare, buckinghamshire, uk) from randomly selected recombinants and used as templates for sequencing . Sequencing was conducted using the 27f and 520r primers, a bigdye terminator cycle sequencing kit (applied biosystems), and a 3130xl genetic analyzer (applied biosystems). All sequences were checked for possible chimeric artifacts by the chimera check program of the ribosomal database project - ii (rdp - ii) and compared to similar sequences of the reference organisms by blast search (30). A 16s rrna gene sequence similarity of 98% was used as the cut - off for positive identification of taxa (operational taxonomic unit less than 98% identity in the 16s rrna gene sequence was the criterion used to identify bacteria at the species level . The sequences were aligned with the clustal x 2.0.12 program (31) and corrected by manual inspection . Nucleotide substitution rates (k nuc values) were calculated (32) after gaps and unknown bases were eliminated . The phylogenetic tree was constructed by the neighbor - joining method (33). Bootstrap resampling analysis sequences for novel phylotypes were deposited in the ddbj database under accession numbers ab538407 to ab538428 . Distance matrices were calculated using the dnadist program within the phylip software package version 3.69 . Three subjects with pi, a clinically healthy implant, and a periodontally diseased tooth were selected . They had not received systemic antibiotics, anti - inflammatory drugs, or oral anti - microbial agents within the last 3 months . The investigation was approved by the ethics committee of the tokyo medical and dental university, and a written informed consent was obtained from all subjects . The following clinical parameters were assessed at six sites per tooth and at six sites per implant (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and distolingual): (1) probing depth (pd), (2) bleeding on probing (bop), (3) suppuration (sup), and (4) gingival index (gi) (27). Intra - oral periapical radiographs (insight dental films, eastman kodak company, sp, japan) were obtained using the parallel technique . Radiographs were analyzed for peri - implant bone loss by the same examiner using the smooth components and threads of the implants as reference points . Based on clinical and radiographic data, a diseased implant, a clinically healthy implant, and a periodontally diseased tooth diseased implants (implants with pi) showed pd5 mm with bop and/or sup and concomitant radiographic bone loss (bone loss more than three threads up to half of the implant length). Healthy implants (h) showed pd<4 mm without bop and sup, and radiographic bone loss . Subgingival plaque samples were obtained from the deepest pockets at the implants with / without pi . In addition, samples from the deepest pockets of the periodontally diseased tooth, not adjacent to the implant were collected . Two weeks before sampling, we performed periodontal examination for all of the residual teeth and implants . Pd, bop, and sup were measured at six points per tooth as pre - examination and together with radiographic evaluation, sampling sites were decided . After 30 s, all paper points were removed and placed in a sterile tube with 1 ml of sterile distilled water . Samples were mixed for 1 min using a vortex mixer . After removing the paper point, the resulting pellet was resuspended in 150 l of lysis buffer from a bacterial dna extraction kit (mora - extract, amr inc ., samples were then incubated for 10 min at 90c and total bacterial genomic dna was isolated using the mora - extract kit . Total bacterial dna was eluted with 200 l of te buffer (amr inc .) And was stored at 20c . 16s rrna gene clone library analysis was performed as described previously (28, 29). Briefly, the primers used for pcr amplification of the 16s rrna gene were 27f (5-agagtttgatcmtggctcag-3) and 1492r (5-tacggytaccttgttacgactt-3). Pcr reaction mixture (100 l) containing 10 l of extracted dna, 2.5 u of takara ex taq (takara bio inc ., otsu, japan), 10 l of 10 ex taq buffer, 8 l of dntp mixture (0.2 mm each), and 50 pmol of each primer . Pcr amplification was performed using a veriti 200 pcr thermal cycler (applied biosystems, foster city, ca, usa) with the following program; 95c for 3 min, followed by 15 cycles of 95c for 30 s, 50c for 30 s, 72c for 1.5 min and a final extension period of 72c for 10 min . Pcr products were purified using a qiaquick pcr purification kit (qiagen, valencia, ca, usa). Purified amplicons were ligated into plasmid vector pcr2.1 and then transformed into one shot invaf competent cells using the original ta cloning kit (invitrogen, san diego, ca, usa). Plasmid dnas were prepared using the templiphi dna amplification kit (ge healthcare, buckinghamshire, uk) from randomly selected recombinants and used as templates for sequencing . Sequencing was conducted using the 27f and 520r primers, a bigdye terminator cycle sequencing kit (applied biosystems), and a 3130xl genetic analyzer (applied biosystems). All sequences were checked for possible chimeric artifacts by the chimera check program of the ribosomal database project - ii (rdp - ii) and compared to similar sequences of the reference organisms by blast search (30). A 16s rrna gene sequence similarity of 98% was used as the cut - off for positive identification of taxa (operational taxonomic unit otu). Less than 98% identity in the 16s rrna gene sequence was the criterion used to identify bacteria at the species level . The sequences were aligned with the clustal x 2.0.12 program (31) and corrected by manual inspection . Nucleotide substitution rates (k nuc values) were calculated (32) after gaps and unknown bases were eliminated . The phylogenetic tree was constructed by the neighbor - joining method (33). Bootstrap resampling analysis . Sequences for novel phylotypes were deposited in the ddbj database under accession numbers ab538407 to ab538428 . Distance matrices were calculated using the dnadist program within the phylip software package version 3.69 . Clinical data of subjects and sites selected for bacterial sampling are summarized in tables 1 and 2, respectively . A total of nine sites (three pi, three periodontitis, and three healthy implants) were selected and collected for subgingival plaque samples . One sample (periodontally healthy implant) was missed in the process of sample preparation, so eight samples were analyzed . A total of 335 sequences from eight samples were subjected to sequence analysis, which revealed 112 species; 51 (46%) were uncultivated phylotypes, of which 22 were novel . The total numbers of bacterial species identified at the sites of pi, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively . Each clone was classified into several clusters corresponding to phylum - level classification (table 3, fig . 1). Microbiota of pi primarily included gram - negative species and the composition was more diverse than that for healthy implants or periodontitis . Also parvimonas micra, peptostreptococcus stomatis, pseudoramibacter alactolyticus, and solobacterium moorei were only observed at pi sites . Fusobacterium nucleatum was identified at all of the pi sites and granulicatella adiacens was identified at two thirds of pi sites; these two species were also detected at periodontitis sites but not at healthy implants . Most of the bacterial species found in the healthy implants were also detected in the pi and periodontitis sites . Phylogenetic tree of bacterial species and phylotypes detected in peri - implantitis (pi), periodontitis (p), and healthy implants (h). Right columns 1pi, 2pi, 3pi, 1p, 2p, 3p, 2h, and 3h represent subject, sample, and the numbers of bacterial species identified at each site (see text). Boxes used to indicate abundance levels, based on total number of clones assayed: not detected (blank box), 15% (black), 610% (yellow), 1120% (green), 2140% (orange), and 40% (red). Clinical data of the subjects clinical information of sampling sites bacterial phyla and genera detected in this study when the diversity and richness of the resident bacterial species were compared between pi and periodontitis, higher values for the shannon index and richness were observed at pi sites (table 4), thus suggesting that the bacterial community at pi sites were more diverse when compared to periodontitis . Comparison of diversity and richness of sequenced clones between peri - implantitis and periodontitis shannon index and richness are estimated based on 2% differences in nucleic acid sequence alignments . Values given in parentheses are 95% confidence intervals, as calculated by the mothur program . In the present study, we identified the bacteria that compose biofilm at sites with pi . It is believed that the source of infecting bacteria on implants is mainly plaque from residual teeth or saliva, and that microbiota around the implants tend to be similar to that of residual teeth (3638). The periodontal status of remaining teeth would thus determine the bacterial composition at pi sites (37, 38). Sites with pi tend to show a more complex microbiota when compared to periodontitis / healthy implant sites and gram - negative anaerobic bacteria are particularly common at such sites . The presence of periodontopathic bacteria is generally considered to be a risk factor for pi, and indeed, many studies have reported the high prevalence of bacteria, such as p. gingivalis and a. actinomycetemcomitans in pi lesions (1520). In contrast, several researchers have argued that pi and/or implant failure does not always harbor periodontopathic bacteria (3941). F. nucleatum is reported to be a pathogen involved in periodontitis and was found at all pi sites in the present study . However, we were unable to confirm the high prevalence of other periodontopathic bacteria in peri - implant / periodontal lesions . It should be emphasized that the sample size was limited and our results do not rule out an association between although numerous species reside in peri - implant lesions when compared to periodontitis sites, potentially important bacteria may have been overlooked as disease pathogens . To our knowledge, this is the first study using the 16s rrna gene clone library technique to analyze the microbiota in pi, to confirm that the biofilm of pi is composed of a greater variety of bacterial species when compared to periodontitis . Bacteria isolated only in pi, such as p. micra, p. stomatis, and p. alactolyticus, have been reported to be present in periodontal and/or endodontal lesions (42, 43). Because most of these bacteria are difficult to grow in culture, they have not yet been characterized by their bacterial properties . Also the chloroflexi, tenericutes, and synergistetes phyla were only detected at pi sites . We considered that the discrepancy of the results was mainly derived from the bacterial sampling method; they used pooled plaque samples taken by curette from four periodontal pockets . Since the number of subjects attended in the studies was small (vartoukian; 10, ours; 3), further studies are necessary for clarifying the role of the phylum synergistetes in peri - implant diseases, moreover for other bacteria . Differences in bacterial diversity between pi and periodontitis lesions may be explained by the characteristics of surfaces to which the bacteria adhere . Surface roughness and free energy (wettability) are thought to have a significant impact on biofilm formation (45) and the higher levels of free energy on the implant surfaces are likely to affect biofilm components . In conclusion, pi biofilms showed a more complex microbiota when compared to periodontitis and periodontally healthy implants, and were mainly composed of gram - negative anaerobic bacteria . Previously established periodontopathic bacteria showed low prevalence and several bacteria were identified as candidate of pathogens in pi, although it is unclear whether the importance of these species is higher when compared to established periodontopathic bacteria . There is no conflict of interest in the present study for any of the authors.
Linear and whorled nevoid hypermelanosis (lwnh) is a rare sporadic pigmentary anomaly, characterized by swirls and streaks of macular hyperpigmentation following the lines of blaschko, without preceding inflammation, and is usually seen in the first 2 years of life . Include zosteriform hyperpigmentation, zosteriform lentiginous nevus, zebra - like hyperpigmentation, reticulate hyperpigmentation of iijima, and nevoid hyperpigmentation following blaschko lines . A 22-year - old male presented to the outpatient department with chief complaints of asymptomatic, dark - colored skin lesions over the body since 10 years of age . According to the patient, the lesions first appeared over both his arms and progressed within the next 2 years to involve the trunk and lower limbs . The lesions darkened with age and no new lesions appeared over the next 9 years; however, over the last 1 year he started noticing multiple white - colored, pinpoint lesions appearing over the hyperpigmented lesions without any preceding skin itching or redness . There was no history of warty lesions or blisters prior to the appearance of these lesions . There was no history suggestive of any recurrent lower respiratory infections or involvement of the cardiovascular and the central nervous systems . The patient described presence of similar dark - colored skin lesions involving the trunk, arms, and legs in his mother without a history of any light - colored patches . Cutaneous examination revealed presence of multiple, whorled, hyperpigmented macules, arranged bilaterally and symmetrically, along the lines of blaschko on the abdomen, chest, and back [figure 1]. Similar lesions were arranged linearly on the extensor and flexor aspects of the arms and legs . Multiple pinpoint depigmented macules were seen along the areas of streaky macular pigmentation [figure 1]. The texture of skin was normal over the streaks, the intervening skin, and over the depigmented areas . Classical whorled, hyperpigmented macules along the lines of blaschko on the trunk with pinpoint areas of depigmentation hematological and routine biochemical tests revealed no abnormalities . Histopathological evaluation of the depigmented lesion showed complete absence of melanocytes [figure 2a]. However, the pigmented macules revealed increased pigmentation of the basal cell layer with melanocytes present up to the mid - epidermis [figure 2b]. No focal areas of pigmentary incontinence were noticed in the dermis . Based on clinicopathological correlation, a diagnosis of lwnh with punctate hypopigmentation was made . However, treatment for depigmented areas with narrow band ultraviolet b radiation and topical corticosteroids was advised . Picture collage showing histopathological examination (a) complete absence of melanocytes in the depigmented area (b) abundance of melanocytes in the hyperpigmented area (h and e, 40) linear and whorled nevoid hypermelanosis is a rare disorder of pigmentation characterized by hyperpigmented macules in a linear or whorled streaky configuration . Onset is within a few weeks of age, with no preceding inflammation or palpable lesion . The usual age of the onset of hyperpigmentation is within the first few weeks of life, which continues to progress for a year or two before stabilization . Clinically, reticulate hyperpigmented macules coalescing to form streaks and whorled areas are seen over the trunk, extremities, and neck following the lines of blaschko . The pigmented streaks display a v - shaped pattern over the spine, an s - shaped or whorled pattern over the anterior and lateral aspects of the trunk, and a linear arrangement over the extremities and genitalia . There is sparing of the face, palms and soles, eyes, and mucous membranes . Central nervous system diseases include microcephaly, arhinencephaly and epilepsy whereas cardiac defects include ventricular septal defect and tetralogy of fallot . Developmental retardation, facial and body asymmetry, deafness, and brachydactyly have been mentioned in the literature in association with lwnh . Histopathology reveals diffuse moderate hyperpigmentation in the basal layer and prominence or vacuolization of melanocytes . Genetic studies suggest somatic mosaicism as a cause for lwnh with mosaic trisomy of 7, 14, 18, 20; x - chromosomal mosaicism has been reported . Dermatoscopic feature mentioned include net like pattern of pigmentation in both linear and whorled parts by naveen et al ., and ertam et al . Described a parallel pattern which consisted of linear or circular arrangement of parallel whorled streaks along lines of blaschko . Monogenic skin disorders are the ones commonly described with lines of blaschko, however, polygenetic skin disorders such as psoriasis, lichen planus, segmental vitiligo, granuloma annulare, etc . Can also present in similar patterns . Literature search reveals an entity called as blaschkolinear vitiligo, which has been described in association with segmental vitiligo, acrofacial vitiligo, and non - segmental vitiligo . Kovacevic et al . Have reported a new entity called as mixed vitiligo of blaschko lines, where they discussed presence of segmental and non - segmental vitiligo in blaschkolinear pattern . Till date, no cases of lwnh with areas of depigmentation have been mentioned in the literature, and to the best of our knowledge, this is most probably the first case report of familial lwnh superimposed by pinpoint spots of depigmentation.
Common variable immunodeficiency (cvid) is the most common form of severe antibody deficiency affecting both children and adults . The characteristic immune defect in cvid is impaired b - cell differentiation with defective secretion of immunoglobulin (ig). The disorder is associated with a broad spectrum of clinical manifestations, including infections, chronic lung disease, gastrointestinal (gi) disease and autoimmune disease . Bacterial infections of the sinopulmonary tract, particularly sinusitis and pneumonia, are experienced by most patients with cvid . Here, a 29-year - old female patient was admitted with complaints of cough, fever, diarrhoea and swelling all over her body . Physical examination revealed massive oedema, fever of 39c, crepitations on the lower lungs, increased bowel sounds and retarded development with weight of 40 kg and height of 145 cm . Pathological: the laboratory results that were out of limits were leukocyte 11 400/l, hemoglobin 10 g / dl, hematocrite 31%, platelets 568 000/l, c - reactive protein (crp) 399 mg / dl, total protein 3 g / dl, albumin 1 g / dl, proteinuria 9 g / day and many leukocytes and fatty acids on direct examination of the faeces . Ig levels were low: igg <33.3 mg / dl (n = 7511560), iga <6.67 mg / dl (n = 82453), igm 7.08 mg / dl (n = 46304). Serum amyloid a deposition was detected on biopsies (figure 1) obtained during gastroduodenoscopy and colonoscopy . Renal biopsy performed to evaluate nephrotic syndrome was also consistent with aa amyloidosis (figure 2). With the history of recurrent infections and low ig levels, she was diagnosed as having cvid leading to secondary amyloidosis . After her hospitalization, her signs and symptoms cleared with antibiotic and antiproteinuric treatment and with antibiotherapy, intravenous ig, antiproteinuric treatment including losartan and cilazapril; oedema and pleural effusion regressed with mild pretibial oedema remaining, crp level declined to 18 mg / dl, proteinuria declined to 7 g / day and albumin level rised to 2.2 g / dl . After resolution of gi symptoms, she was started on colchicines therapy; she is under follow - up with intravenous ig treatment without any infection during the last 10 months . Age of onset is typically after puberty and before 30 years of age, with some evidence of a bimodal distribution demonstrating peaks between 1 and 5 years and between 18 and 25 years . Cvid is a primary immune deficiency disorder characterized by markedly reduced serum levels of igg and low iga or igm, with impaired antibody responses, despite the presence of b cells . However, cvid is associated with a high incidence of inflammatory, autoimmune and malignant conditions, features of more fundamental immune dysregulation . Sinopulmonary infections, including pneumonia, bronchitis and sinusitis, as well as otitis and conjunctivitis, are observed in the majority of patients with cvid . These infections may be acute, chronic or recurrent . Over three - quarters of patients have at least one episode of pneumonia prior to diagnosis . Chronic lung disease is a common problem in patients with cvid and can lead to recurrent hospitalizations, significant morbidity and early death . In a large clinical study of 248 patients, 27% had either bronchiectasis or restrictive or obstructive lung disease . Another study of 224 patients found that 34% had chronic lung disease at the time of diagnosis, which increased to 46% during a mean follow - up of 11 years . The risk factors for the development of chronic lung disease in patients with cvid have not been fully defined . One report of 18 cvid patients found that those with reduced total memory b cells (cd27 + b cells) and very low numbers of switched memory b cells (cd27+igmigd) were more likely to have chronic lung disease . Gi disease is identified in ~20% of cvid patients and may be the presenting disorder in some . Specific disorders include inflammatory bowel disease, sprue - like illness with flat villi, nodular lymphoid hyperplasia, pernicious anaemia, chronic giardiasis, protein - losing enteropathy and nonspecific malabsorption . One biopsy study of gi pathology in 20 cvid patients over a 26-year period found that over one - half of the patients lacked plasma cells throughout the intestinal tract, and 47% showed lymphoid aggregates . We detected deposition of serum amyloid a besides nodular lymphoid hyperplasia in biopsies taken from the stomach, duodenum and colon . Routine laboratory studies are often normal in cvid, in the absence of an associated disorder in addition, modest lymphopenia and a reduced cd4 + level may develop over time . Our case had ig levels at undetectable levels and serious hypoalbuminaemia due to proteinuria and malabsorption . The management of cvid involves sufficient gamma globulin replacement therapy and monitoring for and treatment of associated inflammatory disorders and malignancies . Ig replacement therapy reduces the frequency of most types of infections as in our case, as well as slows the progression of chronic lung disease and offers some protection against autoimmune disorders . The usual initial dosing for intravenous ig is 300400 mg / kg, given every 34 weeks, with the goal of maintaining a trough igg level in the middle of the normal range . Isolated nephrotic syndrome cases responsive to steroid therapy have been reported in the literature associated with cvid . But there is no case with nephrotic syndrome due to amyloidosis documented with renal biopsy . Another patient with nephrotic - range proteinuria (9 g / day) was reported to have amyloid deposition in gastric and duodenal biopsy, but renal biopsy was not performed . Even if intravenous ig treatment may prevent infections and consequently amyloid deposition, insufficient treatment may lead to amyloidosis . Otherwise, with the increasing life expectancy of the patients and resultant increased number of infections, renal amyloidosis may be expected to increase in frequency . Renal amyloidosis in our patient is also thought to be due to delayed diagnosis and gamma globulin treatment . Patients with late diagnosis and insufficient treatment of infections are prone to develop amyloidosis and nephrotic syndrome which worsens the prognosis of the disease which has already high morbidity and mortality rates . Cvid must be kept in mind in patients with recurrent sinopulmonary infections in order to prevent co - morbidities.
Measurement is one of the cornerstones of the scientific research (1). In the health studies, the best and most important indicators for measurement are the quality and quantity of provided services for the patients as well as their satisfaction of received services (2). Patients' satisfaction, which is a key indicator of quality in the healthcare organizations (3), is the responses of the patients receiving the services to the provided services and reflects their overall perceptions of service quality (4). In addition, increasing patients' satisfaction is important because it can ensure the patient participation in the care and services (5). Therefore, the healthcare organizations need to develop and implement quality improvement plans for their survival and success . Measuring quality in the healthcare competitive environment is an undeniable necessity for these organizations, which will improve patient satisfaction (6, 7). High quality of the health sectors and their services is also considered as a desirable goal from the viewpoints of health planners and policymakers because healthy people in any society provide the opportunity for its economic development (8 - 10). Service quality is a strategic factor for healthcare organizations' productivity and is considered as a competitive advantage that should be continuously measured and improved (11). When customers have a good understanding of the quality of health services, they will probably attend the hospital again in the future, if needed, and suggest it to their family and friends (11). Thus, healthcare has a special place among other services because of its risky and precarious nature and therefore, the lack of patients' awareness of received services should be evaluated (12). Generally, measuring the quality of services in the health sector service quality in health has a multi - dimensional structure (10, 13), which was measured in the traditional approach using some objective indicators such as mortality and morbidity rates . Although these indicators are essential tools for assessing and evaluating the quality of clinical services, nowadays it is common to use more subjective assessments and indicators . It can be said that the field of healthcare is moving from providing services to evaluating the quality of services and consequently, the patients' role in defining the quality of services becomes evident more than ever (14). It has led to the increasing uses and high acceptance of the measurement of service quality from the viewpoint of patients (15 - 17). In addition to relying on economic criteria to maintain and improve the quality of health services, managers can use customers' expectations and perceptions as an important tool to determine the healthcare system's weaknesses (18). As a result, service providers are trying to apply client - centered assessment tools (19). There are different measurement models for assessing the quality of services, including kano, fornel and scamper, the european foundation for quality management (efqm), and servqual (20). In the present study, the servqual model, introduced in the mid-1980s by parasuraman et al . This instrument measures the customers' perceptions and expectations of services in five dimensions, including tangibility, reliability, responsiveness, assurance, and empathy . Several studies have been conducted using servqual model, including studies by al - borie and damanhouri (22), camgz - akdag et al . . Chronic kidney disease (ckd) endangers physical health as well as other aspects of health . Therefore, making accurate and comprehensive plans for the rehabilitation of patients affected by ckd is inevitable (30). However, this disease is a global public health concern (31, 32) and the number of patients with the ckd is increasing worldwide . This disease treatment is very costly, especially in developing countries, and these patients are forced to use hemodialysis (hd). They usually receive hd services two to three times a week, and three to four hours in each session . Measuring the quality of services among patients on hd is of paramount importance and can offer appropriate opportunities for improving provided services for these patients . Therefore, it is essential to continuously measure and improve the quality of provided services for this group of patients who spend long hours in hd centers . The present study aimed to measure the quality of provided services for patients with ckd in kerman in 2014 . This cross - sectional descriptive - analytic study was conducted from 23 january 2014 to 14 february 2014 in all four hd centers affiliated to kerman university of medical sciences, including two general hospitals (afzalipour and shafa), javad - ol - aeme specialty clinic, and samen - ol - hojaj charity (a specific patient treatment center). All of patients on chronic hd (n = 195) who were referred to these centers were selected and studied using census method . Patients in all studied centers were admitted for hd in two shifts of six days a week, morning shifts from 9 a.m. to midday and afternoon shifts from 3 p.m. to 6 p.m.; one patient was being admitted per each dialysis bed in each shift . Among the studied patients, 11 patients refused to participate in the study . The frequency of performing hd for referred patients was three times a week each of which took four hours . All of studied patients had attended for hd at least 15 times and therefore, they were completely familiar with the centers and its staff . The required data were collected using the standard questionnaire of servqual model (21), consisting of two parts . The first part included questions regarding the patients' demographic characteristics such as age, sex, marital status, education level, income level, and duration of dialysis . The second part included 28 items to measure the patients' expectations and perceptions of the five dimensions of service quality as follows: tangibility (6 items); the conditions and physical space of the service delivery environment, including equipment, having adorned and groomed staff, furniture, toilets, and bathrooms, payment process, cleanliness and quality of the materials used in the treatment, and the existence of car parking . Reliability (8 items); the ability to provide the committed services dependably and accurately through providing treatment at the predetermined time, listening to the patients' expectations, clear nurses' descriptions of the provided services, disease prevention and the treatment processes, the explanation of the treatment processes, proper maintenance of patients' records, the lack of duplication, and the effectiveness of services . Responsiveness (6 items); the willingness to help customers through decreasing admission time, quick and easy process of providing services, attracting patients' trust, employees' accountability to arranging an appointment for hd, clear physicians' descriptions of patient's disease, and employees' willingness to response to the patients . Assurance (4 items); ability to serve reliably through having polite employees and respecting patients' privacy, employees' awareness of the new medical techniques, ensuring the medical staff's skills, and the center reputation from the patients' viewpoints . Empathy (4 items); the provision of caring, individualized attention to customers through small time interval between admission and the start of dialysis, listening to the patients' comments and ideas, nurses' attention to the patients' needs, and paying attention to the patients' financial costs (33). A five - point likert scale was used to measure the patients' expectations and perceptions of service quality whereby one referred to very poor and five to excellent . Considering the nature of the dialysis centers and their services, it was necessary to make minor changes to the questionnaire . After making those changes, the validity of the questionnaire was approved through getting the opinions of ten faculty members, including four nephrologists, four nurses and two experts in health services management . In addition, the reliability of the questionnaire was confirmed using the inter - item consistency scores (= 0.77 and = 0.70 for patients' expectations and perceptions, respectively). In the expectations section, patients answered to the questions about the ideal or desirable status of services and in the perception section, they answered to the questions about the current status of services . To determine the quality gap, the scores of patients' perceptions of the quality of services provided were compared with the scores of patients' expectations of service quality . If the difference between the patients' perceptions and expectations was positive, it would indicate that the provided services for the patients had been more than their expectations and if it was negative, it would indicate that the provided services for the patients had not meet their expectations . Finally, if there was not any difference between the patients' perceptions and expectations, it would indicate that the provided services was at the level of patients' expectations, i.e. The provided services were at the level of patients' expectations . An approval for conducting this study was received from the ethic committee of kerman university of medical sciences . The verbal consent was obtained from all participants and all of them were assured of the confidentiality of their responses . Moreover, the collected data were analyzed using spss 21.0 (ibm corporation, armonk, ny, usa) through some statistical tests, including independent - samples t test, one - way anova, and paired - samples t test . The results showed that 109 patients were male (59.2%), 117 (63.6%) were married, 112 (60.9%) were older than 40 years, 61 (33.2%) were illiterate, 83 (45.1%) were unemployed, 122 (66.3%) had sufficient income for hd, and 70 (38%) had been treated with hd for one to three years (table 1). Furthermore, the results showed that the means of patients' expectations were more than the current status and their perceptions of the quality of provided services in all dimensions of service quality . In addition, the highest and the lowest means of the patients' perception dimensions were respectively related to assurance (4.30 0.36) and empathy (3.84 0.34). The highest and the lowest means of the patients' expectations dimensions were related to assurance (4.72 0.27) and tangibility (4.30 0.35), respectively . After computing the differences between the means of expectations (ideal status) and the perceptions (the current status), the results revealed that there were gaps in all dimensions . The highest and lowest means of negative gaps were related to empathy (-0.52 0.48) and tangibility (-0.29 0.51). The differences between the patients' perceptions and expectations (gaps) in all five dimensions of hd services quality were statistically significant (p> 0.001) (table 2). In addition, among the patients' demographic characteristics and the five dimensions of service quality, only the difference between the patients' income levels and the gap in assurance was statistically significant (p <0.001); in other words, the decrease in the income levels resulted in the significant decrease in the absolute values of gap means (table 3). Measuring the quantity and quality of provided services to identify their weaknesses is one of the most important and most effective strategies of healthcare managers to improve the quality of services . In addition, due to the effects of services quality on the patients' satisfaction, quality measurement from their viewpoints is considered as an important indicator (16, 17). Accordingly, the present study aimed to measure the quality of provided services for patients with ckd, who were referred to all four hd centers in kerman, using the servqual instrument . The results of the present study showed that patients' expectations were more than the current status of the provided services in all dimensions . In addition, there were negative gaps and statistically significant differences between the means of patients' expectations and their perceptions (gap) in all five dimensions of hd service quality, indicating that the patients' expectations in all five studied dimensions were more than their perceptions of the current status of provided services . Although there were gaps between patients' expectations and perceptions of services, these gaps were not very large, indicating that the studied centers had paid special attention to the quality of provided services and overall, the level of services was acceptable . However, they should make efforts to reach an optimal level . Butt and de run (34), lin et al . (35), bakar et al . (37) concluded that there were negative gaps between patients' perceptions and expectations in all dimensions of service quality, which were in agreement with the results of the present study . The results of the mentioned studies indicate that the provided services in the studied hospitals and centers had not been consistent with the patients' expectations and their managers should do proper planning and priority setting for improving all dimensions of services quality . Therefore, hearing the voice of customers is an important tool in modern organizations management and the studied hospital managers should re - engineer the processes and use the improvement techniques with regard to the patients' feedback and comments . In the present study and among the available gaps in the studied dimensions of quality, as mentioned above, the tangibility had the smallest gap indicating that the studied centers had a clean environment, adorned and groomed staff, adequate physical resources and facilities such as furniture, toilets, and bathrooms, car parking, and modern and updated technologies and equipment, all of which had led to greater patients' satisfaction in this dimension than other dimensions . However, because the hospital physical environment plays an important role in improving the service quality and patients' evaluations of service quality, attractive environment and appropriate hospital hoteling services are considered as one of the most important reasons for referring patients to a hospital (33, 38). Therefore, hospital managers should provide more amenities and facilities based on the patients' needs in order to decrease the gap between patients' perceptions and expectations in the tangibility admission . Lee and yom found that tangibility had the smallest gap, which was in accordance with our results (11). In contrast to our results, zarei et al . Reported the largest and smallest gaps in the tangibility and empathy dimensions, respectively (39). Furthermore, the largest negative gap was in the empathy, indicating that service providers did not have enough attention to the patients' views and comments and did not apply their opinions and comments in their planning and programs . It seems that the high volume of work in the hd wards and downplaying the proper patient - physician relationships had led to physicians low opportunities to express their empathy, listen to, and understand the patients' opinions and comments . Moreover, the large gap in the empathy could be due to physicians, nurses and employees' poor communication with patients . Efforts in this area should, also be made to improve staff behavior and communication with patients . Unlike the results of jabnoun and chaker (40), the results of huang and li's study (41) were similar to our results . Because the services are inherently untouchable, interpersonal interaction during the process of service delivery has an important effect on the patients' perceptions of service quality . In addition, the results of several studies have shown that human factors have greater effect on the patients' perceptions of the quality of services than non - human factors, and interpersonal interaction and relationship is one of the most important factors affecting the patients' perceptions of service quality (42 - 44). Therefore, physicians and staff should recognize and pay attention to the patients' social and emotional needs and wants and should be available for patients when needed . A gap in one dimension can have synergistic effect on other dimensions of service quality and lead to the decrease in those dimensions (45). Therefore, in addition to focusing on dimensions with the largest gap, managers and service providers should consider the improvement of other dimensions . In the present study, the means of service quality dimensions did not have significant associations with sex, age, and marital status . Some of the previous studies have reported higher expectations in women than in men (35, 46), which was inconsistent with our results . In the current study, there was no significant association between the means of service quality dimensions and the patients' education levels; however, the gap in the patients with academic and university education and degrees was larger than that in the illiterate patients . It seems that the patients' expectations had become more reasonable by increasing their education level, and their expectations had been decreased by increasing their knowledge and awareness of treatment processes . (36) showed that the expectations of patients with academic and university education and degrees were higher than that of other patients . In other service quality dimensions, except for reliability, the expectations of patients with sufficient income levels were higher than expectations of patients with insufficient income levels . One explanation might be that the paid hospital costs by the patients with sufficient income levels did not put any considerable pressure on their economic conditions; hence, they expected the hospitals to meet their expectations completely . (36) found that the uninsured patients were less satisfied with the quality of hospital services compared with the insured patients . One of the limitations of the present study was using only patients' perspectives to determine the quality of provided services . It is essential to investigate the viewpoints of physicians, nurses, and other employees on the service quality because most of patients are not fully aware of the treatment processes . Another limitation of the present study was using a questionnaire to determine the patients' perceptions and expectations . Although servqual questionnaire is valid and reliable, the researchers cannot investigate all dimensions of the service quality using only a questionnaire; in that regard, some qualitative studies should also be performed . Overall, the results of the present study showed that the expectations of patients on hd were higher than their perceptions and the level of provided services . The healthcare providers and employees should pay more attention to the patients' opinions and comments and use their feedback and suggestions in order to solve the workplace problems and improve the quality of provided services . Moreover, training the health staff to meet the patients' emotional needs and expectations is recommended . One of the limitations of the present study was using only patients' perspectives to determine the quality of provided services . It is essential to investigate the viewpoints of physicians, nurses, and other employees on the service quality because most of patients are not fully aware of the treatment processes . Another limitation of the present study was using a questionnaire to determine the patients' perceptions and expectations . Although servqual questionnaire is valid and reliable, the researchers cannot investigate all dimensions of the service quality using only a questionnaire; in that regard, some qualitative studies should also be performed . Overall, the results of the present study showed that the expectations of patients on hd were higher than their perceptions and the level of provided services . The healthcare providers and employees should pay more attention to the patients' opinions and comments and use their feedback and suggestions in order to solve the workplace problems and improve the quality of provided services . Moreover, training the health staff to meet the patients' emotional needs and expectations is recommended.
Osteoporosis is a systemic skeletal disorder characterized by low bone strength (arising from both low bone mass and microarchitectural deterioration), which increases the risk of fractures . Osteoporosis is a major public health problem and an important contributor to the global burden of noncommunicable disease . Currently the recommended method for the diagnosis of osteoporosis is bone mineral density (bmd) measurement by dual - energy x - ray absorptiometry (dxa). According to the world health organization criteria, osteoporosis is operationally defined as a bmd that lies 2.5 standard deviations or more below the average value for young healthy women . . Since, due to cost and availability, dxa scans are not recommended for screening purposes, several tools based on known clinical risk factors have been developed to identify those patients with high risk of osteoporosis, in whom actual bmd testing would be most useful in terms of diagnosis, treatment, and followup [3, 4]. Some of these clinical tools, or aids in decision making, include many factors, making calculation of risk cumbersome [5, 6]. Arguably the simplest decision rule is the osteoporosis self - assessment tool (ost) which only takes into account body weight and age, which in adult populations are, respectively, related inversely and directly to the risk of osteoporosis . The ost was developed for predicting risk of femoral neck t - score at or below 2.5 in asian postmenopausal women and later validated for caucasian european and us postmenopausal women . In these populations, the performance of the ost was similar to those of more complex clinical risk assessment tools [3, 1012] although a related tool, called osteorisk, has been validated for latin american postmenopausal women, no direct assessment of the ost has been yet performed in this region . The current prevalence of osteoporosis and the incidence of osteoporotic fractures in latin america are similar to those of southern europe [1416], but lower than those of northern europe and the united states [1, 2]. However, a significant increase in the incidence of osteoporotic fractures is expected to occur in latin america in the next few years, according to a world health organization report . This highlights the need for improving clinical assessment and selection of women for bmd testing . In this report, the performance of the ost in a sample of postmenopausal women from western argentina was assessed . The province of mendoza in western argentina has a population of 1,742,000 inhabitants according to the 2010 census . About 62% of the population lives in greater mendoza, the fourth largest metropolitan area of the country, which includes about 133,000 women aged 50 years or older . The current sample included 4343 women referred to the bone densitometry unit of the nuclear medicine school for a first (diagnostic) dxa scan of lumbar spine and hip . Women with paget's disease, primary hyperparathyroidism, or severe hip osteoarthritis the research protocol was reviewed and approved by the committee of teaching and research of the nuclear medicine school . The study was planned and conducted in full accordance with the current version (2008) of the declaration of helsinki . The height and weight of each patient were measured while she stood without shoes, wearing light clothing . The body mass index (bmi) was calculated as her weight in kg divided by her height in m squared (kg / m). Patients were asked about previous fragility fractures, glucocorticoid, estrogen or bisphosphonate treatment, a diagnosis of rheumatoid arthritis, a history of hip fracture or dxa diagnosis of osteoporosis in their parents, smoking status, alcohol intake and physical activity . Calcium intake, was assessed through a spanish version of the food frequency questionnaire developed and validated by magkos et al . Dxa scans of the lumbar spine (l1l4) and one hip (usually the left) were performed using a lunar prodigy equipment (ge healthcare lunar, madison, wi). Measurements were performed by one of two technicians, both of whom were certified by the international society for clinical densitometry . Stability of the bone densitometer throughout the study (in vitro long - term precision) was checked through daily measurement of a spine phantom according to the manufacturer . Short - term in vivo precision was estimated by dxa scans repeated after repositioning the patient, with two measures at each site in 30 patients, according to the international society for clinical densitometry official positions 2007 . Phantom measurements showed stability of the dxa equipment throughout the study, with a coefficient of variation of 0.5% . The combined in vivo precision for both technicians was 1.5% for the lumbar spine, 1.8% for the femoral neck, and 1.4% for the total hip . Patients were classified as normal, osteopenic, or osteoporotic according to the world health organization criteria, based on the lowest t - score at the lumbar spine, the femoral neck, or the total hip . Reference values were taken from the national health and nutrition examination survey (nhanes iii), which is the recommended reference database for argentine patients . The ost score was calculated as 0.2 (weight in kg age in years) and rounded up to the closest integer . For example, a 64-year - old woman weighing 50 kg has an ost score of 0.2 (50 64) = 2.8, which would be rounded up to 3, and a 52-year - old woman weighing 67 kg has an ost score of 0.2 (67 52) = 3 . Since diagnosis of osteoporosis by dxa is based on a t - score at 2.5 or below at any of the recommended sites (lumbar spine, femoral neck, or total hip), the lowest t - score was taken to dichotomously assign each result to a nonosteoporotic or osteoporotic group . Data were analyzed with the commercial statistical software prism 5.04 for windows and instat3 (graphpad, san diego, ca). The d'agostino and pearson omnibus normality test was routinely used to assess whether data departed significantly from a gaussian distribution . If this was the case, data are presented as median (2575 interquartile range). Comparison of ost scores between women with a dxa diagnosis of osteoporosis (t - score of 2.5 and below at any site) and those without it was performed with mann - whitney's test . Simple linear regression was employed to assess the relationship between ost score and the lowest t - score for each patient (lumbar spine, femoral neck, or total hip). A receiver operating characteristic (roc) curve was used to assess the area under the curve (auc), sensitivity, and specificity . The diagnostic odds ratio was calculated by chi - square test, and results are displayed as mean (95% confidence interval = ci95). The characteristics of the sample are shown in table 1 . Out of 4,343 patients, a total of 2,513 women were classified as nonosteoporotic while the remainder 1,830 women were classified as osteoporotic . Among the main risk factors detected, other than advanced age or low weight, low calcium intake (less than 1000 mg / day) was found in 70% of women, essentially corroborating the result of a previous study in the same population . Fragility fractures were recalled in 16,5% of the patients, sedentarism in 15%, a family history of osteoporosis in 10%, long - term glucocorticoid therapy in 6.2%, and rheumatoid arthritis in 1.8% . Twelve percent of the patients were cigarette smokers at the time of the study, but high alcohol intake was reported by less than 1% . In table 2 the absolute number and the proportion of women whose t - score was at 2.5 or below at the lumbar spine, the femoral neck, the total hip, or a combination of two or all three sites are shown . Of the 1,830 women with diagnosis of osteoporosis, t - scores of 2.5 or below were found in 1,207 at the lumbar spine, in 569 at the femoral neck, and in 1063 at the total hip . These figures correspond to the total number of patients with t - scores at 2.5 or below at each site . For example, the figure of 1,207 for the lumbar spine includes 557 women with t - score at 2.5 or below at the lumbar spine only, plus 125 women with t - score at 2.5 or below at both lumbar spine and femoral neck, plus 266 women with t - score at 2.5 or below at both lumbar spine and total hip plus 259 women with t - score at 2.5 or below at lumbar spine, femoral neck and total hip . For the whole group, ost scores ranged from 11 to + 15 (11 to 7 in osteoporotic and 7 to 15 in nonosteoporotic women). Women with a diagnosis of osteoporosis had significantly lower ost scores than those without it . Median ost scores were, respectively, 0.0 (2 to + 2) versus 2.0 (0.0 to 4.0); p <0.0001 . The result of the roc analysis is shown in figure 1 . Table 3 displays sensitivity and specificity for cut - off values from 3 to 3 . For an ost score cut - off value of 2, the positive predictive value was 52% and the negative predictive value was 79% in the present sample . Figure 2 displays ost scores versus lowest t - scores for the entire sample, showing a significant linear relationship between ost scores and lowest t - scores (p <0.0001). If women with an ost score of 2 or lower are considered at high risk, and those above 2 are deemed at low risk, the unadjusted odds ratio for a diagnosis of osteoporosis by dxa of the high risk group versus the low risk group is 4.06 (ci95 3.51 to 4.71). The auc obtained from a roc analysis can range (expressed as a percentage) from 0 to 100, with 50 being the line of identity . Since sensitivity and specificity are both independent of disease prevalence, the same applies to the auc . Auc at or above 70% are deemed acceptable for a screening test . In the present study, the sensitivity and specificity of any given test vary inversely according to the chosen cut - off value . In previous reports, reviewed by rud et al ., the sensitivity of the ost for prediction of t - scores at 2.5 or below for any region (lumbar spine, femoral neck, or total hip) has a median of 86% (range of 53% to 95%) in white women and 82% (range 79% to 82%) in asian women . In the present study, using a cut - off value of 2, the sensitivity in argentinian women was 83.7%, which is intermediate between the medians for white and asian women . On the other hand, the specificity of the ost for any site has a median of 40% (range 34% to 72%) for white women but a higher median, of 64% (range 60% to 78%), for asian women . The estimated specificity in the present study with a cut - off at 2 was 44%, closer to the specificity for white women than for asian women . The reason why values of sensitivity and particularly the specificity for argentinian women were between those for white and asian women is not clear, but it may be related to the fact that about 80% of the argentine population has european ancestry, with minor but significant contributions from other ethnic groups . One limitation of this study concerns whether the sample is representative of mendoza's postmenopausal women . Participants referred by their physicians for bmd measurement might have more risk factors than postmenopausal women in the general population . In a recent prospective study of 720 postmenopausal women undergoing their first dxa scan, of those below that age, 55% had at least one risk factor (f. d. sarav, unpublished data). Another reason why the sample may not accurately depict the general population of postmenopausal women of great mendoza is socioeconomic status and educational level . Recent estimates place the fraction of the population below the poverty line at about 10% for argentine urban areas . Additionally, according to official statistics, 37% of the population does not have health insurance . Although poor women or those without health insurance can still get a dxa scan through agreements between our center and the public hospital system, in practice their access is limited . These women may differ from the ones included in the present study on their educational level, nutrition, lifestyle, and prevalence of osteoporosis . Most of them consider additional factors other than age and weight, for example, the abone (age, bulk, no estrogen); the osteoporosis risk assessment instrument (orai), which incorporates age range, body weight (dichotomously), and estrogen therapy; the simple calculated osteoporosis risk estimation (score), which includes race other than black, rheumatoid arthritis, nontraumatic fractures, age, weight, and estrogen therapy; and the osteoporosis index of risk (osiris), which takes into account body weight, age, history of nontraumatic fractures, and estrogen therapy . . Found that ost predicted bone mass equally well than orai and score in women from the united states and the netherlands . Similarly, a comparison performed in a large sample of belgian women found that ost performed as well as the more complex risk assessment indices (score, orai, and osiris) in identifying women at low risk of osteoporosis . In a 2004 review article, wehren and siris also stated that ost, the simplest of the instruments, performs as well as more complex tools . Essentially the same was found in a study of 986 postmenopausal moroccan women . In a study of canadian women, the performance of ost was as good as that of orai . In a systematic review, it is stated that ost shows higher accuracy than orai and score concerning the any region the authors noted, however, that overall accuracy is similar in white women, albeit the trade - off between sensitivity and specificity may differ between ost and comparator cdrs (clinical decision rules). A very recent publication compared ost, orai, and abone and reported that ost performed best in us white women . In the american college of preventive medicine position statement on screening for osteoporosis it is stated about the ost, the simplicity of this screening tool and its validation in both genders and in various races account for its popularity and widespread use in selecting patients for confirmatory bmd testing . In the studied sample of postmenopausal women from mendoza, argentina, the ost showed a performance comparable to that reported for european and us white women . The overall performance of the ost was adequate for a clinical screening method simple enough to be used both by patients and physicians . Of course, its use does not preclude careful consideration of other clinical risk factors for osteoporosis.
Recently, a new class of hybrid materials has been generated by attaching dna to organic molecules, polymers, metal complexes, and nanoparticles . Some of the resulting 2d and 3d nanostructures have been used in dna detection and electronic applications; other applications such as drug delivery and therapeutics are emerging . A key factor that enables these applications is the degree of structural control available when dna are linked to other molecules . Types of linkers and linkage direction are crucial for providing the desired control on product distributions of the assemblies and their stability . Recent investigations into the self - assembly of small molecule - dna hybrids in aqueous media have demonstrated that the structures of these assemblies are dictated by several factors, including the number of single - strand (ss) dnas attached to the organic cores, the specific geometry and concentration of the dna strands, and the type (na, k, ca, mg, etc .) And concentration of ions used in aqueous media . We additionally showed that the hydrophobic properties of organic cores is an important parameter to be considered in the self - assembly of the small - molecule - dna hybrids (smdhs). Indeed, richert and co - workers have utilized the hydrophobic properties of stilbenes and anthraquinones linked to dna duplexes to stabilize small - duplex dna - detection probes . In a related study, bergstrom and co - workers end - capped small dna hairpins with rigid, hydrophobic organic molecules to afford enhanced stability . Moreover, perylenedimide (pdi)-dna hybrids have been found to form stable hairpin dimers and larger supramolecular oligomers due to the hydrophobicity of the pdi cores, which leads to pdi pdi stacking via interactions . Despite emerging evidences concerning the critical importance of the hydrophobic interactions between organic cores and dna duplexes in organic - dna hybrids, specifically, the effects of linking hydrophobic organic cores to dna strands through either 3- or 5-ends are still not known although it is clear that such different linkages will lead to different types of hydrophobic interactions such as insertion into the minor groove, intercalation, and stacking in the self - assembly of the small molecule - dna building blocks into dna - hybrid nanostructures . Probing these interactions experimentally is a challenging task, so we decided to combine experimental studies with molecular dynamics (md) simulations at the atomistic level with the goal of unraveling the assembly of two complementary smdhs into a cyclic dimer, the simplest dna - hybrid nanostructure possible . With the continuous improvements in computer technology, utilization of computational methods and tools to describe self - assembly is gaining popularity . While there are different methods available to describe the properties of nucleic acid systems such as melting, mechanical properties, and conformations using coarse - grain simulations, the atomistic details of these systems at long time scales can best be described using atom - based force fields, such as amber and charmm . Previously, we reported the remarkable effects of hydrophobic organic cores in the assembly of small - molecule dna hybrids (smdhs) into caged structures . Our experimental results and computational simulations showed that the final nanostructures assemble in aqueous environments in a manner that minimizes exposure of the hydrophobic surfaces of the organic cores to solvent . These hydrophobic interactions are greatly influenced by the incorporation of multiple noncomplementary deoxythymidine (t) spacers between the core and the dna duplex, as the solvent - accessible surface area (sasa) of the hydrophobic cores is greatly reduced when these spacers are wrapped around the cores . Soon after our report, sleiman and co - workers published a broad study on the self - assembly of cyclic nanostructures made from smdh2 building blocks . They showed that while the small molecule - dna hybrid derived from the flexible organic core 1 (fsmdh2) assembled exclusively into cyclic dimers, that derived from the rigid organic core 2 (smdh2) assembled into a mixture of dimers and higher - order (tetramer, hexamer, etc .) They proposed that the presence of rigid cores at either the 3- or 5-ends of dna duplexes constrains the ways in which these duplexes can assemble and affects the distributions of the final products (dimer, tetramer, hexamer, etc . ). Schematic descriptions of the assembly of cyclic nanostructures (dimer, tetramer, hexamer) from smdh2 building blocks containing organic cores described by the sleiman group (1, flexible; 2, rigid) and us (3, partially rigid). To explain why cyclic dimers are preferred in the case of core 1, sleiman and co - workers invoked a strand - end alignment model that focuses only on the importance of duplex alignment; once the dna duplexes are ideally aligned by the first rigid core, dimers can form if the other ends of these duplexes are properly aligned to accommodate the second rigid core . The orientation of the ends of the dna duplexes was defined as either convergent (i.e., can accommodate the second rigid core) or divergent (i.e., cannot accommodate the second rigid core). While this assumption appears to be a reasonable one, it was based on two cores that are quite different: one (1) that is flexible and moderately hydrophilic and the other (2) that is quite rigid and very hydrophobic . We suspect that a model built upon two such different cores may not accurately reflect the assembly process of smdh2, particularly when the hydrophobic nature of the organic cores can force them to interact very strongly with the bases of the dna component strands as well as with stacked base - pairs of the assembled duplexes in aqueous environments . Furthermore, the magnitude of this hydrophobic interaction would be strongly modulated by the flexibility of the connection points between the cores and the dna component strands . Indeed, we have observed for the smdh3 system that when there is not enough flexibility at the connection points, the drive to minimize exposure of the hydrophobic organic cores to water can sometime become so overwhelming that the system will destabilize some of the dna base pairings in the assembled duplex arms . These observations prompted us to explore the assembly of smdh2 further using the core 3, which is hydrophobic but much more flexible than 2 . Herein, we present a detailed computational study, supported by experimental results, which suggests that both the hydrophobic nature and the flexibility of the organic core play very important roles in the self - assembly of smdh2s into cyclic dimers and higher - order nanostructures . While duplex alignment may be important, the interactions between the cores and the dna duplexes to minimize their sasa comprise a dominant force that cannot be ignored, especially when there is restricted flexibility . Indeed for cyclic dimers involving core 3, when the cores are attached to the 3-ends of the dna component strands, they prefer to insert into the minor groove of the dna duplexes in the product dimers to minimize their sasa . In contrast when cores such as 3 are attached to the 5-ends of the dna component strands, they can only partially insert themselves into the minor groove of the dna duplexes in the product dimers, resulting in much less stable cyclic dimers . Consistent with these insights, smdh2 building blocks containing cores linked to 3-ends of the dna duplexes were found to yield higher percentages of cyclic dimers compared to that obtained from 5-linked smdh2s . The important roles that such hydrophobic interactions play in smdh2 assembly is further supported by the enhanced thermodynamic stability of face - to - face (ff) dimers over analogous cyclic dimers; the former benefits from strong interactions between two overlapping hydrophobic cores . Synthesis of unsymmetric smdh2s was achieved by adding the phosphoramidite core (scheme s1 in the supporting information) to the initial dna arm grown from the surface of a controlled porosity glass bead (cpg) from either the 3- or 5-end of the dna strand, followed by synthesis of the second arm via either 3-normal or 5-reverse phosphoramidite chemistry (scheme s2 in the supporting information). The final dmt - protected products were then cleaved from the solid support, purified by reverse - phase (rp) high - performance liquid chromatography (hplc), and subjected to dmt deprotection to yield the desired smdh2s (table 1). The purities of all smdh2s were ascertained via analytical rp - hplc and their length and base compositions were confirmed via maldi - tof mass spectrometry (see figures s1s14 in the supporting information). Building blocks (smdh2s, table 1) containing two different strands (one of the strands is the reverse sequence of the other to maintain similar thermal properties) were designed to control the formation of cyclic (figure 1) versus ff smdh2 (figure s15 in the supporting information) nanostructures, depending on the orientation of the linkage between the core and the dna strands . The smdh2 assemblies and their controls (table 2) were prepared by combining equimolar amounts of two complementary components (table 1) in tamg buffer (40 mm tris base, 20 mm acetic acid, 7.5 mm mgcl26h2o) and annealing the resulting mixtures using both normal and slow - cooling methods (see section s3 in the supporting information for more details). Amber force field parameters for the organic core were calculated as previously described (section s4 in the supporting information). The amber99 force field with revised and / torsional parameter sets was used to define the dna parameters . All the model dna systems (with sequences described in table 1) were created in b - form using the nucgen module of amber 9 . The smdh2 systems (table 2, entries 15 and 13) were then prepared, where the organic cores were attached at either 3- or 5-ends of the dna sequences (section s5 in the supporting information). Most md simulations were run in a generalized born implicit - solvent model (gb) with 0.3 m salt concentrations . Unrestrained gb md simulations were carried for our three different cyclic dimers (table 2, entries 13) and the cyclic tetramer and hexamer for one smdh2 system (table 2, entry 1, see section s6 in the supporting information for details). Restrained gb md simulations (both normal and annealed) were carried out for cyclic dimers (table 2, entries 13) with varying dna duplex lengths (11, 13, 15, 17, 19, 21, and 23 base pairs) and a model system (used for explicit solvent calculations that were used to calibrate the gb model) consisting of an 11 bp dna duplex with a single organic core attached at either 3- or 5-end of the dna strands (see section s7 in the supporting information). Sasa and root - mean - square deviation (rmsd) analysis for all the md simulations were performed according to our previously published work (see sections s5 in the supporting information for details). The implicit - solvent model used in this study is preferred over explicit - solvent ones due to the large size of our systems, whose computation would be quite long and expensive if explicit - solvent models were used . Compared to explicit - solvent md simulations, the viscosity in implicit - solvent md simulations is low and this accelerates the sampling of md space . Yet, implicit - solvent models are less realistic because they employ empirical parameters to calculate the solvation free energies . To date, implicit - solvent simulations of proteins are well developed while the corresponding simulations of nucleic acids still require improvements . While gb implicit - solvent simulations of regular dna oligomers produce average structures that are in line with explicit - solvent md simulations, the formers can distort the dna backbone and promote fraying effects at terminal base pairs that might not be physical . This is one of the shortcomings of implicit - solvent models and can cause dna to be more flexible than it actually is . Indeed, harris and co - workers showed that the use of implicit solvents in dna minicircle topoisomers with varying lengths display structures that are different from those seen in explicit - solvent simulation, which can be attributed to dna flexibility . One of the reasons for this outcome is the neglect of specific interactions of water molecules with dna in implicit - solvent models (such as the spine of hydration observed in the minor grooves of a - tracks). Thus, the improper description of dna flexibility will show its effects in exotic systems such as in dna minicircles and smdh molecules . As a result, to keep the dna structures in their known native b - form crick and torsional restraints so that the md simulations have a physical meaning . Additionally, explicit - solvent calculations were carried out on our smallest model system (see section s7 in the supporting information for more details) to verify the accuracy of the gb calculations with restraints . Combining equimolar amounts of [5-c-3], [5-c-5], [3-c-3], [5-t3ct3 - 3], [5-t6 - 3], [5-t6 - 5], [5-t3 - 3] with their complements, [5-c-3], [5-c-5], [3-c-3], [5-t3ct3 - 3], [5-t6 - 3], [5-t6 - 5], and [5-t3 - 3], respectively, was expected to result in cyclic structures (figure 1; see also table 2, entries 17) as previously reported . Our earlier study showed that systems with core 3 with noncomplementary t spacers (table 2, entry 4) exclusively formed cyclic dimers at low concentrations of ss - dna (up to 5 m total ss - dna concentration in which each arm s concentration in the smdh2 was calculated separately). However, sleiman and co - workers recently reported that the rigidity of the core and the linkage between the core and the dna strands (33, 55, and 35 orientations; see figure 1, inset b) directly influence the product distributions: systems with flexible core 1 (figure 1, inset a) attached to 3- or 5-end of dna strands mostly formed cyclic dimers, while those with rigid core 2 (figure 1, inset a) afforded a mixture of all cyclic products . Interestingly, page - gel analysis of the [5-c-3]:[5-c-3], [5-c-5]:[5-c-5], and [3-c-3]:[3-c-3] combinations in our present work revealed a mixture of cyclic structures (figure 2) instead of solely cyclic dimers even though our core 3 can be classified as being overall flexible with highly flexible (ch2och2ch2o) arms flanking a rigid 1,3-bis(ethynylphenyl)phenyl component (figure 1, inset a). Nondenaturing page - gel image (6%) of dna assemblies with 5 m total ss - dna concentration (gel was prepared in 1 tamg buffer (40 mm tris base, 20 mm acetic acid, 7.5 mm mgcl26h2o), and run at 4 c for 2 h at a 200 v potential). From left to right: lane 1 = hl5 dna ladder, lane 2 = cyclic-[5-c-3]:[5-c-3] (normal annealing), lane 3 = cyclic-[5-c-3]:[5-c-3] (slow annealing), lane 4 = control-[5-c-3], lane 5 = cyclic-[5-c-5]:[5-c-5] (normal annealing), lane 6 = cyclic-[5-c-5]:[5-c-5] (slow annealing), lane 7 = control-[5-c-5], lane 8 = cyclic-[3-c-3]:[3-c-3] (normal annealing), lane 9 = cyclic-[3-c-3]:[3-c-3] (slow annealing), and lane 10 = control-[3-c-3]. According to the strand - end alignment model, the flexible arms of our core 3 should be long enough to accommodate any strain put on the dna duplexes by the rigid 1,3-bis(ethynylphenyl)phenyl component, favoring the formation of cyclic dimers . However, this was not experimentally observed . Rather, exclusive formation of cyclic dimers is observed when three noncomplementary deoxythymidine spacers (t3) were added to each side of our core 3 ([5-t3ct3 - 3]:[5-t3ct3 - 3]), according to the nondenaturing page - gel analysis (see figure s17 in the supporting information, lane 9). These results, however, are in agreement with our previous report, which concluded that dimer formation is favored when t3 spacers are available to shield the hydrophobic surfaces of the cores from the aqueous media . The nondenaturing page - gel analysis also revealed that there is a higher percentage of dimer formed for [3-c-3]:[3-c-3] compared to the [5-c-5]:[5-c-5] and [5-c-3]:[5-c-3] combination (figure 2, lanes 8, 5, and 2, respectively). To further probe this observation and eliminate the possibility that the formation of cyclic dimers may be affected by the length of the annealing time, we annealed our smdh2 systems (table 2, entries 13) with a cooling rate of 0.01 c / minute in a pcr instrument in the range of 6025 c, which is 34 times slower than our normal annealing protocol (0.34 c / minute in the 6040 c range (figure s16 in the supporting information), where the complete melting of a mixture of cyclic structures occur). Analysis of the nondenaturing page - gel image (figure 2) showed a clear increase in dimer formation for all cases during slow annealing . Moreover, the cyclic dimer formed in high yield (79%) for [3-c-3]:[3-c-3] in slow annealing, much higher than the [5-c-3]:[5-c-3] and [5-c-5]:[5-c-5] combinations (43 and 34%, respectively). The latter afforded the highest percentage of large cyclic structures (31%, figure 2, lane 6) in the slow annealing method compared to the former two (11 and 5%, respectively). Similar observations were also reported by sleiman and co - workers for systems derived from core 2 . Cyclic dimers were formed exclusively in the nondenaturing page - gel analysis when deoxythymidine linkers (t3 and t6, table 2, entries 57) were used in place of organic core 3 (see figure s18 in the supporting information, lanes 35). Our previous computational work showed that hydrophobic interactions between the organic cores and noncomplementary t spacers in smdh3 building blocks play a crucial role in the self - assembly process and the final properties of smdh3-based nanostructures . In such structures, the cores strive to minimize their sasa values through interaction with both the t spacer and the base pairs of the duplexes . When the sasa values were too high for dimers, other structures with lower sasa become dominant . Nevertheless, we were surprised to observe that the directionality of the linkage (3- or 5-) between the core and the duplex dramatically affected the product distributions in the assembly of smdh2-based nanostructures in the absence of noncomplementary t spacers (see the discussion of experimental results). As such, we utilized md simulations to elucidate the details of the self - assembly process . Computationally, we began by examining the simpler dna - hybrid components that constitute the larger cyclic nanostructures (dimers, tetramers, hexamers, etc . ): (1) organic core 3 attached to either 3- or 5-ends of a dna duplex (figure 3a); (2) single organic core attached at 3- and 5-ends of two dna duplexes, control-[5-c-3] (figure 3b); (3) cyclic-[5-c-3]:[5-c-3] structures (dimer structure is shown in figure 3c; tetramer and hexamer structures are shown in figure 1). Initial modeling of (a) organic core 3 attached to a dna duplex, (b) control-[5-c-3], and (c) cyclic-[5-c-3]:[5-c-3] dimer . The simplest component system, consisting of the organic core 3 attached to an 11 bp dna duplex, allows us to investigate how the organic cores attached to either 3- or 5-ends of dna duplexes (see section s7 in the supporting information) interact with these duplexes in an aqueous environment . Crick base pairing and torsional restraints were applied to dna duplexes to keep them in their native b - form since gb implicit solvent md simulations can distort the dna backbone and cause fraying at terminal base pairs (see the discussions below concerning unrestrained and restrained md simulations and section s7 in the supporting information). After 10 ns of md simulations, the 3-linked organic core is already inserted fully into the dna minor groove (figure 4b) while the 5-linked core is only partially inserted (figure 4c). Average sasa values for the 3- and 5-linked organic cores are 153.7 19.6 and 250.8 18.4, respectively, and remain constant over the course of the simulation time (55 ns, figure 4a). Because the 3-ends of dna duplexes are spatially closer to the minor grooves compared to 5-ends, it is easier for a 3-linked organic core to be fully inserted into the dna minor groove . On the other hand, the 5-linked organic core needs to stretch over the terminal base pairs before it can reach dna minor grooves; so it is only partially inserted, has a much larger sasa value, and is less stable in aqueous solution . These results suggest that the differences in the distribution of cyclic nanostructures, as observed in the page - gel experiments (figure 2, lanes 6 versus 9), can be rationalized by how well organic cores are inserted in dna minor grooves to minimize exposure to the aqueous outside environment . To calibrate the stabilities of these restrained gb - based structures, we also performed explicit - solvent calculations for the same model . In these calculations, each final conformation from the gb calculations was solvated with na / cl ions and water molecules and the imposed restraints were removed on the dna duplexes (see section s7 in the supporting information for more details). The simulations show that the 3-linked organic core stays in the dna minor groove while the global dna conformation remains in b - form (figure s22 in the supporting information). It is also found that the organic core in the 5-linked core - dna system is dynamic and does not stay in the dna minor groove for a long time . The terminal base pair is broken, and the organic core stacks within the bases of the distorted terminal side (figure s22 in the supporting information). This clearly shows that dna minor groove insertion is a viable option for 3-linked organic cores to lower their sasa without distorting the dna . This is not the case in 5-linked core - dna systems, where the only option for organic cores to lower their sasa is through stacking within terminal bases after disrupting the canonical base pairing . These results indicate that the restrained implicit - solvent md simulations exhibit similar behavior for the hydrophobic organic cores to the explicit - solvent md simulations without restraints . (a) sasa values of hydrophobic cores attached at 3- (black) and 5-ends (red) of dna duplexes over the course of the simulation . Final structures of 3- and 5-linked 3-dna hybrids are shown in (b, c), respectively . The 3-linked core is fully inserted into dna minor groove compared to the 5-linked core (see figure s22 in the supporting information). We next examined the control-[5-c-3] system (figure 3b) to evaluate potential interactions between organic core 3 and the two dna duplex arms that are linked to it . After 96 ns, restrained implicit - solvent md simulations showed that the organic core already minimized its sasa by inserting itself within the minor groove of one dna duplex and stacking with the terminal dna base pairs of the other duplex (figure 5; see also movie s1 in the supporting information). As a result, the control-[5-c-3] system forms an almost continuous 30 bp dna duplex with a single core stacked in the middle . Molecular surface representations of initial (top) and final (below, after 96 ns) structures of the control-[5-c-3] system obtained from md simulations . The two 15 bp dna duplexes are brought together by the hydrophobic nature of 3 . Two md simulations were carried out; see movie s1 in the supporting information for further details . As we have shown in previous md simulations, the hydrophobic nature of core 3 forces it to minimize its exposed surfaces in an aqueous environment through interactions with the dna duplexes . To simulate the cyclic nanostructures that may form in an experiment, we carried out implicit - solvent md simulations (this is a more feasible option compared to expensive explicit - solvent md simulations for these large systems; see detailed discussion in section s7 in the supporting information) for all possible combinations of cyclic dimers (figure 6) as well as tetramer and hexamer structures (figures 7a - b) for the [5-c-3]:[5-c-3] system without any restraints on dna duplexes (see section s6 in the supporting information for more details). A sasa analysis of the cyclic-[5-c-3]:[5-c-3] dimer, tetramer, and hexamer systems shows that the organic cores 3 in these structures can lower their sasa better in higher - order structures (table 3, entries 13). While the average sasa value of the cyclic-[5-c-3]:[5-c-3] dimer is 200, it is reduced to 134 in the tetramer and 122 in the hexamer (table 3). In the cyclic dimer, the organic cores cannot lower their sasa as much due to strain . As shown above for the 3-linked core model system, the organic cores in cyclic dimers can minimize their sasas through interactions with the dna minor grooves, which severely distort the two dna arms (figure 6). On the other hand, the organic cores in the tetramer and hexamer have additional means to minimize their sasas (figures 7c f), such as stacking with the terminal base pairs of the adjacent dna duplex, as shown for the control-[5-c-3] system (figure 5). Schematic representations of initial (top) and lowest sasa (below) cyclic - dimer structures after 91 ns md simulations . That due to the flexible t linkers, dna deformations in (d, e) are not severe compared to (a c) (see table s2 in the supporting information). Molecular surface representations of the initial (left) and final (middle) structures of the cyclic tetramer (a) and hexamer (b) formed from the [5-c-3]:[5-c-3] system (see figures s20 and s21 in the supporting information). The organic cores 3 are represented in green color and are shown interacting with neighboring dna duplexes in different manners: (c) being sandwiched between neighboring dna duplexes via -stacking, (d) fully inserting into the minor groove, (e) inserting into the minor groove in a distorted manner, and (f) -stacking with distorted terminal base pairs of neighboring dna duplexes (the dna distortions are not as severe in comparison to that observed in the cyclic - dimer structures shown in figure 6). Interestingly, md simulations of the cyclic-[5-t3ct3 - 3]:[5-t3ct3 - 3] dimer structure show average sasa values around 119 (table 3). The rmsd s of the dna duplexes are lower than those for the other cyclic - dimer systems that have only organic cores 3, implying that introducing t3 spacers provides additional flexibility that helps to minimize the sasas of the cores without distorting the dna duplexes (table 3 and figure 6d). Consistently, the percentage of hydrogen bond loss in the cyclic-[5-t3ct3 - 3]:[5-t3ct3 - 3] dimer is only 5% while those for the cyclic-[5-c-3]:[5-c-3], cyclic-[3-c-3]:[3-c-3], and cyclic-[5-c-5]:[5-c-5] dimers are over 20% (table 3). These results are in line with our previous work and page - gel experiments, which show exclusive formation of dimer structures in cyclic-[5-t3ct3 - 3]:[5-t3ct3 - 3] (see figure s17 in the supporting information, lane 9). In the absence of the organic cores 3, exclusive cyclic dimer formation was observed for [5-t6 - 3]:[5-t6 - 3], [5-t6 - 5]:[5-t6 - 5], and [5-t3 - 3]:[5-t3 - 3] (see figure s17 in the supporting information, lanes 35). Similarly, the md simulations show no major dna distortions in [5-t6 - 3]:[5-t6 - 3], with 9% hydrogen bond loss (table 3 and figure 6e). One md simulation was carried out for each of the hexamer and tetramer; four independent md simulations were carried out for cyclic - dimer systems but only the lowest rmsd results are shown (see section s6 in the supporting information for more details). In the unrestrained md simulations of the cyclic dimers discussed above, the dna duplexes can be easily distorted due to the conformational stress put on by the organic cores while trying to minimize their sasas (figures 6a c). One consequence of this is excessive fraying of the terminal base pairs in the dna duplex arms of the dimers (figures 6a c), making it difficult to evaluate their relative stabilities . Thus, restrained md simulations (see detailed discussion in section s7 of the supporting information) were carried out where the dna duplex arms of the dimers were constrained in b - form while their lengths were varied over one helix turn (11, 13, 15, 17, 19, 21, and 23 bp, see section s7 in the supporting information for more details). On average, the total sasa values of the organic cores in the [3-c-3]:[3-c-3] systems are lower than those for the [5-c-3]:[5-c-3] and [5-c-5]:[5-c-5] systems (see figure s23 and table s3 in the supporting information), suggesting that the manner in which the cores are attached to the dna duplexes does matter . While constraining the dna duplex arms gave us a starting point to compare the three different dimer systems via md simulations, these structures may be trapped in local minima and do not accurately reflect the energy landscape of the system . Thus, we performed simulated annealing on all of the 21 smdh2 dimers shown in table s4 in the supporting information (see also section s8 in the supporting information). While the dna structures were kept in b - form conformations with watson crick, torsional, and chirality restraints, the temperature of each system was increased to 3000 k and gradually cooled down to 100 k. for each system, 301 simulated annealing md simulations were run sequentially where the starting structure for each run was taken from the final structure of the previous run . This way, each system was allowed to move away from any potential minimum state and sample other regions in phase space . For each dimer, sasa values of the organic cores were calculated from the simulated annealing md simulations for structures having final restraint energies and duplex rmsd values less than 10 kcal / mol and 10, respectively . Within these structures, sasa values that are uniformly lower than those obtained for the restrained md simulations shown earlier can be obtained for each cyclic dimer (see data in table s3 in supporting information and figure 8; see also figures s23 and s24 in the supporting information), suggesting that simulated annealing offers a more self - consistent basis for comparing the dimers . As discussed above, implicit - solvent simulations of smdh2 can yield unphysical structures if no restraints are imposed on dna to keep them in their native b - form conformation . To see the effects of removal of restraints imposed on dna duplexes in these cyclic dimers, the lowest sasa structures were simulated for over 125 ns without restraints . As expected and similar to the results shown in figure 6, the dna duplexes are distorted from the b - form conformation (table s5 in the supporting information). In some cases, one of the dna duplexes in these cyclic dimers is fully unfolded (see table s5 in the supporting information, bp_17 [5-c-5]:[5-c-5]). Furthermore, the% of hydrogen bonds lost increased with the size of the duplex while the sasa of the organic cores decreased suggesting that dna duplexes are more flexible than they actually are (table s5 in the supporting information). Therefore, to study these types of large dna - hybrid systems with the current implicit - solvent models one is required to impose restraints on dna structures to keep them in b - form conformation so that results have physical meaning . Sasa values of the organic cores in smdh2 cyclic dimers as a function of dna length . The black, red, and green trends represent the results for [3-c-3]:[3-c-3], [5-c-3]:[5-c-3], and [5-c-5]:[5-c-5], respectively, where the lowest sasa values for the organic cores were extracted from simulated annealing md simulations with final restraint energies and duplex rmsd values less than 10 kcal / mol and 10, respectively . As shown in figure 8, there is a clear overall trend, which shows that the dimers with organic cores linked at the 3-ends of the dna strands (cyclic-[3-c-3]:[3-c-3]) are much better at minimizing the sasas of their organic cores than the other two dimer systems . There is a higher chance for the organic cores 3 to be inserted into the minor grooves of the flanking dna duplex in cyclic-[3-c-3]:[3-c-3]. As a result, organic cores can lower their sasas more significantly in these systems than would be possible for the cores in the cyclic-[5-c-5]:[5-c-5] systems, which prevent full minor groove insertion of organic cores . For the [5-c-3]:[5-c-3] cyclic dimer, where the organic cores are linked at both 5- and 3-ends of the dna strands, the cores can be inserted into the minor groove only if the dna orientations are favorable . Not surprisingly, the sasa values for this system lie in the middle of the other two . (for a more detailed discussion of this system, please see section s8 in the supporting information) one interesting result shown in figure 8 is the nonmonotonic behavior observed around base pair lengths 11 and 21 . This phenomenon could be due to the dna helical turn, which is approximately 10 base pairs, suggesting a real connection to the physical system . The sasa of organic cores in the 11 base paired cyclic-[5-c-3]:[5-c-3] system is lower than the cyclic-[3-c-3]:[3-c-3] system . After adding 10 base pairs to the dna sequences, which results in dna duplexes with 21 base pairs, a similar result was observed (figure s8 and table s24 in the supporting information). In cyclic-[5-c-3]:[5-c-3] systems with 11 and 21 dna base pairs the organic cores almost fully insert themselves into the dna minor grooves, which provides an explanation for the nonmonotonic behavior observed in these dna sequences (figure s23 in the supporting information). Note that no such behavior has been observed in cyclic-[5-c-5]:[5-c-5] systems (green curve in figure 8). The idea that the hydrophobic organic cores 3 in smdh2 cyclic dimers can be stabilized in aqueous solutions by inserting into the minor groove can be used to explain the results that we reported above for the slow - cooling experiments of 15 bp smdh2 systems (figure 2). In that experiment, the percentage of cyclic dimers formed in [3-c-3]:[3-c-3] is 79%, much higher than those observed for [5-c-3]:[5-c-3] and [5-c-5]:[5-c-5] (43 and 34%, respectively; see figure 2, lanes 3, 6, and 9). This trend closely follows the simulated annealing md simulation results described in figure 8: a higher proportion of dimers can form when the cores are linked to both 3-ends of the dnas because the cores can be better stabilized by inserting into the minor grooves of the two dna arms . In other smdh2 systems where the cores are linked to 5-ends of dnas, other higher - order structures are formed because there is no predominant stabilization mechanism for the core in the dimer structures . To further test the insights obtained from our md calculations, we designed an smdh system where one side of the cyclic dimer had the organic core 3 and the other side had flexible linkers such as t3-c - t3 and t6 (table 2, entries 810). These systems would also allow us to assess the relative importance of minimizing hydrophobicity versus strand - end alignment . If strand - end alignment is of primary importance, cyclic dimers would form predominantly if one of the linkage sites is flexible (i.e., with t3-c - t3 or t6 linkers) enough to alleviate the strain resulting from the rigid core on the other side . However, as shown in figure 9, the nondenaturing page - gel analyses for [5-c-3]:[5-t6 - 3], [5-c-5]:[5-t6 - 5], and [5-c-3]:[5-t3ct3 - 3] systems, both normal and slow - annealing, all afforded a mixture of dimers, tetramers, and hexamers . That all six experiments shown in figure 9 formed a mixture of products instead of only cyclic dimers points to the importance of the organic core not having the freedom to isolate its hydrophobic surface . Moreover, imagej analysis of the gel image showed that [5-c-3]:[5-t6 - 3] gave higher percentages of cyclic dimer (82% dimer, 14% tetramer, slow cooling, figure 9, lanes 2 and 3) compared to [5-c-5]:[5-t6 - 5] (62% dimer, 29% tetramer, slow cooling, figure 9, lanes 4 and 5), which is consistent with the 3-linkage providing better shielding for the hydrophobic organic cores via minor - groove insertion . In effect, the better that the organic cores insert into the minor groove, the larger is the fraction of cyclic dimers formed . This argument is further supported by the results for [5-c-3]:[5-t3ct3 - 3] (figure 9, lanes 6 and 7), both of which show higher cyclic dimer formation than [5-c-5]:[5-t6 - 5]. Nondenaturing page - gel image (6%) of dna assemblies from an smdh component possessing the organic core 3 and the complementary smdh component possessing a flexible linker (either t3-c - t3 or t6) with 5 m total ss - dna concentration . (gel was prepared in 1 tamg buffer (40 mm tris base, 20 mm acetic acid, 7.5 mm mgcl26h2o), and run at 4 c for 2 h under a 200 v field). From left to right: lane 1 = hl5 dna ladder, lane 2 = cyclic-[5-c-3]:[5-t6 - 3] (normal annealing), lane 3 = cyclic-[5-c-3]:[5-t6 - 3] (slow annealing), lane 4 = cyclic-[5-c-5]:[5-t6 - 5] (normal annealing), lane 5 = cyclic-[5-c-5]:[5-t6 - 5] (slow annealing), lane 6 = cyclic-[5-c-3]:[5-t3ct3 - 3] (normal annealing), lane 7 = cyclic-[5-c-3]:[5-t3ct3 - 3] (slow annealing). The strong hydrophobic interactions between cores can also be invoked to explain the exclusive formation of ff dimers in the [3-c-3]:[5-c-5] and [3-c-3]:[5-t6 - 5] systems (table 2, entries 11 and 12; see also figure s18 in the supporting information, lanes 9 and 10). We attribute this behavior to the combination of reduced configurational entropy and increased ion - cloud sharing that occurs when the organic linkages bring the dna duplexes into close proximity . Notably, the [3-c-3]:[5-c-5] ff dimer has a much higher thermal stability, as illustrated by its higher tm, compared to the cyclic-[5-t3 - 3]:[5-t3 - 3] dimer, whose t3 linker is comparable in size to the core c (figure 10). Indeed, the [3-c-3]:[5-c-5] ff dimer is consistently more stable than all the cyclic dimers that we examined (tm = 52.453 c, table 4, cf entry 10 and entries 79). However, when the linker for one side of the ff dimer was changed to a flexible t6 ([3-c-3]:[5-t6 - 5]), the tm was decreased by 4.7 c (table 4, cf entries 10 and 11). Melting profiles for control, cyclic-, and ff - dimer smdh2 assemblies (5 m) in tamg buffer (40 mm tris base, 20 mm acetic acid, 7.5 mm mgcl26h2o). For accurate comparison to ff dimers, only systems that afford cyclic dimers exclusively are listed . For details, see tables 1 and 2 . The aforementioned results are consistent with our previous work, indicating that hydrophobic interactions between the two organic cores (and that between cores and the dna duplexes) can play a major role in determining the thermal properties of the final hybridized systems . Thus, such hydrophobic interactions must be taken into account in the consideration of product distributions in the assembly of organic - linked dna materials . We note that sleiman and co - workers have also observed similar thermal stability enhancements for ff dimers that are analogous to the one reported herein . However, these tm increases were only attributed to allosteric and chelate cooperativities of the dnas; contributions by hydrophobic interactions were not discussed . Comparing to the cyclic dimers, the increases in thermal stabilities for our ff-[3-c-3]:[5-c-5] and ff-[3-c-3]:[5-t6 - 5] dimer systems can be attributed to two factors: (1) hydrophobic interactions between the cores, and (2) effective extension of the dna helix through the organic core / linkers, which was referred by sleiman and co - workers as chelate cooperativity . The first effect is strongly supported by fluorescent studies in similar ff diphenyl acetylene- and 1,3,5-tris(p - ethynylphenyl)benzene - linked dimer systems . The second factor has been ascribed by leumann and co - workers as due to close structural communications between the two linked helical domains . While chelate cooperativity may be important, our thermal data clearly point to hydrophobic interactions between the cores as a major effect . In summary, we have elucidated the effect of linking hydrophobic organic cores to the 3- and 5- ends of the dna components used in the assembly of smdh2 materials . Md simulations show that the organic cores of these building blocks minimize their hydrophobic surfaces by choosing the best stacking pattern possible when they self - assemble in aqueous media . Computational results indicate a high correlation between the linkage type (3 or 5) and the final sasa of organic cores that can be attributed to the extent that the cores can insert into the dna minor grooves in the duplex arms of the resulting smdh2 . While 3-linked organic cores can insert almost perfectly into the minor groove, 5-linked cores can only insert partially, resulting in less - stable dimers (i.e., with higher sasa). These results can be used to explain why higher percentages of cyclic dimers form in smdh2 materials with 3-linked organic cores in comparison to those with 5-linked cores, as observed experimentally for a broad range of structures: smdh2 materials with 5-linked cores are simply less stable in aqueous media due to the inability of the hydrophobic cores to insert completely into the minor groove . Inadequate shielding of the hydrophobic cores then forces these systems to choose other assembly patterns such as higher - order structures (tetramer, hexamer, etc . ), as shown by nondenaturing page gel analysis . Notably, the important role that hydrophobic organic cores play in the assembly of smdh2 hybrids is strongly supported by comparing the thermodynamic stability of several ff and cyclic dimers: ff - dimer systems consistently have tm s that are several degrees higher than analogous cyclic dimers, suggesting that hydrophobic interactions between the cores as a major factor contributing to stability . Together with the strong interactions observed between 3-linked cores and the minor groove in smdh2, this result opens up the possibility of controlling the product distribution in smdh assembly using the hydrophobic nature of the organic cores in conjunction with the different linking modes (3 and 5) to dna strands . Incorporating these design parameters to the synthesis of small molecule - dna hybrids should expand the range of future applications for dna - based hybrid materials.
Poor aqueous solubility remains a major cause of attrition in the drug development process . Despite theoretical developments, the solubility of druglike molecules still eludes truly quantitative computation . In recent work, we have shown that accurate first - principles calculation is now becoming possible, provided that both the crystalline and solution phases are described by accurate theoretical models . Before this, energy terms from a computed thermodynamic cycle (see figure 1) had been used as descriptors in a multilinear regression model for intrinsic solubility, delivering accuracy much better than from direct computation and comparable with the leading informatics approaches . Since then, sophisticated machine learning techniques have been applied to many problems in the chemical sciences, while, as we have shown, the accuracy of direct computation of hydration energies and solubilities has improved significantly . This led us to revisit the idea of hybrid informatics - theoretical models for solubility . Cheminformatics methods have seen widespread use for property prediction, particularly in the pharmaceutical industry where they have been applied to; aqueous solubility, melting point, boiling point, log p (where p is the partition coefficient between octanol and water), binding affinities, and toxicology predictions . Such methods are usually much quicker than pure chemical theory calculations, making high throughput virtual screening (htvs) a possibility . Some methods have become accessible and easy - to - use web - based tools . However, informatics methods suffer from the difficulty of decomposing the results into intuitive, physically meaningful understanding and cannot reflect the physical details of the system . To understand the underlying physics and chemistry, it is necessary to carry out an atomistic physics - based calculation . The exact nature of the theory varies between these methods and the phase being studied . Crystal structures are often modeled using one of the lattice energy minimizing simulation methods, plane - wave density functional theory (dft) methods, or periodic dft using atom - centered basis sets . The simulation methods often contain empirical parameters, which lowers the cost of these methods significantly, compared to dft . Popular solution - phase models include atomistic simulation methods based on molecular mechanics and dynamics, quantum - mechanical implicit solvation methods (such as the polarizable continuum model (pcm)), and hybrid models (such as the classical statistical mechanics - based reference interaction site model (rism) or hybrid quantum mechanics / molecular mechanics (qm / mm) methods). These methods have the inherent problem for industrial and drug discovery applications of being significantly more computationally intensive than cheminformatics models, which makes high - throughput computation infeasible . The closest thing to an exception among contemporary theoretical models may be 1d rism, which requires only a few minutes of calculation time per compound and has been previously combined with cheminformatics to build the 1d - rism / sdc method . By combining lower levels of theoretical chemistry with cheminformatics, we hope to produce results in good agreement with experiment, but at a lower cost than higher - level theoretical methods, and with higher accuracy than using cheminformatics descriptors alone . A set of 100 broadly druglike organic molecules was assembled with the prerequisites that each molecule should have an available crystal structure in the cambridge structural database (csd) and a well - documented aqueous intrinsic solubility in the literature . Where possible, we prefer experimental solubilities obtained with the cheqsol method, which has been shown to give reproducible results with only small random errors . The possibility of significant systematic errors between different experimental methodologies remains an issue and may possibly limit the accuracy with which modeling - based studies can be validated . A total of 122 potentially useful cheqsol solubilities were obtained from the two solubility challenge papers and downloaded from the web . While noting that several corrections had previously been made, we also corrected or disambiguated the following names: amitriptyline, 5-bromogramine, 5,5-diphenylhydantoin, 4-hydroxybenzoic acid, nortriptyline, and phenanthroline . Of the 122 compounds, 38 had corresponding crystal structures and could be included in our dls-100 dataset . Where a choice existed, we selected the solubility and crystal structure of the least soluble and, therefore, most stable polymorph . For druglike compounds with known crystal structures, one further cheqsol solubility was available from palmer et al . And two from narasimham et al . Crystal structures were selected on the basis of stability, preferring the polymorph with the lowest literature solubility or the lowest lattice energy according to our computations where polymorph - specific experimental information was not available . We also applied the additional pragmatic selection criterion that the asymmetric unit cell should contain only one molecule . Once structures were identified, they were downloaded in either the shelx format (.res) or csd legacy format (.dat). We chose to use chemistry development kit (cdk) molecular descriptors in this study, because these descriptors do not require proprietary software and are applicable to solubility prediction . The cdk is an open source cheminformatics java library . In order to use the cdk molecular descriptors we thus decided to use one principal source for smiles records, selecting the well - annotated database chemspider . Since we are modeling intrinsic solubility, we wish to describe the neutral form of the druglike compound . This remains the case even if a protonated or deprotonated charged form dominates at neutral ph or across the ph range of the cheqsol (or other) experiment . To obtain a smiles string for each molecule in the dls-100 dataset, we wrote a taverna workflow, which uses web services provided by the chemspider database . The workflow is freely available on the myexperiment repository at the following reference . In five cases thus, we instead took the smiles from the solubility challenge web site for cimetidine, pindolol, and phenobarbital, and from wikipedia for griseofulvin and glipizide . Using the resulting 100 smiles, we initially calculated all 268 available nonprotein cdk descriptors for each compound . We found that 145 of these descriptors were either undefined for 2d structures, or had the same value for all 100 compounds; their deletion left 123 remaining descriptors . We took experimentally determined crystal structures of the compounds in our dls-100 dataset as the initial input to our calculations . Dmacrys, a periodic lattice simulation program, was used to perform the crystal structure minimizations and calculate vibrational contributions arising from the crystal . Dmacrys works in conjunction with the gdma2 and gausssian 09 (g09) programs . The output of these calculations gives us the enthalpy of sublimation and crystal portion of the entropy of sublimation . The selected crystal structures were input into dmacrys, which was used to standardize the covalent bond lengths between hydrogens and heavy atoms, as the experimentally determined bond lengths are not accurate, because of the uncertainty in the hydrogen positions obtained by x - ray diffraction, before any calculations were run . Electrostatic interactions were calculated by multipole expansions (obtained using gdma2) of molecular charge distributions calculated at the mp2/6 - 31 g * * level using g09 . Dmacrys carries out a rigid - body minimization of the crystal structure, hence arriving at minimized lattice energies . This lattice energy can be converted to an enthalpy of sublimation by the following formula: enthalpy of sublimation: 1where ulatt is the lattice energy (energy of the crystal assuming the crystal is static and at 0 k relative to infinitely separated molecules) and the 2rt term arises from lattice vibrational energy . The entropy of sublimation was calculated by: entropy of sublimation: 2where srot is the rotational entropy in the gas phase and strans is the entropy of translation in the gas phase . The use of eq 3 makes these assumptions: (i) the rotational and translational entropy of the crystal is minimal, (ii) there is no change in electronic entropy between phases, and (iii) the intramolecular entropy is constant between the two phases . The crystal entropy is calculated by locating the frequencies of the phonon normal modes (lattice vibrations) at the gamma point . This is achieved using lattice dynamics, the results of which are used to calculate the helmholtz free energy (see eqs s2 and s3 in the supporting information). The coordinates of a single molecule were extracted from the minimized lattice and used as input for the gaseous optimization with g09 . Optimizations were carried out at the m06 - 2x and hf levels of theory with a 6 - 31 g * basis set . All solution - phase calculations were carried out with g09 using the self - consistent reaction field (scrf) protocol . We selected the smd (solvation model based on density) implicit solvent model based on previous work . Although rism yielded more - accurate absolute hydration energies than smd in our recent work, smd generated a higher correlation coefficient against experimental results for hydration free energy prediction (r = 0.97 vs r = 0.93) given the parametrized nature of our present model, correlation is more important than absolute agreement, and, hence, smd is a suitable solvation model . Solution - phase calculations were carried out with the same methodologies as used in the gas - phase calculations, m06 - 2x/6 - 31 g * and hf/6 - 31g. Geometry optimization was again carried out, this time taking the gas - phase optimized structure as the starting point . Smd solves for the free energy of solution (ghyd) as a sum of the electrostatic contributions and nonelectrostatic contributions . The electrostatic contributions are calculated by the solution of the nonhomogeneous poisson equation; this equation is a second - order differential equation linking the electrostatic potential, dielectric constant, and charge distribution . The nonelectrostatic contributions of cavitation, dispersion, and solvent structure are calculated as a sum of atomic and molecular contributions using parameters inherent to the smd method . Smd has been shown to provide significant improvements over some other implicit solvent models for datasets containing molecules similar to those used in this study . The hydration free energy is given by eq 4,gibbs free energy of hydration: 4where esolution is the total energy of the system in the smd solvation model and egaseous is the total energy of the system in a vacuum . This scheme typically takes a few hours of calculation time per molecule on two 2.8-ghz 6-core intel xeon x5660 processors . Sublimation energies were calculated in the 1 atm standard state, which is the conventional standard for experimental sublimation energies to be quoted . However, solvation free energies are usually quoted in the ben - naim standard state of 1 mol / l . In this work, g corresponds to the 1 atm standard state, while g corresponds to the ben - naim 1 mol / l standard state (see figure 2). The difference between these two standard states is a constant energy value of 1.89 kcal / mol (7.91 kj / mol). In this work, we calculate the sublimation free energy in the 1 atm standard state and then apply the correction to 1 mol / l in order to be consistent with the hydration free energy calculations; hence, gsolu is in the 1 mol / l standard state for all predictions in this work . The presence of an asterisk () denotes that the values refer to the standard state of 1 our final solution free - energy prediction is then given as the sum of the predicted sublimation and hydration free energies: gibbs free energy of solution: 5 therefore, we have two predictions for each molecule: the first method couples dmacrys with g09 and the smd solvation model at the hf/6 - 31 g level of theory . The second method is dmacrys coupled with g09 and the smd solvation model at the m06 - 2x/6 - 31 g * level of theory . This will be referred to as smd(m06 - 2x). For convenience of comparison with experimental values of solubility, we convert the free energy of solution to log s values, and all experimental solubility values to log s values:6here, r is the universal gas constant and t is the absolute temperature (in kelvin). The conversion of experimental solubility to log s can be found in the supporting information (eq s7). Values for the full dls-100 dataset, including smiles and inchi, can be found in the supporting information (see zip file and dataset). To model the data, we use linear and machine learning regression models: partial least - squares regression, random forest and support vector regression . For reporting the predictive accuracy of these models, we averaged the rmse of log s over a 10-fold cross - validation of the dls-100 dataset . The cross - validation fulfils two purposes in this study: parameter optimization and evaluation of the accuracy of the models on unseen data . To ensure that each test fold of data is truly unseen, the parameter optimization is carried out in a separate layer of cross - validation within the training folds, as we will discuss below . In order to avoid overfitting, the use of multivariate data presents a danger of overfitting machine learning regression models; moreover, redundancy of attributes and correlation within the data add to the risk of reaching misleading conclusions . One is the commonly used standardization method of variable scaling, equalizing the distributions of the variables by normalizing the mean and standard deviation of each column (variable). The advantage of using this method is that it equalizes the prior importance of all the attributes . The second normalization method is principal component analysis (pca), transforming the data into a smaller subspace where the new variables are uncorrelated with each other . The pca data transformation method deals with the redundancy of the data, and places emphasis on the variance of the data . The ability of each principal component to explain the data is measured according to the variance accounted for . Third, we have also fitted each model on the nonpreprocessed raw dataset, for comparison with the results of the two different scaling methods . In this section, a summary of the regression models are presented; detailed explanations can be found in the supporting information . The partial least squares regression (plsr) model design is appropriate in a situation where there is no limit to the x variables or predictors, or where the sample size is small . Moreover, the plsr model is also beneficial for analyzing strongly colinear and noisy data . The goal of a plsr model is to predict the output variable y from the input variables x and to describe the structure of x. for this, plsr finds a set of components from x that are relevant to y; these components are known as latent variables . The intention of plsr is to capture the information in the x - variables that is most useful to predict y. a graphical representation is supplied in the supporting information figure s1(a). Random forest (rf), a method for classification and regression analysis, has very attractive properties that have previously been found to improve the prediction of quantitative structure activity relationship (qsar) data . An ensemble of many decision trees constitutes a random forest, and each is tree constructed using the classification and regression trees (cart) algorithm . The rf method is efficient in handling high - dimensional data sets and is tolerant of redundant descriptors . The main idea in support vector regression (svr) is to minimize the risk factor based on the structural risk minimization from structure theory, to obtain a good generalization of the limited patterns available in the given data . First, the given data d are mapped onto a higher dimensional feature space, using the kernel function k(xi, xj) and then a predictive function is computed on a subset of support vectors . Here, we have used the radial basis kernel function (eq 7) to map the data onto a higher dimensional space . A graphical representation is supplied in the supporting information (figure s1(b)).svr mapping on radial basis kernel function: 7 to evaluate the performance of various machine learning models, we report two statistics: the root mean squared estimate (rmse) and squared pearson correlation coefficient r (not to be confused with the coefficient of determination). Formulas for these are given in the supporting information (eq s5). We have also assessed statistical significance using menke and martinez s method, which we have used previously for similar analysis (see supporting information (eq s6, tables s3s9 for r, and boxes s1s3) for statistical significance). We also analyzed the variable importance for the rf method (see table s17 in the supporting information). Variable importance was calculated in the cart program as implemented in r. in order to compute and compare the performance of the various regression models, we consider rmse scores averaged over a 10-fold cross - validation . In the 10-fold cross - validation, the dataset is randomly split into 10 partitions, where the training set consists of 90% of the data and the test set consists of 10% of the data . The predictivity on the test fold is considered as an external measure to compute the accuracy of the fitted model . The entire process is repeated 10 times in order to cover the entire dataset, with each fold forming the test set on one occasion, and we record the average rmse . The complete design of the workflow is represented in a flowchart (scheme 2); similar workflows have been used for classification in other studies . The complete workflow of this analysis was written in r using the caret package; all scripts are available in the supporting information . In out - of - bag validation, one evaluates the performance of the model by separating training and test data through bootstrap sampling; this is convenient only for the rf method . It is not appropriate to compare rf out - of - bag predictions with other models such as pls and svr, which are not based on bootstrap sampling . So, we used 10-fold cross - validation to evaluate the performance of our various models . For each model, we use 90% of the total data designated as the training set in order to find the optimum values for these parameters . We selected a range incorporating 20 different possible values for each model parameter, in order to select its best value . For each parameter, a further level of 10-fold cross - validation is carried out in order to retrieve the rmse of the models using each possible parameter value . Here, the training portion of 90% of the original data is further split into 10 new folds of 9%, with nine (81% of the original data) being used to build each model and one (9%) as an internal validation; this process of model building and internal validation is repeated to predict each of the 10 possible internal validation folds . This internal cross - validation step is repeated 20 times, once for each possible value of the parameter being assessed . Then, based on the value giving the lowest average rmse score in the internal validation folds, the optimum parameter value is selected . Finally, the model is fitted on the complete training set of 90% of the original data using the selected parameter values . The given 90%:10% split of the data into training and test sets was used to fit the final model for each fold of the main 10-fold cross - validation, once the optimum parameter values have been selected . The average rmse and r values over the 10 folds were considered in order to compare the usefulness of different descriptor sets and to evaluate the performance of the fitted models . The full dls-100 dataset, with the experimental log s values, can be found as supporting information or downloaded from the mitchell group web server (http://chemistry.st-andrews.ac.uk/staff/jbom/group/informatics_solubility.html; see the supporting information (csv_smiles_si.csv and table s1)), which is consistent with the excellent suggestions from walters . The dataset includes csd refcodes, chemspider numbers, smiles, experimental log s values and inchi for all molecules . The log s values in this work come from refs (2, 16, 18, and 19). Where possible, we have selected data obtained from the cheqsol method; where this was not available, we have selected reliable sources using different determination techniques . A good solubility prediction can be considered as a prediction of approximately the same error as that of the experiment . The experimental values have been shown in a number of previous papers to vary considerably . Here, we consider the experimental accuracy limit to be between 0.6 and 1 log s unit (where 1 log s unit represents 5.7 kj / mol at 298 k). Previous work has reported the experimental error in solubility prediction to be as great as 1.5 log s units and, on average, the error to be at least 0.6 log s units . In 2006, dearden noted, as was later reiterated in the solubility challenge, that models with rmse predictions of <0.5 log s units are likely to be overfitted . For a prediction to be useful, it must have an rmse within the standard deviation of the experimental data; otherwise, a trivial prediction using the mean of the experimental data is a more accurate prediction of the log s value . For the dls-100 dataset, first, a purely theoretical prediction, in which no machine learning is used and where predictions are made using only physics - based calculations . Second, theoretical energies are used as the sole descriptors in machine learning models . Third, cheminformatics descriptors, calculated using the cdk, are used as the sole input to machine learning methods . Finally, cheminformatics descriptors and theoretically computed energies are combined as input to machine learning methods . For each of these methods, we present the results and discussion, with comparison between the methods made on the basis of rmse and r (correlation coefficients for cheminformatics and combined models can be found in the supporting information (tables s3s9); rmse values can be found in the supporting information (tables s10s16)). In addition to these results, we have replicated the solubility challenge using 2d molecular descriptors alone . The theoretical methodologies described earlier utilize a thermodynamic cycle to access the free energy of solution . Table 1 shows the r correlation coefficient and the rmse for the predictions made by these methods . Chart 1 shows the linear fit to the data from the smd(hf) method, which has the lower rmse and the higher r correlation coefficient of the two purely theoretical methods . The rmse for the smd(hf) method is nearly three times the suggested criterion of 1 log s unit of error . The situation is even worse for the smd(m06 - 2x) method for which the rmse is just over four times this criterion (see charts s4s6 in the supporting information). Both methods produce results outside the useful prediction criterion of 1.71 log s units . From these results first, it is clear that the given methodologies do not adequately quantify the physics occurring in the solution process (i.e., solid to solution). Second, we can conclude that, if it is possible to explain the underlying structure of these data using a general model, based on the predicted log s values, such a model will be inherently nonlinear . Compared with our previous work, in which theoretical models provided a good prediction of log s, our theoretical methodology here differs only marginally, in the use of mp2 multipoles, and still produces good results (see supporting information (chart s1 and table s2)) for the same 25 molecules in this work (dataset dls-25). The predictions for the additional 75 molecules alone show worse predictions than for the full 100-molecule set presented above (see charts s2 and s3 in the supporting information). The additional 75 molecules therefore appear to form a more difficult dataset to predict . It is likely that improved results can be obtained from purely theoretical calculations, if some of the approximations made here are improved; for example, improved modeling of the solvated phase to more accurately describe the solvent and its effects on the solute could increase accuracy . Also, we note that the intramolecular degrees of freedom are neglected in the dmacrys calculations, and further assumptions are made by using eqs 2 and 3 in the methods section . We subsequently applied machine learning methods to the theoretical energies in order to carry out nonlinear regression analysis . The average rmse scores over 10-fold cross - validation (see the methods section for details) is represented as two - dimensional (2-d) column charts (see charts 2 and 6). Different grayscale column bars represent the different machine learning methods used in this study . The standard deviation is shown as an error bar (black line). The use of the calculated energies as descriptors in the machine learning models yields considerably improved results, compared to those from the predictions made without machine learning . The results now, while still missing the 1 log s unit error criterion, do make useful predictions in which the rmse is within the standard deviation of the experimental data (1.71 log s units). The rf and svr models produce notably better results than pls . Charts 2 and 3 show that the method minimizing the rmse (1.21 log s units) is rf with hf when scaled with pca . An additional point of interest is that the chemical descriptors alone using rf or svr can provide a marginally better prediction of log s than the machine learning methods with only the energies as descriptors . In particular, we noticed that fitting the rf model on data that are scaled to a given mean and standard deviation produces a statistically significant improvement in its prediction with cheminformatics descriptors alone rather than theoretical energies (see the supporting information (boxes s1s3)). In all other cases, this suggests that slightly more useful information about the molecules log s values is conveyed by the cheminformatics descriptors than by the theoretical energies alone (see chart 4). When the descriptors and energies are combined as input for the machine learning methods, we obtain results that are generally only very slightly better than those obtained from cheminformatics descriptors alone . This implies that the theoretical energies contain very little extra useful information not already present in the descriptors . The joint results do present a statistically significant improvement for pls and rf, once scaled by the mean / standard deviation, compared to those for the theoretical energies alone . In light of this, and given that the descriptors alone produce a marginally improved result compared to chemical theory, it is fair to say the cheminformatics descriptors are seen to contain a modest amount of additional information not incorporated in the theoretical energy terms . This suggests that the 123 descriptors of the cheminformatics descriptors and the 10 theoretical energy descriptors convey similar information, with only a small amount of additional information being conveyed by adding the descriptors to the energies and almost no information gained by adding the energies to the set of descriptors . Interestingly, the best result in terms of rmse is from the descriptors with the m06 - 2x energies, which, on their own, produced the worse of the two pure theory results in this work (see charts 5 and 6). The rf model performs particularly well over all descriptor sets, even without any type of scaling, the best rmse result being only 0.13 units outside the 1 log s unit target . The best single prediction, in terms of the rmse, was made by the pls model, using descriptors and the m06 - 2x energies scaled by the standard deviation and the mean, with an rmse of 1.11 0.04 log s units . All of these methods make predictions inside the standard deviation of the experimental data; therefore, all of the predictions are useful . We also note that the rf model shows small but statistically significant improvements with all scaling methods (using the theoretical energies and cheminformatics descriptors combined) when compared to some models trained on the theoretical energies only (see supporting information (boxes s1s3)). This is the only model to show such improvements with all scaling methods in the present work . We analyzed the relative variable importance (see table s17 in the supporting information) and found that x log p (from ref (57)) was consistently rated as the most important feature . X log p is a computed estimate of the base-10 logarithm of the octanol: water partition coefficient (the ratio of concentrations of solute solvated in the two different solvents). This has been seen in many previous studies and is not so surprising given that it provides information specifically about the solvated phase.x log p uses an atom additive model for the prediction of log p. in the supporting information, we include tables (table s17 in the supporting information) displaying the 10 most important descriptors; here, we will briefly comment upon these . We find kier and hall s path and chain indices to be of importance; these quantify the degree of bonding to heavy atoms within a given path or chain length . Broto autocorrelation, which describes the charge and mass distribution along a given path length, is found in the top 10 . Finally, we also note randic s weighted path descriptors, which are used to account for molecular branching . Once the theoretical energies are added to the descriptor set, the free energies of hydration and solution are ranked in the top 10, along with the theoretical log s prediction . Explanations of the molecular descriptors used in this work can be found in ref (61). One may expect that molecular branching would play an important role, because it gives information on the extent and flexibility of the molecule, hence contributing some entropic information . Coupling this descriptor with the kier hall descriptors, information can be acquired on the composition of such chains, in terms of heavy atoms . Again, here, information is imparted concerning the distribution of heavy atoms and electronic factors . For example, the degree of charge separation across a molecule and the localization of charges are important factors in determining particularly enthalpic but also entropic contributions . The theoretical energies in the top 10 are all closely related quantities; it is not surprising that the (purely theoretical) prediction of log s is found in the top 10: since this is the quantity we are trying to predict, it is expected to provide sufficient information to the model to be found in the top 10 . The free energies of solution and hydration provide direct information from electronic structure theory and statistical thermodynamics on the interactions of a given molecule, in a given conformation, within its environment, and on the energetics of phase transitions . As a benchmark, we also present our method s predictions of the solubility challenge set based solely on cheminformatics descriptors (see table 2). As suitable crystal structures are not available for all molecules in the solubility challenge, we could not calculate the theoretical energies . Tables 2 and 3, and chart 7, demonstrate that our method can make predictions for the solubility challenge dataset within the coveted 1 log s unit rmse error and, in fact, makes predictions that are consistent with some commercially available methods and deep - learning methods . A recent publication reported rmse scores of 0.95 log s units for the commercially available package mlr - sc and 0.90 log s units for a deep - learning method . However, these results are not directly comparable with ours, for two reasons . First, our results have been calculated for a 10-fold cross - validation and for the canonical training: test split (see tables 2 and 3). Second, the deep - learning result (rmse = 0.90) given by lusci et al . Is contingent on correcting eight putative errors in the cheqsol solubility data, the most substantial of which is for indomethacin, a compound that has been shown to hydrolyze under alkaline conditions . While we have corrected names and smiles for the solubility challenge set, we have not adjusted any solubility values therein . It is also reasonable to suggest that, using the solubility challenge set as a benchmark, our 100-molecule set could be considered as a, given the improved prediction offered by our method when the solubility challenge set is used instead . Our current work shows that accurate solution free energies are not calculable via the simple theoretical procedure that we present here . A significant portion of the important physics in the solution process is not captured using the approximate methodologies that we utilize in this work . This reaffirms that, currently, qspr methodologies are the most - accurate and time - efficient methods for accurate solution free energy predictions . In addition, we show that state - of - the - art machine learning methods, with a modest number of cheminformatics descriptors, are capable of making solution free - energy predictions that are consistent with those of commercially available programs and newer deep - learning approaches . Here, theoretical energies and cheminformatics descriptors are generally shown to not be complementary for such predictions . Since both sets of descriptors (theoretical energies and cheminformatics descriptors) produce a similar level of accuracy when used alone in the machine learning methods, and little improvement is seen when they are combined, we can conclude that the information conveyed is of a similar nature and that the theoretical energies are, for this reason, a more efficient form of information storage, as 10 descriptors contain equivalent information to 123 molecular descriptors . However, in terms of time, the molecular descriptors are much less expensive to calculate and their use is therefore more time - efficient . Additionally, we note that the rf method has produced promising predictions in this work, with relatively low rmse . This method has consistently produced good results and would be our recommended method to make solubility predictions.
The national cooperative dialysis study, the first randomised controlled study of dialysis dose, defined an adequacy threshold for end - stage chronic kidney disease patients receiving chronic haemodialysis based on the dialyser clearance of urea, a small solute marker of nitrogen turnover, which was defined in terms of a dimensionless parameter known as the normalised urea clearance, or kt / v (k, dialyzer urea clearance; t, dialysis session duration; and v, urea volume distribution). Below a sessional threshold kt / v of 0.9 for standard thrice - weekly schedules, complication - free survival was compromised within months . Subsequent observational studies suggested that higher doses resulted in improved longer - term outcomes, and by consensus the minimum target kt / v was raised to 1.2 . A subsequent prospective randomised controlled study, the hemodialysis (hemo) study, reported that higher doses did not appear to further improve outcome . However, subgroup analysis suggested that women may benefit from higher kt / v doses, fuelling suggestions that using standard kt / v targets to prescribe dialysis may lead to under - dosing in women and small men . These studies suggest that, for standard thrice - weekly therapy, medium - term survival (measured in months) is dependent on achieving a minimum level of small solute removal, as defined by the national cooperative dialysis study . Just as the amount of dialysis delivered to patients with end - stage chronic kidney disease is important in determining survival, it was reported that the dose of intermittent haemodialysis or continuous renal replacement therapy (crrt) was also important in determining survival in patients with acute kidney injury (aki) [8 - 10], although this was not a universal finding . As patients with aki continue to have high mortality, and evidence - based clinical management is somewhat limited, two prospective multicentre trials were designed to investigate the effect of dose of renal replacement therapy on outcome in patients with aki . The veterans affairs / national institutes of health (va / nih) study essentially randomised patients to initially receive either an intensive or less intensive dose of intermittent haemodialysis, or an intensive or less intensive dose of crrt, depending upon severity of illness at the time of randomisation . (during the course of the study patients were switched between treatment modalities according to haemodynamic stability .) During haemofiltration, it is assumed that urea is effectively cleared (to the extent that the concentration in the effluent ultrafiltrate is equal to that of plasma water) so that urea clearance can simply be assessed by the total ultrafiltration volume achieved . In this study, more intensive renal replacement therapy did not show any survival advantage for either the intermittent haemodialysis or crrt groups . However, the minimum haemodialysis target kt / v of 1.2 was somewhat higher than that typically prescribed for patients with aki by the recruiting centres . In addition, there was no survival advantage for the haemofiltration cohort compared to those treated by dialysis . Haemofiltration clears solutes primarily by convection, thus removing a larger spectrum of solutes than haemodialysis, which predominantly clears small water soluble solutes by diffusion . The second study, the renal (randomised evaluation of normal versus augmented level of renal replacement therapy in icu) study, assessed the effect of an augmented dose of crrt (an ultrafiltration rate of 40 ml / kg / h versus 25 ml / kg / h). Once again, this study failed to show any significant effect of dose of convective renal replacement therapy on patient outcomes, although the delivered dosages were less than that prescribed and both small patients (<60 kg) and very heavy patients (> 120 kg) were excluded . Although urea can dissociate to cyanate in plasma water and then form carbamylated products in a reversible fashion, with some analogy to glycosylation, it would appear that toxicity from the accumulation of small nitrogenous solutes is not the major determinant of short - term outcome (days to weeks) in patients with aki . Aki frequently occurs in the setting of multiple organ failure, and mortality remains high, with patient outcome typically dependent upon the severity of the underlying condition and associated co - morbidities . The replacement of organ function may play a critical short - term role in maintaining life in patients already destined by other factors to have the potential to recover . However, urea clearance is only one component of renal replacement therapies . For example, failure to correct persistent volume overload is associated with not only increased post - surgical morbidity, but also increased risk of aki and mortality . Thus, for patients with aki, the adequate removal of even smaller moieties than urea is the principal determinant of the adequacy of renal replacement . These moieties are the neglected uraemic toxins, including potassium, sodium, hydrogen ions and water (figure 1). The consequences of the accumulation of these moieties, hyperkalaemia, pulmonary oedema, and acidosis, may be lethal in minutes . Toxins play a role over time, and as such may require different clinical management strategies . Hemo, hemodialysis; ncds, national cooperative dialysis study; renal, randomised evaluation of normal versus augmented level of renal replacement therapy in icu; va / nih, veterans affairs / national institutes of health . Although the delivery of higher doses of haemofiltration or more frequent haemodialysis did not improve overall outcome, higher volume crrt exchange cycles and more frequent haemodialysis treatments will help correct acidosis, and may be appropriate during the initial resuscitation phase of aki . Correction of volume overload may help explain the positive findings and improved clinical outcomes reported from some of the earlier trials of increased dose of renal support, compared to the more recent va / nih and renal studies, which had similar fluid balance targets.
Carcinoma thyroid, although the most frequent malignancy of the endocrine system, is a rare disease . It accounts for about 1% of all human cancers, with the prevalence in women (59 in 100,000) higher as compared to that in men (24 of 100,000). Differentiated thyroid cancer (dtc) is a slow - growing and treatable disease with good prognosis . However, about 2040% of patients with dtc have recurrent disease and about 5% have distant metastases at presentation . Until recently, treatment of recurrent and metastatic disease consisted of surgery (when feasible), thyroid stimulating hormone (tsh) suppressive therapy with levothyroxine (t4), and radioiodine (i-131) treatment when radioiodine (ri) uptake is present in neoplastic foci . During tumor progression, cellular dedifferentiation occurs in up to 30% of cases, and is commonly accompanied by more aggressive growth, metastatic spread and loss of iodine uptake owing to decreased expression of sodium / iodide symporter (nis). Tsh receptor, whose presence indicates a good prognosis, is lost in such dedifferentiated tumors, rendering tsh suppression therapy ineffective . Patients with dedifferentiated thyroid cancer foci continue to have a much worse prognosis than those with i-131 uptake and lack adequate therapeutic options . Metastatectomy is useful only in few patients who have surgically approachable and solitary / restricted number of sites . While external radiation therapy can palliate metastatic symptoms, it rarely leads to remission . Conventional chemotherapy, i.e., doxorubicin alone or with cisplatin, is very toxic and provides <20% rate of mostly very transient, partial responses . In recent years, research has focused on targeted approaches addressing the pathological characteristics of ri non - avid thyroid carcinoma . It is known that inability to take up i-131 is associated with poor differentiation and increased tumor grade . So far, p53 mutation is the only genetic change clearly shown to correlate with poor differentiation or lacking differentiation . Some recent reports show a significant correlation between the presence of initiating braf mutation and poorer outcome of dtc or loss of function of dtc metastases . The vitamin a (retinol)-derived retinoic acids (ras) are important regulators of a diverse spectrum of physiological processes, including cell proliferation, differentiation, morphogenesis, angiogenesis, and apoptosis . Retinoids inhibit tumor growth and exert several redifferentiating effects: induction of 5-deiodinase, increased expression of nis mrna and of the differentiation marker, alkaline phosphatase, or decreased expression of cd97, which is highly expressed in anaplastic thyroid carcinoma, as well as stimulation of intercellular adhesion molecule-1 synthesis . Experimental studies have shown that ras may increase the expression of nis, type i 5-deiodinase, intercellular adhesion molecule-1 (icam-1) and thyroglobulin (tg), which were known to be decreased or even lost in thyroid cancer cells. [15161820] the results of a few early clinical pilot trials demonstrated that ra may restore ri uptake and decrease the tumor size. [2124] however, the clinical outcomes of 13-cis ra in subsequent studies were disappointing as <20% of patients showed i-131 uptake after ra pre - treatment . We wanted to assess the role of all - trans retinoic acid (atra) as a rediffrentiating and antineoplastic agent in dedifferentiated thyroid tumor patients in our institute . Dtc of follicular cell originserum tg level 10 ng / ml with tsh stimulationri whole - body scintigraphy (wbs) showing no or therapeutically insignificant (<0.3%) uptakeinitial therapy must have included total or near - total thyroidectomy and ri ablation therapy dtc of follicular cell origin serum tg level 10 ng / ml with tsh stimulation ri whole - body scintigraphy (wbs) showing no or therapeutically insignificant (<0.3%) uptake initial therapy must have included total or near - total thyroidectomy and ri ablation therapy for female patients, the following criteria were additionally applied: negative pregnancy test within 1 week of enrollmentpracticing adequate birth control methods negative pregnancy test within 1 week of enrollment practicing adequate birth control methods coexisting second malignancypregnancyabnormal liver function tests, renal function tests, and lipid profile coexisting second malignancy abnormal liver function tests, renal function tests, and lipid profile thirteen cases of dtc with raised tg and/or clinically evident disease and negative wbs were treated with ra (vesanoid, atra) in a dose of 1.5 mg / kg / day for a period ranging between 1.5 and 18 months [tables 1 and 2]. Details of patients and redifferentiation therapy patient characteristics a complete response to the drug was defined as a decrease in serum tg levels to less than 10 ng / ml of thyroid hormone with no evidence of disease on clinical evaluation . A partial response was defined as more than 30% decrease in tg level with no evidence of progression on clinical evaluation . Stable disease was defined as no / 30% change in tg level with no evidence of progression on clinical evaluation . Progressive disease was defined as an increase in serum tg level of> 30% or progression on clinical evaluation or both . Patients were monitored by physical examination, laboratory tests and imaging studies where feasible, on their follow - up visits . Dtc of follicular cell originserum tg level 10 ng / ml with tsh stimulationri whole - body scintigraphy (wbs) showing no or therapeutically insignificant (<0.3%) uptakeinitial therapy must have included total or near - total thyroidectomy and ri ablation therapy dtc of follicular cell origin serum tg level 10 ng / ml with tsh stimulation ri whole - body scintigraphy (wbs) showing no or therapeutically insignificant (<0.3%) uptake initial therapy must have included total or near - total thyroidectomy and ri ablation therapy for female patients, the following criteria were additionally applied: negative pregnancy test within 1 week of enrollmentpracticing adequate birth control methods negative pregnancy test within 1 week of enrollment practicing adequate birth control methods coexisting second malignancypregnancyabnormal liver function tests, renal function tests, and lipid profile coexisting second malignancy abnormal liver function tests, renal function tests, and lipid profile thirteen cases of dtc with raised tg and/or clinically evident disease and negative wbs were treated with ra (vesanoid, atra) in a dose of 1.5 mg / kg / day for a period ranging between 1.5 and 18 months [tables 1 and 2]. Details of patients and redifferentiation therapy patient characteristics a complete response to the drug was defined as a decrease in serum tg levels to less than 10 ng / ml of thyroid hormone with no evidence of disease on clinical evaluation . A partial response was defined as more than 30% decrease in tg level with no evidence of progression on clinical evaluation . Stable disease was defined as no / 30% change in tg level with no evidence of progression on clinical evaluation . Progressive disease was defined as an increase in serum tg level of> 30% or progression on clinical evaluation or both . Patients were monitored by physical examination, laboratory tests and imaging studies where feasible, on their follow - up visits . Age of the patients was between 18 and 65 years with a median of 49 years . Ten patients had papillary while two had follicular and one patient had mixed papillary and follicular thyroid cancer . Tg decreased in 2 patients and increased in 11 patients at the end of therapy . Ri uptake was demonstrable in six patients, though faintly, while seven cases showed no uptake [figures 1 and 2]. Based on clinical and/or biochemical parameters, four patients had progressive disease, eight had stable disease and one patient showed partial response . Of the six patients with ri uptake, three had biochemical progression and the other three had stable disease . I-131 whole body scan before redifferentiation therapy shows no uptake in the neck i-131 whole body scan after 3 months of redifferentiation therapy shows tracer uptake in the neck six out of 13 patients reported minor side effects of nausea and vomiting and 5 patients reported giddiness which eventually subsided . Three patients developed signs of tumor inflammation during the course of therapy, but were asymptomatic for those . Six out of 13 patients reported minor side effects of nausea and vomiting and 5 patients reported giddiness which eventually subsided . Three patients developed signs of tumor inflammation during the course of therapy, but were asymptomatic for those . Surgery and ri therapy result in cure of majority of dtc patients; however, some patients with dtc dedifferentiate, which is associated with loss of iodide uptake ability, more aggressive growth, and metastatic spread, making the tumor resistant to the traditional therapeutic modalities including ri . Patients with advanced dtc at presentation often have dedifferentiated cancers on follow - up . Conventional radiotherapy and chemotherapy have very limited role in the treatment of dedifferentiated thyroid cancer . Aggressive nature of the disease and lack of effective treatment results in dedifferentiated thyroid cancer being responsible for the majority of deaths attributable to thyroid cancer . Several redifferentiating agents and targeted molecules have been studied in the treatment of dedifferentiated thyroid cancer . Besides inducing ri uptake, redifferentiating agents have shown other effects like promotion of apoptosis, growth inhibition and cell cycle regulation . The vitamin a (retinol)-derived ras are important regulators of many physiological processes, including cell proliferation, differentiation, morphogenesis, angiogenesis, and apoptosis . The pleiotropic effects of retinoids are mediated by a nuclear heterodimeric pair of retinoid receptors (rar / rxr). Retinoid - activated rar / rxr heterodimers mediate the transcription of specific gene networks by binding to specific dna response elements and recruiting cofactor complexes which cause the local chromatin structure to alter and engage the basal transcription machinery . Rars and rxrs also integrate a variety of signaling pathways through phosphorylation events and are involved in the control of cell growth, differentiation, and apoptosis . Ra has been successfully used for the treatment of hematological malignancies as well as therapy and chemoprevention of solid cancers including thyroid carcinomas . Cell culture experiments in thyroid carcinoma cell lines showed that ra treatment affects thyroid - specific functions, cell cell or cell matrix interaction, differentiation markers, growth, and tumorigenicity . Ra has an antiproliferative effect on the follicular thyroid carcinoma cell lines ftc-133 and ftc-238 . Furthermore, pre - treatment of these cell lines with ra results in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants . A study by handkiewicz - junak et al in 2009 showed that ra increase radioactive iodine (rai) uptake in thyroid tissue in 17% of the 53 epithelial cell thyroid carcinoma patients studied, whose previous post - therapeutic i-131 scans were negative . Forty - one (77%) patients were evaluable for tg response before and after ra treatment . There was a statistically significant increase in median tg level (60 vs. 90 ng / ml, p<0.05). Zhang et al in 2005 evaluated the effect of atra in 11 patients: iodine uptake was increased in 4 and there was a partial response (pr) of target lesions in 5 patients . Simon et al in 2002 evaluated the response of 50 patients on the basis of reduction in tumor size and tg levels . Thirteen patients showed a clear increase in ri uptake, while eight showed mild increase . Tumor size was assessable in 37 patients, tumor regression was observed in 6 and there was no change in 22 patients . In total, a response was seen in 19 patients (38%). Response to retinoid therapy did not always correlate with increased ri uptake, so the authors assumed other direct antiproliferative effects . Courbon et al in 2006 treated 11 patients with a progressive disease, with 13-cis - ra (1.5 mg / kg / day) over 8 weeks prior to i-131 irradiation . The redifferentiating effect of ra was evaluated by serum tg monitoring during ra treatment and qualitative analysis of iodine uptake on the post - therapeutic whole body scan . Our findings are in agreement with the previously published studies of zhang et al and courbon et al . Ra appears to increase the ri uptake in some patients, but to a mild degree which may not be therapeutically significant . Also, progressive disease was seen in more than 4/13 patients within a short duration . That 8/13 patients showed stable disease and 1/13 showed partial response is a figure which may change on longer follow - up . Firstly, the response criteria based on ri uptake and serum tg levels used in previous studies are not standard . Although ri uptake in the disease site is considered to be beneficial, the long - term clinical implication of such uptake is not known . We used serum tg and not ri uptake as a marker to assess the response . This is also a debatable method as serum tg levels could have decreased or increased with redifferentiation therapy . It could have increased due to redifferentiation of thyroid cancer that leads to increased tg expression by the tumor, or it could have decreased due to reduction of tumor volume . Also, the mean follow - up duration was 6.4 months (median 3.3 months). Thyroid cancers are usually indolent and slow growing, so a follow - up period of 6.4 months may not be long enough to detect the clinical events . Of the six patients in whom ri uptake was reinduced in our study, three had biochemical progression and the other three had stable disease . Thus, mere reinduction of i-131 uptake cannot be justifiably included as a response criterion . We conclude that ra therapy may induce ri uptake and reduce serum tg levels in some patients with dtc, but whether this results in clinically significant response can only be ascertained on long - term follow - up . In our opinion, ra therapy may be beneficial, but definite proof of its efficacy and long - term safety is lacking . Other drugs also need to be evaluated for the treatment of ri negative dtc and clinical or biochemical evidence of disease.
Lag3 is a member of the immunoglobulin superfamily (igsf) and exerts a wide variety of biologic impacts on t cell function.21 lag3 is expressed on cell membranes of natural killer cells (nk),21 b cells,22 tumorinfiltrating lymphocytes (til), a subset of t cells,23 and dendritic cells (dc)24, 25 . The lag3 gene encompasses 8 exons, and the cdna encodes a 498 amino acid membrane protein . Human lag3 is highly homologous with both murine (70%) and pig (78%) lag3.21, 26 lag3 is closely related to cd 4.27 lag3 is located on the human chromosome 12 (12p13.32) adjacent to the cd 4 gene, and its sequence is approximately 20% identical to cd 4.21 the lag3 protein binds a nonholomorphic region of major histocompatibility complex 2 (mhc class ii) with greater affinity than cd 4.28, 29, 30, 31, 32, 33, 34, 35 lag3 is one of the various immunecheckpoint receptors that are coordinately upregulated on both regulatory t cells (tregs) and anergic t cells, and the simultaneous blockade of these receptors can result in an enhanced reversal of this anergic state relative to the blockade of one receptor alone.18 the lag3/mhc class ii molecule interaction leads to the downregulation of cd4 + agspecific t cell clone proliferation and cytokine secretion . T cell mhc class ii molecules downregulate t cell proliferation following lag3 binding and suggest a role for lag3 in control of the cd4 + t cell response.31 lag3 can negatively regulated t cell proliferation, activation and homeostasis . Slag3 is a th1 activity marker in serum that can be detected by elisa.36, 37 slag3 causes dcs to mature 38, 39, 40, 41, 42, 43, 44 and attack tumor cells.43, 44 studies of the mechanisms that underlie monocyte and dc activation38, 40 by slag3 indicate that slag3 induces protein phosphorylation in immature dc that triggers the functional maturation.38, 39 this process requires slag3 binding with mhc class ii.28 beyond the role it plays in a variety of autoimmune diseases, lag3 can also reduce the body's ability to resist infection and promote chronic infection . Lag3 prevents autoimmune disorders in the eye by inducing anterior chamberassociated immune deviation.45 lag3 may regulate the functions of cd4 + and cd8 + t cells during autoimmune diabetes, and limit autoimmunity in diseaseprone environments.46 in bone marrow transplant (bmt) patients, lag3 can regulate cd8 + cells involved in alloreactivity, t cell proliferation and activation after bmt.47, 48 in patients with chronic viral infection, the blockade of both pd1 and lag3 could synergistically activate t cell responses and control the virus.49 lag3 negatively regulates cd8 + t cells in chronic hepatitis c patients.50 in tuberculosis, slag3 is elevated both in healthy people who have been exposed to the bacteria and in tuberculosis patients with good prognoses,51 indicating that slag3 could modulate an antibacterial immune response in mycobacterium tuberculosis.52 in acquired immune deficiency, high expression of lag3 was correlated with impaired invariant natural killer t cell cytokine production for the duration of chronic human immunodeficiency virus (hiv)1 infection and treatment.53, 54 targeting the lag3 pathway has an immune regulatory effect and can enhance immune reconstitution in hivinfected patients.55 vital preclinical studies have demonstrated that lag3 antibodies have potential for cancer immunotherapy (table 1). Dc, dendritic cells; lag3, lymphocyteactivation gene3; slag3, soluble lag3; til, tumorinfiltrating lymphocytes . Lag3 may be an even more promising target in cancer immunotherapy, because antilag3 antibodies can activate t effector cells and affect tregs function.67 many companies are now focusing on the lag3 immune checkpoint in their search for novel approaches to treat malignant tumors and autoimmune disorders, many of which are now in clinical development (table 2). Clinical trials with lag3 lag3 and ctla4 function similarly.19, 68, 69 ctla4 inhibits t cell activation, suppresses t cell receptor signaling, and promotes cell cycle arrest.70 activated lag3/ t cells extend cell cycle progression and increase t cell death . The similarity of function between lag3 and ctla4 may be related to some intersection in their signal transduction pathways . Tetravalent ctla4ig and lag3ig could have a synergistic effect in preventing acute graftversushost disease (gvhd). Lag3 has synergistic action with pd1/pdl1.20, 48, 72 lag3 and pd1 are critical for the prevention of autoimmunity . Their synergistic function reverses autoimmune disease.19 a deficiency of lag3 and pd1 caused lethal myocarditis in a mouse model . The respective ligand receptor interactions between pdl1 and lag3, together with the molecules themselves, synergistically inhibit t cell responses during persistent plasmodium . Blockade of pdl1 and lag3 activated cd 4 + t cells, increased helper t cells and b cells, enhanced protective antibodies and rapidly cleared bloodstage malaria in mice.73 in chronic viral infection, lag3 and pd1 maintain cd8 + t cell exhaustion.18, 49 in vivo research has shown that the blockade of pd1 and lag3 pathways can activate t cells to achieve better viral control compared to either blockade alone.49 coexpression of lag3 and pd1 can induce greater t cell exhaustion and more severe infection.23 pd1 and lag3 signaling pathways can inhibit cd 8 by antigen and cytokine signaling.18 in ovarian cancer, cd8 + til could be negatively regulated by lag3 and pd1 . Cd8+lag3+pd1 + t cells significantly reduced ifn/tnf. Blockade of both lag3 and pd1 could increase specific cd8 + t cells producing cytokine.59 it was also reported that lag3 and pd1 synergistically regulate tcell function, blunting the antitumor immune response . Lag3pdcd1 mice developed an early onset, lethal autoimmune condition, but not a single knockout or wildtype mice . Cytokine analysis revealed high levels of ifn, tnf and mcp1 in the serum of lag3pdcd1 recipients but not a single knockout or wildtype control recipient . Although ctla4, pd1 and lag3 are all negative regulators expressed during tcell activation, high level, dual lag3/pd1 expression is largely restricted to infiltrating til . Thus, lag3/pd1 combinatorial immunotherapy may promote the tumorspecific responses relative to nonspecific or selfantigenspecific immune responses and, thus, may be less toxic than the ctla4 blockade.20 dual antilag3 and antipd1 antibody therapy has a better prognosis than single antibody therapy . Dual knockout mice survive longer than single knockout mice . The strong synergy between the pd1 and lag3 inhibitory pathways another important immune checkpoint, lag3, which is closely related to cd4, can regulate t cell proliferation, activation and homeostasis . Lag3 plays an important role in a variety of autoimmune diseases and promotes chronic infection and cancer . The authors report no conflicts of interest . The authors alone are responsible for the content and writing of the paper.
Hookworm is one of important soil - transmitted helminthes (sth) for humans around the world . Human infection by hookworms induces blood loss, iron - deficiency anemia, and other anemia associated - symptoms and signs . Necator is rather prevalent in tropics and southern subtropics while ancylostoma is in subtropic or northern temperate zones . Worldwidely, n. americanus accounts for the predominant etiology of human hookworm infection, whereas a. duodenale occurs in more scattered focal environments (2). Until the 1970s, sths were highly prevalent in korea, and the population of infected people far outweighed those uninfected . Infections of sth were decreased, however, by a national mass control program in the 1970s to 1990s (3). Only clonorchiasis and sporadic tissue parasitic helminthiases are recognized at present in korea (4). Among sths, hookworm disappeared first, and thus anemia and malnutrition by hookworm infection became negligible in korea in 1980s . The hookworm egg positive rate in korea was 10.7% in 1971, but decreased to 3.7% in 1978, 0.08% in 1985, and 0% in 2004 (3, 5). Of human hookworms, a. duodenale was predominant in korea but n. americanus was recognized in a few localities (6 - 8). The last case of n. americanus infection in korea was reported in 1978 (8). Later, in 1992, an imported case of a. duodenale was reported (9). Herein, we describe a case of an 82-yr - old woman who suffered from severe chronic iron deficiency anemia by necator americanus infection in korea . An 82-yr - old woman was transferred to the national police hospital, seoul, on october 22, 2007, complaining of shortness of breath and dizziness which lasted for years . She lived in uljin - gun (county), a remote rural area of eastern mountain of gyeongsangbuk - do, korea . She had resided in uljin - gun throughout her entire life and had no history of overseas traveling ., she had been treated for anemia including two transfusions in 2006 at a general hospital in seoul . She also visited another general hospital complaining exertional dyspnea, and was treated for pulmonary tuberculosis . On physical examination at admission, she was pale, ill - looking, and undernourished, but her vital signs were stable . Laboratory results revealed overt anemia: rbc 1.6610/l (normal 4.0 - 5.410/l), wbc 3.810/l (normal 4 - 1010/l), hb 3.4 g / dl (normal 12 - 16 g / dl), hct 12.4% (36 - 48%), mcv 74.7 fl (normal 79 - 95 fl), mch 20.5 pg (normal 27 - 33 pg), mchc 27.4 g / dl (normal 32 - 36 g / dl), platelet count, 16210/l (normal 130 - 40010/l), fe 9 g / dl (normal 37 - 145 mg / dl), total iron - binding capacity 318 mg / dl (normal 228 - 428 mg / dl), ferritin 10 ng / ml (normal 10 - 130 ng / ml), neutrophils 56%, lymphocytes 25%, monocytes 11%, and eosinophils 8% . Stool examination was negative for occult blood, but was not examined for parasite eggs . Other biochemistry examinations, including electrolytes, liver and renal functions were within normal limits . Serum elisa against tissue invading helminthiases was negative for clonorchis, paragonimus, cysticercus, and sparganum . Gastroduodenoscopy found hyperemic mucosa and numerous 10-mm long slender reddish moving roundworms in the duodenum (fig . A total of 7 worms were removed by endoscopic biopsy forceps and 5 of them were transferred to the department of parasitology and tropical medicine, seoul national university college of medicine . The worms were round - cylindrical in shape and blood - tinged, and the 5 worms (male: 3, female: 2) measured 10.5 mm (range; 8 - 11 mm) long and 0.5 mm (range; 0.4 - 0.5 mm) wide (fig . The buccal cavity at the anterior end was characterized by one pair of cutting plates . Two male worms showed the copulatory bursa at the posterior end, the dorsal costa of which had a deep notch . After the diagnosis, she was treated with packed rbc transfusion, iron supportive therapy and albendazole medication, 400 mg twice per day for two days . Repeated tests two months after the treatment found her hemoglobin level to be 10.6 g / dl (12 - 16 g / dl) and gastroduodenoscopy found normal mucosa and no n. americanus worms in her duodenum . The present case constitutes evidence of local transmission of hookworm infection in korea in 2007 . The patient had no history of overseas travel and stayed at home for long periods of time . The patient's home is at an isolated remote mountainous village in uljin - gun (county), gyeongsangbuk - do, and she cultivated a small dooryard farm there . The environment of her door - yard must have been favorable for active transmission of hookworm . This transmission of n. americanus may be residual of the past endemicity of hookworm in the locality . However, it is a surprising finding for the recent epidemiology of helminthiasis in korea because most parasitologists and physicians regard that sths were eradicated . The morphological characteristics of the present hookworms were clear and definite enough to identify them as n. americanus . Most human hookworms in korea in the past have been a. duodenale, but n. americanus has also been found in remote agricultural localities in chungcheong - do or gyeongsang - do (6 - 8). Since sths, including hookworm, heavily infected throughout korea in the past, whole rural villages were endemic for the helminthes . Uljin - gun has been also an endemic area of those helminthes, and n. americanus must have been prevalent there . Upgrades of environmental sanitation and mass anthelminthic chemotherapy successfully eliminated sths and their associated diseases in the whole of korea (3, 5). However, the present case allows for the possibility that some remaining endemic foci of sth transmission may be scattered in remote mountainous localities . In the village where the present patient lived, ascaris or trichuris it is necessary to screen infection status of sths in such remote isolated villages in the mountainous zones of korea because other similar cases may live there . Clinical findings of the present case included anemia, dyspnea, and undernourished condition, which are known to be associated with hookworm infection (10). The severe anemia may have been due not only to the hookworm infection, but also to undernourishment and low hematopoietic activity due to her old age . The physician observed hundreds of worms on the duodenal mucosa by endoscopy but recovered only 7 of them . Picking out in addition to endoscopy visuals, the laboratory hematological findings also suggested that the present patient was infected by at least hundreds of hookworms, because n. americanus suck less blood than a. duodenale do (1, 2). In general, infection by a few hookworms is not sufficient to induce clinical anemia . This fact confirmed that the anemia of the present case was caused by heavy infection of the hookworm, n. americanus . Reviewing her medical records, it was unfortunate that her fecal specimen was not examined properly throughout her whole past medical history . She had visited several hospitals but no doctors had suspected hookworm anemia and her feces had not been properly checked for more than 2 yr . The present patient suffered from this disease for at least 2 yr because the possibility of hookworm anemia in this case was completely neglected by physicians . This is the first case of hookworm anemia in korea to be diagnosed by endoscopy . Although most of past endemic sths have disappeared in korea, it is still necessary for physicians to be mindful of the possibility of sths.
A 12-year - old female patient visited the department of oral medicine and radiology, yenepoya dental college, with a complaint of missing anterior teeth since three years ago . She had a history of exfoliation of decidious teeth due to mobility three years ago and there was no eruption of the permanent teeth . There was a history of presence of two mandibular natal teeth which were exfoliated two weeks after birth . Her parents had no consanguineous marriage, however her elder sibling died of similar medical problem . Her medical history revealed that she had visited genetic clinic and they have diagnosed her with ellis - van creveld syndrome . She also had dyspnoea because of congenital lobar emphysema for which she had undergone left upper lobectomy when she was one and half year old . Intraoral examination showed v - shaped notching of the upper lip at the mid line (fig . 2a), absence of mucobuccal fold in maxillary and mandibular anterior region (figs . 2b and c), conical shaped teeth, missing mandibular permanent anterior and retained deciduous mandibular canines, and right lateral incisor teeth (fig . 2c). Chest radiograph showed homogenous opacity in the left upper zone associated with elevation of hilum and left dome of diaphragm suggestive of left upper lobectomy (fig . Antero - posterior view of the legs showed genu valgum, hypoplasia of lateral end of tibia associated with medial tibial diaphysial outgrowth called exostosis, and mild shortening of fibula (fig . Hand - wrist radiograph showed carpal fusion, postaxial polydactyly, and shortening of metacarpal and phalangeal bone with cone shaped epiphysis . There was fusion of capitate and hamate on the right hand and hamate and triquadral on the left hand (fig . A team work of pedodontist, oral and maxillofacial surgeon, and prosthodontist was required to correct the craniofacial and dental defects in this patient . Since the patient was in growing phase, the prosthodontist decided to give acrylic partial dentures for the missing teeth . Oral and maxillofacial surgeon performed frenectomy for the high labial frenum as it was hindering the placement of the partial denture . Ellis - van creveld syndrome (evc) otherwise known as chondroectodermal dysplasia has autosomal recessive inheritance . It is a syndrome found in amish population of pennsylvania in usa, affecting male and female equally.2 familial history may include parental consanguinity or affected siblings or family members . Our patient was the second child of non - consanguineous and normally developed parents, however her elder sibling had died of similar medical problem . These patients die due to either cardiac problem or respiratory distress.2 our case did not have any cardiac problem, however there was congenital lobar emphysema because of her dyspnoea . She had undergone left upper lobectomy for the same reason when she was one and half year old child . The syndrome affects mainly the bones such as the lower limb with genu valgum, which requires surgical correction . The upper limbs show the characteristic bilateral postaxial polydactyly.3,4 our case had all these limb anomalies . Other ectodermal features in this syndrome are the absence or hypoplastic features of finger and toe nails, natal teeth, conical shaped or missing teeth, and absence of labial vestibule because of fusion of upper lip to the gingival margin, leading to the notching of upper lip . This could be due to the continuation of the normal serrated condition of the gingiva from the third to seventh month in the uterine life of the fetus . It is considered as the characteristic and diagnostic feature of this syndrome.5 - 7 all of these features were also present in this case . There are about 40 independent evc / evc2 mutations.8 ruiz - perez and goodship mentioned that the abnormalities in evc syndrome resulted from the tissue specific disruption of the response to hh ligands.9 weyer's syndrome, asphyxiating thoracic dystrophy, and orofacial digital syndrome should be considered in the differential diagnosis of this syndrome . Evc syndrome and weyer's acrodental dysastosis are allelic conditions caused by loss of function mutation in evc and evc2 . These are separated by 2 - 6 kb of genomeric sequence on chromosome 4p16.10 all the features are similar in both conditions except for the fact that there is delay in fusion of mandibular symphysis in acrodental dysastosis.6 patients with evc syndrome and asphyxiating thoracic dystrophy will have identical features in hand, pelvis, and long bones . Presence of cardiac anomalies, nail hypoplasia, fusion of upper lip to gingiva and neonatal teeth in evc syndrome, and renal failure with hypertension in asphyxiating thoracic dystrophy will help in distinguishing these two disorders from each other.6,11 presence of multiple gingivolabial frenula is similar to evc in orofacial digital syndrome . Hypoplastic nasal cartilage, moderate mental retardation, fissured tongue, and ankyloglossia helps to differentiate this from evc syndrome.11 in conclusion, the effective management of this syndrome will require a team which includes pedodontist, oral and maxillofacial surgeon, prosthodontist, clinical geneticist, cardiologist, pulmonologist, orthopaedician, urologist, psychologist, pediatrician, and pediatric neurologist . Since the oral manifestation is one of the characteristic diagnostic features, and it affects the esthetic problem, speech, jaw growth of the child, the dentists have an important role to play in proper management of such cases.
Biofilms, adherent microbial communities embedded in a polymer matrix, are common in nature . However, they are also a persistent cause of hygiene problems in the food industry and in the medical field . Biofilms result from a natural tendency of microbes to attach to biotic or abiotic surfaces, which can vary from mineral surfaces and mammalian tissues to synthetic polymers and indwelling medical devices, and to further grow on these substrates [24]. Candidiasis, caused most frequently by candida albicans, and to a lesser extent by c. glabrata, c. tropicalis, or c. parapsilosis, is often associated with the formation of biofilms on the surface of medical devices and tissues . It is also an opportunistic pathogen of the human body when its proliferation is not controlled by the host immune system . It is one of the most often identified agents in nosocomial infections and is capable of invading virtually any site of the human host, from deep tissues and organs, to superficial sites such as skin and nails, to medical implants and catheters . C. albicans biofilm development has been characterized in various model systems both in vitro and in vivo [79] and consists of distinct phases . The initial step consists of the adhesion of fungal cells of the yeast form to the substrate . It is followed by a phase of cell filamentation and proliferation, which results in the formation of multiple layers of sessile cells of different morphologies, including pseudohyphal and hyphal cells . The next step of maturation results in a complex network of cells embedded in extracellular polymeric material, composed of carbohydrates, proteins, hexosamine, phosphorus and uronic acid, as well as host constituents in natural settings . There is indeed evidence that host glycoproteins, nucleic acids, and cells, such as neutrophils, may participate in the maturity of the matrix, in particular on mucosal sites [1113]. The establishment of the biofilm extracellular matrix (ecm) represents a unique characteristic of biofilms . Quantity and composition of the matrix vary from one species to another and in different sites of infection depending on environmental cues, such as nutrient availability and mechanical stimuli [1417]. Matrix synthesis by candida biofilm cells has been shown to be minimal in static conditions in comparison to dynamic environments, aggravating biofilm formation on mucosal and abiotic sites where there is a fluid flow, such as on the oral mucosa, the urethra, or central venous catheters . The last step, dispersion of cells from a biofilm, plays a key part in the biofilm developmental cycle as it is associated with candidemia and disseminated invasive disease . Pathogenic microbes that build biofilms are potential causes of constant infections that defy the immune system and resist antimicrobial treatment, partly due to the matrix - inherent limited exposure of the cells within a biofilm to these types of immunological and medical arsenals [1922]. Other mechanisms of biofilm resistance have been suggested, such as slow growth, differential regulation of the cell metabolic activity caused by nutrient limitation and stress conditions, and cell density [2325]. In addition, the ability to adhere, as a unique prerequisite to form a biofilm, is a fast process, which makes the prevention of biofilm development difficult with the current antimicrobial tools and strategies . Biofilms are diverse communities and therefore vary depending on the microbe, the surface, and the colonization niche [5, 2630]. This paper gives an update on the recent efforts made in establishing alternative means of eradication and also prevention of candida spp . Biofilms, by developing new models of biofilm formation in flow conditions, as well as high - throughput rapid screening analyses in vitro . Newly developed in vivo models anticipate a shift of interest towards mixed fungal - bacterial biofilms and their role in pathogenesis in mucosal infections in particular . Keeping in mind that there is no unique model representative of all biofilms, it remains quite a challenge to tackle biofilm inhibition . One of the most attractive perspectives is the development of antimicrobe materials, and the latest findings are presented here . Biofilm formation is a multistep growth behaviour that results from complex physical, chemical, and biological processes [31, 32]. Because of the versatility of the milieu in which candida biofilms can develop in the human host, from the oral cavity contributing to dental plaque formation to the blood stream in intravenous catheters and the urinary tract, it seemed necessary to reproduce in vitro as many conditions as possible to establish common and specific characteristics of candida biofilm formation . In that respect, a multitude of in vitro studies has been described that relates to the impact of different types of substrate, nutritional supplies, in flow or static conditions, on adhesion and biofilm properties of several candida species, and recent findings are presented next . An overview of the in vitro models available to study candida biofilms is provided in table 1 . While biofilm formation is a general characteristic of many microbes, biofilm features such as architecture, matrix composition, and resistance to antifungal drugs are species and substrate dependent . And some studies are discussed below, and in particular studies related to candida biofilms formed on dental materials . Interest has indeed grown in investigating the role of candida species and the effect of the type of material in the development of denture stomatitis . For example, in a comparative study, cell counts analyses showed that saliva - coated discs harboured less c. glabrata cells than untreated discs, while the number of c. albicans cells was not affected by the saliva coating . However, both species adhered better on hydroxyapatite (ha) surface than on two other types of dental material, polymethylmetacrylate and soft denture liner . Surprisingly, dual species experiments showed that c. glabrata displayed higher cell counts when grown in the presence of c. albicans than when grown alone . In contrast, hyphal development by c. albicans seemed to be reduced in the presence of c. glabrata in most of the conditions tested . These data may help understand the impact that candida species may have on each other, as mixed species communities are being identified in clinical samples . In another case study, using discs as support for biofilm formation in vitro, ha substrate appeared to be less prone to candida adherence than acrylic denture, porcelain, or polystyrene when not coated with saliva . In addition, the effect of serum and similar materials on biofilm development of c. albicans clinical isolates was also evaluated in vitro . Disc coupons made of polycarbonate, polystyrene, stainless steel, polytetrafluoroethylene (also known as teflon), polyvinyl chloride (pvc), or ha were used in a high throughput assay . For all surfaces tested, however, in absence of serum, teflon supported higher biofilm production than any other material, likely due to its high roughness and hydrophobicity properties . The differential ability to form biofilm of 84 strains from several candida species, including c. albicans, c. glabrata, c. krusei, c. tropicalis, and c. parapsilosis, was assessed on clinical materials, such as teflon and pvc . All species, with the exception of candida glabrata, favoured teflon . In this study, c. glabrata together with c. krusei strains were not highly proficient in forming dense biofilms, as quantified by colony - forming units . Moreover, c. parapsilosis strains showed the least uniformity in the ability to form biofilm, followed by c. tropicalis and c. albicans . While some variability in the ability to form biofilms between strains of c. albicans has been documented in vitro, a study by maccallum et al . Revealed that biofilm formation in vitro did not significantly vary between strains of the four major clades of c. albicans, classified according to single - nucleotide polymorphisms determinations and analysis of dna repeat sequences . However, high variation in the ability to form biofilm among strains of c. parapsilosis and less extensive biofilm formation by c. glabrata specimens has been illustrated in a few studies by crystal violet staining and confocal laser scanning microscopy [3638]. Strain - dependent variation in biofilm formation was also observed among isolates of two genetically nonidentical classes of c. parapsilosis, namely, c. orthopsilosis and c. metapsilosis [39, 53]. All three species could form biofilms, but metabolic activity of biofilm cells differed between strains of the same species . However, conflicting data with different isolates reported the inability of c. orthopsilosis and c. metapsilosis to form biofilm in polystyrene 96-well plate assay in vitro [54, 55]. Biofilm formation among c. parapsilosis sensu strictu strains was also found to vary according to the geographical regions and the body sites from which the isolates came from . Isolates from blood and cerebrospinal fluid seemed more prone to form biofilms than isolates from nails, catheters, and mucosa . Overall, these data suggest a high variability in biofilm ability of strains of c. parapsilosis and related species, perhaps due to inadequate models or to an intrinsic poor ability to establish the biofilm growth by these species . In a calgary biofilm model adapted to candida spp ., c. krusei developed the largest biofilm mass in comparison to c. albicans, c. glabrata, c. dubliensis, and c. tropicalis . This model, allowing 80 biofilms to be formed at once, seemed to be very favourable to c. krusei biofilm development as biofilms of that species constituted of thick multilayered structures composed of pseudohyphal cells, while the other species formed sparse biofilms . In a last example of novel in vitro models of biofilm formation on various soft contact lenses, analyses revealed differences in hyphal content and architecture of the fungal keratitis causative agents fusarium and c. albicans . Polymers such as balafilcon a and galyfilcon a were favourable to filamentous growth of c. albicans, while others such as etafilcon a and lotrafilcon a sustained biofilms formed mainly of yeast cells . In addition, differences in biofilm formation were also observed between peripheral and central regions of the lenses, with dense biofilms formed preferentially in the centres of the lenses . Although a direct relationship between the lens ionic charge and water content and the ability of fungi to form biofilm could not be established, these data confirm previous findings that irregular surface texture of materials affect both cellular morphology and biofilm mass . The physiological specificity of infection sites is also an important factor, and efforts have been made to reproduce some major environmental cues in vitro, such as mimicking the blood flow or the urine . Biofilms grown in synthetic urine medium were comparable to those grown in the commonly used cell culture rpmi medium . And time course studies revealed that the development of both types of biofilm followed a similar pattern, with an initial adherence phase, followed by growth, proliferation, and maturation . The biofilms differed slightly in their architecture, as biofilms grown in synthetic urine medium seemed to be less complex and less dense, with a larger proportion of yeast cells rather than elongated cells . Increased nutritional supply promoted biofilm formation in another model of artificial urine medium, highlighting once again the importance of reproducing as closely as possible the physiological conditions to gain relevant information . C. tropicalis biofilms were also characterized in artificial urine medium, on urinary catheters in a flow model . Cells were able to colonize the catheters in the presence of the artificial urine medium and to detach from these silicone catheters, illustrating their capacity to colonize distal sites . Biofilms grown in static conditions have been predominantly studied, in comparison to flow - based systems, due to a low cost, a rapid processing of large number of samples, and limited technical requirements . However, in order to maintain their niches in dynamic environments, biofilms in vivo endure shear forces generated by the constant flow of physiological fluids . Gene expression analyses revealed only a marginal difference between biofilms grown in static conditions, such as microtiter plates or serum - treated catheters, and those grown in a flow system in microfermentors . Interestingly, the biofilm transcriptomes were not strongly affected by factors such as nutrient flow and aerobiosis, in contrast to the gene expression of free - living cells . However, a few studies indicated that biofilms grown under flow conditions, in cdc reactors or modified robbins devices, contain more extracellular matrix and more biomass [10, 43, 45]. Mature biofilms formed in a flow of replenishing nutrients consist of a dense network of yeast cells, pseudohyphae, and hyphal cells . In a simple flow model, using a silicone strip placed in a conical tube, c. albicans biofilms grew thicker than biofilms grown in static conditions, and grew faster as an 8-hour - grown flow biofilm had similar biomass as a 24-hour - grown static biofilm . The authors speculated that uninterrupted food supply prohibited adverse conditions, such as nutrient starvation and toxic accumulation, and hence promoted rapid cell proliferation . A parallel study, using a rotating disc system (rds) to impose shear forces at physiological levels to biofilms developed on catheter pieces, illustrated similar results as biofilms under shear stress grew thinner but denser than those in no - flow conditions . In the rds model, less cells adhered at first, but by 24 h biofilms displayed similar metabolic activity and dry weight as those obtained in the static model . Suggestions that explained the increased growth rate in shear conditions included an increased rate of maturation in these conditions and a natural selection of more robust cells capable of withstanding the fluid friction by growing faster . Another important aspect of in vitro biofilm modelling is the development of high - throughput systems of particular interest in the large - scale screening of antibiofilm molecules . Most studies so far have made use of the 96-well microtiter plate assay . In this model, biofilms are formed directly on the bottom of the wells, and the quantification method is based on the ability of sessile living cells to reduce tetrazolium salt (xtt) to water - soluble orange formazan compounds . In an effort to upscale biofilm production, a c. albicans biofilm chip system (cabchip) has recently been developed by srinivasan et al . . The high - density microarray platform is composed of more than 700 independent and uniform nanobiofilms encapsulated in a collagen matrix and provides the first miniature biofilm model for c. albicans . Despite the several - thousand - fold miniaturization, the biofilms formed on the chip displayed phenotypic characteristics, such as a multilayer of yeast, pseudohyphae and hyphal cells, and a high level of antifungal drug resistance, consistent with those of biofilms formed by standard methods . However, echinocandins were not proficient to eradicate biofilm in this system, potentially due to their binding to the collagen matrix . In a second generation of the biofilm chip while this system steps - up the number of biofilms that can be produced at once in static conditions, the next step may be to develop high - throughput flow biofilm systems adapted to candida spp . Such a tool has been described based on a device comprised of microfluidic channels that provide fluid flow to 96 individual bacterial biofilms . The effects of antimicrobial agents on the biofilms were rapidly screened, and viability was quantified by fluorescence measurements . These high - throughput techniques will certainly contribute greatly to the discovery of novel antibiofilm molecules . In vivo models are undisputedly required to appreciate the hostile environment that conditions biofilm formation (table 2). A few candida biofilm models, mostly associated to catheter infections, have been developed in several rodents, giving insights on the in vivo biofilm structure and the efficacy of various antifungal agents . The catheter - related in vivo biofilm models resulted in biofilm formation within 24 h and consisted of complex structures of yeast and elongated cells embedded in extracellular matrix, similar to those observed in in vitro model systems . While susceptibility to azoles was reduced in these models, liposomal amphotericin b lock therapy and treatment with caspofungin or chitosan proved to be efficient against in vivo biofilms [64, 65, 71]. Central venous catheter models (cvcs) are also useful for the investigation of the kinetics and occurrence of dissemination of the microorganisms to other organs, demonstrated by colonisation by c. albicans of the kidneys in the rat model . In addition, the development of a cvc model in mice will allow comparison to other modes of infection, in particular to the commonly used disseminated candidiasis by tail vein infection . A murine model for catheter - associated candiduria was recently developed and illustrated the role of candida biofilms in the persistence of the urinary tract infection . It also outlined differences between murine and human catheter - related candiduria in terms of bladder inflammation and fungal burden in the urine . In another catheter - related candida - associated infection model this model, of nondisseminated nature, allowed the study of biofilm development for long periods of time (figure 1) but required the use of immunosuppression treatment of the animals due to the high inflammatory response associated with implant of foreign devices . However, efficacy of the echinocandin anidulafungin, by intraperitoneal injections, was demonstrated against c. albicans biofilm in this in vivo system . These in vivo models are all suited for further study of novel antifungal therapies and for the use of novel material technologies, including less adherent surfaces and material coating with fixed or releasing antifungal agents (see the next section). A relatively cost- and time - effective candida biofilm model on acrylic denture material, which does not require the ex vivo mold process, was illustrated recently . In this rat model, biofilms developed between the hard palate and the denture material, following candida inoculation in that 1 mm space (figure 1). Fungal invasion of the palate and the tongue and neutrophils infiltration also occurred, indicating that the model was consistent with that of acute human denture stomatitis . Interestingly, the denture model offers the possibility to study mixed biofilm structure and behaviour in response to antimicrobial treatments, as the biofilms were composed of both bacterial and fungal cells . Finally, biofilms developed on the denture model were inherently resistant to fluconazole, in accordance with previous findings [8, 72], but also to the echinocandin micafungin, in contrast with previous investigations performed in a different model . A plausible explanation suggested by the authors is that the mixed biofilm nature combined with the specific site of infection, the oral cavity, is the cause of that antifungal resistance . An alternative rat model of candida - associated denture stomatitis recently described differs by the use of animal - fitted devices . In this system, a removable part of the device makes the replacement of the infected device a relatively easy step . Tools and models to study biofilm formation developed on implanted materials are numerous and indicative of the increased medicinal use of such implants . Biofilms formed on live surfaces are much less characterized, yet they are recognized as causing or aggravating numerous chronic diseases . Besides dental plaques, few reports have investigated biofilm development in clinical samples . The oral cavity is an accessible in vivo model for studying protein - surface interactions and has been well characterized for bacterial biofilm . A mucosal model of oropharyngeal candidiasis keratin, originating from desquamating epithelial cells, constituted a large proportion of the biofilm matrix . First evidence was given that epithelial cells, neutrophils, and commensal oral bacteria co - exist within the fungal mucosal biofilm developed on mouse tongue . Bacteria were mostly found on the apical part of the biofilm, and very few were seen to invade the tongue epithelium layer . This model highlights the complexity of mucosal biofilms, as host elements and commensal organisms contribute in an active or passive manner to the structure of the biofilms . C. albicans can also form biofilms on the vaginal mucosa, illustrated by two in vivo and ex vivo models in immunocompetent estradiol - treated mice . C. albicans vaginal biofilms consisted of yeast and hyphal cells embedded in extracellular material, illustrated by cona staining of the interspersed matrix . In the ex vivo model using vaginal explants, no exogenous nutrients were provided, yet biofilms were formed most likely by scavenging host nutrients . Host - pathogen interactions in biofilm settings have not yet been elucidated, but comparison between these models promises to identify model - specific fungal and host elements . The relative contribution and the role of bacteria - candida interactions in the pathogenesis of mucosal infections are yet to be established . However, there is clear evidence that multimicrobial interactions have a central role in the context of human disease . For example, microbial diversity was illustrated in a biofilm - related infection of the urinary tract . Out of 535 clinical samples of urinary catheters, candida spp . Were identified among the 39 different microbial taxa isolated . C. albicans was isolated in 141 samples, and other candida species were present in other 82 samples . Biofilm formation ability of each isolated strain was quantified in vitro, yet not in an artificial urine medium, and cut - off values were used to define no, weak, intermediate, and strong biofilm producers . These data illustrates the fact that, in multispecies biofilms, some have a great potential to cause biofilm - based infections, while others may be more passive members of the structured community . Commensalism, mutual cooperation, and antagonism make the interactions within mixed biofilms complex [78, 79]. A summary of bacteria - candida interactions and their effect on fungal development is provided in table 3 . Bacteria can interact with c. albicans cells within mixed biofilms, and in particular with hyphal cells . The methicillin - resistant gram - positive staphylococcus aureus had the highest hyphal association, in comparison to s. epidermidis, strepococcus pyogenes, pseudomonas aeruginosa, bacillus subtilis, and escherichia coli in decreasing order, respectively . However, interaction between s. aureus and c. albicans did not result in reduced or altered biofilm viability . In another study, addition of bacteria to preformed candida biofilms in vitro had an antagonistic effect on biofilm cell mass, often in a cell - density - dependent manner . With all inoculums tested, p. aeruginosa reduced significantly the fungal biofilm mass when added during the first few hours of biofilm development . In a different experimental assay, . Moreover, simultaneous addition of bacteria and c. albicans cells showed that in all cases fungal adhesion was decreased, whereas bacterial biomasses were not affected . Hypotheses of synergistic relationships between microbes have been suggested, and in particular within mixed biofilm communities . For example, bacterial adhesion was observed on the tongue mucosa of c. albicans - infected animals but not of noninfected animals, in a mucosal model of oropharyngeal candidiasis . Synergistic cooperation can also perturb susceptibility to antimicrobial treatment . For example, s. aureus resistance to vancomycin was enhanced in mixed biofilms with viable c. albicans cells, whereas susceptibility of the fungal cells to the antifungal amphotericin b was not altered . Binding of the fungus to the bacterial cells occurs via the candida - specific adhesin proteins, including als3, eap1, and hwp1, as demonstrated by heterologous expression of these cell wall proteins in the model yeast saccharomyces cerevisiae . The role of adhesins in single- and multispecies biofilm formation is not discussed here but can be found in previous reports [8688]. The current therapies against fungal diseases, employing one of the five classes of antifungals (polyenes, pyrimidine analogues, allylamines, azoles, and echinocandins) administrated orally or intravenously, are not discussed in this paper . Each antifungal compound has advantages and limitations related to its spectrum of activity and mode of action . The susceptibility of candida biofilms to the current therapeutic agents remains low, with the exception of the echinocandins [97, 98]. However, these compounds have been employed in different approaches, such as lock therapy or material coating as releasing agent . These alternative methods and their perspective of usage nosocomial infections associated with medical devices represent a large proportion of all cases of hospital - acquired infections . In particular, insertion of any vascular catheter can result in a catheter - related infection, as microorganisms can colonise catheter external and internal surfaces . Some of the favourite niches of colonisation of candida spp . Include indeed vascular and urinary catheters and ventricular assist devices, which can be accompanied with high mortality rates . Adherence to the catheter surface, facilitated by host proteins such as fibronectin and fibrinogen, can then lead to biofilm formation . The antimycotic lock therapy approach is currently recommended and employed in treating catheter - related bloodstream infections (crbsi), in particular for long - term catheters, according to the infectious diseases society of america guidelines . However, lock therapeutic treatment is pathogen specific as catheter removal is recommended for crbsi caused by candida species and staphylococcus aureus . The lock therapy involves the instillation of high doses of an antimicrobial agent (from 100- to 1000-fold the minimal inhibitory concentration, (mic)) directly into the catheter in order to lock it for a certain period of time (from hours to days). Few reports are currently available on the usage of antifungal lock solutions in clinical practice, but they seem to indicate the curative effect of this kind of treatment [104, 105]. In vitro studies are more prevalent at the moment and seem to also favour the use of antifungal lock therapy to eliminate candida spp . For example, biofilm metabolic activity formed on silicone by c. albicans and c. glabrata could be effectively reduced by a 12 h lock treatment with micafungin (at 100500x mic), which was shown to persist for up to 3 days . Caspofungin had an intermediate effectiveness in the same study, as its activity did not persist as long against c. glabrata biofilms . While these results are promising for potential use of the lock technique to treat infected catheters, 100% biofilm inhibition could not be achieved . Sterilization of catheters was obtained in vivo by lock treatment with amphotericin b lipid complex (ablc) in a rabbit model of catheter - associated c. albicans biofilm . However, in this study, the lock solution was administrated a few hours a day for a prolonged period of time (7 days). Synergistic antibiofilm combinations, used as lock solutions, between classical antimicrobial agents and other compounds such as the mucolytic agent n - acetylcysteine, ethanol, or the chelating agent edta, are also effective against s. epidermidis and c. albicans individual and mixed biofilms . In a similar approach, recent results suggest that the combination of antibacterial agents with gram - positive activity, including doxycycline and tigecycline, with known antifungals, such as amb, caspofungin, and fluconazole, can be useful for the treatment of c. albicans biofilms [110, 111]. The prevention of crbsi has also been the focus of research and randomized controlled trials . In a systematic assessment, showed the clinical effectiveness of cvcs treated with anti - infective agents (ai - cvc) in preventing crbsi . While trials are still required to determine the most cost and clinical - effective anti - infective product, the routine usage of ai - cvc will often be limited if appropriate use of other practical care behaviour is not employed in intensive care units . The echinocandin caspofungin was employed to prevent c. albicans biofilm formation in a biofilm model in mice . C. albicans biofilm formation was greatly reduced in cvcs that had been pretreated for 24 h with high doses of caspofungin . Similarly, the use of chitosan, a polymer isolated from crustacean exoskeletons, as a pretreatment of catheters to prevent c. albicans biofilm formation was validated in a cvc biofilm in vivo model . The use of lock technique or preventive impregnation of antifungals in combating catheter - associated infection seems promising, but not yet convincing on a cost effective point of view as huge doses are still needed to eradicate fungal growth . A developing field of research focuses on the usage of modified materials or coated surfaces to prevent adherence and biofilm development . Implant materials are prone to biofilm formation affecting health in general and duration of the implant in particular . Surface characteristics, such as surface roughness, surface free energy, and chemistry, can influence the type and the feature of the biofilms [114, 115]. For example, c. albicans adhesion is enhanced if the roughness of the denture materials is increased . It is nowadays conceivable that coatings may be engineered to promote selective adhesion, with possible attachment to cell tissue (for implant in bone contact) but not to microbes . They may also address the second phase of biofilm development involving quorum sensing, by inhibiting cell - cell communication signals [117, 118]. Biomaterial modifications as a way to prevent biofilm development have been the focus of intense research, in particular in the field of bacterial biofilms, but the latest findings on their impact on candida biofilms are discussed next . Surface properties of medical devices constitute a major factor contributing not only to the stability in the body but also to their performance and lifetime in vivo and their colonization by microorganisms . In that matter, albumin adhesion is beneficial since it has been shown to prevent binding of microorganisms, while fibrinogen has the opposite effect . Chemical grafting of polyethylene and polypropylene surfaces, functionalized with cyclodextrins, yielded a change in protein adsorption profile of these polymers, by promoting adsorption of albumin and reducing adhesion of fibrinogen to the material surface . In addition, these modified substrates incorporated well the antifungal agent miconazole, leading to reduced biofilm formation by c. albicans in vitro . Modified polyethylene and silicone rubbers proved to be very efficient in inhibiting c. albicans biofilm formation in vitro . These cytocompatible materials were also capable of releasing for several hours considerable amount of an anionic antimicrobial drug, nalidixic acid, suggesting their use as drug - eluting systems . Modifications of polyurethanes dental biomaterials by addition of surface - modifying end groups were successfully employed to manage c. albicans biofilm formation . Increased hydrophobicity resulted in increased metabolic activity of the biofilms grown on polyetherurethane, while they inversely correlated for biofilms formed on polycarbonate surfaces . Addition of 6% polyethylene oxide to elastane 80a showed to be the best combination as no biofilm could be observed on that surface . Modification of the silicone surface of the prostheses has been employed to limit c. albicans colonization, as opposed to incorporation of antimicrobial agents in order to avoid the occurrence of resistance . Silicone disks grafted with c1 and c8 alkyl side chains reduced adherence and biofilm formation of c. albicans by up to 92% . Longer side chains did not show as good results, and combinations of quaternizing agents did not work synergistically either . Similarly, grafting of cationic peptides, such as the salivary peptide hst5 and synthetic variants, onto silicone rubber, inhibited biofilm formation by up to 93%, in a peptide - dependent manner . Fungicidal or fungistatic materials have been employed to fabricate or coat the surfaces of medical devices and have a great potential in reducing or eliminating the incidence of biofilm - related infections . Dental resin material coated with thin - film polymer formulations containing the polyene antifungals nystatin, amphotericin b, or the antiseptic agent chlorhexidine, were used in c. albicans biofilm assays . Biofilm reduction was the greatest on chlorhexidine containing polymers, while the other formulations were much less efficient . Similarly, multilayered polyelectrolyte thin films containing an antifungal -peptide incorporated within the layers of the films inhibited the growth (and hyphal formation) of c. albicans by 74% after 2 h of contact . The polysaccharide dextran is widely used in medicine and is also one of the main components of dental plaque . Cross - linked dextran disks soaked with amphotericin b solutions, described as amphogel, kills fungi within 2 hours of contact and can be reused for almost 2 months without losing its efficacy against c. albicans . This antifungal material is biocompatible and could be used to coat medical devices to prevent microbial attachment . It was recently used for local antifungal therapy in the form of injectable cross - linking hydrogels . It regulates bacterial biofilm dispersal and has also been employed in releasing xerogel to attenuate c. albicans adherence and biofilm formation . The nitric - oxide - based method is still at the experimental level, due to poor water solubility and stability . Coating of medical material surfaces has been employed and tested with several types of coating molecules, including the naturally occurring polymer chitosan and antimicrobial peptides such as histatin 5 (hst5). Surfaces coated with the polymer reduced the viable cell number in biofilms by more than 95%, in the case of c. albicans and also for many bacteria such as staphylococcus aureus . Chitosan, which is proficient against a wide range of pathogenic microbes, disrupts cell membranes as cells settle on the surface . The use of such polymer offers a biocompatible tool for further coating design of medical devices . Acrylic disks precoated with hst5 prove to be efficient in inhibiting biofilm formation of c. albicans, especially in the later stage of development, while biofilm sensitivity to the antimicrobial peptide was the same as the one of free - living cells . The utility and potential of selected peptides, as therapeutic molecules, including the -glucan synthesis inhibitors, the histidine - rich peptides, and the ll-37 cathelicidin family are being determined and could be used as coating compounds against adherence and biofilm formation [133, 134]. Shift from a commensal bacterial biofilm to a more pathogenic biofilm involving candida spp . In the oral cavity for instance is believed to be more influenced by mucosal inflammation and the general well - being of the host than on the nature and surface properties of the material itself . However, development of materials that can fully abolish microbial adherence is a promising perspective against biofilm formation . The discrepancy between antimicrobial coatings killing the biofilm - proficient organisms and antimicrobial releasing coatings to prevent biofilm formation is a current issue . Adhesion and biofilm formation by c. albicans cells can be modulated by physical and chemical signals from the oral bacterium streptococcus gordonii . Indeed, most streptococcus species possess the antigen i / ii, a cell - wall - anchored protein receptor that mediates binding to c. albicans . Moreover, c. albicans hyphal and biofilm development are greatly enhanced by s. gordonii, which also relieved the fungal cells from the repressing effect of the quorum sensing molecule farnesol . Farnesol, a sesquiterpene and signalling molecule produced by c. albicans, represses biofilm formation in vitro . Conversely, tyrosol, a 2-(4-hydroxyphenyl) ethanol derivative of tyrosine, accelerates hypha production in the early stages of biofilm development and is secreted at least 50% more by biofilm cells than by planktonic cells . Several studies demonstrated that farnesol actually increases fungal pathogenicity in animal models, potentially by interfering with normal progression of cytokine induction [138140]. Analogs of farnesol have been identified that fail to induce pathogenicity and yet retain farnesol ability to block hyphal development . While these analogs did not protect mice from candidiasis indeed, a number of molecules with farnesol - like activity, that can induce the shift to the yeast form of growth, have been identified in gram - negative bacteria . For instance, the signalling molecule, homoserine lactone, produced by pseudomonas aeruginosa represses c. albicans filamentation . Uptake of the phenazines generated reactive oxygen species production and led to fungal cell death . In mixed biofilms, binding of the toxins to the fungal cells has a negative influence on c. albicans growth . In a different approach, valle et al . Demonstrated that the use of nonantibiotic molecules, such as polysaccharides, produced by competitive commensal organisms can antagonize biofilm formation . A better knowledge of the microbial community behaviour and in particular of the interaction between commensal and pathogen organisms would help to combat predominance of the infectious or disease causative agents . In this scheme, natural products produced by cells within a biofilm contribute to the dynamic of the community and may play an antiadhesion role for nonwanted other microorganisms . Bacterial lipopolysaccharides also modulate adhesion and biofilm ability of several candida species, in an interspecies - dependent manner . It is not known how mixed populations affect the host immune response in response to infection . The overall population behaviour results from a potential selective advantage to either or both species . Identification and alterations of the communication signals would certainly result in a better understanding of how species coexist and permit a better control of biofilm formation . Targeting quorum sensing molecules or associated signalling mechanisms is an open field of research at present, but the use of quorum quenching enzymes or quorum sensing inhibitors naturally produced by other species could help in the finding of novel antibiofilm agents [148, 149]. With the number of people considered at high risk for microbial infections constantly increasing, immunotherapy seems to offer a great potential despite the complexity of the interaction between the host defence system and the pathogen ., to cause infections depends on a constant and sometimes discontinuous battle between the pathogen and the host immune system . Recognition of candida - specific pathogen - associated molecular patterns (pamps) by dedicated pattern recognition receptors (prrs) such as toll - like receptors and lectins activates the innate effector cells (macrophages, dendritic cells, and neutrophils), which in turn produce a variety of soluble factors, including cytokines and chemokines . However, little is yet known about the interactions between human phagocytes and candida spp . Biofilms, while immunotherapeutic treatment against candidiasis has been undertaken [153, 154]. Chandra et al . Demonstrated that adherent peripheral blood mononuclear cells (pbmcs) enhanced the ability of c. albicans to form biofilm . They also observed that phagocytosis of the fungal cells within a biofilm did not occur while their free - living counterparts were phagocytosed . These data defined the novel concept that candida biofilms seem to have an immunosuppressive effect . Inactivated pbmcs on the other hand did not induce this enhanced growth behaviour, nor did lipopolysaccharide - activated pbmcs, suggesting that the stimulated biofilm formation resulted from (a) candida - biofilm - induced secretory factor(s). Indeed, the cytokine profile of pbmcs following coculture with planktonic or biofilm cells of c. albicans differed greatly, with il-1 as the cytokine most highly overexpressed by contact with biofilms . Supporting these data, a recent study showed that phagocytes alone induced much less damage to biofilms than they did to free - living cells or to resuspended biofilm cells, which lacked the overall structure of biofilms and most of the matrix . Using confocal laser scanning microscopy, katragkou and coworkers deducted that human phagocytes looked like unstimulated cells, presenting a rounded shape when in presence of biofilms . This was also confirmed by a reduced cytokine production in a biofilm - phagocyte coculture, compared to a planktonic cells - phagocytes mix . Phagocytes appeared entrapped within the structured network of cells and matrix and were unable to internalize cells within biofilms . Moreover, c. albicans and c. parapsilosis biofilms were more susceptible to the additive effects between phagocytic host defence and the echinocandin anidulafungin than to each separately and to the combination of the azole voriconazole with phagocytes [156, 157]. These data validate the findings that echinocandins can influence host cell interactions with biofilm . Pathogens have evolved many mechanisms of defence to avoid being recognized by the host environment [159161]. C. albicans can evade immune attack by masking its cell wall -glucan component, a potent pro - inflammatory signature carbohydrate, under a thick layer of mannoproteins . Clear evidence showed that exposing the -glucans by treatment with the antifungal drug caspofungin elicited a stronger immune response . Masking of -glucans depends on a complex network of cell wall remodelling, and targeting these regulatory processes may identify novel antifungal possibilities . For example, disruption of the mapk pathway regulated by the extracellular signal - induced cek1 kinase triggered a greater -glucan exposure, which resulted in an enhanced immune response compared to the wild - type strain . There are conflicting data regarding the role of the -glucan receptor dectin-1, expressed widely on phagocytes, in antifungal immunity . However, studies suggested that dectin-1 is required for fungal killing and induction of early inflammatory responses . These findings are of interest for biofilm recognition by the immune system, as -1,3-glucans are found in high amounts in the extracellular matrix of candida biofilms in vitro and in vivo [10, 12, 165]. Biofilms developed on soft tissue are associated with infiltration of the infected sites by neutrophils, which can then confer innate immune protection . In c. albicans, hyr1, encoding a gpi - anchored cell wall protein, has been shown to confer resistance to neutrophil killing in vitro and in the oral mucosal tissue biofilm model [12, 167]. Immunotherapeutic strategies, such as vaccination, anti - candida antibodies, and cytokine therapy, are under investigation to treat candida infections . However, their applicability in treating biofilm - related infections is still in a preliminary state . In that framework, recent data showed that pretreatment of c. albicans cells with antibodies targeting the complement receptor 3-related protein led to reduced adhesion and biofilm formation in vitro . In another study, anti - c . Albicans antibodies from chicken egg yolk were employed as antiadherent molecules . While the adherence of c. albicans was reduced, biofilm inhibition was only observed in absence of serum, as the activity of the antibody was very much reduced against germ tubes, of which the formation is induced in the presence of serum . In vivo studies of the antibody - based approach the large panel of biofilm models suitable for candida research highlights the diversity of niches in which the fungus can develop ranging from biotic to abiotic surfaces . However, the role and nature of host - pathogen interactions during biofilm formation are only starting to get unveiled . The search for an antibiofilm treatment is a complex subject which requires improved knowledge of the pathogen itself, and also of the host response to adhesion and biofilm formation, the properties of the substrates onto which biofilm develop, and the interactions within microbial communities . The field of chemoinformatics may assist the development of novel antibiofilm compounds, based on already identified good candidate molecules . This approach may also reveal better coating agents for material surfaces that would persist long periods of time in vivo . The use of natural compounds, from dietary plants or probiotics, may also be considered as they are better tolerated by humans.
The overall aim of education is to produce critical thinkers who should be able to evaluate and analyze different types of information gathered in their chosen field of expertise . Contemporary approaches emphasize the active engagement of students in their own learning, learner responsibility, meta - cognitive skills, and a dialogical collaborative model of learning . Self - assessment is considered to be very major component of learning because here the student gathers information and reflects on his own learning . It enhances the potential of learner development as a lifelong learner . Keeping these things in mind, the present study was planned with an objective to assess the impact of self - assessment by medical students on their subsequent academic performance . The present study was conducted in the department of physiology on the first year professional mbbs students . The study was based on evaluation of two theory tests (test i and test ii) on one topic in physiology, consisting of both essay type and short answer questions, administered to all students of the 1 year, at different intervals . The students who missed either test i or test ii were excluded from the study . Afterward, the students were apprised of the process of self - assessment and the way to conduct it . They were asked to gather information on these test topics in 3 days' time so as to self - assess their performance by marking their tests themselves after 3 days under the supervision of teachers . They awarded the marks on a sheet provided to them which was then collected and kept separately . Subsequently, blinding was carried out by hiding the roll numbers on the answer sheets of the students and the same theory tests were marked by the faculty . To ensure ambiguity in marking, one particular answer of all students after this, one to one feedback was given to the students by the evaluators about their attempt and announcement for a retest after 7 days was made . Then, a nonidentical (parallel) theory test on the same topic was given to them, and their performance in the retest was assessed by the same teachers after blinding . Marks awarded by students themselves and by faculty in test i and test ii were then tabulated . The feedback about the perception of students and faculty about this intervention was obtained through prevalidated feedback questionnaires after two theory tests were completed . Statistical analysis was done using spss (spss statistics version 16.0; illinois, chicago). Paired t - test was used to compare test awards by teachers in test i and test ii . Marks obtained by students by self - assessment and those awarded by teachers in test i were compared applying independent t - test . Correlation between self - grading and teacher grading in test i was performed by calculating pearson s correlation coefficient . Feedback was obtained from students and faculty on a 5-point scale, i.e., strongly agree (sa), agree (a), neutral (n), disagree (da), strongly disagree (sda) and analyzed . The study was done to assess the improvement in academic performance of the 1 year mbbs students after the process of self - assessment . Comparison of marks obtained in test i and test ii, both assessed by teachers showed statistically significant increase in marks in test ii (p <0.001). It was found that 74% students showed increase in marks in the second test [figure 1]. Difference of marks obtained by students in test ii as compared to test i another comparison was made between test i self - assessed by students and then the same test i marked by teachers . Seventy - four percent of students over - assessed while 21% under - assessed and 3% had equally assessed themselves as compared to teacher evaluation [figure 2]. However, among the students who over assessed themselves, 34% of the students over assessed only within 10% range of marks . Comparison of student marking showing the departure from teacher marking correlation coefficient (r) was calculated to look for the degree of similarity between teacher and student grades [figure 3] and was found to be statistically significant (r = 0.794, p <0.001). Correlation between student and teacher marking perception of students and faculty regarding the process of self - assessment obtained through feedback questionnaire showed that majority of them agreed to the various attributes and advantages of self - assessment . They also suggested to carry this process of self - assessment from the start of the session . Students also opined that the discussion with peer group after self - assessment should be added . Faculty also perceived it as an effective tool for enhanced self - directed learning for students . They suggested that students should be motivated by the faculty to self - assess themselves . The didactic approach causes little opportunity to contest the medical curriculum and only a part of it is tested in high - stake examinations . The ability to assess one s own work critically is often claimed as a goal of higher education even when self - assessment exercises are not the part of curriculum . According to andrade and du, self - assessment is a process of formative assessment during which students reflect on and evaluate the quality of their work and their learning, judge the degree to which they reflect explicitly stated goals or criteria, identify strengths and weaknesses in their work and revise accordingly . The present study has found significant improvement in the academic performance of students after the process of self - assessment as 74% of the students showed improvement in marks in subsequent test when marks of test i and test ii evaluated by teachers were compared . Students also perceived this process of self - assessment to be helpful in increasing their knowledge and interest in the subject and found it motivating to develop self - directed learning skills . Improved academic performance and learning could be attributed to the fact that the students were curious to judge their own performance through self - grading and hence were motivated to go through the topics again . Self - evaluation is considered to be a very important component of learning as self - identification of his / her learning progress will motivate the student for further learning . In addition, further learning is possible only after recognition of what more needs to be learned . Further, by evaluating the answers themselves, they were able to critically analyze their deficiencies and tried to be careful while attempting the second test . Herbert has also suggested that by this process of self - assessment, students begin to internalize instructional goals and begin to apply them to future efforts . Boud also suggested that self - appraisal is a critical skill helpful for success not only in formal learning activities but also for lifelong learning that is such an essential part of our living in this rapidly and vastly changing world . Further, he suggested inculcation of self - assessment in the curriculum of higher education as formal activity that requires proper exposure as prior education does nsot seem to develop the skill to any extent . This is mainly because traditional assessment is sometimes also regarded as exercise of power by the assessor / examiner over the assesses . Assessment processes in which teacher holds all the power and makes all the choices limit the potential of learner development . Task of self - evaluation shifts the focus from something imposed by someone else to a potential partnership . Further, evan et al . Also concluded that doctors these days must focus on setting targets and goals for themselves and regularly assess their performance . They also suggested that self - assessment should be given the same importance in training as is given to development of communication skills . Although the students tended to over assess themselves while marking, there was a significantly positive correlation between student and teacher marking (r = 0.79), indicating that students have the concept of quality . In a study, sadler and good have also found high correlation between teacher and student gradings . In a similar study by kirov et al ., it was observed that 50% of students in preclinical course of dentistry overestimated their performance . In our study, around half of the students who over assessed themselves did so only in the range of 10% . This being only a single trial, the students can be motivated and made accustomed to carry out self - assessment more meticulously . Such a process can help the students to develop critical skills to judge their own work which is an essential component of lifelong learning and so also have better understanding of the subject . This becomes all the more important during their career as these students will attain some senior positions when normally there is hardly any critical review of their performance . According to antonelli, self - assessment of knowledge and accuracy of skill performance are essential to the practice of medicine and self - directed lifelong learning because now the individuals are themselves responsible for their own continuing professional development . With such an exercise included in the curriculum, there are chances that the desired and required skill of self - assessment be imparted in them right at the time of germination . One limitation of this study was that only one test could be conducted as it was near final examinations of the students . Another limitation found during study was that this process of self - assessment is time - consuming . Furthermore, student dropout at each step also was a limiting factor in this study . A few students found it difficult to self - assess . From this study, we can conclude that self - assessment can increase the interest and motivation level of students for the subject leading to better academic performance and enhanced learning . It can also help in development of critical skills among students to assess and analyze their own work, an essential component of self - directed lifelong learning.
A 67-year - old healthy retired man developed an asymptomatic greenish discoloration of the nail plate of the right toe nail over a period of 2 months . There was a history of chronic nail trauma prior to the discoloration, probably due to tight shoes . Dermatologic examination showed greenish - black discoloration, mild onychodystrophy of the entire nail plate, and distal onycholysis (figure 1). A 68-year - old gardener was seen during summer, after working in wet conditions, for green discoloration of his right finger nail, slight pruritus, and pain . On clinical examination, distal onycholysis and greenish discoloration were noticed on the nail plate starting from the distal edge (figure 2). No fungal coinfection was proved . Cutting the distal part of the nail plate and application of topical gentamicin twice daily for 1 month were followed by positive results . A 56-year - old man, diagnosed 1 year earlier with toenail onychomycosis, in the absence of any treatment, developed greenish discoloration of the entire nail plate . Direct mycologic examination of nail scrapings was positive and culture was positive for trichophyton rubrum . The fungal infection was treated with itraconazole 200 mg / day 14 days per month for 3 months . Bacterial infection was cured after systemic administration of ciprofloxacin 500 mg / day for 3 weeks . A 67-year - old healthy retired man developed an asymptomatic greenish discoloration of the nail plate of the right toe nail over a period of 2 months . There was a history of chronic nail trauma prior to the discoloration, probably due to tight shoes . Dermatologic examination showed greenish - black discoloration, mild onychodystrophy of the entire nail plate, and distal onycholysis (figure 1). A 68-year - old gardener was seen during summer, after working in wet conditions, for green discoloration of his right finger nail, slight pruritus, and pain . On clinical examination, distal onycholysis and greenish discoloration were noticed on the nail plate starting from the distal edge (figure 2). Cutting the distal part of the nail plate and application of topical gentamicin twice daily for 1 month were followed by positive results . A 56-year - old man, diagnosed 1 year earlier with toenail onychomycosis, in the absence of any treatment, developed greenish discoloration of the entire nail plate . Direct mycologic examination of nail scrapings was positive and culture was positive for trichophyton rubrum . The fungal infection was treated with itraconazole 200 mg / day 14 days per month for 3 months . Bacterial infection was cured after systemic administration of ciprofloxacin 500 mg / day for 3 weeks . Bacterial infections of the nails are caused by gram - negative bacteria, usually p. aeruginosa, but can also be caused by klebsiella spp . And gram - positive bacteria like staphylococcus aureus.1,2 p. aeruginosa is the most common pathogen causing bacterial nail infections, although it is rarely reported.3 p. aeruginosa is a gram - negative, aerobic, coccobacillus belonging to the pseudomonadaceae family . These pathogens are widespread in nature, inhabiting soil, water, plants, and animals (including humans). More than half of all clinical isolates produce the blue - green pigments pyoverdin and pyocyanin.2 p. aeruginosa is an opportunistic human pathogen that can produce pulmonary, kidney, and urinary tract infections and even systemic infections . It can be also involved in soft tissue infections, skin and nail infections in immunocompetent subjects and patients with immune deficiency syndrome . P. aeruginosa is not part of normal skin flora, so pseudomonas infections of the intact nail are rare . When infection occurs, p. aeruginosa colonizes moist regions of the skin, axillae, anogenital regions, and retroauricular areas . Predisposing factors must be taken into consideration: onycholysis, onychotillomania, microtrauma to the nail - fold, chronic paronychia, chronic exposure to water, soaps, or detergents, and associated nail disorders, such as psoriasis.4 onycholysis is characterized by separation of the nail plate from the nail bed; it is followed by secondary pseudomonas infection, particularly when the nails are exposed to a warm and moist environment . A number of triggers for onycholysis have been reported, including psoriasis, onychomycosis, yellow nail syndrome, contact dermatitis, medications (doxycycline), endocrine disorders (acquired hypoparathyroidism),5 constant local trauma, especially in elderly . Chloronychia is more common in homemakers, barbers, dishwashers, bakers, and medical personnel, and can be regarded as an occupationally triggered disease.6 small cuttings from the affected nail and/or subungual debris should be sent to the laboratory for investigations . Gram stain coloration is performed presenting gram - negative rods with no particular arrangement; on cetrimide agar medium, p. aeruginosa expresses pyocyanin, a blue - green exopigment, and the colonies are flat, large, and oval, with a characteristic fruity smell . Recently it has been reported a strong relation between fungal and p aeruginosa infection of the nail: fungal infection stimulates bacterial colonization within the nail and overgrowth of p aeruginosa in culture inhibits the isolation of fungus.7 the differential diagnosis includes subungal hematoma, malignant melanoma, infections caused by other pathogens such as aspergillus, candida, and proteus, and chemical exposure to solutions containing pyocyanin or pyoverdine.8 the treatment of chloronychia, especially in elderly people, is difficult in many cases and recommendations based on clinical trials are missing.3 in the past, removal of the entire nail was a therapeutic option, but not nowadays . Treatment consists of cutting off the detached nail plate, brushing the nail bed with a 2% sodium hypochlorite solution twice daily, prevention of repeated immersion by wearing cotton and latex gloves and antibiotics administered topically and orally . Topical silver sulfadiazine, ciprofloxacin, and gentamicin have also been reported to be valuable therapeutic options.9 topical antibiotics (polymyxin b or bacitracin) applied 24 times daily for 14 months have been demonstrated to be effective in immunocompetent patients.6 topical therapy with nadifloxacin applied once daily for several weeks was curative for two patients with acquired immune deficiency syndrome.4 when topical therapies are not preferred by the patient, oral ciprofloxacin should be recommended for 23 weeks . Pseudomonas infections of the nails are treated by fluoroquinolones, when necessary, in comparison with other bacterial nail infections that require culture and sensitivity testing . This is the reason to recognize easily, by simple clinical observation of the color of the nails, especially in aged persons, to avoid unnecessary laboratory investigations and to save time . Treatment with an oral quinolone (ciprofloxacin), particularly in aged patients when laboratory investigations are difficult to perform, could be a valuable option.
In clinical practice, paradoxical clinical results that require alternative strategies may be encountered . Here we present a case of a patient with results of cardiac catheterization that are incompatible with st segment elevation and elevated cardiac enzymes . Although technologically advanced diagnostic examinations are becoming increasingly available, noninvasive bedside ultrasound examination remains to be indispensable . Although cardiac myxomas are relatively uncommon compared with coronary and valve lesions in the elderly, they are easily diagnosed by basic echocardiography; this diagnostic test may be the key to solving puzzling presentations and should a part of most cardiac workups . Stroke is a complication in 10% to 30% of cardiac myxoma patients as found in a case series spanning 20 years in belgium and a 11-year series conducted at the mayo clinic; no cases of cardiac myxoma in either study had concurrent myocardial infarction . Forty cases of myxoma - related myocardial infarction were recorded in a systematic review of published data spanning 32 years . This case report shares our experience with a rare case of cardiac myxoma with concurrent stroke and st - elevation myocardial infarction with normal coronary angiography . Signed consent from the patient was obtained, and approval was granted by the institutional review board of taipei tzuchi hospital for this case report . A 67-year - old unconscious woman presented to the emergency department of our hospital within 30 minutes of collapsing at an outdoor market . She was afebrile with blood pressure of 100/80 mm hg, heart rate of 90 beats per minute, and respiratory rate of 26 breaths per minute on mask oxygen . On physical examination, the patient displayed grimace in response to painful stimuli, pupils were equal and reactive to light, heart beats were regular without obvious murmurs, breath sounds were coarse without wheezing, abdomen was soft and nontender, urine was clear with adequate output into a foley bag, and legs were warm and nonedematous; examination was otherwise unremarkable . Electrocardiogram showed significant st elevation in the inferior and lateral leads (figure 1). Chest x - ray showed lung congestion, minimal effusion in the pericardial and pleural spaces, and a narrow mediastinum . Computed tomography of the brain showed no hemorrhage or detectable ischemia . Although awaiting cardiac catheterization, she regained consciousness and was able to follow commands and move all extremities . However, severe orthopnea with respiratory distress developed, and she was endotracheally intubated . We then performed echocardiography, which revealed an oscillating round mass measuring 3 3 cm inside the left atrium attached by a pedicle to the interatrial septum that was obstructing inflow through the mitral valve during diastole (figure 2). Although in the intensive care unit, the patient's lung edema deteriorated rapidly, resulting in cardiogenic shock that required increased inotropic support and high - setting ventilator support . Echocardiograph of the oscillating left atrial myxoma; surgical specimen of the completely resected atrial myxoma . The myxoma was resected en bloc with its pedicle and adjacent interatrial septum (figure 2). Ataxia was noted during rehabilitation, and magnetic resonance imaging of the brain revealed left cerebellar infarction . Cardiac atrial myxoma is uncommon, with an estimated incidence of <0.1% by autopsy series . Often initially asymptomatic, symptoms arise because of obstructive, embolic, or constitutional events . A 20-year surgical cohort of cardiac myxoma cases in belgium estimated 65.6% of presenting symptoms to be obstructive and 15.6% to be embolic . As in our case, emergent resection may be necessary if cardiogenic shock develops because of mitral valve obstruction . Echocardiography can readily reveal obstruction by a cardiac myxoma, usually of mitral valve inflow, allowing timely determination of severity and necessity for emergent surgical resection . Myxoma - related stroke is relatively more common than myxoma - related myocardial infarction for myxoma - related embolic events . In the aforementioned 20-year myxoma surgical series, brain ischemia was involved in 15.6% of cases, brain and peripheral artery embolic ischemia in 6.2%, and angina in 3.1%, whereas no cases involved myocardial infarction . Myxoma - related coronary artery embolism is rare and much less common than brain embolism . Estimated incidence of myocardial infarction in cardiac myxoma ranges from close to 0% to as high as 10% if coronary emboli is included . A review of cardiac myxoma cases spanning 11 years conducted by the mayo clinic found that 12% of cases had image - evident stroke . Therefore, coexisting stroke and myocardial infarction, as in our case, are exceedingly unusual . Myocardial infarction with normal immediate angiography is rare for cardiac myxoma . In a systematic review of published data from 1970 to 2002, patent coronary arteries were found only on latent angiography (performed more than 2 weeks after the initial event), but not on immediate angiography . Negative latent angiography may be explained by recanalization performed days or weeks after the initial coronary events . Possible explanations may include rapid and spontaneous recanalization, a washout effect of embolic myxoma fragments or induced thrombi, or compromised hemodynamics because of mitral valve obstruction . Echocardiography, a noninvasive and portable examination, is often critical for properly identifying the etiology of cardiac conditions such as acute heart failure . Basic echocardiography, or focused cardiac ultrasound, is easily learned and often sufficient for detecting intracardiac masses or intrathoracic effusion . Our case demonstrates the importance of basic echocardiography for optimizing the management of cardiac cases, and supports the recommendation that basic echocardiography be readily available for most cardiac teams or acute care centers . Our atypical case of normal immediate coronary angiography in myxoma - related myocardial infarction with mitral valve obstruction and multisystem embolism (heart and brain) deserves attention because of the rarity of this combination of presentations . The case stresses the value of basic echocardiography in the efficient and effective management of the patient and the importance of including it in most cardiac workups . In addition, this experience is a reminder for clinicians to include cardiac myxoma in the differential diagnosis list and to manage it timely if found . Although cardiac myxoma may have unexpected presentations, it can be easily diagnosed by basic echocardiography and should not be missed . Once diagnosed, cardiac myxoma requires timely surgical en bloc resection to avoid deterioration resulting from worsening embolism or obstruction.
Major depressive disorder (mdd) is a common disease in patients with coronary heart disease (chd), with a prevalence of 20% to 30% [14] and is associated with worse cardiac prognosis . The american heart association (aha) and american academy of family physicians (aafp) have recommended routine depression screening for cardiac patients, including those suffering an myocardial infarction (mi). However, the uk national institute for health care effectiveness does not recommend the routine depression screening in primary care because of limited / conflicting findings on the optimal screening tools . After screening, further psychosocial assessment of patients who respond positively to the screening tools is considered to effect treatment evaluation . Depression has been categorized by 2 commonly used psychiatric classifications: the diagnostic and statistical manual of mental disorders (dsm - iv) and the international classification of diseases (icd-10). The criteria for the categorization are based on the severity, sub - types of specific symptoms, duration, course, and whether it is secondary to a physical or other psychiatric condition . Mdd is diagnosed if participants meet at least 1 core criterion (depressed mood or anhedonia) and at least 4 additional criteria within the previous 2 weeks . In addition to these 2 diagnostic criteria, several well - established screening tools have been developed and include self - report questionnaires and observer rating scales for depression . Phq-9 is a self - rating instrument for depression developed in the late 1990s for the primary care evaluation of mental disorders (prime - md). It consists of 9 items designed to correspond to the dsm - iv for mdd . The hospital anxiety and depression scale (hads) rating scale has 14 items, 7 of which are designed to measure anxiety (hads - a), and 7 for depression (hads - d). Hads - d was developed to assess depression in medically ill patients and its items focus on the loss of interest and pleasure with somatic features excluded from measurement . Phq-9 is mainly used in north american while hads is more frequently used in europe . Furthermore, phq-9 and hads differ in important ways, such as the exclusion of somatic symptoms in the latter . The beck depression inventory (bdi) is 1 of the most commonly used self - rating scales in the evaluation of depression . Since the development of this tool in 1961, it has been employed in numerous empirical studies . Bdi has recently been updated (the beck depression intervention - ii [bdi - ii]) to better match the current definition of mdd, as it measures symptoms in the preceding 2 weeks, as compared to 1 week in the bdi . Bdi - ii also has fewer items used to assess the somatic symptoms of depression because the somatic symptoms may confound the diagnosis of depression in post - mi patients . The 90-item symptom check list (scl-90) has been proven to be a useful tool for identifying psychiatric symptoms in primary care and research . Ces - d, a 20-item questionnaire with the total score ranging from 0 to 60, has been used extensively in chd patients, demonstrating utility, and a cut - off value of 16 has been found to be adequately sensitive and specific in the identification of chd . The 17-item hamilton depression rating scale (ham - d-17) is an observer rating scale used to evaluate the severity of depression . There is currently no consensus on which commonly used screening tool is best for use in identifying mdd in chd . Furthermore, mdd is under - diagnosed in chd patients because healthcare providers rarely use a standardized screening instrument . To the best of our knowledge, no systematic review has been published to comparatively evaluate the screening tools against the diagnostic criteria from depression in dsm - iv and icd-10 in chd patients . To investigate the performance of screening tools in identifying mdd in chd patients, this systematic review was performed to assess the diagnostic accuracy (focusing on sensitivity and specificity) of various screening tools as compared to the diagnostic criteria . Two reviewers independently searched the following electronic databases in english: ovid medline, embase, psycinfo, scopus, cochrane library, cinahl plus, and web of science before 31 dec 2013 ., following terms were used for searching: coronary heart disease, ischemic heart disease or heart disease; to identify relevant screening tools, a second search was performed using the following terms: tool, measurement, or assessment; and to identify studies related to depression, depress * was used as a term for searching . Then, they performed full - text reviewing on each article meeting the inclusion criteria or having some uncertainties for eligibility . Any disagreements in data extraction and/or specific study inclusion were resolved through consensus by discussion . Inclusion criteria were: participants were diagnosed as having chd and randomly recruited from different healthcare settings, including hospitals and communities . The diagnostic criteria for depression were from dsm - iv of the american psychiatric association or icd-10 of the world health organization . The studies included mdd or severe depressive episode (mde) diagnosed according to corresponding diagnostic criteria, other than minor depression or depressive syndrome . Exclusion criteria were: participants were diagnosed with other cardiovascular diseases, including cardiomyopathy and heart failure other than chd . Studies did not clearly compare the screening tools with diagnostic criteria in dsm or icd . Studies meeting the inclusion criteria were assessed for methodological quality using the quality assessment of diagnostic accuracy studies (quadas), which is a tool used for quality assessment included in systematic reviews . Each study was independently assessed by 2 authors, and data were extracted from articles according to the collecting data items of quadas and cochrane - handbook . The following data were extracted: settings, gender, mean age, stage of depression, screening tools, diagnostic criteria, sensitivity and specificity of screening tools, and numbers of patients with mdd diagnosed by diagnostic criteria . The sensitivity, specificity, likelihood ratios, and predictive values were determined and the binomial 95% confidence intervals were calculated for the sensitivity and specificity with reference manager (revman) version 5.1 from the cochrane collaboration . Two reviewers independently searched the following electronic databases in english: ovid medline, embase, psycinfo, scopus, cochrane library, cinahl plus, and web of science before 31 dec 2013 ., following terms were used for searching: coronary heart disease, ischemic heart disease or heart disease; to identify relevant screening tools, a second search was performed using the following terms: tool, measurement, or assessment; and to identify studies related to depression, depress * was used as a term for searching . Then, they performed full - text reviewing on each article meeting the inclusion criteria or having some uncertainties for eligibility . Any disagreements in data extraction and/or specific study inclusion were resolved through consensus by discussion . Inclusion criteria were: participants were diagnosed as having chd and randomly recruited from different healthcare settings, including hospitals and communities . The diagnostic criteria for depression were from dsm - iv of the american psychiatric association or icd-10 of the world health organization . The studies included mdd or severe depressive episode (mde) diagnosed according to corresponding diagnostic criteria, other than minor depression or depressive syndrome . Exclusion criteria were: participants were diagnosed with other cardiovascular diseases, including cardiomyopathy and heart failure other than chd . Studies did not clearly compare the screening tools with diagnostic criteria in dsm or icd . Studies meeting the inclusion criteria were assessed for methodological quality using the quality assessment of diagnostic accuracy studies (quadas), which is a tool used for quality assessment included in systematic reviews . Each study was independently assessed by 2 authors, and data were extracted from articles according to the collecting data items of quadas and cochrane - handbook . The following data were extracted: settings, gender, mean age, stage of depression, screening tools, diagnostic criteria, sensitivity and specificity of screening tools, and numbers of patients with mdd diagnosed by diagnostic criteria . The sensitivity, specificity, likelihood ratios, and predictive values were determined and the binomial 95% confidence intervals were calculated for the sensitivity and specificity with reference manager (revman) version 5.1 from the cochrane collaboration . After searching ovid medline, embase, psycinfo, scopus, cochrane library, cinahl plus, and web of science with corresponding terms, a total of 3878 articles were identified by the initial screening and included 769 from ovid medline, 411 from embase, 562 from psycinfo, 887 from scopus, 288 from cochrane library, 229 from cinahl plus, and 662 from web of science ., there were no diagnostic criteria for depression, 3 studies were conducted from 3 different viewpoints in the same population, and randomization was not performed in 1 study . Finally, 8 studies were included for further meta - analysis in accordance with the prisma flow chart . We re - analyzed the data of included studies with the methodology, the summary receiver operating characteristic (sroc), and the bivariate approach . In 8 studies included, there were 10 self - reporting questionnaires (such as phq-2, phq-9, phq categorical algorithm, hads - d, bdi, bdi - ii, bdi - ii - cog, ces - d, scl-90, 2 simple yes / no items) and 1 observer rating scale (ham - d). In some self - reporting questionnaires, the data were insufficient to calculate the sroc and the pooled statistic sensitivity and specificity of each screening tool and different cut - off values . Thus, the positive lr, negative lr, positive predictive value (ppv), negative predictive value (npv), and prevalence were calculated using revman 5.1 . As shown in table 2, for mdd alone, the sensitivity and specificity of different screening tools at the validity and optimal cut - off point varied from 0.34 [0.19, 0.52] to 0.96 [0.78, 1.00] and from 0.69 [0.65, 0.73] to 0.97 [0.93, 0.99], respectively . The quadas was used to assess the quality of included studies, in which the patient spectrum, diagnostic criteria, disease progression bias, verification bias, clinical review bias, incorporation bias, test execution, study withdrawals, and indeterminate results were evaluated . Assessment of the spectrum bias showed that participants in 8 studies were representative of chd patients . Although 5 different diagnostic criteria, such as the revised clinical interview schedule (cis - r), the mini international neuropsychiatric interview (mini), the structure clinical interview for dsm - iv axis disorders (scid), the diagnostic interview schedule (dis) and the structure clinical interview (sci), were used in the 8 studies, patients met the criterion standard for mdd according to dsm - iv or icd-10 . Ideally, the results of the index test and the reference standard should be collected from the same patients at the same time . If this is impossible and a delay occurs, misclassification may be present due to spontaneous recovery or disease progression . Assessment of disease progression bias showed 6/8 studies had high quality . In addition, there was no partial verification bias, differential verification bias, or incorporation bias . Sufficient description of index test and the reference standard were present in all the studies . Assessment of review bias showed 4/8 studies were blinded between index test and reference standard, which can be used in practice when test results are interpreted . There was explanation of withdrawals and no report of test results that were intermediate or could not be interpreted in any of the studies . After searching ovid medline, embase, psycinfo, scopus, cochrane library, cinahl plus, and web of science with corresponding terms, a total of 3878 articles were identified by the initial screening and included 769 from ovid medline, 411 from embase, 562 from psycinfo, 887 from scopus, 288 from cochrane library, 229 from cinahl plus, and 662 from web of science ., there were no diagnostic criteria for depression, 3 studies were conducted from 3 different viewpoints in the same population, and randomization was not performed in 1 study . Finally, 8 studies were included for further meta - analysis in accordance with the prisma flow chart . We re - analyzed the data of included studies with the methodology, the summary receiver operating characteristic (sroc), and the bivariate approach . In 8 studies included, there were 10 self - reporting questionnaires (such as phq-2, phq-9, phq categorical algorithm, hads - d, bdi, bdi - ii, bdi - ii - cog, ces - d, scl-90, 2 simple yes / no items) and 1 observer rating scale (ham - d). In some self - reporting questionnaires, the data were insufficient to calculate the sroc and the pooled statistic sensitivity and specificity of each screening tool and different cut - off values . Thus, the positive lr, negative lr, positive predictive value (ppv), negative predictive value (npv), and prevalence were calculated using revman 5.1 . As shown in table 2, for mdd alone, the sensitivity and specificity of different screening tools at the validity and optimal cut - off point varied from 0.34 [0.19, 0.52] to 0.96 [0.78, 1.00] and from 0.69 [0.65, 0.73] to 0.97 [0.93, 0.99], respectively . The quadas was used to assess the quality of included studies, in which the patient spectrum, diagnostic criteria, disease progression bias, verification bias, clinical review bias, incorporation bias, test execution, study withdrawals, and indeterminate results were evaluated . Assessment of the spectrum bias showed that participants in 8 studies were representative of chd patients . Although 5 different diagnostic criteria, such as the revised clinical interview schedule (cis - r), the mini international neuropsychiatric interview (mini), the structure clinical interview for dsm - iv axis disorders (scid), the diagnostic interview schedule (dis) and the structure clinical interview (sci), were used in the 8 studies, patients met the criterion standard for mdd according to dsm - iv or icd-10 . Ideally, the results of the index test and the reference standard should be collected from the same patients at the same time . If this is impossible and a delay occurs, misclassification may be present due to spontaneous recovery or disease progression . In addition, there was no partial verification bias, differential verification bias, or incorporation bias . Sufficient description of index test and the reference standard were present in all the studies . Assessment of review bias showed 4/8 studies were blinded between index test and reference standard, which can be used in practice when test results are interpreted . There was explanation of withdrawals and no report of test results that were intermediate or could not be interpreted in any of the studies . In this study, 8 studies conducted in chd patients from primary care settings and hospitals were systemically reviewed . The sensitivity and specificity of screening tools were different from the diagnostic criteria in identifying mdd . The screening tools had the highest sensitivity without affecting the specificity in identifying the greatest number of true - positives . For screening, sensitivity should be maximized when choosing a screening tool for depression so that cases are not missed . In our study, ces - d (16) and scl-90 (25) had high sensitivity and npv as compared to other screening tools, with the sensitivity of 93% and 96%, respectively, and npv of 97.54% and 99.24%, respectively . However, the low ppv of these 2 tools (54.79% and 32.35%, respectively) meant that only about half of patients who have a positive result on the screening meet the diagnostic criteria for major depression . Thus, any patient who has positive results on depression screening should be followed up to confirm the diagnosis of depression . For diagnosis, the ppv depends, in part, on the prevalence of the disorder in the population . Due to the relatively low number of depressed patients as compared to non - depressed patients in all the studies, the ppvs (22.99% to 70.59%) were much lower than npvs (84.31% to 79.24%). A cut - off value of 10 on phq-9 had the sensitivity of only 54% in 2 studies, but its high specificity (90% and 91%) and high ppv (57.58% and 60.20%) mean that patients who screen positive do not need a follow - up for confirming the diagnosis of depression . It has been found that up to 30% of patients having stable heart disease also develop depression [14]. The prevalence of mdd in chd patients in the included studies varied from 4.38% to 22.05% . The study with the lowest prevalence (4.38%) was performed in chd patients from primary care settings and is similar to the 12-month prevalence of 4% to 7% in the communities, as previously reported . The optimal cut - off value is another important factor in the comparisons of the accuracy among various measures . For phq-9, the optimal cut - off value of 10 for the identification of mdd was consistent in 2 included studies . A study on the phq-9 indicated that a lower cut - off value (8) resulted in an increased sensitivity with only modest reduction in the specificity when compared with the recommended cut - off value (10). Some optimal cut - off values were lower than generally recommended, particularly in the screening for mdd . Lowering the cut - off value substantially improves the sensitivity of these tools while retaining the specificity, thereby improving their usefulness in screening for depression in chd patients . In the study of haddad et al ., results showed that the performance of hads - d at a standard cut - off value (8) was weaker, with a sensitivity of 53% and specificity of 91%, a large proportion of depression patients were not diagnosed under this condition, and the satisfactory performance was found when the cut - off value was 5 or above (8) with the sensitivity of 81.3% and specificity of 76.7% . However, there were no subgroups in the assessment of mdd and the accuracy of hads - d was not evaluated . The other 3 studies on hads - d showed that the recommended cut - off value was 5 in 2 studies and 4 in 1 study . The sensitivity of 77%, 86%, and 87%, respectively, and the specificity of 69%, 75%, and 75%, respectively, were comparable among 3 studies . For bdi - ii, cut - off values were different in 3 included studies . The recommended cut - off value for bdi - ii (14) was used in the studies of bunevicius et al . And frasure - smith et al ., and their results showed it was effective to screen mdd with good sensitivity (89% and 74%, respectively) and specificity (91% and 78%, respectively). In the study of huffman et al ., the cut - off value of 14 resulted in sensitivity of 88.2% and specificity of 84.2% . However, a bdi - ii score of 16, which had equivalent sensitivity (88.2%) and better specificity (92.1%), was recommended for mdd . Both cut - off values resulted in very few cases with a false - negative (npc=98.13% for 16 and npv=97.96 for 14), which is important for a good screening tool . Using a cut - off of 16, 62.5% of patients with a positive result in screening were found to have mdd, and the cut - off value of 14 had a lower ppv (45.45%). Depression is under - recognized in chd patients because healthcare providers rarely use standardized screening tools and there is no consensuses which of the available screening tools should be used . In 2008, the aha recommended systematic screening using phq-2 for depression in all chd patients . However, this guideline is challenged because there remains a paucity of evidence that systematic screening for depression is helpful to improve the outcomes of chd patients [3840]. Patients with false - positive results in the screening are at increased risk for receiving unnecessary anti - depressive treatment . Studies have demonstrated that although primary providers can provide effective therapies without referral for up to 75% of patients with depression, most patients are unrecognized or inappropriately treated . After appropriate referral, psychosocial interventions may improve the physiological function and decrease the cardiovascular morbidity and mortality in chd patients . First, meta - analysis was not performed to assess the pool - statistic such as sensitivity, specificity, and predictive values because of the small number of each tool in studies that met the inclusion criteria . Second, studies included in this review only targeted the identification of mdd rather than minor depression, depression syndrome, and depression of different severities . Apart from mdd, minor depression also has an influence on the morbidity and mortality of chd patients . Third, there are different semi - structured methods used to determine the interview - based diagnosis, including cis - r, mini, acid, and dis, all of which have different diagnostic accuracies . Another limitation is the lack of cost - effectiveness analysis in the identification of mdd, and thus whether the cost also influences the false - positives of screening tools is still unclear . In the absence of systematic screening to recognize (and thus treat) chd is difficult for the front - line clinicians in the in - patient and primary case settings . To the best of our knowledge, this is the first systematic review in which various depression screening tools were compared with the diagnostic criteria in identification of mdd among chd patients . In our study, phq-9 (10), bdi - ii (14 or 16), and hads - d (5 or 4) when the performance of a screening tool is evaluated, the high sensitivity and npv should be balanced with the high specificity and ppv, which will provide useful guidance for the application of appropriate tools and optimal cut - off value in the identification of depression in chd patients . Taking into account psychometric properties and ease of use, effective screening tools should be integrated into clinical care . After screening, further diagnosis,
We encountered an adult patient (index case, case 1) with unique benign unilateral cerebral cortical encephalitis manifesting with generalized epileptic seizure and seropositivity for mog antibody in 2014 . To explore any other cases with similar features, we identified 24 consecutive patients diagnosed with steroid - responsive encephalitis of unknown etiology seen at tohoku university hospital from 2008 to 2014 . The patients were older than 20 years and were followed for more than 19 months . We defined steroid - responsive encephalitis of unknown etiology as cases with encephalopathy (epileptic seizure, abnormal behavior, disturbance of consciousness, or focal brain symptoms) that responded to corticosteroid therapy and could not be explained by fever, systemic illnesses, or postictal symptoms . Additional criteria included abnormal brain mri and csf findings during the acute phase that were compatible with encephalitis and not indicative of alternative cns diseases . Sera and csf were collected during the acute phases and were stored at 80c . In some cases, we conducted live cba for mog antibody based on our previous reports with modification (we used anti - human igg1 as the secondary antibody to avoid nonspecific binding). Briefly, full - length mog - expressing or mog - nonexpressing stable cell lines were incubated with a 1:16 dilution of serum and then incubated with a 1:400 dilution of alexa fluor 488 mouse anti - human igg1 antibody (a10631; thermo fisher scientific, rockford, il). After cell immunostaining, 2 investigators (r.o . And t.t . ), who were blinded to patients' data, judged mog antibody positivity by comparing the staining results of mog - expressing and mog - nonexpressing cells . In mog antibody positive samples, the antibody titers were calculated by consecutive twofold dilutions to ascertain the maximum dilution with positive staining . Simultaneously, m23-aqp4 antibody in the serum was tested by live cba using alexa fluor 488 goat anti - human igg (a11008, thermo fisher scientific) as the secondary antibody . Anti - nmda receptor (nmdar) antibody, anti--amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (ampa) antibody, anti - leucine - rich glioma - inactivated protein 1 (lgi1) antibody, anti - contactin - associated protein 2 (caspr2) antibody, and anti--aminobutyric acid receptor type b receptor (gabab) antibody in the csf were tested by indirect immunofluorescence using commercially available kits (euroimmun, lbeck, germany). This study was approved by the institutional ethics committee, and all patients provided written informed consent . We encountered an adult patient (index case, case 1) with unique benign unilateral cerebral cortical encephalitis manifesting with generalized epileptic seizure and seropositivity for mog antibody in 2014 . To explore any other cases with similar features, we identified 24 consecutive patients diagnosed with steroid - responsive encephalitis of unknown etiology seen at tohoku university hospital from 2008 to 2014 . The patients were older than 20 years and were followed for more than 19 months . We defined steroid - responsive encephalitis of unknown etiology as cases with encephalopathy (epileptic seizure, abnormal behavior, disturbance of consciousness, or focal brain symptoms) that responded to corticosteroid therapy and could not be explained by fever, systemic illnesses, or postictal symptoms . Additional criteria included abnormal brain mri and csf findings during the acute phase that were compatible with encephalitis and not indicative of alternative cns diseases . Sera and csf were collected during the acute phases and were stored at 80c . In some cases, we conducted live cba for mog antibody based on our previous reports with modification (we used anti - human igg1 as the secondary antibody to avoid nonspecific binding). Briefly, full - length mog - expressing or mog - nonexpressing stable cell lines were incubated with a 1:16 dilution of serum and then incubated with a 1:400 dilution of alexa fluor 488 mouse anti - human igg1 antibody (a10631; thermo fisher scientific, rockford, il). After cell immunostaining, 2 investigators (r.o . And t.t . ), who were blinded to patients' data, judged mog antibody positivity by comparing the staining results of mog - expressing and mog - nonexpressing cells . In mog antibody positive samples, the antibody titers were calculated by consecutive twofold dilutions to ascertain the maximum dilution with positive staining . Simultaneously, m23-aqp4 antibody in the serum was tested by live cba using alexa fluor 488 goat anti - human igg (a11008, thermo fisher scientific) as the secondary antibody . Anti - nmda receptor (nmdar) antibody, anti--amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (ampa) antibody, anti - leucine - rich glioma - inactivated protein 1 (lgi1) antibody, anti - contactin - associated protein 2 (caspr2) antibody, and anti--aminobutyric acid receptor type b receptor (gabab) antibody in the csf were tested by indirect immunofluorescence using commercially available kits (euroimmun, lbeck, germany). This study was approved by the institutional ethics committee, and all patients provided written informed consent . In addition to the index case, 3 other patients were found to be seropositive for mog antibody . All of these patients were male, and the median onset age was 34 years (range 2339). All of the patients experienced epileptic seizures, 3 showed abnormal behavior, 2 patients had on (figure 1), and 1 patient had dysuria, but no patient had myelopathy . In csf examinations, the median cell count was 83/l (range 29311), and the median protein concentration was 46 mg / dl (range 3586). Myelin basic protein (mbp) in csf was not elevated in any of the 3 patients (cases 1, 2, and 3) whose csf samples were available, although they were positive for mog antibody in the csf . On brain mri examination, all 4 cases showed unilateral hemispheric cortical hyperintense lesions in fluid - attenuated recovery (flair) imaging (figure 2a and figure 3, a c, f h, j none of the mog antibody negative patients in our cohort of encephalitis patients showed such flair - hyperintense cortical lesions . The 4 mog antibody positive patients were treated with high - dose iv corticosteroids and antiepilepsy drugs, and they fully recovered . We also screened for other encephalitis - related autoantibodies, including aqp4, nmdar, ampa, lgi1, caspr2, and gabab antibodies, but negative results were obtained for all of the cases . Clinical features of 4 patients with unilateral cortical encephalitis positive for the mog antibody hyperintensities on axial short t1 inversion recovery (a, c) and gadolinium enhancement (b, d) were seen in the right optic nerves in cases 1 (a, b) and 2 (c, d), suggesting unilateral optic neuritis . When hospitalized with epileptic seizure, the right hemispheric cortical region in case 1 was fluid - attenuated inversion recovery hyperintense (a) (arrowheads) and partially gadolinium enhanced . Meanwhile, hyperintensities in the cortical region were less evident on diffusion - weighted (b), apparent diffusion coefficient (c), t2-weighted (d), t1-weighted (e), and gadolinium enhancement on t1-weighted (f) mri . On admission, fluid - attenuated inversion recovery (flair) hyperintensity was seen in the unilateral cerebral cortex in cases 1 (a c), 2 (f h), 3 (k n), and 4 (p r) (arrowheads). Brain spect showed hyperperfusion in those cerebral cortical regions with flair hyperintensity in cases 1 (d), 2 (i), and 4 (s). However, the flair hyperintensities in the cortical regions disappeared after more than 2 years (e, j, o, t). A 38-year - old man developed right eye pain and visual loss . His visual acuity (va) was 30/200, and the critical flicker frequency (cff) was 16.4 hz (normal> 35) in his right eye . He presented with a right relative afferent pupillary defect and color vision defect in the right eye . Regarding visual evoked potentials (vep), the amplitude of p100 was reduced with prolonged latency in the right eye (120.6 ms). This patient was diagnosed with idiopathic on (figure 1, a and b) and treated with high - dose iv methylprednisolone (himp) (1,000 mg / d for 3 days) followed by an oral prednisolone (psl) taper . Seven months later, the patient acutely developed loss of consciousness and generalized tonic seizure and was admitted to our hospital . On admission, he was alert, but his left hand was weak due to todd palsy . A csf study showed mild pleocytosis and elevated interleukin-6 (il-6; 72.6 pg / ml, normal <4.0 pg / ml). Glutamic acid decarboxylase (gad) antibody, thyroid peroxidase (tpo) antibody, and thyroglobulin (tg) antibody results were negative in the serum, but the mog antibody test was positive in the serum (1:512) and in the csf (1:32) (table 1). Eeg showed rhythmic slow waves in the right cerebral hemisphere but no epileptic discharge in the interictal stage . Brain mri scanned on the day of epilepsy onset showed flair hyperintensity in the right hemispheric cortical region, and the cortical layer was mildly swollen (figure 3, a slight hyperintensity in the regions of diffusion - weighted imaging (dwi) and t2-weighted imaging (t2wi) were seen but were less evident than in the flair image (figure 2, a we started carbamazepine (400 mg / d) and lamotrigine (25 mg / d), but 1 week later, the patient developed delirium, paranoia, hallucination, and anorexia . Subsequently, we made a presumptive diagnosis of autoimmune encephalitis to start himp therapy, and his symptoms disappeared within a few days . He continued oral prednisolone (15 mg / d, then gradually tapered to 4 mg / d in 18 months), carbamazepine, and lamotrigine and experienced no relapse thereafter . At 26 months after discharge a 36-year - old man let out a strange noise and lost consciousness for several minutes, resulting in a one - car accident when he was driving a car . He was admitted to a local hospital that day and was treated with carbamazepine (400 mg / d). Brain mri taken on admission showed a flair - hyperintense area in the right parietal cortex . After admission, he twice developed a generalized tonic seizure, right eye pain with visual loss, and dysuria . The cff was 25 hz, and vep revealed prolonged p100 latency (128.4 ms). Cbc and blood biochemistry results were normal, while the mog antibody test was positive in the serum (1:2,048) and in the csf (1:4) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The csf study showed mild pleocytosis and moderately elevated il-6 (840 pg / ml). The eeg results were normal when the patient was in the interictal stage . On mri examination 3 weeks after onset, the right optic nerve was swollen, short t1 inversion recovery hyperintense, and gadolinium - enhanced (figure 1, c and d). Flair hyperintensity in the right hemispheric cortex was seen, and the corresponding cortical layer was slightly swollen (figure 3, f h) and partially gadolinium - enhanced . Meanwhile, hyperintensity was less evident in dwi and t2wi in the cortical region, but brain spect showed hyperperfusion in the region (figure 3i). Whole - body pet - ct showed no abnormal uptake suspicious of malignancy or inflammation . With a presumptive diagnosis of autoimmune encephalitis four weeks after admission, the patient was discharged without any symptoms . Oral psl (25 mg / d, gradually tapered off in 2 years) and carbamazepine (400 mg / d) was continued, and he did not experience any relapse . At 40 months after discharge, the mog antibody titer was reduced (1:128) and brain mri showed no residual lesions (figure 3j). A 23-year - old man with involuntary movement of the left hand was diagnosed with epilepsy and was treated with carbamazepine (400 mg / d) with no apparent effect . One month later, he developed a generalized tonic seizure that lasted for 1 hour . Although the cbc and blood biochemistry results were normal, the mog antibody test was positive in the serum (1:256) and in the csf (1:16) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The eeg examination revealed rhythmic slow waves in the right hemisphere, especially in the right parietal region, but no epileptic discharge was seen in the interictal state . Brain mri scanned 1 month after the onset of epilepsy showed flair hyperintensity in the right hemispheric cortical region (figure 3, k n). Abnormalities in the region in dwi, t2wi, and gdt1wi were equivocal . Tests for cytomegalovirus antigen in the blood and mycobacterium tuberculosis (quantiferon) were negative, and pcr for herpes simplex virus (hsv), gram stains, and culture results were negative in the csf . However, because we could not rule out cns infectious disease, we initially treated the patient with iv ceftriaxone, isoniazid, ethambutol, acyclovir, fluconazole, and dexamethasone (33 mg / d). His symptoms disappeared soon after the treatment, and we suspected autoimmune encephalitis rather than cns infection . Four weeks after admission, he was discharged with no symptoms, but oral prednisolone (15 mg / d, gradually tapered off in a year) and carbamazepine (600 mg / d) were continued . Eighteen months later, he had not experienced a relapse, and the mog antibody was undetectable in the serum . A 38-year - old man was admitted to a local hospital with headache and abnormal behavior (he was unable to dress himself). After admission, he experienced a generalized tonic seizure, and he was transferred to our hospital . Because the cause of the generalized tonic seizure was unknown despite a diagnostic workup, he was discharged 1 week later (carbamazepine was continued). However, 35 months after the first admission, he developed a generalized tonic seizure, which initially involved the right hand . The cbc and blood biochemistry were normal, but the mog antibody test was positive in the serum (1:1,024) (table 1). Brain mri scanned 4 days after the second episode of epilepsy showed flair hyperintensity in the left hemispheric cortical region (figure 3, p pcr for hsv, gram stains, and culture results were negative in the csf . After admission, his symptoms became worse (agitation and violent behavior) despite the administration of sedatives . We suspected autoimmune encephalitis and started himp, after which time he became asymptomatic and was discharged . He continued carbamazepine (300 mg / d) and experienced no relapse . At 84 months after discharge, he was mog antibody - negative . A brain mri taken 72 months after discharge was normal (figure 3 t). His visual acuity (va) was 30/200, and the critical flicker frequency (cff) was 16.4 hz (normal> 35) in his right eye . He presented with a right relative afferent pupillary defect and color vision defect in the right eye . Regarding visual evoked potentials (vep), the amplitude of p100 was reduced with prolonged latency in the right eye (120.6 ms). This patient was diagnosed with idiopathic on (figure 1, a and b) and treated with high - dose iv methylprednisolone (himp) (1,000 mg / d for 3 days) followed by an oral prednisolone (psl) taper . Seven months later, the patient acutely developed loss of consciousness and generalized tonic seizure and was admitted to our hospital . On admission, he was alert, but his left hand was weak due to todd palsy . A csf study showed mild pleocytosis and elevated interleukin-6 (il-6; 72.6 pg / ml, normal <4.0 pg / ml). Glutamic acid decarboxylase (gad) antibody, thyroid peroxidase (tpo) antibody, and thyroglobulin (tg) antibody results were negative in the serum, but the mog antibody test was positive in the serum (1:512) and in the csf (1:32) (table 1). Eeg showed rhythmic slow waves in the right cerebral hemisphere but no epileptic discharge in the interictal stage . Brain mri scanned on the day of epilepsy onset showed flair hyperintensity in the right hemispheric cortical region, and the cortical layer was mildly swollen (figure 3, a slight hyperintensity in the regions of diffusion - weighted imaging (dwi) and t2-weighted imaging (t2wi) were seen but were less evident than in the flair image (figure 2, a we started carbamazepine (400 mg / d) and lamotrigine (25 mg / d), but 1 week later, the patient developed delirium, paranoia, hallucination, and anorexia . Subsequently, we made a presumptive diagnosis of autoimmune encephalitis to start himp therapy, and his symptoms disappeared within a few days . He continued oral prednisolone (15 mg / d, then gradually tapered to 4 mg / d in 18 months), carbamazepine, and lamotrigine and experienced no relapse thereafter . At 26 months after discharge a 36-year - old man let out a strange noise and lost consciousness for several minutes, resulting in a one - car accident when he was driving a car . He was admitted to a local hospital that day and was treated with carbamazepine (400 mg / d). Brain mri taken on admission showed a flair - hyperintense area in the right parietal cortex . After admission, he twice developed a generalized tonic seizure, right eye pain with visual loss, and dysuria . The cff was 25 hz, and vep revealed prolonged p100 latency (128.4 ms). Cbc and blood biochemistry results were normal, while the mog antibody test was positive in the serum (1:2,048) and in the csf (1:4) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The csf study showed mild pleocytosis and moderately elevated il-6 (840 pg / ml). The eeg results were normal when the patient was in the interictal stage . On mri examination 3 weeks after onset, the right optic nerve was swollen, short t1 inversion recovery hyperintense, and gadolinium - enhanced (figure 1, c and d). Flair hyperintensity in the right hemispheric cortex was seen, and the corresponding cortical layer was slightly swollen (figure 3, f h) and partially gadolinium - enhanced . Meanwhile, hyperintensity was less evident in dwi and t2wi in the cortical region, but brain spect showed hyperperfusion in the region (figure 3i). Whole - body pet - ct showed no abnormal uptake suspicious of malignancy or inflammation . With a presumptive diagnosis of autoimmune encephalitis four weeks after admission, the patient was discharged without any symptoms . Oral psl (25 mg / d, gradually tapered off in 2 years) and carbamazepine (400 mg / d) was continued, and he did not experience any relapse . At 40 months after discharge, the mog antibody titer was reduced (1:128) and brain mri showed no residual lesions (figure 3j). A 23-year - old man with involuntary movement of the left hand was diagnosed with epilepsy and was treated with carbamazepine (400 mg / d) with no apparent effect . One month later, he developed a generalized tonic seizure that lasted for 1 hour . Although the cbc and blood biochemistry results were normal, the mog antibody test was positive in the serum (1:256) and in the csf (1:16) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The eeg examination revealed rhythmic slow waves in the right hemisphere, especially in the right parietal region, but no epileptic discharge was seen in the interictal state . Brain mri scanned 1 month after the onset of epilepsy showed flair hyperintensity in the right hemispheric cortical region (figure 3, k n). Abnormalities in the region in dwi, t2wi, and gdt1wi were equivocal . Tests for cytomegalovirus antigen in the blood and mycobacterium tuberculosis (quantiferon) were negative, and pcr for herpes simplex virus (hsv), gram stains, and culture results were negative in the csf . However, because we could not rule out cns infectious disease, we initially treated the patient with iv ceftriaxone, isoniazid, ethambutol, acyclovir, fluconazole, and dexamethasone (33 mg / d). His symptoms disappeared soon after the treatment, and we suspected autoimmune encephalitis rather than cns infection . Four weeks after admission, he was discharged with no symptoms, but oral prednisolone (15 mg / d, gradually tapered off in a year) and carbamazepine (600 mg / d) were continued . Eighteen months later, he had not experienced a relapse, and the mog antibody was undetectable in the serum . A 38-year - old man was admitted to a local hospital with headache and abnormal behavior (he was unable to dress himself). After admission, he experienced a generalized tonic seizure, and he was transferred to our hospital . Because the cause of the generalized tonic seizure was unknown despite a diagnostic workup, he was discharged 1 week later (carbamazepine was continued). However, 35 months after the first admission, he developed a generalized tonic seizure, which initially involved the right hand . The cbc and blood biochemistry were normal, but the mog antibody test was positive in the serum (1:1,024) (table 1). Brain mri scanned 4 days after the second episode of epilepsy showed flair hyperintensity in the left hemispheric cortical region (figure 3, p pcr for hsv, gram stains, and culture results were negative in the csf . After admission, his symptoms became worse (agitation and violent behavior) despite the administration of sedatives . We suspected autoimmune encephalitis and started himp, after which time he became asymptomatic and was discharged . He continued carbamazepine (300 mg / d) and experienced no relapse . At 84 months after discharge, he was mog antibody - negative . A brain mri taken 72 months after discharge was normal (figure 3 t). His visual acuity (va) was 30/200, and the critical flicker frequency (cff) was 16.4 hz (normal> 35) in his right eye . He presented with a right relative afferent pupillary defect and color vision defect in the right eye . Regarding visual evoked potentials (vep), the amplitude of p100 was reduced with prolonged latency in the right eye (120.6 ms). This patient was diagnosed with idiopathic on (figure 1, a and b) and treated with high - dose iv methylprednisolone (himp) (1,000 mg / d for 3 days) followed by an oral prednisolone (psl) taper . Seven months later, the patient acutely developed loss of consciousness and generalized tonic seizure and was admitted to our hospital . On admission, he was alert, but his left hand was weak due to todd palsy . A csf study showed mild pleocytosis and elevated interleukin-6 (il-6; 72.6 pg / ml, normal <4.0 pg / ml). Glutamic acid decarboxylase (gad) antibody, thyroid peroxidase (tpo) antibody, and thyroglobulin (tg) antibody results were negative in the serum, but the mog antibody test was positive in the serum (1:512) and in the csf (1:32) (table 1). Eeg showed rhythmic slow waves in the right cerebral hemisphere but no epileptic discharge in the interictal stage . Brain mri scanned on the day of epilepsy onset showed flair hyperintensity in the right hemispheric cortical region, and the cortical layer was mildly swollen (figure 3, a slight hyperintensity in the regions of diffusion - weighted imaging (dwi) and t2-weighted imaging (t2wi) were seen but were less evident than in the flair image (figure 2, a we started carbamazepine (400 mg / d) and lamotrigine (25 mg / d), but 1 week later, the patient developed delirium, paranoia, hallucination, and anorexia . Subsequently, we made a presumptive diagnosis of autoimmune encephalitis to start himp therapy, and his symptoms disappeared within a few days . He continued oral prednisolone (15 mg / d, then gradually tapered to 4 mg / d in 18 months), carbamazepine, and lamotrigine and experienced no relapse thereafter . At 26 months after discharge a 36-year - old man let out a strange noise and lost consciousness for several minutes, resulting in a one - car accident when he was driving a car . He was admitted to a local hospital that day and was treated with carbamazepine (400 mg / d). Brain mri taken on admission showed a flair - hyperintense area in the right parietal cortex . After admission, he twice developed a generalized tonic seizure, right eye pain with visual loss, and dysuria . The cff was 25 hz, and vep revealed prolonged p100 latency (128.4 ms). Cbc and blood biochemistry results were normal, while the mog antibody test was positive in the serum (1:2,048) and in the csf (1:4) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The csf study showed mild pleocytosis and moderately elevated il-6 (840 pg / ml). The eeg results were normal when the patient was in the interictal stage . On mri examination 3 weeks after onset, the right optic nerve was swollen, short t1 inversion recovery hyperintense, and gadolinium - enhanced (figure 1, c and d). Flair hyperintensity in the right hemispheric cortex was seen, and the corresponding cortical layer was slightly swollen (figure 3, f h) and partially gadolinium - enhanced . Meanwhile, hyperintensity was less evident in dwi and t2wi in the cortical region, but brain spect showed hyperperfusion in the region (figure 3i). Whole - body pet - ct showed no abnormal uptake suspicious of malignancy or inflammation . With a presumptive diagnosis of autoimmune encephalitis oral psl (25 mg / d, gradually tapered off in 2 years) and carbamazepine (400 mg / d) was continued, and he did not experience any relapse . At 40 months after discharge, the mog antibody titer was reduced (1:128) and brain mri showed no residual lesions (figure 3j). A 23-year - old man with involuntary movement of the left hand was diagnosed with epilepsy and was treated with carbamazepine (400 mg / d) with no apparent effect . One month later, he developed a generalized tonic seizure that lasted for 1 hour . Although the cbc and blood biochemistry results were normal, the mog antibody test was positive in the serum (1:256) and in the csf (1:16) (table 1). The gad antibody, tpo antibody, and tg antibody tests were negative in the serum . The eeg examination revealed rhythmic slow waves in the right hemisphere, especially in the right parietal region, but no epileptic discharge was seen in the interictal state . Brain mri scanned 1 month after the onset of epilepsy showed flair hyperintensity in the right hemispheric cortical region (figure 3, k n). Abnormalities in the region in dwi, t2wi, and gdt1wi were equivocal . Tests for cytomegalovirus antigen in the blood and mycobacterium tuberculosis (quantiferon) were negative, and pcr for herpes simplex virus (hsv), gram stains, and culture results were negative in the csf . However, because we could not rule out cns infectious disease, we initially treated the patient with iv ceftriaxone, isoniazid, ethambutol, acyclovir, fluconazole, and dexamethasone (33 mg / d). His symptoms disappeared soon after the treatment, and we suspected autoimmune encephalitis rather than cns infection . Four weeks after admission, he was discharged with no symptoms, but oral prednisolone (15 mg / d, gradually tapered off in a year) and carbamazepine (600 mg / d) were continued . Eighteen months later, he had not experienced a relapse, and the mog antibody was undetectable in the serum . A 38-year - old man was admitted to a local hospital with headache and abnormal behavior (he was unable to dress himself). After admission, he experienced a generalized tonic seizure, and he was transferred to our hospital . Because the cause of the generalized tonic seizure was unknown despite a diagnostic workup, he was discharged 1 week later (carbamazepine was continued). However, 35 months after the first admission, he developed a generalized tonic seizure, which initially involved the right hand . The cbc and blood biochemistry were normal, but the mog antibody test was positive in the serum (1:1,024) (table 1). Brain mri scanned 4 days after the second episode of epilepsy showed flair hyperintensity in the left hemispheric cortical region (figure 3, p pcr for hsv, gram stains, and culture results were negative in the csf . After admission, his symptoms became worse (agitation and violent behavior) despite the administration of sedatives . We suspected autoimmune encephalitis and started himp, after which time he became asymptomatic and was discharged . He continued carbamazepine (300 mg / d) and experienced no relapse . At 84 months after discharge, he was mog antibody - negative . A brain mri taken 72 months after discharge was normal (figure 3 t). In the index case (case 1), we tested for mog antibody because the patient had unilateral benign on rather than unilateral cortical encephalitis with epileptic seizure . Then, we tested for mog antibody in our cohort of 24 consecutive adult cases of corticosteroid - responsive encephalitis of unknown etiology and identified 3 additional patients with mog antibody positivity . Unexpectedly, these 3 mog antibody positive patients also had unilateral cortical encephalitis with epileptic seizure as seen in the index case, and there were no cases of unilateral cortical encephalitis with epileptic seizure without mog antibody positivity in our cohort . The unilateral cortical lesions best depicted by flair images were unique and appeared distinct from brain lesions previously described in mog antibody positive diseases including adem . The unilateral cortical lesions in our cases 14 needed to be differentiated from seizure - induced brain mri abnormalities . Such brain mri abnormalities induced by epileptic seizure are localized in the cortical / subcortical regions, hippocampus, basal ganglia, white matter, or corpus callosum, and they are readily visible on dwi due to cytotoxic changes . However, the mri findings in our cases were much more clearly seen in flair images than in dwi and adc findings (figure 1, a pleocytosis in the csf and a favorable response to himp suggested that the unique unilateral cortical lesions were inflammatory, and hyperperfusion on spect corresponding to the cortical flair hyperintensity supported the inflammatory nature and epileptogenicity of the swollen cortical lesions in the acute phase . We also ruled out a variety of autoantibody - mediated or immune - mediated encephalitides (table 2) before we concluded that the unilateral cortical encephalitis with epileptic seizure in our cases was unique . Rasmussen encephalitis (re) is described as unilateral cerebral cortical encephalitis, similar to that observed in our patients . However, re is clinically characterized by focal epilepsy, progressive hemiplegia, and cognitive decline with unilateral hemispheric focal cortical atrophy in the chronic stage, and corticosteroid and other anti - inflammatory therapies are only partially effective . The lesion distribution in our 4 patients was also dissimilar to the brain mri abnormalities in cases of encephalitis with seizure associated with nmdar antibody, vgkc antibody, gad antibody, and antithyroid antibodies, and our patients were negative for those autoantibodies . Likewise, the clinical and neuroimaging features of our cases were distinct from limbic encephalitides with positivity for gad, lgi1, gabab, or ampa antibodies and from the brain syndrome previously described in nmosd . Differential diagnosis of autoimmune or immune - mediated encephalopathy flair - hyperintense lesions localized at the cerebral cortex or sulcus, similar to the findings observed in the present cases, can develop in various cns diseases including meningitis, subarachnoid hemorrhage, leptomeningeal metastasis, acute infarction, and moyamoya disease . In a review of such mri abnormalities, the left temporo - occipital cortical flair - hyperintense lesions in a 23-year - old man with the diagnosis of meningitis appeared to be similar to the brain mri findings in our cases . More recently, numa et al . Reported a case of a 37-year - old woman who was diagnosed with adem when she was 4 years old and developed on followed by recurrent adem 33 years later . She was mog antibody positive, and brain mri showed unique cortical flair - hyperintense lesions in the left temporal and frontal lobes . Thus, unilateral cortical encephalitis in mog antibody positive patients, as in the 4 cases in our study, may have been previously unnoted as a distinct phenotype . The relationship between mog antibody and the unilateral cerebral cortical encephalitis observed in our cases remains unclear . Two of our patients had benign unilateral on, in which mog antibody is often detected, while cases 3 and 4 lacked such characteristics of cns diseases such as on, letm, nmosd, or adem . Thus, unilateral cerebral cortical encephalitis may be another characteristic manifestation of mog antibody positive patients . Although some cases of mog antibody associated diseases fulfill the diagnostic criteria of seronegative nmosd, the spectrum of mog antibody associated diseases is obviously wider than nmosd . In the near future, mog antibody associated diseases may be recognized as a distinct clinical entity of inflammatory demyelinating diseases of the cns . In addition, in a few brain - biopsied cases of tumefactive brain lesions with mog antibody positivity, pathologic examinations revealed active inflammatory demyelination with deposition of immunoglobulins and complement or ms type ii pathology . Moreover, we recently reported high csf - mbp levels without elevated csf glial fibrillary acidic protein levels, an astrocytic damage marker, in mog antibody positive patients . However, in 3 of our mog antibody positive cases whose csf - mbp levels were measured during the acute phase, there was no elevation in csf - mbp despite the extensive cortical involvement and csf pleocytosis . Thus, it is also possible that mog antibody itself may not be directly associated with the unilateral cerebral cortical encephalitis with epileptic seizure in our patients and that another autoimmune disorder coexisting with mog antibody positivity might be responsible for the encephalitis . In fact, mog antibody can be detected in some patients with other autoantibody - associated encephalitides such as nmdar antibody positive encephalitis . In addition, a pathogenic autoantibody may be generated years before the clinical onset of disease, as seen in aqp4 antibody positive nmosd . Accordingly, cns lesions associated with mog antibody may possibly develop later in the disease course of cases 3 and 4 . Therefore, an unknown autoantibody might be associated with the unilateral cerebral cortical encephalitis with epileptic seizure in a fraction of mog antibody positive cases although we need to perform immunohistochemistry or immunofluorescence with rodent brain tissue slices and the sera and csf from the patients as an attempt to see whether there are any antibody reactivities to the cerebral cortical tissues . Because our patient cohort was small and derived from a single university hospital, the results should be verified in prospective, larger - scale, multicenter studies . In addition, we analyzed only adult patients in the present study, and it is important to determine whether mog antibody positive unilateral cerebral cortical encephalitis with epileptic seizure also occurs in children . Therefore, at this point, it is premature to discuss the frequency of mog antibody positive unilateral cerebral cortical encephalitis in corticosteroid - responsive encephalitis of unknown etiology . However, since we experienced 6 cases of nmdar antibody associated encephalitis and 1 with vgkc antibody associated encephalitis during the same period (20082014), unilateral encephalitis with mog antibody may not be so uncommon . Taken together, we report a form of benign unilateral cerebral cortical encephalitis with epileptic seizure in 4 adult patients with mog antibody positivity . The pathogenesis of this condition appears to be immune - mediated or autoantibody - mediated, although the clinical, mri, and laboratory features differ from those in previously described mog antibody associated cns diseases and known autoantibody - mediated encephalitides . Another autoantibody that coexists with mog antibody may be responsible for this type of encephalitis . R.o . Analyzed the data and wrote the paper, substantial contribution to the study conception, acquisition, analysis, and interpretation of data for the work, writing the manuscript, drafting and correction of all versions of the manuscript including figures, tables, and references, completion of the work to be submitted, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Substantial contribution to the conception and design of the work, as well as supervision of the acquisition, analysis, and interpretation of data for the work, revised several versions of the manuscript critically for important intellectual content, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Substantial contribution to the conception and design of the work, as well as supervision of the acquisition, analysis, and interpretation of data for the work, revised several versions of the manuscript critically for important intellectual content, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Contribution to the plan of the work, acquisition, analysis, interpretation of data for the work, and drafting the original manuscript related to the case, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . . Substantial contribution to the conception and design of the work, as well as supervision of the acquisition, analysis, and interpretation of data for the work, revised several versions of manuscript critically for important intellectual content, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Substantial contribution to the conception and design of the work, as well as supervision of the acquisition, analysis, and interpretation of data for the work, provided final approval of the version to be published, agreed to be accountable for all aspects of the work . Substantial contribution to the conception and design of the work, as well as supervision of the acquisition, analysis, and interpretation of data for the work, supervision of the manuscript preparation, revised several versions of the manuscript critically for important intellectual content, final responsibility and approval of the version to be published, agreed to be accountable for all aspects of the work . This study was partially supported by a grant - in - aid for scientific research from the japan society for the promotion of science (kakenhi), the health and labour sciences research grant on intractable diseases (neuroimmunologic diseases) from the ministry of health, labour and welfare of japan . I. nakashima received travel funding and speaker honoraria from biogen japan, tanabe mitsubishi, and novartis pharma; is on the editorial board for multiple sclerosis international; and received research support from lsi medience corporation . T. takahashi received speaker honoraria from biogen idec and cosmic corporation and received research support from cosmic corporation . Y. takai received research support from ministry of education, culture, sports, science and technology of japan . Sato served on the advisory board for merck, teva, and shire; received speaker honoraria from novartis, genzyme, merck - serono, biogen, teva, bayer, and roche; is an associate editor for arquivos de neuropsiquiatria; and received research support from ministry of education, culture, sports, science and technology in japan, japan society for the promotion of science, and capes / brasil . T. misu received speaker honoraria from bayer schering pharma and biogen idec japan; and received research support from ministry of education, culture, sports, science and technology, ministry of health, labor and welfare of japan . H. kuroda received research support from ministry of education, culture, sports, science and technology of japan . M. aoki received research support from japanese ministry of health labor and welfare, japanese ministry of education, culture, sports, science and technology . K. fujihara serves on the advisory boards for bayer schering pharma, biogen idec, mitsubishi tanabe pharma corporation, novartis pharma, chugai pharmaceutical, ono pharmaceutical, nihon pharmaceutical, alexion pharmaceuticals, and medimmune; has received travel funding and speaker honoraria from bayer schering pharma, biogen idec, eisai inc, mitsubishi tanabe pharma corporation, novartis pharma, astellas pharma inc ., takeda pharmaceutical company limited, asahi kasei medical co., daiichi sankyo, and nihon pharmaceutical; is on the editorial board for clinical and experimental neuroimmunology; is an advisory board member for sri lanka journal of neurology; and received research support from bayer schering pharma, biogen idec japan, asahi kasei medical, the chemo - sero - therapeutic research institute, teva pharmaceutical, mitsubishi tanabe pharma, teijin pharma, chugai pharmaceutical, ono pharmaceutical, nihon pharmaceutical, genzyme japan, ministry of education, science and technology of japan, and ministry of health, welfare and labor of japan.
Corneal disorders are commonly initiated from inflammation, trauma, systemic disease, as well as pathological changes from adjacent tissues, which could eventually result in impaired vision, even blindness due to vascularization conjunctivalization, keratinization, corneal scarring, and opacification . In some severe cases, corneal reconstruction is a series of techniques for restoring the integrity and transparency of the cornea, and mainly refers to surgical techniques such as cornea transplantation, limbal stem cell (lsc) transplantation and amnion transplantation, and autologous oral mucosal epithelial transplantation . Other techniques involving biologic methods that aim to supply or stimulate the differentiation of lscs have been widely investigated . Recently, there is mostly evidence from in vivo or in vitro studies for using mesenchymal stem cells (mscs) in corneal reconstruction . Clinical trials are not available at the moment, and only one case has been reported for mscs in humans . Accumulated studies on the role of mscs in corneal reconstruction provided amplified additional evidence that mscs indeed modify the corneal microenvironment, though the exact mechanisms are still unknown . Related studies on the roles of mscs in the cornea are reviewed here to summarize the possible mechanisms and shed additional light . Although originally identified in the bone marrow, mscs have been found in many other tissues, including the adipose, heart, wharton s jelly, dental pulp, peripheral blood, cord blood, menstrual blood [9 - 11], fallopian tube, and limbal stroma of the human eye . These cells have self - renewal ability as undifferentiated cells and could differentiate into lineages of mesenchymal tissues, including bone, cartilage, fat, muscle, and marrow stroma . Under certain conditions, these cells could transdifferentiate into neurons or cardiac muscle cells [16 - 19]. Because of the low expression of major histocompatibility class ii (mhc ii) under unstimulated conditions and the absence of costimulatory molecules such as cluster of differentiation 40 (cd40), cluster of differentiation 40 ligand (cd40l), b71, and b72 on cell surface [20 - 24], mscs could escape the monitoring of the immune system and infuse into an allogeneic host without being rejected . Mscs can be administered directly to the cornea [26 - 31], or by carriers, such as the amniotic membrane [32 - 35] or fibrin gels . For clinical applications, the regenerative / reparative potential and the immune - suppressive capacity of mscs are the current areas of research focus . Many clinical studies on mscs led to positive results, which focused on the effects of mscs on regenerative medicine, preventing graft rejection and controlling graft versus host disease (gvhd). Mscs can transdifferentiate into other kinds of cells, including cardiomyocytes and neuronal cells [16 - 19]. However, hypotheses still need further evidence to establish that mscs play a role in corneal reconstruction . Gu demonstrated that mscs can differentiate into corneal epithelial - like cells in vivo and in vitro . In vivo, rabbit mscs (rb - mscs) were suspended in fibrin gels and transplanted onto the surface of naoh damaged rabbit corneas . As a result, the damaged corneal surface was restored after the rb - mscs were transplanted . Rb - mscs also participated in the healing process of the naoh injured corneal epithelium and expressed cytokeratin 3 (ck3), a corneal epithelial - specific marker . In vitro, rb - mscs differentiated into cells with a morphological and molecular phenotype of corneal epithelial - like cells that were positive to ck3 another in vivo study demonstrated that mscs have the ability to differentiate into corneal epithelial cells in experimental limbal stem cell deficiency rabbits . The expression of certain stem cell markers, such as adenosine 5'-triphosphate - binding cassette member 2 (abcg2), 1-integrin, and connexin 43, in the cornea epithelium after mscs transplantation indicated that mscs maintained stem cell characteristics; some mscs even transdifferentiated into epithelial progenitor cells . The in vivo study on human mscs (hmscs) found that hmscs could survive and migrate into the cornea stroma after being transplanted onto the surface of the alkali - burned rabbit cornea . Not only did the hmscs differentiate into the corneal epithelium, but also some even migrated into the corneal stroma and differentiated into cells other than epithelia . Another in vivo study found that when mscs were intrastromal - transplanted into keratocan - null (kera) mice, the cells survived in the cornea without evoking an immune and inflammatory response and expressed keratocan in the host kera mice . The investigators speculated that these corneal intrastromal - transplanted mscs may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes . Similarly, another in vivo study showed that intrastromal - transplanted umbilical mscs could survive similar to a keratocyte phenotype in the mouse corneal stroma . In an in vitro study, after coculture with corneal stromal cells (cscs), the induced mscs expressed positive staining for ck12 with the corneal epithelial cell characteristics confirmed with scanning electron microscopy . In addition, in vivo, the induced mscs had remarkable effects on treating the corneal alkali burn and reconstructing the corneal surface in a rat limbal stem cell deficiency model . In contrast, some researchers believed that mscs could not transdifferentiate into corneal epithelial cells in vivo . An in vivo study compared the transplantation of mscs with lscs concluded that mscs and lscs could assist the reconstruction of the damaged corneal surface in a rat corneal chemical burn model . However, the therapeutic mechanism was not associated with the epithelial differentiation from mscs because there was no sufficient evidence to support that mscs could differentiate into corneal epithelial cells . A later in vivo study had the same conclusion in a rat corneal chemical burn model . To a certain extent, the lack of sufficient evidence may be because keratocytes do not have specific markers and share many markers with mscs . Our in vivo study also found that subconjunctival injected mscs could not migrate into the injured cornea and transdifferentiate into corneal epithelial cells in a rat corneal alkali burn model . This may be closely related to how the mscs were administered . Therefore, it is still unclear whether mscs play a role in corneal reconstruction by the transdifferentiation effect, and the hypothesis requires further investigation . Mscs could ameliorate the inflammation in different damaged tissues, such as dextran sulfate sodium - induced colitis, acute kidney injury, and lung injury . The anti - inflammatory effect of mscs on the cornea was demonstrated on a rat corneal chemical burn model . The isolated hmscs from healthy donors grew and expanded on the amniotic membrane, followed by transplanting the membrane onto rat corneas 7 days after the chemical burns . Four weeks after transplantation, inflammation factors such as cluster of differentiation 45 (cd45), interleukin 2 (il-2), and matrix metalloproteinase-2 (mmp-2) decreased in the hmsc transplantation model detected with immunofluorescent stain . These findings indicated that the therapeutic effect of the damaged rat cornea treated with hmscs might be partially due to the inhibition of inflammation . An in vivo study further certified the anti - inflammatory effect of mscs on the cornea by detecting more inflammatory related factors . In this study, mscs were applied to the cornea directly without using the amniotic membrane as a carrier . Mscs decreased the expression of il-2 and interferon- (ifn-) in the rat cornea after chemical injury . However, increased expression of interleukin-10 (il-10), transforming growth factor-1 (tgf-1), and interleukin-6 (il-6) was also detected . In our study, we investigated the effects of subconjunctivally injected mscs in the acute stage of an alkali - burned rat cornea . After mscs were subconjunctivally injected, the infiltrated cd68 macrophages in the alkali - burned cornea were significantly decreased . The mrna expression levels of macrophage inflammatory protein-1 alpha (mip-1) and tumor necrosis factor - alpha (tnf-) were also downregulated . We speculate that mscs inhibit macrophage infiltration by suppressing the expression of macrophage chemokine mip-1 . In another in vivo study the investigators found that hmscs were effective in reducing corneal opacity and inflammation after either intraperitoneal or intravenous administration following cornea chemical injury . Regarding a specific mechanism, they found chemical injury to corneal epithelial cells could activate hmscs to secrete tnf- stimulated gene / protein 6 (tsg-6) in vitro, a multipotent anti - inflammatory protein . In addition, in vivo, the anti - inflammatory effects of hmscs were largely abrogated by knockdown of tsg-6 . Therefore, the authors speculated that systemic administration of hmscs reduced inflammatory damage to the chemically burned cornea primarily by secreting anti - inflammatory protein tsg-6 in response to injury signals from the damaged cornea . In summary, the anti - inflammatory effects of mscs on the cornea are undoubtedly apparent . However, the underlying mechanisms require clarification . Many studies found that mscs were good activators for angiogenesis, and mscs could secrete vascular endothelial growth factor (vegf) in an ischemia or tumor model [48 - 52]. However, mscs seemed to have an opposite effect on corneal angiogenesis . Some in vivo studies found that applying mscs on the cornea could effectively inhibit inflammation - related angiogenesis after chemical injury . Meanwhile, mmp-2, an inflammation - related proangiogenic factor, was significantly downregulated after mscs treatment . However, the level of vegf was similar between the control and msc - treated cases in an in vivo study of a rat corneal chemical burn model . In our in vivo study, we found that the level of vegf was downregulated after the msc subconjunctival injection in the acute stage of rat alkali - burned corneas . In vitro, the coculture of human corneal epithelial cells (hcecs) and hmscs upregulated the level of vegf . Furthermore, the hmscs constitutively expressed mmp-2 and tsp-1 . At the same time, hmscs significantly suppressed the secretion of mmp-9 from hcecs . Vegf, mmp-2, and mmp-9 are proangiogenic factors in the cornea, and tsp-1 is an antiangigenic factor, which could inhibit vegf - induced angiogenesis by cd36 activation [54 - 56]. Therefore, tsp-1 appears to be an antiangiogenic factor, which opposes the proangiogenic effect of vegf on the cornea in vivo . Similarly, mscs could also play a role in host versus graft disease (hvgd) [59 - 61]. A study on skin transplantation found that applying mscs could prolong baboon skin graft survival in vivo . In an in vivo study, mscs were efficient in heart transplantation by prolonging semiallogeneic heart graft survival, rather than a fully mhc - mismatched heart graft in a heart transplant mouse model . This study described a time dependency characteristic of mscs that the infusion of mscs was effective in prolonging graft survival when being used before transplantation, and partially effective during transplantation, but inefficient merely one day after transplantation . In the case of corneal transplantation, one of the most common causes of corneal allograft failure is irreversible rejection . In an in vivo study, allogeneic rat mscs were applied for 2 h topically to the transplanted corneas immediately after operation . Unfortunately, the survival of the corneal grafts was not significantly prolonged, though the il-6 and il-10 levels were significantly increased in the rejected grafts after the mscs were applied . This research proved that topical application of allogeneic rat mscs does not prolong corneal xenograft survival effectively in a pig - to - rat model . However, in a following in vivo study, the researchers performed orthotopic corneal allotransplantation using c57bl/6 mice (h-2) as donors and balb / c (h-2) as recipients . The researchers demonstrated that preoperative intravenous injection of hmscs decreased early surgically induced inflammation and reduced the activation of antigen - presenting cells (apcs) in the cornea and draining lymph nodes (dlns). These results suggested that hmscs improved the survival of corneal allografts without engraftment and primarily by secreting tsg-6 that acts by aborting early inflammatory responses . Moreover, in another in vivo study of the rat corneal allograft rejection model, which was established by using wistar rats as donors and lewis rats as recipients, postoperative intravenous injection of mscs, rather than preoperative intravenous injection, prolonged graft survival time . The authors also found that injecting mscs reduced th1 proinflammatory cytokines and elevated the secretion of il-4 from t lymphocytes . Therefore, we speculate that suppressing corneal transplantation rejection by injecting mscs depends on the timing and route of administration . Corneal fibroblasts (activated stromal keratocytes) are thought to be the key underlying mediator of this sight - compromising response . The conditional medium from mscs (mscs - cm) inhibited the wound healing activities of corneal fibroblasts in vitro . Therefore, certain factors secreted by mscs appear to have therapeutic value in corneal repair . Intravenously injected mscs engrafted to the injured cornea and promoted wound healing, by differentiation, proliferation, and synergy with hematopoietic stem cells in an in vivo study of the rabbit alkali burn model . The mscs homed in on local sites and then differentiated into myofibroblasts due to the local tissue microenvironment . In another in vivo study, corneal injury in mice was induced with thermal cauterization, and then the mscs were systematically administered . The authors found that the mscs homed in on the injured cornea and survived there whereas homing toward the normal cornea did not occur . In the setting of corneal injury, mscs administration elicited significant and rapid corneal epithelial regeneration . Our study showed that subconjunctival injection of mscs significantly accelerated corneal wound healing in alkali - burned corneas . Another study showed that hmscs acted as a source of feeder cells in vitro for cultivating transplantable corneal epithelial cell sheets . In this study, hmscs expressed keratinocyte growth factor (kgf) and hepatocyte growth factor (hgf), soluble growth factors required for epithelial cell proliferation . Recently, a case report demonstrated, for the first time, a patient with post - traumatic persistent sterile corneal epithelial defect treated with topical application of autologous adipose - derived mscs . The mscs were transferred into the bottom of the ulcer using an insulin syringe with a 27-g needle attached . An in vitro study tried to determine whether mscs could be induced to transdifferentiate into hcecs . This was done by evaluating whether mscs could be injected and home in to a corneal endothelial injury site . In this study, this effect was obtained . This study along with those involving the corneal epithelium showed that mscs have potential therapeutic value in treating corneal epithelial and endothelial injuries . Mscs have potential therapeutic value in corneal reconstruction since they have anti - inflammatory and modulatory effects on corneal angiogenesis based on results obtained with several animal models . Furthermore, mscs are useful in suppressing corneal transplantation rejection and facilitating corneal wound healing . Additional animal model research is needed to address questions regarding how to transdifferentiate mscs into corneal epithelial cells, the most appropriate route and time for applying mscs for different kinds of corneal reconstruction, the specific mechanisms, and so on . Before mscs can be tested in a clinical setting, these uncertainties must be resolved, and additional insight gained into how their use elicits such beneficial effects . Although originally identified in the bone marrow, mscs have been found in many other tissues, including the adipose, heart, wharton s jelly, dental pulp, peripheral blood, cord blood, menstrual blood [9 - 11], fallopian tube, and limbal stroma of the human eye . These cells have self - renewal ability as undifferentiated cells and could differentiate into lineages of mesenchymal tissues, including bone, cartilage, fat, muscle, and marrow stroma . Under certain conditions, these cells could transdifferentiate into neurons or cardiac muscle cells [16 - 19]. Because of the low expression of major histocompatibility class ii (mhc ii) under unstimulated conditions and the absence of costimulatory molecules such as cluster of differentiation 40 (cd40), cluster of differentiation 40 ligand (cd40l), b71, and b72 on cell surface [20 - 24], mscs could escape the monitoring of the immune system and infuse into an allogeneic host without being rejected . Mscs can be administered directly to the cornea [26 - 31], or by carriers, such as the amniotic membrane [32 - 35] or fibrin gels . For clinical applications, the regenerative / reparative potential and the immune - suppressive capacity of mscs are the current areas of research focus . Many clinical studies on mscs led to positive results, which focused on the effects of mscs on regenerative medicine, preventing graft rejection and controlling graft versus host disease (gvhd). Mscs can transdifferentiate into other kinds of cells, including cardiomyocytes and neuronal cells [16 - 19]. However, hypotheses still need further evidence to establish that mscs play a role in corneal reconstruction . Gu demonstrated that mscs can differentiate into corneal epithelial - like cells in vivo and in vitro . In vivo, rabbit mscs (rb - mscs) were suspended in fibrin gels and transplanted onto the surface of naoh damaged rabbit corneas . As a result rb - mscs also participated in the healing process of the naoh injured corneal epithelium and expressed cytokeratin 3 (ck3), a corneal epithelial - specific marker . In vitro, rb - mscs differentiated into cells with a morphological and molecular phenotype of corneal epithelial - like cells that were positive to ck3 . Another in vivo study demonstrated that mscs have the ability to differentiate into corneal epithelial cells in experimental limbal stem cell deficiency rabbits . The expression of certain stem cell markers, such as adenosine 5'-triphosphate - binding cassette member 2 (abcg2), 1-integrin, and connexin 43, in the cornea epithelium after mscs transplantation indicated that mscs maintained stem cell characteristics; some mscs even transdifferentiated into epithelial progenitor cells . The in vivo study on human mscs (hmscs) found that hmscs could survive and migrate into the cornea stroma after being transplanted onto the surface of the alkali - burned rabbit cornea . Not only did the hmscs differentiate into the corneal epithelium, but also some even migrated into the corneal stroma and differentiated into cells other than epithelia . Another in vivo study found that when mscs were intrastromal - transplanted into keratocan - null (kera) mice, the cells survived in the cornea without evoking an immune and inflammatory response and expressed keratocan in the host kera mice . The investigators speculated that these corneal intrastromal - transplanted mscs may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes . Similarly, another in vivo study showed that intrastromal - transplanted umbilical mscs could survive similar to a keratocyte phenotype in the mouse corneal stroma . In an in vitro study, after coculture with corneal stromal cells (cscs), the induced mscs expressed positive staining for ck12 with the corneal epithelial cell characteristics confirmed with scanning electron microscopy . In addition, in vivo, the induced mscs had remarkable effects on treating the corneal alkali burn and reconstructing the corneal surface in a rat limbal stem cell deficiency model . In contrast, some researchers believed that mscs could not transdifferentiate into corneal epithelial cells in vivo . An in vivo study compared the transplantation of mscs with lscs concluded that mscs and lscs could assist the reconstruction of the damaged corneal surface in a rat corneal chemical burn model . However, the therapeutic mechanism was not associated with the epithelial differentiation from mscs because there was no sufficient evidence to support that mscs could differentiate into corneal epithelial cells . A later in vivo study had the same conclusion in a rat corneal chemical burn model . To a certain extent, the lack of sufficient evidence may be because keratocytes do not have specific markers and share many markers with mscs . Our in vivo study also found that subconjunctival injected mscs could not migrate into the injured cornea and transdifferentiate into corneal epithelial cells in a rat corneal alkali burn model . Therefore, it is still unclear whether mscs play a role in corneal reconstruction by the transdifferentiation effect, and the hypothesis requires further investigation . Mscs could ameliorate the inflammation in different damaged tissues, such as dextran sulfate sodium - induced colitis, acute kidney injury, and lung injury . The anti - inflammatory effect of mscs on the cornea was demonstrated on a rat corneal chemical burn model . The isolated hmscs from healthy donors grew and expanded on the amniotic membrane, followed by transplanting the membrane onto rat corneas 7 days after the chemical burns . Four weeks after transplantation, inflammation factors such as cluster of differentiation 45 (cd45), interleukin 2 (il-2), and matrix metalloproteinase-2 (mmp-2) decreased in the hmsc transplantation model detected with immunofluorescent stain . These findings indicated that the therapeutic effect of the damaged rat cornea treated with hmscs might be partially due to the inhibition of inflammation . An in vivo study further certified the anti - inflammatory effect of mscs on the cornea by detecting more inflammatory related factors . In this study, mscs were applied to the cornea directly without using the amniotic membrane as a carrier . Mscs decreased the expression of il-2 and interferon- (ifn-) in the rat cornea after chemical injury . However, increased expression of interleukin-10 (il-10), transforming growth factor-1 (tgf-1), and interleukin-6 (il-6) was also detected . In our study, we investigated the effects of subconjunctivally injected mscs in the acute stage of an alkali - burned rat cornea . After mscs were subconjunctivally injected, the infiltrated cd68 macrophages in the alkali - burned cornea were significantly decreased . The mrna expression levels of macrophage inflammatory protein-1 alpha (mip-1) and tumor necrosis factor - alpha (tnf-) were also downregulated . We speculate that mscs inhibit macrophage infiltration by suppressing the expression of macrophage chemokine mip-1 . In another in vivo study, the investigators administered hmscs to the chemically injured rat cornea . The investigators found that hmscs were effective in reducing corneal opacity and inflammation after either intraperitoneal or intravenous administration following cornea chemical injury . Regarding a specific mechanism, they found chemical injury to corneal epithelial cells could activate hmscs to secrete tnf- stimulated gene / protein 6 (tsg-6) in vitro, a multipotent anti - inflammatory protein . In addition, in vivo, the anti - inflammatory effects of hmscs were largely abrogated by knockdown of tsg-6 . Therefore, the authors speculated that systemic administration of hmscs reduced inflammatory damage to the chemically burned cornea primarily by secreting anti - inflammatory protein tsg-6 in response to injury signals from the damaged cornea . In summary, many studies found that mscs were good activators for angiogenesis, and mscs could secrete vascular endothelial growth factor (vegf) in an ischemia or tumor model [48 - 52]. However, mscs seemed to have an opposite effect on corneal angiogenesis . Some in vivo studies found that applying mscs on the cornea could effectively inhibit inflammation - related angiogenesis after chemical injury . Meanwhile, mmp-2, an inflammation - related proangiogenic factor, was significantly downregulated after mscs treatment . However, the level of vegf was similar between the control and msc - treated cases in an in vivo study of a rat corneal chemical burn model . In our in vivo study, we found that the level of vegf was downregulated after the msc subconjunctival injection in the acute stage of rat alkali - burned corneas . In vitro, the coculture of human corneal epithelial cells (hcecs) and hmscs upregulated the level of vegf . Furthermore, the hmscs constitutively expressed mmp-2 and tsp-1 . At the same time, hmscs significantly suppressed the secretion of mmp-9 from hcecs . Vegf, mmp-2, and mmp-9 are proangiogenic factors in the cornea, and tsp-1 is an antiangigenic factor, which could inhibit vegf - induced angiogenesis by cd36 activation [54 - 56]. Therefore, tsp-1 appears to be an antiangiogenic factor, which opposes the proangiogenic effect of vegf on the cornea in vivo . Similarly, mscs could also play a role in host versus graft disease (hvgd) [59 - 61]. A study on skin transplantation found that applying mscs could prolong baboon skin graft survival in vivo . In an in vivo study, mscs were efficient in heart transplantation by prolonging semiallogeneic heart graft survival, rather than a fully mhc - mismatched heart graft in a heart transplant mouse model . This study described a time dependency characteristic of mscs that the infusion of mscs was effective in prolonging graft survival when being used before transplantation, and partially effective during transplantation, but inefficient merely one day after transplantation . In the case of corneal transplantation, one of the most common causes of corneal allograft failure is irreversible rejection . In an in vivo study, the immunomodulatory effects of mscs were investigated with orthotopically transplanted pig corneas in rats . Allogeneic rat mscs were applied for 2 h topically to the transplanted corneas immediately after operation . Unfortunately, the survival of the corneal grafts was not significantly prolonged, though the il-6 and il-10 levels were significantly increased in the rejected grafts after the mscs were applied . This research proved that topical application of allogeneic rat mscs does not prolong corneal xenograft survival effectively in a pig - to - rat model . However, in a following in vivo study, the researchers performed orthotopic corneal allotransplantation using c57bl/6 mice (h-2) as donors and balb / c (h-2) as recipients . The researchers demonstrated that preoperative intravenous injection of hmscs decreased early surgically induced inflammation and reduced the activation of antigen - presenting cells (apcs) in the cornea and draining lymph nodes (dlns). These results suggested that hmscs improved the survival of corneal allografts without engraftment and primarily by secreting tsg-6 that acts by aborting early inflammatory responses . Moreover, in another in vivo study of the rat corneal allograft rejection model, which was established by using wistar rats as donors and lewis rats as recipients, postoperative intravenous injection of mscs, rather than preoperative intravenous injection, prolonged graft survival time . The authors also found that injecting mscs reduced th1 proinflammatory cytokines and elevated the secretion of il-4 from t lymphocytes . Therefore, we speculate that suppressing corneal transplantation rejection by injecting mscs depends on the timing and route of administration . Corneal fibroblasts (activated stromal keratocytes) are thought to be the key underlying mediator of this sight - compromising response . The conditional medium from mscs (mscs - cm) inhibited the wound healing activities of corneal fibroblasts in vitro . Therefore, certain factors secreted by mscs appear to have therapeutic value in corneal repair . Intravenously injected mscs engrafted to the injured cornea and promoted wound healing, by differentiation, proliferation, and synergy with hematopoietic stem cells in an in vivo study of the rabbit alkali burn model . The mscs homed in on local sites and then differentiated into myofibroblasts due to the local tissue microenvironment . In another in vivo study, corneal injury in mice was induced with thermal cauterization, and then the mscs were systematically administered . The authors found that the mscs homed in on the injured cornea and survived there whereas homing toward the normal cornea did not occur . In the setting of corneal injury, mscs administration elicited significant and rapid corneal epithelial regeneration . Our study showed that subconjunctival injection of mscs significantly accelerated corneal wound healing in alkali - burned corneas . Another study showed that hmscs acted as a source of feeder cells in vitro for cultivating transplantable corneal epithelial cell sheets . In this study, hmscs expressed keratinocyte growth factor (kgf) and hepatocyte growth factor (hgf), soluble growth factors required for epithelial cell proliferation . Recently, a case report demonstrated, for the first time, a patient with post - traumatic persistent sterile corneal epithelial defect treated with topical application of autologous adipose - derived mscs . The mscs were transferred into the bottom of the ulcer using an insulin syringe with a 27-g needle attached . An in vitro study tried to determine whether mscs could be induced to transdifferentiate into hcecs . This was done by evaluating whether mscs could be injected and home in to a corneal endothelial injury site . In this study, this effect was obtained . This study along with those involving the corneal epithelium showed that mscs have potential therapeutic value in treating corneal epithelial and endothelial injuries . Mscs have potential therapeutic value in corneal reconstruction since they have anti - inflammatory and modulatory effects on corneal angiogenesis based on results obtained with several animal models . Furthermore, mscs are useful in suppressing corneal transplantation rejection and facilitating corneal wound healing . Additional animal model research is needed to address questions regarding how to transdifferentiate mscs into corneal epithelial cells, the most appropriate route and time for applying mscs for different kinds of corneal reconstruction, the specific mechanisms, and so on . Before mscs can be tested in a clinical setting, these uncertainties must be resolved, and additional insight gained into how their use elicits such beneficial effects.
Noroviruses (novs), a group of small, round - structured rna viruses constituting the norovirus genus in the family caliciviridae, infect both humans and animals . Human novs cause epidemic acute gastroenteritis, affecting millions of people and claiming over 200,000 lives annually worldwide . At present structurally, novs are nonenveloped, containing an outer protein capsid that encapsulates the single - stranded, positive sense rna genome of 7.7 kb . The nov capsid is made from a single major structural viral protein, vp1 . Crystallography of recombinant nov - like particles (vlps) reveals a t = 3 icosahedral symmetry consisting of 180 copies of vp1 organized into 90 dimers . Vp1 is divided into two major domains, the shell (s) and the protruding (p) domains . The s domain forms the interior, icosahedral shell; while the p domain forms the dimeric protrusions extending outward from the shell . The p domain can be further divided into p1 and p2 subdomains, corresponding to the legs and the head of the arch - like protrusion, respectively . The p2 subdomain forms the outermost surface of the capsid with highly variable sequence, responsible for the virus human novs are difficult to study due to the lack of an efficient cell culture system and a small animal model . Currently, research into nov host interactions relies on various nov subviral particles . Expression of full - length vp1 results in vlps that are structurally similar to an authentic virus . Furthermore, expression of various subdomains results in smaller subviral particles or complexes . For example, production of the s domain forms s particles, corresponding to the interior shell of the capsid, while expressions of the p domains with or without modifications can form p dimers, 12-mer small p particles, or 24-mer p particles . In addition, various glutathione s - transferase (gst)-p domain fusion proteins have been shown to form polyvalent complexes owing to the dimeric and oligomeric features of the gst and the p domain . These vlps, p particles and p complexes retain the basic structures of the capsid or p dimer, recognize host ligands and, thus, have been used as tools or models for the study of nov host interactions . Human novs recognize histo - blood group antigens (hbgas) as attachment factors or receptors, which play an important role in the host susceptibility of nov infection, as shown by both human challenge studies and outbreak investigations . Hbgas are oligosaccharides linked to membrane proteins or lipids as glycoprotein or glycolipid that are distributed extensively on the surfaces of red blood cells and mucosal epithelia . They are also present as free oligosaccharides in biological fluids, such as saliva or milk . Human novs interact with hbgas in a strain - specific manner, whereby a number of nov - hbga binding patterns involved in all abo, lewis and secretor / nonsecretor types have been identified . The structural basis of these interactions have been elucidated by x - ray crystallography of nov p dimers in complex with hbga oligosaccharides . However, it has been observed that some human novs, such as va115 (gi.3), desert shield virus (gi.3) and noda485 (gii.1), do not bind any hbgas . A human challenge study of snow mountain virus (smv, gii.2) did not reveal a dependence of host susceptibility on hbga type, despite the fact that the smv vlp recognizes only the b antigen . In addition, a recent study showed that nov vlps of ueno 7k (gii.6) and noda485 binds caco-2 cells and human small intestinal epithelium biopsy in a hbga - independent manner . These data suggest that hbgas may not be the only receptors for human novs . For example, using thin - layer chromatography and quartz crystal microbalance with dissipation monitoring, larson et al . Reported binding of gii.4 vlps to galactosylceramide and hbga glycosphingolipids that were purified from human meconium samples . Takeda and co - workers demonstrated that vlps of gii novs bound heparan sulfate on the cell surface, while belliot and co - workers showed that gii.4 vlps recognized sialic acid - containing carbohydrates, such as sialyl lewis x (le), sialyl - lacto - n - fucopentaose, sialyl - lacto - n - tetraose, and sialyl - lacto - n - neotetraose, with affinities comparable to those of hbga ligands . Using saturation transfer difference nuclear magnetic resonance spectroscopy, peters and co - workers detected the interactions between gii.4 vlps and the sialic acid moiety of sialyl le and sialyl le . However, they also found that carbohydrates containing sialic acid, but not fucose, e.g. 3-sialyllactose and 6-sialyllactose, do not exhibit detectable binding with the vlp . The results of these studies, taken together, imply that sialic acid - containing oligosaccharides could also be ligands of human novs . In fact, sialic acid - containing oligosaccharides have been shown to be ligands or receptors for some animal caliciviruses (cvs), including murine nov (mnv1), feline calicivirus (fcv) and a porcine sapovirus (psav, cowden strain). However, solid evidence to establish the ligand status of sialic acid for human cvs (human novs and human sapoviruses) is lacking . Here, we report the first experimental evidence that human novs recognize sialic acid - containing glycosphingolipids (gangliosides). The catch - and - release electrospray ionization mass spectrometry (car - esi - ms) assay was used to screen a library of gangliosides against the p particle of human nov va387 (gii.4). The affinities of 13 gangliosides for the p dimer of va387 and of a second human nov strain, va115 (gi.3), were measured using the direct esi - ms assay . Using a competitive esi - ms assay, the proxy protein method, the highest affinity ligand, gm3, was subjected to additional binding measurements and the affinities for both the va387 p particle and vlp were determined . Notably, the ganglioside affinities measured for nov va387 are comparable to those of known hbga oligosaccharide receptors . Enzyme - linked immunosorbent assays (elisa) provided additional evidence that both strains of novs exhibit binding to sialic acid - containing oligosaccharides . The vlps of va387 (gii.4) were produced in insect cells (sf9) through a recombinant baculovirus containing the gene encoding va387 vp1 (genbank accession number ay038600, molecular weight (mw) of monomer 58,887 da) as described previously . The resulting vlps were purified by sucrose gradient . Va387 p particles (24-mer, mw 865,036 da), p dimers (mw 69,312 da), and gst - p domain fusion proteins were produced based on the p domain sequences (residues 222539) of vp1 via e. coli as reported in our previous studies . The gst gene fusion system (ge healthcare life sciences, piscataway, nj) with plasmid vector pgex-4t-1 was used for the p proteins expression . Preparations of va115 (gi.3) vlps and p particles were attempted based on the vp1 sequences (genbank accession number ay038598) and the established procedure described above, but the yields for both particles were found to be very low . The p dimers (mw 67,712 da) and the gst - p fusion proteins of va115 were produced in high yield (> 20 mg l bacteria) through the same procedure as used for the production of the p proteins of va387 . Formations of the 24-mer p particles, p dimers, and the gst - p polymers were analyzed by gel - filtration chromatography via a superdex 200 size exclusion column (ge healthcare life sciences) controlled by an akta fast performance liquid chromatography system (fplc, model 920, ge healthcare life sciences). A single chain fragment (scfv, mw 26,539 da) of the monoclonal antibody se1554 was produced using recombinant technology as described elsewhere . A recombinant fragment of the c - terminus of human galectin-3 (gal-3c, mw 16,330 da) was generously provided by prof . C. cairo (university of alberta). Bovine ubiquitin (ubq, mw 8565 da) was purchased from sigma - aldrich canada (oakville, canada). The proteins were concentrated and exchanged into an aqueous 200 mm ammonium acetate (ph 7) using vivaspin 0.5 ml centrifugal filters (sartorius stedim biotech, gttingen, germany) with a mw cutoff of 10 kda and stored at 80 c until use . The structures of the oligosaccharides and glycoconjugates used in this study are shown in figure s1 (supporting information). The 17 ganglioside and globoside oligosaccharides (gm3, gm2, gm1a, gm1b, gd3, gd2, gd1a, gd1b, gt3, gt2, gt1a, gt1c, fucosyl - gm1, asialo - gm2, asialo - gm1, gb3 and gb4) were purchased from elicityl sa (crolles, france). H type 3 trisaccharide, a type 3 tetrasaccharide, and b type 3 tetrasaccharide were a gift from prof . Each solid compound was dissolved in ultrafiltered milli - q water (millipore, ma) to give a 1 mm stock solution . Polyacrylamide (paa)-conjugated neu5ac, 6-sialylacnac, and gm3 trisaccharide were purchased from vector lab (burlingame, ca). All of the esi - ms assays were carried out on a synapt g2s quadrupole - ion mobility separation - time - of - flight (q - ims - tof) mass spectrometer (waters, manchester, u.k .) The car - esi - ms and direct esi - ms assays were performed in negative ion mode, whereas the proxy protein esi - ms assay was implemented in positive ion mode ., 0.68 mm i.d .) Pulled to 5 m using a p1000 micropipette puller (sutter instruments, novato, ca). A platinum wire was inserted into the nanoesi tip, and a capillary voltage was applied to carry out esi . The source parameters for both negative and positive ion modes were: capillary voltage 0.8 kv (negative ion mode) or 1.0 kv (positive ion mode), source temperature 60 c, cone voltage 60 v (negative ion mode) or 35 v (positive ion mode), trap voltage 5 v, and transfer voltage 2 v. data acquisition and processing were performed using masslynx software (version 4.1). The car - esi - ms assay was performed to identify carbohydrate ligands of the nov va387 p particle . Ions corresponding to ligand - bound p particle were isolated using the quadrupole mass filter . The quadrupole was set to transmit a broad mass - to - charge ratio (m / z) window (approximately 200 m / z units), which allows for the simultaneous passage of free and ligand - bound p particle complexes at a given charge state . Protein ligand complexes were subjected to collision - induced dissociation (cid) in the trap region of the synapt g2s by increasing the trap voltage from 5 to 200 v. argon (1.42 10 mbar) was used to carry out cid in the trap region . In most instances, the deprotonated ligands released from the complexes could be identified from their mws . Where required, ims was used to separate the released isomeric ligands . For ims separation a wave height of 35 v was used, and the wave velocity was ramped from 2000 to 500 m s. in all cases a helium flow rate of 150 ml min and a nitrogen flow rate of 40 ml min were used . The arrival time distributions (atds) for the released ligands were compared to reference atds, which were measured for the deprotonated carbohydrates produced directly from solution . The direct esi - ms assay was used to quantify the affinities of the carbohydrate ligands for the nov p dimers of va387 and va115 . At least four different initial ligand concentrations were used for each oligosaccharide tested, and the binding measurements were carried out in triplicate . A complete description of the data analysis method employed to calculate the intrinsic association constants (ka, int) can be found elsewhere . Briefly, the abundance ratio (ri) of the ligand - bound protein (pli), bound to i molecules of l, to free protein (p) measured by esi - ms (after correction for nonspecific ligand - protein binding) is taken to be equal to the equilibrium concentration ratio in solution, eq 1:1 assuming the protein has h independent and identical binding sites, ka, int can be expressed by eq 2:2where [p]0 and [l]0 are the initial concentrations of the protein and ligand, respectively, and f is the fraction of occupied binding sites, eq 3:3 in the case of the p dimer, which has two equivalent binding sites, ka, int can be found using eq 4:4 the proxy protein esi - ms assay was used to quantify the affinities of gm3 trisaccharide for nov va387 p particle and vlp . A complete description of the data analysis method employed to calculate ka, int can be found elsewhere . Briefly, a proxy protein (pproxy), which binds to l with a known affinity, is used to monitor the extent of l binding to p. specifically, in the presence of p, the abundance ratio rproxy (= [pproxyl]/[pproxy]) will quantitatively reflect the concentration of l bound to p and ka, int can be evaluated using eq 5:5where the initial concentrations of target protein ([p]0), proxy protein ([pproxy]0) and ligand ([l]0) as well as the association constant for binding of pproxy to the ligand (ka, pproxy) are known; [p]m,0 is the initial concentration of binding sites in the target protein, i.e., [p]m,0 = h [p]0 . Paa - conjugated neu5ac, 6-sialylacnac and gm3 trisaccharide were dissolved in 1x pbs (ph 7.4). They were diluted and coated on a 96-well microtiter plate at concentration of 2 g ml and stored at 4 c overnight . After blocking with 5% nonfat dry milk, nov vlp, p particle, or gst - p domain fusion proteins as well as gst (negative control) at 50 ng l were added and incubated for 2 h at 37 c . The ligand - bound nov vlp and p proteins were detected by homemade guinea pig hyperimmune serum against va387 vlp and va115 p protein (1:3000), respectively, followed by horseradish peroxidase (hrp)-conjugated goat antiguinea pig immunoglobulin g (igg, 1:3000; icn, aurora, oh). The signals were displayed using a tmb kit (thermo fisher scientific, rockford, il). Evidence of ganglioside binding to novs was initially revealed through the screening of a small (20 components) carbohydrate library against the p particle (24-mer, mw 865,036 da) of nov va387 (gii.4) using the car - esi - ms assay . The library consisted of the oligosaccharides of 17 glycosphingolipids, gm1a, gm1b, gm2, gm3, gd1a, gd1b, gd2, gd3, gt1a, gt1c, gt2, gt3, fucosyl - gm1 (referred to as fuc - gm1), asialo gm1, asialo gm2, gb3 and gb4 as well as three known hbga oligosaccharide ligands, h type 3 trisaccharide (referred to as h3), a type 3 tetrasaccharide (a3), and b type 3 tetrasaccharide (b3). The intrinsic affinities of the hbga ligands range from 700 to 1500 m. the car - esi - ms assay was carried out by first incubating the p particle with the carbohydrate library, followed by direct esi - ms analysis of the mixture . Because of the high mw of the p particle, the identity of the bound ligands could not be established directly from the mass spectrum . Instead, using a quadrupole mass filter set to pass a range of mass - to - charge - ratio (m / z) ions, all of the ligand - bound p particle ions at a given charge state were isolated and then activated (heated) using cid to release the ligands (as ions) from the complex . Given that carbohydrates have relatively low gas - phase acidities and are able to effectively compete with proteins for negative charge, accurate mass analysis, alone or in combination with ion mobility separation (ims), which separates ions based on size and shape, allowed for positive ligand identification . Shown in figure 1a is a representative esi mass spectrum acquired in negative ion mode for an aqueous ammonium acetate (200 mm, ph 7, 25 c) solution of p particle (3 m) and the carbohydrate library (10 m each). From the mass spectrum it can be seen that the p particle exists predominately as a 24-mer, with a charge state distribution ranging from 60 to 65 . Signal corresponding to an 18-mer is also present, although at lower abundance, with a charge state distribution of 51 to 54 . Due to the high mw of the p particle and the formation of adducts during the esi process, it was impossible to resolve the ions corresponding to free p particle and its complexes with one or more oligosaccharide ligands . However, cid, performed using a 200 m / z wide isolation window centered at 14,350 to pass ions corresponding to the 61 charge state of the p particle, led to the appearance of singly deprotonated ions of the three hbga oligosaccharides as well as gm3 (m / z 632.2), gm2 (m / z 835.3), gd3 (m / z 923.3), gm1a and/or gm1b (m / z 997.3), and fuc - gm1 (m / z 1143.4) (figure 1b). Ions corresponding to the singly deprotonated gd2 (m / z 1126.4) and the doubly deprotonated ions of gd1a and/or gd1b (m / z 644.1) were also detected, although at low abundance (figure 1b). Abundant multiply charged protein monomer ions, pm at n = 1023, were also evident (figure 1b). Implementation of the car - esi - ms assay using other charge states of the p particle complexes produced similar results (figure s2, supporting information). Ion mobility separation of the released ligands revealed evidence that both gm1a and gm1b are released from the p particle, with gm1a being more abundant (figure s3a, supporting information). The doubly deprotonated ions of gd1a and gd1b could not be differentiated using optimized ims conditions (figure s3b, supporting information), and therefore, it was not possible to establish whether one or both oligosaccharides bind to the p particle directly from these measurements . Instead, the car - esi - ms assay was applied to solutions containing p particle (3 m) and 10 m of gd1a or gd1b . These data revealed that only gd1a binds to the p particle under these solution conditions (figure s4, supporting information). The car - esi - ms results provide compelling evidence that the p particle of va387 exhibits a broad specificity for mono- and disialylated gangliosides . However, there is a clear preference for gm3, and the addition of saccharides to gal (e.g., gm1 or gm2) or sia (e.g., gd3, gd2 or gd1b) decreases binding, compared to gm3 . These data, combined with affinities measured for ganglioside oligosaccharides, vide infra, suggest that the sia - gal - glc moiety represents the dominant recognition epitope for this nov . (a) esi mass spectrum acquired in negative ion mode for an aqueous ammonium acetate solution (200 mm, ph 7 and 25 c) of nov va387 p particle (3 m) and a 20-component (10 m each) carbohydrate library consisting of the oligosaccharides of gm1a, gm1b, gm2, gm3, gd1a, gd1b, gd2, gd3, gt1a, gt1c, gt2, gt3, fuc - gm1, asialo gm1, asialo gm2, gb3, and gb4 as well as the h3, b3, and a3 oligosaccharides . (b) cid mass spectrum measured for the 61 charge state of the free and ligand - bound p particle . Based on the relative abundances of the released oligosaccharide ligands measured by car - esi - ms (figure 1) it would appear that the affinities of the ganglioside ligands are similar to those of the highest affinity hbga oligosaccharides . However, this conclusion is predicated on the assumption that the release efficiency of the bound - ligands is essentially independent of structure . Because of the presence of the sialic acid, it is possible that gangliosides (which are likely deprotonated in the gaseous complexes) are preferentially released from the p particle due to a lower activation energy resulting from coulombic repulsion . Therefore, it was important to measure the affinities directly . In order to do this, the affinities were measured using the direct esi - ms assay, which has been shown to provide reliable ka values for many protein carbohydrate interactions . Affinities were measured for the oligosaccharides of 13 gangliosides (gm3, gm2, gm1a, gm1b, gd3, gd2, gd1a, gd1b, gt3, gt2, gt1a, gt1c, and fuc - gm1) for the va387 p dimer (mw 69,312 da). A reference protein (pref) was used in all cases to correct the mass spectra for the occurrence of nonspecific carbohydrate a representative esi mass spectrum acquired for an aqueous ammonium acetate solution (200 mm, ph 7, 25 c) of va387 p dimer (12 m) and gm3 trisaccharide (80 m) is shown in figure 2a as well as the distribution of ligand - bound p dimer after correction for nonspecific binding . From the esi - ms data, ka, int values were calculated for each oligosaccharide (table 1). Affinities were also measured for a3, b3, and h3 and shown to agree well with the reported values (table s1, supporting information). Inspection of the ka, int values reveals that, of the tested gangliosides, gm3 exhibits the highest affinity for the va387 p dimer, which is consistent with the results of the car esi - ms measurements, vide supra . Moreover, the ka, int (1500 m) is identical, within experimental error, to that of b3 (1500 150 m). Of the 12 other gangliosides investigated, nine bind weakly (ka, int <500 m) and three (gd1b, gt3 and gt1a) do not show any detectable binding . Notably, the quantitative binding data obtained for the p dimer agree qualitatively with the relative affinities inferred from the car - esi - ms measurements performed on the p particle . Moreover, all ligands with affinities> 100 m were detected in the car - esi - ms measurements (table s2, supporting information). (a) esi mass spectrum acquired in negative ion mode for aqueous ammonium acetate solution (200 mm, ph 7 and 25 c) of nov va387 p dimer (p2, 12 m), gm3 trisaccharide (80 m) and pref (4 m). Another minor form of p dimer (p2, mw 74,080 da) was also detected with lower abundance . Inset, normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding . (b) esi mass spectrum acquired in negative ion mode for aqueous ammonium acetate solution (200 mm, ph 7 and 25 c) of nov va115 p dimer (p2, 12 m, mw 67,712 da), gm3 trisaccharide (80 m), and pref (4 m). Inset, normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding . Nb = no binding detected . To demonstrate the relevance of the affinity data acquired for the p dimer, affinity measurements were also carried out for gm3 trisaccharide binding to the va387 p particle and vlp (180-mer, mw 10.5 mda). An adaptation of the proxy protein esi - ms method, which combines direct esi - ms binding measurements and competitive protein binding, was used to evaluate the affinities . A recombinant fragment of the c - terminus of human galectin-3 (gal-3c, mw 16,330 da), which contains a carbohydrate recognition domain and interacts with a -galactoside moiety, served as the proxy protein (pproxy). Importantly, gal-3c binds to gm3 trisaccharide with an affinity of (1.20 0.02) 10 m. the extent of binding of gm3 trisaccharide to gal-3c, as determined by esi - ms, in the presence of known concentrations of the target protein (p particle or vlp) allowed for a quantitative measure of gm3 binding to the target . Esi - ms measurements were performed on aqueous ammonium acetate solutions (160 mm, ph 7 and 25 c) of pproxy (3.0 m), pref (1.0 m), gm3 trisaccharide (40 m), and either p particle, at concentrations ranging from 0 to 7.2 m (corresponds to monomer concentration of 0172.8 m), or vlp, at concentrations ranging from 0 to 570 nm (monomer concentration of 0102.6 m). Representative esi mass spectra acquired in positive ion mode in the absence and presence of nov vlp (570 nm) are shown in figure 3a and 3b, respectively . The distributions of ligand - bound pproxy, following correction for nonspecific ligand binding, are also given . Inspection of the distributions reveals a measurable decrease in the extent of gm3 trisaccharide binding to gal-3c upon addition of vlp . The dependence of the extent of gm3 trisaccharide binding to pproxy on vlp concentration is shown in figure 3c . Binding measurements performed on solutions containing p particle yielded qualitatively similar results (figure s5, supporting information). Analysis of the pproxy binding data acquired in the presence of vlp or p particle using the procedure outlined in experimental section yields gm3 affinities of 2600 200 and 5500 600 m for the p particle and vlp, respectively . The slight differences in the magnitude of the affinities measured for the binding of a common carbohydrate ligand to the p dimer, p particle, and vlp of a nov (the first such data set to be reported), likely reflect subtle differences in the structure of the carbohydrate binding site presented by these related protein complexes . These differences notwhithstanding, the present results suggest that the p dimer can serve as a surrogate of the vlp for carbohydrate binding studies . Representative esi mass spectra measured in positive ion mode for aqueous ammonium acetate solutions (160 mm, ph 7 and 25 c) of pproxy (gal-3c, 3.0 m), pref (ubq, 1.0 m), and gm3 trisaccharide (40 m) without (a) or with (b) nov va387 vlp (570 nm, 180-mer). Insets show the fraction of free and gm3-bound pproxy, after correction for nonspecific ligand binding . (c) plot of the abundance ratio of gm3-bound pproxy to free pproxy (rproxy) versus vlp concentration . The solution conditions for each measurement were the same as in (a), but with the addition of vlp . It has been proposed that nov va387 has a binding interface that recognizes hbgas through the -l - fuc epitope as the major binding interaction and either the -d - galnac or -d - gal epitope as a minor binding interaction . However, these core recognition elements are missing in the ganglioside ligands identified in the present study . Therefore, it is of interest to establish whether the ganglioside ligands interact with the nov through the hbga binding site or through a distinct ganglioside binding site . It is not possible to answer this question through competitive binding measurements carried out using a ganglioside oligosaccharide (e.g., gm3 trisaccharide) and va387 p dimer in the presence of varying concentrations of a hbga oligosaccharide ligand due to the low affinities of these ligands . Instead, future efforts will rely on x - ray crystallography to establish whether va387 nov has distinct binding sites for hbga and ganglioside ligands . The aforementioned binding data reveal that nov va387 binds to mono- and disialylated gangliosides, with affinities comparable to those of the highest affinity hbga oligosaccharide ligands . To demonstrate that this is not an isolated example of a human nov that recognizes gangliosides, the affinities of the 13 ganglioside oligosaccharides for the p dimer of nov va115 (gi.3 genotype), which does not bind to human hbgas, were also measured (table 1). A representative esi mass spectrum acquired for an aqueous ammonium acetate solution (200 mm, ph 7, 25 c) of nov va115 p dimer (12 m) and gm3 trisaccharide (80 m) is shown in figure 2b as well as the distribution of ligand - bound p dimer after correction for nonspecific binding . Notably, the va115 p dimer binds to all 13 oligosaccharides tested, and overall, the affinities are slightly higher than those for va387 . Additional evidence for the recognition of sialic acid by human novs comes from elisa measurements carried out on the va387 vlp, p particle, and gst - p fusion protein as well as va115 gst - p fusion protein, with paa - conjugated neu5ac, 6-sialylacnac, and gm3 trisaccharide . As shown in figure 4, it is curious that the va387 vlp exhibited weaker binding than that of the p particle to the three glycoconjugates and the cause of the weaker binding is, at this time, unknown . Nevertheless, the fact that all three assemblies of nov capsid protein exhibit a similar binding pattern to the three glycoconjugates (gm3> 6-sialylacnac> neu5ac) validate their applications as models for nov ligand interaction . Moreover, comparing the binding of gst - p fusion protein of va387 to that of va115 indicates that the sialic acid - containing glycoconjugates have slightly higher affinities for va115, consistent with the esi - ms data . These results, together with those from esi - ms, suggest both -(2,3)- and -(2,6)- linked sialic acids as critical motifs in va387 and va115 binding, similar to what has been reported for mnv1 and psav . It is important to point out that, although sialic acid - containing oligosaccharides have been identified as receptors for an animal nov (mnv1) and two other animal cvs (fcv and psav), human novs generally recognize gangliosides in addition to hbgas . Furthermore, human novs differ greatly from mnvs in many other important aspects, including host tropism (human vs mouse), clinical manifestation (with vs without diarrhea / vomiting), and pathogenesis . Binding of nov vlp, p particles, and gst - p fusion protein of va387 as well as gst - p fusion protein of va115 to paa - conjugated gm3 trisaccharide, 6-sialylacnac, and neu5ac in 1x pbs (ph 7.4). Gst, which does not show binding to any of the three glycoconjugates, served as a negative control . Taken together, the results of esi - ms and elisa measurements performed on two human novs representing two different genogroups (gi and gii) provide the first experimental evidence of interactions between human novs and gangliosides and sialic acid - containing glycoconjugates . Notably, the affinities measured for the oligosaccharides of the ganglioside ligands by esi - ms are comparable in magnitude to those reported for the oligosaccharides of known hbga receptors . These experimental data demonstrate sialic acid - containing oligosaccharides as alternative (to hbgas) ligands for human novs and suggest a new mechanism of human nov host interaction, one that involves hbga and sialic acid - containing oligosaccharide receptors and co - receptors for attachment and penetration into host cells and opens a new direction in human nov research . Further studies to characterize the role of cell surface sialic acids / gangliosides in the early stage of viral infection and its potential coordination with hbgas for viral attachment and/or entry are needed.
Decompression illness is caused by intravascular or extravascular gas bubble formation following rapid reduction in environmental pressure as seen with diving accidents . It comprises two different pathophysiological syndromes: decompression sickness and arterial air embolism caused by pulmonary barotrauma . Decompression sickness can occur when a diver's time at depth leads to supersaturation of dissolved gases (usually nitrogen) and bubble formation during decompression . When the rate of ambient pressure reduction exceeds the rate of inert gas washout from tissue, formation of gas bubbles in the extravascular tissue may lead to tissue injury, vessel obstruction and organ dysfunction . Arterial air embolism occurs when expanding gas ruptures alveolar capillaries (pulmonary barotrauma) and enters the arterial circulation as a result of rapid decompression . This is typically seen with rapid uncontrolled ascents especially in the setting of breath - holding or the presence of an underlying lung disease (with reduced compliance). Typical manifestations include cerebral symptoms (weakness, numbness, confusion or unconsciousness, seizure, visual disturbances, headaches and vertigo) when bubbles rupture into vessels, or respiratory symptoms (pleuritic chest pain due to pneumothorax or pneumomediastinum) when bubbles rupture into pleural spaces . However, other sites including muscles, mesenteric circulation, cardiovascular system and, as shown in the following case, the kidneys may also be involved . We report the first case of acute kidney injury caused by arterial air embolism injury in a 27-year - old caucasian recreational diver following three rapid ascents . He was spear fishing on surface supply hookah at a depth of 15 metres for around 15 min when strong currents took him up to the surface whilst he was holding his breath attempting to reload his speargun . He had another two attempts to rescue his gear from the seafloor, but he was taken up by the swell each time . During the third ascent he became confused and when reaching the surface, his vision became blurred and he was unable to move his limbs . On arrival at the local hospital he was normotensive and his neurological symptoms resolved within a few hours . Due to initial pleuritic chest pain, a computer tomography was performed which showed normal lung parenchyma and no evidence of pneumothorax or pneumomediastinum . Nine hours after the incident, he developed diffuse abdominal pain with an initial lactate 10.8 mmol / l . Further imaging to look for ischaemic gut was not performed as he responded well to initial therapy consisting of 100% oxygen, analgesia with paracetamol and fentanyl (two doses, each 25 g intravenously) and intravenous hydration and his lactate level returned to normal within a few hours . Due to rapidly evolving acute kidney injury, he was transferred to a tertiary hospital for hyperbaric therapy on day 2 post - diving accident (initial creatinine 120 mol / l, 987 mol / l on day 2 and peak at 1210 mol / l on day 4). The diagnosis of ischaemic - induced acute tubular necrosis (atn) was supported by a high fractional urinary sodium excretion of 5.5%, elevated lactate dehydrogenase [486u / l (125250)], normal urine sediment and urinary tract ultrasound and a mag3 scan in keeping with atn (figure 1). The absence of myoglobinuria and only moderately elevated creatine kinase [maximum 893u / l (30170)] made rhabdomyoloysis - induced atn unlikely . He received supportive care with intravenous hydration, sodium bicarbonate combined with a total of seven sessions of hbo and recovered fully without needing dialysis (figure 2). 1.tc-99m-mag3 scan performed on day 4 post - diving accident: the first two panels show the perfusion of both kidneys over 32 s (2 s / frame) and the third panel shows tracer concentration (reflecting renal function) over 16 min (2 min / frame). The top graph (renogram) shows normal perfusion of the left and right kidney and the bottom graph illustrates tracer retention in the left and right kidney in keeping with acute tubular necrosis . 2.time line of creatinine (mol / l) and clinical symptoms . Tc-99m - mag3 scan performed on day 4 post - diving accident: the first two panels show the perfusion of both kidneys over 32 s (2 s / frame) and the third panel shows tracer concentration (reflecting renal function) over 16 min (2 min / frame). The top graph (renogram) shows normal perfusion of the left and right kidney and the bottom graph illustrates tracer retention in the left and right kidney in keeping with acute tubular necrosis . Decompression illness due to arterial air embolism following rapid ascent is a serious diving complication and can present with multi - organ involvement including the kidneys as demonstrated in this case . Upon rescue onto the support boat the patient was positioned supine whilst the boat was rocking in the waves, which may have contributed to embolization at multiple sites, including the central nervous system, the mesenteric circulation, as suggested by elevated lactate and abdominal pain and the kidneys . The diagnosis of decompression illness is made on clinical grounds since gas bubbles are rarely detectable on imaging . The most common symptoms of decompression sickness are localized pain, numbness / paresthesia and muscular weakness, typically presenting within the first 24 h, whereas symptoms of air embolism from pulmonary barotrauma occur immediately upon ascent [4, 5]. Although no diagnostic blood test exists for air embolism, widespread air embolization has been reported to result in elevated serum creatine kinase of up to 900 u / l, as was the case in our patient . The distinction between pulmonary barotrauma and decompression sickness is sometimes difficult and generally unnecessary as recompression is the treatment of choice for both . When hyperbaric therapy is not readily available, the best first aid treatment comprises administration of 100% oxygen and supportive care . However, early transfer to a hyperbaric treatment unit is important as symptoms may evolve over time as shown in our patient . Hyperbaric oxygen (hbo) leads to quick elimination of gas bubbles by reducing bubble size via increased pressure (boyle's law), increasing the inert gas partial pressure gradients between tissue and alveolar gas (increased nitrogen washout) and also oxygenates ischaemic tissue and exerts an anti - inflammatory effect [1, 7, 8]. The addition of non - steroidal anti - inflammatory drugs such as tenoxicam may decrease the number of hbo sessions required as shown in a randomized trial, but their use does no longer make part of the routine treatment and was not an option for our patient in the setting of acute kidney injury . Hbo results in complete resolution of symptoms in most cases, whereas mild residual symptoms may persist in a few and serious complications in a minority of cases . The primary goal, however, remains the prevention of pulmonary barotrauma, a potentially fatal complication of diving . Divers should be screened during dive medical for conditions predisposing to barotrauma (airway obstruction, bullae, reduced lung compliance) and diving courses emphasize the importance of controlled ascent and avoiding breath - holding . A.v . : patient diagnosis and management, literature search and writing of the paper; j.j . : patient management; a.w . : patient management; literature search and writing of the paper; n.b . : patient management; literature search and writing of the paper; and p.f . : management, literature search and writing of the paper.
This study is an extension of a report on patients with type 1 diabetes at children's hospital of new orleans (14) and was approved by the institutional review board at louisiana state university health sciences center, new orleans, louisiana . Glucose data were downloaded from patient meters at each clinic visit . Meter model and sampling protocols varied by patient preference and insurance provider . An average of three glucose measurements per day were recorded in a study using a similar self - monitoring protocol (7). A1c was measured by national glycohemoglobin standardization program (ngsp)-approved immunoassays (15) at the children's hospital (184 patients) or by commercial laboratories that presumably also used ngsp - approved methods (18 patients, including 4 low-, 7 moderate-, and 7 high - hgi subjects). A population regression equation {a1c (%) = [0.021 mbg (mg / dl)] + 4.3, r = 0.57} was derived using mean mbg and mean a1c from 202 patients collected at 1,612 clinic visits as described elsewhere (14). The same data were used to calculate hemoglobin glycation index (hgi) and to divide patients into low-, moderate-, and high - hgi groups . Predicted a1c values were calculated at each clinic visit by inserting mbg into the regression equation . Hgi values were calculated by subtracting predicted a1c from observed a1c measured at the same clinic visit . Patients were divided into low-, moderate-, and high - hgi groups based on mean hgi tertile (33%) rank (low hgi, <0.41, n = 67; moderate hgi, 0.41 to 0.26, n = 68; high hgi,> 0.26, n = 67). Eag was calculated by inserting observed a1c into the adag linear regression equation (eag [mg / dl] = [28.7 a1c (%)] 46.7, r = 0.92) (1). A mean blood glucose index (mbgi) that quantifies the difference between mbg and eag was calculated by subtracting observed mbg from eag . Descriptive statistics and linear regression analyses were generated using graphpad prism v. 4.03 (graphpad software, san diego, ca). Glucose data were downloaded from patient meters at each clinic visit . Meter model and sampling protocols varied by patient preference and insurance provider . An average of three glucose measurements per day were recorded in a study using a similar self - monitoring protocol (7). A1c was measured by national glycohemoglobin standardization program (ngsp)-approved immunoassays (15) at the children's hospital (184 patients) or by commercial laboratories that presumably also used ngsp - approved methods (18 patients, including 4 low-, 7 moderate-, and 7 high - hgi subjects). A population regression equation {a1c (%) = [0.021 mbg (mg / dl)] + 4.3, r = 0.57} was derived using mean mbg and mean a1c from 202 patients collected at 1,612 clinic visits as described elsewhere (14). The same data were used to calculate hemoglobin glycation index (hgi) and to divide patients into low-, moderate-, and high - hgi groups . Predicted a1c values were calculated at each clinic visit by inserting mbg into the regression equation . Hgi values were calculated by subtracting predicted a1c from observed a1c measured at the same clinic visit . Patients were divided into low-, moderate-, and high - hgi groups based on mean hgi tertile (33%) rank (low hgi, <0.41, n = 67; moderate hgi, 0.41 to 0.26, n = 68; high hgi,> 0.26, n = 67). Eag was calculated by inserting observed a1c into the adag linear regression equation (eag [mg / dl] = [28.7 a1c (%)] 46.7, r = 0.92) (1). A mean blood glucose index (mbgi) that quantifies the difference between mbg and eag was calculated by subtracting observed mbg from eag . Descriptive statistics and linear regression analyses were generated using graphpad prism v. 4.03 (graphpad software, san diego, ca). In our original description of this study population (14) we reported that the mean sd values of glycemic variables for the low-, moderate-, and high - hgi groups, respectively, were: mbg, 186 31, 195 28, and 199 42 mg / dl; a1c, 7.6 0.7, 8.4 0.7, and 9.6 1.1%; and hgi, 1.0 0.4, 0.1 0.2, and 1.1 0.9% . The present analysis used a1c from that study to calculate mean eag for the low, moderate, and high hgi groups, respectively, which were: 163 20, 193 19, and 230 31 mg / dl . Figure 1 compares eag and mbg in the population and in the different hgi groups and shows that mean eag and mean mbg were similar when compared in the population or in the moderate - hgi group . In contrast, eag underestimated mbg by an average of 12% (23 mg / dl) in the low - hgi group and overestimated mbg by 16% (31 mg / dl) in the high - hgi group . The average difference between eag and mbg in these groups represented an a1c difference of about 1% based on the slope of the adag regression equation . Linear regression analysis of hgi versus mbgi for all 202 patients showed that mbgi (the mean difference between eag and mbg) for individual patients was significantly positively correlated with mean hgi {mbgi (mg / dl) = [28.7 hgi (%)] + 1.9, r = 0.91, p <0.0001}. Disagreement between eag and mbg . Mean eag and mean self - monitored mbg were compared in all 202 patients in the population and separately by hgi group . Mbg was similar to eag in the population and in the moderate - hgi group, higher than eag in the low - hgi group and lower than eag in the high - hgi group . Dividing the study population into hgi groups automatically produces subpopulations with similar mbg but different a1c . Because eag is calculated from a1c, it is not surprising that eag systematically under- or overestimates mbg in some patients . The adag study concluded that a1c could be reliably translated into eag based on the linear relationship between a1c and mean blood glucose measured by continuous glucose monitoring in a mixed population of diabetic and nondiabetic subjects (1). This conclusion assumes that all population variation in a1c is either random or due to variation in blood glucose concentration . However, numerous reports of biological variation in a1c (713) indicate that this assumption is false . We previously developed hgi to quantify biological variation in a1c due to factors other than blood glucose concentration and showed that hgi was quantitatively consistent within individuals over time, different between individuals, normally distributed and positively correlated with risk for complications (7,8,14). The fact that many patients have hgi values that are always positive or always negative indicates that hgi measures systematic a1c bias between individuals . The present study clearly demonstrates that this systematic a1c bias makes eag a systematically biased estimate of mbg downloaded from patient glucose meters in high- and low - hgi patients . It is important to emphasize that the present study used routine a1c and mbg data typical of that available in most diabetes clinics . If a1c is reported as eag, patients and clinicians will be confronted with significant discrepancies between eag and self - monitored mbg, which will confound interpretation of glycemic control . Furthermore, treating patients based on eag alone could result in inappropriate medical decisions (2). 1, if low - hgi patients are intensively managed to a low eag target, their mbg would presumably remain above the target, inadvertently leaving these patients at unnecessary risk for chronic complications . Conversely, intensive management could drive mbg in high - hgi patients below the eag target, which presumably would increase their risk for hypoglycemia . We conclude that translating a1c into eag produced biased estimates of mbg downloaded from patient glucose meters in low- and high - hgi patients . However, because mbgi (the difference between eag and mbg) was positively correlated with hgi, eag derived using the carefully determined adag regression equation may have clinical value for assessing biological variation in a1c . Either hgi or mbgi could prove clinically useful for more comprehensive risk assessment and personalized patient care.
Oral health is an essential component of general health and overall well - being of an individual . Oral cavity and its surrounding structures that are free of any diseases is indicative of good oral health . This not only makes a person look and feel good, it is equally relevant in maintaining oral functions . Because professional (dental) students specialize in preventive information and health promotion, it is important that their own oral health knowledge, attitude, and practice are adequate . Moreover, medical students are far more likely to encounter underserved and vulnerable populations than dental students . They should have optimal knowledge regarding oral health so that they can provide the required oral health education and guide or refer to a dental surgeon as and when required . Thus, oral health care needs to be addressed by the combined efforts of dental and medical professionals and should be integrated into comprehensive health - promoting strategies and practices . Because today's students will provide health services in the future and will be responsible for public oral health education, it is important to study their oral health knowledge, attitude, and practice . The present study aims at comparison of oral health knowledge, attitude, and practices among dental and medical students of kiit university in bhubaneswar, odisha, india . Bds and mbbs students were invited from kalinga institute of dental sciences and kalinga institute of medical sciences, bhubaneswar, respectively, to participate in this survey using a self - administered structured questionnaire written in english and validated through a pilot study during the academic session 20162017 . It was voluntary participation, and informed consent was obtained from those who participated in the study . The questionnaire consisted of 27 questions designed to evaluate the oral health knowledge, attitude towards the dental surgeon and dental treatment, and practices in relation to oral health among the bds and mbbs students . The questionnaire was organized into 4 parts: the first part elicited information on the demographic attributes of students including age, gender, and year of study . The second part assessed the participant's oral health knowledge and included 18 questions on purpose of tooth brushing, type of tooth brush, time interval for change of tooth brush, flossing, knowledge about the cause and prevention of tooth decay, common dental diseases including gum disease, effect of soft drinks on teeth, bad breath, role of tobacco, oral cancer, and importance of oral health on general health . The third part was used to elicit their attitude towards the dental surgeon and dental treatment and comprised 5 questions regarding regular visit to a dental surgeon, attitude towards dental care, cost associated with treatment, and preferred place of visit in terms of dental clinic or dental hospital . The last part assessed the practices in relation to oral health by using 4 questions regarding their visit to dental surgeon, materials used and frequency of brushing, and oral hygiene aids used in addition to tooth brushing . The students were asked to respond to each item according to the response provided in the questionnaire . Responses included multiple - choice questions in which the students were instructed to choose only one appropriate response from a provided list of options . One hundred and fifty completely filled questionnaires from dental students and 150 from medical students were collected and analyzed . The obtained data were analyzed using the statistical package for the social sciences (spss) software for windows version 20.0 (ibm spss - chicago, il: spss inc . ). The mann whitney u test was used to compare the knowledge, attitude, and practice related to oral health in both groups . The t - test was used to compare the mean percentage scores for knowledge, attitude, and practices among dental and medical students . Correlation between knowledge, attitude, and practice were examined by karl pearson's correlation coefficient method . A p value of 0.05 was used as a cut - off level for statistical significance . The obtained data were analyzed using the statistical package for the social sciences (spss) software for windows version 20.0 (ibm spss - chicago, il: spss inc . ). The mann whitney u test was used to compare the knowledge, attitude, and practice related to oral health in both groups . The t - test was used to compare the mean percentage scores for knowledge, attitude, and practices among dental and medical students . Correlation between knowledge, attitude, and practice were examined by karl pearson's correlation coefficient method . A p value of 0.05 was used as a cut - off level for statistical significance . One hundred and fifty completely filled questionnaires from dental students (mean age: 20.19 1.46) and 150 from medical students (mean age: 21.00 2.05) were collected . Age and gender distribution of the participants a total of 92.67% of the students (96.67% dental and 88.67% medical) knew that the purpose of tooth brushing was to prevent tooth decay and gum disease, and the difference was statistically significant (p = 0.0080). Eighty - two percent of the dental students believed brushing with fluoridated toothpaste twice a day prevents tooth decay whereas only 66.67% of the medical students were aware about the fact; the values were statistically significant (p = 0.0020). In terms of knowledge regarding flossing, 88% dental and 64% medical students had heard about flossing, and the difference was highly significant (p = 0.00001). Awareness regarding chronic trauma increases the chance for oral cancer showed statistically significant difference (p = 0.0380) among dental (54.67%) and medical (42.67%) students . Almost all dental and medical students were aware that a soft toothbrush is preferable over hard bristles, smoking and/or tobacco chewing can cause oral cancer, and general health is related to oral health . More than 50% of the respondents knew about the interval for changing tooth brush, tooth decay and gum disease to be the most common dental diseases, improper brushing and consumption of sugary foods and soft drinks as the cause of tooth decay, presence of cavity as an indication of tooth decay, gum bleeding and how to prevent it, cause of bad breath, and smoking and tobacco chewing increases the chance for tooth loss . Table 2 shows the comparison of dental and medical students in each question of knowledge (numbers are only correct answers) by mann comparison of dental and medical students in each question of knowledge (numbers are only correct answers) by mann - whitney u test a total of 89.33% of the dental students believed that regular visit to dental surgeon is necessary whereas only 54% medical students agreed on the fact; the difference was highly significant (p = 0.00001). Medical students felt that dental surgeons give more importance to treatment rather than prevention in contrast to dental students who believed that both are given equal importance . Eighty percent of the students (85.33% dental and 74.67% medical) said they did not avoid or delay a dental visit due to cost, and the results were significant (p = 0.0050). Dental hospital and private clinic were almost equally voted as the preferred place of visit for treatment . Comparison of dental and medical students in terms of attitude towards the dental surgeon and dental treatment a total of 84% dental and 72% medical students had visited a dentist at least once in their lifetime, and the difference was statistically significant (p = 0.0120). Almost 97% of the students (94% dental and 99.33% medical) used toothbrush and tooth paste to clean their teeth, and the value was statistically significant (p = 0.0060). More than 70% of students did not use any other oral hygiene methods in addition to tooth brushing . Comparison of dental and medical students in terms of practice of oral health is presented in table 4 . Comparison of dental and medical students in terms of practice of oral health significant difference only in terms of knowledge was seen when mean percentage scores were compared between dental and medical students [table 5]. Comparison of dental and medical students mean percentage scores for knowledge, attitude, and behavior of students by t - test the study showed that female students (both dental and medical) had better oral health knowledge and showed better oral health practice than male students, and the difference was statistically significant [table 6]. Comparison of male and female dental and medical student s mean percentage scores for knowledge, attitude, and behavior by t - test correlation between knowledge, attitude, and practice was examined by karl pearson's correlation coefficient method . A positive linear relationship was found between attitude and knowledge, practice, and knowledge whereas a negative linear relationship was found between practice and attitude of dental students . Among medical students, a positive linear relationship was found between attitude and knowledge, practice and knowledge, and practice and attitude [table 7]. Health professionals play a pivotal role in providing knowledge regarding oral health and its significance among general public . Dental and medical students should possess high level of awareness of self oral health care so that this attitude can be instilled among patients and community at large . The methodological strength of the present study was that it was the first formal assessment of oral health knowledge, attitude, and practices among dental and medical students conducted in bhubaneswar city with an adequate sample size . The scores of knowledge, attitude, and practice of dental students was compared with that of the medical students, and it was noted that the mean knowledge score was significantly higher among dental students than medical students, which is in agreement with the results of similar study by rong ws et al ., al - batayneh et al ., saran et al ., and al kawas et al . A total of 77.33% of the dental students brushed twice a day compared to 53.33% of the medical students, hence indicating better oral hygiene measures adopted by dental students ., 34% of health sciences students brushed twice daily and 45% once a day . On the contrary, a higher percentage (65.33%) of both dental and medical students in our study brushed twice whereas a lower proportion (31.33%) brushed once daily . On the other hand, baseer et al . Reported that 77.9% of the health professionals brushed their teeth once in the morning . The university of nairobi showed that 27.5% of the dental and 39% of the medical students brushed twice daily, and the values were much lower than those reported in our study . Eighty - two percent of our dental students felt that tooth decay can be prevented by brushing with fluoridated toothpaste, and this is almost compatible with the findings of the studies by cebeci et al . Who reported it to be 79% and ahamed et al . Who reported it to be 81% among clinical dental students . According to our study, 96.67% students (141 dental and 149 medical) used toothbrush and toothpaste as cleaning aids . Similar results were reported by baseer et al . In a kap study among health professionals in riyadh . A total of 32.3% of turkish dental students flossed regularly while 16% of our dental students flossed . Only 79.33% of our study participants felt that there was a role of sugary foods in causing tooth decay, whereas ansari et al . In his study found that it to be 93.8%; thus, showing higher level of awareness compared to our study participants . On the other hand, both dental and medical students in our study were much more aware of the effect of soft drinks on teeth as compared to studies by ansari et al . Most of the dental (92.67%) and medical (87.33%) students considered oral health to be important in maintaining good general health, and this is compatible with the findings of the study by usman et al . (dental 96%, and medical 80%). On the other hand, 80% dental and 67% medical students in kanpur city considered oral health to be a part of overall health . The question of knowledge regarding smoking and tobacco chewing as the cause of oral cancer showed much higher frequency of correct answers among both dental (98%) and medical (94%) students in our study, and this was consistent with the findings of frola and barrios . On the other hand, reported that only 78% of the dental students were aware about the cause of oral cancer . In terms of visiting a dentist, 84% of dental students in our study had visited once in their lifetime in contrast to 30% and 32% reported by peker et al . And neeraja r et al . Respectively . This was significantly lower than that reported among jordanian students (86%) as reported by al omari et al . Mani et al . Reported that 92.4% of the dental students disagreed in terms of not worrying much about visiting a dentist, which was in accordance with our study . Reported only 8% of the dental students were not much worried about visiting a dentist as compared to 30% of the medical students who were . On the contrary, usman et al . Reported 88.3% and ansari et al . Reported 60% of the medical, dental, and paramedical students to have visited a dentist at least once in their lifetime a total of 89.33% of our dental students and 81% of medical students felt the necessity of regular visits to a dental surgeon . On the contrary, reported that 46% of the medical students put off going to the dentist until they had toothache as compared to 20% of dental students . Reported that 75% of medical students, 86% of paramedical students, and 69% of dental students put off visiting a dentist till they encountered a dental problem . Our study showed female participants to be better than their male counterpart in terms of knowledge and practice . Nanakorn et al ., kassak et al ., halboub et al . And rashid et al ., and contrary to the studies reported by ahamed et al ., ostberg and fukai reported female dental students to have better oral health attitudes than males . This has been attributed to the positive self care attitudes for internal psychological reasons to improve their appearance and self esteem . On the contrary, tseveenjan et al . Several studies have shown oral health knowledge and attitude to be high among the dental students because it forms a significant part of their curriculum, and hence, positively influences their attitude and behavior . Because good oral health is essential for good general health, medical education should include oral health as an integral component of their curriculum . Moreover, our study showed that though dental students had better knowledge and attitude towards oral health, rightly practicing it remains a concern . On the only limitation of this study is that, even though the confidentiality is maintained, scores depends on self - reported data, which may be over- or underreported due to social desirability . Several areas such as knowledge regarding association of chronic trauma and oral cancer, reasons for a delayed dental visit including cost factor, and use of other oral hygiene methods in addition to tooth brushing still remain unexplored, which warrant the need for conducting more such studies . Moreover, the present study is limited to a questionnaire . To study the effect of oral health education among dental and medical students, cross - sectional and longitudinal comparisons clinical examination of the students to substantiate the answers to the questionnaire would be more desirable . In the present study, we found that oral health knowledge of dental students were better compared to medical students as oral health is a significant component of the dental curriculum . Had better oral health knowledge and took better care of their teeth than male students . Further emphasis on oral health is necessary in undergraduate training to improve oral health knowledge, attitude, and practice among the students . These students who are the future providers of dental and medical care will act as role models for oral health education among individuals and community at large.
Primary ovarian cancers tend to spread, at first, within the peritoneal cavity and the omentum, and are frequently associated with ascites, a fluid rich in growth factors and tumor cells disseminated from the primary cancer that fills the peritoneum . Tumor spread to more distant sites, including the contralateral ovary and bilateral ovarian cancers almost always represents a primary tumor and its metastasis rather than dual primary cancers . The parallel evolution model suggests that cells with metastatic potential separate from the primary tumor at an early stage in development and evolve independently from the primary tumor . The ovary is a site for a wide range of tumors, both primary and metastatic . Colorectal metastasis from primary ovarian carcinoma account for approximately 4.0% and an isolated rectal metastasis is very rare . Identification of the correct primary tumor is necessary for an optimal management, including, specific chemotherapy (ct) in advanced stages . Whereas, ovarian adenocarcinomas respond to platinum based ct, cases of colonic adenocarcinomas are candidates for 5-fluorourocil based ct . A 50-year - old female patient presented with bilateral adnexal lump and dysfunctional uterine bleeding with altered bowel habits . Computed tomography of the abdomen and pelvis revealed a bilateral adnexal lump situated in the ovaries of sizes 108 cm and 43 cm, and a lobular mass of 8 cm at the rectum with peritoneal implants (figure 1). She underwent a total hysterectomy, a bilateral adnexectomy, and a bilateral iliac lymphadenectomy for the bilateral ovarian lumps . Gross features of the ovaries showed capsular breach with presence of solid and cystic areas on cut sections . The microscopic examination revealed a bilateral papillary serous adenocarcinoma with psammomatous calcification (figure 2). The surgical findings revealed an 8.0 cm submucosal tumor in the rectum and invasion from the serosal side . The tumor primarily involved the rectal muscularis propria and subserosa with only focal invasion of the rectal mucosa . The pathological findings of the ovarian tumor specimen were consistent with those of the hemicolectomy specimen . The patient was staged as t3cn1m1 and stage iv, according to tnm and figo classification respectively . Figure 1computed tomography of the abdomen revealed a bilateral adnexal lump situated in both the ovaries with peritoneal implants . Computed tomography of the abdomen revealed a bilateral adnexal lump situated in both the ovaries with peritoneal implants . Figure 2carcinoma ovary shows papillary serous adenocarcinoma with psammomatous calcification (hematoxylin & eosin 40). Carcinoma ovary shows papillary serous adenocarcinoma with psammomatous calcification (hematoxylin & eosin 40). Figure 3colorectal carcinoma shows moderately differentiated adenocarcinoma (hematoxylin & eosin 40). Colorectal carcinoma shows moderately differentiated adenocarcinoma (hematoxylin & eosin 40). Immunohistochemical staining was positive for cytokeratin 7 (figure 4) and negative for cytokeratin 20 (figure 5). This staining pattern was consistent with that of colorectal metastasis from the original ovarian lesion and immunohistochemistry supported the histological interpretation of a metastatic ovarian carcinoma . Post adjuvant 6 cycles of specific ct in the form of cisplatinum (50 mg) and 5-fluorourocil (100 mg) were administered to our patient and after 6 months of follow up period, she is doing well . Figure 4positive immunohistochemical staining for cytokeratin 7 (immunostain cytokeratin 7 40). Figure 5negative immunohistochemical staining for cytokeratin 20 (immunostain cytokeratin 20 40). Haraoka et al . In an autopsy series, reported that colorectal metastases present with an ovarian origin in about 6.0% of cases and represent about 30.5% of all ovarian cancers . Their patients ranged in age from 34 to 77 years with an average age of 58.8 years . The location was ascending colon in 4 patients, transverse in 2, descending in 5, sigmoid in 5, and rectum in 5 . The tumor appearance was protruding in 13 cases, ulcerative in 2, and a submucosal tumor type in 1 of the 14 cases in which the macroscopic appearance was reported . In 9 of the 14 cases in which the tumor protruded, distinguishing colorectal metastasis from ovarian carcinoma and primary colon cancer based on the macroscopic appearance is often difficult . Colorectal metastasis was observed to be synchronous with ovarian carcinoma in 7 patients in a study by loy et al .,, elevated levels of tumor markers such as ca-125 and cea may help to make a correct diagnosis . However, up to 15% of the cases of ovarian carcinoma were present without any elevated levels of serum ca-125 . Reported a cytokeratin 7 positive / cytokeratin 20 negative immunophenotype to be nearly 100% specific for an ovarian origin . Conversely, a cytokeratin 7 negative/ cytokeratin 20 positive immunophenotype was seen in 94% of the tumors of colonic origin . In our case, the colonic tumor was positive for cytokeratin 7 and negative for cytokeratin 20, a pattern consistent with an ovarian origin rather than a colonic one . In addition, a comparison of the patient's primary ovarian cancer with the colorectal mass revealed that the two tumors have similar histological features . The spread of ovarian cancer to the colorectum can occur in four different ways, hematogenous spread, lymphatic spread, direct invasion and peritoneal dissemination . The most plausible explanation of colorectal involvement in ovarian adenocarcinomas is through intra - peritoneal seedling . Hematogenous spread occurs in advanced peritoneal disease . In a substantial number of autopsy cases of advanced ovarian cancers, reed et al . Identified peritoneal involvement in 83100% cases, with large intestinal involvement in 5060% cases . In most cases of bowel metastases, the serosa is affected initially and then invasion extends from serosal and subserosal tissues into the muscularis propria and mucosa of the bowel wall . In the present case, the tumor mainly involved the rectal muscularis propria and subserosa with only focal invasion of the rectal mucosa and invasion of the retroperitoneum . Certain morphological features indicative of metastasis from colorectum include garland - like tumor necrosis, segmental destruction of glands and absence of squamous metaplasia, whereas cribriform growth patterns and intra - luminal dirty necrosis are indicators of ovarian adenocarcinomas . However, the problem exists in sorting out these tumors, including in cases mucinous adenocarcinomas, when there is simultaneous involvement of the ovary and the colorectum at similar (synchronous) or at different (metachronous) times . Showed that morphologically, metastatic tumor deposits in the colorectum retained the morphology of ovarian papillary serous cystadeocarcinoma with foci of psammomatous calcification in 6 of their 11 cases . Absence of garland - like tumor and necrosis were pointers towards an ovarian primary, apart from the clinical context of a preceding ovarian tumor in 81.8% such cases . It is thus important to identify whether or not a tumor has an ovarian origin using immunohistochemical markers, since the occurrence of gastrointestinal metastases from ovarian cancer is very rare . As described lately, specific cytokeratin immunemarkers like cytokeratin 7 and cytokeratin 20 have been documented to be helpful in resolving these dilemmatic situations of ovarian involvement by colorectal carcinoma and vice versa . Zighelboim et al . Described a single case of atypical sigmoid metastasis from a high - grade ovarian adenocarcinoma, using differential expression of ck7 and 20 . O'hanlan et al . Have reported that a longitudinal negative margin of 25 cm in the resected bowel along with a wedge resection of mesentery, including paracolic and intermediate - level nodes might be indicated to achieve optimal debulking of gastrointestinal metastases from ovarian carcinomas . Our patient underwent total hysterectomy, bilateral adnexectomy, and bilateral iliac lymphadenectomy with left hemicolectomy and postoperative chemotherapy . In cases of colorectal involvement by ovarian adenocarcinomas and vice versa, it can be difficult to ascertain an exact primary . In such cases, apart from complete clinical details, histomorphological features like garland - like necrosis, desmoplasia and psammomatous calcification are useful pointers towards exact primary and along with differential expression of cytokeratins 7 and 20 by immunohistochemistry can be helpful in solving these dilemmas.
The treatment of anterior cruciate ligament (acl) injuries has spawned a great deal of research . Far fewer studies have focused on the management of this condition in the pediatric and adolescent patient . Consequently, the management strategy for acl injuries in this population is not as clearly elucidated as it is for their skeletally mature counterparts . . Looked at insurance claims over a five - year period for soccer players aged 5 to 18 years and noted knee injuries constituted 22% of all injuries, while acl injuries accounted for 31% of the total knee injury claims . The risk for complete acl tears rises as children mature secondary to increased skeletal rigidity . Conversely, tibial spine avulsions and partial acl tears constitute a greater percentage of total acl injuries in preadolescents than in older children, due to the ability of the immature skeleton to absorb force at this age . Nonetheless, complete acl tears comprise a major portion of acl injuries in younger children [13]. Kellenberger and von laer similarly noted in a review of 330 patients with knee injuries that 80% with tibial eminence avulsions were less than 12 years old, whereas 90% of the patients with nonosseous acl lesions were over 12 . From a gender standpoint, it has been shown that as females approach skeletal maturity, and certainly upon reaching it, they have a higher risk of acl rupture (two to eight times) than do males . However, while still skeletally immature, the reverse is true with boys having the higher incidence of the two sexes [2, 5]. Historically, significant debate regarding the proper management of acl injury in the skeletally immature patient has existed . Two basic options are available, surgical reconstruction and conservative management, each with their own potential sequelae . Theoretically, there has been concern that operative management would violate the growth plate resulting in concomitant growth disturbance and angular or rotational deformity of the limb [610]. Consequently, many patients have been treated conservatively via activity modification and bracing with postponement of surgery until skeletal maturity [1113]. It has been shown that 21100% of children sustaining acl injuries will have coexistent meniscal damage [11, 1418]. Additionally, this age group is poorly compliant with activity restrictions leading to a significant risk of further meniscal injury or articular cartilage damage [11, 15, 18, 19]. In a recent systematic literature review, vavken and murray identified only 1 study with level ii evidence and 10 with level iii evidence on this topic . This paper aims to discuss acl tears in the skeletally immature patient, specifically focusing on the natural history, nonoperative management, operative management including complications, and prevention of these injuries . In the pediatric and adolescent patient, the location of the acl injury is an important determinant of management . Common in this population, tibial spine avulsion injuries, if nondisplaced, can be treated nonoperatively with satisfactory outcomes . However, displaced avulsion injuries require arthroscopic reduction and internal fixation [21, 22]. Similarly the extent of the acl tear is important to differentiate . Nonsurgical management of partial tears may yield acceptable results in this population when paired with a structured rehabilitation program . However, children and adolescents with greater than 50% tears of the acl have been shown to have poor outcomes if not surgically reconstructed and may progress to a complete tear . For complete tears of the acl, nonoperative treatment generally carries an unfavorable prognosis . It has been shown in numerous studies to lead to increased intra - articular damage in the form of meniscal tears and damage to the cartilage [11, 12, 15, 16, 24]. In a study of 39 pediatric and adolescent patients with an average age of 13.6 years at injury, millett et al . Retrospectively compared acute surgical reconstruction (less than 6 weeks from time of injury), to chronic reconstruction (more than 6 weeks after injury). 36% of patients in the chronic group sustained medial meniscal tears versus only 11% of acute group, which led the authors to support early operative intervention . . Retrospectively looked at 56 patients who sustained an acl tear while skeletally immature and compared those who had open physes at reconstruction with those whose reconstruction was delayed until skeletal maturity . . A higher rate of medial meniscal tears in the delayed group, (41%) compared to the open physes group (16%) was noted . In addition, a higher rate of meniscectomy and lower subjective outcome scores were noted in the delayed group . No growth disturbances were found in either group . Also, in a recent comparative study, streich et al . Compared 28 children with intraligamentous tears of the acl, all were tanner stage 1 or 2 with a mean age of 11 years at time of treatment . Interestingly, the surgery group was selected to include only patients who had concomitant damage to the meniscus or articular cartilage . The nonoperative group included only isolated acl ruptures . At a mean follow - up time of 70 months, the patients had grown an average of 20.3 cm with no evidence of leg length inequality or angular deformity in either group . However, the surgical group had significantly better clinical and functional results than did the nonoperative group . Additionally, 58% of the nonoperatively managed patients went on to require surgical intervention due to persistent instability . A recent systematic review found only 1 study that showed no increase in secondary intra - articular injury in conservatively treated patients in whom surgery was delayed until skeletal maturity . The identified study by woods and o'connor retrospectively compared two groups of adolescents with acl rupture . One group of 13 adolescents with a mean age of 13.8 years at time of injury, presented with open physes . Surgery was delayed until skeletal maturity and performed at a mean of 70 weeks following injury . The other group of 116 adolescents had a mean age of 15.0 years at time of injury, presented at various time intervals after acl rupture, and were skeletally mature on presentation . The skeletally mature group was not intentionally delayed and had a mean time interval from injury to surgery of 14.1 (0.3355.1) weeks . No significant difference with respect to overall additional knee injuries, meniscal injuries, and articular cartilage injuries was noted between the delayed patients and the skeletally mature patients . The authors attributed the lack of additional knee injuries in the delayed group to strict adherence to nonoperative treatment including, complete abstention from sports activities and daily use of an acl brace . One self - described limitation of this study was a lack of statistical power due to small sample size . Examined acl rupture in children 12 years of age and younger comparing 20 nonoperatively treated patients to 6 delayed reconstruction patients at a minimum of 2-year postinjury or postoperative followup respectively . Copers, if they had returned to preinjury activity level and performed above 90% in all hop tests, or as noncopers . Of the nonoperative group 65% returned to preinjury activity level and 50% were classified as based on the large number of copers in the nonoperative group and relatively low number of meniscal injuries, a treatment algorithm based on functional and patient subjective measures was suggested that could identify patients who could be allowed to participate in their desired activities until skeletal maturity when acl reconstruction could be considered . Due to the substantial amount of literature showing risk of further damage to the joint and recurrent instability requiring surgical intervention, prolonged nonoperative therapy for complete acl rupture remains controversial . In addition, from a compliance standpoint, the pediatric and adolescent population will likely have significant difficulty with stringent activity restrictions . However, if nonoperative treatment is chosen, the protocol should include bracing of the affected knee, restriction of sports participation and other activities involving jumping and pivoting, and structured physical therapy and rehabilitation . It traces a course from its proximal attachment on the posteromedial portion of the lateral femoral condyle and travels distally and medially to attach in front of and lateral to the tibial spine on the anterior tibia [28, 29]. Though the acl is proportionally smaller in the pediatric and adolescent populations compared to the adult population, the anatomic landmarks remain the same . Acl reconstruction aims to restore stability and functionality to the knee by recreating the native anatomy . The primary concern with acl reconstruction in the skeletally immature patient is disruption of the tibial or femoral physis with resultant growth disturbance and deformity of the joint . Approximately two - thirds of the length of the lower extremity is derived from growth at the knee joint, specifically from the distal femoral and proximal tibial physes . The distal femoral growth plate is actually the largest and fastest growing physis in the human body accounting for roughly 70% of the length of the femur and 40% of the length of the entire lower limb . Similarly the proximal tibial growth plate contributes 55% of tibial length and 25% of leg length . On average, the two growth plates add approximately 1 cm and 0.6 cm of length, respectively, to the lower extremity per year . They do so until final skeletal maturation takes place, usually between age 1416 years in girls and 1618 years in boys [31, 32]. The transphyseal technique for acl reconstruction is the standard operative method for treating adult patients . Consequently, when adolescents are nearing skeletal maturity, it is commonly accepted that they may be managed as adults . Mccarroll et al . Reported good to excellent results in a cohort of 60 athletes with a mean age of 14.2 years, using transphyseal acl reconstruction with bone - patellar tendon - bone (btb) graft . Of note, btb grafts are typically avoided in the skeletally immature patient as growth arrest can be induced from bone bridges resulting from insertion of the bony portion of the graft across the physis . For this reason, kocher et al . Advocated the use of soft - tissue grafts in acl reconstruction of skeletally immature pubescent adolescents . In their study of 59 patients with a mean age of 14.7 years, excellent functional results were reported with a low revision rate and minimal growth disturbance using transphyseal acl reconstruction with autogenous hamstring grafts . Mcintosh et al . Described good clinical results and return to previous activity level in patients with wide open physes who had undergone transphyseal reconstruction . Even in tanner stage 1 or 2 patients, two studies have shown satisfactory results with transphyseal procedures in patients with a mean age of 12.1 years and 11 years, respectively [25, 35]. No growth disturbance was noted in either study, and only one patient was noted to have an angular deformity, which was not deemed to cause any functional impairment . When utilizing transphyseal techniques in these patients, of paramount importance is the avoidance of fixation devices or hardware crossing the physis . As an alternative to the transphyseal approach, a number of physeal sparing techniques, both intra - articular and extra - articular, have been described . Theoretically, these techniques should minimize the risk of growth disturbance or angular deformity by avoiding violation of the physis . Though a number of retrospective studies exist with the majority achieving excellent results, there is a scarcity of prospective or comparative data that would advocate the superiority of one method over the other . One of the first to use a physeal sparing approach, macintosh and darby in 1976 described good results using a portion of the iliotibial band looped around the lateral femoral condyle, through the knee and attached to the proximal tibial metaphysis distally to reconstruct the acl . A recent systematic review identified 6 studies using modifications of this physeal sparing, extraosseous reconstruction technique, and showed no growth deformity at an average 47.3 month followup in patients with a mean age of 12.1 years . 5 preadolescents (tanner stage 1) at a minimum of 4-year followup demonstrated excellent stability and no leg length discrepancy or angular deformity . Other studies have also shown all - epiphyseal techniques to be safe and efficacious [38, 39]. A hybrid of physeal sparing and transphyseal approaches, partial transphyseal techniques utilize only one tunnel through the physes thereby limiting, in theory, the possibility for growth disturbance . Several studies utilize this method, which has been described both with tunnels drilled only through the femoral epiphyses and with tunnels drilled only in the tibia . Both techniques have demonstrated satisfactory results [14, 17, 40, 41]. Growth disturbance and angular deformity after acl reconstruction in the skeletally immature patient have been a primary area of concern for surgeons treating patients in this demographic . In animal studies, various technical factors have been associated with physeal injury and subsequent growth disturbance, including fixation of the graft near or across the physis, increased tunnel diameter in relation to physeal diameter [7, 9], overtensioning of the graft [6, 42, 43], placement of bone blocks across the physis, inadequate filling of the tunnels with graft material [45, 46], and tunnel malposition . An extensive discussion of these technical aspects is beyond the scope of this review . In a survey of the herodicus society and the acl study group, kocher et al . Identified 15 reported cases of growth disturbance / angular deformity in human patients . The main factors associated with these cases were hardware fixation across the physis and spanning the physis with graft bone plugs . Large tunnel size was also associated with these undesirable outcomes . Though growth deformities have been clearly demonstrated in scientific studies using animal models, the vast majority of these reports in skeletally immature humans are from case studies and survey data [8, 48, 49]. Vavken and murray in their recent systematic review of acl tears in the skeletally immature patient identified 31 studies (479 total patients) of acl reconstruction with at least 1 transphyseal tunnel and noted only 3 angular deformities and 2 limb - length discrepancies . They identified 6 reports (106 total patients) of extraphyseal reconstruction with no growth deformities described . In comparison, the same systematic review identified 12 articles (476 total patients) reporting nonoperative management with a mean of 50.2% of patients who required later surgical stabilization due to unstable knees with severe meniscal and cartilage damage . When comparing operative techniques, few studies actually contrast the various operative procedures . In a recent review, . Identified 13 case series of various acl reconstruction methods in 187 tanner stage i, ii, and iii patients . They concluded that there was no clinical difference between transphyseal and physeal sparing techniques as both produced excellent clinical results in tanner stage ii and iii patients with a very low incidence of growth abnormalities in either group . Due to a lack of studies in tanner stage this included 55 studies and 935 patients with a mean age of 13 years, and a mean followup of 40 months . They determined the rate of leg - length discrepancy or axis deviation to be 1.8% (95% confidence interval [ci], 0% to 3.9%). Also of note, transphyseal techniques were associated with a lower risk of leg - length discrepancy or axis deviation than were physeal sparing procedures (1.9% versus 5.8%; relative risk, 0.34; 95% ci, 0.14 to 0.81). The authors theorized that this phenomenon may have resulted from drilling close to the growth plate in the physeal sparing techniques potentially leading to heat damage and early closure . Though there may be little that can be done to prevent traumatic (contact) acl injuries, discrete risk factors do exist for noncontact acl tears . Specifically, neuromuscular, anatomical, hormonal, shoe - surface interaction, and environmental causes have been identified as potential risk factors for acl rupture . Mandelbaum et al . Conducted a prospective non - randomized controlled trial over the course of two years in female soccer players aged 1418 years . They examined the effectiveness of a neuromuscular and proprioceptive training program, including education, stretching, strengthening, plyometrics, and sports - specific agility drills, on the incidence of acl injuries and noted a significant decrease in the treatment group over the two year period . Interestingly, distefano et al . Conducted an acl prevention study on 65 soccer athletes with a mean age of 10 years . They compared pediatric specific and traditional training programs to non - trained controls with the hypothesis that the age specific program would produce the best results . However, the youth athletes saw no significant improvement versus the controls in the pediatric program, whereas significant improvements in balance and vertical jump height were noted with the traditional program . From a training standpoint specific training areas include balance, lower extremity strength and stretching, body awareness, and core strength and control . These facets of a neuromuscular program work to reduce risk factors such as landing force and knee valgus moments as well as increasing advantageous, protective muscle activation . However, further research is required in order to validate the effectiveness of any proposed intervention program . Managing acl tears in the skeletally immature patient is a complicated and at times challenging undertaking . As such, it should be undertaken only by a surgeon with experience treating pediatric and adolescent injuries of this nature . Two basic choices exist: (1) conservative management with or without delayed reconstruction or (2) early reconstruction . While data can be found to support both modes of care, an overwhelming preponderance of the literature supports early operative intervention for complete acl tears in this population . Operative intervention has consistently been shown to increase knee stability and decrease the risk of further damage to the meniscus and articular cartilage with minimal risk of growth disturbance . Conservative or delayed operative care should only be considered in the most compliant patients with uncomplicated injuries . As there is little data supporting one surgical technique as superior, patient age and surgeon familiarity and comfort should guide the choice.
Most reported cases have documented the development of significant complications, since it is difficult to diagnose this rare entity preoperatively . The literature is reviewed and the radiologic findings and management of this rare entity are discussed . A 90-year - old woman was referred to our hospital for acute appendicitis complicated with right incarcerated groin hernia . She initially presented to a local hospital with right lower quadrant abdominal pain for one day . The abdomen and pelvic ct with intravenous and oral contrast showed swelling of the appendix with perifocal fat stranding and cecal wall thickening (figure 1a). There was initial improvement, however, a painful and irreducible lump in the right groin developed three days thereafter . On referral, a previously performed ct revealed the finding of a fluid - contained mass with stranding of surrounding fat in the right groin lateral and inferior to the pubic tubercle . Neither the appendix nor cecum was in close proximity to the mass (figure 1b). The appendix was inflamed and severely adherent to the cecum and terminal ileum but there was neither perforation nor abscess . The femoral hernia neck was constricted, and there was no herniation of the abdominal viscera (figure 2a). A separate groin incision was made, and phlegmonous inflammation of the distended femoral hernia sac was found (figure 2b). Dissection was carried out to free the entire circumference of the hernia sac and the neck . The hernia sac was excised, and the neck was closed with suture - ligation . Mcvay hernioplasty was performed . On opening, the sole content of the hernia sac was pus . The reported incidence of appendicitis within a groin hernia is 0.13% of all cases of acute appendicitis.1 it most frequently occurs in the right inguinal and right femoral hernias . Rene jacques croissant de garengeot, in 1731, first reported a case of acute appendicitis within a femoral hernia.2 since then, there have been only scattered case reports of appendicitis within femoral hernias . The clinical presentation usually suggests an incarcerated or strangulated femoral hernia, and acute appendicitis is incidentally found at operation . With the increased use of ct for evaluating patient with atypical presentation of incarcerated hernia and lower abdomen peritonitis, there have been occasional reports in which the diagnosis of hernial appendicitis is made preoperatively . The typical ct features include a tubular structure within the hernia sac with surrounding fat stranding and low position of the cecum or close proximity to the hernia sac.35 this prospective ct diagnosis may be helpful in establishing appropriate treatment strategy . Our case illustrates a very rare variety of femoral hernia appendicitis . The appendix was not contained in the hernia sac . Instead zuckerkandl first described this phenomenon in 1891, albeit within an inguinal hernia sac.6 as with any peritoneal recess, the hernia sac may contain pus secondary to any intraperitoneal infectious process . The pus causes distention and inflammation of the sac, which was mistaken for strangulated hernia . The appendix may be either in close proximity to or distant from the hernia sac . There is usually no evidence of intestinal obstruction, which will raise suspicion of strangulated omentum or richter s hernia . Once the diagnosis of strangulated femoral hernia is made, most surgeons prefer direct surgical intervention to avoid considerable risk of postoperative complications . Thus, the diagnosis of appendiceal pus - containing femoral hernia is made only during the operation . Despite wide availability of ct, the ct features of pus - containing femoral hernia is almost never reported in the literature . In our patient, the ct showed an inflammatory fluid - filled mass lateral and inferior to the pubic tubercle, a typical location of acute appendicitis, and no evidence of intestinal obstruction . The differential diagnosis is acute appendicitis complicated with a rare complication of pus - filled femoral hernia or a richter s hernia . As illustrated in our case, the awareness of the ct findings facilitated the decision on a treatment strategy . Although drainage of purulent content or appendectomy via hernia sac has been reported in many cases in which diagnosis is not expected, it carries a high rate of infectious complications . Separate laparotomy and groin incision would be very helpful in complete dissection and direct excision of the hernia sac . There are some controversies surrounding the method of femoral hernia repair in cases of hernial appendicitis or pus - containing femoral hernia . However, if no spillage of infectious content occurs, mesh repair is a reasonable alternative . In conclusion, selective use of ct for evaluating an atypical case of incarcerated femoral hernia may be helpful in early diagnosis and decision on the treatment strategy . The ct features of typical acute appendicitis and incarceration of inflammatory fluid - filled hernia sac without evidence of intestinal obstruction should raise suspicion of this rare entity.
Diabetes mellitus (dm) is a metabolic consequence of a decrease in insulin production and/or activity characterized by hyperglycemia and vascular and nerve impairment . The macroangiopathy and, above all, the microangiopathy are the most important pathogenic consequences of the excess of glucose in the blood . We can distinguish two main types of diabetes: type 1 diabetes (t1d) in which the main cause is a deficiency of insulin production due to self - destruction of the pancreatic beta - cells and type 2 (t2d) in which the initial insulin resistance leads, with time, to an insulin deficiency . Diabetic retinopathy (dr) is a serious and frequent complication of diabetes resulting from damage to the retinal microvasculature . The retinal cells primarily involved in dr are both endothelial and neuronal cells . With time, especially if the glycemic control is not adequate, diabetes causes a weakening of the walls of smaller vessels that results in the formation of microaneurysms and then edema, bleeding, and microinfarcts (ischemia). The new vessels grow in a chaotic way by destroying nervous tissue, causing increasingly serious bleeding and promoting retinal detachment . The prevalence of dr is directly proportional to the prevalence of dm . To date, approximately 366 million people worldwide have diabetes and this number is expected to increase . The incidence of the disease is increasing exponentially in developing countries [2, 3]. Dr is generally considered a disease of the retinal vessels but has been rarely approached as a real neurosensory disorder in which the visual impairment results not only from a microvascular alteration but also from a nervous impairment (diabetic encephalopathy). Ocular symptoms (such as a slow and gradual decrease in visual acuity, metamorphopsia, and a sudden loss of vision in one eye) occur when the dr has reached a very advanced and irreversible stage: the diagnosis is often too delayed . There is indication for specific techniques such as the optical coherence tomography or oct (in particular in the presence of a macular edema) and the intravenous fluorescein angiography (ivfa), which, however, is an invasive examination (it needs an intravenous injection of a contrast medium). In recent years, psychophysical and electrofunctional exams are having an increasing use because several studies have shown the sensitivity of these methods in identifying signs of the disease already in the preclinical phase . Over the past two decades, the advent of new neurophysiological techniques to assess retinal and brain (optic tract) function, such as electroretinography (erg) and the measurement of visual evoked potentials (vep), allowed a more detailed study of the visual function and of the effects that dm can have on it . The standard flash erg is an electrofunctional test able to evaluate the bioelectrical massive retinal response to an unstructured light stimulus (flash). It allows us to test the operation of the entire surface of neuroretina, limited to the photoreceptor and outer plexiform layers . The potentials recorded reflect many events that relate to different types of cells: photoreceptors, bipolar cells, amacrine cells, and mller cells . According to the international society for clinical electrophysiology of vision (iscev), the standard erg examination (table 1) consists of a minimum of 5 different surveys: scotopic erg (dark adapted eye and weak flash), massive combined erg (dark eye and strong flash), oscillatory potentials, photopic erg (erg cone with strong flash and light adapted eye) and flicker - erg (with a quickly repeated stimulus). Each component of the erg is characterized by the following parameters: the latency (the time that elapses between the start of the stimulus and the beginning of the response), the implicit time (the time, expressed in milliseconds, between the start of the stimulus and the peak of the response), and then the amplitude (e.g., the voltage wave). Tzekov and arden already in 90s emphasized the importance of light adapted flash erg and oscillatory potentials in understanding the pathophysiology of dr and light adapted flash erg and oscillatory potentials usefulness in predicting progression from nonproliferative to the more sight - threatening stages (preproliferative or proliferative). In a research of yamamoto et al . Flash erg has been used to study the responses of cones in 31 diabetics (15 of them had no signs of retinopathy). Results showed, in diabetics with or without retinopathy, an early involvement of type s cones sensitive to blue light (the amplitude of the b - wave was reduced) which appear to be more susceptible to hypoxic damage . However, oscillatory potentials (ops) are considered the most relevant electroretinographic test for dr diagnosis . They are 4/5 waves of small amplitude and high frequency that overlap the ascending phase of the b - wave [10, 11]. These waves seem to reflect the activity of the negative feedback exerted by the amacrine cells towards bipolar and ganglion cells . The oscillatory potentials are excellent markers of trophic disorders of the retina and, therefore, frequently they are absent in diabetic patients even in a preclinical stage of retinopathy [4, 12]. Op-2 and op-3, in particular, tend to disappear early when the foveal and parafoveal area are affected while op-4 disappears in more extensive injuries . Luu et al ., in an attempt to correlate the changes in the erg with the caliber of the retinal vessels of patients without clinical signs of dr, have shown a reduction in the amplitude of the oscillatory potentials and slower implicit time; the scotopic erg has also allowed them to detect a predominant involvement of the rods . An increase in the activity of mller cells has been demonstrated in mice with streptozotocin - induced diabetes (the streptozotocin is a substance toxic to pancreatic beta - cells; a single injection of 6070 mg / kg is sufficient to cause an insulin - dependent diabetes in 48 hours). This phenomenon resulted in an alteration of ops, a reduction of amplitude, and an increase in latency . Using the same type of laboratory animals, in 2011 wright et al . Have postulated the possible role of glutathione (gsh) in the genesis of electroretinographic alterations: indeed there were noted correlations between gsh and all erg parameters (with the exception of b - wave implicit times), not significantly altered by the presence of hyperglycemia . Neurovascular coupling is a physiological process adjusting the nervous microcirculation blood flow in response to neuronal activity . The flicker - erg stimulation (30 hz frequency) was used in healthy subjects to study this process: indeed, it induces a greater activity of nerve cells and, therefore, a microvascular response due to release of no (nitric oxide) and other vasodilatory substances by excited neurons and by endothelial cells [1720]. Several studies, using instruments able to evaluate the response of retinal vasculature as the dynamic vessel analyzer (dva; imedos, jena, germany), have shown that, in diabetic subjects without signs of retinopathy, there is a reduction of the retinal vessels vasodilator capacity in response to flicker stimulation [21, 22]. Probably this is the basis of the reduced oxygen supply to the retina in diabetic subjects and this impairment seems to be directly proportional to the degree of retinopathy, if it is present . Therefore, at the genesis of altered responses to flicker stimulation in diabetic subjects, several mechanisms seem to contribute: on the one hand there is the damage to neurons and photoreceptors; on the other hand there is the microvascular damage itself, which (causing hypoxic injury) establishes a sort of vicious cycle against the retinal cells . Recently, the miniganzfeld stimulator retimax by cso (scandicci, florence, italy) is under implementation with specific analytical software for dr (diabetic retinopathy test, drt). The drt is based on 30 hz flicker stimulation and allows evaluating both the amplitude and latency showing, for each parameter, the standard deviation (sd) compared to normative values present in a database (based on the age of the patient). It provides a measure of retinal and macular integrity especially when the changes are minimal and dysfunction is localized in a small area . The mferg reflects the function of a wide part of the posterior pole (4050 degrees), and the result obtained groups together a set of weak amplitude responses (10 volts) mainly elicited by the first two retinal layers (photoreceptor layer and outer plexiform - bipolar layer). Searching some predictive risk factors for the development of dr, numerous research groups have used the mferg . The reason for this interest is the discovery that in the retina occur neuronal alterations (and thus functional ones) much earlier than vascular impairment, which is already an indication of anatomical damage: mferg allow correlating very accurately a functional deficit with the part of retina affected . The parameters considered in the various studies have been the implicit time (it) and the amplitude (amp) of p1 main wave . Among the most relevant studies in this sense, harrison et al . Showed the sensitivity of mferg in the early detection of retinal areas affected by dr, correlating functional alterations (increase of it and reduction of amplitude) with the anatomical damage . They monitored 46 eyes of 23 patients using a grid dividing retina into 35 zones: the most altered areas at mferg examination were, during the follow - up, the first to develop a macular edema . Similar approach, but with a longer lasting follow - up, had the study of ng et al . : results found were comparable even with a lower sample size of subjects examined (18 patients). Evaluating mferg in younger people (adolescents with t1d), observed an increased susceptibility (and particularly an increased it) of the nasal retina compared to other areas, as also holm and adrian have demonstrated in adults: these findings indicate that the nasal retinal area is the most vulnerable to diabetic damage, and mferg can be very useful for early evaluation . Similar target study in 2010 of lakhani et al . Examined mferg in 48 adolescents with t1d without dr and 45 controls . Considered parameters were glycated hemoglobin (hba1c) levels, time since diagnosis of diabetes, age at diagnosis, age at testing, and sex . The researchers recorded standard (103 hexagons) and slow - flash (61 hexagons) mferg and found that a poor long - term glycemic control is associated with an increase of localized neuroretinal dysfunction areas . Therefore, latest researches have demonstrated that mferg reveals local retinal dysfunction in diabetic eyes before the onset of retinopathy, in direct proportion to the degree of clinical abnormality . In particular, the analysis of p1 it variations improves the test sensitivity since is the first parameter to be altered . Hard exudates, especially, seem to prolong p1 implicit time compared to healthy eyes and independently of macular thickness . Other authors did comparisons with particular programs, as the m1m2 paradigm or the photopic negative response, and, even in these cases, the ability of the mferg to identify the damaged areas of the retina in the preclinical phase has been confirmed . In a very recent research wright et al . Also used the spatial - temporal partial least squares (pls - st), a multivariate analysis that improves the data derived from modern imaging techniques . Using data derived from all points earned, the st - pls allows a rigorous statistical evaluation of changes in the waveform and signal distribution related to retinal function . The results of the traditional techniques of analysis were compared with that of the st - pls: the first revealed, in subjects suffering from t1d without dr, variations of the it but not of the amplitudes and, in addition, the spatial position of these changes has not been identified . In contrast, using the st - pls, researchers found significant variations between groups and they could highlight the spatial position of these changes on the retinal map, confirming that the changes in retinal function in dm occur before the onset of clinical disease . The mferg examination has proved to be very useful in the preclinical phase but less suitable in the follow - up of patients with dr after medical intervention or laser surgery, especially in comparison with other methods of examination such as the oct and the ivfa . However many results are conflicting, also because of the subjects heterogeneity and the differences in the techniques used . For example, durukan et al . Found that mferg cannot be performed to evaluate retinal functionality after the treatment of diabetic macular edema (dme) with intravitreal injections of triamcinolone acetonide, probably because of the irreversible macular damage . On the other hand, du et al have documented a reduction in the amplitude of p1 wave after a treatment with laser photocoagulation and leozappa et al . Have evaluated the mferg 1 week and 1, 3, and 6 months after surgery (standard three - port pars plana vitrectomy with peeling of inner limiting membrane) in 25 eyes of 21 patients with dme: both researches have considered mferg useful for predicting functional prognosis . The pattern electroretinogram (perg) detects the functionality of the innermost retinal layers (ganglion cells and fibers). Perg is measured by using conjunctival or skin electrodes that do not alter vision, and visual stimulus is constituted by a structured pattern (typically a chessboard) in which white and black elements alternate with a regular frequency . Caputo et al . Examined 42 patients with t1d with a number of microaneurysms (highlighted by fluorescein angiography) from 0 to 4 and a disease duration less than 11 years . Perg results showed the amplitude of n95 wave significantly reduced in diabetics compared to control subjects of the same age, and significant differences were found between controls and diabetics without retinopathy, controls, and diabetics with retinopathy and between diabetic patients with early retinopathy versus diabetics without retinal impairment . In addition, the amplitude resulting inversely correlated with the duration of the disease . Because of the sensitivity of the method in detecting the activity of retinal ganglion cells, perg has been also intensively used in diabetic subjects with suspected glaucoma or ocular hypertension . The amplitude of n95 wave was altered in diabetic subjects with suspected glaucoma compared to controls, even when the visual field examination was normal . A further application of this test, recently showed by ozkiri, was to evaluate the functional recovery after treatment of diabetic macular edema with intravitreal injections of bevacizumab . After 1 and 3 months, the author found an increase in both visual acuity and the amplitude of p50 wave in 35 eyes treated with bevacizumab at a concentration of 2.5 mg . The focal erg, also called foveal erg or focal macular erg (fmacerg), is mainly used for the evaluation of foveal cones . Usually it is registered in an on - off modulation at low (e.g., 8 hz) and high frequency (e.g., 41.4 hz). . Showed an increase of implicit time and a reduction in the amplitude of the ferg in 26 patients with t2d but without any ophthalmoscopic sign of retinopathy compared with 52 healthy controls . They also showed a significant correlation between these changes and the duration of the disease rather than the values of glycated hemoglobin (index of glycemic control)., however, have undergone 60 subjects affected by t1d to the analysis of ferg using a small stimulus (9 degrees) and a frequency of 8 hz . The analysis of harmonics revealed an alteration of f2 wave which resulted from reduced amplitude in diabetics with mild or even absent retinopathy compared to healthy controls of the same age . A statistically significant correlation with such alterations has been demonstrated both with the duration of the illness and with glycemic control . The visual evoked potentials (veps) are defined as changes in the bioelectric potentials of the occipital cortex evoked by visual stimuli . They are generated by complex neurosensory events related to the translation and transmission of nerve impulses along the optic tract, from the photoreceptors to the occipital cortex . They can be elicited with pattern or with flash stimuli . As pointed out by the recommendations of the international federation of clinical neurophysiology (ifcn) and international society for clinical electrophysiology of vision (iscev), it is extremely important to use standardized methods in order to standardize and share data between individual laboratories (table 2). The pattern vep is constituted by a set of electrical responses evoked by the variation of luminance contrast of a structural stimulus (typically a chessboard) projected on a tv screen and detected with specific electrodes placed on the scalp . The flash vep, instead, is constituted by a set of electrical responses evoked by a light stimulus of short duration and high intensity . The response of optic nerve fibers to this type of stimulus is different from response to a pattern stimulus: in this case it has nothing to do with the ability of discrimination (visual acuity) but more roughly leads information of brightness (magnocellular system) and movement . The main parameters of veps that can be evaluated are latency, amplitude, topography, and shape of the wave . Several external factors such as technical characteristics, cooperation of the patient, fixing, attention, sex, age, transparency of the optical mediums, and the size of the pupil may alter, more or less significantly, the examination . However, the amplitude and the latency of the p-100 wave are the most reliable indicators of clinically significant alterations of the visual pathway . A significant reduction in amplitude and increased latency of veps was found in both types of dm without signs of retinopathy . Several studies involving patients with various degrees of retinopathy found a strong correlation between retinal neovascularization (proliferative dr) and abnormal veps, attributed to a substantial damage of the ganglion cells and the retinal nerve fiber in diabetic subjects [5153]. Have recently emphasized the role of the veps in identifying signs of damage to the retinal ganglion cells before the onset of clinical signs of the disease in 40 diabetic patients including 20 subjects with nonproliferative diabetic retinopathy (npdr) and 20 others without any retinopathy on fundus oculi and compared to 40 age- and sex - matched normal nondiabetic controls . The pathophysiology of central nervous system dysfunction in patients with dm is not completely understood, but it certainly has a multifactorial etiology . Probably vascular and metabolic factors are involved similarly to what happens in diabetic peripheral neuropathy, in which the ischemia and reduced protein synthesis cause the loss of nerve fibers . In support of a common pathogenetic hypothesis between peripheral and central neuropathy, some authors argue that subjects with peripheral damage have abnormalities of the veps higher than those without signs of peripheral nerve involvement . It also seems that such damage is related to duration of disease rather than glycemic control . What appears quite clear is that the damage to central neurons is very early compared to the retinal one [56, 57]. Recently more complex methods such as multifocal vep (mfvep) have also been used to try to correlate the alteration of evoked potentials with specific retinal areas . Wolff et al . Found significant mfvep implicit time (it) differences between controls and all patients with diabetes, controls, and diabetics without retinopathy and between controls and diabetics with retinopathy . In the retinopathy group, the mferg it was more frequently abnormal than mfvep it . Considering those findings, it would be recommended to assist veps with flash and pattern electroretinogram (perg) in order to confirm the existence of an involvement of the outer retina and therefore exclude a direct involvement of the inner retina and/or of the visual pathway . Retinopathy, as a major complication of diabetes, has clearly an important role in the genesis of visual dysfunction . However, as has been widely documented, several anomalies occur in the retina and in visual pathways long before structural alterations may be clinically detected . Visual abnormalities in diabetes must be approached in a broader sense, considering the visual function as a complex sensory system . The techniques described allow the evaluation of this system in the various stages of the visual process and have an important role in both in clinic and research settings . Complete knowledge of the function and the electrophysiology of neuroretina allows having a deeper understanding of the effects of diabetes on the central nervous system, area that in this field has traditionally received less attention than the peripheral ones . The purpose of this small review is to enhance the use of these diagnostic methods in everyday clinical practice improving the approach to the patient care (table 3). For a long time a repeatable, cheap, quick, and objective test for the screening of dr has been searched . Although with some technical limitations and quite high costs, the erg, and the study of oscillatory potentials and mferg in particular, have definitely proved to be a valuable and objective tool for the early diagnosis of the disease and potentially for the ophthalmologic follow - up of the diabetic patient . Vep examination, with the analysis of the p-100 wave, assesses the visual function from the retina to the visual cortex and, therefore, provides important information about the function of the optic pathway . The greatest and most regrettable limitation of these diagnostic techniques is represented by the still low uptake . The latest researches data presented in this review can encourage both the research and above all the use in daily clinical practice.
E. coli cultures were grown in lb liquid media or on lb agar plates with antibiotics when necessary (50 g / ml kanamycin, 50 g / ml apramycin, 25 to 50 g / ml chloramphenicol, 50 g / ml carbenicillin) at 37 c with shaking . Streptomyces liquid precultures were grown in tryptic soy broth with the appropriate antibiotics (50 g / ml kanamycin, 50 g / ml apramycin, 25 g / ml nalidixic acid), inoculated into ssm media (1% soytone, 1% soluble starch, 2% maltose, 0.5% trace element solution, ph 5.7) and grown at 30 c with shaking . Streptomyces were also grown on mannitol soya flour medium plates (2.0% agar, 2.0% mannitol, 2.0% soya flour) with appropriate antibiotics (50 g / ml kanamycin, 50 g / ml apramycin, 25 g / ml nalidixic acid). Salinispora were grown in a1 media (1.0% starch, 0.4% yeast extract, 0.2% peptone, and 2.8% sea salt) at 30 c with shaking . The method of tar capturing used in this study has been previously reported . The isolation of high - quality genomic dna from salinispora was performed according to standard procedures . The capture vector, pcap01, containing yeast arsh4/cen6 and trp1 marker, e. coli puc ori, streptomyces c31 integrase gene(int), its attachment site (attp) and origin of dna transfer (orit), aph(3)ii gene (kan / neo resistance), and two 1 kb regions flanking the gene cluster on either side was used to capture and propagate the enterocin gene cluster . The vector pcap01 with the captured enc gene cluster from s. pacifica cnt-150 was named pbb01 . The plasmid pbb01 and its derivatives were conjugated into s. lividans tk23 and s. coelicolor m1146 using a standard triparental mating method with e. coli et 12567/pbb01 and e. coli et 12567/pub307 . After 6 days of growth in ssm media, the cultures were extracted with four volumes of etoac and concentrated in vacuo . The extracts were then dissolved in 100 l of mecn and analyzed by hplc using a luna 5u c18 column (150 mm 4.60 mm, 5 m beads) with a linear gradient of 2100% mecn in water with 0.1% tfa over 30 min with a flow rate of 0.7 ml / min . Hplc - hr - esi - msms analysis of all extracts was carried out on an agilent 1290 q - tof (2002000 m / z, 20 kev). The data were subjected to the molecular networking workflow and analyzed as described previously . The inactivation of the acyl - coa synthetase encoding gene, enc16, in the heterologous host strain s. coelicolor m1146-pbb01 was done using -red recombination with an apramycin - resistant marker (aac(3)iv) as described previously.
Students working toward their dentistry course learn how to prescribe medication during their studies, but, despite its importance, they generally do not learn about it in a systematized way . Traditionally, brazilian dental students take basic pharmacology courses in their first few semesters that teach the pharmacokinetics and pharmacodynamics knowledge needed to understand pharmacotherapy . However, a true understanding of the context in which medications are prescribed comes in daily clinical practice and is not incorporated into basic pharmacology coursework . In general, students learn through observing their teachers and more experienced colleagues, acquiring practice in prescribing as they progress in their programs . They are introduced to fields requiring clinical observation from their fourth semester onwards . In brazil, dentistry courses are usually organized into 10 semesters taken over five years (although there are some courses organized according to year). Patients' individual characteristics (i.e., age, gender, and sociocultural profile) and medical histories (i.e., comorbidities and use of other medication) are often not considered . There are very few studies in the literature concerning prescription errors in dentistry, particularly by undergraduates . Moreover, the lack of training in prescription and management of drug interactions has precipitated issues with identifying patients' problems and the therapeutic objective . According to the world health organization (who)'s recommendations, prescriptions should identify the professional, the patient, and the mode of administration, as well as the medicine's pharmaceutical form, its dosage, frequency of use, duration of treatment together with patient guidance and information . Extreme care should be taken to avoid errors in medical prescriptions as they may not only lead to difficulties and mistakes in dispensing medicines, but may also result in incorrect drug use that can make treatments ineffective or unsafe, which increases risks and healthcare costs . Since the dental surgeon is a prescriber and needs to use medicines as part of his or her clinical dental practice, lack of adequate training for students is an important matter that impacts directly on the quality of medical prescriptions provided . Although there are data pointing to flaws in the training of prescribers, many teaching professionals, particularly those involved in clinical courses, have alleged that students do develop the ability to prescribe over the course of their education . Therefore, the objective of this study was to evaluate changes in the pattern of prescription over the dentistry course study in a brazilian 5-year program . Students in their fourth to tenth semester of studying dentistry at university brasilia during the period from november 2010 to january 2011 who had completed their basic pharmacology coursework were enrolled in this study . All students volunteered after signing an informed consent form (approval no . 112/2010, institutional ethics committee). We asked the students to self - evaluate their knowledge of clinical pharmacology as good, average, poor, or very poor . Then, we asked each participant to use an appropriate prescription form to prescribe paracetamol to control pain in a sample case of a dental extraction without complications . We assigned a maximum score of 5 points to each prescription, where the score was the sum of subscores from three main areas: identification (1 point), pharmaceutical direction (2.5 points), and user information (1.5 points). The identification subscore (1 point) included correct identification of the prescriber (0.5 point) and the patient (0.5 point). The pharmaceutical direction subscore (1.5 point) included pharmaceutical form (0.5 point), concentration (0.5 point), and quantity to be purchased (0.5 point). And the user information subscore (2.5 points) included mode of administration (0.5), posology (0.5), length of treatment (0.5), warnings (0.5), date and span of prescription (0.5). We expressed the data as median values (95% ci) and conducted comparisons using the kruskal - wallis test followed by the dunn's post - test test . Students in their fourth to tenth semester of studying dentistry at university brasilia during the period from november 2010 to january 2011 who had completed their basic pharmacology coursework were enrolled in this study . All students volunteered after signing an informed consent form (approval no . 112/2010, institutional ethics committee). We asked the students to self - evaluate their knowledge of clinical pharmacology as good, average, poor, or very poor . Then, we asked each participant to use an appropriate prescription form to prescribe paracetamol to control pain in a sample case of a dental extraction without complications . We assigned a maximum score of 5 points to each prescription, where the score was the sum of subscores from three main areas: identification (1 point), pharmaceutical direction (2.5 points), and user information (1.5 points). The identification subscore (1 point) included correct identification of the prescriber (0.5 point) and the patient (0.5 point). The pharmaceutical direction subscore (1.5 point) included pharmaceutical form (0.5 point), concentration (0.5 point), and quantity to be purchased (0.5 point). And the user information subscore (2.5 points) included mode of administration (0.5), posology (0.5), length of treatment (0.5), warnings (0.5), date and span of prescription (0.5). We expressed the data as median values (95% ci) and conducted comparisons using the kruskal - wallis test followed by the dunn's post - test test . In total, we evaluated 92 dental students, grouped according to their year in the program (2year, n=12; 3year, n=32; 4year, n=28; 5year, n=20). The mean age of the study participants was 22 years old (1.54). The cohort was 73% women and 27% men . In their self - evaluation of their knowledge of clinical pharmacology, the majority (62%) of only 10% described their knowledge as " good ", whereas 26% and 2% described their knowledge as " poor " and " very poor ", respectively . Figure 1 summarizes how the quality of prescriptions varied by year in the program . As expected, there was an improvement in prescription quality in relation to students' time in the program (p<0.0001). We observed a significant difference between scores achieved by 1-year and 2-year students and scores achieved by students in their final two years . Second - year students had a median prescription quality total score of 2.0 points [(1.5 - 2.5), p<0.005], whereas students in their final (5) year of training had a median prescription quality total score of 3.5 points (3.4 - 3.8). However, this variation was not continuous, as there was no significant improvement between the 2 and 3years, or between the 4 and 5 years . Total score achieved by dentistry students for their paracetamol prescriptions in observing in detail the mistakes made by the students, we noted that failures to identify the patient or the prescriber were common . Although the 5-year students generally performed better than their peers with fewer years in the patient / prescriber identification subscore [0.6 (0.50 - 0.8)], that score differed significantly only versus 2-year students [0.3 (0.1 - 0.5); p<0.05] (figure 2). Partial score for patient and prescriber identification, obtained from the paracetamol prescriptions produced by dentistry program students with respect to form of presentation, quantity, and dose / posology of the prescription, we observed that students in their final two years had scores similar to each other [4year 1.4 (1.2 - 1.5); 5year 1.4 (1.3 - 1.5)]. Their scores were significantly higher than those achieved by students in their 2[0.6 (0.4 - 0.9)] and 3 years [0.7 (0.5 - 0.9)], which did not differ from each other (figure 3). Partial score for information about the medicine (generic name, concentration, posology) on paracetamol prescriptions produced by dentistry program students students in their 5year of study generally performed better than the other students in use instructions, information, and patient warnings about using paracetamol . They scored significantly higher [1.5 (1.4 - 1.6)] than 2-year [1.0 (0.8 - 1.3)] and 3-year [1.1 (1.3 - 1.6)] students (figure 4). Partial score for warnings, use instructions, and side effects on paracetamol prescriptions produced by dentistry program students inclusion of correct drug indications based on scientific evidence and an appropriately completed prescription are important parameters in the quality of pharmacotherapy delivery . Our study showed that there was an aggregation of knowledge about drug prescribing over the dentistry course program; however, important deficiencies were still present at the end of training . Clinical topics are introduced during the 2and 3 years of the program, albeit with a low level of complexity . At this stage, students have the greatest difficulty with making prescriptions, which was most evident in terms of information about dosage / posology . This problem diminishes but does not disappear in the more advanced stages of the program, when students receive more practice at prescribing . Nevertheless, we observed that the acquisition of knowledge was not continuous in that there were no significant differences between the performance of 4-year versus 5-year students in the general quality of their prescriptions . The most common prescription flaws were failure to identify the patient, lack of information about the mode of administration or possible side effects, and absence of non - pharmacological directions . A study conducted by rauniar, et al . (2008) evaluated the prescribing abilities of 258 1-and 2-year students in medicine and dentistry following a three - hour interactive session in clinical pharmacology using the objective structured practical examination . They evaluated two main components, the first of which concerned the prescriber (identification of the prescriber and patient, diagnosis, and signature) and the second of which concerned the drug (correct selection, dosage / posology, and quantity). In terms of prescriber data, 2-year students in both programs made more mistakes than 1-year students in either program . However, in terms of the information provided about the drugs, 2-year students in medicine had a higher level of accuracy than 1year students, while the opposite pattern emerged for the dentistry students . They reported that students acquired limited prescribing abilities in the pre - clinical stages of their programs . Our results provide a clear demonstration that students acquire knowledge up to a certain point (apparent plateau at 4year) and that they finish their training with important deficiencies that may influence their future actions in professional practice . A prior analysis of prescriptions, conducted by dental surgeons working in public health units in brazil, showed that prescriptions commonly contained abbreviations and/or illegible letters and often lacked information about mode of administration, the total quantity of the drug being prescribed, dosage / posology, duration, and/or guidance about the proposed treatment . Professionals who have completed their degrees should not make these mistakes, and it is very likely that the propensity for such mistakes may be traced back to gaps in their pre - doctoral preparation . Although a study conducted with medical students showed low levels of perception about the importance of this question and a lack of awareness of the mistakes made, the students in our study appeared aware of their difficulties . Curiously, while most of the students evaluated their knowledge of clinical pharmacology as " average " (62%), a considerable number evaluated their knowledge as " poor " (26%). Lack of knowledge on the name of the drug, its posology, and duration of treatment, doubts about how to fill out the prescription form, and uncertainty about the correct indication and possible allergies were the main difficulties indicated by students who were in their final year of the dentistry program at a mexican university . Still, the students themselves are capable of recognizing that they had difficulties and doubts that made them feel insecure about prescribing . Researchers have discussed the issue of the lack of integration of clinical knowledge into the basic knowledge curriculum, as well as the issues of insufficient time being devoted to teaching clinical pharmacology, and the lack of specific clinical training . Based on our study's results, we suggest that there should be more emphasis on teaching good prescribing practices to dentistry program students on a progressive and continual basis from the early semesters, when students have their first contact with clinical fields, through the final semester . To improve student involvement and motivation, teaching strategies should include internet tools in instructional units about prescribing based on scientific evidence as well as discussion of clinical cases and problem - based learning . Although dentistry students show a general improvement in their prescribing performance, deficiencies remain even in advanced students, particularly in relation to uses of the drug being prescribed . The data suggest that teaching of good prescription practices should extend throughout the later phases of pre - professional dental education.
The main feedstocks which present paramount importance for the application of lipases are fats and oils . Such materials are primarily composed of triglycerides, which are glycerol esters with saturated and unsaturated fatty acids, from vegetable, animal, or microbial origins . One of the distinguishable characteristics between fats and oils is the occurrence of unsaturated and saturated fatty acids in the triglycerides: higher saturated fatty acids content (as examples in figure 1), higher melting point, and the presence of remaining solids at room temperature are characteristics of a fat; on the other hand, oils usually present higher occurrence of unsaturated fatty acids, remaining in liquid state at room temperature . In addition to triglycerides, vegetable oils can present di- and monoglycerides, free fatty acids (ffas), phosphatides, and unsaponifiable matter, such as carotenoids, phytosterols, tocochromanols, chlorophyll, triterpenic alcohols, and hydrocarbons [14]. The role of fats and oils in plants is related to energy reserve, regarding their occurrence in seeds, and protection against water loss (by wax formation) and against mechanical injuries (by hormone generation), when such components appear in the leaves and fruits [2, 5]. Worldwide production of fats and oils was estimated in 174.6 million tons for the season 2010/2011 . From that, 86% represent vegetable oils (table 1), with soybean, palm, rapeseed, and sunflower seed as the major resources [6, http://lipidlibrary.aocs.org/, 2011]. In brazil, some oilcrops, such as castor bean (ricinus communis), jatropha (jatropha curcas), crambe (crambe abyssinica), macaw palm (acrocomia aculeata), and oiticica (licania rigida), have been explored as alternatives for biodiesel production due to their high tolerance to drought and frost, higher productivity on low - fertility soils, and great potential for the sustainable development of brazilian northeast [7, http://www.ruralbioenergia.com.br/, 2009]. Moreover, the use of raw materials with appropriated physicochemical characteristics and widely available enables cost reduction for the production of the biofuel, since the feedstock cost represents 7088% of the final price of biodiesel . For the selection of a proper raw material for use as substrate for the production of biodiesel the fatty acids profile varies greatly between fats and oils and can be referred by distinct nomenclatures (table 2). It can comprise from high concentration of saturated fatty acids, like in palm seeds, such as coconut (cocos nucifera), palm kernel (elaeis guineensis), and babassu (orbignya oleifera) (table 3), as well as animal fats (table 4), to high content of monounsaturated fatty acids, commonly in oleaginous fruits (table 5). Certainly, there are some exceptions of typical profiles, such as castor bean oil, which has a high content of ricinoleic acid; crambe, with high quantity of erucic acid; palm, with similar quantities of saturated and unsaturated fatty acids (table 3). Additionally to the vegetal and animal sources of lipids and fats presented in tables 35, fatty acids can also come from microbial origin . As recently revised by li et al ., yeasts from cryptococcus, lipomyces, rhodosporidium, rhodotorula, trichosporon, and yarrowia genera, as well as filamentous fungi and bacteria, can reach 53% of lipids content in its dry mass, with evidence for major appearance of palmitic and oleic acids . In another work, the profiles of fatty acids of the microalgaes spirulina sp . The authors observed the prevalence of saturated fatty acids (lauric, miristic, palmitic, and stearic acids), with contributions of up to 46% for the total fatty acids content . These diversified profiles of fatty acids from different origins contribute for the generation of biofuels with different properties . For example, the higher the size of fatty acid hydrocarbon chain, the higher the cloud point and the cold filter plugging point . Therefore, due to the necessity of heating before ignition, it becomes difficult the use of a biodiesel with such characteristic in regions with low environment temperature . Another factor concerning the use of unsaturated fatty acids for the production of biodiesel is that the fewer the double bonds in the molecules, the higher the cetane number of the biofuels (which, in turn, means a better quality of their combustion). This can cause some inconvenience due to the biofuel oxidation, degradation, and polymerization (resulting in low cetane number or formation of solid residues), if improperly stocked or transported . Then, in general, a biodiesel with high quantities of esters derived from monounsaturated fatty acids (e.g., oleic or ricinoleic acids) presents better results as a fuel . Animal fat processing is named rendering, where carcasses with fatty material are heated with hot water or steam to release fats, with subsequent separation by centrifugation or by surface removal . The vegetable oil processing is comprised of some steps, including mechanical pretreatment (cleaning, sorting, and comminution), heating, dehydration, mechanical pressing and/or solvent extraction, miscella distillation, meal desolventization, and refining [1, 9]. For biodiesel production, the oil refining processes play an important role in the yield of the conversion steps, since oil impurities, such as water, phosphatides, and pigments, can affect the conversion of triglycerides to esters due to excessive emulsification of the reaction mixture and difficulties in biodiesel separation, amongst others [1, 9]. Such approach can contribute to the profits of an industrial plant, thus bettering the viability of biodiesel . As a classical example, soybean meal generated during soybean oil extraction is already used for protein and isoflavones extraction, and its main phospholipid, lecithin, separated in the degumming step, is used as natural emulsifier . . 3.1.1.3) and act on ester bonds of several compounds, with acylglycerols being the most proper substrates, catalyzing reactions of hydrolysis, synthesis, and trans- and interesterification (figure 2). Lipases are more active in insoluble substrates, especially triglycerides made of long - chain fatty acids with over 10 carbon atoms, while esterases are active in soluble substrates, especially simple esters, such as ethyl acetate and triglycerides made of short - chain fatty acids with less than six carbon atoms . Esterases follow michaelis - menten kinetics, while lipases need a minimum substrate concentration to show high activity levels . Due to the similarity of the catalytic triad found in lipases compared to those observed in serine proteases, the most widely accepted hypothesis is that the mechanism of lipase catalysis is similar to that of serine protease catalysis . It is believed that the kinetic mechanism of lipases does not depend on the type of reaction being catalyzed (hydrolysis, acidolysis, transesterification, etc . ). The reaction begins with a nucleophilic attack on the carbon from the ester bond of the susceptible substrate by hydroxyl group in the serine residue of the active site, forming an acyl - enzyme complex and releasing alcohol from the lipid . Later, the acyl - enzyme complex is hydrolyzed, releasing the lipase regenerated . Figure 3 shows the stages of the reaction catalyzed by the lipase and its intermediates . Furthermore, characteristics such as stability in the presence of organic solvents, no necessity of cofactors for their action and high enantioselectivity, turn lipases into a group of enzymes with one of the major technological interests [2830]. Lipases occur widely in nature and can be produced by many microorganisms and higher eukaryotes . In animals, lipases obtained from pig and human pancreas they are engaged in several lipid metabolism steps, including fat digestion, adsorption, reconstitution, and in lipoproteins metabolism . In plants, lipases are present in higher plants seeds, as castor bean and canola (brassica napus). They are also found in several plants' energy reserve tissues [28, 3133]. However, for the production of industrial enzymes, microorganisms are the preferred source, once they have shortest generation time, high yield of conversion of substrate into product, great versatility to environmental conditions and, simplicity in genetic manipulation and in cultivation conditions . Due to habitats' multiplicity, microorganisms usually produce various lipases types, with distinct specificity regarding to substrate utilization and also to optimum ph and temperature range . Lipases can be produced by bacteria, filamentous fungi, and yeasts, allowing these microorganisms to use lipids from animal or vegetable origin as carbon and energy sources for their growth . Though many microorganisms have been reported in literature as lipase producers, the genera candida, rhizopus, and pseudomonas are considered the main industrial sources of lipases . The use of lipases in industry is still limited by the cost of commercial enzymes, especially when large quantities of enzyme are required and when the final product is of low added value . The use of solid - state fermentation (ssf) as a production system is one way of reducing enzyme production costs, especially because agroindustrial waste can be used as a culture medium . A comparative economic analysis showed that the production of lipase from penicillium restrictum by ssf is more economically feasible than its production by submerged fermentation (smf), with a production cost for the former being found to be 68% lower and a payback time of 1.5 years . Other advantages of producing enzymes by ssf have been highlighted alongside the reduced production costs . In studies of lipase production by the fungus penicillium restrictum using ssf and smf, different significant physiologies were observed between the two systems when simple (oleic acid and glucose) and complex (olive oil and starch) sources of carbon were used, with a reduction in catabolite repression being observed for ssf . Lipases from different microorganisms have been produced using ssf with different solid wastes, such as lipase from penicillium restrictum in babassu cake [55, 56]; lipase from p. simplicissimum in babassu cake, soybean cake, and castor bean cake [5761]; lipase from candida rugosa in rice flour; lipase from rhizopus homothallicus in sugarcane bagasse [63, 64]; lipase from aspergillus niger in wheat bran and sesame seed cake [65, 66]; lipase from rhizopus rhizopodiformis and rhizomucor pusillus in olive oil cake and sugarcane bagasse; lipase from rhizopus oligosporus in a variety of cakes . These lipases were produced by ssf on a bench scale, mostly using tray bioreactors, and yielded high productivity rates . There are no pre - established procedures in the literature for predicting the performance and design of ssf bioreactors . For this reason, large - scale systems have generally been developed from the results obtained from bench - scale or pilot systems . Ideally, a large - scale system should operate in the same way and with the same performance as a bench - scale system although this is often not the case for ssf processes . The main limiting factor on scaling up such processes is heat transfer, which depends on the stage of fermentation, and the design and operation mode of the bioreactor [7072]. Some mathematical models have been developed to describe the growth kinetics of the microorganisms under different operating conditions and to describe heat and mass transfer in tray bioreactors, fixed - bed bioreactors [70, 74], rotating drum bioreactors [75, 76], shaking reactors, and fluidized bed reactors . These models could be used as inputs for designing the scale - up of such systems . In addition to the reduction of the costs related to fermentation step for industrial - scale production of lipases, the strategies to recover and purify lipases must also be as low as possible and should also be rapid, give high yields, and ideally be easy to scale up . Lipases are enzymes that are known to be strongly hydrophobic, because of the presence of alkyl groups on the surface of their structure . Generally, a good first step for lipase purification is the use of hydrophobic - interaction chromatography . Normally, prepurification involves precipitation with ammonium sulphate, and ion - exchange chromatography and gel filtration are also widely used [7880]. Studied the production and purification of a thermostable alkaline lipase from bacillus thermocatenulatus in escherichia coli . The purification stages were done in butyl sepharose (hydrophobic bed) and tsk g3000 (gel filtration), giving a purification factor of 125 and a yield of 32% . A lipase from aspergillus niger f044 was purified by precipitation with ammonium sulphate, deae - sepharose ff (ion exchange), and sephadex g-75 (gel filtration). A yield of 33% a lipase from penicillium simplicissimum produced by submerged fermentation was purified in a five - step process . First, the culture was concentrated using a 10 kda membrane, then it was precipitated with ammonium sulphate . After concentration and prepurification, the sample was injected in sequential chromatography steps on phenyl sepharose cl-4b (hydrophobic interaction), ultrogel aca-54 (gel filtration), and hydroxyapatite (ion exchange). The resulting purification factor was 788, and the yield was 20% . In order to purify a lipase from penicillium camembertii u-159, isobe et al . A sequence of chromatography steps followed, using deae - sepharose (ion exchange), amino octyl sepharose (hydrophobic interaction), hydroxyapatite (ion exchange), and concanavalin - a sepharose (affinity). Pool of lipases from p. simplicissimum produced by ssf using babassu cake as a culture medium . The process undertaken by means of sequential immobilization in hydrophobic supports (butyl, phenyl, and octyl agarose) resulted in three fractions with distinct thermal stability, specificity, and enantioselectivity properties . Depending on the source, lipases can present molar mass ranging from 20 to 75 kda, enzymatic activity at ph between 4 and 9 and at temperatures since 27 until 70c . Lipases are usually stable in neutral aqueous solutions at room temperature, presenting, in most cases, an optimal activity at 3040c . However, its thermostability varies considerably depending on the origin, and, according to castro et al ., microbial lipases present the best thermostability . Most commercial lipolytic preparations are composed by a mixture of various isozymes, in different proportions, such as those obtained from candida rugosa, pseudozyma (formerly candida) antarctica, rhizopus niveus, and chromobacterium viscosum, among others . Each isoform has different properties (e.g., molar mass, specificity, stereoselectivity, glycosylation extension, and substrate preference) [28, 84, 85]. The main sources of lipases and their properties are described in table 7 . For industrial applications this enzyme can present specificity regarding the substrate (fatty acid or alcohol), including the differentiation of isomers . Nonspecific lipases (such as those produced by candida rugosa, staphylococcus aureus, chromobacterium viscosum, thermomyces lanuginosus, and pseudomonas sp . ): they cleave acylglycerol molecules randomly generating ffas and glycerol, as well as mono- and diglycerides as intermediates . In this case, the products are similar to those produced by chemical catalysis, but with less thermodegradation, due to the lower temperature used for the reaction, when compared to chemical processes [29, 46, 86]. 1,3-specific lipases (e.g., from aspergillus niger, mucor javanicus, rhizopus delemar, rhizopus oryzae, yarrowia lipolytica, rhizopus niveus, and penicillium roquefortii): they release fatty acids from positions 1 and 3 of a glyceride and from, for this reason, products with different compositions of those obtained by nonregioselective lipases, or even by chemical catalysts . Generally, the hydrolysis of triglycerides to diglycerides is much faster than those into monoglycerides [29, 46, 87]. Fatty acid - specific lipases: they act specifically on the hydrolysis of esters, which have long - chain fatty acids with double bonds in cis position on carbon 9 . Esters with unsaturated fatty acids, or without double bond in carbon 9, are slowly hydrolyzed . This type of specificity is not common among the lipases and probably the most studied example of this case is the lipase from geotrichum candidum [29, 46, 8789]. The study of substrate specificity is also of great importance for the application of lipases in biodiesel production, since it is a valuable input for the selection of the proper enzyme based on the composition of the raw material . Evaluated the substrate specificity of an acidic lipase produced by penicillium simplicissimum, observing the highest activities on tricaprin (c8: 0) and tricapryin (c10: 0), which were 83 and 92% higher, respectively, than those detected in the model substrate (olive oil). Lipases can also be stereospecific, where one of the isomers of a racemate is hydrolyzed preferentially over another, or even the formation of one isomer can be catalyzed selectively from prochiral precursors such as meso - diester or meso - diol compounds . Some examples are lipases from burkholderia cepacia, pseudozyma antarctica, candida rugosa, and rhizopus delemar [88, 89]. The main technology for biodiesel production in brazil and in the world is homogenous alkaline transesterification (or alcoholysis). In this reaction, an alcohol (usually methanol or ethanol), with a molar basis, is added to the oil or fat and, in the presence of a catalyst (brnsted acids or bases), a mixture of glycerin and alkyl esters of fatty acids is generated, which is called biodiesel (figure 4). However, alkaline catalysts, especially sodium hydroxide, became dominant for the production of biodiesel, due to their lower costs and faster kinetics [9, 23, 91, 92]. However, homogenous alkaline transesterification presents some disadvantages over enzyme - catalyzed processes, such as the need of raw materials (refined oils and alcohols) virtually free of fatty acids, phosphatides, and water; excess of alcohol and catalyst to avoid reversible reactions, which in turn makes difficult the separation of biodiesel and glycerin . Therefore, alternative catalysts have been studied, such as organic bases, metallic complexes, oxides, aluminosilicates, and enzymes . Their main characteristics are that they are easily recycled and the absence of soap formation, which facilitates the products separation at the end of alcoholysis [9193]. The use of biocatalysis has, therefore, advantages over chemical processes, and these include esterification of both triglycerides and fatty acids; generation of a cleaner glycerol; reuse, mostly in the case of an immobilized lipase utilization . However, some problems still need to be resolved, as high cost of lipases and possible inhibition in the presence of short - chain alcohols, glycerol, and other impurities in the raw material [23, 91, 93, 94]. In the case of biocatalysis, the schematic flowsheet of figure 4(a) can also be applied, but it can also be necessary to use immobilized enzymes, for the reasons shown above . Due to kinetic disadvantages, it can be necessary also to use more sequential reactors in order to achieve the residence time of the feedstock in the presence of the enzymes, for a desired conversion (figure 4(b)). For biodiesel production by enzymatic catalysis, some factors should be considered and some topics should be covered, which can be divided into aspects for current and prospective approaches . Higher yields are achieved for biodiesel production when refined oil is used, compared to crude oils . This is due to the presence of phospholipids in the nonrefined oil, which affect the interaction between lipase and substrate, since they possibly occlude the pores of the support, in the case of using an immobilized enzyme . Therefore, at least the oil degumming step should be conducted before transesterification reactions, in order to obtain a better production of biodiesel [23, 91]. The oil degumming is traditionally done using chemical and physical processes, such as water degumming, ultrafiltration, and mainly acid (phosphorous or citric) treatment [95, 96]. However, since the 1990s, enzyme - catalyzed degumming has been reported as a potential alternative to the conventional processes, and this comprises the use of phospholipases, which are classified into four groups . 3.1.1.4) catalyze the cleavage of ester bonds in phospholipids, thus releasing ffas and contributing for the increase of the overall yield of biodiesel . 3.1.4.4), but these are involved in the breakdown of phosphate bonds in phospholipids and do not contribute to the increase in ffas content of the oil . Enzymatic degumming is done at mild temperature (4045c) and ph of about 4.55.0, for a period of 24 h [97, 99]. The use of free enzymes for biodiesel production results in technical limitations, and it is practically unreliable, due to impossibility of their recovery and reuse, which in turn increases the production costs of the process, besides promoting the product contamination with residual enzyme . These difficulties can be overcome by the use of these enzymes in its immobilized form, allowing the reuse of biocatalyst several times, reducing costs, and further improving the quality of the product . There are several techniques cited for lipases immobilization, such as adsorption, covalent bonding, entrapment, encapsulation, and cross - linking, but they will not be discussed in details in this paper, since there is a recently published review focusing on this issue . In this context, nielsen et al . Revised technical and economic aspects of biodiesel production, concluding that for enzyme - catalyzed biodiesel viability, using immobilized lipases, the enzymes must be stable for the production of 12007400 kg of biodiesel for each kg of enzyme preparation, depending on the substrate source and lipase used . The confinement or physical location of an enzyme in a given region of a defined space, while maintaining their catalytic activity, which can be used repeatedly and continuously due to the ease of its separation from the reaction medium, comprises its immobilization . The catalytic activities of enzymes and other features may change depending on the type of the immobilization technique (adsorption, covalent bound, entrapment, encapsulation, and cross - linking) and the interaction strength between enzyme and carrier used which may, in some cases, cause structural distortions in the protein . Still, the catalytic activity of the enzyme in a particular medium can be changed by increasing or decreasing stirring due to the support fragmentation by interaction between agitation system and support . Thus, it is possible to occur some activity loss during transesterification reaction, even when immobilized lipases are used, and this is more probably due to enzyme leaching than to enzyme inactivation . On the other hand, if such leaching does not occur and the enzyme remains bound to the support, the increase of contact surface may help in raising mass transfer, thereby increasing the efficiency of the enzyme as a catalyst . Lipases from different sources have been immobilized and used in biodiesel production, but the most commonly reported were obtained from pseudozyma antarctica and thermomyces lanuginosus [8, 103]. The use of fixed bed reactors with immobilized lipases is a more suitable solution for continuous production of biodiesel, since the enzymes suffer lower shear stress compared with the batch process . Pandey reported the methanolysis of a waste oil mixture (containing 1980 ppm of water and 2.5% ffas), using immobilized lipase from c. antarctica, and considering 3 steps of substrate addition . The highest biodiesel yield observed was 90.4% . In another example, the same author reported the ethanolysis of a fat in a recirculating packed bed reactor (flow of 1.8 lh) using a phyllosilicate sol - gel - immobilized lipase from burkholderia cepacia, and in this case, a yield of 96% was observed . Over the subsequent four cycles evaluated, the yield was maintained in at least 90% . The enzymatic production of biodiesel can be performed using organic solvents (usually hexane, heptanes, or petroleum ether) or simply using the mixture of substrates (lipids and alcohol) depending on the size of the chain of alcohol . If methanol or ethanol is used, a solvent can facilitate the oil solubility in alcohol and also decrease the viscosity of the reactional mixture, but there will be an additional cost for its removal (distillation or extraction) after the reaction [8, 94, 102]. Knothe et al . Reported the biodiesel production from sunflower oil using petroleum ether as solvent with immobilized lipase from pseudomonas fluorescens, reaching 99% yield when the alcohol used was methanol, ethanol, or 1-butanol . In the absence of soetaert and vandamme reported the use of the lipases from mucor miehei and c. antarctica in the transesterification of various oils using hexane as solvent and found that the lipase from m. miehei is more efficient in converting primary alcohols (methanol, ethanol, propanol, and 1-butanol) with yields between 95 and 98%, whereas lipase from c. antarctica is more proper for the conversion of secondary alcohols (isopropanol and 2-butanol) with yields between 61 and 84% . In the absence of the solvent, the yields of methyl and ethyl esters decreased, particularly when methanol was used, with yield reduction up to 19% . The use of solvents with intermediate polarity (such as t - butanol, 1,4-dioxane) has been suggested to achieve a better dissolution of the alcohol for transesterification (particularly methanol, due to its higher inhibitory effect over lipases) and oil, without affecting the lipase activity . Reported the use of t - butanol as a solvent for the transesterification of cottonseed oil using the commercial preparation novozym 435 for 24 hours at 55c and achieved a yield of 97%, maintaining the lipase activity by 95% of the initial activity during 500 h of continuous operation . The molar ratio between substrates is a variable with large influence on the biodiesel synthesis . Excess of alcohol, in relation to the stoichiometric ratio of 3/1, is used to ensure higher reaction rate and to minimize diffusional limitations . However, excessive levels of alcohol, mainly those with short chains, may inhibit the enzyme by increasing the polarity of the medium, which reduces the stabilization and removes the water layer associated with the immobilized enzymes . The optimal ethanol to oil ratio was 6/1, where 93% conversion was achieved after 480 min of reaction . When increased ethanol to oil ratios (9/1 and 12/1) were used, the conversion, considering the same time of catalysis, dropped to 51 and 55%, respectively . Hence, the gradual addition of alcohol can maintain lipase stable for a longer period . Pandey reported that the gradual addition of methanol (in 3 steps, every 16 h of reaction), maintaining the same enzyme in the bioreactor, resulted in a yield of 98% of biodiesel, and that the conversion was kept over 95% during 50 cycles . Another aspect that may accelerate the methanolysis is the preincubation of lipase in ester or oil . The addition of silica to the reaction medium provides a positive effect on yield, due to the absorption of glycerol and water, thus reducing lipase inhibition . Is the use of 6% (w / w) of silica - gel in the reaction mixture along with the commercial preparation lipozyme tl . Another possibility would be to remove the glycerol by dialysis or its dissolution in isopropanol or t - butanol [102, 105]. As the use of methanol and ethanol can promote lipase inhibition, the use of alternative donors of acyl group, such as methyl acetate, ethyl acetate, and propan-2-ol, is being studied, since their use avoids the production of glycerol as a by - product, which blocks the porous support and lipase active sites . Novozym 435 was tested for biodiesel production using several oils and the donors of acyl groups cited above, and the results observed were yields always above 90% [53, 91]. However, the control of water content in the reaction system is important for some reasons: lipase requires a minimum amount of water to maintain its active conformation; an excess of water may promote the hydrolysis of the substrate and generate diffusion limitations of substrate, thus reducing the biodiesel yield; the water can influence negatively the reaction when methanol or ethanol is used but does not affect the reaction when higher - chain alcohols are considered [93, 94, 102]. Reported the use of lipase from pseudomonas cepacia immobilized on polymeric sol - gel matrix in the transesterification of tallow oil at 40c for 1 h using a mixture of 10 g of fat, 3 g of lipolytic preparation, 3 g of methanol or 5 g ethanol, and different amounts of water . The authors observed that when water concentrations below 0.2% were used, the conversion was significantly decreased and, after the reuse of lipase during 11 cycles, the activity was decreased by 10% . Pandey reported the use of lipase from chromobacterium viscosum in the transesterification of jatropha oil with a 10% enzyme dosage . When the biocatalyst in a free form was used, it was observed a yield of 62%, whereas when the enzyme was immobilized on celite 545, 71% of yield was achieved . By adding 1% of water to the free enzymatic preparation and 0.5% of water to the immobilized enzyme, the biodiesel yields raised to 73% and 92%, respectively . Generally, the higher the temperature, the higher the reaction rate of alcoholysis or transesterification, until reaching the temperature of inactivation of lipases (usually above 60c). This approach is valid mainly for systems in which the enzyme is used just once or few times . When enzyme reuse is considered, high temperatures, which can be suitable for short - term use of the enzymes, may be not the most proper, since the half - life time of lipases decreases with increases in temperature . Matassoli et al . Investigated the influence of temperature in ethanolysis of crude palm oil catalyzed by lipozyme tl i m (3% w / w) with a molar ratio ethanol / oil of 3/1 and gradual addition of ethanol, observing the best result at 50c . For the evaluation of the effect of temperature on lipase - catalyzed biodiesel production, the authors investigated soybean biodiesel production using the commercial product novozym 435, within the range of 4570c, observing the highest yield (92%) when 65c was used . The amount of enzyme added to reaction is also an important factor for biodiesel production, because it affects reaction rate (typically, the higher the enzyme dosage, the faster the reaction), but there is a limit in which the addition of enzyme does not alter anymore the rate of product formation or that the amount turns the process more economically prohibitive . In this context, enzyme - catalyzed biodiesel production was investigated using dosages of c. antarctica lipase b (novozym 435) from 1 to 20% (w / w). The authors observed the highest conversion of triglycerides to ethyl esters (93%) when 10% (w / w) of the immobilized enzyme was used . Regarding the effect of lipase specificity, pandey reported the use of some specific and non - specific lipases (from c. rugosa, p. cepacia, and p. fluorescens) in biodiesel production . The non - specific lipases promoted the highest yields of methyl esters when a molar ratio of 3/1 of methanol / oil was used . Specific lipases need gradual addition of methanol to achieve high yields (between 80 and 90%), and this is probably due to acyl migration of sn-2 to sn-1, which occurs spontaneously in glycerides . For reduction of enzymatic processes costs, some researchers have studied microbial immobilization, such as fungal mycelia, bacteria, and yeasts cells, for their use as whole cell catalysts, taking advantage of functional proteins at the cell surface . From all whole cell support immobilization techniques, the most used is that named porous biomass support particles (bsps) because it does not require chemical additives or cell preproduction; aseptic handling is unnecessary; higher enzyme production and rate of substrate mass transfer within bsp; the particles are reusable and resistant to mechanical shearing; the bioreactor scale - up is easy and presents lower costs compared to bioreactors used in other methods [8, 104]. The first example for biodiesel production using whole cell as biocatalysts was the rhizopus oryzae mycelium immobilized in polyurethane foam . The growth conditions were optimized regarding the production of intracellular lipase, as well as pretreatment methods and water content during methanolysis . The addition of substrates (olive oil or oleic acid) to the culture medium significantly improved lipase activity of the whole cell catalyst . The results for the obtainment of methyl esters of soybean oil using this catalyst at 32c for 72 h (8090% yield), when the addition of methanol to the system was implemented intermittently in the presence of 1020% water, were very similar to those described with the use of extracellular lipases . Aiming to stabilize the r. oryzae cells, it was tested a cross - linking treatment with 0.1% glutaraldehyde, keeping the lipase dosage unaltered for 6 cycles . The yield of methyl ester varied between 70% and 83%, along 72 h of experiment . Without this treatment, the lipase activity decreased reaching a yield of 50% in the sixth batch [8, 91, 104]. In order to achieve higher yields of biodiesel using cells immobilized in bsp, soetaert and vandamme used fixed bed systems . To increase the interfacial area between the reaction mixture and the whole cells, the former was emulsified by sonication before each batch cycle . When a gradual addition of methanol (ratio 4/1, methanol / oil) was conducted at a flow rate of 25 lh, a yield of 90% the authors attributed this decrease to the cell removal from the bsp, since it was detected a decrease of 56% in the cell concentration in the bsp between the first and the tenth batches . As further examples, other freeze - dried whole cells, such as from r. chinensis mycelium, s. cerevisiae (containing intracellular r. oryzae lipase, rol), and recombinant s. cerevisae expressing the lipase gene of r. oryzae ifo 4697 (cell surface rol), have been used as biocatalysts for the production of methyl biodiesel from soybean oil . In the absence of solvent, the yields observed for the cited examples were 86, 71, and 78%, respectively, after 165 h of reaction at 37c [8, 102]. . Showed that it is possible to decrease the costs associated to the synthesis of enzyme - catalyzed biodiesel, by using the fermented solids produced by cultivating burkholderia cepacia lteb11 on a mixture of sugarcane bagasse and sunflower seed meal . The authors used this fermented solids to catalyze the ethanolysis of soybean oil aiming to produce biodiesel in a fixed - bed reactor in a cosolvent - free system . Although the use of whole cells does not require many of the steps related to the downstream process of biodiesel production, such as the isolation and purification of the enzyme after fermentation, processes in which the transesterification reaction is done by using immobilized enzymes or cells present at least one notable difference, which is the reaction time . Fukuda et al . Reported the use of novozym 435 in a continuous process, for 7 h, and they observed yields of 9294% in terms of methyl esters . When a fed - batch operation was considered, the yield was 87%, after 3.5 h of reaction with methyl oleate . The authors also compared the process performance by using whole cells in a fed - batch operation mode, and observed the necessity of 70 h of reaction in order to obtain yields of 8090% of biodiesel . In the last case, the utilization of t - butanol as solvent would possibly reduce the reaction time, due to higher efficiency of mass transfer in the system . Some waste oils, by - products from vegetable oils processing, may also be suitable raw materials for biodiesel production . These oils usually present high contents of ffas, and some examples are the sunflower oil and corn oil, which have 55.6% and 75.3% of ffas, respectively . Pandey reported the esterification and transesterification of these oils in the presence of hexane using immobilized lipase from c. antarctica and observed yields of 64% and 50% of methyl esters, while maintaining the lipase stable for over 100 cycles . Hou and shaw reported that the esterification of acid oils is much faster than the transesterification of nonacid oils . In the former case, it was necessary only 3 h of reaction and 1% of lipase for the esterification of ffas, where a yield of 95% was achieved, whereas for the latter case, the same yield was observed only after 30 h of methanolysis and using a higher enzyme dosage (4%). One disadvantage of the esterification reaction is the formation of water as a by - product, which often inhibits the reaction of triglycerides . One possible solution to this is to conduct the reaction in two separate stages: first, esterification of the ffas in the mixture, with the evaporation of the generated water; then the methanolysis of the triglycerides . In the first step, the molar ratio of methanol / ffa should be low, such as 1/2 and low quantity of enzyme (0.5% w / w) is needed . In the second step, on the other hand, the molar ratio between methanol and triglycerides should be changed to 1/1, and the enzyme quantity should be increased to about 6% (w / w). Hydroesterification is a process that combines two basic processes, the hydrolysis of triglycerides and the esterification of fatty acids, in sequential reactions, in order to produce biodiesel . Studied the use of residual cooking oil for biodiesel production by enzymatic hydrolysis accompanied by chemical esterification . The c. rugosa lipase used completely hydrolyzed the oil after 10 h of reaction . The ffas were converted into biodiesel by chemical esterification using amberlyst 15 (acidic styrene divinylbenzene), and a 99% conversion into biodiesel was obtained after 2 h. in this work, there was a loss of enzyme activity, and the hydrolysis yield was decreased to 92% after five batch cycles . Cavalcanti - oliveira et al . Studied the use of a lipase from thermomyces lanuginosus (tl 100 l) in the hydrolysis of soybean oil in a hydroesterification process . This stage was followed by the esterification of the ffas with methanol, which was catalyzed by niobic acid in pellets . They obtained 92% conversion of the ffas into fatty acid methyl esters after only 1 h of incubation . Sousa et al . Studied the lipase from jatropha seeds for the hydrolysis of different raw materials for biodiesel production using hydroesterification strategy . The best conversions were obtained using soybean oil and jatropha oil, obtaining up to 98% of ffa after 2 h. the esterification of the ffas from the jatropha oil with methanol was catalyzed by niobic acid in pellets, obtaining up to 97% conversion into biodiesel after 2 h. the biodiesel obtained from this process fulfilled all the legal requirements for its commercial use . The use of enzyme catalysts (lipases) in biodiesel production is being increasingly studied because of the advantages that these catalysts present over chemically catalyzed and noncatalytic processes . Some of the advantages offered by the use of lipases are lower energy consumption; lower thermal degradation of substrates and products; versatility in the use of raw materials, including possibility to use acid oils without the decrease of process efficiency; easier purification of the alkyl esters (biodiesel) and separation of the coproduct (glycerol), especially if immobilized enzymes or whole cells are used; environmental benefits, due to biodegradability of the catalyst . Nevertheless, some process conditions should be taken into account in order to have a feasible enzyme - based technology for the production of biodiesel, and these include the establishment of descriptive correlations between the enzyme dosage and the substrate source, in order to rationalize enzyme usage and optimize costs; deep study of reaction conditions and their optimization; the selection of a proper biocatalyst which can be reused and maintain its stability over several cycles; product recovery strategies, especially when a cosolvent is used in the reaction . The enzyme - based production of biodiesel is still under development, and it seems that there is a tendency for the use of conventional technologies as a new application for lipases, such as their immobilization in magnetic nanoparticles, microwave and ultrasound - assisted transesterification, esterification in pressurized fluids, and transesterification in supercritical fluids . Although technical aspects of such strategies may lead to conversion improvement, economical considerations must be investigated in more details.
The study was conducted in 100 households of 9 affected villages of engil district on the edge of herat city . Households were included if they were located on 3 randomly selected transect lines and if members of the households owned either cattle or sheep . Because there are no reliable estimates on seroprevalence of cchf in afghanistan, we estimated prevalence of immunoglobulin (ig) g to be 7% (2,3). A sample size of 160 persons was required to detect this prevalence at the 95% confidence level . Allowing for cluster sampling at the household level, 320 persons was the target sample size (90 households, assuming an average of 3.5 persons> 15 years of age per household). From each household, all members> 15 years were surveyed, if they gave consent . Sheep and cattle were selected as livestock types, and 1 or 2 animals per household were randomly selected . Inclusion criteria for humans were residing in a livestock - owning household, giving informed consent, being> 15 years of age, willing to answer the risk factor questionnaire, and willing to give 5 ml of blood . For collection of blood samples and vector specimens from sheep and cattle, permission was given by the head of household . A standardized, structured, and pretested questionnaire that covered individual data for each participant, including personal details, exposure variables, and self - reported disease history, was used . Animal data were collected from each household and included each animal s origin, tick exposure, age, and sex . Blood samples from human participants were collected by trained health workers according to standard procedures . Blood samples were centrifuged at room temperature at the local laboratory on the day of collection . Serum was separated, frozen, and transported to kabul for storage and onward shipment . A sandwich / indirect elisa detected specific igg at a 1:100 dilution for all human samples by using the vector - best diagnostic kit, (vector - best, novosibirsk, russia). We have previously compared this kit with an in - house elisa (us army medical research institute for infectious diseases, fort detrick, md, usa), and the results were comparable in all samples tested (l. mustafa et al ., unpub . We used an in - house elisa using the ibar 10200 strain of cchf as antigen (us army medical research institute for infectious diseases) and anti - species igg horseradish peroxidase conjugated (kpl inc ., gaithersburg, md, usa) to test for igg in all animal serum specimens . Data were analyzed by using stata version 8 (statacorp, college station, tx, usa). The primary outcome was seropositivity among household members, with secondary outcomes being seropositivity in animals and the presence of virus in ticks . Exposure factors for seroprevalence were identified on an a priori basis and appropriate measures of statistical significance were applied to detect differences at the 95% confidence level . The study was approved by the ethics boards of namru-3 and the afghan public health institute, afghanistan . In total igg seroprevalence was 37/330 (11.2%, 95% confidence interval [ci] 8.015.1). Of all the potential explanatory variables, only 2 factors were associated with an elevated risk of igg positivity: daily contact with cattle (33/264 [12.5%] vs. 1/52 [1.8%]; = 5.1, p = 0.02) and exposure to raw animal skins (24/144 [16.7%] vs. 12/176 [6.8%]; = 7.7, p = 0.006). Self reported clinical illness (fever) occurred in 55% of participants over a 5-month reporting period . Among the participants, 20.8% reported that they had had an illness involving bleeding from teeth, gums, and or other parts of the body, but this event was not associated with igg positivity or with age group . These results suggest that the risk for cchf exposure is uniformly high among the population . The oldest age group shows an approximate lifetime risk for exposure and seroconversion of 17% (95% ci 10.2%25.8%). Ninety - two cattle and 40 sheep were included, and serologic analysis of their blood samples was conducted . Seroprevalence was 79.1% (95% ci 69.0%87.1%) among cattle and 75.0% (95% ci 57.0%88.5%) among sheep . Prevalence was uniformly high regardless of age, sex, or origin of the animals, suggesting that the disease is highly endemic in the livestock population . Among our sample, ticks (n = 259) from domestic animals were predominantly adult hyalomma marginatum (94.6%). Engorged females were found more on cattle than on sheep (43% and 27%, respectively). Seroprevalence in this population of animal owners is higher than in other reported studies from the region (3,4), and the risk for exposure appears approximately uniform . The route of transmission to humans is either through the bite of ticks or through contact with infected animals or animal products . This second route of transmission is probably more important in iran and afghanistan than tick - borne transmission . Control of cchf requires control of the disease vector, and surveillance is necessary to ensure optimum timing of interventions such as livestock dipping or sponging because tick abundance is highly seasonal . Further research on rates of antibody acquisition among humans and animals, virus transmission dynamics, and effectiveness of disease control measures is required . Cchf is a regional public health concern of larger than previously acknowledged significance and requires control mechanisms from both the health and agriculture sectors.
Although ring - opening polymerizations (rops) provide a method to transform cyclic monomers into well - defined, functional, and degradable polymers, it has been challenging, in general, with some exceptions, to overcome difficulties that are often experienced in the attempt to introduce functional moieties within the cyclic monomer structures and with incompatibilities of these desired functionalities during the polymerizations . Chemistries such as azide alkyne huisgen cycloaddition, diels alder reaction, and radical - mediated thiol ene / yne reactions have revolutionized polymer chemistry, allowing for efficient and reliable routes toward postpolymerization modifications in the design of complex and functional polymeric materials . Combined with the extraordinary efficiency in the generation of stable covalent linkages by click-type reactions in biomaterials, acid - labile functionalities, such as acetals, ortho esters, and hydrazones, are also of major interest in contemporary materials design, especially toward biomedical applications . In spite of the wide utilization of acetals / thio acetals in synthetic organic chemistry (e.g., as hydroxyl protecting groups), the most intriguing characteristic of acetal / thio acetal functionalities originates from their unique property of behaving as a dynamic covalent bond, i.e., being cleavable upon exposure to acidic conditions such as those found in the gastrointestinal tract, tissue at sites of inflammation and in tumors, and in endosomal and lysosomal compartments, while being relatively stable at the normal physiological ph . In this context, acetals / thio acetals are appealing for their incorporation into biomacromolecules, where the dynamic covalent linkages can bridge to biologically active molecules and, ultimately, release them at a targeted site in a controlled manner . Furthermore, introduction of hydroxyl functionalities by controlled cleavage of acetals / thio acetals would improve the hydrophilicity of a given polymer, which has been limited conventionally due to their incompatibility with the polymerization processes . The inherent acid - labile property of acetals has been introduced to the backbone or the side chains of functional polymers or cross - linkers in the preparation of cross - linked nanoparticles . For instance, among several elegant works, reports on acetal - linked linear polymers or dendrimers have elucidated the cleavability as well as the chemical stability of acetal linkages in a broad spectrum of polymeric systems, e.g., degradable, biocompatible dextrans, by frechet et al . ; hydroxyl - group protection during anionic polymerization and hyperbranched polyethers with acetal backbone linkages by frey et al . ; acetal - based degradable shell cross - linked polymeric micelles by wooley et al . ; introduction of vinyl ether side chain moieties along poly(ethylene glycol) backbones by wurm et al . And brocchini et al . ; and acetal - linked backbone - cleavable aba - type triblock copolymers by ni et al . Here, our interest is the application of acid - labile acetal / thio acetal linkages to the side chains in degradable polymeric systems for their potential use as drug delivery carriers . Recently, acetal - linked prodrug micellar nanoparticles and their ph - triggered controlled drug release from the side chains have been highlighted by zhang et al . And zhong et al however, the polymeric nanoconstructs used in these studies were hydrocarbon - based, nondegradable backbone systems polymethacrylates and poly(acrylic acid), respectively which limit their application as potential biomaterials because of the possible long - term accumulation in the human body . A major effort of our group has been to develop polyphosphoesters (ppes) tailored by manipulation of pendant functional groups on the pentavalent phosphorus atoms and to integrate the ppe system into new classes of degradable, biocompatible polymeric nanomaterials . Particularly, introduction of reactive vinyl ether moieties to degradable, biocompatible ppes and their utilization to conjugate a library of hydroxyl- or thiol - containing biologically active molecules via multiple types of conjugation chemistries are envisioned to expand the breadth of this chemical approach in the development of functional biomaterials . Herein, we report the organocatalyzed rop of ethylene glycol vinyl ether - containing cyclic phosphotriester monomer, as an initial fundamental study toward unique degradable, functional polymer materials . After demonstrating the well - controlled homopolymerization of the monomer with predetermined molecular weights and narrow molecular weight distributions, the resulting vinyl ether - functionalized ppe scaffold was conjugated with hydroxyl- or thiol - containing model small molecules via three different types of conjugation chemistries thiol ene click reaction, acetalization, or thio acetalization reaction revealing efficient routes for postpolymerization modifications of functional polymers . Furthermore, amphiphilic diblock copolymers containing the ethylene glycol vinyl ether - functionalized ppe as a hydrophobic block segment, also prepared by rop, afforded well - defined micelles that showed a ph - dependent hydrolytic core degradability of both backbone and side chains in aqueous solutions . The degradation products, as identified by mass spectrometry, were found to be nontoxic toward two standard cell lines: raw 264.7 mouse macrophages and ovcar-3 human ovarian adenocarcinoma cells . Hence, this polymeric system can have potential applications in the development of nanomedical devices for a variety of biomedical applications . A novel ethylene glycol vinyl ether - functionalized cyclic phosphotriester monomer, 2-ethylene glycol vinyl ether-1,3,2-dioxaphospholane 2-oxide (evep), 1, was synthesized by following a typical condensation method: coupling of ethylene glycol vinyl ether (eve) to 2-chloro-3-oxo-1,3,2-dioxaphospholane (cop) in the presence of triethylamine (tea) in dichloromethane (dcm) at 4 c (scheme 1). It was critical to purify the monomer rigorously, by removing the residual starting materials completely, because of the potential for undesired dual initiation by residual eve and the possible formation of random / branched (co)polymers by remaining reactive cop during the polymerization process . Multiple purification methods, including silica gel column chromatography, (vacuum) distillation, extraction, and precipitation, were attempted, but decomposition of the reactive cyclic monomer and/or incomplete removal of the impurities were inevitable in all cases . Therefore, use of an exact stoichiometric equivalence of reagents was attempted, and then the purification process was applied for removal of the tea salts by a series of recrystallization in dcm, precipitation in diethyl ether, and filtration . Even though tetrahydrofuran (thf) could be used as a solvent, the quantitative conversion of eve and cop to 1 was supported by h, c, and p nmr spectroscopy (figure s1). By c nmr spectroscopy, the methylene carbon of the hydroxymethyl group of eve resonating as a singlet (h - decoupled) at 61.2 ppm disappeared upon coupling to cop to result in a doublet (j(p, c)) having a coupling constant of 10.2 hz at 66.1 ppm for the methylene carbon of the new phosphoester linkage . In addition, consumption of the cop was observed by p nmr spectroscopy as replacement of the cop phosphorus resonance at 23.20 ppm with that for the monomer at 17.91 ppm . Although the conversion to monomer appeared to be complete, h nmr analysis indicated that there were residual solvents and tea salts that were not removed (96% purity). The rop of 1 was performed in a glovebox at ambient temperature . For the kinetic study, 1 and benzyl alcohol (bnoh) (molar ratio of 100:1) were mixed in dcm, and the polymerization began with the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (dbu) (molar ratio to initiator of 3:1). After being stirred for a predetermined period of time, an aliquot of the reaction mixture was collected, immediately quenched by adding a solution of excess benzoic acid in dcm, and then analyzed by p nmr spectroscopy . A portion of the collected samples was precipitated into diethyl ether prior to injection into the gel permeation chromatography (gpc) instrument (figure s2). Although routine decoupled p nmr spectroscopy has complications that may limit the quantitative value, the distinct resonance frequencies of the phosphorus nuclei of the monomer vs the polymer provided the best opportunity (relative to h or c nmr spectroscopy) to monitor the polymerization . Therefore, the conversion was estimated from p nmr spectroscopy by comparing the integral ratio of two distinct peaks of monomer 1 at 17.91 ppm and homopolymer, pevep, at 0.68 ppm, on crude polymerization aliquots . Once the polymerization was quenched and worked up, h nmr spectroscopy end - group analysis of the degree of polymerization was found to be in agreement with the monomer conversion calculated from the p nmr data . The kinetic study displayed a rapid initial polymerization rate, in which the monomer conversion reached at 23% within the beginning 3 min . However, the maintenance of linearity of mn vs monomer conversion suggested a living rop up to 79% conversion (figure s2b). The consistent low pdis (1.04) until the monomer conversion reached at 79% indicated that there was minimal adverse transesterification of the ppe backbone during the polymerization process . A kinetic plot of ln([m]0/[m]) vs polymerization time illustrated pseudo - first - order kinetics (figure s2a), which is a typical characteristic of rop . The same molar ratio used for the kinetic study was applied for a scaled - up production of 2 . The purification of 2 by precipitation in diethyl ether was insufficient to remove the residual starting materials and benzoic acid completely . Silica gel column chromatography was also attempted, but instability of the ppe backbone was problematic . Dialysis (mwco 68 kda) of the reaction mixture against organic solvents, switching from meoh to dcm, was the most assured purification method among those tested . Dialysis in basic aqueous solutions (e.g., carbonate buffer at ph 78) was also feasible, but the complete removal of water was challenging, which is imperative for the following postpolymerization modification reactions . Being consistent with the kinetic study, quenching the polymerization at a predetermined time (9 min), targeting at 50% conversion, yielded the predicted molecular weight and pdi . The degree of polymerization (dpn) calculated based on p nmr spectroscopy - determined monomer conversion was in agreement with that calculated from chain - end analysis by h nmr spectroscopy, i.e., by comparisons of the integrals of proton resonances of the benzyl group (7.437.34 or 5.08 ppm, labeled as a or b, respectively, in figure 1a) of the initiated chain end to those of the distinct double bonds (6.49 or 4.06 ppm, labeled as f or g, respectively, in figure 1a) or protons on the substituents to the phosphorus atom (3.943.86 ppm, labeled as e in figure 1a) of 2, which was indicative of retention of the vinyl groups (figure 1a). In addition, one distinct p resonance confirmed the stability of the degradable ppe backbone during rop of 1 and the work - up process of 2 . H (300 mhz, cd2cl2, ppm) and p (121 mhz, cd2cl2, ppm, inset) nmr spectra of (a) 2, (b) 3, (c) 4, and (d) 5 . Ene click reaction with thiol - containing model small molecule, 2-(2-methoxyethoxy)ethanethiol . Ene click chemistry is a robust and versatile method that tolerates a variety of functional groups in achieving a high degree of functionalization on vinyl groups . Herein, this efficient chemistry was applied to demonstrate the presence and chemical availability of vinyl groups on 2 . To verify the integrity of the ppe backbone in the presence of radicals during uv irradiation, a mixture of 2 and 2,2-dimethoxy-2-phenylacetophenone (dmpa) in methanol - d4 (meod4) was irradiated under uv light (365 nm, 6 w) for several hours, as a preliminary control reaction . P nmr spectroscopy confirmed that the polymer backbone was intact under these conditions . Accordingly, an excess of 2-(2-methoxyethoxy)ethanethiol relative to the vinyl ether bonds was employed with dmpa in meoh and uv irradiation for 1 h to ensure a high coupling efficiency and to avoid undesired cross - linking reactions between the double bonds along the backbone . The purified products were obtained by conducting precipitation in diethyl ether followed by sequential dialysis (mwco 68 kda) against meoh and dcm . Comparison of the h nmr spectra of 1 and 2 before and after thiol ene click reaction, as shown in figures 1a and 1b, respectively, verified the disappearance of the vinyl proton resonance (6.49 ppm, labeled as f in figure 1a) and the corresponding appearance of two distinct proton resonances of methoxy (3.33 ppm, labeled as f in figure 1b) and -protons adjacent to the sulfur atom (2.782.69 ppm, labeled as g and g in figure 1b) in the thio ether functional group . Gpc analysis of 3 clearly showed a peak shift to shorter elution time, relative to 2, with a monomodal peak having a pdi of 1.07 after thiol ene click reaction (figure s3). Using commercially available 4-methylbenzyl alcohol and a catalytic amount of p - toluenesulfonic acid (ptsa), the presence and chemical availability of vinyl ether moieties of 2 were demonstrated via acetalization . Initially, to confirm the integrity of the ppe backbone in the presence of alcohols and strong acidic catalyst, ptsa (pka 2.8 (water)), a mixture of 2 and 2 equiv of 4-methylbenzyl alcohol (relative to the absolute number of vinyl ether bonds) or that of 2 and 0.2 equiv of ptsa (in relation to the absolute number of vinyl ether bonds) or that of 2, 4-methylbenzyl alcohol, and ptsa in n, n - dimethylformamide - d7 (dmf - d7) was allowed to stir for a period of time, as a preliminary control reaction . An aliquot of each reaction solution was collected at a predetermined time, quenched by the addition of an excess of tea, and then evaluated by p nmr spectroscopy . P nmr spectra confirmed the intact ppe backbone in the presence of either 4-methylbenzyl alcohol or ptsa for several hours . However, the combination of 2, 4-methylbenzyl alcohol, and ptsa in the mixture solution was accompanied by undesired degradation / transesterification along the ppe backbone structure after 6 min of reaction, as confirmed by p nmr spectroscopy . Accordingly, a scaled - up production of 4 was conducted using the same molar ratio as used for the preliminary control reaction, and the acetalization reaction was quenched at 5 min by the addition of an excess of tea to ensure the intact ppe backbone structure . With respect to the purification, employed an extraction method to purify their acetal - linked linear poly(ethylene glycol) by using water; however, this extraction method was not compatible with the instability of the ppe backbone in our system . In addition, the complete removal of ptsa and/or 4-methylbenzyl alcohol was not achieved by precipitation of the desired polymer in organic solvent, such as diethyl ether . More importantly, this precipitation method was not desirable, especially for acetal - bearing polymers as a result of the possible cross - linking side reactions by trans - acetalization in the presence of a trace amount of ptsa . Thus, sequential dialysis (mwco 68 kda) against two different organic solvents, dmf and dcm, was conducted for purification . Finally, the desired product, 4, was obtained after removal of the organic solvents, as confirmed by h and p nmr spectroscopy (figure 1c). The conversion percentage of vinyl ethers to acetals was calculated from chain - end group analysis by h nmr spectroscopy, i.e., by comparison of the integrals of proton resonances of the benzyl group (7.447.32 ppm, labeled as a in figure 1c) of the initiated chain end to those of two distinct acetal linkages, methyl (1.33 ppm, labeled as j in figure 1c) and methylene (4.874.75 ppm, labeled as i in figure 1c), or 4-methyl protons on the benzyl substituents (2.32 ppm, labeled as m in figure 1c). According to the chain - end analysis by h nmr spectroscopy, approximately 18% of the initial vinyl ether groups were converted into the acetal linkages, ca . The hydrolysis side reaction was probably attributed to a trace of water present in ptsa and/or highly viscous ppe . It is noteworthy that the complete disappearance of vinyl proton resonances was observed with prolonged reaction time (12 h), which could have enhanced the acetal conversion percentage . However, quenching the reaction at the optimal time, 5 min according to the preliminary reaction, was essential for the integrity of the ppe backbone . This backbone stability was verified by the existence of one distinct p resonance peak at 0.68 ppm, observed by p nmr spectroscopy . Gpc analysis of 4 was not available in thf because of its polar nature, conferred by the newly formed hydroxyl groups on the side chains . The same reaction protocol for the acetalization reaction was applied to the thio acetalization of 2 . The h nmr spectrum of thio acetal - bearing pevep50, 5, is shown in figure 1d . The conversion percentage of vinyl ethers to thio acetals was calculated from chain - end group analysis by h nmr spectroscopy, i.e., by comparisons of the integrals of proton resonances of the benzyl group (7.447.32 ppm, labeled as a in figure 1d) of the initiated chain end to those of the two distinct thio acetal linkages, methyl (1.51 ppm, labeled as j in figure 1d), methylene (4.71 ppm, labeled as i in figure 1d), or 4-methylbenzyl protons (2.31 ppm, labeled as m in figure 1d). By h nmr chain - end analysis, approximately 8% of the initial vinyl groups were found to be converted into the thio 36% of the repeat units underwent hydrolysis of the side chains to present hydroxyl groups . The intact ppe backbone was affirmed by the presence of one distinct p resonance peak at 0.67 ppm, as measured by p nmr spectroscopy . Similar to the acetal linkage - bearing polymer, 4, the polar nature endowed by the newly formed hydroxyl groups on the side chains did not allow for gpc analysis . Based on the kinetic study of 2, amphiphilic diblock copolymer, mpeg44-b - pevep33, 6, was prepared by rop using the molar ratio 100:1:3 of 1, -methoxy--hydroxy poly(ethylene glycol) 2000 da (mpeg44-oh), and dbu, respectively (scheme 2). The polymerization was quenched at 6 min by the addition of a solution of excess of benzoic acid in dcm, and the desired diblock copolymer product was obtained after sequential dialysis (mwco 68 kda) against organic solvents, meoh and dcm . The dpn calculated based on p nmr spectroscopy - determined monomer conversions was in agreement with that calculated from chain - end analysis by h nmr spectroscopy, i.e., by comparisons of the integrals of the proton resonance of the methyl group of the initiated chain end (3.33 ppm, labeled as a in figure 2a) to that of the double bond (6.49 or 4.06 ppm, labeled as f or g, respectively, in figure 2a) or -protons to the vinyl ether oxygen atom (3.943.86 ppm, labeled as e in figure 2a) of the pevep block segment . Additionally, one distinct p resonance peak at 0.71 ppm confirmed the intact pevep block backbone structure during the chain extension by rop of 1 and the work - up process of 6 . Gpc analysis of 6 showed a distinct peak shift to lower elution time from that of the macroinitiator, mpeg44-oh, after polymerization, with a monomodal peak with a pdi of 1.09 (figure 2b). (a) h (300 mhz, cd2cl2, ppm) and p (121 mhz, cd2cl2, ppm, inset) nmr spectra of 6 . (b) gpc traces of macroinitiator, mpeg44-oh, and diblock copolymer 6, mpeg44-b - pevep33, as a function of elution time (min). The tg values of the prepared polymers varied, as measured by differential scanning calorimetry (dsc), depending on the side chain substituents (table 1). The conjugation of the 2-(2-methoxyethoxy)ethanethioether groups onto the side chains induced a decrease of the tg value from 39 c of 2 to 64 c of 3 after the thiol ene click reaction . The slight increase of tg value of 4 and 5, 27 and 31 c, respectively, as compared with that of 2, 39 c, was ascribed to the rigidity and interactions of the aromatic rings . Meanwhile, the tg value of the diblock copolymer with the extended ethylene glycol backbone units, 6, was complicated to analyze but appeared to give only a single tg value, similar to that of 2, 38 c . In addition, no tm for the peg block segment was observed for the diblock system; therefore, an extensive investigation against the homopolymers, mpeg44-oh and pevep50, and a mpeg44-oh / pevep50 blend was conducted . These studies confirmed that the pevep block fully suppresses peg crystallinity in the diblock system, while the physical blend does little to suppress peg crystallization (full dsc traces for all systems upon heating and cooling are presented with detailed discussions in the supporting information (figure s9)). The self - assembly behavior of the amphiphilic diblock copolymer, mpeg44-b - pevep336, was studied by direct dissolution in nanopure water . A high concentration of 6 could be dispersed in nanopure water or buffer (> 15 mg / ml) without a significant turbidity or precipitation, which allowed for analyses to be performed across a broad range of concentrations and also serves as a promising criterion for its use as a drug delivery carrier . The morphology and surface charge of the resulting micellar nanoparticles, 7, were characterized by dynamic light scattering (dls), transmission electron microscopy (tem), atomic force microscopy (afm), and -potential measurements (figure 3 and figure s4). Dls and -potential analyses indicated narrow and monomodal size distributions (pdi = 0.114) with almost neutral charges and a negligible difference at ph 5.0 and 7.4, 4.52 and 7.64 mv, respectively . The tem images also revealed uniform nanoparticles with an average diameter of 39 5 nm . Although the hydrodynamic and dry - state diameters measured by dls and tem, respectively, were in agreement, afm indicated significant deformation of the micelles upon deposition and drying on the mica substrate . 3 nm height and 40 7 nm diameter indicate flattening of the micelles, which is predicted to occur based upon the fluid shell and core components, each being composed of a highly viscous polymer, peg (tg = 17 c) and pevep (tg = 39 c, table 1 and figure s9). The afm data are useful qualitatively; however, further quantitative analysis is complicated by the presence of substantial amounts of polymer debris and agglomerations of 7 across the substrate (figure 3c, d), which are also indicators of the fluidity of the micellar assemblies . (a) dls results of 7: dh(intensity) = 49 7 nm, dh(volume) = 46 7 nm, and dh(number) = 44 6 nm (pdi = 0.114). (b) tem image of 7: dav = 39 5 nm, after counting more than 150 nanoparticles . Afm height image (c) and three - dimensional image (d) of 7: dav = 40 7 nm, after counting more than 100 nanoparticles . The phosphoester linkages of ppes can be cleaved by spontaneous hydrolysis and/or enzymatic degradation; the hydrolysis rate of the phosphoester linkages is known to be highly ph - dependent . In this study, we postulated that the exposure of micelles, 7, in acidic aqueous environment would induce a dual degradation of the phosphoester backbone linkages and the vinyl ether side chain moieties of the pevep block segment . Hence, the stability of the phosphoester backbone linkage and the vinyl ether functionality of 7 were monitored in deuterated buffer solutions by p and h nmr spectroscopies, respectively, in parallel to measuring the micelle size and the intensity by dls . Furthermore, the collected degradation products were identified using electrospray ionization (esi), gas chromatography (gc), and matrix - assisted laser desorption / ionization time - of - flight (maldi - tof) mass spectrometry . The backbone stability of the pevep segment in d2o at ph values of 5.0 and 7.4 and temperature of 37 c was monitored by p nmr spectroscopy (figure 4). The integral ratio of the p resonance of the intact pevep segment at 0.71 ppm to that of the newly emerging peaks was analyzed . The ph - dependent degradation of the pevep backbone linkages became apparent by the disappearance of p resonance corresponding to the intact pevep backbone, coincident with the appearance of new p resonances as a result of hydrolytic degradation at ph 5.0, whereas there was no significant change in the pevep p resonance when the samples were incubated at ph 7.4 . (a) rate of the pevep backbone degradation of 7 at ph 5.0 (black line) or ph 7.4 (red line) at 37 c as a function of time, as measured by a comparison of the integrals of initial to the newly appeared p nmr resonances . Changes in the p nmr resonance of pevep backbone of 7 at ph 5.0 (b) and 7.4 (c) at 37 c over a period of time . To compensate for potential complications in the detection of vinylic proton resonances of the micelle core, we evaluated the ph - dependent hydrolytic reactivity of the vinyl ether functionalities by observing both the polymer side chain functionalities and the small molecule hydrolysis product using h nmr spectroscopy . To determine the reactivity of the acid - labile vinyl ether functionality, the integral ratio of a distinct vinyl proton resonance of the micelles at 6.49 ppm to the newly appeared proton resonance of acetaldehyde, one of the hydrolysis products of the vinyl ether functionality, at 9.60 ppm was compared in d2o at ph values of 5.0 and 7.4 at 37 c over a period of time (figure 5). For the micellar nanoparticles in ph 5.0 aqueous solution at 37 c, both the gradual disappearance of vinyl proton resonance intensity and the appearance of an acetaldehyde proton resonance signal were clearly observed within 1 day, and the equal ratio of these two different proton resonances was reached after 14 days . By contrast, the vinyl proton resonance of the micelles in ph 7.4 aqueous solution at 37 c remained consistent without any observable generation of acetaldehyde over 39 days . Indeed, this observation bolstered our hypothesis that the spontaneous cleavage of the vinyl ether moieties occurred in ph 5.0 aqueous solution simultaneously with the ppe backbone degradation, but not at ph 7.4 . Rate of formation of acetaldehyde or disappearance of vinyl proton resonance at ph 5.0 (a) and ph 7.4 (b) and at 37 c as a function of time, as measured by a comparison of the integrals of vinyl and acetaldehyde proton resonance . Transition of proton resonances of acetaldehyde and vinyl groups at ph 5.0 (c) or ph 7.4 (d) and at 37 c over a period of time . It was hypothesized that hydrolysis of the ppe backbone would decrease the proportion of the hydrophobic: hydrophilic block segment ratio and that hydrolysis of the side chain vinyl ether groups would increase the hydrophilicity of the ppe backbone; thereby, each would weaken the micelle assemblies . In order to demonstrate the effects of ph - dependent hydrolytic degradation of the polymer on the behavior and stability of the micelles, 7, aqueous buffer solutions containing 6 at ph 5.0 and 7.4 were incubated at 25 and 37 c, and their degradation profiles were assessed by measuring the changes in hydrodynamic diameter and the intensity of light, scattered by micelles, when measured by dls over a period of time (figure 6). Overall, as predicted from the nmr degradation studies, the micelle assemblies in ph 5.0 aqueous solutions, at both 25 and 37 c, became unstable within 1 day, and the nanoparticles were undetectable within 7 and 2 days, respectively . The swelling behavior of the micelles upon hydrolysis of pevep segment was accounted for by the diffusion of water into the core region (figure 6a). Interestingly, the unstable and dissociated micelles did not cause the formation of visible precipitates, and thus, the micelle solutions remained clear during monitoring . Also, in agreement with the lack of backbone or side chain hydrolysis observed by nmr spectroscopy, the nanoparticle sizes in ph 7.4 aqueous solutions at 25 c remained consistent over 39 days (figure 6b). Surprisingly, when the samples were incubated at ph 7.4 and 37 c, large aggregates formed after 20 days, which persisted until day 43 when particles were no longer detectable . The lack of changes in the nmr data over the same period of time and conditions suggests that transesterification reactions may be a possible chemical change that produced subsequent morphological changes . In accordance with these observations, the intensity of light, scattered by the nanoparticles, as measured by dls, was also dependent on the ph of water and temperature (figure 6c). In the case of nanoparticles in ph 5.0 aqueous solutions, both at 25 and 37 c, the signal intensities became weaker rapidly, reaching below 20% as compared to the initial intensity, and finally were undetectable within a week . The signal intensity of light scattered by micelles in ph 7.4 aqueous solution at 37 c decreased gradually over a period of month . Meanwhile, there was no significant change in the signal intensity for the micelles in ph 7.4 aqueous solution at 25 c throughout the monitoring period . Taken together, the acidity of solutions was the primary determinant of the micelle stability . Changes in the hydrodynamic diameter of micelles at ph 5.0 (a) or ph 7.4 (b) and at a temperature of 25 c (black line) or 37 c (red line) over a period of time . (c) changes in the relative intensity of micelles in different environments, ph and temperature, over a period of time . The average values and their standard deviations, from three measurements, are shown . There have been several reports where the hydrolytic or enzymatic degradation behavior of ppe - containing micelles were studied by using titration, nmr spectroscopy, gpc, and/or dls methods; however, to the best of our knowledge, the identification of the actual degradation products of ppe has not been performed directly . In this study, we successfully identified the degradation products of ppe qualitatively using electrospray ionization (esi), gas chromatography (gc), and matrix - assisted laser desorption / ionization time - of - flight (maldi - tof) mass spectrometry . For this study, nanoparticles in aqueous solution at ph 5.0 were incubated at 37 c until they were not detectable by dls, and the complete disappearance of p resonance signal from the intact pevep backbone was confirmed by p nmr spectroscopy . The mixture solution containing the degradation products was analyzed by using esi, gc, and maldi - tof ms . The presence of oligomers and phosphoric acids was confirmed by esi ms (figure 7), demonstrating the hydrolytic degradability of phosphoester linkages of ppe . Tandem mass spectrometry was performed on most of the precursor ions as a way of verifying the chemical structures of these ions . The ms / ms product ions are listed along with the structures in figure 7 . Two series of oligomers (f and g series) that differ by 44 da were observed (figure 7). In order to verify that the f series were not the fragments of the g series, i.e., by losing vinyl alcohol (m / z 44), ms / ms of the lower mass precursor ions m / z 309 and 353 were performed (figures s5 and s6). The predominant fragment ion for m / z 309 was 141 while that for m / z 353 was 185, thus both losing a neutral repeating unit, 168 . In addition, a loss of ethylene glycol was observed for both ions . However, unlike the precursor ion m / z 353, m / z 309 lost vinyl dihydrogen phosphate to produce ion e. this major difference implied that the end group of the f series oligomers is 2-hydroxyethyl dihydrogen phosphate, while that of g series oligomers is bis(2-hydroxyethyl) hydrogen phosphate . Therefore, the f series were not the fragment ions from the g series . Because of its unionizable nature by electrospray ionization, the presence of ethylene glycol, as one of the degradation products, was confirmed by gc ms with electron ionization (ei) (figure s7). Neither vinyl- nor vinyl ether - containing compounds were detected using gc ms, which agreed with our observation by h nmr spectroscopy, shown in figure 5 . Again, this absence of vinyl or vinyl ether functionalities within the degradation products substantiated our hypothesis of a simultaneous hydrolysis of the vinyl ether moieties during the degradation process of the ppe backbone . Finally, maldi - tof ms analysis of the mixture of the degradation products verified the presence of the intact peg block segment with a single distribution having a spacing of 44 da, corresponding to a peg repeat unit (figure s8). Mass spectra in negative ion mode; m / z range of 1002000 (a) and 50180 (b). The cytotoxicities of the parent micelles, 7, and their degradation products, 8, were evaluated toward two cell lines, raw 264.7 mouse macrophages and ovcar-3 human ovarian adenocarcinoma cells, at a concentration range from 3 to 3000 g / ml for 24 h (figure 8). Both 7 and 8 maintained high cell viability over the range of the tested concentrations in both cell lines . We have previously observed low cytotoxicity and immunotoxicity of ppe - based micelles with different surface charges, their shell cross - linked analogues, and their degradation products even though we were not able to identify the degradation products at that time . These ppe - based nanoparticles are expected to have broad implications in clinical nanomedicine as alternative vehicles to those involved in several of the currently available medications, with precise control over their molecular structures and overall architectures . Cytotoxicity of the parent micelles of mpeg44-b - pevep337 (black line) and their degradation products 8 (red line) at a concentration range of 33000 g / ml for 24 h in raw 264.7 mouse macrophages (a) and ovcar-3 human ovarian adenocarcinoma cells (b). In conclusion, a novel polyphosphoester with ethylene glycol vinyl ether side chain functionality was developed as a versatile template for postpolymerization modifications, and its degradability and biocompatibility were investigated . A well - defined (pdi 1.05) homopolymer with vinyl ether side chain functionality was prepared by conducting rop using an organocatalyst, dbu . The kinetic study of this homopolymerization revealed an excellent controllability during rop with predetermined molecular weights and narrow molecular weight distributions . Subsequently, the vinyl ether side chain moieties displayed chemical availability and reactivity upon conjugation with hydroxyl- or thiol - containing model small molecules via three different types of conjugation chemistries thiol acetalization reaction resulting in modified polymers that contained either stable thio ether or hydrolytically labile acetal or thio acetal linkages . Despite the relatively low conversion percentages observed during acetalization and thio acetalization, ca . 18 and 8%, respectively, we anticipate that these degrees of conjugation efficiency would be adequate to achieve a sufficient loading of diagnostic and/or therapeutic molecules into this nanoparticle system . Meanwhile, amphiphilic diblock copolymers, mpeg44-b - pevep33, were also prepared by rop, and they afforded well - defined micelles with a narrow and monomodal size distribution in water . The degradation study of the prepared micelles demonstrated a full acid - catalyzed hydrolytic degradation behavior of both the side chain functionalities and the backbone linkages . Finally, the parent micelles and their degradation products, as identified qualitatively by mass spectrometry, were found to be nontoxic toward raw 264.7 mouse macrophages and ovcar-3 human ovarian adenocarcinoma cells . The fundamental understanding of selective hydrolysis of the vinyl ether and/or acetal / thio acetal moieties for the introduction of hydroxyl groups to the ppe system, which was conventionally limited to a cyclic ppe monomer, as a potential protecting group strategy is currently under investigation . Moreover, incorporation of biologically active molecules into these pevep - based functional, degradable polymers via the presented conjugation chemistries is underway.
In every field of dentistry, awareness regarding the importance of preserving tooth tissue is becoming evident . The current odontologic era is characterized by an increasing move toward less invasive treatment and preventive dentistry . The search for a more gentle, comfortable, and conservative caries excavation has led to the development of methods which aim at providing minimal thermal changes, less vibration and pain, and removal of infected dentine only . Laser, chemomechanical excavation, and air abrasion are successful in overcoming these problems . The chemomechanical caries removal (cmcr) technique stands out among other alternative methods as it is a nonaggresive excavation method which uses a chemical gel that is said to remove only the infected dentine where collagen is degraded, maintaining the demineralized portion that is capable of being remineralized and repaired . This approach is based on principle of minimal invasive dentistry that involves application of substances such as caridex, carisolv, papacarie, and carie - care for the removal of carious dentin . The use of carisolv introduced in the mid 90's as a chemical agent to remove caries is broadly discussed in the dental literature . Although carisolv was quite a success in the field of dentistry, certain drawbacks have also been reported which includes requirement of customized instruments, more time and its high cost - making it available for just a privileged few . In 2003, a research project in brazil developed a papain - based gel to universalize the use of chemomechanical method, and the new formula was commercially known as papacarie . The union of these three components confers antibiotic, bacteriostatic, and anti - inflammatory properties to this agent . Papain is an endoprotein from the proteolytic cysteine family that acts only upon damaged tissue, since plasma antiprotease is not present in the infected tissue, preventing papain's proteolytic action in tissues considered normal . Chloramine is a compound containing chlorine and ammonia with antibiotic and disinfecting properties, used for the irrigation of root canals . Papacarie was found to be easy to manipulate, simple and cheap, as well as effective in removing infected tissues . Carie - care is a more recent, minimally invasive method for chemo - mechanical dentine caries removal, developed by uni - biotech pharmaceuticals private limited, chennai, india in collaboration with vittal mallya scientific research foundation . Even this is a gel - based formulation containing a purified enzyme, derived from the plant carica papaya along with the benefits of clove oil . Cys-25 then performs a nucleophilic attack on carbonyl carbon which frees the amino terminal of the peptide, the enzyme is then deacylated by a water molecule and releases the carboxy terminal portion of the peptide . Studies have been done using various cmcr agents namely 5% sodium hypochlorite, gk101, caridex, carisolv and papacarie . However, there is a need to evaluate the efficacy of a more recent material like carie - care. The extent of carious dentine excavation, the time taken by each technique and even the microbiological aspect needs to be evaluated as it plays an important role in the progression of dentinal caries . Hence, the present study was designed to evaluate and compare the efficacy of newer enzymatic approaches, papacarie and carie - care as cmcr agents . This in vitro study was aimed to evaluate the efficacy and effectiveness of caries removal by two cmcr agents papacarie and carie - care. The study was carried out in the department of pedodontics and preventive dentistry in association with department of oral and maxillofacial pathology, st . The response or dependent variables were time required to remove dentine measured in seconds (efficiency) and the detection of bacteria after carious removal and dentinal tubule destruction (efficacy). Materials used in the study a sample size of 30 was determined by statistician by resource equation method ., in which caries distribution is done according to site and stage of progression of the lesion . This concept of sites / stages (si / sta) replaced the classification of black and promoted a medical model of conservative dentistry in clinical practice . The extracted teeth having site 2 (carious lesions at contact area of interproximal surfaces of the crown) and stage 3 (large dentin lesion with extended and frank cavitation) carious lesion were included in the study . The exclusion criteria were deep carious lesion with pulp exposure or potential for pulp exposure [figure 2]. Extracted primary molars each tooth was sectioned mesiodistally in the center of the carious lesion using diamond discs mounted in a straight handpiece so that the two halves (buccal and lingual or palatal) had equal sized carious lesions . The two specimens obtained from each tooth were alternatively grouped into two allotting thirty specimens each to papacarie and carie - care for caries excavation so as to avoid selection bias . The caries excavation was done according to the manufacturer's instructions . For both groups, the carious lesion was covered with gel and left undisturbed for 30 s. when the gel was cloudy, it was removed by scraping gently with the spoon excavator without application of any vertical pressure, after which some more gel was applied on the carious lesion and scraped as the gel turned cloudy and the process was continued until the gel was no longer cloudy . The gel was then rinsed and the cavity was then wiped with a moistened cotton pellet . The time taken for procedures was measured from start of caries removal till the cavity was confirmed to be free of caries with the help of a stop watch and was recorded . After caries removal, the tooth samples were decalcified in 10% formic acid at normal room temperature . [figure 3] the decalcified samples were then washed in water, then the teeth were dehydrated in ascending degrees of ethanol (70 - 100%), they were then cleared in xylene and later embedded in paraffin [figure 4]. During the experiment, the teeth were sectioned into 5 m thickness sections serially and were stained with eosin and hematoxylin to check for the presence of bacterial deposits microscopically and dentinal tubule destruction using conventional light microscope by a single operator to reduce bias [figures 5 and 6]. Decalcification of specimens specimens embedded in paraffin visible bacteria in the histological section of carie - care sample no bacteria evident in the histological section of papacarie sample the independent variable investigated in this experiment was the method of carious dentine removal . The response or dependent variables were time required to remove dentine measured in seconds (efficiency) and the detection of bacteria after carious removal and dentinal tubule destruction (efficacy). A sample size of 30 was determined by statistician by resource equation method ., in which caries distribution is done according to site and stage of progression of the lesion . This concept of sites / stages (si / sta) replaced the classification of black and promoted a medical model of conservative dentistry in clinical practice . The extracted teeth having site 2 (carious lesions at contact area of interproximal surfaces of the crown) and stage 3 (large dentin lesion with extended and frank cavitation) carious lesion were included in the study . The exclusion criteria were deep carious lesion with pulp exposure or potential for pulp exposure [figure 2]. Each tooth was sectioned mesiodistally in the center of the carious lesion using diamond discs mounted in a straight handpiece so that the two halves (buccal and lingual or palatal) had equal sized carious lesions . The two specimens obtained from each tooth were alternatively grouped into two allotting thirty specimens each to papacarie and carie - care for caries excavation so as to avoid selection bias . The caries excavation was done according to the manufacturer's instructions . For both groups, the carious lesion was covered with gel and left undisturbed for 30 s. when the gel was cloudy, it was removed by scraping gently with the spoon excavator without application of any vertical pressure, after which some more gel was applied on the carious lesion and scraped as the gel turned cloudy and the process was continued until the gel was no longer cloudy . The gel was then rinsed and the cavity was then wiped with a moistened cotton pellet . The time taken for procedures was measured from start of caries removal till the cavity was confirmed to be free of caries with the help of a stop watch and was recorded . After caries removal, the tooth samples were decalcified in 10% formic acid at normal room temperature . [figure 3] the decalcified samples were then washed in water, then the teeth were dehydrated in ascending degrees of ethanol (70 - 100%), they were then cleared in xylene and later embedded in paraffin [figure 4]. During the experiment, the teeth were sectioned into 5 m thickness sections serially and were stained with eosin and hematoxylin to check for the presence of bacterial deposits microscopically and dentinal tubule destruction using conventional light microscope by a single operator to reduce bias [figures 5 and 6]. Decalcification of specimens specimens embedded in paraffin visible bacteria in the histological section of carie - care sample no bacteria evident in the histological section of papacarie sample the mean time taken for complete caries removal was 385.8 s for papacarie and 427.13 s for carie - care, which is slightly more . Paired t - test showed significant difference between the mean time taken for caries removal for both the groups [table 1]. Comparison of time taken in seconds between inter - comparative groups there is no significant dentinal tubule destruction between papacarie and carie - care groups, thus indicating both are conservative [table 2]. Comparison of both groups with respect to dentinal tubule destruction carie - care group showed more amount of bacterial emanents when compared with papacarie . Fischer's exact test showed significant results with carie - care group containing more amount of bacterial remnants following caries excavation [table 3]. Although the use of burs in both high speed and low - speed handpieces for caries removal conventionally allows faster treatment, they may remove sound tooth structure as well, which may weaken the remaining tooth structure, as well as cause pulpal trauma . Philosophies of dental treatment change with time and now there is more than ample evidence provided by research for a reappraisal of the traditional approaches to caries treatment . The chemo - mechanical caries removal technique has generated great interest among dental researchers due to its concept of saving unaffected tooth structure while guaranteeing the removal of the denatured collagen stage of carious dentine . Although the two layers of infected and affected dentine can be differentiated by fuschin staining, the removal of infected dentin is operator and technique sensitive method . Similarly, the caries detector dyes cannot specify correctly dentine removal in the cavity preparations on the pulpal surface of deep cavities and at the amelo - dentinal junction . Further, in this present era of esthetic and adhesive dentistry, any remaining color or stain is unacceptable . Thus, the best alternative is preserving remineralizable tissue and prevention of overexcavation of the cavity . There is also a need to evaluate and compare the antimicrobial efficacy and efficiency in caries removal of these newer enzymatic approaches available commercially such as papacarie and carie - care. The present in vitro study was conducted with the objective to evaluate the efficacy (bacterial remnants and dentinal tubule destruction) and efficiency (time taken) of caries removal using papacarie and carie - care. The extracted teeth were selected based on the modified classification of mount and hume, which help determine the type of treatment (prevention, healing, re - mineralisation, or invasive intervention), and assist clinicians in selecting appropriate restorative materials . Carious teeth with site 2 and stage 3 lesion were included as cmcr agents can dissolve only dentinal caries structure . The selection bias was avoided by excavating the corresponding cavity halves as this minimizes differences in the excavation results due to variations in the extension, depth, localization, and structure of the caries lesion . As microflora is one of the main etiological factors in caries occurrence, it is essential to reduce the microbial counts in caries lesions . Apart from this, the efficacy in caries removal is also of interest . Papain, the main ingredient of papacarie, is an enzyme similar to human pepsin and has got bactericidal and bacteriostatic properities . Elindt demonstrated that papain acts only on infected tissues since infected tissues lack plasmatic anti - protease called a1 antitrypsin, this is present only in sound tissues which inhibit protein digestion . The infected dentin does not contain a1 antitrypsin enzyme, so this allows partially degraded molecules to be broken by papain . Carie - care, a more recent and also economical than papacarie, is a gel based formulation . Clove oil is a natural analgesic and is also known to have anesthetic properties . In the present investigation, evaluations for bacterial remnants and dentinal tubule destruction were done histologically by hematoxylin and eosin staining of the sectioned samples which were decalcified in 10% formic acid . Formic acid was used as it gives good results with minimal soft tissue shrinkage and minimal loss of tissue when compared with nitric acid which shows crumbling of tissue . Bacteria are the most common cause of dental caries and, for this reason, it is important to eliminate the largest possible numbers of bacteria during the removal of carious tissue . In the present study, the manufacturer's instructions were adopted, and caries removal ceased when the gel attained a clear (nonturbid) appearance . Papacarie excavation resulted in a highly significant reduction for all tested viable bacteria when compared to carie - care. As found in this in vitro investigation, among the two agents, the time taken for caries removal by carie - care was found to be more which is 427.13 s when compared to papacarie (385.8 s). Papacarie exerts an inhibitory action on cariogenic bacteria and the time taken for removal of caries was about 6 min which is in accordance with the study conducted by motta et al . In addition to papain, both papacarie and carie - care contain chloramines that are used to chemically soften the carious dentin . The chlorination affects the secondary or quarternary structure of collagen, by disrupting hydrogen bonding and thus facilitating caries removal . Papacarie was found to have no ability to affect the sound collagen fibers in the inner affected and normal dentin, as papain can digest only dead cells ., who concluded that papacarie is significantly more efficient in reducing the residual cariogenic bacteria in the dentin of primary teeth when compared to carisolv . Have shown clinical efficacy of caries removal is best with airotor and the microbiological efficacy (residual cariogenic bacteria) of caries removal was almost comparable with airotor and chemo - mechanical methods of caries removal . However, both papacarie and carie - care exhibited no dentinal tubule destruction after caries excavation which shows their minimal invasive method in preserving the underlying affected dentin for remineralization . The drawbacks of this study are this is an in vitro study, so the features such as pain during caries removal and patient's comfort levels could not be monitored . Further large - scale, well - designed randomized controlled trials are needed to substiantiate the clinical outcome of the present study . The following conclusions were drawn from the study: the mean time taken for caries removal was found to be more for carie - care when compared to papacariemore amount of bacterial remnants was present after excavation with carie - careboth papacarie and carie - care were found to be conservative as no dentinal tubule destruction was evident . The mean time taken for caries removal was found to be more for carie - care when compared to papacarie more amount of bacterial remnants was present after excavation with carie - care both papacarie and carie - care were found to be conservative as no dentinal tubule destruction was evident . Since this is an in vitro study, the clinical significance of these findings can only be determined with further studies assessing the clinical outcome of these chemo - mechanical methods in caries excavation . Within the limitations of the present study, we find the chemo - mechanical caries removal to be an adequate alternative to the conventional rotary instruments . However, the greater time requirement represents a substantial barrier to its wider use by clinicians.
Institutional review board approval (irb0005239) was obtained as well as written consent from the mothers of both patients to use photographs of their children for publication . A 3-month - old girl was referred to our tertiary eye clinic with a large capillary hemangioma covering her forehead, nose and the left side of her face [fig . 1]. She could only open the left eyelids to a vertical fissure height of 1 mm but was able to fix and follow with both eyes . Capillary hemangioma covering the left forehead, periocular area, nose, cheek, and upper lip as well as medial right upper lid at presentation oral prednisone (2 mg / kg) and intralesional corticosteroid injections both failed to clear the visual axis while surgery and radiotherapy were considered inappropriate due to their unacceptable risk profile . The successful off - label use of ibi prompted us to use a test dose for her left upper lid and, 1 month later, the left palpebral fissure had noticeably increased in size . The visible tumor was subsequently treated with monthly ibi using a dose of 0.5 mg / kg bleomycin diluted in a volume of normal saline equivalent to the estimated volume of the lesion . Treatment was discontinued after nine injections over 10 months since the final result was very satisfactory [fig . 2]. Ten months later, after nine injections of intralesional bleomycin, the left visual axis is completely cleared and all treated areas show marked improvement a 10-week - old girl was referred to our clinic with a right periocular capillary hemangioma after earlier debulking surgery had not cleared the visual axis . Examination revealed a normal left eye as well as normal anterior and posterior segments on the right with complete mechanical ptosis due to the hemangioma [fig . 3]. Given the failure of surgical intervention, monthly ibi treatments of 0.5 mg / kg diluted in normal saline were commenced and no adverse effects related to the treatment were noted . Two months after debulking surgery, a small notch, representing inadequate clearance of the visual axis, is visible in the right upper lid after five ibis, the right marginal reflex distance matched that on the left [fig . 4] and the patient was orthophoric with no evidence of amblyopia . After five intralesional bleomycin injections, the right visual axis has been cleared completely and regression of the remaining tumor has commenced no deterioration has been noted in either patient up to 12 months after the discontinuation of treatment . A 3-month - old girl was referred to our tertiary eye clinic with a large capillary hemangioma covering her forehead, nose and the left side of her face [fig . 1]. She could only open the left eyelids to a vertical fissure height of 1 mm but was able to fix and follow with both eyes . Capillary hemangioma covering the left forehead, periocular area, nose, cheek, and upper lip as well as medial right upper lid at presentation oral prednisone (2 mg / kg) and intralesional corticosteroid injections both failed to clear the visual axis while surgery and radiotherapy were considered inappropriate due to their unacceptable risk profile . The successful off - label use of ibi prompted us to use a test dose for her left upper lid and, 1 month later, the left palpebral fissure had noticeably increased in size . The visible tumor was subsequently treated with monthly ibi using a dose of 0.5 mg / kg bleomycin diluted in a volume of normal saline equivalent to the estimated volume of the lesion . Treatment was discontinued after nine injections over 10 months since the final result was very satisfactory [fig . 2]. Ten months later, after nine injections of intralesional bleomycin, the left visual axis is completely cleared and all treated areas show marked improvement a 10-week - old girl was referred to our clinic with a right periocular capillary hemangioma after earlier debulking surgery had not cleared the visual axis . Examination revealed a normal left eye as well as normal anterior and posterior segments on the right with complete mechanical ptosis due to the hemangioma [fig . 3]. Given the failure of surgical intervention, monthly ibi treatments of 0.5 mg / kg diluted in normal saline were commenced and no adverse effects related to the treatment were noted . Two months after debulking surgery, a small notch, representing inadequate clearance of the visual axis, is visible in the right upper lid after five ibis, the right marginal reflex distance matched that on the left [fig . After five intralesional bleomycin injections, the right visual axis has been cleared completely and regression of the remaining tumor has commenced no deterioration has been noted in either patient up to 12 months after the discontinuation of treatment . Bleomycin was first isolated in 1966 by umezawa from a soil fungus, streptomyces verticillus . The main mechanism of action of bleomycin is dna cleavage via oxidative damage caused by free radicals which form when its metal binding core is oxidized . Bleomycin also induces apoptosis in rapidly growing cells and has a sclerosing effect on the vascular endothelium which makes it useful in the proliferating phase of vascular neoplasms . Additional mechanisms include blocking the cell cycle at g2, degrading cellular rna and the induction of tumor necrosis factor . Many investigators have reported the successful use of ibi in treating hemangiomas and lymphangiomas in various anatomic locations . One conservative estimate indicates that 56.2% of lesions treated with ibi will undergo 70100% regression although other reports indicate a significantly higher success rate . Basal cell carcinoma, kaposi sarcoma, keratoacanthoma and skin metastases of malignant melanoma have also responded well to ibis . Based on previous publications, we used the following technique and dosages: general anesthesia for all injections.dose: 0.5 mg / kg (recommended range: 0.2 to 0.9 mg / kg per injection if aged under 1 year).volume: the dose in milligrams calculated above is then diluted in a volume of normal saline roughly equivalent to the volume of the lesion; for example, if the lesion measures 1 2 2 cm = 4 cm, then the calculated dose is diluted in 4 ml normal saline.a multipuncture technique is used with a 23-gauge needle, entering through normal skin and advancing into the lesion.local pressure is applied for 10 min.oral analgesics are prescribed post-injection.review and retreatment are done every 4 weeks as needed . Dose: 0.5 mg / kg (recommended range: 0.2 to 0.9 mg / kg per injection if aged under 1 year). Volume: the dose in milligrams calculated above is then diluted in a volume of normal saline roughly equivalent to the volume of the lesion; for example, if the lesion measures 1 2 2 cm = 4 cm, then the calculated dose is diluted in 4 ml normal saline . A multipuncture technique is used with a 23-gauge needle, entering through normal skin and advancing into the lesion . Pulmonary fibrosis is the most important dose - dependent complication of systemic bleomycin therapy but has not been reported after ibis . In one study, no spill - over of bleomycin into the bloodstream could be demonstrated after ibis which could imply that the risk of developing pulmonary fibrosis after ibis is small . Bleeding, ulceration, cellulitis, eschar formation, hypopigmentation, transient alopecia and flu - like symptoms may also occur . Lymphangitis, flagellate hyperpigmentation, and raynaud's phenomenon have been reported but are rare . The side - effect profile of ibis therefore compares favorably with that of conventional treatment modalities . Based on our experience, we would like to suggest that ibis also warrant consideration in the treatment of children with eyelid hemangiomas where conventional modalities have been unsuccessful or where treatment with beta - blockers may be contraindicated . This could represent a significant step forward in the management of a condition that can prove very difficult to treat.
Hailey - hailey disease (hhd), otherwise known as familial benign chronic pemphigus is a rare autosomal dominant, acantholytic disease with incomplete penetrance that is characterized by recurrent blisters, plaques, and erosions often accompanied by a burning or pruritic sensation . It is caused by a mutation in the atp2c1 gene on chromosome 3q21-q24, which encodes a disrupted golgi associated ca atpase . This mutation induces abnormal intracellular ca signaling which promotes premature keratinocyte proliferation leading to inappropriate desmosomal protein production causing failed keratinocyte adhesion and acantholysis, typically at flexural regions and friction prone sites . We report a 64-year - old female with a 37-year history of severe hailey - hailey disease involving her whole body . The patient s hhd has been unresponsive to common therapies used in the management of hhd . In 2005, a then 55-year - old caucasian female presented with clinical signs and symptoms of hhd, which had been active for 32 years . The patient initially developed symptoms of hailey - hailey disease in 1973 in her axillae and inframammary folds as well as her pubic region (namely her labia). She also reported that hhd lesions have previously appeared on her arms, neck, back, abdomen, popliteal regions, and oral mucosa . The presence of white bands on her fingernails, a rare manifestation of the disease, has also been documented . In 2003, a second biopsy was performed and confirmed that the inframammary lesions were in fact hhd lesions . Most recently, the patient presented with erythematous and crusted erosions and erupted bullae on her pubic region and middle back (figure 1). Since the patient s initial presentation and diagnosis, several different treatments have been administered for the management of the disease and its symptoms including: steroid and non - steroidal anti - inflammatory treatments (systemic, topical and intra - lesional), cyclosporin, methotrexate, dapsone, botulinum toxin a, fraxelated co2 laser to affected regions, and a variety of oral and topical antimicrobial therapies with minimal relief . Of the treatments given the patient s disease and symptoms were best controlled by oral doses of prednisone during times of exacerbated symptoms with doses of 30 - 50 mg per os . However, attempts to taper the prednisone to doses less than 10 mg were met with the recurrence of symptoms of the disease . Recent studies have reported the use of narrowband uvb or alitretinoin as successful independent therapeutic options in the treatment of hhd . Considering the persistent nature of the patient s disease a combination therapy of alitretinoin, an oral retinoid agent known as toctino (30 mg per os daily), and narrowband uvb therapy was started . The narrowband uvb therapeutic dose range was determined by the patient s skin type . For our patient, the narrowband uvb range began at 0.200 j / cm with an initial exposure time of 30 seconds . Uvb therapy was administered twice weekly and was to be continued until a total of 30 treatments (approximately 4 months) had been reached . Treatment was administered using the professional full body unit by ultralite enterprises (ultralite enterprises, lawrenceville, ga, usa). Based on the patient s positive response to the treatment, the uvb dose was increased by increments of 0.020 j / cm each visit until a dose of 0.294 j / cm was reached . The treatment doses administered throughout the patient s treatment were within a safe range as the minimum erythematous dose described by hamada et al . The course of treatment undertaken in this case is unique because existing literature describes the treatment of hhd with oral alitretinoin while the patient was tapering prednisone . The patient described in this report began the combination alitretinoin - narrowband uvb treatment within days of prednisone cessation . The patient reported that the lesions found on her pubic region and middle back improved within the first 2 - 3 weeks of starting this new combination alitretinoin and narrowband uvb treatment . Upon follow - up 6 weeks after beginning this new treatment course, a marked increase in healing bullae and tissue on the patient s back were noted . Residual rubor from uvb burns experienced during the fourth uvb session were also noted . Only after the fourth uvb treatment did the patient present with rubor from uvb burns, which led to the cessation of the uvb treatment, but continued use of oral alitretinoin as a mono - therapy . Healing hhd lesions could also be seen in the patient s pubic area at this time with no new lesions noted (figure 2). No other topical or systemic treatments were used throughout this treatment period . Currently, a mono - therapy of oral alitretinoin has maintained remission of the disease for 14 weeks since the beginning of the initial combination alitretinoin and narrowband uvb treatment course . No other treatments or interventions have been used, and no new lesions have been experienced with the alitretinoin mono - therapy . Patients usually experience a relapsing - remitting course of the disease . For many patients, a family history of hhd lesions favoring the axillae, chest, neck, genital areas, and other flexural regions are usually noted . Secondary infection with candida and/or staphylococcus is often noted and considered to be a common complication of the disease . Involvement of the vulva, conjunctiva, and mucosae are considered rare manifestation of the disease . There is currently no cure for hhd, however, treatment for managing the symptoms are available . In some cases, antibiotics, antifungal agents, as well as systemic, topical, and intralesional corticosteroids have proven effective for the management of hhd . Furthermore, other agents such as cyclosporine, retinoids, botulinum toxin a, and dapsone have also proven to be effective in some cases, and ineffective in others . According to sardy and ruzicka, the patient, whose case is described, presented with a severe manifestation of hhd with recurrent bullae, erythematous patches, and erosions . The patient has experienced hhd lesions on many different areas of her body including her genital region, neck, back, and oral mucosa, to name a few . For this patient, hhd has been a part of her life for almost 40 years . Recently, the successful remission of hhd with narrowband uvb has been discussed . In this patient this may be attributed to the oral alitretinoin, a retinoid agent that has been shown to induce photosensitivity . The case report by sardy and ruzicka showed successful treatment with alitretinoin in a patient with hhd . The treatment was accompanied by a continued course of oral prednisone, which was later tapered while treatment with oral alitretinoin persisted . Conversely, in our patient s case, the combination daily oral alitretinoin (30 mg) and narrowband uvb therapy described, which resulted in the successful remission of the patient s hhd, was started after the tapering and complete cessation of prednisone use . Further, considerable and sustained clinical improvement of the patient s hhd lesions has been noted with the administration of the daily oral alitretinoin therapy alone . Presently there has been no need for any treatment with further uvb, prednisone, or any other systemic or topical agents as the patient s disease appears to be in remission . For the first time in almost 40 years the patient has found relief and an effective treatment for her hhd . In conclusion, we suggest that conjunctive therapy of oral alitretinoin with narrowband uvb therapy be considered as a therapeutic option for the treatment of hhd to be followed by a mono - therapy of alitretinoin . The use of oral alitretinoin and narrowband uvb therapy should be explored further . In addition, the efficacy of oral alitretinoin as a mono - therapy for hhd should be explored.
The extent to which computerized provider order entry (cpoe) systems significantly hinder or assist clinical work efficiency has been a subject of debate and investigation for over 20 years . Yet the evidence base to address this issue remains sparse.13 zheng and colleagues4 recently drew attention to the poor and inconsistent methods used in conducting time and motion studies to measure the impact of systems on work patterns . Controlled studies to measure the effects of cpoe on doctors work are rare and we identified no published controlled studies of the impact on hospital nurses work.1 2 4 studies in ambulatory care59 and speciality areas in hospitals, such as the icu,10 11 predominate . Despite considerable developments in cpoe systems, the body of evidence regarding the impact of these systems on doctors and nurses work on general hospital wards is relatively sparse and reliance continues to be placed on results from important early studies.5 12 13 significant advances have been made in understanding the ways in which systems may disrupt and enhance work patterns and qualitative studies have played a vital role in understanding the complex socio - technical issues associated with integrating information systems into clinical work.1419 however, concerns of clinicians regarding the impact that systems have on the efficiency of their work continue to be raised and are a significant barrier to adoption.2022 prior to the imminent introduction of an electronic medication management system (emms) (with electronic prescribing and medication administration records (emar)), we interviewed 50 hospital clinicians (doctors and nurses) and managers to determine their expectations of system introduction.23 work practice change associated with the system was the most strongly and frequently raised issue by all groups.23 specific issues often center on the perceived increased time it takes for doctors to prescribe medications and for nurses to perform medication administration using a computer compared to paper medication charts.7 22 24 as a result of this, there are concerns that there will be less time for clinicians to spend on direct patient care activities.20 these beliefs are often in contrast to the system benefits promoted to clinicians, namely that information systems will improve efficiency and patient care.21 25 time savings are anticipated by re - distributing time across medication tasks.12 for example, while it may take longer to type a prescription compared to writing it, the ability to modify existing orders rather than prepare new orders and to use ordersets is expected to save time for example, improving the legibility of orders is expected to reduce nurses time spent clarifying orders . However, there are few data available to indicate whether time savings made in a particular sub - task are offset by time losses in other sub - tasks to make medication practice more or less efficient overall . Qualitative studies have revealed that many doctors and nurses report both improved and worsening efficiency.2628 improved sharing of medication information is possible with cpoe, but reduced face - to - face communication between clinicians has also been identified as a potentially negative impact of system introduction, but there is little quantitative evidence to clarify this issue.15 29 we aimed to undertake a large controlled time and motion study of hospital doctors and nurses to measure whether the proportions of time spent in medication tasks, direct care, and communication with each other significantly changed following the introduction of an emms with electronic prescribing and emar . We used a controlled before and after study design and used a direct time and motion observational approach . Data were collected from staff on four wards (respiratory, renal / vascular, and two acute geriatric medical wards) in a 400-bed major public hospital in sydney . We recruited a sample of 70 nurses and 59 doctors on the wards and collected data as outlined in table 1 . Nurses of all classifications were included: registered nurses (rn) (new graduates, rn with 24 years of experience, and rn with 5 + years of experience) and clinical nurse specialists (cns). Approximately 80% participation by eligible nurses was obtained in both the pre- and post - periods . Details of study samples in each study period cns, clinical nurse specialists; emms, electronic medication management system; rn, registered nurses . We sought to include all medical registrars, residents, and interns on the study wards . Physician specialists were excluded as they defer the vast majority of prescribing ordering tasks to more junior staff . A major challenge for the recruitment of doctors was the transitory nature of the medical workforce . For example, in the pre - period we identified a possible study population of 38 doctors of whom nine refused to participate (four because they did not want to be observed and five for a range of reasons such as about to go on leave and pending resignation or transfer). Nine who had agreed to participate were transferred from the study wards prior to observation, leaving 20 who participated . Thus, approximately 69% (20/29) of eligible doctors participated, although 76% had agreed (29/38) to do so . In the post - period there was a gap of approximately 2.6 years between pre- and post - data collection due to a delay in system implementation on the intervention wards . Post - data collection occurred at least 9 months after system implementation to ensure clinicians were familiar with the system . For the nurses study, two wards acted as controls and one (a geriatric ward) was the intervention ward (the fourth ward was initiating implementation and thus was excluded). For the doctors study, two wards acted as controls and two (both geriatric wards) were intervention wards . At baseline all wards had a cpoe system for ordering diagnostic tests and viewing results as well as ordering diets, transport, porters, and allied health consultations . The cpoe system did not allow clinical documentation . In the pre - intervention period, there was no intermediate transcription step between a prescriber's order and the final medication chart entry, as is the case in some countries . They will notate or contact the prescribing physician if changes are required . However, this step is not a pre - requisite for nurses to administer medications once a physician has documented an order . This process did not change after emms implementation . On the intervention wards in the post - period, the cerner millennium powerorders system, allowing electronic medication management functions, was integrated with the hospital's existing cpoe system in november 2007 . Prescribers were required to use the system to prescribe medications in the post - period . Prescribing is mainly by menu selection of pre - prepared order sentences which are triggered upon drug selection and which can be modified by the prescriber . Care sets allow for a group of related orders to be selected and ordered simultaneously with a single click . Active decision - support at the time consisted of allergy alerts and drug drug interaction alerts set at the most severe level . During the intervention period intervention wards had access to both computers on wheels (cows) and fixed computers (desk pcs) to access the emms . All nurses and doctors were invited to participate via information sessions followed by a direct approach . Rosters (schedules) from each ward were used to calculate the full - time equivalents for each nurse classification (rn new graduate, rn 24 years, rn 5 + years, and cns) and medical classification (intern, resident, and registrar). The sampling strategy was prepared in order to ensure all hours of the work day and weekdays were sampled proportionately to ensure the sample was representative . Representative sampling was used to determine the number of minutes that participants needed to be observed for each hour of the day for each classification . Following signed consent, nurses and doctors were assigned a study identification number, and demographic information regarding their age, classification, and length of experience was collected . We did not store staff names once a unique identifier was assigned and did not match data for individual staff in the pre- and post - periods . Observers randomly allocated nurses to a list for each observation session according to the sampling strategy . If a nurse at the top of the list was not working that day, observers selected the next nurse on the list . A similar approach was used for doctors, but on occasion pre - arranged times were requested as doctors were more mobile and locating them in the hospital could be difficult without this step . Nurses were observed between the hours of 7:00 and 19:00 and doctors from 8:00 to 20:00 on weekdays . The work observation method by activity timing (wombat) method was applied.3033 this is a technique for undertaking direct observational studies of health professionals . Using software on a handheld computer, observers capture multi - dimensional aspects of work and communication patterns . The wombat tool automatically captures all time data related to tasks and also details interruptions to work and multi - tasking (ie, tasks conducted in parallel). For example, details of tasks conducted in parallel, such as documenting and answering a question from a colleague, could be recorded and time - stamped automatically . Research teams interested in using this software should contact the corresponding author (jiw). Online supplementary appendix 1 gives the definitions for each of the tasks reported in this paper . This classification was developed following extensive observations and pilot testing.34 35 the method has been applied in australian studies of health professionals31 36 and was most recently validated in canadian studies of intensive care clinicians.32 37 the observers shadowed clinicians for an average of 1 h blocks, recording data using the handheld computer . For each task, the data collector recorded with whom the nurse / doctor completed the task, the information tools used, and any interruptions to work (defined as ceasing a task in order to respond to an external stimuli) or tasks completed in parallel (multi - tasking) tasks are continually recorded and include work, social / personal (including breaks), and activities carried out in transit . When the participant nurse / doctor engaged with patients, visitors, or other health professionals, the nurse / doctor was asked to introduce the observer and seek permission to continue . Several dummy observation sessions were undertaken as part of observer training conducted over 23 weeks . Inter - rater reliability tests were performed with two data collectors simultaneously, but independently, observing a clinician and comparing data . Kappa scores38 for task classification were> 0.85 throughout data collection, indicating high levels of agreement between observers . On average inter - rater reliability observation sessions the study was approved by the human research ethics committees of the university of new south wales and the study hospital . To assess changes in time spent on direct care and medication tasks for each profession, we calculated the proportion of total observed time in each task category by study period (pre / post) and group (control / intervention wards). We further examined medication sub - tasks for doctors and nurses as proportions of the total time spent in medication tasks . The difference in changes post - emms in intervention groups relative to control groups was defined as the differences in the proportions (p) over time between control and intervention groups, that is, (pintervention_post pintervention_pre)(pcontrol_post pcontrol_pre). We compared the difference in changes post - emms between intervention and control groups in each task category using the z test for proportions with the level of significance set at p<0.05 . Other descriptive statistics for number, average length, and frequency of tasks in each task category were also presented by study period and group . Information sources (eg, desk pc, cow) used in medication tasks were determined by calculating the proportions of total medication task time undertaken using each source . Time spent in interactions was examined in terms of both proportion of total observation time in professional communication, and the proportion of overall task time completed with others . We used a controlled before and after study design and used a direct time and motion observational approach . Data were collected from staff on four wards (respiratory, renal / vascular, and two acute geriatric medical wards) in a 400-bed major public hospital in sydney . We recruited a sample of 70 nurses and 59 doctors on the wards and collected data as outlined in table 1 . Nurses of all classifications were included: registered nurses (rn) (new graduates, rn with 24 years of experience, and rn with 5 + years of experience) and clinical nurse specialists (cns). Approximately 80% participation by eligible nurses was obtained in both the pre- and post - periods . Details of study samples in each study period cns, clinical nurse specialists; emms, electronic medication management system; rn, registered nurses . We sought to include all medical registrars, residents, and interns on the study wards . Physician specialists were excluded as they defer the vast majority of prescribing ordering tasks to more junior staff . A major challenge for the recruitment of doctors was the transitory nature of the medical workforce . For example, in the pre - period we identified a possible study population of 38 doctors of whom nine refused to participate (four because they did not want to be observed and five for a range of reasons such as about to go on leave and pending resignation or transfer). Nine who had agreed to participate were transferred from the study wards prior to observation, leaving 20 who participated . Thus, approximately 69% (20/29) of eligible doctors participated, although 76% had agreed (29/38) to do so . In the post - period there was a gap of approximately 2.6 years between pre- and post - data collection due to a delay in system implementation on the intervention wards . Post - data collection occurred at least 9 months after system implementation to ensure clinicians were familiar with the system . For the nurses study, two wards acted as controls and one (a geriatric ward) was the intervention ward (the fourth ward was initiating implementation and thus was excluded). For the doctors study, two wards acted as controls and two (both geriatric wards) were intervention wards . All wards had a cpoe system for ordering diagnostic tests and viewing results as well as ordering diets, transport, porters, and allied health consultations . The cpoe system did not allow clinical documentation . In the pre - intervention period, there was no intermediate transcription step between a prescriber's order and the final medication chart entry, as is the case in some countries . They will notate or contact the prescribing physician if changes are required . However, this step is not a pre - requisite for nurses to administer medications once a physician has documented an order . This process did not change after emms implementation . On the intervention wards in the post - period, the cerner millennium powerorders system, allowing electronic medication management functions, was integrated with the hospital's existing cpoe system in november 2007 . Prescribers were required to use the system to prescribe medications in the post - period . Prescribing is mainly by menu selection of pre - prepared order sentences which are triggered upon drug selection and which can be modified by the prescriber . Care sets allow for a group of related orders to be selected and ordered simultaneously with a single click . Active decision - support at the time consisted of allergy alerts and drug drug interaction alerts set at the most severe level . During the intervention period, intervention wards had access to both computers on wheels (cows) and fixed computers (desk pcs) to access the emms . All nurses and doctors were invited to participate via information sessions followed by a direct approach . Rosters (schedules) from each ward were used to calculate the full - time equivalents for each nurse classification (rn new graduate, rn 24 years, rn 5 + years, and cns) and medical classification (intern, resident, and registrar). The sampling strategy was prepared in order to ensure all hours of the work day and weekdays were sampled proportionately to ensure the sample was representative . Representative sampling was used to determine the number of minutes that participants needed to be observed for each hour of the day for each classification . Following signed consent, nurses and doctors were assigned a study identification number, and demographic information regarding their age, classification, and length of experience was collected . We did not store staff names once a unique identifier was assigned and did not match data for individual staff in the pre- and post - periods . Observers randomly allocated nurses to a list for each observation session according to the sampling strategy . If a nurse at the top of the list was not working that day, observers selected the next nurse on the list . A similar approach was used for doctors, but on occasion pre - arranged times were requested as doctors were more mobile and locating them in the hospital could be difficult without this step . Nurses were observed between the hours of 7:00 and 19:00 and doctors from 8:00 to 20:00 on weekdays . The work observation method by activity timing (wombat) method was applied.3033 this is a technique for undertaking direct observational studies of health professionals . Using software on a handheld computer the wombat tool automatically captures all time data related to tasks and also details interruptions to work and multi - tasking (ie, tasks conducted in parallel). For example, details of tasks conducted in parallel, such as documenting and answering a question from a colleague, could be recorded and time - stamped automatically . Research teams interested in using this software should contact the corresponding author (jiw). Online supplementary appendix 1 gives the definitions for each of the tasks reported in this paper . This classification was developed following extensive observations and pilot testing.34 35 the method has been applied in australian studies of health professionals31 36 and was most recently validated in canadian studies of intensive care clinicians.32 37 the observers shadowed clinicians for an average of 1 h blocks, recording data using the handheld computer . For each task, the data collector recorded with whom the nurse / doctor completed the task, the information tools used, and any interruptions to work (defined as ceasing a task in order to respond to an external stimuli) or tasks completed in parallel (multi - tasking). Tasks are continually recorded and include work, social / personal (including breaks), and activities carried out in transit . When the participant nurse / doctor engaged with patients, visitors, or other health professionals, the nurse / doctor was asked to introduce the observer and seek permission to continue . Several dummy observation sessions were undertaken as part of observer training conducted over 23 weeks . Inter - rater reliability tests were performed with two data collectors simultaneously, but independently, observing a clinician and comparing data . Kappa scores38 for task classification were> 0.85 throughout data collection, indicating high levels of agreement between observers . On average inter - rater reliability observation sessions lasted 35 min each . The study was approved by the human research ethics committees of the university of new south wales and the study hospital . To assess changes in time spent on direct care and medication tasks for each profession, we calculated the proportion of total observed time in each task category by study period (pre / post) and group (control / intervention wards). We further examined medication sub - tasks for doctors and nurses as proportions of the total time spent in medication tasks . The difference in changes post - emms in intervention groups relative to control groups was defined as the differences in the proportions (p) over time between control and intervention groups, that is, (pintervention_post pintervention_pre)(pcontrol_post pcontrol_pre). We compared the difference in changes post - emms between intervention and control groups in each task category using the z test for proportions with the level of significance set at p<0.05 . Other descriptive statistics for number, average length, and frequency of tasks in each task category were also presented by study period and group . Information sources (eg, desk pc, cow) used in medication tasks were determined by calculating the proportions of total medication task time undertaken using each source . Time spent in interactions was examined in terms of both proportion of total observation time in professional communication, and the proportion of overall task time completed with others . On all wards there was no significant change after emms introduction in the proportion of time nurses and doctors on the intervention wards spent in direct patient care activities relative to control ward clinicians (p=0.23 and p=0.08, respectively; table 2). Doctors on the control wards in the post - period spent 19.7% (average of 3.1 min / task; 2 h/10 h shift) of their time in direct patient care (excluding medication tasks) and those on the intervention wards 25.7% (average of 2.6 min / task; 2.6 h/10 h shift). Nurses spent 22.1% of their time in direct care activities on the control wards in the post - period and 26.1% on the intervention wards (table 2). Task time distribution for doctors before (pre) and after (post) electronic medication management system (emms) implementation * tasks which made up the remainder of clinicians time were: social / personal time, documentation (other than medication - related), transit time, administration, responding to pages, indirect care, and supervision / education . Percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post there was an overall significant temporal change in the proportions of time spent in direct care across all wards not associated with emms introduction . Over the 2.6 years of the study (the time from pre- to post - data collection) the proportion of time spent in direct patient care significantly increased for doctors from 15.0% (95% ci 12.7% to 17.3%) in the pre - period to 22.6% (95% ci 20.9% to 24.3%) in the post - period (p<0.0001), and for nurses from 20.2% (95% ci 18.2% to 22.1%) in the pre - period to 24.2% (95% ci 22.4% to 26.0%) in the post - period (p=0.003). Following the introduction of the emms on the intervention wards there was no significant change in the proportions of time spent by doctors or nurses on medication - related tasks (table 2) relative to clinicians on the control wards (p=0.4 and p=0.28, respectively). Doctors in the post - period on the control wards spent 7.4% (average of 49.6 s / task; 44.4 min/10 h shift) of their time on medication tasks and those on the intervention wards 8.5% (average of 45.1 s / task; 51 min/10 h shift). Medication tasks consumed a greater proportion of nurses overall time taking 23.7% (approximately 2 h in an 8.5 h shift) of their time on the control ward in the post - period and 22.6% (1.9 h / shift) on the intervention wards (table 2). We examined time distribution for specific types of medication - related tasks to assess if time was redistributed across tasks following emms introduction (tables 3 and 4). Both doctors and nurses on the intervention wards significantly increased the proportion of time spent reviewing medications using the emms compared to their colleagues on the control wards (p=0.01 and p<0.0001, respectively). However, no overall changes occurred in the time proportions of medication tasks since this increase was offset by (non - significant) decreases in nearly all other medication - related tasks (tables 3 and 4). Time spent on individual medication tasks by doctors * * excludes the medication - related tasks of administration, transcribe, and find medication chart, as these categories had fewer than 10 tasks in each period . Percentage difference in change over time (pre vs post) between intervention and control groups, time spent on individual medication tasks by nurses * * excludes low frequency tasks of find order, and clarify or order medication, as these categories had fewer than 10 tasks in each period . Percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post time doctors and nurses on all wards experienced an increase in the percentage of time devoted to medication tasks . For doctors, it increased from 7.0% (95% ci 6.4% to 7.5%) in the pre - period to 7.9% (95% ci 7.3% to 8.6%) in the post - period (p=0.03), while for nurses, it increased from 20.2% (95% ci 19.4% to 21.1%) in the pre - period to 23.1% (95% ci 21.7% to 24.5%) in the post - period (p=0.001). We examined the percentage of medication tasks performed with different information sources before and after emms introduction . Nurses on the intervention wards in the post - period significantly reduced their reliance on patients paper medical records as part of the medication process . Before emms introduction, nurses spent 89.0% (95% ci 81.0% to 97.0%) of medication task time (and 88% of all medication tasks) using a paper record, which declined to only 14.4% (95% ci 12.4% to 16.4%) after emms introduction (11% of medication tasks), while there was a much smaller change on the control wards, from 79.6% (95% ci 75.5% to 83.6%) of medication time to 66.9% (95% ci 62.0% to 71.8%). There was a significant relative reduction in the percentage of medication time where paper medical records were used of 61.9% on the intervention wards after emms introduction compared to the control wards (p<0.0001). On the intervention wards after emms introduction, 52.5% (95% ci 48.9% to 56.0%) of medication tasks involved a cow (63% of medication task time) and 2.5% (95% ci 2.0% to 3.1%) a desk pc (3% of medication task time). No computers were used in the pre - emms period and no cows were available on the control wards in the post - period . We found no significant change before and after emms introduction in the use of paper medical records during medication tasks by intervention ward doctors relative to the control wards (p=0.6). However, intervention ward doctors completed a significantly greater percentage of their medication task time using a desk pc compared to the control ward doctors (27.0% vs 15.2%; p=0.022) and used a cow for 22.0% (95% ci 17.7% to 26.4%) of medication task time . Desk pcs were used on the control wards during medication tasks, for example, to access pathology results from the cpoe system . There was no significant change on the intervention wards in the proportion of time nurses and doctors spent in professional communication following the introduction of the emms (p=0.57 and p=0.8; table 2). There was also no temporal change in the proportion of time spent on professional communication for either doctors (pre: 33.2% (95% ci 28.6% to 37.9%) vs post: 37.1% (95% ci 35.0% to 39.3%); p=0.13) or nurses (pre: 23.8% (95% ci 20.9% to 26.7%) vs post: 21.4% (95% ci 19.5% to 23.4%); p=0.17). In the post - period, nurses spent about 1.8 h/8.5 h shift of their time on professional communication, while doctors spent about 3.7 h/10 h shift . However, when we examined task time spent alone and with others, we found that doctors on the intervention wards in the post - period spent significantly more time with patients (6.3% increase; p=0.009), more time with other doctors (21.4% increase; p=0.003), and significantly less time working alone (14.8% decrease; p=0.0003) compared to doctors on the control wards (table 5). Doctors on the intervention wards spent a significantly greater proportion of their time with other doctors, largely due to an increase in the frequency of interactions (tasks per hour) (table 5). Nurses on the intervention wards spent a significantly lower proportion of time working with doctors following emms implementation compared to nurses on the control wards (4.2% decrease; p=0.0001; table 6). This was due to both fewer interactions (tasks per hour) and shorter interactions (mean task time) (table 6). Frequency and proportion of time doctors interacted with others during tasks before (pre) and after (post) electronic medication management system (emms) implementation * percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post frequency and proportion of time nurses interacted with others during tasks before (pre) and after (post) electronic medication management system (emms) implementation * percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post on all wards there was no significant change after emms introduction in the proportion of time nurses and doctors on the intervention wards spent in direct patient care activities relative to control ward clinicians (p=0.23 and p=0.08, respectively; table 2). Doctors on the control wards in the post - period spent 19.7% (average of 3.1 min / task; 2 h/10 h shift) of their time in direct patient care (excluding medication tasks) and those on the intervention wards 25.7% (average of 2.6 min / task; 2.6 h/10 h shift). Nurses spent 22.1% of their time in direct care activities on the control wards in the post - period and 26.1% on the intervention wards (table 2). Task time distribution for doctors before (pre) and after (post) electronic medication management system (emms) implementation * tasks which made up the remainder of clinicians time were: social / personal time, documentation (other than medication - related), transit time, administration, responding to pages, indirect care, and supervision / education . Percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post there was an overall significant temporal change in the proportions of time spent in direct care across all wards not associated with emms introduction . Over the 2.6 years of the study (the time from pre- to post - data collection) the proportion of time spent in direct patient care significantly increased for doctors from 15.0% (95% ci 12.7% to 17.3%) in the pre - period to 22.6% (95% ci 20.9% to 24.3%) in the post - period (p<0.0001), and for nurses from 20.2% (95% ci 18.2% to 22.1%) in the pre - period to 24.2% (95% ci 22.4% to 26.0%) in the post - period (p=0.003). Following the introduction of the emms on the intervention wards there was no significant change in the proportions of time spent by doctors or nurses on medication - related tasks (table 2) relative to clinicians on the control wards (p=0.4 and p=0.28, respectively). Doctors in the post - period on the control wards spent 7.4% (average of 49.6 s / task; 44.4 min/10 h shift) of their time on medication tasks and those on the intervention wards 8.5% (average of 45.1 s / task; 51 min/10 h shift). Medication tasks consumed a greater proportion of nurses overall time taking 23.7% (approximately 2 h in an 8.5 h shift) of their time on the control ward in the post - period and 22.6% (1.9 h / shift) on the intervention wards (table 2). We examined time distribution for specific types of medication - related tasks to assess if time was redistributed across tasks following emms introduction (tables 3 and 4). Both doctors and nurses on the intervention wards significantly increased the proportion of time spent reviewing medications using the emms compared to their colleagues on the control wards (p=0.01 and p<0.0001, respectively). However, no overall changes occurred in the time proportions of medication tasks since this increase was offset by (non - significant) decreases in nearly all other medication - related tasks (tables 3 and 4). Time spent on individual medication tasks by doctors * * excludes the medication - related tasks of administration, transcribe, and find medication chart, as these categories had fewer than 10 tasks in each period . Percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post time spent on individual medication tasks by nurses * * excludes low frequency tasks of find order, and clarify or order medication, as these categories had fewer than 10 tasks in each period . Percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post doctors and nurses on all wards experienced an increase in the percentage of time devoted to medication tasks . For doctors, it increased from 7.0% (95% ci 6.4% to 7.5%) in the pre - period to 7.9% (95% ci 7.3% to 8.6%) in the post - period (p=0.03), while for nurses, it increased from 20.2% (95% ci 19.4% to 21.1%) in the pre - period to 23.1% (95% ci 21.7% to 24.5%) in the post - period (p=0.001). We examined the percentage of medication tasks performed with different information sources before and after emms introduction . Nurses on the intervention wards in the post - period significantly reduced their reliance on patients paper medical records as part of the medication process . Before emms introduction, nurses spent 89.0% (95% ci 81.0% to 97.0%) of medication task time (and 88% of all medication tasks) using a paper record, which declined to only 14.4% (95% ci 12.4% to 16.4%) after emms introduction (11% of medication tasks), while there was a much smaller change on the control wards, from 79.6% (95% ci 75.5% to 83.6%) of medication time to 66.9% (95% ci 62.0% to 71.8%). There was a significant relative reduction in the percentage of medication time where paper medical records were used of 61.9% on the intervention wards after emms introduction compared to the control wards (p<0.0001). On the intervention wards after emms introduction, 52.5% (95% ci 48.9% to 56.0%) of medication tasks involved a cow (63% of medication task time) and 2.5% (95% ci 2.0% to 3.1%) a desk pc (3% of medication task time). No computers were used in the pre - emms period and no cows were available on the control wards in the post - period . We found no significant change before and after emms introduction in the use of paper medical records during medication tasks by intervention ward doctors relative to the control wards (p=0.6). However, intervention ward doctors completed a significantly greater percentage of their medication task time using a desk pc compared to the control ward doctors (27.0% vs 15.2%; p=0.022) and used a cow for 22.0% (95% ci 17.7% to 26.4%) of medication task time . Desk pcs were used on the control wards during medication tasks, for example, to access pathology results from the cpoe system . There was no significant change on the intervention wards in the proportion of time nurses and doctors spent in professional communication following the introduction of the emms (p=0.57 and p=0.8; table 2). There was also no temporal change in the proportion of time spent on professional communication for either doctors (pre: 33.2% (95% ci 28.6% to 37.9%) vs post: 37.1% (95% ci 35.0% to 39.3%); p=0.13) or nurses (pre: 23.8% (95% ci 20.9% to 26.7%) vs post: 21.4% (95% ci 19.5% to 23.4%); p=0.17). In the post - period, nurses spent about 1.8 h/8.5 h shift of their time on professional communication, while doctors spent about 3.7 h/10 h shift . However, when we examined task time spent alone and with others, we found that doctors on the intervention wards in the post - period spent significantly more time with patients (6.3% increase; p=0.009), more time with other doctors (21.4% increase; p=0.003), and significantly less time working alone (14.8% decrease; p=0.0003) compared to doctors on the control wards (table 5). Doctors on the intervention wards spent a significantly greater proportion of their time with other doctors, largely due to an increase in the frequency of interactions (tasks per hour) (table 5). Nurses on the intervention wards spent a significantly lower proportion of time working with doctors following emms implementation compared to nurses on the control wards (4.2% decrease; p=0.0001; table 6). This was due to both fewer interactions (tasks per hour) and shorter interactions (mean task time) (table 6). Frequency and proportion of time doctors interacted with others during tasks before (pre) and after (post) electronic medication management system (emms) implementation * percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post frequency and proportion of time nurses interacted with others during tasks before (pre) and after (post) electronic medication management system (emms) implementation * percentage difference in change over time (pre vs post) between intervention and control groups, that is, diff=(pintervention_post implementation of the emms was not associated with significant changes in the proportions of time doctors and nurses spent on direct patient care or medication - related tasks, relative to their colleagues on the control wards . As the hospital already had in place cpoe for orders other than medications, we were able to specifically examine the impact of the addition of this module on work . The results provide little support for arguments that emms use results in redistribution of time away from direct patient care or towards medication tasks . Examination of medication sub - tasks showed that increases in time spent, for example, reviewing medications in the system, were compensated by reduced time in other areas . Increased time spent reviewing patients medication charts may be a potentially positive work practice change for patient care, or indicate that it took longer to scroll through medication charts to find information relative to paper charts . These results suggest that emms use may result in a redistribution of time within and across medication - related tasks . There are limited comparative data, and differences in settings, work task classifications, and methods further hinder comparisons between studies . Early research, such as that by bates et al12 published in 1994, showed that following cpoe introduction time spent by doctors ordering increased . They found medical interns significantly increased ordering time from 5.3% to 10.5% (p<0.001) and surgical house officers from 6.4% to 15.5% (p<0.001). However, they also found that the use of ordersets took less time using cpoe compared to paper . In a more recent before and after study of cpoe, zheng et al40 observed medical residents for 68 h in a pediatric intensive care unit . They found no significant change in the time doctors spent in direct care or ordering . Further, we provide some of the first such data on nurses work patterns following cpoe introduction . Cpoe systems and clinicians computer skills are likely to have substantially improved over time and both may have contributed to reducing the potential negative impact of cpoe on clinicians time . Intervention ward doctors spent a significantly greater proportion of time with patients and other doctors . Interactions with patients may have increased as a result of the use of cows at patients bedsides, particularly during ward rounds . The increased time with other doctors may have been due to increased use of a shared doctors room on wards in which several pcs are located . These hypotheses are consistent with observations from our previous studies of doctors use of computing devices on wards with emms at this site.41 we observed that paper medication charts were often reviewed and completed at the central nurses work station . However, the emms moved these tasks to either the cows or the pcs in the doctors rooms . This physical shifting of work to another location may also have contributed to the observed significant decrease in interactions nurses had with doctors post - emms compared to control ward nurses . Interestingly, zheng et al40 also found that doctors spent a greater proportion of time interacting with patients (increasing from 1.18% to 4.05%; p<0.05) after compared to before cpoe implementation . However, as there was no control group in that study, it is not possible to be certain if this was associated with the cpoe system or a temporal change . We found significant temporal changes in the proportion of time both nurses and doctors spent on direct patient care and medication tasks in the post - period . Very few previous studies have measured changes in nurses time in relation to the introduction of an emar system.2 a study42 on one surgical ward in a uk hospital reported that following the introduction of a closed - loop medication system (comprising e - prescribing, automatic dispensing, bar code identification, and emar), medication administration rounds were significantly shorter, but nurses spent a significantly greater proportion of time on medication tasks outside these rounds . That study timed medication rounds and then used work sampling over 20 h to determine time on medication tasks outside rounds, but did not consider time in non - medication tasks such as direct care or professional communication . Importantly, we are able to link the patterns of work observed in our study with previously published prescribing error rates from these same hospital wards during the same study periods . A review of the medication charts of 1948 patient admissions from these wards before and after emms implementation showed prescribing error rates significantly declined with emms by 66.1% (from 6.25 errors per admission (95% ci 5.23 to 7.28) to 2.12 (95% ci 1.71 to 2.54); p<0.0001), with no significant change in the prescribing error rates on three control wards.43 thus doctors on these emms wards achieved significant improvements in medication safety while devoting the same proportion of their time to medication and direct patient care tasks compared to doctors on the control wards where no improvement in prescribing error rates was observed . Time and motion data are very valuable in addressing specific concerns that clinicians have about reduced efficiency and decreases in time spent on patient care associated with cpoe . Cpoe systems induce strong emotional reactions44 and questions raised about efficiency may be conflated with apprehension about other underlying matters such as shifts in roles and responsibilities which are often less well articulated.4547 gaining an understanding of the dimensions of these concerns requires both quantitative and qualitative studies.48 our results suggest concerns about dramatic reductions in efficiency are unfounded . Studies of this type in real - world clinical settings are complex and subject to bias from different sources . In relation to published research on this topic, our study has made advances in addressing methodological issues identified as problematic in previous studies.40 our study complies with the criteria outlined in the stamp guidelines40 in relation to observational work study designs . We applied a controlled design which allowed us to account for temporal changes in patterns of work . Hospital staff did not identify any significant policy or practice changes which occurred only in the intervention or control wards during this period . It is possible that other factors which we did not measure may have influenced our results . The large sample sizes, a sampling strategy to reflect both staff composition and work at different times of the day, and our ability to account for multi - tasking are particular strengths of the study . Limitations of our study include that we only examined weekday work . However, we found that at baseline there were no significant differences in task time distributions for direct care, medication tasks, or professional communication by ward . A further potential limitation of the study was that both intervention wards were acute geriatric wards . However, it is important to note that almost all admissions to these wards were via the emergency department . Also this hospital has no general medicine service and therefore we believe that the findings are likely to be generalizable to general wards . Patients on these wards have a high number of medications and often complex medication regimens . Thus, we believe these wards with heavy prescribing and medication administration loads are good test cases regarding the impact of the emms on staff efficiency . We used a direct observational approach which lends itself to the hawthorne effect whereby participants may change their behavior while being observed . The extended length of our study reduced the chance of sustained behavioral change, particularly on busy hospital wards . Further, observational studies of clinicians in situ have suggested that the extent of behavior change is minimal.34 49 50 we had high participation rates for nurses (80%) and doctors (69%). It is possible that doctors and nurses who did not participate were systematically different from the remaining population in terms of the factors under investigation, namely the amount of time that doctors and nurses spend particularly on medication tasks, and that these clinicians work was affected in different ways to clinicians in the study . One of the most frequent reasons for not participating in the study was that a doctor was about to go on leave or move to another ward . The large sample sizes guard against the likelihood that this would be a significant form of sampling bias in this study . As indicated in the methods section, we did not match nurses in the pre- and post - data analysis as ethics approval prohibited retaining individual nurse identification to allow repeated measures analysis . For doctors we collected data on tasks undertaken beyond their official shift times, but there were insufficient data in this category to assess whether there had been any significant change after emms introduction . We calculated kappa scores on multiple occasions to test for the reliability of task classification . Methods for applying kappa scores for assessing both task and time concurrently in time and motion studies have not been developed . We are currently developing these methods and such techniques would strengthen future time and motion studies . Empirical evidence to demonstrate the ideal length of time after the introduction of clinical information systems to assess impact on work practices is lacking . The long time period between pre- and post - data collection in our study is both a potential strength and weakness of the study . These systems can take a long time to bed - down and there have been criticisms that measurements taken too soon after system implementation may not reflect what will become however, we also acknowledge that a longer follow - up period may introduce the potential for other significant factors to confound the results . Studies of this type in real - world clinical settings are complex and subject to bias from different sources . In relation to published research on this topic, our study has made advances in addressing methodological issues identified as problematic in previous studies.40 our study complies with the criteria outlined in the stamp guidelines40 in relation to observational work study designs . We applied a controlled design which allowed us to account for temporal changes in patterns of work . Hospital staff did not identify any significant policy or practice changes which occurred only in the intervention or control wards during this period . It is possible that other factors which we did not measure may have influenced our results . The large sample sizes, a sampling strategy to reflect both staff composition and work at different times of the day, and our ability to account for multi - tasking are particular strengths of the study . Limitations of our study include that we only examined weekday work . However, we found that at baseline there were no significant differences in task time distributions for direct care, medication tasks, or professional communication by ward . A further potential limitation of the study was that both intervention wards were acute geriatric wards . However, it is important to note that almost all admissions to these wards were via the emergency department . Also this hospital has no general medicine service and therefore we believe that the findings are likely to be generalizable to general wards . Patients on these wards have a high number of medications and often complex medication regimens . Thus, we believe these wards with heavy prescribing and medication administration loads are good test cases regarding the impact of the emms on staff efficiency . Hawthorne effect whereby participants may change their behavior while being observed . The extended length of our study reduced the chance of sustained behavioral change, particularly on busy hospital wards . Further, observational studies of clinicians in situ have suggested that the extent of behavior change is minimal.34 49 50 we had high participation rates for nurses (80%) and doctors (69%). It is possible that doctors and nurses who did not participate were systematically different from the remaining population in terms of the factors under investigation, namely the amount of time that doctors and nurses spend particularly on medication tasks, and that these clinicians work was affected in different ways to clinicians in the study . One of the most frequent reasons for not participating in the study was that a doctor was about to go on leave or move to another ward . The large sample sizes guard against the likelihood that this would be a significant form of sampling bias in this study . As indicated in the methods section, we did not match nurses in the pre- and post - data analysis as ethics approval prohibited retaining individual nurse identification to allow repeated measures analysis . For doctors we collected data on tasks undertaken beyond their official shift times, but there were insufficient data in this category to assess whether there had been any significant change after emms introduction . We calculated kappa scores on multiple occasions to test for the reliability of task classification . Methods for applying kappa scores for assessing both task and time concurrently in time and motion studies have not been developed . We are currently developing these methods and such techniques would strengthen future time and motion studies . Empirical evidence to demonstrate the ideal length of time after the introduction of clinical information systems to assess impact on work practices is lacking . The long time period between pre- and post - data collection in our study is both a potential strength and weakness of the study . These systems can take a long time to bed - down and there have been criticisms that measurements taken too soon after system implementation may not reflect what will become however, we also acknowledge that a longer follow - up period may introduce the potential for other significant factors to confound the results . Hence, the importance of applying a controlled design . This is one of the few controlled studies of the impact of emms on hospital clinicians task time distribution . The results demonstrate that clinicians concerns related to reduced efficiency and time away from direct clinical care as a result of emms implementation voiced in interviews and focus groups23 were not realized . Most importantly, significant improvements were achieved with reductions in prescribing error rates, while maintaining similar patterns of task time distribution relative to colleagues on control wards . However, the implications of reduced interactions between doctors and nurses after emms implementation for safe medication administration should be a focus of future investigations.
Orbital apex syndrome is a condition in which the oculomotor nerve, trochlear nerve, abducens nerve, and ophthalmic branch of the trigeminal nerve are damaged . Orbital apex syndrome can be caused by a variety of inflammatory, infectious, neoplastic, iatrogenic/ traumatic, and vascular conditions.1 we present our findings in a patient with orbital apex syndrome associated with herpes zoster ophthalmicus . Magnetic resonance imaging (mri) showed inflammatory changes that extended to the cavernous sinus and orbital apex . Treatment with intravenous prednisolone 60 mg followed by tapering and vidarabine antiviral therapy markedly improved the extraocular palsies and optic nerve dysfunction . These changes an 81-year - old woman attended a neighborhood hospital because of nausea and loss of appetite of 10 days duration . The internist found that the patient was severely dehydrated, and she was admitted to our hospital on the same day . On the following day, a rash developed along the first division of the trigeminal nerve, and she had a fever of about 38c . Examination of the cerebrospinal fluid showed 90 cells/l, with a predominance of lymphocytes and a protein level of 75 mg / dl . Igg for varicella zoster virus in the cerebrospinal fluid was positive on enzyme immunoassay and the unit value was 12.8, but igm for varicella zoster virus was negative . Mri and computed tomographic images of the head showed no signs of meningitis or edematous changes in the cerebral parenchyma . Herpetic encephalitis induced by varicella zoster virus was suspected from the clinical findings, so intravenous vidarabine 600 mg / day was initiated in combination with sulbactam + defoperazone sodium and betamethasone 4 mg . The patient still had severe dehydration due to insufficient fluid intake, and her serum creatinine was elevated to 0.96 mg / dl (normal range 0.470.79 mg / dl). Thus, we did not use acyclovir systemically because it could have worsened her renal dysfunction . Stay, there was a reduction of the corneal reflex, and herpes keratoconjunctivitis was diagnosed . Treatment with antiviral ointment, acyclovir, and betamethasone eye drops were started . On day 17 of the hospital day, she developed ptosis and total ophthalmoplegia in the right eye (figure 1a). The pupillary diameters were 4.5 mm od and 2.5 mm os, and the light reflex in the right eye was absent (figure 1b). Central critical fusion frequency was 29 hz od and 31 hz os, and the intraocular pressures were 17 mmhg od and 14 mmhg os . Goldmann kinetic perimetry showed a slight constriction of the central visual field in the right eye (figure 1d). Enhanced t1-weighted mri showed enhancement of the optic nerve and orbital apex (figure 2a and b). From these clinical findings, ie, orbital inflammation affecting the orbital apex and igg for varicella zoster virus in the cerebrospinal fluid, the patient was diagnosed with orbital apex syndrome due to varicella zoster virus reactivation.2 after the patient was transferred to our hospital for further treatment, intravenous prednisolone 60 mg / day was started in combination with vidarabine 600 mg / day . The ocular symptoms improved soon after initiation of steroid therapy . The limitation of adduction and supraduction improved from day 1 after initiation of therapy . Repeated mri showed a decrease in enhancement of the right optic nerve and the tissues around the orbital apex (figure 2c and d). Twelve weeks after onset, diplopia at the primary position disappeared, and the best - corrected visual acuity in the right eye improved to 1.0 (figure 3). Twenty weeks after onset, eye movements were almost completely recovered, except for abduction (figure 4). However, the anisocoria still remained, despite the patient taking prednisolone 15 mg / day . Herpes zoster ophthalmicus can be triggered by reactivation of varicella zoster virus that is dormant in the trigeminal nerve ganglia . Reactivation of varicella zoster virus can be triggered by aging, an immunocompromised host, trauma, surgery, iatrogenic immunosuppression, tuberculosis, syphilis, and radiation therapy . Half of herpes zoster ophthalmicus cases have ocular complications, including blepharitis, keratoconjunctivitis, iritis, scleritis, and acute retinal necrosis.3 neurological complications, such as ophthalmoplegia and optic neuritis, are rare, but are responsive to antiviral or steroid treatment . Ophthalmoplegia was found in 3.5%10.1% of two large herpes zoster ophthalmicus series.3,4 among the cases with extraocular nerve palsies, oculomotor nerve palsy is the most frequent and abducens nerve palsy the second most frequent.5,6 several cases of orbital apex syndrome have been reported in association with more severe herpes zoster ophthalmicus.712 shirato et al reported a 71-year - old man who developed orbital apex syndrome 12 days after onset of herpes zoster ophthalmicus.7 t1-weighted, gadolinium - enhanced mri showed diffuse enhancement of the orbital fat, ocular muscles, and optic nerve on the right side . Five months after the onset of herpes zoster ophthalmicus, the extraocular palsies and ptosis were slightly improved, but there was no improvement in visual acuity . Kattah and kennerdell reported two cases of orbital apex syndrome associated with herpes zoster ophthalmicus.8 both cases developed extraocular palsies and optic nerve dysfunction one and five days after onset of rash . After oral prednisolone 80 mg, one of the patients recovered fully from the neurological complications, and the other patient had an improvement in ophthalmoplegia and optic neuritis, but the left eye progressed to endophthalmitis followed by corneal perforation . Krasnianski et al reported a case of orbital apex syndrome with complete ophthalmoplegia due to orbital myositis and optic neuritis two days after the onset of herpes zoster ophthalmicus . 10 the case was a 67-year - old woman who was treated with acyclovir 750 mg for 10 days with prednisolone 100 mg intravenously for three days, and then the prednisolone was tapered . Mri with gadolinium enhancement showed marked thickening of all the external ocular muscles and optic nerve sheath on the right side . Bourke and pyle reported a 63-year - old woman with herpes zoster ophthalmicus who developed complete ptosis, total ophthalmoplegia, and loss of visual acuity 10 days after the rash developed.11 treatment consisted of intravenous methylprednisolone 500 mg / day for three days, followed by a two - week course of oral prednisolone reducing by 80 mg / day . The ocular movements slowly improved . The day following initiation of therapy, supraduction, infraduction, and incyclotorsion visual acuity also increased from finger counting to 6/24 on the next day, and further improved to 6/18 soon thereafter . Dhingra et al9 reported a case of orbital apex syndrome associated with herpes zoster ophthalmicus and multiple myeloma . The signs and symptoms of herpes zoster ophthalmicus developed after the patient had completed chemotherapy . Although systemic acyclovir and prednisolone 30 mg slightly improved the visual acuity and disturbances of eye movements, the ptosis, anisocoria, and partial ophthalmoplegia did not improve . Saxena et al12 also reported a case of human immunodeficiency virus associated with herpes zoster ophthalmicus . A 29-year - old woman developed severe anterior uveitis, optic neuritis, complete ptosis, and ophthalmoplegia due to myositis 14 days after the onset of a facial rash . Highly active antiretroviral therapy and oral acyclovir 800 mg five times a day were started . Five days later, oral prednisolone 1 mg / kg / day was combined with highly active antiretroviral therapy and acyclovir therapy . By the end of four weeks, visual acuity and extraocular motility were markedly improved . In most of the earlier reports, and in our patient, orbital apex syndrome developed within 14 days of onset of herpes zoster ophthalmicus and was relatively responsive to steroid therapy, but may need more than 60 mg of prednisolone . The outcomes of visual function were good, although the duration of treatment may need to be from four weeks to one year . Sanjay et al summarized the clinical characteristics and course of 20 well documented cases of herpes zoster ophthalmicus presenting with complete ophthalmoplegia.13 herpes zoster ophthalmicus preceded the ophthalmoplegia as orbital apex syndrome by a mean interval of 9.5 days . Sixty - five percent of these cases recovered completely or partially after antiviral and steroid therapy, although anisocoria remained in most cases, as in our patient . In consideration of our case and the earlier reports, combined antiviral and steroid therapy should be effective in cases of orbital apex syndrome if the steroid therapy is used for at least four months . The pathological mechanism for ophthalmoplegia in cases of herpes zoster ophthalmicus histopathological studies have demonstrated significant perivascular and perineural inflammation of the ocular tissues, including the optic nerve, cavernous sinus, superior orbital fissure, and retina.14 earlier histological studies on autopsy specimens have suggested two possible pathological mechanisms, ie, reactivation of varicella zoster virus in the trigeminal ganglion, which then invades the cavernous sinus and superior orbital fissure,5 and a second mechanism might be lymphocytic infiltration of the affected nerves by sensory offshoots of the trigeminal nerves to all the motor nerves of the eye.15 naumann et al showed that inflammatory cells infiltrated the orbital apex along the long posterior ciliary vessels and nerves in 21 enucleated eyes affected by herpes zoster ophthalmicus, indicating that the neuropathy arises from an occlusive vasculitis.16 thus, the ophthalmoplegia and optic neuritis are suggested to be due to direct invasion by varicella zoster virus or by an inflammatory reaction and occlusive vasculitis after virus invasion . Lexa et al reported a case of herpes zoster ophthalmicus with optic neuritis, total ophthalmoplegia, and cerebellar manifestations.14 enhanced mri showed enhancement around the optic nerve sheath, which corresponded with the post mortem findings of invasion of inflammatory cells into the sheath of the optic nerve and ischemic changes in the optic nerve axons . Cases of herpes zoster ophthalmicus with optic neuritis have been reported, and mri and computed tomography show direct spread of granulomatous inflammation into the orbital apex.8,11 immunohistological examination of an autopsy specimen in a case of human immunodeficiency virus infection showed direct invasion of varicella zoster virus into the retina and optic nerve.17 our patient responded well to steroids and antiviral therapy, although the time needed to achieve significant resolution was more than five months . However, treatment for herpetic encephalopathy, with which our patient was initially diagnosed, might not have been long enough to improve symptoms . Mri findings showing enhancement of the optic nerve might reflect a direct inflammatory reaction rather than occlusive vasculitis . In conclusion, herpes zoster ophthalmicus is a relatively common disease in clinical ophthalmological practice, but is rarely accompanied by neurological complications . Orbital apex syndrome should be considered among the more severe ocular complications of herpes zoster ophthalmicus . If combined antiviral and steroid therapy is initiated early, it can be expected that visual dysfunction will recover . Therefore, ophthalmologists should be careful not to miss the neurological complications, and be especially vigilant during the 14 days after onset of herpes zoster ophthalmicus.
In february 2007, the board and the assembly of deans of medical schools of the dutch federation of university medical centres decided to institute a project group to revise the 2001 medical education blueprint . The main reasons for undertaking this project were developments in medical scientific disciplines, such as biotechnology and genetics, and developments in medical education, such as the introduction of the bachelor - master structure and the ongoing modernization of postgraduate speciality training programmes . In revising the 2001 blueprint the project group was specifically asked to establish the attainment levels of both the bachelor and the master programme in medicine, to consider whether the canmeds model or a similar competency profile could also be serviceable in revising the new framework for medical education and to reconsider the level of detail of the revised 2001 blueprint, aiming to find formulations that are realistic and testable and that facilitate external accountability for programme content . Framework was thought to reflect bettter the nature of the document). On august 12, 2009, the 2009 framework for undergraduate medical education in the netherlands was presented to the dutch minister for health . A translation in english is available from the website of the dutch federation of university medical centres (http://www.nfu.nl/fileadmin/documents/raamplan2009engelstalige_versie.pdf). The 2009 framework consists of nine chapters: introductionexplanationdevelopments in dutch and european legislationstages, cycles and levels in medical education in the netherlandsmaster s degree programme in medicine: target profilemaster s degree programme in medicine: competenciesmaster s degree programme in medicine: issues relating to illness and healthbachelor s degree programme in medicine: profile and learning outcomesbasic sciences in the medical curriculum developments in dutch and european legislation stages, cycles and levels in medical education in the netherlands master s degree programme in medicine: target profile master s degree programme in medicine: competencies master s degree programme in medicine: issues relating to illness and health bachelor s degree programme in medicine: profile and learning outcomes basic sciences in the medical curriculum in an appendix the final document also includes a skills list . In 2002, the bachelor - master structure was introduced in the dutch system for higher education, establishing bachelor s degree and master s degree programmes as independent study programmes . This change followed in the wake of the signing of the bologna declaration in 1999 . This declaration, signed by 29 european countries, aims to promote greater overall convergence in higher education in europe, based on a two - cycle degree structure . In the transitional period, permission to use the protected title of physician and qualification to perform certain actions reserved for physicians are linked to registration in the individual health care professions act register . Registration is only open to those who have completed the degree programme in medicine and therefore to those who, following the introduction of the bachelor - master structure, have obtained the master of science (msc) degree on completion of the master s degree programme in medicine . The project group decided to define students learning outcomes upon completion of the master s degree programme in terms of competencies . A competency is defined as the ability to adequately perform a professional activity in a specific, authentic context, using an integrated body of knowledge, understanding, skills and professional behaviour . After careful consideration, the canmeds model was chosen, because, firstly, this model involves an excellent and useful division into physicians roles and competencies in a variety of professional situations, and secondly, this model is also used in the framework of the modernization of postgraduate specialty training . Together, the seven roles or competency domains in the canmeds model constitute the target profile in chapter 5 of the 2009 framework (see table 1 (tab . The profile is elaborated in terms of key competencies and their constituent sub - competencies . As the project group felt that bachelor students in medicine do not yet operate in an authentic professional setting, the bachelor s degree programme is described in terms of knowledge, skills, and behaviour and not competencies . Knowledge and understanding relate to the basic sciences, the foundation of medicine in the natural sciences, and aspects of behavioural and social sciences . The 2001 blueprint included a list of clinical conditions which comprised about 330 clinical conditions in all . Issues relating to illness and health was more appropriate to the wide - ranging field in which today s physicians are operating, as a fair share of physicians work is not directly patient or disease related . The list of issues described in chapter 7 has been subdivided into several categories . The first and most voluminous one comprises the complaints that induce patients to consult their physician . The next two, smaller, categories deal with findings upon physical and additional examinations . The introduction of a list of issues meant that the list of clinical conditions that was included in the 2001 blueprint was not to return in the 2009 framework: on the one hand, the contemplation of each issue would involve a series of clinical presentations anyway, and, on the other hand, a list of clinical conditions would require ceaseless maintenance in the wake of ongoing developments in medical disciplines . In the 1994 blueprint and the 2001 blueprint, the project group felt that the aspect of required knowledge had been somewhat eclipsed by lists of problems, clinical conditions and skills . Right from the start, the project group wished to give the basic sciences a more prominent place in medical education . The model of competencies selected by the project group also carried the risk of outlining required knowledge only implicitly . Therefore, in the 2009 framework, a separate chapter is devoted to basic sciences in the medical curriculum dealing with both the foundation of medicine in the natural sciences and aspects of the behavioural and social sciences . Spot - checking at the eight dutch medical faculties showed that all medical faculties still used this list, sometimes with minor adjustments . After discussion in the project group, the skills list from the previous blueprint, with minor adjustments, was added to the appendices to the 2009 framework . The project group was asked to pay special attention to finding formulations that are realistic and testable and that allow external accountability for medical education programme content . This prompted the project group to clearly define the competencies in terms of levels that are to be attained . Medical education in the netherlands involves a training sequence with two stages that medical students pass through in succession . The first stage of this training sequence comprises two cycles: a three - year bachelor s degree programme in medicine, followed by a three - year master s degree programme in medicine . In the second stage of the training sequence, following upon completion of the bachelor and master programmes, medical graduates undertake postgraduate specialist training to become medical specialists in primary or secondary healthcare . Having completed that stage, medical practitioners then engage in lifelong learning activities both in practice and in formal continuing education courses . The levels linked to the successive stages can be called starter level, beginning practitioner level and experienced practitioner level . After completion of their bachelor programme in medicine, medical students can be characterized as starters . These students have mastered a basic body of knowledge and understanding of scientific disciplines that are relevant to their subsequent professional practice . They have also acquired a set of basic skills and are able to show appropriate professional behaviour in training - related situations . They are able to apply knowledge, skills and behaviour in dealing with issues that involve a relatively low level of complexity . After completion of the master programme in medicine, medical graduates can be characterized as beginning practitioners . Newly graduated physicians demonstrate a basic competence in practice, possess an integrated body of knowledge, skills and professional behaviour and can handle issues involving higher levels of complexity . Medical graduates are capable of performing independent consultations but only perform these under supervision during postgraduate medical training . After a number of subsequent years of further schooling, training and experience in medical specialist training in the discipline of their choice, physicians operate independently without supervision . An element of independent practice is participation in interprofessional coaching activities and lifelong continuing - education courses . In the 2009 framework, the levels of proficiency to be achieved in the bachelor and master programmes in medicine are defined in a five - level structure (see table 2 (tab . The project group also had to consider the level of difficulty of the issues that are presented to students . On the one hand, these are medical factors, such as typical and atypical presentations or co - morbidity problems; on the other hand, these are contextual factors from the psychosocial model . The level of difficulty is then codetermined by the availability of standard solutions to a problem and the applicability of protocols and guidelines . The level of difficulty also lies in the degree to which different physician roles must be integrated in dealing with the issue in question . Graduates in medicine must be able to handle issues involving varying - including higher - levels of difficulty, with the proviso that the degree of required supervision increases with the degree of difficulty of the case presented . In this respect, there is one sub - competency incorporated in the role of medical expert that is of particular importance: students ability to recognize and name the personal limits to their knowledge and skills and to decide in time if, and, if so, when a third party needs to be called in, including their supervisor . The 2009 framework states that graduates in medicine must have mastered all issues related to illness and health that are included in the list at least at level ii . Some of the issues, depending on the specifics of individual training programmes, will be included in additional competency testing at higher levels . The formulations of sub - competencies specify the levels (iii, iv, or v) that have to be attained . To illustrate the assignment of levels some examples are given in table 3 (tab . Major changes are the definition of competencies, the indication of the levels of competency to be attained and the introduction of a list of issues related to health and disease . The 2009 framework will be used as the basis for accreditation and for a revision of the legislation that outlines the educational requirements of physicians . Laan, md phd, is professor of rheumatology and medical education, radboud university medical centre, institute for medical education, nijmegen, the netherlands.r.r.m . Leunissen, msc, is senior staff member, radboud university medical centre, institute for medical education, nijmegen, the netherlands.c.l.a . Van herwaarden, md phd, is professor of pulmonology and former dean of the radboud university medical centre, nijmegen, the netherlands . Laan, md phd, is professor of rheumatology and medical education, radboud university medical centre, institute for medical education, nijmegen, the netherlands . Leunissen, msc, is senior staff member, radboud university medical centre, institute for medical education, nijmegen, the netherlands . Van herwaarden, md phd, is professor of pulmonology and former dean of the radboud university medical centre, nijmegen, the netherlands.
With over 60 million people affected, copd is a major global health problem leading to substantial morbidity and mortality.1 in addition to the disease burden, copd requires a major shift in patients daily life as they need to adhere to drug treatment, implement lifestyle changes, monitor signs and symptoms, and apply decision making on early treatment of exacerbations to prevent complications.2 interventions to improve this self - management behavior in copd patients have been receiving increasing attention and generally involve patient education and teaching skills to patients for monitoring their condition, carrying out medical regimens, and changing their health behavior.3 a recent systematic review found positive effects on a range of outcomes, including health - related quality of life (hrqol), dyspnea, and health care utilization.4 although the evidence favors self - management interventions, there seems to be a large heterogeneity in the effects of these interventions . Findings from five randomized trials, all based on the self - management program living well with copd,5 were contradictory and have raised questions about large scale implementation of self - management interventions in copd patients . The first two trials reported large positive effects on respiratory - related hospitalization5 and the combined endpoint of respiratory - related hospitalization and emergency department visit,6 but these promising findings could not be replicated in subsequent studies in the uk7 and the netherlands.8 the fifth trial even reported higher mortality rates among patients in the self - management group and recruitment was terminated prematurely.9 several researchers have postulated hypotheses in an attempt to explain the different outcomes of these five trials . The diversity among interventions, study populations, follow - up time, and outcome measures across these five trials compromise a generalization to real life . Patient factors might matter more than assumed to date and it has been suggested that adherence to and uptake of self - management interventions are better in specific subgroups of patients.10 currently, however, evidence on which subgroups are more likely to benefit from or respond negatively to self - management interventions is lacking . With this knowledge, clinicians might be able to target self - management interventions at those patients who benefit most . Identification of such patient subgroups in individual trials is complicated, as these usually lack power . A meta - analysis of individual patient data (ipd) enables a more reliable subgroup analysis with sufficient power due to the large numbers of patients included and by allowing a similar definition of subgroups across studies.11 collecting the ipd from different trials also enables standardized statistical analyses and inclusion of data on available but unreported endpoints, which has additional advantages for analyzing the main effects of self - management interventions . The present ipd meta - analysis aims to summarize the evidence on the effectiveness of copd self - management interventions on relevant outcomes, including hrqol, hospitalization, and mortality, with a particular focus on identifying subgroups of patients with copd who are most likely to benefit from self - management interventions . This ipd meta - analysis was conducted according to the guidelines in the cochrane handbook for systematic reviews of interventions12 and followed a prespecified protocol.13 we searched the electronic databases pubmed, embase, cochrane central register of controlled trials, psycinfo, and cinahl from january 1985 through june 2013 and scrutinized the reference lists of identified relevant systematic reviews . With no general agreement on an operational definition of self - management interventions, an international group of self - management research experts set out to reach consensus on the criteria for defining self - management intervention . There is general agreement on the multifaceted nature of self - management interventions.3,4,14 therefore, self - management interventions were defined as interventions providing information to patients and including minimally two of the following components: 1) stimulation of sign / symptom monitoring, 2) education in problem solving skills (ie, stress / symptom management), enhancement of 3) medical treatment adherence, 4) physical activity, 5) smoking cessation, or 6) dietary intake . The emphasis for each component had to be on enhancing the patient s active role and responsibility . Therefore, interventions focusing on pulmonary rehabilitation were not considered eligible for this meta - analysis . Studies were selected by two researchers working independently (nhj and hw) and included if they 1) met the requirements of the definition of self - management intervention above, 2) had a randomized trial design with concealed allocation to treatment, 3) included patients with an established diagnosis of copd, 4) compared the self - management intervention to usual care or another self - management intervention, 5) reported data on one or more of the selected outcomes, 6) followed patients for at least 6 months, and 7) were reported in english, dutch, french, german, italian, portuguese, or spanish . Methodological quality was assessed by two researchers independently (nhj and hw) using three relevant criteria from the risk of bias tool from the cochrane collaboration:12 1) random concealed allocation to treatment, 2) intention - to - treat analysis, and 3) absence of other major sources of bias (eg, high drop - out rates, risk of contamination). Studies that scored a high risk of bias on one or more criteria were defined as high risk of bias . Those studies were included, but their impact on the results was assessed in a sensitivity analysis . The principal investigators of selected studies were invited to participate in this ipd meta - analysis and share their de - identified trial data . Data from each trial were checked on range, extreme values, internal consistency, missing values, and consistency with published reports . Details on requested variables, data management, project management, and ethical considerations can be found in the published protocol.13 the main outcomes of this study included: hrqol at 6 and 12 months (as measured with chronic respiratory questionnaire15 or st george respiratory questionnaire16), respiratory - related hospitalization (time - to - first - event, within 6 and 12 months), all - cause hospitalizations (time - to - first - event, within 6 and 12 months), and mortality (time - to - event, within 6 and 12 months). Additional outcomes analyzed were generic quality of life (qol), as measured with the short form health survey,17 and total days of respiratory - related and all - cause hospital stay since enrollment at 6 and 12 months . Clinically relevant potential effect modifiers (ie, variables, such as sex or age) were prespecified based on the self - management literature and availability of data across trials and presented in table 1 (along with the baseline data). Based on teleconferences with the principal investigators, we decided to collect data on baseline exacerbation frequency, in addition to the potential effect modifiers prespecified in the protocol.13 the principal investigators of the individual trials were involved in the process of designing a detailed plan for statistical analysis and agreed upon this prior to data analysis . Missing values were imputed within studies only using multiple imputation by chained equations (overall 2.7% missing data, except 33.7% for hrqol follow - up data).18 for each study, 25 multiple imputed datasets were created and used for the primary analyses . Within these analyses, a one - stage approach was used, that is, simultaneously analyzing all observations while accounting for clustering of observations within studies.19 results of imputed datasets were pooled using rubin s rules.20 all analyses were carried out according to the intention - to - treat principle . For time - to - event endpoints, effects of self - management were quantified by estimating hazard ratios using cox proportional hazard models, including a frailty term to account for clustering within studies . The continuous outcomes (hrqol and generic ol) were quantified by standardized mean differences (smd) between intervention arms and analyzed using linear mixed effects models . Using the smd, results are converted to a uniform scale representing the intervention effect relative to the observed variability in one study before pooling the results of different studies . Binary outcome data (mortality, respiratory - related, and all - cause hospitalization) were analyzed with log - binomial mixed effects models, which estimated risk ratios (rrs) or odds ratios (ors) in case of nonconvergence of a model, respectively . To correctly model the presence of overdispersion in the count data of total days of hospital stay, all mixed effects models included a random intercept and a random slope for the treatment effect to take clustering at study level into account . To assess whether the effect of self - management was modified by patient characteristics, the aforementioned models were extended with interaction terms for the patient characteristics included in table 1 . The independent variables in the models were random intercept, random slope, allocation to self - management, patient characteristic, and interaction term (treatment allocationpatient characteristic). This was performed for each patient characteristic separately . All effect modifiers with p<0.10 for the interaction (likelihood ratio test) in the univariable analysis were included in a multivariable model to estimate the effect of self - management within subgroups independent of other potential effect modifiers . Effect modification was considered significant if the interaction term showed p<0.05 in the final model . As a sensitivity analysis, we investigated the potential of retrieval bias (ie, bias due to selective inclusion of studies in the ipd meta - analysis) by pooling the published main effects of studies for which ipd were unavailable with the main effects of included studies in a random effects meta - analysis . To assess the impact of studies of lower methodological quality on the main effects, an additional sensitivity analysis was performed, including only studies with a low risk of bias . Three additional sensitivity analyses were performed to assess the robustness of findings from the subgroup analyses: 1) a complete case analysis was carried out to assess the effect of imputing data, and analyses were repeated by 2) excluding older studies (recruitment before 2001) and 3) excluding the largest trial.6 all analyses were performed in r for windows version 3.1.1 (r development core team . Released 2013 . We searched the electronic databases pubmed, embase, cochrane central register of controlled trials, psycinfo, and cinahl from january 1985 through june 2013 and scrutinized the reference lists of identified relevant systematic reviews . With no general agreement on an operational definition of self - management interventions, an international group of self - management research experts set out to reach consensus on the criteria for defining self - management intervention . There is general agreement on the multifaceted nature of self - management interventions.3,4,14 therefore, self - management interventions were defined as interventions providing information to patients and including minimally two of the following components: 1) stimulation of sign / symptom monitoring, 2) education in problem solving skills (ie, stress / symptom management), enhancement of 3) medical treatment adherence, 4) physical activity, 5) smoking cessation, or 6) dietary intake . The emphasis for each component had to be on enhancing the patient s active role and responsibility . Therefore, interventions focusing on pulmonary rehabilitation were not considered eligible for this meta - analysis . Studies were selected by two researchers working independently (nhj and hw) and included if they 1) met the requirements of the definition of self - management intervention above, 2) had a randomized trial design with concealed allocation to treatment, 3) included patients with an established diagnosis of copd, 4) compared the self - management intervention to usual care or another self - management intervention, 5) reported data on one or more of the selected outcomes, 6) followed patients for at least 6 months, and 7) were reported in english, dutch, french, german, italian, portuguese, or spanish . Methodological quality was assessed by two researchers independently (nhj and hw) using three relevant criteria from the risk of bias tool from the cochrane collaboration:12 1) random concealed allocation to treatment, 2) intention - to - treat analysis, and 3) absence of other major sources of bias (eg, high drop - out rates, risk of contamination). Studies that scored a high risk of bias on one or more criteria were defined as high risk of bias . Those studies were included, but their impact on the results was assessed in a sensitivity analysis . The principal investigators of selected studies were invited to participate in this ipd meta - analysis and share their de - identified trial data . Data from each trial were checked on range, extreme values, internal consistency, missing values, and consistency with published reports . Details on requested variables, data management, project management, and ethical considerations can be found in the published protocol.13 the main outcomes of this study included: hrqol at 6 and 12 months (as measured with chronic respiratory questionnaire15 or st george respiratory questionnaire16), respiratory - related hospitalization (time - to - first - event, within 6 and 12 months), all - cause hospitalizations (time - to - first - event, within 6 and 12 months), and mortality (time - to - event, within 6 and 12 months). Additional outcomes analyzed were generic quality of life (qol), as measured with the short form health survey,17 and total days of respiratory - related and all - cause hospital stay since enrollment at 6 and 12 months . Clinically relevant potential effect modifiers (ie, variables, such as sex or age) were prespecified based on the self - management literature and availability of data across trials and presented in table 1 (along with the baseline data). Based on teleconferences with the principal investigators, we decided to collect data on baseline exacerbation frequency, in addition to the potential effect modifiers prespecified in the protocol.13 the principal investigators of the individual trials were involved in the process of designing a detailed plan for statistical analysis and agreed upon this prior to data analysis . Missing values were imputed within studies only using multiple imputation by chained equations (overall 2.7% missing data, except 33.7% for hrqol follow - up data).18 for each study, 25 multiple imputed datasets were created and used for the primary analyses . Within these analyses, a one - stage approach was used, that is, simultaneously analyzing all observations while accounting for clustering of observations within studies.19 results of imputed datasets were pooled using rubin s rules.20 all analyses were carried out according to the intention - to - treat principle . For time - to - event endpoints, effects of self - management were quantified by estimating hazard ratios using cox proportional hazard models, including a frailty term to account for clustering within studies . The continuous outcomes (hrqol and generic ol) were quantified by standardized mean differences (smd) between intervention arms and analyzed using linear mixed effects models . Using the smd, results are converted to a uniform scale representing the intervention effect relative to the observed variability in one study before pooling the results of different studies . Binary outcome data (mortality, respiratory - related, and all - cause hospitalization) were analyzed with log - binomial mixed effects models, which estimated risk ratios (rrs) or odds ratios (ors) in case of nonconvergence of a model, respectively . To correctly model the presence of overdispersion in the count data of total days of hospital stay, all mixed effects models included a random intercept and a random slope for the treatment effect to take clustering at study level into account . To assess whether the effect of self - management was modified by patient characteristics, the aforementioned models were extended with interaction terms for the patient characteristics included in table 1 . The independent variables in the models were random intercept, random slope, allocation to self - management, patient characteristic, and interaction term (treatment allocationpatient characteristic). All effect modifiers with p<0.10 for the interaction (likelihood ratio test) in the univariable analysis were included in a multivariable model to estimate the effect of self - management within subgroups independent of other potential effect modifiers . Effect modification was considered significant if the interaction term showed p<0.05 in the final model . As a sensitivity analysis, we investigated the potential of retrieval bias (ie, bias due to selective inclusion of studies in the ipd meta - analysis) by pooling the published main effects of studies for which ipd were unavailable with the main effects of included studies in a random effects meta - analysis . To assess the impact of studies of lower methodological quality on the main effects, an additional sensitivity analysis was performed, including only studies with a low risk of bias . Three additional sensitivity analyses were performed to assess the robustness of findings from the subgroup analyses: 1) a complete case analysis was carried out to assess the effect of imputing data, and analyses were repeated by 2) excluding older studies (recruitment before 2001) and 3) excluding the largest trial.6 all analyses were performed in r for windows version 3.1.1 (r development core team . Released 2013 . Seven studies were not included in this ipd meta - analysis.9,2126 we could not contact the investigators of three studies;21,23,24 for two studies, the investigators could not obtain approval from their local institutional review board;9,22 the data from one study were no longer available;25 and investigators of one study could not participate due to time constraints.26 the investigators of the other 14 studies participated in this ipd meta - analysis, resulting in the inclusion of data on 3,282 patients . Not all studies measured the same baseline characteristics; only sex, age, and forced expiratory volume in 1 second in% of predicted were assessed in all studies . Patients had a mean age of 68.1 years (9.6) and a mean forced expiratory volume in 1 second in% of predicted of 47.7% (18.9%). Apart from dyspnea classification, all baseline variables were well balanced between control and intervention groups . Table 2 presents the characteristics of included studies.58,2736 seven studies recruited participants in a clinic or hospital setting,57,27,29,30,32 five studies in general practice,8,28,31,34,36 and two in both settings.33,35 the sample size of studies ranged from 5330 to 743 patients.6 self - management interventions varied across studies: a majority included an action plan and consisted of individual sessions with a nurse, and some involved group contacts . Duration of interventions ranged from 1 day31 to 24 months.8 self - management interventions improved hrqol at 12 months (smd 0.08, 95% confidence interval [ci] 0.000.16), but not at 6 months (smd 0.05, 95% ci 0.05 to 0.15) (table 3). The interventions improved time to first respiratory - related hospitalization (hazard ratio 0.79, 95% ci 0.660.94). Although there was no clear effect on respiratory - related hospitalization within 6 months, there was a significant risk reduction at 12 months (rr 0.77, 95% ci 0.640.93). Self - management interventions improved the time to first all - cause hospitalization (hazard ratio 0.80, 95% ci 0.690.90) and risk of hospitalization within 6 months (rr 0.81, 95% ci 0.670.97) and 12 months (rr 0.84, 95% ci 0.730.96). Figure 1 shows the effects across studies for hrqol, respiratory - related and all - cause hospitalization, and mortality . Sensitivity analyses of including the published effects of studies for which no ipd were available resulted in similar effects of the self - management interventions (supplementary material). No effects were observed on the additional outcomes of generic qol or total days in hospital (supplementary material). The final models in the prespecified subgroup analysis revealed no consistent effect modification by any patient characteristic across all relevant outcomes (table 4), but the effect on specific outcomes differed according to some of the patient characteristics we studied . A positive effect of self - management interventions was observed in males (or 0.61, 95% ci 0.410.90) compared to females on the outcome respiratory - related hospitalization within 6 months (or 1.24, 95% ci 0.762.02; interaction p=0.006). Patients with severe airflow limitation showed a reduced risk on all - cause hospitalization within 6 months when allocated to the intervention (rr 0.71, 95% ci 0.580.88), while there was no treatment effect in patients with 50% forced expiratory volume in 1 second in% of predicted (rr 1.02, 95% ci 0.781.34; interaction p=0.016). Obese patients showed the most protective effects of self - management interventions on respiratory - related hospitalization within 6 months (or 0.44, 95% ci 0.270.72; interaction p=0.038) and mortality within 6 months (or 0.35, 95% ci 0.111.10; interaction p=0.026). Finally, patients with baseline moderate self - efficacy scores showed the largest reduction in risk on respiratory - related hospitalization within 12 months (or 0.39, 95% ci 0.210.75) compared to patients with low (or 0.85, 95% ci 0.461.59) or high levels of self - efficacy (or 0.89, 95% ci 0.471.71; interaction p=0.036). Additional analyses for generic qol and total days in hospital did not reveal different insights (supplementary material). Subgroup analysis according to exacerbation frequency was impossible due to too diverse data collection at baseline and comparison of subgroups in individual trials did not reveal consistent effects across studies (supplementary material). Sensitivity analyses to assess the robustness of the subgroup effects yielded similar findings to the primary analysis . Self - management interventions improved hrqol at 12 months (smd 0.08, 95% confidence interval [ci] 0.000.16), but not at 6 months (smd 0.05, 95% ci 0.05 to 0.15) (table 3). The interventions improved time to first respiratory - related hospitalization (hazard ratio 0.79, 95% ci 0.660.94). Although there was no clear effect on respiratory - related hospitalization within 6 months, there was a significant risk reduction at 12 months (rr 0.77, 95% ci 0.640.93). Self - management interventions improved the time to first all - cause hospitalization (hazard ratio 0.80, 95% ci 0.690.90) and risk of hospitalization within 6 months (rr 0.81, 95% ci 0.670.97) and 12 months (rr 0.84, 95% ci 0.730.96). Figure 1 shows the effects across studies for hrqol, respiratory - related and all - cause hospitalization, and mortality . Sensitivity analyses of including the published effects of studies for which no ipd were available resulted in similar effects of the self - management interventions (supplementary material). No effects were observed on the additional outcomes of generic qol or total days in hospital (supplementary material). The final models in the prespecified subgroup analysis revealed no consistent effect modification by any patient characteristic across all relevant outcomes (table 4), but the effect on specific outcomes differed according to some of the patient characteristics we studied . A positive effect of self - management interventions was observed in males (or 0.61, 95% ci 0.410.90) compared to females on the outcome respiratory - related hospitalization within 6 months (or 1.24, 95% ci 0.762.02; interaction p=0.006). Patients with severe airflow limitation showed a reduced risk on all - cause hospitalization within 6 months when allocated to the intervention (rr 0.71, 95% ci 0.580.88), while there was no treatment effect in patients with 50% forced expiratory volume in 1 second in% of predicted (rr 1.02, 95% ci 0.781.34; interaction p=0.016). Obese patients showed the most protective effects of self - management interventions on respiratory - related hospitalization within 6 months (or 0.44, 95% ci 0.270.72; interaction p=0.038) and mortality within 6 months (or 0.35, 95% ci 0.111.10; interaction p=0.026). Finally, patients with baseline moderate self - efficacy scores showed the largest reduction in risk on respiratory - related hospitalization within 12 months (or 0.39, 95% ci 0.210.75) compared to patients with low (or 0.85, 95% ci 0.461.59) or high levels of self - efficacy (or 0.89, 95% ci 0.471.71; interaction p=0.036). Additional analyses for generic qol and total days in hospital did not reveal different insights (supplementary material). Subgroup analysis according to exacerbation frequency was impossible due to too diverse data collection at baseline and comparison of subgroups in individual trials did not reveal consistent effects across studies (supplementary material). Sensitivity analyses to assess the robustness of the subgroup effects yielded similar findings to the primary analysis . This ipd meta - analysis of 14 randomized trials showed that self - management interventions exerted positive effects in copd patients on respiratory - related and all - cause hospitalization . Self - management interventions also resulted in small improvements on hrqol at 12 months, but had no effect on hrqol at 6 months or on mortality . One novel aspect from this study was the prespecified subgroup analyses, which did not show a consistent pattern across health outcomes of subgroups of patients benefiting most from the self - management interventions . The main effects reported by the present study are in line with a recent cochrane review on self - management trials in copd patients.4 like the present study, the authors did not find an effect of self - management on mortality . However, the follow - up period of 12 months may have been too short to elicit an effect on this outcome . Although the cochrane review applied a wider definition of self - management interventions and could include all eligible trials (n=23 vs n=14 in this ipd meta - analysis, respectively), we were able to include more recently conducted studies (n=6) of which some have cast doubts on the usefulness of self - management in copd patients.7,8 by including data from these recent studies as well as performing a sensitivity analysis, including the published results of the prematurely terminated trial,9 the present study provides more extensive evidence that self - management interventions elicit positive effects in copd patients and can be considered safe . However, the positive effects observed for hrqol at 12 months should be considered modest improvements, and no effects were observed at 6 months . It also remains questionable whether the statistical difference that we observed is a clinically important difference for copd patients . Furthermore, it remains questionable whether our findings also apply to copd patients recently discharged from hospital . A recently published systematic review on self - management interventions in this group of patients found that positive effects were limited to hrqol,37 but the authors applied rather wide inclusion criteria for the interventions, resulting in the inclusion of many interventions with only a limited self - management component compared to the present study . The novel aspect of the present study compared to the previously conducted systematic reviews was the prespecified subgroup analysis . This subgroup analysis revealed larger effects of self - management interventions in males, patients with more severe airflow limitation, patients with moderate levels of self - efficacy, and obese patients, but only on some outcomes . To date, differential effects of self - management interventions in subgroups of copd patients have scarcely been examined . One study included in this ipd meta - analysis analyzed response of subgroups of copd patients to the self - management intervention on hospitalization or death.7 the preplanned subgroup analyses did not show any evidence of differential effects, but the authors found that only 42% of intervention group subjects learnt to self the successful self - managing patients had significantly reduced hospitalization rate.7 the present ipd meta - analysis, with more power to perform subgroup analyses, suggested larger effects of self - management interventions on respiratory - related hospitalization as well as mortality at 6 months in obese patients . Although effect modification by body mass index has not yet been analyzed in copd patients in the context of self - management interventions, evidence is starting to emerge that overweight or obese patients encompass a specific phenotype of copd patients.38 it is possible that this particular phenotype of copd patients responds differently to self - management interventions . Our analyses only revealed differential effects of obesity on the outcomes respiratory - related hospitalization at 6 months and mortality at 6 months . Effects at 12 months were in a similar direction, but these were not statistically significant . Previous efforts to assess the influence of body mass index on effectiveness of pulmonary rehabilitation have also yielded inconsistent results.39,40 although our subgroup analysis was prespecified13 and yielded several statistically significant findings, the high number of analyses increases the chance of false - positive findings . With no consistent pattern across multiple health outcomes, the subgroup results should be interpreted with caution.41 limiting self - management support to specific patient subgroups cannot be recommended at this stage and further research is therefore needed to confirm the observed subgroup effects for other health outcomes . Reassuringly, there were no indications in our analyses that certain subgroups of patients responded in a negative way to the self - management interventions . To our knowledge, the present study is the first to pool and reanalyze the original data of a large number of randomized trials on self - management interventions in patients with copd and transcends the previously conducted systematic reviews.4,37 an ipd meta - analysis is a resource intensive approach, given the time and efforts needed for collecting and merging the raw patient data.42 as a result, no articles published after june 2013 were included for analysis . The high response rate of principal investigators (66.7%), large number of patients included (n=3,282), prespecified statistical plan, and close collaboration with the principal investigators through regular teleconferences contribute to the robustness of our findings . First, in spite of numerous efforts to contact and convince the principal investigators of all eligible studies, we could not obtain the data of seven randomized trials, including the prematurely aborted trial.9 however, the sensitivity analysis of pooling the published results of those trials with the main effects of included studies showed that this did not alter our findings (supplementary material). Second, we assumed all interventions to be homogeneous self - management interventions in our analyses, but the included self - management intervention designs differed from each other in terms of dose, mode, and content . Without consistent evidence for subgroups of patients benefiting across various health outcomes, we could hypothesize that specific subgroups of patients only respond better to particular components of interventions (ie, action plans in self - management interventions). Future research addressing various interventions is needed to identify what type of intervention works for whom . Nevertheless, the reported main effects on hrqol at 12 months, and respiratory - related and all - cause hospitalization were consistent across cultures and health care settings . This indicates that, despite their diversity, self - management interventions exert positive effects, even in different formats and differing patient populations . Third, this ipd meta - analysis was highly dependent on data that were previously collected . Exacerbation frequency has attracted considerable attention in recent years,43 but due to the diverse data collection across studies, the quality of available data on baseline exacerbation rate was too low to enable a pooled analysis of this patient characteristic . This emphasizes the urgent need for a uniform operational definition of exacerbations within the field of copd research.44 for similar reasons, we could not study other potentially relevant variables, such as global initiative for chronic obstructive lung disease stage, coping style, disease perception, and adherence . Previous studies have shown that even though adherence to self - management treatment is a challenge for a majority of patients enrolled in randomized trials, the patients who actually applied those new self - management skills showed better outcomes.7,45 this suggests that emphasis should be placed on patients ability to apply self - management guidelines and subsequently change their behavior as this is a prerequisite for better outcomes . Collection of data on intervention delivery, treatment adherence, and behavior change in randomized trials, particularly on complex interventions, such as self - management, is indispensable to identify patients most likely to adhere to the self - management interventions and in whom these interventions may improve prognosis.46 self - management interventions exert positive effects in patients with copd on respiratory - related and all - cause hospitalization and modest improvement of hrqol at 12 months, but do not show an effect on mortality . These benefits seem similar across the subgroups of patients studied as subgroup analysis did not reveal a consistent pattern across different health outcomes . Our findings support implementation of self - management strategies in practice, but targeting self - management interventions at specific subgroups of patients cannot be recommended based on the current evidence.
This question is still unanswered in terms of the use of ddt (dichlorodiphenyltrichloroethane), a persistent organic pollutant used for malaria control in many parts of africa . In zambia, malaria is still the leading cause of morbidity and mortality accounting for 36% of all hospital admissions with the majority being pregnant women and children . In response to this high malaria burden, the government of zambia through the ministry of health developed an integrated vector management (ivm) strategy [1, 2]. This strategy focused on increasing coverage of indoor residual spraying (irs) activities in addition to distribution of insecticide - treated nets (itns), expansion of environmental management, and larviciding . In 2000, a private mining company, konkola copper mines, was the first to reintroduce ddt as one of its main chemicals for irs after more than two decades since its use had been discontinued in zambia . This followed the total ban of ddt in the united states of america and other developed countries in the early 1970s due to its negative environmental effects . The apparent success of this programme in one mining town and the fact that irs with ddt was the principal method by which malaria was eradicated or in some cases significantly reduced in many countries in the world led the zambian government in 2003 to initiate irs activities initially in five pilot districts [1, 5]. Several world health organisation- (who-) endorsed insecticides, including ddt, which was applied only to unplastered surfaces, were used for this activity . Several studies have shown that ddt causes thinning of bird egg shells and finds its way into the food chain if improperly handled [68]. It is an endocrine disruptor and has been shown to cause other human health effects ranging from reproductive failure to increased incidences of different cancers [4, 925]. Its main action is through the disruption of the neurological functions of the brain by disturbing neurotransmitters, mimicking th4, a thyroid hormone, and disturbing the internal signalling system of synapses [21, 2535]. Locally, 239,758 kg of ddt has been used in zambia since 2000 with no documented studies being conducted on environmental and human exposures . Previous studies on ddt exposure in other settings involved historically exposed communities and looked mainly at its metabolites dde and ddd . The few studies done in south africa focused on reproductive outcomes in occupationally exposed males and some in women [9, 15]. Given the potential harm that ddt has been shown to cause in various locations around the world, coupled with the hiv pandemic and poverty situation in zambia, it is highly likely that these effects will be more pronounced in the zambian population . So far, no study has been carried out to ascertain the fate of the recently applied ddt in both the environment and human health . To this effect, we aimed to carry out an exposure assessment in selected areas of zambia in order to quantify the prevalence of ddt and its metabolites in soil and water around communities where it was recently used . This work is part of a broader research project which aims to link recent ddt exposure to neurodevelopmental outcomes in children of the targeted populations and also evaluate other factors that may be associated with the observed outcomes . The study areas comprised chawama, chongwe, and mongu, selected based on history of past exposure to ddt in the last three years . Chawama was randomly selected for this study as it was one of the pilot areas where ddt was applied in 2004 and has a very high malaria burden compared to the rest of lusaka . It is a periurban area in the center of lusaka situated at 15.35 south latitude, 28.7 east longitude, and 1069 meters above the sea level . Chongwe, on the other hand, is a rural town east of lusaka situated at 15.35 south latitude, 28.7 east longitude, and 1069 meters above the sea level . It was also randomly selected among among two other areas, kafue and mumbwa, which are rural districts within a 200 km radius to lusaka, as it was one of the areas where ddt was applied commencing in 2008 . In contrast, mongu is a town with a mixed urban and rural population located west of lusaka situated at 15.25 south latitude, 23.13 east longitude, and 1018 meters above the sea level . It was conveniently selected as the control area because there is no report of ddt application as part of government irs activities . Mongu was selected as the reference area while chawama and chongwe are the areas where ddt was applied . The sampling for soil and water in all the study areas was done during july 2012 . The inclusion criterion for the areas with recent application was that the residences should have been sprayed at least three times with ddt in the last 10 years . A total of 14 soil samples were collected in chongwe (3), chawama (7), and mongu (4). In order to discern recent exposure characteristics, samples were obtained from the top soil at a depth of 25 cm using bucket and tube augers depending on the type of soil present in the area . At each sampling point, 500 g of the soil sample was collected and placed in a nontransparent paper bag which was put into a plastic bag to avoid leakage . In order to ensure representativeness of the soil samples in the study areas, a zigzag sampling pattern was employed covering the entire sampling area in each community as illustrated in figure 1 . A total of 14 water samples were collected in chongwe (3), chawama (7), and mongu (4), respectively . The samples were obtained from various drinking water sources such as shallow wells, open streams, and communal taps . These samples were obtained using 1-litre water sample bottles which had been triple - rinsed with wash grade acetone / hexane mixture . The bottles had tightly fitted caps and lids to avoid spillages and enough space was allowed at the neck of the bottle to facilitate air exchange . The samples were transported to the mass spectrometry unit in the department of chemistry at the university of botswana under the united nations transport of dangerous goods (untdg) regulations of 2011 where they were kept in a cold room at ~4c until they were extracted and analysed . A slightly modified aoac method 2007.01 adapted by nagel [39, 40] using twenty (20) grams of soil was weighed into a centrifuge tube, and 12 ml of water was added and shaken for 4 hours after which 20 ml of acetonitrile was added for homogenization . The supernatant was thereafter quantitatively transferred to a second centrifuge tube containing 6 g mgso4, 1.5 g nacl, 1.5 g na3citrate dihydrate, and 750 mg na2hcitrate sesquihydrate . Sample cleanup was achieved by using dispersive solid phase extraction using cartridges that contained 150 mg primary secondary amine (psa), 400 mg c18ec, and 900 mg magnesium sulfate of between 98.5 and 101.5% purity . In each extraction, 6 ml of supernatant was transferred into an spe cartridge and shaken for 1 minute before centrifugation for 2 minutes at 4500 rpm . The organic layer was filtered using a syringe and a membrane filter (0.45 mm) and then evaporated under nitrogen to 1 ml . The concentrate was transferred into a 2 mlgc vial and was ready for gas chromatography - mass spectrometry (gc - ms) analysis . The standard solid phase extraction (spe) method of conditioning, loading, washing, and elution was used for the extraction of ddt from the water samples . This was achieved using florisil spe cartridges and an agilent vacuum manifold (vacelut sps 24). The procedure was a slight modification of a method developed in the laboratory for the determination of pcbs in transformer oil . Eight (8) millilitres of hexane was added to condition the cartridge followed by 8 ml of deionised water at 1 ml / min . This was followed by loading 20 ml of the water sample at a flow rate of 0.5 ml / min . The cartridge was allowed to dry for another 20 minutes to allow for interaction of the ddt with the stationary phase . Five (5) millilitres of hexane was eluted through the cartridge to wash off any nonpolar analytes that could have bound strongly to the stationary phase . Elution of the ddt was accomplished by running 10 ml of a 5: 5 n - hexane: acetone mixture . The resulting eluate was blown gently under a stream of nitrogen to concentrate it to 0.5 ml and transferred to gc vial for gc - ms analysis . An agilent (palo alto, ca, usa) 5975 c series gas chromatograph - quadrupole mass spectrometer (gc - qms) system equipped with a selective mass detector was used for analysis . The system has capability to perform ion manipulation in the full scan and selected ion monitoring (sim) modes . It also has two ionization modes, that is, electron ionization (ei) and chemical ionization (ci). In this study, helium was used throughout as a carrier gas at a flow rate of 1 ml / min . The oven program for the gc was as follows: the column was held at 80c for 2 min and then ramped at 10c / min up to 270c for a total run time of approximately 30 minutes . The mass spectrometer was operated in the negative chemical ionization (nci) mode for soil analysis while the electron ionization (ei) mode was used for water sample extracts . The automated mass spectral deconvolution and identification system (amdis) by the national institute for standards and technology (nist) in conjunction with microsoft excel was used for data analysis . Extraction efficiencies were determined by analysis of spiked sample extracts with the analytes of interest . For quality control and assurance, procedural blanks were extracted with n - hexane and subjected to the same extraction procedure as the other samples . Descriptive statistics were used to describe data and the term tddt was used to refer to the sum of all ddt and its metabolites dde and ddd . In the presentation of the results, however, a distinction was made as to which ddt metabolite was being referred to . Written permits were acquired from the ministry of mines and mineral resources to export the soils and water into botswana for analysis . A summary of validation results derived from 6 point calibration curves with corresponding regression equations is presented in table 1 . From table 1 low detection limits were also estimated in both soil and water samples and when these are compared to the 1 g / l which is the world health organization (who) maximum recommended limit (mrl) for ddt and its derivatives in drinking water, they were found to be low enough for analysis . Results of concentrations of ddt and its metabolites in the study areas are summarized in figure 2 . Figure 2 shows that all analytes were present in all the soils of chawama and chongwe . However, they were all found to be below the lods in mongu which was our reference area . The concentrations varied in all the study areas but the highest amounts of ddt at 25.8 ng / g were determined around a rural homestead of chongwe followed by 25.7 ng / g in soils collected very close to the exterior walls of a house in chawama township . Ddt accounted for 12% of the tddt in the soils sampled . In some areas, no ddt above the limits of detection was detected in water samples from mongu, which was the reference sampling site . In general, the lowest amount of ddt accounted for 4% of the tddt while the highest concentration at 511 ng / g was detected around a pit latrine in chawama township translating into 75% of tddt . Ddt levels were found in significant levels in both water and soils in the study areas of zambia . These high levels were most prominent in sites with a history of ddt exposure through the irs program for malaria control . No ddt above the limits of detection was found in the reference area . In soil from the two zambian study areas, the median concentrations were twice or higher than those reported in spain, uganda, and south africa among selected countries where they were 46, 59, and 43 ng / g, respectively [4244]. It is possible that there might be inherent sampling errors and that, coincidentally, both sampled sites with recent exposure could have higher ddt concentrations as found in this study . However, given that every effort was made to randomly select the locations and sampling sites, it is unlikely that these sampling errors could be important enough to explain these results . We therefore reasonably argue that what was found in this exposure assessment could be from recent exposure . Ddt has not been sprayed in zambia since 2010 when evidence of mosquito resistance to it began to emerge . Ddt and its metabolites can persist in soil for 215 years and therefore the period of recent exposure still falls within the half - life brackets for ddt . The soil sampling protocol restricted the depth at which the soil samples were collected to the a and e horizons which are generally about 0.64.5 m below the surface and typically lose minerals and chemicals due to leaching over time . Showed that ddt and other pesticides metabolize faster at varying rates depending on the soil type due to environmental factors such as prevailing climatic conditions, ph of the soil, and the action of microorganisms . It has been shown to degrade even faster in temperate climates such as the one found in zambia . Ddt has a much longer half - life of up to 150 years in water, and due to its lipophilic nature it tends to gravitate towards organic material and other such fatty tissues . Its very high concentrations in the water bodies where the samples were collected were a surprising and alarming result . The median concentrations of ddt were found to be more than two hundred times higher than those recorded in nigeria and south africa at less than 0.368 and 2 this is against the background in which the who has recommended a maximum of 1 g / l per 0.01 mg / kg of body weight calculated at the assumption that a 10 kg child drinks up to 1 litre per day . These results corroborate the findings from the 2010 environmental council of zambia (ecz) audit of irs activities which showed lapses in the implementation of environmental safeguards during the spraying exercises . No ddt and its metabolites above detectable limits were detected in the reference area in both soil and water samples . Mongu was not included in the irs program due to its proximity to the zambezi river, a source of livelihood and nutrition for the local communities . Despite the historical application of ddt in the 1940s to the 1980s for other purposes in mongu, the sampling of only elluvium a top soil could have masked residues which most likely may be present in the lower strata . Given the rapid velocity of the water in the zambezi river and the high water table in mongu, it is highly unlikely that ddt could have remained in the aquatic system . This coupled with the presence of many aquatic species and other organic materials in the water bodies in this area could have resulted in the ddt sequestering itself in them due to its lipophilicity . These results are clinically significant given the bioaccumulation and biomagnification characteristics of ddt as it travels up the food chain [7, 8, 47, 48]. Several studies in various settings of the world have shown that ddt in plants is taken up through the roots and when these plants are consumed by both humans and animals, the ddt remains sequestered in these species' adipose tissue . This is also true when ddt sequesters itself in aquatic species which are edible to humans and animals . These studies also show that the primary exposure route for humans to ddt is through ingestion of contaminated foods and water . The high tddt concentrations found in the study sites and subsequent ddt contamination burden may be an indication of challenges associated with environmental monitoring of such pollutants especially in resource poor settings already plagued with high malaria incidences among other public health challenges (table 2). Given that these highly exposed areas are also often inhabited by very poor populations most of whom are women and children, this poses an ethical dilemma to decision makers on the cost - effectiveness of reintroducing ddt . Speculations were raised by several scholars on the effectiveness of the reintroduction of ddt for malaria control . This is based on results of studies such as this one and that conducted in south africa also which showed that ddt had contaminated the soils, water, and livestock of previous irs communities . The presence of ddt and its metabolites in environmental samples from soils and water of selected study areas has been demonstrated . Given that the breakdown products dde and ddd are more stable in the environment and human matrices and have been implicated in dire effects, urgent action is required . This calls for more investment in surveillance and environmental monitoring in order to develop effective remediation solutions that will rapidly break down this ddt and thereby remove it from the food chain . Furthermore, the cradle - to - grave principle of waste management must be applied to this dilemma as the cost of ddt reintroduction is currently being borne by the public and given the persistent nature of ddt even unborn children will suffer the consequences of this lapse in environmental stewardship . Hard choices driven by appropriate leadership may have to be made which favour a win - win situation for current and future generations.
The most common ocular adverse event following the use of cyclosporine a (csa) 0.05% ophthalmic emulsion is ocular burning (17%). Other adverse effects that have been reported include conjunctival hyperemia (1 - 5%), discharge, epiphora, eye pain, foreign body sensation, pruritus, stinging and blurred vision . Here, we report a specific side effect of csa, namely eye drop - induced eyelash elongation in a patient with refractory giant papillary conjunctivitis . A 32-year - old female with giant papillary conjunctivitis on the left eye, who had undergone papillectomy 3 years previously and was refractory to topical steroid therapy, was treated with csa 0.05% ophthalmic emulsion (restasis) 4 times a day, preservative - frees artificial tears and gentamicin ophthalmic solution in the left eye . After 5 months of topical csa treatment, elongated eyelashes of her left eye were observed without other adverse effects . Although hypertrichosis and trichomegaly have been documented in the literature as side effects of systemic csa, topical csa 0.05% eye drop - induced elongated eyelashes have not been reported, and we believe ophthalmologists should be mindful and inform patients about this specific side effect . A-32-year - old asian female patient, who had received conjunctival papillectomy on the left eye in 2002, visited our ophthalmic outpatient department in 2007 complaining of a swollen eyelid and discharge from her left eye . The ocular examination revealed notable injected conjunctiva, especially over the superior part, and multiple papillae with injected and engorged vessels on the left eye (fig ., she was started on topical steroid medications including fluorometholone 0.1% and prednisolone acetate 1% ophthalmic solutions for 2 months . However, steroid treatment was discontinued due to poor response and an elevated intraocular pressure . Therefore, her treatment was switched to topical cyclosporine a (csa) 0.05% ophthalmic emulsion (restasis) 4 times a day, preservative - free artificial tears and gentamicin ophthalmic solution . After 5 months of topical csa treatment, she came back to our clinic complaining of elongated and darkened eyelashes on her left eye without other adverse effects (fig . 2). According to the patient, no systemic medications such as calcium channel blockers, erythropoietin or minoxidil were used during the treatment period . Csa is a hydrophobic, cyclic polypeptide produced as a metabolite by the fungus tolypocladium inflatum . Csa functions as an immunomodulating agent that binds to cyclophilin, a cytoplasmic protein, thus interrupting the signaling for interleukin (il]-2 production, in addition to inhibiting the proliferation of cd4 t lymphocytes . It also has direct inhibitory effects on both eosinophil and mast cell activation, which has established its role in the treatment of allergic inflammation [2, 3]. In the early 1980s, meanwhile, csa eye drops were also prescribed for patients with inflammatory ocular surface disorders, particularly dry eye syndrome and severe allergic keratoconjunctivitis . The major side effect of systemic csa is nephrotoxicity that is reversible with dosage reduction . Other documented adverse reactions to systemic csa include mild hepatotoxicity, hypertension, dose - dependent hypertrichosis and trichomegaly, tremor, infection, gum hyperplasia, gastric irritation symptoms and neuropathies . On the other hand, adverse events following the use of csa 0.05% ophthalmic emulsion include ocular burning (17%], conjunctival hyperemia (1 - 5%], discharge, epiphora, eye pain, foreign body sensation, pruritus, stinging and blurred vision . However, to our knowledge, no csa ophthalmic emulsion - induced hypertrichosis has been reported in the literature to date . Suggested that csa induces telogen follicles to enter an anagen growth phase, implying a role of csa in regulating the hair follicle immune system and its cellular components through the release of inhibitory / stimulatory cytokines [8, 9]. The experiments also indicated that the rate of anagen induction is dependent on the dose, time course, and method of administration . In summary, although rarely encountered, csa 0.05% ophthalmic emulsion may induce the growth of eyelashes, and we believe ophthalmologist should be mindful and inform patients treated with topical csa about this side effect.
During the past two decades, medical research has provided substantial evidence supporting the hypothesis that length of gestation and birth weight affect infant mortality and childhood morbidity (gortmaker, 1979; showstack, budetti, and minkler, 1984). Infants weighing less than 2,500 grams (or 5.5 pounds) have a mortality rate that is 40 times greater during the neonatal period than infants weighing more than 2,500 grams (mccormick, 1991). Not only do infants weighing less than 750 grams have lower survival rates, but they have an increased risk of serious neurologic and developmental impairment (hack and fanaroff, 1989). Despite the importance of birth weight in birth outcome, the primary cause of perinatal mortality in the united states is preterm birth (kleinman and madans, 1991). Although this is an issue of individual medical importance, it is also a matter of national policy concern . Even though infant mortality rates by birth - weight category in the united states are among the lowest in the developed world, the overall infant mortality rates are among the highest . This statistical anomaly is because of the higher rates of preterm infants born in the low - birth - weight categories (behrman, 1987). Perhaps even more troubling joyce (1990) estimated that by 1990 the percentage of low - weight births among black females in new york city would exceed the rates of 20 years earlier, with most of the increase in the late 1980s . Although data limitations make conclusions tentative, joyce offered the increase in substance abuse, particularly cocaine and crack, as the most likely cause of the increased incidence of low birth weight . The challenge to medical practitioners is to develop programs that reduce the incidence of preterm delivery and low birth weight, especially among females of lower socioeconomic status, both white and black . Evidence seems to indicate that a comprehensive prenatal care program focusing on prematurity prevention may be able to reduce the incidence of low birth weight among females of all ages (buescher et al ., 1988). In fact, early prenatal care (beginning in the first trimester) among white teenagers has been shown to be associated with a 27-percent reduction in low - weight births (frank et al ., 1989). Although the association between prenatal care and birth outcome is indisputable, there is still no clear cut causal relationship between the two . Although proponents of prenatal care programs stress the potential cost savings, estimates vary widely depending on the population studied and the methodology used . Murray and bernfield (1988) have estimated that the annual cost savings of adequate prenatal care is approximately $230 per mother (1986 dollars). This includes the cost savings from neonatal intensive care and rehospitalization within the first year . Monmaney (1988) reported that a virginia program, if adopted statewide, could save the state almost $50 million annually by reducing the incidence of certain types of mental retardation due to low birth weight . If this program were adopted nationally, it would save between $14,000 and $30,000 for every low - birth - weight baby avoided . Lifetime and aggregate estimates of savings tell an even more dramatic story . The national commission to prevent infant mortality (1991) has estimated the cost of lifetime custodial care of low - birth - weight babies to be as much as $500,000 per child . Additionally, this report estimated that 80 percent of the females at high risk for low - birth - weight babies can be identified in the first prenatal visit . The congressional office of technology assessment (1987) has estimated the cost of caring for babies who weigh less than 1,140 grams (2.5 pounds) at birth to be an average $140,000 per patient, bringing the annual cost of neonatal intensive care in the united states to a total of $1.5 billion . A survey conducted by the institute of medicine and reported by droste (1988) estimated that for every dollar spent on prenatal care, $3.38 is saved in the cost of caring for low - birth - weight infants . Despite the evidence that high quality prenatal care is associated with improved pregnancy outcomes (and lower overall costs), only 76 percent of all pregnant females receive care in their first trimester . For black and hispanic females, the corresponding figure is 61 percent (health resources and services administration, 1991). If the cost savings have not been overstated, utilization of prenatal care programs appears to be at suboptimal levels . Previous research into the cost effectiveness of prenatal care has been limited because of the lack of individual cost data . Most of the studies previously cited use birth certificate data to examine the relationship between prenatal care and birth outcome, and payment rate schedules to estimate cost savings . This study develops a simple model of birth outcome measured by the infant's birth weight . From this model, the hospital cost differential between females who received prenatal care and those who did not is estimated . An estimate of the cost differential can be more accurate than before because of the availability of a detailed microdata base that contains individual observations on birth outcome and hospital costs incurred . Data for this study were provided by hillcrest baptist medical center and scott and white hospital . More than 7,000 records for infants and mothers were obtained, representing virtually all births in mclennan county from june 1987 through july 1989 . The procedure for matching babies to mothers resulted in the loss of fewer than 20 records for the period under study . For each record, the relevant demographic data, including age, race, marital status, and zip code, were obtained . Actual hospital procedures were also recorded, controlling for cesarean delivery, premature labor, and whether the infant died or was discharged to the home or to another hospital with a neonatal intensive care unit (i.e., scott and white hospital in temple, texas). Finally, mothers who did not receive prenatal care were identified from a survey conducted by the nursing staff of the hospital nursery at the time of admittance into labor and delivery . Prenatal care is described as any type of medical care received by a prospective mother, such as physician visits or any organized prenatal program provided by a medical practitioner . Mean birth weight for mclennan county babies was 3,365 grams (7.4 pounds), compared with that of the california study cohort reported by showstack, budetti, and minkler (1984) of 3,388 grams (also 7.4 pounds). Factors important in determining birth outcome (kessel et al ., 1984) are: the ethnic group and marital status of the mother (60.6 percent white and 70.1 percent married); the percentage of the population in the high - risk age groups (13.7 percent are either under age 18 or over 34 years of age); the type of delivery (20.7 percent cesarean); the percentage premature (4.9 percent); the percentage of multiple births (6.2 percent); and the percentage of females who received no prenatal care (5.4 percent). The data reveal that non - white infants are smaller (3,265 grams versus 3,430 grams), and non - white mothers are younger (23.3 years versus 25.8 years of age) and more likely to be unmarried (52.4 percent versus 15.2 percent). The first task was to examine the relationship between prenatal care and birth outcome . The mean birth weight for babies whose mothers received prenatal care was 3,380 grams (7.4 pounds). Those babies whose mothers received no prenatal care weighed an average of 3,100 grams (6.8 pounds). These mean birth - weight differences remain when the data are divided according to race and marital status . Table 2 presents the characteristics according to race and marital status . In all eight categories, the differences ranged from 105 grams for non - white married females to 379 grams for white single females . The distribution of birth weights shows the same basic pattern: females with prenatal care are more likely to give birth to babies weighing more than 2,500 grams and less likely to have babies weighing less than 1,500 grams . The odds of having a low - birth - weight baby are substantially higher for females who do not receive prenatal care . Using the approach suggested by wartenberg and northridge (1991) for calculating an odds ratio, females who receive no prenatal care are 2.68 times more likely to give birth to a low - birth - weight infant (one weighing less than 2,500 grams) than females who receive at least some care . In fact, white females increase their risk of having a low - birth - weight infant 3.92 times by failing to obtain prenatal care; the increase for non - white females is only 1.85 times . Several confounding factors may contribute to it, including intracategory differences in socioeconomic status, alcohol and cigarette use, and drug abuse . The small sample sizes for the no - care groups may also play a role . At any rate, there is no way to know for sure because data on these variables were not collected . Another observation worth noting is the apparent association between prenatal care and the likelihood of cesarean delivery . Does prenatal care increase the odds of having a cesarean section, or is some other mechanism at work? The high incidence and related causes of cesarean deliveries have been the object of considerable medical research (taffel, placek, and liss, 1987; myers and gleicher, 1988). It is unlikely that females who receive prenatal care have a higher incidence of factors that are the primary indicators for cesarean section (i.e., previous cesarean section, dystocia, breech presentation). One avenue worth future exploration is the impact of defensive practices by caregivers to avoid possible malpractice lawsuits . Females who received prenatal care had fewer babies transferred to acute - care facilities, fewer infant deaths, and a higher incidence of cesarean deliveries . Although this does not rule out intrinsic differences between females who receive and those who do not receive prenatal care, it does demonstrate a clear association between prenatal care and birth outcome within narrowly defined demographic cohorts . Because other factors also contribute to differences in birth weights, ols regression was used to adjust for the following characteristics . Table 3 presents the regression results of birth weight (measured in ounces) on these explanatory variables . The data suggest that the lack of prenatal care has a negative effect on birth outcome . Even after adjusting for the other independent variables, babies born to mothers who received no prenatal care weighed about 145 grams (5.09 ounces) less than those whose mothers received prenatal care . Increased maternal age is associated with bigger babies . For each additional year of the mother's age at delivery, the baby's weight increases by 6 grams (0.20 ounces). The use of age categories, though not reported in table 3, displays a similar pattern . Females who are under 18 years of age give birth to babies who weigh an average of 60 grams (2.2 ounces) less than those of females between 18 and 34 years of age . The age coefficient for females more than 35 years of age is insignificant, indicating that the relationship between age and birth weight is likely to be non - linear . Age may serve, in part, as a proxy for birth order, with a higher incidence of first births (and thus smaller babies) to those in their early teens . For example, it is well documented that single females have a higher incidence of cigarette smoking than married females . Multiple births reduce birth weight by 659 grams (23.06 ounces) and premature delivery is associated with birth weights that are 943 grams (33.02 ounces) lower . After adjusting for all these characteristics, non - white females still give birth to babies who weigh 80 grams (2.79 ounces) less than white females . Additionally, when the population is divided into white and non - white cohorts, the regression results are quite similar . The impact of multiple births is more pronounced on white than non - white babies . Birth weights are 740 grams (26 ounces) lower for white multiple births and only 456 grams (16 ounces) lower for non - white multiple births . White premature babies weigh 884 grams (31 ounces) less than those born at term; non - white premature babies weigh 1,026 grams (36 ounces) less than those born at term . One of the more interesting differences is the impact of prenatal care between the two groups . White females who receive prenatal care give birth to babies who weigh 326 grams (11.49 ounces) more than those who do not . The effect of prenatal care on non - white birth outcome is much less pronounced . Non - white females who receive prenatal care give birth to babies who weigh about 172 grams (6.05 ounces) more . As previously stated, prematurity and its resulting low birth weights are major contributing factors leading to complications that result in higher costs, such as transfers to intensive - care unit (icu) facilities . There was a much higher incidence of prematurity, low birth weights, and transfers to acute - care facilities among females who did not receive prenatal care . Only 4.60 percent of the females who received prenatal care experienced premature labor, whereas 9.50 percent of those who did not receive prenatal care delivered prematurely . Transfers to acute - care facilities involved 0.71 percent of the babies whose mothers received prenatal care, and 1.85 percent of those whose mothers did not receive prenatal care . Infant mortality was more pronounced among mothers who did not receive prenatal care; 2.11 percent of their babies died in the hospital, compared with 0.38 percent of those babies born to mothers who received prenatal care . Hospital charges for infants with prenatal care are on average $1,198.42 less than those without prenatal care ($1,045.69 versus $2,244.11). The regression equation for hospital charges was estimated using birth weight (equation 2.1) as an independent variable, and birth - weight categories (equation 2.2). Three birth - weight categories were defined: bwt1 for normal birth weights greater than 2,500 grams, bwt2 for low birth weights from 1,500 to 2,500 grams, and bwt3 for very low birth weights less than 1,500 grams . As expected, birth weight and hospital charges are negatively associated . The hospital charge for the infant the use of birth - weight categories in estimating equation 2.2 shows a somewhat different perspective on this relationship . Other things equal, coefficient estimates indicate that infants in the bwt2 category (from 1,500 to 2,500 grams) had charges that were $1,065.41 lower than normal - birth - weight infants (more than 2,500 grams). This may be because of the large proportion of infants in this category that can be classified small - for - term, weighing between 2,240 and 2,500 grams . The added expense for very low - birth - weight infants (less than 1,500 grams) was $13,638.32 because of the medical complications evident in extremely low - birth - weight infants . Infants who were transferred had charges that were more than $48,091 higher than those who were not . Goodness of fit as measured by r is greater than .23, depending on the specification of the equation . Mean birth weight for mclennan county babies was 3,365 grams (7.4 pounds), compared with that of the california study cohort reported by showstack, budetti, and minkler (1984) of 3,388 grams (also 7.4 pounds). Factors important in determining birth outcome (kessel et al ., 1984) are: the ethnic group and marital status of the mother (60.6 percent white and 70.1 percent married); the percentage of the population in the high - risk age groups (13.7 percent are either under age 18 or over 34 years of age); the type of delivery (20.7 percent cesarean); the percentage premature (4.9 percent); the percentage of multiple births (6.2 percent); and the percentage of females who received no prenatal care (5.4 percent). The data reveal that non - white infants are smaller (3,265 grams versus 3,430 grams), and non - white mothers are younger (23.3 years versus 25.8 years of age) and more likely to be unmarried (52.4 percent versus 15.2 percent). The mean birth weight for babies whose mothers received prenatal care was 3,380 grams (7.4 pounds). Those babies whose mothers received no prenatal care weighed an average of 3,100 grams (6.8 pounds). These mean birth - weight differences remain when the data are divided according to race and marital status . Table 2 presents the characteristics according to race and marital status . In all eight categories, the differences ranged from 105 grams for non - white married females to 379 grams for white single females . The distribution of birth weights shows the same basic pattern: females with prenatal care are more likely to give birth to babies weighing more than 2,500 grams and less likely to have babies weighing less than 1,500 grams . The odds of having a low - birth - weight baby are substantially higher for females who do not receive prenatal care . Using the approach suggested by wartenberg and northridge (1991) for calculating an odds ratio, females who receive no prenatal care are 2.68 times more likely to give birth to a low - birth - weight infant (one weighing less than 2,500 grams) than females who receive at least some care . In fact, white females increase their risk of having a low - birth - weight infant 3.92 times by failing to obtain prenatal care; the increase for non - white females is only 1.85 times . Several confounding factors may contribute to it, including intracategory differences in socioeconomic status, alcohol and cigarette use, and drug abuse . The small sample sizes for the no - care groups may also play a role . At any rate, there is no way to know for sure because data on these variables were not collected . Another observation worth noting is the apparent association between prenatal care and the likelihood of cesarean delivery . Does prenatal care increase the odds of having a cesarean section, or is some other mechanism at work? The high incidence and related causes of cesarean deliveries have been the object of considerable medical research (taffel, placek, and liss, 1987; myers and gleicher, 1988). It is unlikely that females who receive prenatal care have a higher incidence of factors that are the primary indicators for cesarean section (i.e., previous cesarean section, dystocia, breech presentation). One avenue worth future exploration is the impact of defensive practices by caregivers to avoid possible malpractice lawsuits . Females who received prenatal care had fewer babies transferred to acute - care facilities, fewer infant deaths, and a higher incidence of cesarean deliveries . Although this does not rule out intrinsic differences between females who receive and those who do not receive prenatal care, it does demonstrate a clear association between prenatal care and birth outcome within narrowly defined demographic cohorts . Because other factors also contribute to differences in birth weights, ols regression was used to adjust for the following characteristics . Table 3 presents the regression results of birth weight (measured in ounces) on these explanatory variables . The data suggest that the lack of prenatal care has a negative effect on birth outcome . Even after adjusting for the other independent variables, babies born to mothers who received no prenatal care weighed about 145 grams (5.09 ounces) less than those whose mothers received prenatal care . Increased maternal age is associated with bigger babies . For each additional year of the mother's age at delivery, the baby's weight increases by 6 grams (0.20 ounces). The use of age categories, though not reported in table 3, displays a similar pattern . Females who are under 18 years of age give birth to babies who weigh an average of 60 grams (2.2 ounces) less than those of females between 18 and 34 years of age . The age coefficient for females more than 35 years of age is insignificant, indicating that the relationship between age and birth weight is likely to be non - linear . Age may serve, in part, as a proxy for birth order, with a higher incidence of first births (and thus smaller babies) to those in their early teens . For example, it is well documented that single females have a higher incidence of cigarette smoking than married females . Multiple births reduce birth weight by 659 grams (23.06 ounces) and premature delivery is associated with birth weights that are 943 grams (33.02 ounces) lower . After adjusting for all these characteristics, non - white females still give birth to babies who weigh 80 grams (2.79 ounces) less than white females . Additionally, when the population is divided into white and non - white cohorts, the regression results are quite similar . The impact of multiple births is more pronounced on white than non - white babies . Birth weights are 740 grams (26 ounces) lower for white multiple births and only 456 grams (16 ounces) lower for non - white multiple births . White premature babies weigh 884 grams (31 ounces) less than those born at term; non - white premature babies weigh 1,026 grams (36 ounces) less than those born at term . One of the more interesting differences is the impact of prenatal care between the two groups . White females who receive prenatal care give birth to babies who weigh 326 grams (11.49 ounces) more than those who do not . The effect of prenatal care on non - white birth outcome is much less pronounced . Non - white females who receive prenatal care give birth to babies who weigh about 172 grams (6.05 ounces) more . As previously stated, prematurity and its resulting low birth weights are major contributing factors leading to complications that result in higher costs, such as transfers to intensive - care unit (icu) facilities . There was a much higher incidence of prematurity, low birth weights, and transfers to acute - care facilities among females who did not receive prenatal care . Only 4.60 percent of the females who received prenatal care experienced premature labor, whereas 9.50 percent of those who did not receive prenatal care delivered prematurely . Transfers to acute - care facilities involved 0.71 percent of the babies whose mothers received prenatal care, and 1.85 percent of those whose mothers did not receive prenatal care . Infant mortality was more pronounced among mothers who did not receive prenatal care; 2.11 percent of their babies died in the hospital, compared with 0.38 percent of those babies born to mothers who received prenatal care . Hospital charges for infants with prenatal care are on average $1,198.42 less than those without prenatal care ($1,045.69 versus $2,244.11). The regression equation for hospital charges was estimated using birth weight (equation 2.1) as an independent variable, and birth - weight categories (equation 2.2). Three birth - weight categories were defined: bwt1 for normal birth weights greater than 2,500 grams, bwt2 for low birth weights from 1,500 to 2,500 grams, and bwt3 for very low birth weights less than 1,500 grams . The hospital charge for the infant was lowered by $10.24 for every ounce the baby weighed . The use of birth - weight categories in estimating equation 2.2 shows a somewhat different perspective on this relationship . Other things equal, coefficient estimates indicate that infants in the bwt2 category (from 1,500 to 2,500 grams) had charges that were $1,065.41 lower than normal - birth - weight infants (more than 2,500 grams). This may be because of the large proportion of infants in this category that can be classified small - for - term, weighing between 2,240 and 2,500 grams . The added expense for very low - birth - weight infants (less than 1,500 grams) was $13,638.32 because of the medical complications evident in extremely low - birth - weight infants . Infants who were transferred had charges that were more than $48,091 higher than those who were not . Goodness of fit as measured by r is greater than .23, depending on the specification of the equation . Although the results of this study do not demonstrate a causal relationship between prenatal care and birth outcome, they do suggest an association between prenatal care and positive birth outcome . The independent effect of prenatal care on birth weight, adjusted for differences in other regressors in equation 1, is 145 grams (5.09 ounces). In other words, even after adjusting for other differences, infants born to females who receive prenatal care weigh about 145 grams more than those whose mothers do not receive prenatal care . Referring to table 5, these babies also are less likely to fall into the low- and very low - birth - weight categories (10.23 percent versus 17.42 percent), proportionately fewer are born prematurely (4.60 percent versus 9.50 percent), the incidence of transfer to an acute - care facility is less than one - half (0.71 percent versus 1.85 percent), and the incidence of early death is much lower (0.38 percent versus 2.11 percent). The main contribution of this study is that it brings into the analysis for the first time cost information based on actual hospital charges rather than estimates based on surveyed prices . The predicted value of the cost of care can be determined using the results presented in table 4 from equation 2.1 . Babies whose mothers received prenatal care have a predicted hospital cost of $1,064.61, compared with $2,068.66 for those whose mothers did not receive care prior to the onset of labor a difference of $1,004.05 . The basis of the cost savings associated with prenatal care seems to be in the associated lower incidence of extremely low - birth - weight babies among females who receive prenatal care . As previous studies have indicated (e.g., lennie, klun, and hausner, 1987), low - birth - weight infants have significantly higher medical expenses than normal - birth - weight infants . Table 6 provides a breakdown of the average hospital charges and proportion of births in each of the three birth - weight categories . For females who received prenatal care, the hospital charges for low - birth - weight infants (1,500 to 2,500 grams) were more than 4 times those of normal - birth - weight infants . Very low birth weights resulted in charges of more than 33 times those for normal birth weights . For females who did not receive prenatal care, low - birth - weight infants had almost 6 times the charges of normal - birth - weight infants, and very low - birth - weight infants had charges of more than 70 times normal . Fortunately, only 5.72 percent of the births to females with prenatal care fall into the two low - birth - weight categories . However, this contrasts with more than 14 percent of the births in these low - birth - weight categories to females who received no prenatal care . Although it is unreasonable to expect that low and extremely low birth weights will be eliminated completely, it seems reasonable to expect that were they to receive prenatal care, the distribution of birth weights for the mothers who received no prenatal care would converge toward that of the mothers who received prenatal care . Using this as a working assumption, if the 364 mothers who received no prenatal care had the same birth - weight distribution as the 6,344 mothers who received prenatal care, their average hospital charges would fall from $2,297.42 to $926.19, a reduction of $1,371.23 . Note that this calculation holds constant the average charges within each category, to allow for differences in the distribution of charges within each category . This figure is actually $103.31 less than the charges for those infants whose mothers had prenatal care . One reason for this phenomenon may be that women in this category had fewer cesarean deliveries and thus the infants with normal birth weights had shorter average hospital stays . Females delivering normal - sized babies had lower cesarean - section rates than females delivering extremely low - birth - weight babies (23.0 percent versus 40.7 percent for white females; 17.2 percent versus 20.6 percent for non - white females). If the rate for non - white females adjusts to the higher rate for white females within each birth - weight category, overall the non - white group would have 154 more cesarean deliveries . Using the coefficient on length of stay from equation 2.1 in table 4 (i.e., 317.90), the average cost would increase by approximately $20 per infant for every day the average stay increased . Thus the average stay for babies delivered by cesarean section would have to be more than 5 days above the average for normally delivered babies . The use of population - based data is an important addition to the analysis of cost - of - care questions . Although the study sample is regionally isolated and too small to make sweeping generalizations, the demographic characteristics of the mclennan county population are representative of those of metropolitan areas across the country . This includes age and ethnic composition, socioeconomic characteristics, and, unfortunately, drug use and abuse . A savings of $1,371.23 per birth for this group of women translates into a group savings of more than $499,000 . With more than 3.8 million births annually in the united states, if the same percentage of females fail to receive prenatal care nationally (5.43 percent), this translates to 208,000 births for this group . Prenatal care for this group could potentially save $285 million nationally in hospital charges in the perinatal period alone . Thus, to the extent that prenatal care can be provided for less than $1,371 per patient, there will be a net system savings because of the better care . Despite the evidence that prenatal care is associated with desirable birth outcomes, although the incidence of low and very low birth weights, premature labor, transfers to acute - care facilities, and early death was significantly greater for those women who had no prenatal care, the combined impact on total cost of care is not large in an absolute sense . This is not meant to imply that prenatal care is unimportant from an economic perspective . Because the most important prenatal visit is the first one, it is important that it be early in the pregnancy . If the medical data gathered during this examination can identify those women who are most at risk for premature labor and its associated low birth weights, then these women can be targeted for special treatment . Prenatal care cannot control for the socioeconomic and environmental differences that result in poor birth outcome . However, it has proven its worth in identifying factors that affect birth outcome, such as cigarette smoking, alcohol consumption, drug use, and poor diet . Once these confounding factors have been identified, a strict regimen can be prescribed to eliminate or reduce the compromising activity . By carefully screening prospective mothers' medical histories, factors such as health status, emotional well - being, and attitudes toward the pregnancy further study should be undertaken to determine whether prenatal care is an important factor in preventing prematurity, or whether it is merely a proxy for health status or some other socioeconomic consideration . More information on medical histories, occupation, and education is needed to better understand this relationship.
The purpose of ergonomics applied to dentistry is to obtain means and systems to decrease physical and mental stress, as well as prevent diseases related to dental practices . Work - related musculoskeletal disorders (wmsd) have increased in recent decades and are factors that commonly lead to professionals being unable to work, with financial and medical consequences . In the case of dentists, incorrect posture is a cause of cervical column, neck, and shoulder disorders, besides hand neuropathies, due to the continuous use of dental instruments and tools . Alexopoulos, stathi and charizani (2004) observed that 52% of the greek dentists who participated in a related study, reported at least one osteomuscular disorder . Adapting working positions to ergonomic rules is one of the measures to prevent wmsd, which can significantly contribute to a reduction in physical effort, thereby bringing benefits to the daily routine of professional activities . Another consideration is equipment design which is extremely important in terms of decreasing muscular fatigue and, consequently, reducing physical and mental stress, as highlighted by parsell, et al . Posture - comfort stools with arm supports can offer musculoskeletal benefits when cavity preparations are carried out by dentists . (2006) reported that it is possible to reduce wmsd with ergonomic adaptations to the clinical mirrors . (2003) also reported an association between an increase in the hours of use the ultrasonic scaler, the instrument of choice for calculus debridement, and an increase in tendonitis in the upper extremities resulting from dental hygiene procedures . Rgis - filho (1997) demonstrated, by the muscular biomechanics expressed by kinematic registrations (working angles), kinetic (muscular torque) and electromyographic (neuromuscular activity graphically represented by the electrical activity of the muscle), that endodontics, and more specifically root canal preparation, is a dental practice that commonly causes wmsd, surpassing the clinical procedures of extractions, scaling and curettage, as well as amalgam condensation . (1998) reported that of a group of 192 dentists 79.2% had fatigue and pain in the hands and forearms, which occurred with greater intensity during or after root canal preparation . With the aim of reducing these symptoms, endodontic instrument handles manufactured according to ergonomic principles have been proposed, since larger diameters facilitate the gripping of the instruments and reduce muscular electromyographic activity . As in the case of several other medical and dental specializations, endodontics has evolved in recent years, setting forward greater precision, reduced possibility of mistakes and injuries, less discomfort to the patients and faster procedures . Some authors have suggested that, in addition to these factors, root canal preparation with nickel - titanium (niti) instruments is associated with the least stress and operating fatigue, especially due to the decrease in working time when compared with manual instrumentation with stainless steel . The development of endodontic instruments made from niti alloy has the potential to enhance the shaping of narrow, curved canals, a procedure which has proved difficult to achieve using stainless steels instruments because of their inherent stiffness . The objective of this study was to verify, in vivo, the results obtained from the kinematic kinetics, and electromyographic records in endodontists during the preparation of simulated root canals using rotary and manual instrumentation techniques . Eight right - handed healthy subjects, four females and four males, between 23 and 39 years of age, participated in this study . All of them were endodontists with clinical experience accumulated over at least 2 years . Each endodontist, seated on a dental stool in the " 12 o'clock " position, prepared two simulated canals with 20 mm in resin blocks with diameters corresponding to an instrument size 20 and curvatures of 40 (crinodonto produtos race 25.02 rotary ni - ti (fkg, la chaux - of - fonds, orbe, switzerland) and flexo - file 25.02 (dentsply - maillefer, ballaigues, orbe, switzerland) instruments were used . Prior to the use of the above - described instruments, the resin blocks were attached to a lathe machine and prepared . In the cervical third, the preparation was carried out with rotary pre - race instruments 40.10 and 35.08 (fkg, reads chaux - de - fonds, orbe, switzerland), following the crown - down instrumentation procedure, where the dentist essentially works from the crown of the tooth, shaping the canal towards the apex . Flexo - files 40.02, 35.02 and 30.02 were used by the manual group, race 25.06 and 25.04 instruments by the rotary group . The purpose was to reach the working length (wl=20 mm) with the 25.02 instrument . Once the wl was reached, 10 repetitions of 3 seesaw movements each were carried out while data was collected on muscular activity over 4-s periods . The reading of the movements would be very extensive if it were done throughout the whole preparation procedure, using all the instruments, thence, only the last instrument employed for each preparation was used (25.02, which correspond to 0.25 mm diameter in the initial tip with 0.02 mm taper at each millimeter in the active part of the instrument). That was true for the manual method, where the instrument is handled with fingers tips, as well as for the rotating instrument . In the rotary method, the instruments, which are driven by electric or pneumatic motors, make a continuous 360 rotation . Instrumentation steps for manual and rotary methods electromyographic (emg) data were collected using surface electrodes attached to the skin over the muscle of interest . The skin was cleaned to adherence of the electrodes and detection of the emg ., boston, ma, usa) were attached over the flexor carpi radialis, extensor carpi radialis, brachioradialis, biceps brachii, triceps brachii, middle deltoid, and upper trapezius . The electrodes were positioned in parallel with the muscular fibers, at approximately two centimeters from each muscle motor point (between the motor point and the tendon). The emg signals were amplified (x2000), band - pass filtered (20 - 450 hz) and recorded . The data were digitized at 12 bits and collected by an ibm computer at 1000 hz . For the analysis of the movements, x, y, and z coordinates were recorded utilizing led (light emission diode) markers fastened to the temporomandibular joint, to the hip (over the iliac crest), shoulder (lateral portion of acromion), elbow (lateral epicondyle), wrist (lister's tubercle), and hand (metacarpal head). The infrared signal emission of these markers was captured at a frequency of 100 hz, by a three - dimensional optotrak 3020 optical system (digital northern inc ., the resulting electromyographic emg signals in millivolts (mv), and the x, y, and z positional data were synchronized by an odau ii - optotrak data acquisition unit ii, and mathematically treated using matlab code (math works inc ., version 6.0). The emg signals were rectified, filtered (low - pass at 20 hz using a second - order butterworth filter), and normalized by the maximum voluntary isometric contraction (mvic) of each subject . The averaged emg of the mvic was calculated within the 500 - 1000 milliseconds (ms) interval from the beginning an isometric contraction . For all mvic tests, the linear displacement of the markers, the joint torque generated by the shoulder, elbow, and wrist, as well as deviations, angular velocity, and accelerations, were also calculated . The anthropometric data, center of mass, and moment of inertia of the segments were calculated based on the weight and sex of the subject, according to zatsiorsky's model, modified by de leva (1996). Based on these data, the joint torque of the shoulder, elbow, and wrist were calculated through inverse dynamics and later normalized to each subject's body weight . The joint torque averages (nm / kg) for the shoulder, elbow, and wrist, as well as the emg signal normalized values were calculated at intervals of 100 ms each, starting from the beginning of the movement of the wrist assessed by measuring the angular displacement . The student t - test for paired samples was applied according to the experimental design and type of variable, considering that this study involved a parametric variable and two experimental groups . For this analysis, the average of the angular displacement, joint torques and emg across all subjects during the movement was calculated across intervals of 100 ms, for each instrumentation method . After, the methods were compared through the paired test, because the same individuals carried out the instrumentation with both methods . The resulting electromyographic emg signals in millivolts (mv), and the x, y, and z positional data were synchronized by an odau ii - optotrak data acquisition unit ii, and mathematically treated using matlab code (math works inc ., version 6.0). The emg signals were rectified, filtered (low - pass at 20 hz using a second - order butterworth filter), and normalized by the maximum voluntary isometric contraction (mvic) of each subject . The averaged emg of the mvic was calculated within the 500 - 1000 milliseconds (ms) interval from the beginning an isometric contraction . For all mvic tests, the linear displacement of the markers, the joint torque generated by the shoulder, elbow, and wrist, as well as deviations, angular velocity, and accelerations, were also calculated . The anthropometric data, center of mass, and moment of inertia of the segments were calculated based on the weight and sex of the subject, according to zatsiorsky's model, modified by de leva (1996). Based on these data, the joint torque of the shoulder, elbow, and wrist were calculated through inverse dynamics and later normalized to each subject's body weight . The joint torque averages (nm / kg) for the shoulder, elbow, and wrist, as well as the emg signal normalized values were calculated at intervals of 100 ms each, starting from the beginning of the movement of the wrist assessed by measuring the angular displacement . The student t - test for paired samples was applied according to the experimental design and type of variable, considering that this study involved a parametric variable and two experimental groups . For this analysis, the average of the angular displacement, joint torques and emg across all subjects during the movement was calculated across intervals of 100 ms, for each instrumentation method . After, the methods were compared through the paired test, because the same individuals carried out the instrumentation with both methods . A significance level of 5% was established . It was ascertained that there were differences between the results obtained with the use of manual and rotary techniques with regard to the variables of the minimum (min . ), maximum (max . ), and angular extension (ae) techniques for the shoulder, and low and maximum techniques for the wrist . Distribution of the sample based on maximum (max), minimum (min) angular displacement and angular excursion (ae) between the articulations over all repetitions across all subjects regarding the direction of joint movement . This angle are of the flexion / extension for shoulder and elbow (external angle), and radial / ulnar deviation for wrist the average measured joint torque values for the shoulder, elbow, and wrist (nm / kg) are given in table 2 . It can be observed that the instrumentation method did not influence any of the analyzed variables in a statistically significant way . The use of the rotary technique led to a higher uniformity of the results obtained regarding the torque values during the time intervals . The manual technique showed high oscillations during the time intervals analyzed (figure 2). Joint torque values normalized to each subjects body weight for the shoulder, elbow, and wrist (nm / kg) during the moviments for the preparation of simulated root canals with manual and rotary instruments . The positive and negative values represent the clockwise and counterclockwise directions respectively average and standard error across all subjects of the normalized electromyography (emg) activity of the extensor carpi radialis (a), brachioradialis (b), flexor carpi radialis (c), biceps brachii (d), upper trapezius (e) and middle deltoid (f) during all intervals calculated for each instrumental method normalized emg values expressed as a percentage of mvic are given in table 3 . It was observed for the manual method that there was a higher emg activity in the extensor carpi radialis, flexor carpi radialis and brachioradialis muscles, and lower emg activity in the middle deltoid and upper trapezius compared with the rotary method . The biceps brachii and triceps brachii did not show statistically significant differences in relation to the preparation methods (figure 3). Comparison between the manual and rotary methods according to the average and standard error across all subjects of the joint torque (nm / kg) for the wrist (a), elbow (b) and shoulder (c), during all intervals calculated (each 100 ms) for each instrumental method it was ascertained that there were differences between the results obtained with the use of manual and rotary techniques with regard to the variables of the minimum (min . ), maximum (max . ), and angular extension (ae) techniques for the shoulder, and low and maximum techniques for the wrist . Distribution of the sample based on maximum (max), minimum (min) angular displacement and angular excursion (ae) between the articulations over all repetitions across all subjects regarding the direction of joint movement . This angle are of the flexion / extension for shoulder and elbow (external angle), and radial / ulnar deviation for wrist the average measured joint torque values for the shoulder, elbow, and wrist (nm / kg) are given in table 2 . It can be observed that the instrumentation method did not influence any of the analyzed variables in a statistically significant way . The use of the rotary technique led to a higher uniformity of the results obtained regarding the torque values during the time intervals . The manual technique showed high oscillations during the time intervals analyzed (figure 2). Joint torque values normalized to each subjects body weight for the shoulder, elbow, and wrist (nm / kg) during the moviments for the preparation of simulated root canals with manual and rotary instruments . The positive and negative values represent the clockwise and counterclockwise directions respectively average and standard error across all subjects of the normalized electromyography (emg) activity of the extensor carpi radialis (a), brachioradialis (b), flexor carpi radialis (c), biceps brachii (d), upper trapezius (e) and middle deltoid (f) during all intervals calculated for each instrumental method it was observed for the manual method that there was a higher emg activity in the extensor carpi radialis, flexor carpi radialis and brachioradialis muscles, and lower emg activity in the middle deltoid and upper trapezius compared with the rotary method . The biceps brachii and triceps brachii did not show statistically significant differences in relation to the preparation methods (figure 3). Comparison between the manual and rotary methods according to the average and standard error across all subjects of the joint torque (nm / kg) for the wrist (a), elbow (b) and shoulder (c), during all intervals calculated (each 100 ms) for each instrumental method with the advent of rotary instruments, general dentists and endodontists can carry out treatments faster and with a better final quality of the preparations . Rotary instruments, however, should allow less physical effort and a lower probability of injury for dental professionals . However, no study has ever compared rotary and manual techniques with regard to the muscular and biomechanical aspects of these devices . Bramson, smith and romagnoli (1998) verified that the repetitive movement of an articulation has a limit before the appearance of wmsd and that depending on the extent to which this limit is exceeded the risk of wmsd is classified as low, medium or high (figure 4). Through the comparative kinematic analysis performed in this study, the angle displacements for the wrist and elbow verified during the preparations using rotary or manual instruments angular movement of the articulation limit before the appearance of work - related musculoskeletal disorders (wmsd) classified as low, medium or high risks by bramson (1998) the joint torque represents the muscular action on the articulation added to the passive elements of which it is composed, such as ligaments, tendons, and articulation capsules . Through the results obtained, we can observe that, despite the lack of a statistically significant difference between the root canal preparation techniques, the quality analysis of the behavior of the shoulder, elbow, and wrist joint torques revealed a greater uniformity of values during the intervals for the rotary instrumentation, allowing for a more homogeneous effort without high peaks . On the other hand, the analysis revealed many oscillations in joint torque behavior for the manual instruments in the three articulations, thus suggesting the need for recruitment of a greater number of muscles to generate such torques and execute the task . It can be observed, through the emg results, that there was a difference between the instrumentation techniques for most of the muscular groups under analysis . Except for the middle deltoid and trapezius, where use of rotary instrumentation resulted in a higher emg level, the explanation for the occurrence of a higher emg activity during use of the manual instrumentation lies in the very fact that there is a need greater apprehend of instruments to execute the task . The wrist and the elbow position are fundamental for this execution . During use of rotary instruments, there is the need to hold the equipment, but less effort is required to penetrate and work inside the root canals . In this case the professionals employ a different motor strategy, using the muscles closer to the shoulder . It is important to emphasize that the continuous activity of both the extensor carpi radialis and flexor carpi radialis observed in this study with the manual technique, as well as of the brachioradialis muscle, upper trapezius and middle deltoid with the rotary instrumentation, characterize a static contraction . The tension generated by the static contraction may contribute to the alteration of microcirculation in the muscle, thus determining the appearance of painful points called " trigger points ", characteristic of myofascial pain . Myofascial pain causes more muscular tension, which can lead to poor blood circulation, which yields more pain, thence generating a vicious cycle . Myofascial pain affects various regions of the body, but one of the most common points is the trapezius muscle region, which is important for the head posture positioning, as well as for the execution of the scapulae function . The muscular recruitment for performing articular movements for root canal preparation with either the rotary or manual techniques is distinct . Nevertheless, the rotary instruments presented less difficulty in modulating the joint torque in each articulation, thus, presenting a greater uniformity of movements . Concerning the ergonomic aspect, alternating rotary and manual instrumentation for root canal preparation might contribute to a decrease in the occurrence of wmsd . In other words, alternating the use of the manual and rotary techniques would produce a better balance in the activation of the muscles, because the motor strategies are different . Different motor strategies can be found when there is variability of the tasks and when pain is present, and they can be a mechanism of protection against wmsd . Based on the methodology used and the results obtained, it can be concluded that: the angular displacement of the wrist and elbow during root canal preparation using rotary and manual instruments were classified as low risk in terms of the occurrence of wmsd; there were no significant differences between the kinetic records for the two instrumentation techniques; a higher level of muscular activity was verified in the majority of the muscles assessed (extensor carpi radialis, flexor carpi radialis, brachioradialis and triceps brachii), although the motor strategy was shown to be distinct for each of the two techniques tested.
Direct immunoflourescence (dif) has become an indispensable diagnostic tool in the diagnosis of immunobullous lesions of the skin and has been widely used to supplement the clinical and histologic features of various vesiculobullous disorders . The utility of this technique is limited by cost, site, and time of the biopsy, technical and tissue processing factors, history of treatment and the nature of the disease . This study was undertaken to set up the immunofluorescence facility in our department and to evaluate the practical utility of dif in immunobullous dermatoses at our institution as a university granted research project . The objectives of this study was to evaluate the sensitivity of dif in immunobullous dermatoses and to study the pattern of dif . The study also aimed to correlate dif with clinical and histologic findings and to analyze discrepancies . A cross - sectional study was undertaken at the department of pathology and dermatology of a tertiary care hospital in western india from august 2010 to september 2012 . One hundred patients, of any age and sex, with strong clinical suspicion of an immunobullous lesion were included in the study . Patients with a known infective etiology of the bullous lesions were excluded . After taking written informed consent of the patients, two biopsies were performed in each case; one from the fully developed vesiculobullous lesion (lesional biopsy) and the other from the perilesional area within 2 cm diameter of the lesion (perilesional biopsy). Histopathological examination (hpe) finding were used as a further aid in the diagnosis whereas the perilesional biopsy, placed in normal saline / phosphate buffered saline was immediately taken to the laboratory for dif . The tissue was embedded in the cryostat embedding medium, frozen, and cut at 5 m thickness at 22c . Sections were taken on poly l lysine coated glass slides, fixed with ether - alcohol mixture, and air dried for 10 min . Later the slides were rinsed in phosphate buffered saline at ph 7.3 for 15 min . The sections were treated with fluorescein isothiocyanate labeled and optimally diluted (1:40) antisera, i.e., igg, iga, igm, and c3 respectively . The slides were incubated in wet chamber for 1 h in dark room, washed with phosphate buffered saline pbs and mounted with glycerine jelly . Dif was reported based on nature and site of immune deposits, semi quantitative grading of strength of fluorescence and pattern of immune complex deposits . Statistical methods used to analyze this study include, percentages, ratio, sensitivity, specificity, and positive and negative predictive values . Statistical methods used to analyze this study include, percentages, ratio, sensitivity, specificity, and positive and negative predictive values . Ages of our patients ranged from 16 years (a girl diagnosed as pemphigus vulgaris) to 87 years (a male diagnosed as bullous pemphigoid [bp]) with a mean age of 57 years . The maximum (33%) cases belonged to the age group of 41 - 50 years, pemphigus group being maximum (27%). Fluid filled lesions (95%) were the most common clinical finding followed by erosion and crusting (73%). Itching (63%) was the most common symptom followed by pain (57%) and burning (55%). Subcorneal blister was seen in 12%, subepidermal in 28%, and suprabasal in 60% of the cases . Histopathological diagnosis of various immunobullous lesions [figures 1a, 1b, 3a 4a, 5a, 6a, 7a, 7b] along with corresponding dif findings are depicted in table 1 . (h and e, 100), (b) blister contains eosinophils and few neutrophils . H and e, 400) direct immunoflourescence of pemphigus vulgaris (a) lacelike intercellular space deposition of igg, (b) lacelike ics deposition of c3 pemphigus foliaceus (a) subcorneal blister with dyskeratotic acantholytic granular cells . Secondary cleft formation seen in the mid - level of epidermis (h and e, 100), (b) direct immunofluorescence shows intercellular space deposition of igg in upper dermis bullous pemphigoid (a) subepidermal blister formation with an inflammatory infiltrate in the bullous cavity . (h and e, 100), (b) direct immunoflourescence shows linear deposition of igg in the basement membrane zone linear iga bullous dermatosis (a) neutrophils along the dermo - epidermal junction (h and e, 400), (b) direct immunoflourescence showing linear deposits of iga along the dermoepidermal junction dermatitis herpetiformis (a) subepidermal bulla (h and e, 400), (b) direct immunoflourescence showing granular deposits of iga along the dermal papillae pemphigus erythematosus (a) subcorneal bulla containing acantholytic cells . (h and e, 100), (b) subcorneal bulla contains neutrophils . (h and e, 100) histopathological diagnosis and dif findings of immunobullous lesions on dif examination, intercellular space (ics) deposition of igg was present in 51 out of 58 cases of pemphigus vulgaris resembling fishnet pattern [figure 2a]. All the cases of pemphigus foliaceus showed ics deposition of igg in upper dermis [figure 3b]. Majority (18/25) of bp showed linear igg and c3 deposition in the basement membrane zone (bmz) [figure 4b]. Single patient of linear iga disease showed linear deposition of iga along the bmz [figure 5b]. Both cases of dermatitis herpetiformis (dh) had granular iga deposited along the bmz [figure 6b]. Single case of pemphigus erythematosus showed granular deposition of igg at ics and also showed igm deposition along the bmz [figures 8a and b]. Direct immunoflourescence of pemphigus erythematosus (a) deposition of igg in intercellular space (arrow), (b) granular deposition of igm at dermoepidermal junction (arrow) as regards the 11 dif negative cases, seven were of pemphigus vulgaris (three had no epidermis) and four were in remission being on long - term cyclophaphamide pulse and steroid therapy . Negative immunofluorescence may be viewed as the state of immunological remission in pemphigus because most patients with negative immunofluorescence did not have relapse after discontinuation of the treatment . A total of 4 cases with specific histopathological findings of bp had negative dif due to lack of epidermis in one and technical / sampling errors in the three cases . Dif was diagnostic in all the pemphigus variants including the pemphigus foliaceus (12%) and the pemphigus vegetans (1%). Dif also helped to confirm the diagnosis of dh, linear iga dermatosis and pemphigus erythematosus in these solitary cases respectively . The autoimmune bullous lesions result from an immune response against adhesion molecules of epidermis or bmz . There is clinical overlap among various groups of bullous diseases for example: linear iga dermatosis may mimic bp or dh . Hpe reveals the site of formation of bulla, the presence of infiltrate and its composition . A differential diagnosis is generated on the basis of combination of findings in the biopsy specimen . Subepidermal bullous diseases have overlapping hpe findings and hence their diagnosis on hpe findings alone is difficult . At present, diagnosis of autoimmune bullous disorders is based on immunological and molecular findings rather than clinical and hpe diagnosis alone . The ideal site for the biopsy specimen depends on the type of disorder . For bullous diseases, dif is performed using the perilesional skin that is normal appearing skin immediately adjacent to a lesion . The immune deposits are partially or completely degraded in inflamed or blistered skin and dif may be negative . The differential diagnosis of a dif test depends on primary site of immune deposition, the class of immunoglobulin or other type of immune deposits, number of immune deposits and if multiple, the identity of the most intense deposits and deposition in other sites besides the main site . With these parameters, the most frequent disorder in our study was pemphigus vulgaris and the finding is in accordance with other regional and international studies . Male to female ratio was 1:1.7, which is comparable to the findings of shamim et al . Dif findings in pemphigus vulgaris showed igg positivity in ics in fishnet pattern in 51% . Dif is positive in 90 - 100% of patients with active disease if an appropriate biopsy specimen has been obtained . Occasionally, the fluorescence may be limited to or most intense at the level of the epidermis that is involved with blister formation that is lower epidermal layer for pemphigus vulgaris and superficial epidermal layers in pemphigus foliaceus . This variation in the intensity of fluorescence at various layers of the epidermis may be caused by differences in the relative amounts of the target desmosomal proteins for each of the two diseases, namely desmoglein 1 for pemphigus foliaceus, and desmoglein 3 for pemphigus vulgaris . Immunofluorescence is required to differentiate staphylococcal scalded skin syndrome (ssss) from pemphigus foliaceus, since a small number of acantholytic cells are seen in case of the former ssss . The lesions of pemphigus foliaceus can become impetiginized, which produces pustules as in impetigo and subcorneal pustular dermatosis . Pemphigus vulgaris and vegetans have similar dif findings and hence need to be differentiated by clinical and hpe characteristics . The combination of ics and bmz deposition of igg is seen in pemphigus erythematosus, in which immunopathologies of pemphigus foliaceus and lupus erythematosus co - exist . We had one such case, where the patient had clinical features of systemic lupus erythematosus, her rheumatoid factor and anti - nuclear antibody tests were positive and dif revealed findings of pemphigus and lupus erythematosus . Thus, dif helped to confirm the diagnosis of senear - usher syndrome, a rare variant of pemphigus foliaceus . Bp affects the elderly during their fifth to seventh decade of life, with an average age of onset being 65 years . Nearly, 48% of the patients were in the age group of 61 - 70 years . The reported sex ratios in bp have varied from 2.4:1 to 1:5.75 in different studies due to variation in the sample size . In our present study, herpes gestationis, linear iga dermatosis and epidermolysis bullosa acquisita show subepidermal bulla with neutrophil rich infiltrate, which can be confused with bp . However, dif helps to differentiate these conditions from bp . In our present study, only two cases of dh were seen both being males aged 37 years and 38 years . Nicolas et al . Reported dh most commonly between the age of 25 - 55 years . Inchara reported four cases of dh . But none of the four biopsy proven cases showed immunoglobulin deposits . In absence of characteristic, dif pattern they recommended to use a combination of the clinical and the serological data . Differential diagnosis for deposition of igg and/or c3 at the bmz includes bp, cicatricial pemphigoid, pemphigoid gestationis, epidermolysis bullosa acquisita, and bullous systemic lupus erythematosus . C3 deposits with significantly higher intensity than igg favor the pemphigoid group of diseases such as bp, cicatricial pemphigoid, and pemphigoid gestationis . In bp and pemphigoid gestationis, the deposition consists predominantly and occasionally exclusively (especially in pemphigoid gestationis), of c3 . They are present in continuous, fine, and linear patterns . In bullous systemic lupus erythematosus, approximately 60% of cases reveal bmz deposition of igg indistinguishable from that of epidermolysis bullosa acquisita . Differentiation between bullous systemic lupus erythematosus and epidermolysis bullosa acquisita is based on the presence of serological evidence of systemic lupus erythematosus . In these subepidermal bullous lesions, dif / indirect immunoflourescence if on salt split skin yields higher rate of positivity in contrast with conventional dif . In bp, patterns of fluorescence was in the roof (40.60%), floor (9.4%), and combined roof and floor (50%). On indirect immunoflourescence, positivity was almost doubled with salt split technique (68%) as compared to routine methods (36%). Our study revealed 90% ppv and 94% sensitivity and the sensitivity was 84% in bp . False negativity was seen in 7/58 cases of pemphigus vulgaris and 4/25 cases of bp group . This was mainly due to technical errors like lack of epidermis in the biopsy (n = 4), exposure to light (n = 1), improper site of the skin biopsy (n = 1) and longer stay of the biopsy in the normal saline (n = 1). Total 4 histologically proven cases of pemphigus vulgaris, who were on the treatment revealed the dif negativity . This finding was against the previous documentation, which states that dif is a very reliable diagnostic test for pemphigus, which becomes positive at an early stage and remains positive for a long period after clinical remission . Hence, in the absence of the characteristic dif pattern, one needs the combination of clinical, histologic, and immunologic data to support the definite diagnosis of different immunobullous disorders . However, a diagnosis based solely on the clinical or histologic findings may not be accurate . Dif is extremely helpful in confirming a suspected diagnosis and in distinguishing among closely related cases of immunobullous lesions . Our study concludes that the dif is an essential for diagnosing autoimmune blistering diseases with the clinical and the histopathological overlap . However, larger studies with proper selection of cases and judicious use are mandatory to optimize its utility . Dif helped in confirmimg the diagnosis of bullous lesions with the clinical and histopathological overlap.in this study, a rare case of senear - usher syndrome could be diagnosed due to dif.sampling errors contributed to fn results . Dif helped in confirmimg the diagnosis of bullous lesions with the clinical and histopathological overlap . In this study, a rare case of senear - usher syndrome could be diagnosed due to dif.
Depending on the nature of their practice, radiologists have variable degrees of direct contact with patients, but all radiologists must be able to communicate well with other medical and non - medical staff and with their patients . Their professional role also requires that they communicate their radiological findings and opinions clearly and concisely, both verbally and in written reports . Introduction . The patients need to know the name of the radiologist, and his / her professional role in their care . It is advisable to ensure that the patients are also aware of who else is in the room at the time of their examination, e.g. Radiological trainee / nurse / radiographer, and their role(s).establishing the purpose of the examination . This may entail taking a short clinical history relevant to the clinical request.explanation and consent . Here local protocols and procedures are likely to be in place for explaining the procedure to the patients and obtaining either verbal or written consent to undergo the examination, even though these may vary between countries . The patients must be fully informed in order to give their consent, and must therefore have the opportunity to voice any concerns, or ask any questions they may have before the examination is carried out . It is important that the radiologist is satisfied that the patients have willingly given appropriate informed consent.communication of findings to patients . Local custom and practice may determine by whom and under what circumstances, radiological findings are communicated to patients . All doctors, as well as radiologists, must answer patients questions truthfully, trying not to cause premature or unnecessary alarm, and not entering into conversations about treatment options which are outside their area of knowledge or expertise . There will be times when radiologists will be directly asked questions which lead to them having to give bad news to the patients . The patients will expect radiology staff to maintain their medical confidentiality, and not to communicate any details about their case with others unless this is directly relevant to their care.when things go wrong . If an error has been made, or a complication arisen, some evidence suggests that it is better for all concerned if appropriate discussion takes place between the medical staff involved and the patients . Although medico - legal considerations may affect local policy for disclosure and admission of fault, no attempt should be made to deliberately hide any facts related to the case . The patients need to know the name of the radiologist, and his / her professional role in their care . It is advisable to ensure that the patients are also aware of who else is in the room at the time of their examination, e.g. Radiological trainee / nurse / radiographer, and their role(s). Establishing the purpose of the examination local protocols and procedures are likely to be in place for explaining the procedure to the patients and obtaining either verbal or written consent to undergo the examination, even though these may vary between countries . The patients must be fully informed in order to give their consent, and must therefore have the opportunity to voice any concerns, or ask any questions they may have before the examination is carried out . It is important that the radiologist is satisfied that the patients have willingly given appropriate informed consent . Local custom and practice may determine by whom and under what circumstances, radiological findings are communicated to patients . All doctors, as well as radiologists, must answer patients questions truthfully, trying not to cause premature or unnecessary alarm, and not entering into conversations about treatment options which are outside their area of knowledge or expertise . There will be times when radiologists will be directly asked questions which lead to them having to give bad news to the patients . The patients will expect radiology staff to maintain their medical confidentiality, and not to communicate any details about their case with others unless this is directly relevant to their care . When things go wrong . If an error has been made, or a complication arisen, some evidence suggests that it is better for all concerned if appropriate discussion takes place between the medical staff involved and the patients . Although medico - legal considerations may affect local policy for disclosure and admission of fault, no attempt should be made to deliberately hide any facts related to the case . The provision of an accurate and timely radiological report is part of a process which requires reliable communication between the referrers, radiology department and reporting radiologists . All have duties in respect of clear communication.departmental responsibilities . This includes contact details for all referrers and clarity about who is responsible for the clinical care of the patients and how they may be contacted, including in an emergency . Departments (including administrative assistants / medical staff involved in booking / accepting an appointment) should ensure that referrer contact details are available for all patients referred for imaging or interventional procedures.referrer responsibility . Referrers have a duty to ensure that accurate patient information is available on the request card or electronic referral, with relevant clinical information and a clear indication of the reason for the investigation, together with their own contact details . The referrers also have a duty to alert radiology departments to safety issues which may affect the examination, such as mr safety hazards and contrast contraindications . Clear examination checklists and if an individual refers a patient for a radiological test or procedure, this carries with it a duty to ensure that he or she reads the written report which is generated unless he or she has clearly delegated this task to someone else appropriate.radiologists responsibilitiescontactability . Radiologists must be contactable for queries related to the appropriateness of different imaging modalities and questions / clarifications related to their reports . It is not possible to always be available, but clear lines of communication within the radiology department, and who is responsible for answering queries, should be apparent to referrers.direct verbal communication with referrers . This may be necessary in an emergency situation when imaging findings indicate that urgent action needs to be taken . Appropriate local protocols about the circumstances under which direct communication will be initiated, and how it is carried out will ensure that both radiologists and referrers are clear about what the radiologist will undertake as their duty of care . When a radiological finding is communicated verbally, the name of the person contacted should be recorded . A formal written report should still be issued, including details of how the report was verbally communicated and to whom [4, 5].clinical radiological meetings . Meetings between referrers / clinical teams can be very useful to focus discussions about patient management . These will often involve images which have already been reported being reviewed . Sometimes interpretation will be different because additional relevant clinical information is available and sometimes an error or misinterpretation in the original report will be revealed . Where there is a difference of opinion from the original report, appropriate action should be taken, such as adding an addendum to the report, or following the local protocol for dealing with error or discrepancy in the reporting opinion.written communication ., reports should be structured into sections on clinical details, technique, imaging findings and conclusion . Perfect report because this will depend on referrer expectation as well as radiologists varying opinions, but there is evidence that long free text reports which do not reach a clear conclusion are those that are least favoured by referrers . Departmental responsibilities . This includes contact details for all referrers and clarity about who is responsible for the clinical care of the patients and how they may be contacted, including in an emergency . Departments (including administrative assistants / medical staff involved in booking / accepting an appointment) should ensure that referrer contact details are available for all patients referred for imaging or interventional procedures . Referrers have a duty to ensure that accurate patient information is available on the request card or electronic referral, with relevant clinical information and a clear indication of the reason for the investigation, together with their own contact details . The referrers also have a duty to alert radiology departments to safety issues which may affect the examination, such as mr safety hazards and contrast contraindications . Clear examination checklists and the teaching of junior staff can be helpful to promote this . If an individual refers a patient for a radiological test or procedure, this carries with it a duty to ensure that he or she reads the written report which is generated unless he or she has clearly delegated this task to someone else appropriate . Radiologists responsibilitiescontactability . Radiologists must be contactable for queries related to the appropriateness of different imaging modalities and questions / clarifications related to their reports . It is not possible to always be available, but clear lines of communication within the radiology department, and who is responsible for answering queries, should be apparent to referrers.direct verbal communication with referrers . This may be necessary in an emergency situation when imaging findings indicate that urgent action needs to be taken . Appropriate local protocols about the circumstances under which direct communication will be initiated, and how it is carried out will ensure that both radiologists and referrers are clear about what the radiologist will undertake as their duty of care . When a radiological finding is communicated verbally, the name of the person contacted should be recorded . A formal written report should still be issued, including details of how the report was verbally communicated and to whom [4, 5].clinical radiological meetings . Meetings between referrers / clinical teams can be very useful to focus discussions about patient management . Sometimes interpretation will be different because additional relevant clinical information is available and sometimes an error or misinterpretation in the original report will be revealed . Where there is a difference of opinion from the original report, appropriate action should be taken, such as adding an addendum to the report, or following the local protocol for dealing with error or discrepancy in the reporting opinion.written communication ., reports should be structured into sections on clinical details, technique, imaging findings and conclusion . Perfect report because this will depend on referrer expectation as well as radiologists varying opinions, but there is evidence that long free text reports which do not reach a clear conclusion are those that are least favoured by referrers . Radiologists must be contactable for queries related to the appropriateness of different imaging modalities and questions / clarifications related to their reports . It is not possible to always be available, but clear lines of communication within the radiology department, and who is responsible for answering queries, should be apparent to referrers . Direct verbal communication with referrers . This may be necessary in an emergency situation when imaging findings indicate that urgent action needs to be taken . Appropriate local protocols about the circumstances under which direct communication will be initiated, and how it is carried out will ensure that both radiologists and referrers are clear about what the radiologist will undertake as their duty of care . When a radiological finding is communicated verbally, the name of the person contacted should be recorded . A formal written report should still be issued, including details of how the report was verbally communicated and to whom [4, 5]. Clinical radiological meetings . Meetings between referrers / clinical teams can be very useful to focus discussions about patient management sometimes interpretation will be different because additional relevant clinical information is available and sometimes an error or misinterpretation in the original report will be revealed . Where there is a difference of opinion from the original report, appropriate action should be taken, such as adding an addendum to the report, or following the local protocol for dealing with error or discrepancy in the reporting opinion . Written communication ., reports should be structured into sections on clinical details, technique, imaging findings and conclusion . Perfect report because this will depend on referrer expectation as well as radiologists varying opinions, but there is evidence that long free text reports which do not reach a clear conclusion are those that are least favoured by referrers . It is a sign of strength and not weakness to seek a second opinion from an expert, subspecialist, or just another colleague when unsure about how to interpret an examination . Equally, it should be readily given when requested, as individuals should be willing to help colleagues for the benefit of patients.feedback to others on differences of opinion or error . This should be done promptly but sensitively for patient protection, and to reduce future error . For best practice, clear local protocols on how an addendum is added to a report, and how error is notified to the referrer, patient and reporting radiologist should be in place . Collecting series of others errors without alerting the individual that they are making those errors so that they can learn and improve their practice is not ethical as it does not help to reduce error . Ideally there should be departmental mechanisms for sharing examples of error in a blame - free meeting environment, preferably with the images viewed by the group with the same information as was available at the time of reporting, and the reporter not named to the group . This ensures that there is group learning from error, and repeated errors can be identified to increase general awareness of pitfalls in interpretation . There are guidelines available on precisely how these meetings can be conducted to make them effective, blame - free and educational for all . It is a sign of strength and not weakness to seek a second opinion from an expert, subspecialist, or just another colleague when unsure about how to interpret an examination . Equally, it should be readily given when requested, as individuals should be willing to help colleagues for the benefit of patients . Feedback to others on differences of opinion or error . This should be done promptly but sensitively for patient protection, and to reduce future error . For best practice, clear local protocols on how an addendum is added to a report, and how error is notified to the referrer, patient and reporting radiologist should be in place . Collecting series of others errors without alerting the individual that they are making those errors so that they can learn and improve their practice is not ethical as it does not help to reduce error . Ideally there should be departmental mechanisms for sharing examples of error in a blame - free meeting environment, preferably with the images viewed by the group with the same information as was available at the time of reporting, and the reporter not named to the group . This ensures that there is group learning from error, and repeated errors can be identified to increase general awareness of pitfalls in interpretation . There are guidelines available on precisely how these meetings can be conducted to make them effective, blame - free and educational for all . - workers should be treated with respect and courtesy . In the case of personal or professional disagreement, this should be dealt with calmly, and if necessary, proper employment processes and complaints procedures used.teamwork . Radiologists work closely with other staff within the radiology department, including radiographers, nurses and clerical and secretarial staff . They have a particularly close working relationship with radiographers, and their roles and responsibilities, particularly in respect of communication with patients, may overlap . There should be clarity on the duties of each; for example, explaining procedures to patients and asking relevant safety questions . Radiologists should listen to the opinions and concerns expressed by other staff members . There is evidence that this reduces error and helps to prevent medical accidents.education . Whenever possible, radiologists should convey to other staff the rationale for choosing a particular investigation, and the clinical importance of following / adjusting examination protocols . Teaching and educating other staff will help to improve the quality of the service as a whole.confidentiality . Communication with other staff should preserve the patient s right to confidentiality and cases should not be discussed amongst staff unless relevant to their individual care or for teaching . Staff members who become patients themselves share the same right to confidentiality as all other patients . All staff and co - workers should be treated with respect and courtesy . In the case of personal or professional disagreement, this should be dealt with calmly, and if necessary, proper employment processes and complaints procedures used . Teamwork . Radiologists work closely with other staff within the radiology department, including radiographers, nurses and clerical and secretarial staff . They have a particularly close working relationship with radiographers, and their roles and responsibilities, particularly in respect of communication with patients, may overlap . There should be clarity on the duties of each; for example, explaining procedures to patients and asking relevant safety questions ., radiologists should convey to other staff the rationale for choosing a particular investigation, and the clinical importance of following / adjusting examination protocols . Teaching and educating other staff will help to improve the quality of the service as a whole . Communication with other staff should preserve the patient s right to confidentiality and cases should not be discussed amongst staff unless relevant to their individual care or for teaching . Staff members who become patients themselves share the same right to confidentiality as all other patients . When teaching, those learning should be treated fairly and with respect and not singled out for criticism.teaching should be focused to the needs of learners and evaluated with feedback from them . Radiologists who have significant teaching responsibilities may benefit from undertaking specific training to enhance their teaching skills when teaching, those learning should be treated fairly and with respect and not singled out for criticism . Teaching should be focused to the needs of learners and evaluated with feedback from them . Radiologists who have significant teaching responsibilities may benefit from undertaking specific training to enhance their teaching skills this involves deciding what the standard of achievement should be, then sampling and comparing the results with the expected performance . If the standard is not reached, remedial action should be taken and then a re - audit carried out to ensure that the expected improvement has been achieved . Examples include: the information on imaging requests, patient perception of the quality of communication, completion of consent forms, contractibility of staff, completion of report addenda, audit of teaching quality, access to confidential patient information on pacs, etc . Audit is a powerful tool to improve all aspects of the working of a radiology department and the quality of care it provides to patients . Good communication ensures better and safer outcomes for patients, and a more satisfactory working environment for staff.
This review focuses on the opioid growth regulatory system and its implications for the pathobiology of diabetes . Zimmerman's honor jointly sponsored by the american academy of ophthalmology and the american association of ophthalmic oncologists and pathologists, chicago, illinois, october 19, 2014 . Zimmerman was the founder of modern ophthalmic pathology having served at the armed forces institute of pathology for 52 years . He was mentor to many practicing ophthalmic pathologists and the recipient of numerous national and international honors ., it emphasizes its implications for the pathobiology of diabetic complications including impaired wound healing, abnormal corneal sensitivity, and dry eye . Figure 1 highlights two of the main and opposing characters in this story: the naturally occurring opioid growth factor (ogf), [met]-enkephalin, and its pharmacologic antagonist, naltrexone (ntx). They bind to specific receptors and they perform various biologic functions including analgesia, cardiovascular control, respiration, behavior, learning and memory, emotion, and cell division and growth . This review focuses on the latter function of regulation of growth and cell division by the opioid growth regulatory system . This system, also called the ogf - ogfr axis, is comprised of two major components: opioid growth factor (ogf) itself and its specific opioid growth factor receptor (ogfr). It is a pentapeptide with the sequence tyr - gly - gly - phe - met . Its action is to depress cell division when bound to the other key component of the opioid growth regulatory system, the specific opioid receptor for ogf, not surprisingly termed ogfr . The ogfr has been cloned and sequenced in the human, rat, and mouse . Ogf bound to this specific receptor is the only such opioid that has an effect on cell division . Ogf is tonically produced so that usually its level in tissues is neither maximized nor minimized . As a result of this characteristic, manipulation of the opioid growth regulatory system, either by the addition of exogenous ogf or by blocking its receptor, can decrease or increase cell division, respectively . Ogf usually is produced in an autocrine or paracrine manner, meaning that it is manufactured by the cells that will be modulated by it or by their neighbors . Nevertheless, systemic levels may be of importance for diabetic complications [1, 2]. The opioid growth regulatory system is truly an ancient cellular regulatory mechanism that has been conserved from bacteria to humans [3, 4]. This system can modulate growth and development in embryologic, normally dividing, healing, and even neoplastic tissues (basically, any cell that has the potential to divide). The authors' observations suggest that it does not overdrive cell division in tissues that have attained contact inhibition of cell division . When ogf is bound to its specific receptor, ogfr, cell division is suppressed . Figure 2 demonstrates several ways in which the relationship between ogf and its receptor can be manipulated to regulate cell division . For example, addition of exogenous ogf or an increase in the number of its receptors downregulates cell division . Conversely, one can increase cell division by decreasing the interaction of ogf with its receptor, either by decreasing the production of ogf or its receptor or by utilizing a blocking agent, like the strong opioid antagonist, naltrexone (ntx), to directly block ogf - ogfr interaction . The presence of the opioid growth regulatory system has been demonstrated in the corneal epithelium of all vertebrate orders including mammals, birds, reptiles, amphibians, and fish, some of which are demonstrated in figure 3 . Over the past 25 years, the authors' research team has delineated the role of the opioid growth regulatory system in the homeostasis and healing of ocular tissues . More recently, as will be discussed shortly, it has been shown to play a role in the pathobiology of diabetic ocular complications, such as depressed epithelial wound healing, abnormal corneal sensitivity, and dry eye, and in the nonocular complication of delayed healing of diabetic cutaneous wounds . In the corneal epithelium, ogf appears to be produced in an autocrine manner . For example, immunohistochemical examination of the corneal epithelium in the peripheral cornea, limbus, and conjunctiva has demonstrated the presence of preproenkephalin, the precursor to ogf, within the corneal epithelium in these regions thereby supporting the autocrine production of ogf by the corneal epithelial cells . As seen in figure 4, corneal explants in culture demonstrate that the opioid growth regulatory system modulates the outgrowth of homeostatic corneal epithelium, with exogenous ogf retarding and disorganizing the outgrowth and cell division of the epithelium and ntx accelerating outgrowth in reference to control explants without altering the normal outgrowth pattern [7, 8]. If the opioid growth regulatory system can modulate corneal epithelial migration and cell division in tissue culture, what is its impact on homeostatic corneal epithelium in vivo? Figure 5 documents the ability of treatment with ogf to suppress dna synthesis in the cornea of the living rat . (please note that all findings or data cited in this review are significant at a minimum of p <0.05, but, for the sake of brevity, no specific significance values will be presented except as cited in figures and their captions .) If blockade of the opioid growth regulatory system positively impacts epithelial outgrowth in tissue and organ culture and increases dna synthesis in vivo, how would it impact corneal epithelial wound healing? Indeed, treatment with either systemic or topical ntx results in an increased rate of rat corneal epithelial wound healing [911]. As illustrated in figure 6, either intraperitoneal or topical ntx significantly increases the rate of reepithelialization of standardized rat corneal epithelial wounds . Similarly, rabbit corneal epithelial wound healing also is increased by blockade of the opioid growth regulatory system by topical ntx [911]. Using the gene gun, one can specifically determine the role of the interaction of ogf and its receptor (ogfr) in regulating epithelial wound healing by delivering sense or antisense ogfr cdna into corneal epithelial cells (figure 7) [12, 13]. Overexpression of ogfr results in delayed wound healing of rat corneal epithelial abrasions and suppression of ogfr production using antisense cdna results in expedited wound healing . Is the increased corneal epithelial wound healing that is achieved through manipulation of the opioid growth regulatory system accompanied by proliferative abnormalities in the epithelium? In order to answer this question, animals were treated in vivo for one week with ntx [8, 14]. Figure 8 demonstrates that dna synthetic cells increased by 6985% in response to ntx treatment . Cellular packing density was increased; however, no toxicity or proliferative pathology was seen . There was negligible apoptosis or necrosis . Just as the opioid growth regulatory system regulates epithelial wound healing in animals, studies of organ cultured human corneas subjected to epithelial wounds demonstrated its impact on human epithelial wound healing . Figure 9 illustrates accelerated human corneal epithelial healing of organ cultured corneas grown in culture medium supplemented with 10 m ntx . Conversely, supplementation of culture medium with ogf suppresses epithelial wound healing (figure 9). Finally, ogf and ntx impact cultured human corneal dna synthesis as one might anticipate with increased synthesis resulting from ntx treatment and dna synthesis suppression from ogf supplementation . Diabetes is the leading cause of blindness among working - age adults in the united states . In 2012, 29.1 million americans or 9.3% of the population had diabetes . The prevalence of diabetes rises to 25.9% of american seniors . Among the complications of diabetes are associated with keratopathy that is reflected in delayed corneal epithelial wound healing [1719], abnormal corneal sensitivity [1822], and dry eye [20, 21, 23, 24]. Elevated levels of ogf, [met]-enkephalin, have been found in the plasma of diabetic patients [2527]. Elevated ogf levels also have been found in genetically obese diabetic (db / db) mice, which are used as a model for type 2 diabetes [2830]. Moreover, ogf and ogfr have been found in the corneal epithelium in diabetic animals . It was postulated that abnormalities of opioid regulation could contribute to the complications of diabetes and that blockade of the opioid growth regulatory system by ntx might reverse or ameliorate these complications . The relevance of the opioid growth regulatory system to the following diabetic corneal complications: delayed epithelial wound healing, abnormal corneal sensitivity, and dry eye will be discussed separately . During the course of their disease, seventy percent of diabetics will suffer from diabetic keratopathy, which includes recurrent erosion, delayed wound healing, edema, and even ulcers [3234]. These complications may occur spontaneously or follow specific insults, such as ocular surgery [3639]. Immunocytochemistry confirms the presence of ogf and ogfr in the corneal epithelium of diabetic animals . In order to determine whether blockade of the opioid growth regulatory system would improve epithelial wound healing in diabetes, treatment with intraperitoneal ntx twice daily resulted in a marked increase in the rate of corneal reepithelialization compared to untreated control animals . Figure 10 illustrates the impact of ntx treatment on the rate of corneal epithelial healing in untreated diabetic rats . Untreated diabetic animals healed at a rate that was significantly worse than that in normal controls . On the other hand, diabetic ntx - treated animals healed at a rate equal to that of the normal controls . Therefore, having demonstrated the ability of ntx to increase cell proliferation, it is not surprising that ntx treatment increased dna synthesis in unwounded diabetic rat corneas 4-fold in the basal epithelium of the peripheral cornea, 3.5-fold in the limbal region, and 8-fold in the conjunctiva compared to control animals . Does glucose control improve epithelial wound healing in diabetic rats and if so does ntx have an insulin - like effect on corneal epithelial wound healing? In order to answer these questions, corneal epithelial wound healing in untreated diabetic rats was compared to that in animals treated with insulin minipumps . At 40 hrs after wounding, untreated diabetic (db) rats had significantly larger residual epithelial defects than the controls (either nondiabetic or db - insulin - treated rats). This and other studies demonstrated clearly that intensive therapy with insulin, leading to normoglycemia in rats with diabetes, does prevent the delay in wound healing of ocular surface epithelium observed in poorly controlled diabetic animals . Given that systemic control of diabetes facilitates corneal epithelial wound healing, does topical insulin have an effect independent of systemic glucose control? In rats that have been diabetic for 9 or 11 weeks, topical insulin was administered four times daily for 7 days to wounded corneas . Diabetic animals treated with vehicle alone had wounds that were 35% larger than those in healthy vehicle - treated animals [40, 41]. Topical insulin treatment resulted in epithelial wounds that were 19% to 60% smaller than diabetic vehicle - treated ones . There was no insulin effect on healthy rat epithelium, and there was no effect on corneal thickness, iop, apoptosis, or serum glucose levels . Thus, topical insulin treatment is effective in reversing the delayed epithelial wound healing characteristic of diabetic animals . What is the effect of ntx treatment on corneal epithelial wound healing in diabetic animals? In preparation for testing ntx treatment for epithelial defects, a toxicity study of topical ntx was performed in insulin - controlled diabetic rats (figure 11). There was no difference from normal rats or insulin - treated diabetic controls in iop, corneal thickness, endothelial cell number, or epithelial apoptosis, necrosis, or organization . Similarly, topical ntx proved as effective as intraperitoneal treatment for more rapidly healing epithelial defects in diabetic animals, and topical insulin was equally effective and safe as topical ntx for this purpose [40, 41, 43]. Combining topical insulin and topical ntx was not more effective than either one used independently [40, 41, 43, 44]. In short, there is a possibility that insulin and ntx have their effect through similar mechanisms in diabetic animals or that each medication has the potential to maximize epithelial wound healing in these animals, leaving no opportunity for further increase by the complementary modality . Furthermore, insulin has no effect on epithelial proliferation in normal animals, and ntx has no impact on blood glucose levels in diabetic animals . These data were compared to that involving the healing of corneal epithelial wounds in untreated or systemic insulin - treated diabetic rats given topical ntx (figure 12). In both treatment groups, topical ntx significantly increased the rate of epithelial wound healing in contrast to the situation when insulin and ntx are combined in topical administration . One possible explanation for the difference in results obtained relative to the route of insulin administration (systemic versus topical) may be the inability of systemic insulin to reach the tear film in a concentration equivalent to that obtained with topical administration . One should note that although ntx has been discussed relative to induced type 1 diabetes, it also is effective in facilitating corneal epithelial wound healing in obese db / db mice with type 2 diabetes on a genetic basis . The potential toxicity of ntx applied topically four times daily for 7 days in concentrations of 10 to 10 m was evaluated in vivo in intact and abraded corneas of insulin controlled or uncontrolled diabetic rats . Ocular surface morphology, intraocular pressure, corneal thickness, and corneal sensitivity were evaluated . No toxicity from ntx treatment was found . In summary, in diabetic animals, topical ntx restores corneal epithelial wound healing to levels comparable to systemically or topically insulin - treated animals without apparent epithelial toxicity . Diabetic corneal neuropathy, particularly as assessed by confocal microscopy, correlates with peripheral neuropathy [4652]. Corneal aesthesiometry (measuring corneal sensitivity to touch using progressively stiffer filaments, which are von frey hairs) also can be helpful in the clinical assessment of diabetic corneal neuropathy [20, 50, 53, 54]. Corneal nerve damage can be induced by obesity related to diet or to type 2 diabetes . Moreover, diabetic corneal nerve injury is repairable as evidenced by the fact that corneal nerve regeneration has been demonstrated after simultaneous kidney and pancreas transplantation and other therapies . In order to evaluate the reversibility of corneal diabetic neuropathy, rats having eight weeks of induced diabetes were treated with 1 or 5 days of four times daily ntx at 10 m concentration (figure 13) [42, 59]. Corneal sensitivity was restored to normal levels beginning one hour after termination of drug exposure and extending for at least 4 days thereafter . Conversely, control diabetic animals maintained sensitivity scores that were 1.5- to 2.0-fold less than both the normal and ntx - treated groups . The ntx effect resulting in normalization of corneal sensitivity ended after 120 hrs, for animals treated for one day, and 192 hrs following discontinuation of a 5-day treatment period with four times daily ntx . At those respective time points, corneal sensitivity reverted to being 1.9-fold less than normal animals and comparable to control diabetic animals . Ntx also is effective in restoring corneal sensitivity to normal levels in obese db / db type 2 diabetic mice . In summary, topical ntx treatment restores corneal sensitivity to normal levels in both type 1 and type 2 diabetic rats . These findings implicate the opioid growth regulatory system in the pathobiology of diabetic corneal neuropathy and are consistent with other studies cited above, which suggest that diabetic corneal neuropathy can be reversible . Dry eye is more common in diabetic patients and correlates with poor glycemic control [21, 23, 60]. Moreover, diabetic dry eye is more common in individuals with diabetic retinopathy of increased severity . Apparently normal rats have periods during which there is a spontaneous decrease in tear production (figure 14). It was determined that one drop of 10 m ntx restores tear production to normal levels for up to 48 hrs in such animals . If right and left eyes are compared after one drop of 10 m ntx in the right eye, there is no effect on the contralateral eye . Conversely, neither one drop of 10 m ntx nor vehicle had any impact on tear production that already was at a normal level . There was no difference in corneal sensitivity during periods of normal or reduced tear production over a 20-fold difference in force using von frey hairs, which are used to test for corneal sensitivity to touch . Although ntx has no ability to raise tear production in rats with normal tear secretion, one drop of 10 m ogf, [met]-enkephalin, significantly reduces tear production in rats with initially normal levels (figure 15). Thus, ntx blockade of the opioid growth regulatory system appears to have the ability to raise tear production to normal levels in nondiabetic rats having a period of depressed tear production . These data support the concept of opioid growth regulatory system modulation of tear production even in nondiabetic rats . Dry eye was evaluated in rats having type 1 diabetes of 8-week duration treated with four times daily topical ntx at 10 m concentration . Untreated diabetic rats had tear production reduced by 32% to 53% compared to normal or to ntx - treated animals . In contrast, diabetic rats treated with ntx had tear production similar to normal rats extending for at least 3 days following the termination of treatment . By 96 hours after termination of treatment, tear production had decreased again to 22% to 59% less than normal animals, thereby emphasizing how effective the previous ntx treatment had been . Figure 16 illustrates tear production in wild - type or db / db type 2 diabetic mice given one drop of 10 m ntx (a) or only vehicle (b). Note the rise in tear production to normal levels with ntx treatment until about 72 hrs after treatment (a). (b) on abnormal tear production at all times tested . In summary, these findings support a role for opioid growth regulatory system in the pathobiology of abnormal diabetic tear production in that ntx treatment restores tear production to normal levels for up to 3 days following discontinuation of the therapy in type 1 or type 2 diabetic rats . The research reported above demonstrates that the opioid growth regulatory system is important in the pathobiology of diabetic ocular surface disease, in that manipulating the system through blockade of the ogfr with ntx restores corneal epithelial wound healing, ocular sensation, and tear production to normal levels in diabetic animals . Nevertheless, the question arises as to whether these findings are only of localized importance restricted to the ocular region or whether they are manifestations of a systemic abnormality of the opioid growth regulatory system secondary to diabetes . Delayed or incomplete healing of cutaneous wounds, particularly foot ulcers, is an important systemic diabetic complication . For example, diabetic foot ulcers are said to be one of the most costly and devastating complications of diabetes mellitus and affect 15% of diabetic patients during their lifetime . Therefore, the impact of opioid growth regulatory system blockade on cutaneous wound healing in diabetic rats was evaluated to test the effectiveness of ntx on this important and quantifiable indicator for the systemic complications of diabetes . The following research was performed by jessica immonen, a graduate student then and now doctor immonen, at the department of anatomy at rocky mountain university of health professions, as the basis for her masters and doctorate degrees . Working with drs . Immonen evaluated the impact of ntx 10 m, ntx 10 m, or ntx 10 m in sorenson's phosphate buffer, lubricant, moisturizing cream, or dimethylsulfoxide applied to the skin surface three times daily on dna synthesis in the skin of normal or type 1 diabetic rats (figure 17). She discovered that cutaneous dna synthesis in unwounded normal rats was elevated by 43% to 132% in response to ntx in any of the four vehicles compared to normal baseline . Ntx applied three times daily topically to dorsal skin of db rats elevated labeling index (li) by 103147% in lubricant and by 8589% in moisturizing cream . Note the dna synthesizing cells in the photomicrographs in figure 17 . A model of 6 mm full - thickness cutaneous wounds was used to investigate the healing response to one of the previously tested concentrations of ntx, 10 m, in either moisturizing cream (mcn) or vehicle alone (figure 18). Within 3 days of treatment initiation, normal rats treated with once or three times daily ntx had wounds 30% and 11% smaller at the respective dosages than control animals . Diabetic animals treated with ntx in moisturizing cream had wounds 13% to 57% smaller than diabetic controls . When normal (n) rats with standard skin wounds were treated three times daily with 10 m ntx in moisturizing cream, they healed 626% faster than normal control rats . Diabetic ntx - treated rats had wounds 6289% smaller than diabetic controls (figure 19). Diabetic control animals have delayed expression of fibroblast growth factor-2 (fgf-2) and vascular endothelial growth factor (vegf). Similar findings are noted for the expression of -smooth muscle actin (sma) in capillaries . In summary, topical ntx accelerates cutaneous wound closure, at least in part, by stimulating expression of angiogenic factors within healing tissue of diabetic animals . Obviously, there is the potential for ntx treatment to have a significant impact on facilitating the initial closure of such wounds in diabetic patients . Ntx has a more pervasive impact on the overall process of wound healing beyond just wound closure . For example, birefringence of sirius red - stained healing granulation tissue revealed increased collagen formation and maturation in ntx - treated animals (figure 20). Finally, inadequate wound healing at 60 days after wounding in diabetic control animals is further demonstrated by reduced tensile strength in comparison to control nondiabetic animals or to ntx - treated diabetic wounds, which have tensile strength similar to normal controls . It must be noted that ntx treatment of standardized cutaneous wounds in the spontaneously diabetic db / db mouse model of type 2 diabetes also results in an increased labeling index and more rapid wound closure comparable to normal levels (figure 21). It is interesting that, in these db / db mice, epithelium was hyperplastic in the skin of unwounded ntx - treated normal and db rats compared to their counterparts (figure 22). In summary, opioid growth regulatory system blockade by ntx significantly and positively impacts cutaneous wound healing in diabetic animals thereby demonstrating its involvement in the pathobiology of systemic, nonocular diabetic complications . The opioid growth regulatory system is a phylogenetically ancient growth regulatory system that has been conserved across multiple existing animal phyla and, specifically, in ocular tissue . It regulates cell division in all cell types capable of dividing that have been tested to date including normal, healing, embryologic, and neoplastic tissues . The function of the opioid growth regulatory system appears to be disordered in diabetic animals, and its function can be restored with ntx treatment to normalize corneal epithelial wound healing, corneal sensitivity, and tear production in models of both type 1 and type 2 diabetes . Moreover, studies by our team relative to cutaneous wound healing in diabetic animals further support the hypothesis that the opioid growth regulatory system is disordered relative to wound closure, wound maturation, and the restoration of tissue tensile strength in nonocular tissue, specifically the skin . Thus, our findings support the hypothesis that the function of the opioid growth regulatory system is diffusely and abnormally impacted by diabetes . Where do we go from here? Naltrexone has been shown to be well tolerated in short - term ocular application in healthy human volunteers . Further studies leading to the topical use of ntx in wound healing are required . Moreover, in our opinion, a more global study, to determine the impact of ntx therapy on the prevention of systemic complications of diabetes, is indicated.
Osteoporosis is a disease characterized by the structural deterioration of bone and low bone mass . The world health organization defines osteoporosis as a bone mineral density (bmd) value of 2.5 standard deviations or more below the young adult mean.1 an estimated 10 million patients in the united states have osteoporosis, and its prevalence is expected to continue to rise in the world population.2 degenerative spondylolisthesis and spinal stenosis, as well as vertebral compression fractures, have been reported at an increased rate in patients with osteoporosis,3 and the disease requires special consideration for any older patient undergoing spine surgery . Obtaining optimal fixation in the osteoporotic spine is technically challenging; osteoporosis may also be associated with a poor fusion rate, with rates as low as 56% in elderly patients.4 however, for appropriately selected patients, the presence of osteoporosis is not a contraindication to surgical intervention . Very little available literature exists specifically evaluating the clinical and radiographic outcomes in patients with osteoporosis undergoing transforaminal lumbar interbody fusion (tlif). In fact, to our knowledge, no previous study has examined the effect that osteoporosis may have on rates of interbody cage subsidence, migration, pedicle screw loosening, or iatrogenic fractures in patients undergoing instrumented tlif . Therefore, we sought to compare the radiographic and clinical outcomes of tlif in patients with and patients without osteoporosis . After institutional review board approval, we retrospectively reviewed all patients aged 50 years or older undergoing instrumented tlif for any indication at one of three institutions between july 2004 and june 2014 . Only patients with immediate postoperative and follow - up radiographs and computed tomography (ct) scans available for evaluation of the interbody cage were included in the study . We excluded all patients without at least a 6-month follow - up ct scan . Utilizing a technique similar to that described by lee et al,5 we measured ct hounsfield units (hus) on the lumbar vertebrae for all included patients using preoperative ct scans . Patients were then stratified as either osteoporotic or nonosteoporotic based on average lumbar vertebral body hu values . The hu cutoff values were previously determined from a separate study at our institution and found to correlate with bmd obtained from dual - energy x - ray absorptiometry (dexa) scans (p.m. formby et al, personal communication). Patients with hu 112.4 were considered osteoporotic, and those with hu> 112.4 were considered nonosteoporotic . Plain radiographs and ct scans were evaluated for evidence of implant subsidence, migration, interbody fusion, iatrogenic fracture, or loosening of posterior pedicle screw fixation . Patient medical records were then reviewed for postoperative symptoms, which were further subdivided into persistence of symptoms or recurrence of symptoms . We used the student t test to evaluate continuous variables and mid - p exact tests for dichotomous data, and p values 0.05 were considered statistically significant . Twenty - five (19.5%) of these patients were designated as osteoporotic based on the postoperative ct hu measurements versus 103 (80.4%) patients without osteoporosis . We excluded 40 patients who did not have at least a 6-month follow - up ct imaging, leaving 18 (20.5%) patients with osteoporosis and 70 (79.5%) patients without osteoporosis . These patients had a mean radiographic follow - up of 35.8 27.9 months . The mean age was significantly higher in the osteoporotic group (65.2 years) versus the nonosteoporotic group (56.9 years, p <0.0001). Males comprised 50 and 64.3% of the osteoporotic and nonosteoporotic patient groups, respectively, with a combined mean age of 58.5 7.9 years . We found no significant differences between the osteoporotic and nonosteoporotic groups with regard to sex (p = 0.28), body mass index (27.6 versus 29.9, p = 0.12), number of identified medical comorbidities (2.3 versus 1.8, p = 0.15), total number of vertebral levels fused (1.9 versus 1.8, p = 0.56), or fusion rate (83.3 versus 88.6%, p = 0.56). There were no statistically significant differences between groups for gross implant migration in the coronal or sagittal planes (11.1 versus 1.4%, p = 0.11), pedicle screw loosening (22.2 versus 8.6%, p = 0.14), revision surgery (16.6 versus 14.3%, p = 0.78), or postoperative symptoms (44.4 versus 50.0%, p = 0.69; tables 1, 2). There were no statistically significant differences between the osteoporotic and nonosteoporotic groups in persistent or recurrent symptoms, though the rate of recurrence in the nonosteoporotic group was three times higher (persistent symptoms: 38.9 versus 30.0%, p = 0.47, respectively; recurrent symptoms: 5.6 versus 18.6%, p = 0.18, respectively). Abbreviations: bmi, body mass index; ca / vit d, calcium / vitamin d; dexa, dual - energy x - ray absorptiometry; hu, hounsfield units . Note: the only significant differences between the osteoporotic and nonosteoporotic groups were for mean patient age and hu measurements . Note: the rates of interbody cage subsidence, iatrogenic fracture, and overall complication rates were higher in the osteoporotic group . Significantly higher rates of subsidence (5.6 2.4 [72.2%] versus 5.2 1.9 [45.7%], p = 0.05) and iatrogenic fractures (16.7 versus 1.4%, overall, the osteoporotic population had significantly higher radiographic complication rates compared with patients without osteoporosis (77.8 versus 48.6%, p = 0.03; table 2). As the average life expectancy continues to increase in the united states, spine surgeons should expect to manage a greater proportion of elderly patients with degenerative spinal conditions . Concomitant with increasing age is an associated increase in the prevalence of osteopenia and osteoporosis . In a study of patients over the age of 50 years undergoing spinal surgery in south korea, chin et al found osteopenia and osteoporosis rates of 41.4 and 51.3%, respectively, based on dexa.3 despite the growing population of elderly patients, however, few studies have evaluated the role that low lumbar bmd may have in the complication rates and overall outcomes of patients undergoing lumbar interbody fusion . We found that a minority of patients (21.5%) undergoing tlif had any dexa information available at the time of surgery, despite advancing age and clinical risk factors for osteoporosis . Utilizing hu measurements to assess lumbar vertebral body bmd, we found that of the patients with hu values consistent with osteoporosis, only 27.8% had any workup for osteoporosis prior to or following their tlif . This finding is concerning, particularly because we found a higher overall radiographic complication rate in patients with osteoporosis undergoing tlif, and it suggests that patients are undergoing spinal fusion surgery without being appropriately evaluated for low bmd . Although there were few patients with osteoporosis in this study, we found an increased incidence and amount of cage subsidence (fig . 1), a higher rate of iatrogenic fractures, and more overall radiographic complications in patients with osteoporosis . Though our data did not reach statistical significance, we also found a greater propensity for implant migration and posterior pedicle screw loosening . (a) immediate postoperative and (b) 2-year follow - up sagittal computed tomography (ct) scans of a 53-year - old man who underwent l4l5 transforaminal lumbar interbody fusion . The patient had hounsfield unit (hu) measurements on immediate postoperative ct scan consistent with osteoporosis (average l4 hu = 105.7). Interbody cage subsidence into the superior end plate of l5 with interspace collapse is evident (9.9 mm), with evidence of increased fusion mass in the anterior column . However, at 26-month clinical follow - up, he had no recurrence of symptoms . However, it is important to note that the increased overall radiographic complication rate did not necessarily portend worse clinical outcomes for patients with osteoporosis . Patients with osteoporosis and patients without osteoporosis underwent similar rates of revision surgery (16.7 versus 14.3%, respectively), and patients without osteoporosis actually had a higher prevalence of postoperative symptoms following tlif, particularly when stratifying the constellation of symptoms based on persistence versus subsequent recurrence of symptoms: we found that the rate of recurrent symptoms was higher in the nonosteoporotic group (18.6 versus 5.6%, respectively), although this finding also did not reach statistical significance . It is unclear, however, why this trend was observed, and it may be related to the fact that we only included patients with six - month or later postoperative ct scans in our study, which may have biased our clinical data with an increased proportion of patients with postoperative symptoms . Despite a higher interbody cage subsidence rate in the osteoporotic group, which in turn would theoretically lead to loss of foraminal height and eventual recurrence of radiculopathy, there did not appear to be any association between osteoporosis and recurrence of symptoms . We are unaware of any other studies specifically evaluating postoperative radiographic complications in patients with osteoporosis undergoing tlif . Previous studies have drawn conflicting conclusions regarding whether elderly patients are at increased risk for postoperative complications following lumbar fusion . Carreon et al found a 79.6% rate of postoperative complications in patients 65 years old at the time of posterior lumbar instrumented fusion.6 however, cassinelli et al found a much lower rate of major complications (3%) and minor complications (31%) in a similar cohort of patients 65 years undergoing instrumented and noninstrumented lumbar spine arthrodesis.7 similarly, cavagna et al evaluated patients> 65 years undergoing instrumented lumbar fusions and found good clinical outcomes and no serious postoperative complications or reoperations in this cohort.8 however, they did find a 10.3% rate of implant failure (two screws and two rods failed) at 2-year or later follow - up . Glassman et al investigated clinical outcomes of patients 65 years old compared with those <65 years old undergoing single - level instrumented posterolateral lumbar arthrodesis; the authors found that the older population had similar clinical and health - related quality - of - life improvements when compared with the younger cohort.9 however, the older cohort experienced significantly increased rates of serious (38 versus 17%) and overall adverse postoperative events (56 versus 36%), although there was no increased risk of revision or reoperation between these groups . Likewise, okuda et al did not find major differences in clinical outcomes between patients 70 years old and those <70 years old undergoing instrumented lumbar interbody fusion.10 these authors found no increased risk or instrument failure between groups, although they did find lower rates of fusion and higher rates of collapsed union and delayed union in the elderly group.10 inherent to any small retrospective study, the possibility of type ii statistical error limits the generalizability of our analysis . The proportion of patients with osteoporosis in our study was low, which could be a source of bias . Our study was also limited by the quality of the medical records, as well as available radiographs and ct scans . In addition, despite a minimum age of 50 years for inclusion in this study, our patients were still relatively young and healthy, with few comorbidities and a mean age of only 58.5 years . We looked at a heterogeneous group of patients with disparate preoperative diagnoses undergoing tlif for any reason, which could contribute to bias in this study . The use of hu for measurement of bone density has not been validated in the literature, with few reports of its use to date . Further prospective studies are needed to evaluate the role that osteoporosis plays in complication rates and overall clinical outcomes in patients undergoing lumbar fusion surgery . In conclusion, we found significantly increased rates of cage subsidence, iatrogenic fractures, and overall radiographic complications in patients with osteoporosis undergoing tlif . However, these radiographic complications did not predispose patients with osteoporosis to worse clinical outcomes, and larger, prospective studies are needed to further evaluate lumbar fusion surgery in patients with osteoporosis.
Light - cured composite resins have been considered a material of major importance in restorative dentistry due to their esthetic properties . However, achievement of clinical and esthetic success with this material involves several aspects, including the curing technique . The process of curing of composite resins occurs in three main phases: pre - gel, gel point and post - gel . During the pre - gel phase, the material may flow and undergo molecular rearrangement, in order to compensate shrinkages forces . During this phase thereafter, the resin passes from the flow state (pre - gel) to the viscous state (post - gel), which establishes its gel point . During the post - gel phase, the resin presents a high modulus of elasticity, loses its flowing ability and transmits the stress yielded by polymerization shrinkage to the tooth - restoration interface5,15,16 . At this stage, there is a predominance of cross links in the polymer structure . Studies have demonstrated that curing technique may influence the polymerization shrinkage of composite3,4,8 - 11,15 . The incomplete curing of composite resins is associated to a reduction in their mechanical properties and biocompatibility, increased content of residual monomers and altered clinical performance due to esthetic impairment, with high tendency to surface staining and possibility of marginal leakage . Therefore, different curing techniques have been suggested to overcome the problems related to polymerization shrinkage, especially postoperative sensitivity and marginal leakage3 - 7: stepped technique (2 stages) low light intensity is applied for a determined period, followed by high light intensity for a certain additional period . Ramped technique (progressive) initial low light intensity is applied, which is gradually increased for a certain period until it reaches a high final value that s maintained until completion of exposure . Pulsed - delay technique (delayed pulse) initial low light intensity is applied for a certain period, which is sufficient to allow curing of the surface . Surface finishing and polishing should be performed during this period, followed by a second exposure at a higher light intensity . The aim of these techniques, which comprise initiation of light - curing at a low light intensity and a delay time, is to allow the occurrence of a more evident pre - gel phase, which would provide a low rate of conversion of monomers and thus allow material flow, yielding low internal stress from shrinkage and providing a good marginal adaptation . At the final stage of these techniques, completion of curing at a high light intensity would provide a proper degree of conversion, which is required for the achievement of satisfactory physical and mechanical properties4,6,7,10,11,18 . The measurement of microhardness is an indicator of the mechanical, physical and biological properties of a restorative material7 . Application of this test in depth as a parameter for analysis of curing is justified, since studies have indicated a good correlation between the knoop hardness number (khn = kg / mm) and infrared light spectroscopy, a direct method that evaluates the degree of conversion of monomers6 . The aim of this study was to evaluate in vitro the effect of four light - curing techniques on the depth of cure of a composite resin by microhardness testing . The null hypothesis was that there is no difference between different curing techniques among different depths of cure from the composite resin surface . To test this null hypothesis, a factorial study 44 was conducted and the factors under study were cure technique at 4 levels: traditional / continuous (t), ramped (r), stepped (s) and pulse - delay (pd), and depth of cure at 4 levels 0.1, 1.0, 2.0 and 4.0 mm from the lighted surface, resulting in 16 groups . The specimens were longitudinally sectioned, polished and microhardness measurements were done at established distances from the surface . The specimens were fabricated in cylindrical cavities (3.0 mm of diameter 7.0 mm of height) prepared in polyvinyl chloride (pvc) plates (figure 1). Ten specimens were fabricated for each light - curing method: traditional (continuous), ramped (exponential), stepped (two stages) and pulsed - delay (delayed pulse). The traditional curing technique was applied with the curing lite 2500 unit (3m / espe). The intensity was kept continuous at 600 mw / cm for a constant and continued period of 40 s. the ramped technique is one of the functions of the optilux 501 unit (demetron, kerr, orange, ca, usa). By this function, the light intensity applied was exponential, reaching 1000 mw / cm during the 10 initial s, followed by additional 10 s at this intensity . The stepped technique (s) was applied with the vip unit (bisco inc ., the light intensity employed for this technique was followed by two stages, being the first at low intensity and the last at a high light intensity . The values of this intensity were 200 mw / cm for 10 s and 600 mw / cm for 30 s, respectively . The pulsed - delay technique comprised treatment at high and low light intensities with a time interval . The intensities applied were 100 mw / cm for 5 s, followed by a time interval of 3 min and a final light - curing at 600 mw / cm for 30 s. after curing, the pvc plates were longitudinally sectioned throughout the specimens with diamond discs (diamond wafering blade, 15hc #11 - 4244; buehler ltd ., lake bluff, il, usa) coupled in a saw (the cut surfaces of the specimens were finished and polished in a polishing machine (arotec apl 4; arotec ind . So paulo, sp, brazil), with serial silicon carbide papers of decreasing abrasiveness (grits 320, 400, 600 and 1200) and felt (6 m, 3 m, 1 m) with a diamond suspension (buehler metadi; buehler ltd .) With grits corresponding to felts . The specimens were stored under moist conditions at 37c for 24 h before the microhardness determination . Depth of cure was determined by a microhardness tester (future tech with software fm - ars) with a 25 g load for 5 s. this device provided the knopp hardness (kg / mm). For each half of specimen was made three indentations, resulting in six indentations per specimen . They were made at the depths of 0.1, 1.0, 2.0 and 4.0 mm from the surface where the polymerization was made (figure 1). The six indentations were averaged (n=10 for each depth). The ratio of hardness at 1.0, 2.0 and 4.0 mm from the surface related to the value at 0.1 mm was calculated and the value 0.80 (80%) is considered acceptable . The variables under study were cure technique at 4 levels and depth in the specimen at 4 levels with 10 repetitions, resulting in a sample number of 4 4 10 . Data were subjected to two - way anova at the significance level of 5%, for observation of the relationship between microhardness, depth and interaction between the light - curing methods investigated, followed by tukey test ., cary, nc, usa) was used for the analysis and the significance level set at 5% . The null hypothesis was that there is no difference between different curing techniques among different depths of cure from the composite resin surface . To test this null hypothesis, a factorial study 44 was conducted and the factors under study were cure technique at 4 levels: traditional / continuous (t), ramped (r), stepped (s) and pulse - delay (pd), and depth of cure at 4 levels 0.1, 1.0, 2.0 and 4.0 mm from the lighted surface, resulting in 16 groups . The specimens were longitudinally sectioned, polished and microhardness measurements were done at established distances from the surface . The specimens were fabricated in cylindrical cavities (3.0 mm of diameter 7.0 mm of height) prepared in polyvinyl chloride (pvc) plates (figure 1). Ten specimens were fabricated for each light - curing method: traditional (continuous), ramped (exponential), stepped (two stages) and pulsed - delay (delayed pulse). The traditional curing technique was applied with the curing lite 2500 unit (3m / espe). The intensity was kept continuous at 600 mw / cm for a constant and continued period of 40 s. the ramped technique is one of the functions of the optilux 501 unit (demetron, kerr, orange, ca, usa). By this function, the light intensity applied was exponential, reaching 1000 mw / cm during the 10 initial s, followed by additional 10 s at this intensity . The stepped technique (s) the light intensity employed for this technique was followed by two stages, being the first at low intensity and the last at a high light intensity . The values of this intensity were 200 mw / cm for 10 s and 600 mw / cm for 30 s, respectively . The pulsed - delay technique comprised treatment at high and low light intensities with a time interval . The intensities applied were 100 mw / cm for 5 s, followed by a time interval of 3 min and a final light - curing at 600 mw / cm for 30 s. after curing, the pvc plates were longitudinally sectioned throughout the specimens with diamond discs (diamond wafering blade, 15hc #11 - 4244; buehler ltd ., lake bluff, il, usa) coupled in a saw (the cut surfaces of the specimens were finished and polished in a polishing machine (arotec apl 4; arotec ind . So paulo, sp, brazil), with serial silicon carbide papers of decreasing abrasiveness (grits 320, 400, 600 and 1200) and felt (6 m, 3 m, 1 m) with a diamond suspension (buehler metadi; buehler ltd .) With grits corresponding to felts . The specimens were stored under moist conditions at 37c for 24 h before the microhardness determination . Depth of cure was determined by a microhardness tester (future tech with software fm - ars) with a 25 g load for 5 s. this device provided the knopp hardness (kg / mm). For each half of specimen was made three indentations, resulting in six indentations per specimen . They were made at the depths of 0.1, 1.0, 2.0 and 4.0 mm from the surface where the polymerization was made (figure 1). The six indentations were averaged (n=10 for each depth). The ratio of hardness at 1.0, 2.0 and 4.0 mm from the surface related to the value at 0.1 mm was calculated and the value 0.80 (80%) is considered acceptable . The variables under study were cure technique at 4 levels and depth in the specimen at 4 levels with 10 repetitions, resulting in a sample number of 4 4 10 . Data were subjected to two - way anova at the significance level of 5%, for observation of the relationship between microhardness, depth and interaction between the light - curing methods investigated, followed by tukey test . The sas program (version 8.02, sas institute inc ., cary, nc, usa) was used for the analysis and the significance level set at 5% . Knoop microhardness mean values recorded for the specimens using the four study methods, as well as the standard deviation and percent curing in relation to the most superficial region are presented in table 1 . Up to the depth of 2.0 mm, all curing techniques showed an acceptable relative hardness compared to the 0.1 mm value (0.80) (table 1). Different letters indicate statistically significant differences (p<0.05); uppercase letters compare the effect of depth of cure for each curing method (within columns) and lowercase letters compare curing methods for each depth of cure (within lanes). [microhardness ratio relative to the 0.1 mm value] there was a statistically significant effect for the interaction method of cure distance from the surface and for the isolated factors (p<0.05). For all depths of cure, the traditional method provided higher knoop microhardness values compared to the new light - curing methods, stepped and pulsed (p<0.05). The ramped method was similar to the traditional method at depths of 0.1 and 1.0 mm (p>0.05) but lower values at the deepest depth (p<0.05). The microhardness values decreased according to the distance from the external surface (p<0.05), irrespective of the curing method used . Ideally, the degree of curing should be the same throughout the depth of the restoration, and the ratio of hardness (more external / more internal) should be equal to 1 or a close value . When a composite resin increment is cured, light passes through its interior and loses intensity due to dispersion, leading to a lower curing effectiveness14 . This light dispersion inside the bulk of material leads to the difference in microhardness between the external and internal surfaces . Light - curing of composite resins is considered adequate when this proportion is equal to or higher than 80% (n=0.80)7,14 . With regard to the mean knoop microhardness values in depth and the methods evaluated, the ratio of curing was higher than that reported by those authors up to the depth of 2.0 mm (table 1). These data confirm the recommendation that resin increments should be limited to a maximum thickness of 2.0 mm for achievement of satisfactory curing3,4,6,7,10,14,15 . Concerning the effectiveness of curing of the new modulated techniques, the results of the present study indicated that they are favorable for curing, as demonstrated by the knoop microhardness test, when these techniques were applied using the same curing times, intensities and materials investigated, according to other studies3,4,7,15 - 18 . Regarding the new light - curing techniques, the ramped method was able to cure 2.0 mm of composite resin in a shorter time (20 s), even at a lower light density, when compared to the traditional method . Considering that the higher the degree of conversion, the higher the microhardness, the present outcomes confirm those of bouschlicher, et al.3 . Similarly to the present microhardness values for the ramped and traditional techniques up to 2 mm in depth, those authors found a similar degree of conversion between these two methods, which was able to reduce the conversion rate up to 1 mm below the most external surface by the ramped method . However, this did not significantly affect the total conversion of the material with use of both methods . Initially, the lower conversion rate with the ramped method allowed a reduction in the stress rate and maximum shrinkage stress, with no damage to the physical properties of the restorative material6,12,13,15,17 . Other studies investigated the forces yielded during polymerization shrinkage and demonstrated that the pre - gel phase is extremely fast in composite resins cured by high light intensities as used in conventional continuous (600mw / cm - 40 s), which therefore is a negative aspect of this technique8,10,12,17 . The microhardness values achieved by the stepped and pulsed methods were lower than those obtained with the traditional method . Despite the numerical difference between groups as seen in table 1, those methods provided satisfactory curing, as demonstrated by de wald, et al.7 . And rueggeberg, et al.14 . In order to validate the mechanical behavior of a composite resin and, probably from a clinical point of view, it could not be consider as an isolated element to affect the behavior of the restoration . With regard to the stepped technique, the results found by bouschlincher, et al.2, comprising comparison of the force intensity and maximum shrinkage force, did not demonstrate any difference between the traditional and stepped methods . Despite the initial low light intensity, curing techniques may give rise to reduce polymerization shrinkage due to stress relief by allowing flow to occur during setting3,4,8, some material's chemical and physical properties could be negatively affected, producing a polymer mechanically more fragile . A recent study2 found that low power density applied to the composite resin at the initial periods of polymerization reaction resulted in polymers with increased susceptibility to softening in ethanol, in spite of achieving a comparable degree of cure to that of the conventional continuous technique . It has been hypothesized that slow start polymerization are probably associated with relatively few centers of polymer growth, generating a small number of free radicals, resulting in a more linear polymeric structure, with lower cross linking density, as evidenced by reduced glass transition temperature and increased susceptibility to ethanol degradation2 . Although the pulsed technique yielded lower microhardness values than those achieved by the traditional method, due to the lower light density, the material also presented an acceptable degree of curing (n=0.80)7,14 . The outcomes of other studies7,10,14 - 18 corroborate this finding, with the report that initial low light intensity does not lead to a considerable decrease in the mechanical properties . Moreover, specific literature has demonstrated that light - curing techniques comprising initial low light intensity provided better marginal sealing than those that use high light intensity1 - 6,9 - 12 . Further clinical and laboratory studies on the force and shrinkage vectors and the gaps yielded by the characteristic inherent to this material should be conducted, in order to determine the real performance of curing techniques in relation to shrinkage and performance of composite resin restorations in a long - term basis . Composite resin curing by the 4 light curing methods provided decreasing means of microhardness in relation to the surface, according to the increase in depths analyzed; 2 . At the depth corresponding to the resin increment recommended by the manufacturer (2.0 mm), all curing techniques provided satisfactory composite hardness; 3 . The use of initial low light intensity for curing composite resins does not compromise the microhardness up to 2.0 mm depth.
Diffuse blue - gray skin discoloration has been reported in several conditions such as addison s disease, argyria, hemochromatosis and polycythemia vera.1 riehl melanosis is characterized by brown - violet pigmentation on sun - exposed areas, erythema, and pruritus.2 erythema dyschromicum perstans is either idiopathic or acquired, typically occurs in those younger than 40 years, and first presents with erythematous macules that slowly turn slate gray resulting in gray - blue hypermelanosis.2 finally, end - stage metastatic melanoma can produce a blue - gray to brown hue to the body.2 in addition, drugs including minocycline, amiodarone, zidovudine, and bleomycin have been reported to cause cutaneous darkening (table 1). Minocycline is a semi - synthetic tetracycline antibiotic that turns black when oxidized, and can produce discoloration of the skin, nails, oral mucosa, conjunctiva, teeth, bones, and thyroid gland.3 three types of minocycline - induced cutaneous hyperpigmentation have been described:3 type i is the most common, and is associated with blue - black discoloration in areas of previous inflammation and scarring . Type ii most commonly affects the legs and is characterized by blue - gray pigmentation of previously normal skin . Type iii is the least common and is characterized by diffuse muddy - brown discoloration predominantly on sun - exposed skin . Any patient receiving more than 100 grams of minocycline can develop discoloration.4 the pigment deposition is the result of a drug metabolite - protein complex chelated with calcium, or an insoluble minocycline - melanin complex.5 minocycline hyperpigmentation occurs in 2.4% to 14.8% of patients on chronic treatment, most commonly for acne and rosacea.5 in a study of 700 patients on high - dose long - term minocycline treatment for acne (100 mg daily, 100/200 mg on alternate days, or 200 mg daily for 10.5 months), the only side effect that was significantly greater in patients taking higher doses (cumulative dose greater than 70 g) compared with lower doses was pigmentation (p <0.01).6 its anti - inflammatory effects are helpful for rheumatoid arthritis, immunobullous disease and other inflammatory diseases.3 the incidence of minocycline pigmentation is higher in patients treated for autoimmune diseases and may be more common with increasing age.3 q - switched lasers use high energy, nanosecond pulsing and are available in 3 wavelengths for drug - induced pigmentation, including the ruby 694 nm, the alexandrite 755 nm, and the nd: yag infrared 1064 nm.7 there have been reports of the alexandrite laser leading to the resolution of type ii minocycline induced hyperpigmentation.6 other reports have shown efficacy of the ruby laser for minocycline facial and leg pigmentation.8,9 a single study compared the yag and ruby lasers in the treatment of minocycline pigmentation and the ruby laser was found to be more effective.9 however, there are no studies comparing the 3 q - switched lasers for effectiveness and patient comfort in the treatment of minocycline - induced hyperpigmentation . A 70-year - old caucasian male presented with a one - year history of progressive worsening blue - gray discoloration of the face . Physical examination revealed macular, non - blanching, diffuse blue - gray hyperpigmentation on the forehead, temples, cheeks, nose, and chin sparing the oral mucosa . The patient was taking 100 mg of minocycline orally twice daily for 3 years (total 219 grams) to suppress symptoms of rheumatoid arthritis . A 0.4 0.3 0.3 cm punch biopsy demonstrated mild perivascular lymphocytic infiltrates with increased pigment deposition in the basal layers of the epidermis (figure 1). The patient was spot - treated with 3 q - switched lasers (1064 nm, [palomar spectrum rd1200] 755 nm, and 694 nm [both syneron - candela alex trivantage]) to evaluate which laser would achieve the best results in removing pigmentation with minimal discomfort . The 1064 nm laser was set at 1.6 joules (j) with a 5 mm spot size . The 755 nm laser used a fluence of 5.5 j with a 4 mm spot size . We chose the 755 nm laser for treatment based on its significant improvement with minimal pain, discomfort, and downtime for the patient . The patient received two full - face treatments spaced 2 weeks apart with the 755 nm at 5.5 j and 4 mm spot size and one follow - up spot treatment . These treatments led to complete resolution of hyperpigmentation and the patient was completely satisfied with the result (figure 4). This patient had type iii minocycline - induced hyperpigmentation on the sun - exposed skin of the face after taking 219 grams of minocycline over 3 years . Of the 3 q - switched lasers tested, the 755 nm laser was effective in reversing pigmentation with minimal patient discomfort after 2 treatments . Type iii minocycline - induced hyperpigmentation is less likely to respond than types i and ii.3 it is not known exactly how laser therapy removes the pigment associated with minocycline use, but is thought to result from fragmentation of the intracellular and extracellular pigmentation and drainage through the lymphatic system.2 the recommended minocycline dose for acne is 100200 mg daily . Many patients treated for a year or two will reach a cumulative dose of over 100 g. according to the us food and drug administration (fda), there is a manufacturing delay of tetracycline leading to a shortage of the drug.10 as a result, physicians are forced to use alternative medications such as doxycycline.10 therefore, we may see a greater incidence of minocycline - induced hyperpigmentation as minocycline prescribing increases . Fortunately, we can utilize the alexandrite 755 nm laser to remove pigmentation associated with the use of this drug . A recent case reported successful treatment with the alexandrite laser, with the patient deciding to continue minocycline therapy and returning 3 years later with recurrence to receive another laser treatment.11
Three months after suffering a stroke, 25% of surviving patients remain wheelchairbound, and in 60% of subjects, walking ability and speed are markedly reduced2 . One of the main impairments after stroke is reduced muscle strength on the side contralateral to the brain lesion3 . The relation between paretic knee muscle strength on the contralateral side and gait ability after a stroke has been investigated in many studies1, 3,4,5,6,7,8,9,10,11,12,13,14 . A moderate to strong relation is generally observed between knee muscle strength in the paretic limb and gait performance4,5,6,7,8,9,10,11,12,13 . However, muscle strength on the side ipsilateral to the lesion can also be affected after stroke3, 15 . Harris et al.15 suggested that the development of ipsilateral muscle weakness was associated with immobility after stroke and weight loss due to swallowing difficulty and nutritional insufficiency . In addition, throughout the aging process, older people demonstrate an overall decline in muscle mass caused not by stroke but by aging16,17,18 . This generalized loss of skeletal muscle is considered a major factor leading to the development of impairment in muscle strength for older adults18 . Regardless of the occurrence of stroke, lower limb weakness has been identified as an important risk factor for walking in older adults19,20,21,22 . Muscle weakness in the bilateral lower limbs caused by aging may have already been increasing before a stroke23 . Thus, paretic and nonparetic lower limb weakness of older stroke patients is complex as a result of the multidimensionality of the challenges caused both by stroke and aging24, 25 . This muscle weakness associated with aging is most obvious in areas such as japan, the united states, and europe, which have dramatically aging populations26,27,28 . Very few studies have assessed the contribution of strength in nonparetic lower limbs to gait performance1, 3, 29,30,31,32 . Furthermore, the findings are controversial: three studies1, 29, 31 reported no significant relation between knee muscle strength in the nonparetic lower limb and gait performance, whereas a further three found a significant relation3, 30, 32 . The association between strength and functions may be curvilinear; a critical amount of strength is needed for normal performance of specific activities33 . Above this threshold level of strength below the threshold, there should, theoretically, be a stronger relation between strength change and change in performance . A major aim of stroke rehabilitation is optimization of the recovery of muscle strength to regain walking ability34,35,36,37 . However, the relation between strength in the nonparetic lower limb and gait performance has attracted much less attention, and the results are conflicting . As the threshold level of strength to predict walking independently is unknown in older stroke patients, it is difficult to predict the level of muscle strength that allows independent walking in older patients with hemiparesis . If bilateral knee muscle strength could be used to predict independent walking, training to regain gait performance would become more evidence - based in aging societies . Therefore, this study was designed to assess the relations between bilateral knee extension strengths and gait performance in older subjects with poststroke hemiparesis and to predict gait performance by both paretic and nonparetic knee extension strength . Taking into consideration for previous studies on knee extension strength and walking ability3, 30, 32, we hypothesized that there is a significant relationship between bilateral knee extensor strength and gait performance and that both paretic and nonparetic lower limb strength could be used to predict independent walking in older stroke patients . To our knowledge, this is the first study to demonstrate the predictive values of both paretic and nonparetic lower limb strength with respect to gait independency . The eligibility criteria included hemiplegia of the lower extremities, absence of severe consciousness disorder, ability to sit up with a backrest for more than 30 minutes, ability to push against a dynamometer with the nonparetic lower limb after physical guidance, a period of less than 2 months since the stroke event, absence of severe cardiorespiratory insufficiency, and willingness to participate in the study . The average value and standard deviation (sd) of normalized knee extensor strength in 20 patients with poststroke hemiplegia were assessed to determine the sample size . There is a strong relation between force as measured by equipment and body weight in strength measurements23 . The variability (relative dispersion) of the force scores was reduced by normalization against body weight . Thus, the torques (nm) determined by force (n) and lower leg length (m) were normalized by the ratio of body weight (nm / kg) to predict gait ability by strength measurement . The average normalized knee extensor strength in the nonparetic lower limb of the 20 subjects was 1.33 newton - meters / kg (nm / kg; sd, 0.37 nm / kg). For the 9 subjects who could walk independently, it was 1.51 nm / kg (sd, 0.37 nm / kg), and for the 11 subjects who required assistance, it was 1.18 nm / kg (sd, 0.40 nm / kg). Five percent of the average normalized knee extensor strength for the 9 subjects who could walk was 0.08 nm / kg (8% difference), and the standard effect size was 0.30 . Sample size was based on a desired 90% statistical power to detect an 8% difference in normalized knee extensor muscle strength against body weight (nm / kg), with a two - sided of 5% . A sample size of 234 was derived by insertion of 1-power (0.90), (0.05), and standard effect size (0.30) values into the hulley matrix38 . The authors therefore planned to recruit about 234 people with poststroke hemiplegia for this study . The study was approved by the kawasaki municipal tama hospital institutional committee on human research . All subjects and their families were briefed about the aims of the study and the testing procedure prior to participation . Written informed consent was obtained from each subject . Knee extension strength was assessed with a tas mt-1 handheld dynamometer (anima, tokyo, japan). The dynamometer pad is 5555 mm, and its front side is curved to fit the shape of the areas to be measured on the extremities . The measurement range of this dynamometer is 0.1 to 999.9 n, with a recording interval of 0.1 n. a handheld dynamometer may be used to quantify maximal strength and may offer several advantages over free weights, including ease of transport, time efficiency, and low cost . The intraclass correlation coefficients, used to characterize the reliability of the strength tests using the handheld dynamometer, ranged from 0.84 to 0.99, which is considered good39, 40 . Furthermore, use of a handheld dynamometer provides a reliable and valid means of measuring muscle strength in patients with brain damage41,42,43,44 . Prior to strength testing, the tester took the subject s leg and guided it in the appropriate direction in accordance with the testing protocol to familiarize the subject with the feeling of pushing against the dynamometer . Strength of the knee extensor muscles was then assessed bilaterally using the tas mt-1 . For knee extensor assessment, subjects were seated in a hard chair with their knees flexed 90 and their arms on their thighs . The dynamometer was placed perpendicular to the leg just above the malleoli . During all tests, the dynamometer was kept stable by the examiner using both hands and/or the subject s leg and was fixed by a belt to keep the knee flexed 90. subjects were told to push against the dynamometer by attempting to straighten their leg . They were asked to build force gradually to a maximum voluntary effort . Gait ability was precisely defined by the functional independence measure (fim) locomotion item45 . The fim locomotion item was chosen because of its widespread use at rehabilitation facilities and ease of scoring . In this study, when the fim locomotion item score of a subject was 6 points or more (modified independence, in which the use of a cane and orthosis was accepted for 50-m gait, or complete independence), the subject was considered to be able to walk . To determine the association between normalized knee extensor muscle strength in paretic and strength in the nonparetic lower limb, then, patients were classified into 2 groups: those who scored 6 points or more for the fim locomotion item and those who scored less than 6 points . Discriminant analysis was performed to identify the combination of knee extension strength of both limbs that discriminates best between possibility and impossibility of gait and to clarify the contribution of the paretic and nonparetic lower limbs to gait performance . After discriminant analysis, paretic and nonparetic knee extension strengths, adjusted by the degree of a standardized discriminant coefficient, were integrated in the strength index . The strength index was modeled as a simple regression, and parameter estimates were assessed for goodness of fit to the model: strength index = 1 x+ 2 y (1, standardized discriminant coefficient of paretic lower limb; x, normalized knee extension strength of paretic lower limb; 2, standardized discriminant coefficient of nonparetic lower limb; y, normalized knee extension strength of nonparetic lower limb). The threshold level for prediction of independence was judged as the point where both the negative and positive predictive values were high46,47,48 . Between january 2005 and january 2009, 238 consecutive stroke inpatients from kawasaki municipal tama hospital (kanagawa, japan) were enrolled in the present study . Characteristics of patients who met the inclusion criteria are presented in table 1table 1.characteristics of patients satisfying the eligibility criteria (n=238)age (y)70.9 10.5gender (n)men138women100diagnosis (n)infarction196hemorrhage42time post stroke at assessment (d)8.2 6.9paralysis side (n)right125left113sensory disturbance (n)tactile sense87deep sense19ataxia (n) 37aphasia (n) 33unilateral spatial neglect (n)24lower leg length (m)0.39 0.04knee extensor muscle strength (nm)52.4 40.7values are shown as the mean sd . The mean age of participants was 70.9 years (sd, 10.5 years). 196 and 42 were diagnosed with cerebral infarction and cerebral hemorrhage, respectively, 125 patients had right hemiplegia, and 113 patients had left hemiplegia . The average time since stroke event was 8.2 days (sd, 6.9 days). Mean body weight was 57.6 kg (sd, 11.6 kg). Strength of the knee extensor muscles in the paretic lower limb of the 238 subjects in this study ranged from 0.00 to 321.0 nm (average, 52.4; sd, 40.7 nm). That in the nonparetic lower limb ranged from 7.1 to 336.0 nm (average, 72.6; sd, 39.9 nm). Strength of the normalized knee extensor muscles in the paretic lower limb ranged from 0.00 to 3.82 nm / kg (average, 0.90; sd, 0.62 nm / kg). That in the nonparetic lower limb ranged from 0.10 to 4.00 nm / kg (average, 1.24; sd, 0.58 nm / kg). The correlation coefficient (r) between normalized knee extensor muscle strength in the paretic lower limb and that in the nonparetic lower limb was 0.73 (pearson correlation coefficient, p <0.0001). Values are shown as the mean sd discriminant analysis was carried out to determine which paretic and nonparetic knee extension strength had a weighted impact on differentiating between the ability and inability to walk . The discriminant analysis classified the difference between the ability and inability to walk (eigenvalue, 0.49; wilks lambda, 0.67;, 93.85; df, 2; p <0.0001). Two variables contributed to classification of the ability of stroke patients to walk, which was performed with a standardized discriminant coefficient (knee extensor muscle strength in the paretic lower limb, 1.32; that in the nonparetic lower limb, 0.55). Thus, paretic and nonparetic knee extension strengths were integrated in the strength index: strength index = 1.32 x + 0.55 y, where x is the normalized knee extension strength in the paretic lower limb and y is the normalized knee extension strength in nonparetic lower limb . After discriminant analysis, the threshold level for prediction of independence was judged as the point where both the negative and positive predictive values were high . A threshold level of 2.0 provided the best balance between positive and negative predictive values for the strength index (fig . 1fig . 1.prediction of gait performance by predictive value curvethe threshold level for prediction of independence was judged as the point where both the negative (open symbols) and positive predictive values (filled symbols) were high . The curve for the negative and positive predictive values indicated that a strength index of 2.0 would provide the best balance for gait performance (positive predictive value, 0.74; negative predictive value, 0.69).). Prediction of gait performance by predictive value curve the threshold level for prediction of independence was judged as the point where both the negative (open symbols) and positive predictive values (filled symbols) were high . The curve for the negative and positive predictive values indicated that a strength index of 2.0 would provide the best balance for gait performance (positive predictive value, 0.74; negative predictive value, 0.69). In the present study, a relation between both paretic and nonparetic lower limb strength and gait performance was discovered . Our results indicated that (a) the paretic lower limb strength was correlated with the nonparetic lower limb strength, (b) both the paretic and nonparetic knee extension strengths were predictors of gait performance in older patients with poststroke hemiparesis, (c) the paretic knee extension strength affected gait performance more than the nonparetic strength, and (d) the strength indices of the paretic and nonparetic strengths combined with the weighted impact for gait performance could predict gait performance . A strength index of 2.0, meaning that the prediction of gait performance was deduced from the bilateral knee extension strengths, provides the best balance between positive and negative predictive values . About 75% of patients with a strength index of more than 2.0 could walk independently . However, 70% of patients with a strength index of 2.0 or less could not walk independently . A previous study suggested that there was a moderate to strong relation between paretic knee extension strength and gait performance4,5,6,7,8,9,10,11,12,13, whereas the relation between nonparetic knee extension strength and gait performance was controversial1, 3, 29,30,31,32 . However, the abovementioned studies recruited subjects who could walk without supervision or physical assistance and assessed gait speed as gait performance . An additional new observation in the present study was that both the paretic and nonparetic lower limb strengths affected the ability to walk independently . In our study, the predictive value of combined bilateral knee extension strength for gait independency was clearly demonstrated . Moreover, we provided evidence indicating that paretic lower limb strength is correlated with nonparetic lower limb strength in this study . Throughout the aging process, people demonstrate an overall decline in muscle mass17 . This generalized loss of skeletal muscle is considered a major factor leading to the development of impairments in muscle strength for older adults18 . This muscle weakness associated with aging is obvious in regions such as japan, the united states, and europe, where society is dramatically aging26,27,28 . Our results implied that throughout the aging process, older people demonstrate an overall decline in muscle mass caused not by stroke but by aging . Although progressive resistance training is an appropriate intervention and assessment for improving gait performance and bilateral lower limb strength in older people with poststroke hemiparesis, comparatively little research has focused on the training effect of combined paretic and nonparetic lower limbs of older patients with stroke50 . Our study demonstrated the threshold level of the strength index for subjects with poststroke hemiparesis who could ambulate independently . Therefore, the period required to reach the threshold level in resistance training programs may be estimated by further research . In the future, a prospective cohort study is necessary to identify predictors of recovery of independent gait ability after stroke . In addition, isometric evaluation with a handheld dynamometer utilizes a mode of contraction different from that used in training, as isotonic contraction is most commonly used for exercise training . Thus, use of a handheld dynamometer is limited by lack of specific training . Future studies need to assess whether handheld dynamometers can measure changes in strength after resistance training with the same precision as those measured by isotonic testing . Engardt et al.51 noted that patients learned to use the nonparetic leg to compensate for the weakness of the paretic leg in the early phase of rehabilitation . In previous studies, patients might have already learned to use the nonparetic leg to compensate for the weakness of the paretic leg in their gait because a long period had passed since the onset of stroke1, 3, 29,30,31,32 . It is still difficult to predict the contribution of both the paretic and nonparetic lower limb strengths to gait performance in older patients with poststroke hemiparesis . However, about 25% of patients with a strength index of more than 2.0 could not walk independently . When patients with a knee extension strength over the threshold level are unable to walk independently, learning to use the nonparetic and paretic legs might be a useful strategy for the patient to adopt . Activities of daily living are considered behavioral chains of component actions; such chains have been learned and performed since childhood52 . A patient with hemiplegia cannot walk by means of the behavioral chains used by a healthy person and thus has to learn new behavioral chains to walk independently . There is a growing body of evidence indicating that locomotor treadmill training with partial body - weight support may be an effective method of improving gait quality in the acute stage of stroke53, 54 . The intensity of resistance training and skill training can be decided by the threshold level of strength determined in this study . Future studies are needed to assess whether changes in muscle strength measured using a handheld dynamometer can reflect the ability of a subject to perform activities of daily life after resistance and skill training . It has been reported that stroke patients cannot perform at higher angular velocities due to spastic antagonist restraints55 . However, isometric strength has been shown not to be affected by antagonist muscle spasticity56 . Our investigation evaluated the relationship between isometric bilateral knee extension strength and gait performance; as a result, antagonist muscle spasticity would have had little effect on agonist muscle strength . However, because spasticity of knee extensor and flexor muscles in stroke patients was not examined in this study, further research is needed to investigate the relationship between lower limb strength and spasticity and gait performance.
The use of robotic - assisted laparoscopic surgery in minimally invasive gynecology has been rapidly increasing since the da vinci robotic platform (intuitive surgical inc, sunnyvale, ca, usa) was approved for this purpose by the food and drug administration in 2005 . The rate of robotic hysterectomy is estimated to have increased from as low as 0.5% in 2005 to as high as 22% in 2010 . This technology addresses the ergonomic challenges of traditional laparoscopy by providing a 3-dimensional view, wristed instruments, and diminished tremor for more precise movements, and potentially offers more patients the benefits of laparoscopy, including shorter hospital stays, decreased intraoperative blood loss, faster recovery, and decreased wound infections . As robotics represents an increasing percentage of minimally invasive procedures in gynecology, the debate over the relative benefits of robotic vs conventional laparoscopic surgery is driven by questions of cost, safety, credentialing, and medicolegal issues . Robotic surgery presents surgical teams with a radical departure from the operating room (or) culture of open surgery and even conventional laparoscopy . The large footprint of the platform and its complexity changes the demands on the or team and may adversely affect efficiency . Team members are separated in space and lack face - to - face communication . Teamwork and communication have been highlighted as 2 essential components of or safety . To date, few studies have investigated the inherent communication challenges in robotic surgery . The purpose of this study was to evaluate the association between the quality of communication and surgical outcomes, specifically operative time, blood loss, and perioperative complications . We conducted a prospective questionnaire - based pilot study from march 1 through may 31, 2013, at a university - affiliated tertiary care medical center . Surgeons, circulating nurses, and surgical technicians involved in robotic gynecologic surgery were invited to participate . The anesthesia team was excluded because of frequent personnel turnover during case assignments, precluding adequate study participation . All participants signed an informed consent and were assigned a study identification number by the study administrator to maintain anonymity . Circulating nurses and surgical technicians assigned to those cases were core members of the robotic surgical team . Minimally invasive surgery fellows and obstetrics and gynecology residents were also involved in the study . All robotic gynecologic surgeries for benign indications during the study period were included . At the end of each surgery the survey was based on 2 validated questionnaires: the safety attitudes questionnaire and the psychometric testing of interpersonal communication skills questionnaire . We focused on the following aspects: individual communication skills, teamwork, efficiency, and provider satisfaction . In addition, participants were asked to identify the major factors affecting communication during the case: noise level in the or, console - to - bedside communication problems, lack of nurse availability, lack of instrument availability, lack of participant familiarity with the procedure, other, or none . In addition, operative details and outcomes were gathered, including estimated blood loss (ebl), operative time, and perioperative complications . Statistical analysis was performed using sas 9.3 (sas institute, inc ., cary, nc, usa). In addition, to assess the overall perception of the quality of communication, we subjected the 13 survey questions to a principal component analysis, creating a composite quality of communication (cqoc) score . Multivariate linear regression models were used to assess the relationship of cqoc to surgical outcomes . In the absence of prior similar studies, a sample size could not be determined a priori . Thirty - two or team members participated in the study, and 108 surveys were completed . The average response rate was 60% (105/174), with a lower rate in fellows (9/22; 41%) and a higher rate in circulating nurses (34/43, 79%; p = .025). Response rates stratified by professional category n, total number of participants per professional category . Significance attributed to the difference in response rate between fellows and circulators only . A total of 24 hysterectomies, 11 robotic myomectomies, 3 sacrocolpopexies with or without hysterectomy, 1 advanced endometriosis resection, and 1 trachelectomy were included in the study . Spearman correlation between individual quality - of - communication survey items and surgical outcomes showed few significant associations . Higher quality - of - communication scores on the item, others misunderstood me because they misinterpreted my words or actions, correlated with a longer room - in to room - out time (p <.01). Similarly, higher scores on the item, i asked others to repeat themselves because i didn't understand / hear their message the first time, correlated with a greater ebl (p <.05). Analysis of the cqoc score showed an association between the quality of communication in the or and both cut - to - close time and ebl, when controlling for body mass index, prior abdominal surgery, type of procedure, and uterine weight in separate multivariate linear regression models . A higher cqoc score was significantly associated with a greater ebl (p = .010) and a longer operative time (p = .045). The coefficients presented in table 3 demonstrate that for every 1-sd increase in the perceived deficit in quality of communication, there was an additional 51-ml of ebl and a 31-min increase in operative time . Correlation between quality of communication and surgical outcomes nonstandardized coefficients of correlation . Controlled for body mass index, prior major abdominal surgery, and uterine weight . We compared how the different team members judged the quality of communication in the or, and we found that, for most questions, surgeons rated communication to be worse than did circulating nurses and surgical technicians . For example, surgeons agreed with the statement, steps took longer than necessary because i or others had to repeat / clarify what they were saying, at significantly higher rates than did nurses and surgical technicians (mean 1.4, sd 0.6 vs. 0.8, sd 0.8; p <.001). Despite a difference in mean cqoc scores between surgeons vs. circulating nurses and surgical technicians, both groups' responses showed that higher cqoc scores were associated with a longer operative time and a greater ebl . The most commonly reported factors that negatively affected the quality of communication were the following: the noise level in the or (28/36; 78%), console microphone / console - to - bedside communication (23/36; 64%); and the lack of familiarity of participants with the procedure (22/36; 61%) (table 4). There was no significant difference between team members' report of factors that negatively affect the quality of communication . There were a total of 5 perioperative complications: 1 cystotomy, 1 conversion to laparotomy, 1 suspected postoperative transient ischemic attack, and 2 readmissions for postoperative abscesses . However, we did not find any associations via logistic regression between cqoc and perioperative complications, (p>.999). Factors affecting communication at least 1 participant reported the issue as occurring in each case . We present an evaluation of the association between the quality of communication and surgical outcomes in robotic surgery . Our study demonstrated that poor quality of communication is associated with a longer operative time and a higher ebl . Our survey was based on 2 validated questionnaires including both experienced and inexperienced team members . Our results illustrate the complexity of communication in the robotic or and demonstrate for the first time the differences encountered in the perception of communication between team members stratified by their role . Robotic surgery has introduced unique and novel challenges to the workflow of a surgical team that have never been experienced before . The surgeon and surgical assistants more specifically, surgeons operate while sitting at the surgeon's console, and their field of vision focuses on the 3-dimensional viewer . Several meters away from the surgeon's console, the large robotic patient cart (the platform that attaches to the patient and holds the operating instruments) often obscures the surgeon's view of their assistants at the patient's bedside . The physical distance and obstacles create an auditory, visual, and physical barrier between team members, potentially hampering efficient communication . Surgical teams have traditionally heavily relied on a multitude of nonverbal communication tools, such as body language and eye contact, to anticipate the next step in the workflow of a given surgery . It is not until surgeons encounter auditory, visual, and physical barriers that they realize how much they have been relying on those cues for efficient communication . Cao and taylor reported that the complexity of the robotic setup causes a communications breakdown in the robotics or and potentially deleterious effects on team function, decision - making, and flow of information . They demonstrated that group tasks were executed with greater efficiency and accuracy in a simulated robotic cholecystectomy in which subjects used scripted speech patterns to communicate with team members . Webster and cao concluded that facilitated team communication can ease adaptation to new technologies that disrupt customary workflow . Our data indicate that decreased quality of communication is attributable to modifiable factors: a high level of noise in the or, problems with console microphone and console - to - bedside communication, and lack of familiarity of participants with the procedure . The high level of noise in the room may result from both the substantial background noise generated by the vision console and team members speaking loudly to communicate across significant distances . The current built - in da vinci audio system available may not address properly the communication challenges experienced by team members . Engineering solutions to these communication barriers may decrease provider mental load and improve surgical outcomes . Our data suggest a direct relationship between the team members' experience and quality of communication . When nurses and trainees who are inexperienced in robotic procedures participate in these cases, it may negatively affect team functioning . Adoption of systematic approaches to integrating new team members into the robotic or may avoid deleterious consequences caused by poor team dynamics . There is an expanding body of literature on evaluating the effect of these modifiable factors on teamwork, communication, and provider mental load based on the assumption that suboptimal working environment may affect surgical outcome . The current study demonstrated that this assumption has merit . Recognizing the importance of further defining this relationship, randell and colleagues are undertaking a large - scale study to understand and improve communication and teamwork in robotic surgeries . Finally, our study showed that, not only did both surgeons and nurses share the same perception regarding teamwork, but surgeons were more critical of team performance . Prior studies evaluating attitudes regarding patient safety in the or have shown that nurses tend to be more critical than physicians and that physicians have a tendency to have limited insight regarding their team members' perception of performance, safety, and teamwork . The finding in our study that surgeons are more critical or concerned than nurses regarding team integration may reflect a loss of sense of control experienced by the robotic surgeon, located distant from the patient and the rest of the team . It was 1 institution's experience and had a small sample size, thus limiting the generalizability of the data . Whereas response rate differed by type of participant and may represent response bias, there were no significant differences in the perception of communication, stratified by role . Although we cannot determine a cause - and - effect relationship, this study does establish a clear relationship between the complexity of surgery and poor communication in robotic gynecologic surgery and provides a rationale for further comprehensive study . More objective methods of assessing communication effectiveness, such as videotaped analysis, can be employed in addition to subjective assessment in future studies . Our study demonstrated that the diminished quality of communication reported by or team members is associated with more adverse metrics of patient outcomes . Ambient noise, audio clarity, and team members' inexperience, all contributed to lower communication scores . Overcoming the communication and teamwork obstacles introduced by robotic surgery may increase patient safety . Future studies are needed to assess interventions for improvement in communication and teamwork in the robotics or.
Transarterial chemoembolization (tace) is an established local therapy for managing unresectable hepatocellular carcinoma (hcc) in patients with advanced cirrhosis and confers significant survival benefits.1,2 iodized oil is usually used as a carrier of most anticancer agents to achieve preferential uptake and persistent deposit of the chemotherapeutic agent within the hcc nodules in conventional tace . Most anticancer agents used in tace are hydrophilic and usually prepared as emulsions mixed with iodized oil before intraarterial administration.3 these hydrophilic agents may cause rapid release of the active anticancer components in the bloodstream after they accumulate in the target lesions; therefore, these agents may not be expected to yield persistent and sufficient anticancer drug levels in the plasma against tumor growth.4 miriplatin (miripla; dainippon sumitomo pharma, osaka, japan) is a third - generation platinum derivative and has been developed recently for transarterial treatment of hcc.5 unlike other hydrophilic anticancer agents, miriplatin contains myristates as lipophilic side chains, which combine with the carrier ligand of platinum; therefore, it is easily dissolved in iodized oil without the need for emulsification.6 following intra - arterial administration, the miriplatin - iodized oil suspension accumulates in the target tumor, and continuous antitumor effects caused by gradual release of active platinum compounds are expected.68 in an early phase ii trial, 56% of the patients treated with miriplatin via transarterial chemoinfusion therapy were shown to achieve a complete response without major adverse events.4 in a randomized late phase ii study in patients with unresectable hcc, the miriplatin suspension demonstrated similar therapeutic efficacy to a zinostatin stimalamer suspension; further, it caused less hepatic vascular injury than the latter.9 these clinical studies suggest that miriplatin is a promising alternative to conventional hydrophilic chemotherapeutic agents such as cisplatin or doxorubicin for treating unresectable hcc . Miriplatin was approved for clinical use and covered by public health insurance in japan in january 2010 . Thereafter, we initiated the clinical use of miriplatin instead of epirubicin hydrochloride (farmorubicin; pfizer japan, tokyo, japan) for every tace procedure for treating hcc at our institute . In this study, we retrospectively compared the local tumor control rate of miriplatin - iodized oil suspension with that of epirubicin - iodized oil emulsion and evaluated the prognostic factors affecting local tumor progression in the targeted tace for hcc . Consecutive patients with unresectable hcc who had received targeted tace with miriplatin or epirubicin as the sole therapy between april 2008 and july 2011 at our institution were considered for inclusion in this study . Each patient was required to meet the following criteria: no previous treatment for the lesions under study, a child - pugh classification of a or b, total serum bilirubin level of <3 mg / dl, no portal venous thrombus in the main trunk, no previous history of chemotherapy with platinum derivatives, an interval of at least 4 weeks after the cessation of any previous anticancer therapy, segmental or more distal chemoembolization, and no more than three intrahepatic lesions . The diagnosis of hcc was confirmed from previous imaging findings as well as by the elevated levels of serum tumor markers . Serum -fetoprotein level of 20 ng / ml and/or serum des - carboxy - prothrombin level of 40 mau / ml were considered as positive tumor markers . Serum -fetoprotein levels were measured by latex photometric assay (lpia 100; mitsubishikasei, tokyo, japan) and serum des - carboxy - prothrombin levels were measured by the electrochemiluminescence immunoassay method (picolumi pivka - ii; eisai, tokyo, japan). The detection limits for serum -fetoprotein and des - carboxy - prothrombin levels were 2 ng / ml and 5 mau / ml, respectively . The size and number of tumors were determined from the cone - beam computed tomography (ct) images obtained during each tace session . Because we replaced epirubicin with miriplatin for all tace procedures at our institution from june 2010 onwards, 64 patients with 79 lesions received epirubicin treatment between april 2008 and june 2010 and another 47 patients with 66 lesions received miriplatin treatment between june 2010 and july 2011 . This study was carried out in accordance with the guidelines of our institutional review board, and written informed consents were obtained from all patients for tace using miriplatin or epirubicin . The miriplatin - iodized oil suspension was prepared by dissolving 70 mg of miriplatin into 45 ml of iodized oil (lipiodol ultrafluid; terumo, tokyo, japan), while the epirubicin - iodized oil emulsion was prepared by dissolving 10 mg epirubicin into 1 ml of iopamidol (iopamiron 370; bayer schering pharma, osaka, japan) and then mixing that into 12 ml of iodized oil . The maximum dose for a single tace session was limited to 140 mg for miriplatin and 50 mg for epirubicin . The actual dose was determined based on the size and number of target tumors and the liver function of the patient . All the tace procedures were performed using the same angiographic system (innova 3100; ge healthcare, waukesha, wi) by the same two interventional radiologists, who have more than 10 years experience in hepatic vascular interventions . First, an appropriate microcatheter was coaxially inserted through a 4-f catheter via the femoral artery and placed into the tumor - supplying artery . Second, a cone - beam ct image was obtained by injecting iopamidol from the microcatheter to confirm whether the target tumor was actually located within the treatment area . After the tumor location was confirmed, each hepatic area containing the target tumors was embolized with gelatin particles (gelpart; nippon kayaku, tokyo, japan) after infusion with the appropriate concentration of chemoth erapeutic agents . Administration of the drugs and gelatin particles was terminated when the tumor vessels were completely filled with the drugs and the tumor stain disappeared on angiographic imaging . Seven days after the tace session, the initial iodized oil uptake was assessed by unenhanced ct by using a 16-multidetector ct scanner (somatom sensation; siemens medical solutions, forchheim, germany). Uptake of the iodized oil was graded as good, fair, or poor by a single reader, on the basis of its accumulation in the tumor . The uptake was scored as good when complete and dense uptake occurred throughout the tumor, fair when inhomogeneous uptake occurred throughout the tumor, and poor when there was evidence of failure of uptake . Tumor progression was judged using triphasic contrast - enhanced ct or magnetic resonance (mr) imaging . Follow - up ct / mr imaging was performed every 13 months, depending on previous imaging findings and the laboratory data . Recurrence was evaluated by the evidence of abnormal early enhancement with washout in the delayed phase of each imaging modality . We statistically compared the patient profiles, tumor characteristics, and treatment procedures between the miriplatin and epirubicin groups using fisher s exact test or unpaired t test . The local control rate was calculated from the date of the tace session to the last date on which tumor progression was documented (or the date of death of the patient). Factors affecting local tumor control were first subjected to univariate analysis with the log - rank test . The parameters subjected to univariate analysis were: patient sex, age, etiology, child pugh class, clinical stage, previous treatment history, tumor size, serum -fetoprotein level, serum des - carboxy - prothrombin level, treatment area, number of treated tumors, iodized oil dose, initial iodized oil uptake, and drug administered . Consecutive patients with unresectable hcc who had received targeted tace with miriplatin or epirubicin as the sole therapy between april 2008 and july 2011 at our institution were considered for inclusion in this study . Each patient was required to meet the following criteria: no previous treatment for the lesions under study, a child - pugh classification of a or b, total serum bilirubin level of <3 mg / dl, no portal venous thrombus in the main trunk, no previous history of chemotherapy with platinum derivatives, an interval of at least 4 weeks after the cessation of any previous anticancer therapy, segmental or more distal chemoembolization, and no more than three intrahepatic lesions . The diagnosis of hcc was confirmed from previous imaging findings as well as by the elevated levels of serum tumor markers . Serum -fetoprotein level of 20 ng / ml and/or serum des - carboxy - prothrombin level of 40 mau / ml were considered as positive tumor markers . Serum -fetoprotein levels were measured by latex photometric assay (lpia 100; mitsubishikasei, tokyo, japan) and serum des - carboxy - prothrombin levels were measured by the electrochemiluminescence immunoassay method (picolumi pivka - ii; eisai, tokyo, japan). The detection limits for serum -fetoprotein and des - carboxy - prothrombin levels were 2 ng / ml and 5 mau / ml, respectively . The size and number of tumors were determined from the cone - beam computed tomography (ct) images obtained during each tace session . Because we replaced epirubicin with miriplatin for all tace procedures at our institution from june 2010 onwards, 64 patients with 79 lesions received epirubicin treatment between april 2008 and june 2010 and another 47 patients with 66 lesions received miriplatin treatment between june 2010 and july 2011 . This study was carried out in accordance with the guidelines of our institutional review board, and written informed consents were obtained from all patients for tace using miriplatin or epirubicin . The miriplatin - iodized oil suspension was prepared by dissolving 70 mg of miriplatin into 45 ml of iodized oil (lipiodol ultrafluid; terumo, tokyo, japan), while the epirubicin - iodized oil emulsion was prepared by dissolving 10 mg epirubicin into 1 ml of iopamidol (iopamiron 370; bayer schering pharma, osaka, japan) and then mixing that into 12 ml of iodized oil . The maximum dose for a single tace session was limited to 140 mg for miriplatin and 50 mg for epirubicin . The actual dose was determined based on the size and number of target tumors and the liver function of the patient . All the tace procedures were performed using the same angiographic system (innova 3100; ge healthcare, waukesha, wi) by the same two interventional radiologists, who have more than 10 years experience in hepatic vascular interventions . First, an appropriate microcatheter was coaxially inserted through a 4-f catheter via the femoral artery and placed into the tumor - supplying artery . Second, a cone - beam ct image was obtained by injecting iopamidol from the microcatheter to confirm whether the target tumor was actually located within the treatment area . After the tumor location was confirmed, each hepatic area containing the target tumors was embolized with gelatin particles (gelpart; nippon kayaku, tokyo, japan) after infusion with the appropriate concentration of chemoth erapeutic agents . Administration of the drugs and gelatin particles was terminated when the tumor vessels were completely filled with the drugs and the tumor stain disappeared on angiographic imaging . Seven days after the tace session, the initial iodized oil uptake was assessed by unenhanced ct by using a 16-multidetector ct scanner (somatom sensation; siemens medical solutions, forchheim, germany). Uptake of the iodized oil was graded as good, fair, or poor by a single reader, on the basis of its accumulation in the tumor . The uptake was scored as good when complete and dense uptake occurred throughout the tumor, fair when inhomogeneous uptake occurred throughout the tumor, and poor when there was evidence of failure of uptake . Tumor progression was judged using triphasic contrast - enhanced ct or magnetic resonance (mr) imaging . Follow - up ct / mr imaging was performed every 13 months, depending on previous imaging findings and the laboratory data . Recurrence was evaluated by the evidence of abnormal early enhancement with washout in the delayed phase of each imaging modality . We statistically compared the patient profiles, tumor characteristics, and treatment procedures between the miriplatin and epirubicin groups using fisher s exact test or unpaired t test . The local control rate was calculated from the date of the tace session to the last date on which tumor progression was documented (or the date of death of the patient). Factors affecting local tumor control were first subjected to univariate analysis with the log - rank test . The parameters subjected to univariate analysis were: patient sex, age, etiology, child pugh class, clinical stage, previous treatment history, tumor size, serum -fetoprotein level, serum des - carboxy - prothrombin level, treatment area, number of treated tumors, iodized oil dose, initial iodized oil uptake, and drug administered . We performed targeted tace for 56 hepatic areas of 66 hcc nodules in 47 patients of the miriplatin group and 72 hepatic areas of 79 hcc nodules in 64 patients of the epirubicin group . The mean dose of anticancer agents used for a single tace session was 50.5 (range, 10100) mg and 17.5 (range, 540) mg for miriplatin and epirubicin, respectively . The patient profile, tumor characteristics, and treatment procedures in both the groups are summarized in table 1 . There were no significant differences in any of the parameters investigated between the two study groups . Although no significant difference was found, the values of the mean iodized oil dose (p = 0.060) and the number of treated tumors (p = 0.071) differed between the groups . More iodized oil was used and fewer tumors were treated in a single tace session for the miriplatin group compared to the epirubicin group . The median follow - up periods were 150 days (range, 25472 days) for subjects receiving miriplatin and 340 days (range, 331183 days) for those receiving epirubicin . The overall recurrence rates were 39.3% (26 of 66 study lesions) and 31.6% (25 of 79 study lesions) for the miriplatin and epirubicin groups, respectively . The median periods between the tace and local tumor progression were 122.5 days (range, 28459 days) for 26 recurring lesions treated with miriplatin and 222 days (range, 33808 days) for 25 recurring lesions treated with epirubicin . The local control rate was significantly higher in the epirubicin group than in the miriplatin group (log - rank test, p <0.001) (figure 1). The local control rates at 6 months and 1 year were 70.7% and 44.8% for the miriplatin group and 83.4% and 69.2% for the epirubicin group, respectively . Pugh class (p = 0.042), serum -fetoprotein levels (p <0.001), serum des - carboxy - prothrombin levels (p = 0.019), iodized oil dose (p = 0.005), initial iodized oil uptake (p = 0.036), and drug administered (p <further, multivariate analysis revealed that the independent factors affecting local control rate were the serum -fetoprotein level (<20 ng / ml vs 20 ng / ml; p <0.001), drug administered (epirubicin vs miriplatin; p = 0.002), and child pugh class (a vs b; p = 0.042) (table 3). Miriplatin is a novel anticancer agent that is specifically designed for the intraarterial treatment of hcc . This highly lipophilic agent is anticipated to exert antitumor effects only if it is appropriately delivered in a suspension of iodized oil and adequately accumulated in the target site over prolonged periods of time . Despite the promising results of miriplatin observed in the phase ii trials4,9 as well as the early clinical experience with this drug1012 in tace for treating hcc, our study demonstrated that tace with miriplatin had significantly higher local tumor progression than that with epirubicin in patients with matched profiles, similar tumor characteristics, and treatment procedures . The local control rates of a single session of targeted lipiodol - tace for small hcc tumors (<5 cm in diameter) were reported to be 66.8%74.4% at 1 year.13,14 these results are comparable to the results of our study, where the local control rate of targeted tace for the epirubicin group was 69.2% . It is noteworthy that the local control rate of the miriplatin group (44.8%) was considerably lower than that of the epirubicin group reported in our study and in previous studies . Recently, a study from miyayama et al15 reported that the local recurrence rate was significantly higher for miriplatin compared to epirubicin with mitomycin c in the lipiodol - based superselective tace for hcc . The reported local control rates for the miriplatin group (5 months, 76.5%; 10 months, 32.7%) were in compliance with the results of our study . Since both of the studies were performed independently, the inferiority of miriplatin to epirubicin was confirmed from our study . Our study also revealed that miriplatin usage was selected as an independent factor associated with inferior local control after tace for hcc in the multivariate cox analysis, and the hazard ratio of using miriplatin instead of epirubicin was estimated to be 2.53 (p = 0.002). One reason for this poor local tumor control with miriplatin could be the reduced vascular damage caused by miriplatin . Miriplatin itself causes less vascular damage than zinostatin stimalamer,4 the only oil - soluble anticancer agent approved for the intraarterial treatment of hcc in japan . Therefore, repeated use of miriplatin for the same hepatic area is clinically feasible without inducing major vascular occlusion or arterioportal shunting . On the other hand, tace carried out with an anthracycline anticancer agent such as epirubicin or doxorubicin has been reported to lead to a high incidence of vascular damage and occlusion of the hepatic artery.16,17 indeed, vascular damage is frequently observed in hepatic arteries of the treatment area after chemoembolization with epirubicin - iodized oil emulsion, hampering subsequent intra - arterial treatment by inducing extrahepatic arterial supply to the previously treated lesions . However, since some vascular damage may prevent early recanalization of tumor vessels and feeders, using miriplatin for tace may result in earlier restoration of blood supply to the tumors and earlier washout of the iodized oil from the tumor site . To prevent early recanalization of the tumor vessels, it might be useful to add other vascular - toxic anti - cancer agents in the miriplatin - iodized oil suspension . The viscosity of the miriplatin - iodized oil suspension may be higher than that of the epirubicin - iodized oil emulsion . In addition, we found that the oil droplets of the miriplatin - iodized oil suspension delivering to the tumor were generally much larger than those of the epirubicin - iodized oil suspension . The higher viscosity and larger chemotherapeutic droplets of the miriplatin - oil suspension may have resulted in unintentional early occlusion of narrow tumor feeders before the iodized oil could completely accumulate in the entire tumor . Although the level of iodized oil accumulation was confirmed on follow - up ct at 1 week after tace, complete distribution of the hyperattenuated area over the treated tumor might not imply complete uptake of the iodized oil in the entire tumor . Gelatin particles used as an embolic material in this study did not obliterate tumor feeders permanently . This may be because the embolic material gradually degraded in the serum and was absorbed in the plasma over several weeks . Some contrast material injected with the gelatin particles and/or the iodized oil might have been transiently deposited in the lesions until the restoration of blood supply at the tumor site . Therefore, the therapeutic efficacy of the miriplatin - iodized oil suspension might be improved by reducing the chemotherapeutic viscosity and further fragmenting the oil droplets of the mixture, either by emulsifying the suspension with a contrast agent or by heating . Another reason for inferior local tumor control with miriplatin may be the low serum platinum concentrations due to an excessively slow release of active platinum from the tumor site where the miriplatin - iodized oil suspension is accumulated . Miriplatin exerts an antitumor effect only when active platinum is released from the lipophilic chemical complex containing myristates as the leaving groups . In a previous study on rat hepatic tumors, only 6% of the total platinum was reported to be released into the surrounding parenchyma at 28 days after intraarterial chemoinfusion of miriplatin - iodized oil suspension in the tumors.6 moreover, the total platinum levels in the plasma of patients receiving miriplatin at 20120 mg / body was approximately 300-fold lower than those reported in previous studies of intraarterial administration of 40100 mg cisplatin / body.4,18 the maximum plasma concentration time ranged from 1837 days for the miriplatin study, which was much longer than the 1060 minutes observed in the cisplatin study.4 therefore, sufficient plasma concentration of total platinum against the tumor progression is expected only when the miriplatin is retained in the target tumor for prolonged periods . The total platinum levels in the plasma and in the liver parenchyma might not have been adequate for the hcc lesions treated with miriplatin in this study . Repeated administration of miriplatin for the same lesions might increase the level of the serum platinum concentration, consequently increasing the antitumor effects of miriplatin . In this study, one patient who received miriplatin in the tace for hcc died of acute interstitial pneumonia 42 days after the therapy . The patient had previously been treated with epirubicin - iodized oil tace three times for the treatment of other hcc lesions . No adverse events were documented in the previous tace procedures with epirubicin, and the patient had no history of allergy . The patient received 58 mg of miriplatin and 3.3 ml of iodized oil for two intrahepatic lesions located at hepatic segment vii and started to complain of general fatigue 3 days after the therapy . Miriplatin may have caused the acute interstitial pneumonia; however, the exact reasons remain unknown because the patient s family did not consent to an autopsy . Eosinophilia is the most frequently observed hematological toxicity of miriplatin.4,10,19 the eosinophil counts of this patient were not elevated after the therapy, but there is a possibility that an allergic reaction toward miriplatin induced acute interstitial pneumonia . No other major adverse events were observed in the present study, and the hepatic toxicities of both groups were mild and within acceptable limits . This third - generation platinum compound does not elicit cross - resistance to cisplatin.20 repeated use of cisplatin often causes drug resistance and allergic reactions such as anaphylaxis.10,21 the risk of allergic reactions increases from the third session of tace with cisplatin.21 therefore, miriplatin can be considered as a second - line chemoembolization agent in patients who exhibit hypersensitivity or resistance to cisplatin . Cisplatin, which is a first - generation platinum compound, is associated with renal toxicity, whereas miriplatin is known to cause less renal dysfunction in comparison,10 suggesting that the latter drug is safe for use even in patients with unstable renal function without requiring excessive hydration before and after the therapy . Further, the use of miriplatin is associated with fewer adverse events as compared to cisplatin, including nausea, appetite loss, pain, anorexia, and fever.10 combining miriplatin and other anticancer agents would potentially enhance the therapeutic efficacy of miriplatin and reduce probable adverse effects . In conclusion, tace for hcc using a miriplatin - iodized oil suspension resulted in inferior local tumor control compared with an epirubicin - iodized oil emulsion in patients with matched profiles, tumor characteristics, and treatment procedures . In the multivariate cox proportional analysis, miriplatin usage for tace for hcc was found to be an independent factor associated with inferior local control rate bearing a hazard ratio of 2.53 when compared to epirubicin usage . However, further investigation is required to evaluate the long - term therapeutic efficacy of miriplatin.
The es / pnet group of tumors are primary malignant tumors of bone and soft tissue that consist of small round tumor cells . These uncommon and highly malignant tumors primarily affect children and young adults, but can occur in any age group . Extra - skeletal es / pnet is rare in comparison with skeletal es / pnet . Extra - skeletal es / pnet is a rare disease that may develop in soft tissues at any location . Primary thoracopulmonary es / pnet is the single most common form of extra - skeletal es / pnet, but primary mediastinal location of the extraskeletal es / pnet is extremely rare . And tumors show aggressive features with a high incidence of local recurrence and distant metastases . In our search for the literature, es / pnet presented with multiple mediastinal masses has not been reported previously . In our case report, we review a unique case of this rare form of extra - skeletal es / pnet presented as separate masses in different mediastinal compartments . Ethics committee approval is not included as it is commonly accepted that case reports do not require such approval . Because our work did not use patients data that would allow identifying them, informed consent is not necessary . A 42-year - old korean man visited our outpatient clinic with pleuritic left chest pain since a month . He also complained frequent and vigorous cough and chest pain was aggravated when coughing and breathing . Serum lactate dehydrogenase (ldh) level was increased of 487 u / l (normal range, 0250 u / l). Otherwise, the results of initial laboratory tests including complete blood count, chemistry panel, and urine analysis revealed no remarkable abnormality . On initial chest radiographs after admission showed moderate amount of left pleural effusion with contralateral mediastinal shifting and obliteration of left cardiac and diaphragmatic margins on frontal view (figure 1a and b). There were seen large contour bulging mass opacities in left lower hemithorax on lateral view (arrows in figure 1b), which were seen as well - defined masses overlapped with left cardiac shadow on left side down decubitus view with fluid shifting (arrow in figure 1c). Chest posterior anterior (a), left lateral (b), and left decubitus (c) views show moderate amount of left pleural effusion . Left lateral (b) and left decubitus (c) views reveal anterior and posterior mediastinal contour bulging mass opacities (black arrows in b and c), which are obscured on chest posterior anterior view (a). Findings of pre- and postcontrast - enhanced chest ct (figure 2a e) revealed heterogeneously enhancing (2080 hounsfield unit) well - defined 2 separate large masses, which were located in each anterior and middle mediastinum of the left lower hemithorax . The larger one of the masses in left anterior mediastinum (black asterisk in figure 2) was measured about 15.0 9.0 8.0 cm, which showed broad contact with the left chest wall anteriorly, mass effect with compression resulting in contour deformity of the adjacent left cardiac chambers posteromedially on axial scan (figure 2b) and inversion of the left diaphragm inferiorly on coronal scan (figure 2c). The other separate smaller mass in the middle mediastinum of the left lower hemithorax (white asterisk in figure 2) was measured about 6.0 6.6 6.8 cm, which was located between posterior wall of the left ventricle and left lateral wall of the descending thoracic aorta on axial and coronal scans (white asterisk in figure 2b and d). Intervening mediastinal fat plane between those masses and adjacent mediastinal structures seemed to be preserved (black arrows in figure 2b) with compression and contour deformity of the left cardiac chambers and no definite evidence of direct tumor invasion . There was seen moderate amount of left pleural effusion with mild pleural enhancement (figure 2b, d, e). Axial pre- (a) and contrast - enhanced (b) chest ct scan with mediastinal window images show 2 separate masses in left anterior (black asterisk) and posterior mediastinum (white asterisk), which show heterogenous enhancement with low - density areas after intravenous contrast administration (b). Extrinsic compression with contour deformity of the left cardiac chambers and left lateral wall of descending thoracic aorta is seen; however, intervening fat plane between the masses and mediastinal structures seems to be preserved (black arrows in b). Moderate amount of left pleural effusion is noted (b, d, e). Coronal (c, d) and sagittal (e) reconstruction images show 2 separate masses in left anterior (black asterisk in c and e) and posterior (white asterisk in d and e) mediastinum and well demonstrated anatomical relationship of the 2 mediastinal masses . Chest magnetic resonance imaging (mri) was not performed in our patient and initial radiologic differential diagnoses included tumor of thymic origin with metastasis, malignant lymphoma, malignant neurogenic tumors, malignant germ cell tumor with metastasis, malignant mesenchymal tumors based on the findings of chest ct scan . Subsequently performed pet - ct scan showed increased maximum standardized uptake value (suvmax 8.3) of fdg for each of the anterior and middle mediastinal masses (figure 3a d). (a, b) show increased fluorodeoxyglucose (fdg) uptakes (maximum standardized uptake value (suvmax, 8.3) in the anterior and posterior mediastinal masses (each asterisk in a, b). Torso maximum intensity projection images (c, d) reveal increased fdg uptake in each of the anterior (arrow in c) and posterior (arrow in d) mediastinal masses . Fdg = fluorodeoxyglucose . Based on imaging findings on chest ct and pet - ct scans, after multidisciplinary discussion with the attending physician, chest radiologist, and thoracic surgeon, we decided a surgical approach with excision of the mediastinal masses for the proper diagnosis and management . The surgical approach with lateral thoracotomy and excision of the mediastinal masses was performed . On operation field, there were seen highly vascularized anterior and middle mediastinal masses in left lower hemithorax . Those masses were tightly adhered to adjacent pleura, pericardium, diaphragm, and lungs . The left anterior mediastinal mass was more than double adult fist - sized with lobulation, which tightly attached to left 6th costal cartilage . Upper portion of the left anterior mediastinal mass was well encapsulated, whereas lower portion of the tumor was easily ruptured with necrosis . A separate left middle mediastinal mass was identified and which was well encapsulated with less than adult fist - sized and rugged surface . Those left anterior and middle mediastinal masses revealed their blood supply from intercostal arteries and also small feeding arteries from the descending thoracic aorta, respectively . The patient well recovered with no immediate and delayed postoperative complication . On gross histopathologic examination, the tumors were seen as multilobulated, friable, soft masses, totally weighing 654 g and measuring 19 13 6 cm in the larger one . On cross section, the cut surface has a gray - yellow and gray - tan appearance with multifocal areas of necrosis (figure 4). Gross specimen of the left anterior mediastinal mass . Grossly the tumor reveals a multilobulated, friable, soft mass with areas of multifocal necrosis (black arrows). Microscopically, the tumors revealed lobular growth pattern with hemorrhage and extensive necrosis on low magnification (figure 5a). The tumor was composed of uniform round blue cells with inapparent, small nucleoli, and scanty cytoplasm (figure 5b). On immunohistochemical stain, the tumor cells showed strong membranous staining for cd99 (figure 5c). These findings were consistent with es / pnet and histologic findings were identical in the 2 separate masses . On microscopy, the tumor shows lobular growth pattern with extensive necrosis (black arrows) (40, h&e) (a). The tumor is composed of uniform round blue cells with inapparent, small nucleoli, and scanty cytoplasm (400, h&e) (b). On immunohistochemical stain, the tumor cells show strong membranous staining for cd99 (400, cd99) (c). With consideration of aggressive behavior of the tumor, postoperative chemotherapy was recommended . Four months later, chest ct and pet - ct scans were performed in our out - patient clinic for routine postoperative follow - up evaluation . The follow - up chest ct scan again revealed a recurrent mediastinal mass at paracardiac area of the left lower hemithorax (asterisk in figure 6a), which showed increased fdg uptake on pet - ct scan (not shown). Also a focal osteolytic lesion was newly appeared on left 9th posterior rib suggesting bone metastasis (white arrow in figure 6b). Follow - up contrast - enhanced chest ct scan 4 months after the mediastinal tumor excision in a 47-year - old man . Axial contrast - enhanced chest ct scan performed on postoperative 4-month follow - up shows a recurrent heterogeneously enhancing left anterior mediastinal mass (asterisk) in the left paracardiac area (a). A newly appeared focal osteolytic metastatic lesion (arrow) ewing sarcoma / primitive neuroectodermal tumors represent a family of high - grade small round cell tumors, which have neuroectodermal origin and show varying degrees of neuronal differentiation . The ewing sarcoma family of tumors includes classic ewing sarcoma of bone, extra - skeletal ewing sarcoma, askin tumors of the chest wall, and primitive neuroectodermal tumors of bone or soft tissues . The family of es / pnet tumors can be united as a tumor entity by the presence of a reciprocal translocation of the long arms of chromosomes 11 and 22, t (11;22)(q24;q12), so - called ews (ewing sarcoma protein)-friend leukemia virus integration-1 (fli-1) fusion gene . These highly malignant tumors are reported commonly in children and young adults, which show 80% of patients being in their first 2 decades of life . And 80% of these tumors occur in the skeletal system, which tend to involve the diaphysis or metaphyseal - diaphyseal regions of long bones . Extraskeletal es / pnets are quite rare than skeletal es / pnets and common location of extraskeletal es / pnets is thoracopulmonary area . Among the thoracopulmonary es / pnets, primary mediastinal es / pnets are very rare . In our search for the radiology literature, only 7 cases of primary es / pnets of the anterior or middle mediastinal location have been reported . To our knowledge, es / pnet presented with multiple mediastinal masses in different mediastinal compartments has not been reported . The classic histologic finding of es / pnet is made up of solid sheets of small round blue cells with hyperchromatic nuclei and scant eosinophilic cystoplasm (figure 5b). Then, it is hard to differentiate the es / pnet from similar other small round blue cell tumors such as embryonal rhabdomyosarcoma, neuroblastoma, and lymphoma by a standard microscope alone . Immunohistochemical staining is helpful in differentiating es / pnet from other small round blue cell tumors . The es / pnet group of tumors has a common cell surface glycoprotein marker, cluster of differentiation 99 (cd99), which is a product of major histocompatibility complex class i - related (mic) 2 gene . And positive finding of other markers, including fli-1, neuron - specific enolase (nse), vimentin, molecular genetic techniques, such as fluorescent in - situ hybridization (fish), reverse transcriptase polymerase chain reaction (rt - pcr) can be used for definitive diagnosis for the es / pnet . Identification of the t (11;22)(q24;q12) chromosomal translocation (ews - fli1 gene fusion) is highly specific for es / pnet, because> 90% of the tumors show this gene rearrangement . On chest ct scan, es / pnet usually manifests as a large unilateral heterogeneously enhancing mass, often with low attenuating necrosis or cystic areas and high attenuating hemorrhage . The tumor tends to displace rather than encase adjacent structures such as vessels, the trachea / bronchi, or the mediastinum, which finding also can be well recognized in our case . Calcification of the mass is relatively rare and can be seen in 10% of the tumors . Interestingly, distant metastasis is rare at the time of diagnosis in the thoracopulmonary es / pnet, but after treatment, including excision, chemotherapy, and local radiation therapy, the tumor shows tendency of frequent local recurrence and distant metastasis as seen in our case . In case of thoracopulmonary es / pnet, the tumor is frequently associated with adjacent rib or sternum destruction and pleural effusion . In our case, the mediastinal tumors revealed relatively typical ct features, which include heterogeneously enhancing large masses with areas of low attenuation corresponding tumor necrosis on gross specimen . No calcification was identified and there was no lymph node metastasis at the time of initial diagnosis . Our case presented as separate 2 synchronous masses in each anterior and middle mediastinum at initial diagnosis . We think it is not certain whether those 2 separate mediastinal masses are synchronous multiple primary tumors or a malignant tumor with metastasis . And unfortunately, local tumor recurrence was identified on postoperative 4-month follow - up chest ct scan in our patient, in whom adjuvant chemotherapy and radiation therapy were not performed . Magnetic resonance imaging similarly shows a large heterogeneous mass with typically isointense to slightly hyperintense on t1-weighted image and heterogeneously hyperintense on t2-weighted image when compared to skeletal muscle . Prominent areas of high - signal intensity on t1- and t2-weighted images represent hemorrhage and necrosis . The tumors show variable enhancement patterns after intravenous contrast material administration, depending on tumor size, degree of necrosis, and hemorrhage . . Combination of surgical excision, chemotherapy and radiotherapy is known to standard treatment for the es / pnet group of tumors . Although optimal combination of chemotherapeutic agents is yet to be established, combination therapy including vincristine, adriamycin, cyclophosphamide, and actinomycin d (vaca) is known to standard first - line treatment . Neoadjuvant chemotherapy can also be considered to eliminate micrometastases and reduce the size of primary bulky tumor mass before surgical excision . Askin et al reported median survival of only 8 months after the diagnosis of the tumor . However, recent studies show significant improvement in 5-year overall survival of exceeding 60% to 65% for localized es / pnet with a combination of surgical excision, intense chemotherapy, and high - dose radiotherapy . Most important prognostic factors are known as tumor size, presence of distant metastasis at presentation, and surgical resection margin . Resection margins were free from tumor cells, but postoperative adjuvant chemotherapy and radiation therapy were not performed in our case due to patient's refusal of treatment and the clinical course of the patient well represents highly malignant characteristics and behavior of the es / pnet as previously reported . Based on radiologic findings in our case of extra - skeletal es / pnet manifested as multiple mediastinal masses and previous literature review, the imaging feature of the tumor is relatively typical appearance of malignant tumors although which is nonspecific . Therefore, it is difficult to differentiated es / pnet from other malignant mediastinal tumors, especially in the case of anterior mediastinal location . In our routine practice, more common anterior mediastinal tumors, including thymic carcinoma, malignant lymphoma, nonseminomatous germ cell tumor, share similar imaging findings and confirmative histopathologic diagnosis is essential for definite diagnosis and management . Although the mediastinal es / pnet is a rare tumor entity in adult patient, it should be considered as a differential diagnosis for malignant mediastinal tumors and which can be manifested as multiple masses in a patient . Understanding this rare entity of extra - skeletal es / pnet and characteristic imaging findings can help radiologists and clinicians to approach proper diagnosis and better management for this highly malignant tumor.
In recent years, corneal specialists have shown a fast growing interest in collagen crosslinking using the photosensitizer riboflavin and ultraviolet a light as a new therapeutic alternative for the treatment of progressive keratoconus . The aim of this treatment is to modify the biomechanical properties of the cornea, thus increasing its rigidity and preventing the progression of keratoconus.1 promising results have been published recently, and data from the dresden clinical study have shown stabilization of the disease, even after 5 years.2,3 it is necessary to follow strict treatment settings and patient eligibility criteria to avoid serious ocular side effects . The patient s cornea must be irradiated with a small peak - like sector of the ultraviolet a spectrum (370 nm) with 3 mw / cm irradiance for 30 minutes, which corresponds to a dose of 5.4 j / cm.3 the ultraviolet a irradiance must be checked before each treatment using an ultraviolet a meter to ensure that the optimal irradiance dose is delivered . The photosensitizing riboflavin 0.1% solution must be applied 5 minutes before irradiation and every 5 minutes during irradiation . It is also essential to perform preoperative pachymetry on each patient to exclude those with extended areas of less than 400 m of stromal thickness . We describe how to perform collagen crosslinking in the protocol setting using a slightly modified ultraviolet a dermatological lamp and preparing the riboflavin 0.1% solution inhouse . We used a slightly modified 370 nm ultraviolet a lamp (uv 109a, waldmann, villingen - schwenningen, germany) designed for dermatological treatment (figure 1a), covered with a black plastic adhesive tape leaving a small window through which ultraviolet light can reach the cornea (figure 1b). The desired irradiance of 3 mw / cm is tested with a ultraviolet a meter (waldmann) before each treatment, thus establishing the optimal distance from the ultraviolet a lamp to the cornea . The lamp was held by a clamp mounted on an intravenous pole (figure 2). Patients corneas were irradiated with 3 mw / cm of ultraviolet a (370 nm) for 30 minutes . The riboflavin 0.1% solution was prepared just before treatment by mixing 10 mg of riboflavin-5-phosphate (guinama, valencia, spain) with 10 ml of dextran 20% solution . The solution was introduced into an empty 15 ml balanced salt solution bottle (alcon laboratories, fort worth, tx, usa) wrapped in aluminum foil to avoid exposure of the riboflavin to ambient light (figure 3) through a 0.22 m sterilization filter (millipore corporation, bedford, ma, usa). The riboflavin 0.1% solution was applied 5 minutes before irradiation and every 5 minutes during irradiation . Thus far, we have successfully treated 12 patients with progressive keratoconus using this ultraviolet a lamp and riboflavin 0.1% solution (figure 4) prepared inhouse . The riboflavin solution was also easily prepared in the clinical pharmacy of the hospital by trained personnel . One important issue to be mentioned is the fact that the limbus should be protected against irradiation, and a round 8 mm hole in the tape that covers the lamp could be made for this purpose . The price of a commercially available ultraviolet a lamp designed for collagen crosslinking is approximately 15,000, and the riboflavin solution prepared inhouse costs approximately 60 per patient . The price of the slightly modified ultraviolet a dermatological lamp we used is approximately $400, the riboflavin 0.1% solution we prepared inhouse costs $2.20 per patient, and the ultraviolet light meter price costs around $200 . This provides ophthalmologists worldwide with an affordable alternative device for collagen crosslinking, especially for those in developing countries or places where resources are limited.
More than 40,000 people die from falls globally.1 adults aged> 65 years have the highest risk of death or severe injury after falls . Approximately 30% of older people fall each year and the frequency exponentially increases with age.24 falls not only profoundly impact a persons health but also bring huge economic burden . For older people, falls accounted for 10%15% of emergency visits and up to 50% of severe injury - related hospitalizations.5,6 the occurrence of falls results from a complex interaction of multiple determinants, including older age, gender, chronic illness, poor vision, lack of exercise, impaired cognition, environmental factors, and lower socioeconomic level.7 the environmental factor, responsible for 30%50% of falls for older people, might be the most crucial among these factors because it encapsulates the interaction of individual factors with the surroundings.8 different residential environments accordingly result in distinct risk of falls for older people . A previous study has reported that older adults living in rural areas had 36% higher odds of self - reported past - year fall - related injuries than those living in urban areas (odds ratio = 1.36, 95% confidence interval [ci] = 1.061.75).9 as rural and urban older people have numerous health - related disparities, leading to disproportionate incidence of health problems in rural older people, we are interested in whether the mortality after falls also varies between rural and non - rural areas.10,11 however, nonfatal falls result in considerable morbidity, including dependence, immobilization, and decreased function.12 it has been reported that the 1-year mortality rate was 22% in older people with a hip fracture after a fall and was 24.5% in those with a cervical fracture after a fall.13 therefore, to understand the potential factors predisposing the death after falls would be helpful for developing appropriate fall prevention programs . In this study, we investigated the mortality rate after falls of rural and non - rural older people and explored the risk factors of mortality after falls among older people . Control study by using the data from the national health insurance research database (nhird) in taiwan . The nhird comprises the medical records from the single - payer national health insurance (nhi) program, which covers more than 99% of residents in taiwan . In this study 1) a rural group, was identified from the inpatient database of yilan county from 2006 to 2009 ., 3,897 older people were hospitalized due to accidental falls and, therefore, were grouped as the rural group . 2) a non - rural group, was extracted from a longitudinal cohort database, comprising 128,359 older inhabitants from all townships in taiwan . Among these older people, 5,541 were hospitalized for accidental falls and were grouped as the non - rural group . Both groups were followed up for 4 years after falls for the all - cause mortality . According to the criteria adopted by the nhird, the rurality of taiwan townships is defined by the following characteristics: population density, ratio of people with educational levels higher than college, ratio of people older than 65 years, ratio of people who are agriculture workers, and the number of physicians per 100,000 people . These criteria have led to the classification of yilan county of taiwan as a rural area.14 this study was ethically approved by the institutional review board in national yang - ming university hospital . The diagnoses of hospitalization were coded by the international classification of diseases, ninth revision, clinical modification (icd-9-cm). If the number of falling episodes was more than two during the study period, only the first one would be taken as the index episode . However, due to the high and full coverage of nhi, people in taiwan only exit insurance when they die . Three major variables were considered as the potential risk factors of mortality after falls, including demographic factor, comorbidity, and medications . The comorbidity was evaluated by the charlson comorbidity index to reflect the severity of disease because older people tend to have multiple chronic disorders . We coded the ambulatory prescriptions 3 months prior to falls according to the anatomical therapeutic chemical classification (level 4) and calculated the number of medications . The standard of inappropriate medication use was the 2012 beers criteria, which provides the list of drugs that should be avoided in older people . It would be considered as inappropriate medication use if an older person took any drugs that are on the list 3 months before their fall . The continuous variables were presented as mean (standard deviation) and the categorical variables were presented as count (percentage). To compare the characteristics between the two groups, a two - sampled t - test and chi - square test were used for continuous variables and categorical variables, respectively . A logistic regression model was used in a multivariate analysis for risk factors of mortality after falls . All databases were managed by microsoft sql server 2012 (redmond, wa, usa), and the statistical analyses were performed using ibm spss statistics for windows, version 19.0 (ibm corporation, armonk, ny, usa). Control study by using the data from the national health insurance research database (nhird) in taiwan . The nhird comprises the medical records from the single - payer national health insurance (nhi) program, which covers more than 99% of residents in taiwan . In this study 1) a rural group, was identified from the inpatient database of yilan county from 2006 to 2009 ., 3,897 older people were hospitalized due to accidental falls and, therefore, were grouped as the rural group . 2) a non - rural group, was extracted from a longitudinal cohort database, comprising 128,359 older inhabitants from all townships in taiwan . Among these older people, 5,541 were hospitalized for accidental falls and were grouped as the non - rural group . Both groups were followed up for 4 years after falls for the all - cause mortality . According to the criteria adopted by the nhird, the rurality of taiwan townships is defined by the following characteristics: population density, ratio of people with educational levels higher than college, ratio of people older than 65 years, ratio of people who are agriculture workers, and the number of physicians per 100,000 people . These criteria have led to the classification of yilan county of taiwan as a rural area.14 this study was ethically approved by the institutional review board in national yang - ming university hospital . The diagnoses of hospitalization were coded by the international classification of diseases, ninth revision, clinical modification (icd-9-cm). If the number of falling episodes was more than two during the study period, only the first one would be taken as the index episode . However, due to the high and full coverage of nhi, people in taiwan only exit insurance when they die . Three major variables were considered as the potential risk factors of mortality after falls, including demographic factor, comorbidity, and medications . The comorbidity was evaluated by the charlson comorbidity index to reflect the severity of disease because older people tend to have multiple chronic disorders . We coded the ambulatory prescriptions 3 months prior to falls according to the anatomical therapeutic chemical classification (level 4) and calculated the number of medications . The standard of inappropriate medication use was the 2012 beers criteria, which provides the list of drugs that should be avoided in older people . It would be considered as inappropriate medication use if an older person took any drugs that are on the list 3 months before their fall . The continuous variables were presented as mean (standard deviation) and the categorical variables were presented as count (percentage). To compare the characteristics between the two groups, a two - sampled t - test and chi - square test were used for continuous variables and categorical variables, respectively . A logistic regression model was used in a multivariate analysis for risk factors of mortality after falls . All databases were managed by microsoft sql server 2012 (redmond, wa, usa), and the statistical analyses were performed using ibm spss statistics for windows, version 19.0 (ibm corporation, armonk, ny, usa). Among the 52,544 older inhabitants of a rural area in taiwan, 3,897 people experienced at least one hospitalization due to an accidental fall during 20062009 . The frequency in the rural area was significantly higher than that in the non - rural area (7.4% [3,897 of 52,544] vs 4.3% [5,541 of 128,359], p<0.001). The comparison of the rural group and the non - rural group was presented in table 1 . The rural group was significantly younger (76.4 years vs 77.8 years, p<0.001), had a greater charlson comorbidity index (2.2 vs 2.1, p<0.001), and took more medications (13.7% vs 12.5%, p<0.001) than the non - rural group . The proportion of inappropriate medication use was also higher in the rural group (38.7% vs 36.6%, p=0.07), but the difference did not achieve statistical significance . By further analyzing the hospitalization of older people in the rural area, although the causes of fall - related hospitalization differed between the rural and non - rural groups, it did not achieve a statistically significant difference . The most frequent complication in the rural area was femur fracture (23.8%), followed by head injury (14.1%) and upper extremity fracture (13.0%). The most frequent cause of fall - related hospitalizations in a non - rural area was a hip fracture (24.9%), followed by upper extremity fracture (15.7%) and other fractures (15.5%). The 4-year cumulative all - cause mortality rate after a fall in the non - rural group was 23.4% (95% ci = 22.124.7), which was significantly higher than that of the rural group (8.8%) (95% ci = 7.99.8). The independent risk factors of mortality after falls included male, older age, non - rural area, severer comorbidity, higher number of medications taken, and presence of inappropriate medication use . After adjusting for age, gender, comorbidity, number of medications, and inappropriate medication use, the rural group had a significantly lower risk of mortality after falls than the non - rural group (adjusted odds ratio = 0.32, 95% ci = 0.280.37, p<0.001) (table 2). Rural and non - rural areas are distinct in terms of socioeconomic condition, surrounding environment, and the accessibility to health services.15 the urban rural difference also leads to some health - related disparities.10,11,16 it has been noted that rural older people fall more frequently than urban older people . But seldom have studies compared the mortality after falls for older people between different places of residence . Additionally, as the risk factors of falls have been analyzed thoroughly, it would be helpful to further understand the potential factors predisposing the mortality after falls.1,8,9,1719 in this study, we found that the rural older people had a significantly lower mortality, even after more frequent falls . The first was the lower incidence of fractures in the rural area, which has been similarly reported in many countries.2026 most investigators suggested that the difference of fracture incidence could be explained by the higher bone mineral density (bmd) of a rural population.2123,2730 a population - based 15-year cohort study of females aged 50 years in sweden reported that the rural population had significantly higher bmd, lower prevalence of osteoporosis, and lower incidence of hip fractures.21 a study in norway, which included 7,333 females aged 65 years, found that urban females had a higher rate of forearm fractures, and the authors commented that this was due to the lower forearm bmd of urban females than that of rural females.22 therefore, as fracture is the major cause of fatal falls, the rural population might have less morbidity and less mortality even after more frequent falls because of their higher bmd and their lower incidence of fractures.3133 the second was the rural urban difference of industrial distribution . According to the government statistics in taiwan, the percentage of the agricultural population in yilan county at the end of 2009 was approximately 25%, which was almost twice than that in all of taiwan (13%).34,35 the rural group in this study consequently comprised of farmers who usually spend the majority of their time undertaking outdoor physical activity even after falls . Previous studies have revealed that early mobilization in older people after fractures could enhance functional recovery and reduce mortality, and mobility function was the independent risk factor for recurrence falls.3638 therefore, the rural older people might recover earlier after falls and have less morbidity and mortality than the non - rural older people . Previous studies report that environment - related factors and weather could impact the occurrence of falls in older people.8,39 according to government statistics in taiwan, there were more single storied houses but less buildings with elevators in yilan county.15 additionally, the area of a house is larger and the weather is more wet and humid in yilan county.15,40 the different home surroundings, environments, and weather between the rural and urban areas consequently lead to different etiologies of falls . For example, rural older people might have more chance of slipping outdoors or falling from indoor stairs, but less chance of stumbling inside their home or on a street . These different etiologies of falls, which usually result in diverse types of injuries, could explain the rural urban difference of mortality after falls . In addition to the place of residence, this study also noted that males, comorbidity, number of medications taken, and the use of inappropriate medication were also the independent risk factors of mortality after falls . The results were concordant with the reported predictors of death after a hip fracture in older people.4143 as hip fractures accounted for the major cause of death after falls, it is reasonable to find similar risk factors of mortality between falls and hip fractures . 1) the fall episode was identified by fall - related hospitalization and fall - related outpatient care was included . Therefore, selection bias might not be avoided and the incidence of falls would be underestimated . 2) some fall - related variables were not taken into consideration, for example, the reasons of falls and the place of falls . Several individual factors, which might impact on the prognosis of falls, were also not included, such as education level, body mass index, and bmd . 3) due to the limitation of the claim database, we did not calculate the frequency of multiple injuries . Rural older people had a higher frequency of fall - related hospitalizations but lower mortality after falls than non - rural older people . The place of residence is an independent risk factor of death after falls for older people.
Gck - mody (gck diabetes, glucokinase diabetes, or mody2) is a monogenic condition caused by heterozygous mutations in the gene encoding glucokinase (gck). It is characterized by chronic, lifelong, and mild hyperglycemia present from birth, and less than 50% of patients fulfil the criteria for overt diabetes . The increased risk of atherosclerotic vascular disease as compared with healthy individuals without diabetes is known in subjects with impaired glucose tolerance, patients with type 2 diabetes, and patients with metabolic syndrome [3, 4]. Noninvasive imaging techniques, such as carotid intima - media thickness (cimt) measurements, may help to stratify this risk of atherosclerosis, as well as the risk of myocardial ischemia: the cimt has been shown to independently predict coronary events in type 2 diabetes and cardiovascular diseases . However, the situation in gck - mody is likely to be different from that in type 2 diabetes: patients with gck - mody have increased fasting blood glucose and relatively low 2-hour post - ogtt blood glucose without other components of metabolic syndrome or insulin resistance . Such a blood glucose profile has been shown to be associated with lower rates of cardiovascular mortality among patients with type 2 diabetes . Importantly, niskanen et al . Demonstrated that components of insulin resistance syndrome, including hyperinsulinemia after an oral glucose load, serum lipid abnormalities, and elevated blood pressure, are major determinants of cimt in patients with diabetes . The risk of macrovascular complications in gck - mody is considered low, but the data are scarce . We aimed to evaluate the carotid intima - media thickness (cimt) as an indicator of this risk in gck - mody patients aging 35 years or older and their unaffected relatives, who share a similar environment and lifestyle . We studied 27 patients from 20 czech families with genetically confirmed gck - mody (age 3575 years; median 46 years) and 24 unaffected family members (siblings, parents, and partners) representing the control group (age 3579 years; median 50 years). Each of the 20 participating families contributed 1 to 3 patients with gck - mody and 1 to 3 control individuals matched by age and gender . Control individuals with fasting blood glucose more than 5.6 mmol / l (100 mg / dl) and/or with a known history of diabetes were excluded from the study . The identification of families with gck - mody has been reported previously [9, 10]. The study protocol was approved by the ethics committee of the 3rd faculty of medicine, charles university in prague, czech republic . The structured assessment included a questionnaire, anthropometric examination and blood sampling for biochemical analysis . High - resolution b - mode carotid ultrasonography (using phillips iu22 ultrasound) was performed to measure the cimt of the distant wall for 1 cm lengths of the carotid bifurcation and the internal carotid and right and left common carotid arteries . The mean cimt values of 10 sites were combined in an unweighted average to produce an overall cimt . The upper normal limit of cimt was set to 0.7 mm [12, 13]. The patient history of coronary heart disease and ischemic stroke was obtained from medical records . The clinical and demographic characteristics of gck mutation carriers and control individuals were compared using welch's two - sample t - tests (continuous variables) and fisher's exact tests (categorical variables). Mixed linear regression models with cimt, blood pressure, and serum creatinine as outcomes were used to estimate and test the adjusted effects of gck mutation status . Age, gender, and mutation status no significant differences in baseline characteristics were found between patients and control individuals, with the exception of fasting blood glucose (gck - mody 7.6 mmol / l, sd 1.2 (136.4 mg / dl); controls 5.3 mmol / l, sd 0.3 (95.4 mg / dl); p <0.0001) and glycated hemoglobin hba1c (gck - mody 6.9% (sd 1.0), 52 mmol / mol (sd 10); controls 5.7% (sd 0.4), 39 mmol / mol (sd 3); p <0.0001) (table 1). The prevalence of smokers and hypertensive patients was similar in both samples (p = 1). The measured cimt values for participants with and without gck mutations are shown in figure 1 . The mean cimt was 0.707 mm (range: 0.41.1) in gck - mody patients and was 0.692 mm (range: 0.41.1) in healthy control individuals . According to the published recommendations [12, 13] after adjusting for age, gender, and family status, the estimated mean difference in cimt between patients and healthy individuals increased slightly to 0.049 mm (95% ci from 0.026 to 0.123; p = 0.19). As expected, the estimated trends of mean cimt indicated a moderate increase in cimt with age and mutation status (see regression lines plotted in figure 1). Carotid plaques (local intima - media thickening exceeding 1 mm and protruding into the lumen) were identified in 7 (25.9%) patients and 3 (12.5%) control individuals (p = 0.1), but all of these plaques were hemodynamically insignificant . Myocardial changes typical of ischemic heart disease described on echocardiography and/or ecg were detected in three of 27 patients with gck - mody and two of the 24 healthy control individuals (p = 0.866). Three study participants had suffered from myocardial infarction (two with gck - mody and one control individual) (p = 0.895) in the past, and two had suffered from ischemic stroke (one with gck - mody and one control individual). A similar proportion of participants (35%) from both groups were treated for hypertension with one or more antihypertensive drugs . Four of the 27 patients with gck - mody (14.8%) were treated with oral hypoglycemic agents, and one was treated with insulin . To the best of our knowledge, the present study is the first case - control study including the cimt measurements of gck - mody patients older than 35 years . The results are consistent with the mild natural course of gck - mody and confirm that gck mutation is not associated with an increased risk of developing macroangiopathic complications . The 95% confidence interval of the cimt difference, adjusted for age, family, and gender, is 0.026 to + 0.123 mm, indicating that the possible increase in cimt associated with gck mutation is low and most likely clinically insignificant . The absence of serious chronic microvascular complications in gck - mody was observed by page et al . And velho et al . [16, 17], who described proliferative retinopathy in less than 4%, proteinuria in 6%, and peripheral neuropathy in 5% of patients with hyperglycemia at more than 5 years after diagnosis . A major problem with assessing diabetic complications in those patients is the differentiation between patients who only have gck - mody and those who develop type 2 diabetes in addition to gck - mody . It is generally assumed that patients with gck - mody do not necessarily develop insulin resistance or dyslipidemia in the natural disease course, and their glucose tolerance remains stable over many years . Nevertheless, carrying a gck mutation does not protect against the development of type 2 diabetes, which occurs at a similar prevalence in gck - mody patients and in the general population . It has been reported that metabolic syndrome and insulin resistance are the major components of atherosclerosis risk in patients with diabetes and also in individuals without diabetes with insulin resistance [3, 20, 21], whereas the role of hyperglycemia in cardiovascular disease associated with type 2 diabetes is less clear . In contrast with patients with type 2 diabetes, patients with gck - mody have mild hyperglycemia without other components of metabolic syndrome or insulin resistance . Therefore, our study adds to the accumulating evidence that chronic mild hyperglycemia without additional components of metabolic syndrome has a milder effect on the development of macrovascular complications compared with the same glycemic levels associated with metabolic syndrome components . Admittedly, more subtle effects on cimt would remain undetected, as the present study is moderately sized . The numbers of available patients in other studies are, however, comparable a detailed analysis of the effects of p.gly299arg mutation in the gck gene was limited to a single large pedigree, whereas another study reporting selected metabolic parameters and history data included 35 families, but these subjects were not examined at a single centre, and the data did not include cimt [16, 17]. Thus, the number of participants may reflect a compromise between the depth of the acquired data and the subjects' willingness to undergo a complicated set of investigations . Patients with gck - mody exhibit only a small increase in glucose levels after oral glucose loading . By contrast, patients with type 2 diabetes have relatively high 2-hour glucose levels (as a proxy for postprandial glucose levels), indicating that postprandial glucose levels could be the most pathogenic glycemic factor for developing micro- and macrovascular complications . The cimt has been shown to correlate more strongly with postprandial glycemia than with fasting hyperglycemia . Additionally, the serum hs - crp levels are lower in gck - mody patients than in patients with type 2 diabetes . However, a common variant in the pancreatic gck promoter has been shown to influence the risk of diabetes complications . Showed that the a allele at c.30g> a of gck was associated with an increased risk of coronary artery disease in not only patients with type 2 diabetes but also individuals who did not have diabetes, albeit with a much weaker association (or = 1.27; 95% ci 1.021.59). Additionally, the snp rs4607517, which is in linkage disequilibrium with c.30g> a, has been associated with fasting glucose in genome - wide association studies . The association between components of the fasting glucose genetic risk score (represented by five snps, including rs4607517) and cimt has been described with an increment of 0.0048 mm in carriers . In conclusion, our data indicate that the natural course of mild lifelong hyperglycemia is associated with a low risk of developing diabetic macrovascular complications . However, patients with gck - mody should take steps to reduce the risk of developing classical type 2 diabetes in addition to gck - mody that is, avoid obesity and maintain a high level of physical activity . Our data support a conservative therapeutic approach for hyperglycemia in nonpregnant patients with gck - mody . Other risk factors for micro- and macrovascular complications should be treated according to present guidelines.
Approximately 10%15% of chronic smokers get lung cancer (lc) and around 20% develop chronic obstructive pulmonary disease (copd). Age, smoking history, and impaired lung function have been identified as key risk factors, although host susceptibility factors cannot been excluded . Cross - sectional studies show that the prevalence of copd is around 50% of those diagnosed with lc, although the prevalence might change depending on the patient s age, sex, and smoking exposure.15 in recent decades, it has been described that copd is an indicator of greater risk of respiratory complications and that it significantly increases the risk of cardiac arrhythmias and supraventricular tachycardia in patients undergoing lung resection surgery.68 for this reason, it is not surprising that the assessment of copd in patients with lc has great interest mainly in patients eligible for surgery, since the mortality rates are significantly higher in patients with lc who have other pulmonary comorbidities and therefore higher risk of postoperative pulmonary complications.9,10 so far, most lc studies regarding copd have been focused on the early stages of the disease, trying to prevent complications and mortality related to surgery.9,11,12 despite these advances in surgery and the introduction of new radiotherapy techniques on these days, most lc patients are being treated with chemotherapy or new tyrosine kinase inhibitors, which is the standard treatment for most patients with lc regardless of whether they have copd.13 while it is relatively well recognized that after resection, the prognosis of those with copd is worse than that of those without copd,10,14 in patients with lc not subject to surgery due to advanced stages of the cancer, it is unknown whether copd impacts in the prognosis when they are treated with chemotherapy and/or tyrosine kinase inhibitiors . The objective of our study is to analyze the clinical characteristics and survival rates in patients with lc and copd, and to compare these to the patients without airflow obstruction . Patients with lc (number [n]=471) were consecutively recruited between january 2006 and october 2013 following referral to a specialist lc clinic at a local tertiary hospital (guadalajara, spain). These patients were older than 35 years (range: 3595 years), and the diagnosis was confirmed by histological or cytological specimens in all cases . Nonsmokers with lc were also included, and those cases of primary lc with the following pathological diagnoses were finally selected for analysis: adenocarcinoma; squamous cell carcinoma; small cell cancer; and nonsmall cell lung cancer (not otherwise specified, including large cell carcinoma). We used postbronchodilator spirometry (masterlab; jager ag, wrzburg, germany) and subjects were classified as having copd according to global initiative for chronic obstructive lung disease (gold) staging with a ratio of forced expiratory volume in 1 second (fev1) to forced vital capacity (fev1/fvc) of <0.7 . Predicted values for lung function variables are from the european community for coal and steel.15 each subject s information was recorded using a standardized database that included demographics, pulmonary function tests, image techniques, blood analysis, type of tumor, anatomical extension, treatment side effects, and survival . The patients with poor performance status (eastern cooperative oncology group score 4) for which only the best supportive care was recommended, and those who moved from our city during follow - up were excluded . All included patients gave their informed consent before entry into the database, and the study was approved by the local ethics committee (guadalajara ethics committee, guadalajara, spain). For this study, we focused our analysis on patients with advanced stages at diagnosis without surgery (stages 3b and 4). Patients with early - stage cancer and surgical treatment were not included in the study . All of the patients were treated according to gold guidelines for copd16 and according to the national comprehensive cancer network guidelines, regardless of whether they had copd.13 in most cases, first - line therapy included cisplatin or carboplatin in combination with any of the following agents: paclitaxel; gemcitabine (squamous carcinoma); etoposide (small cell carcinoma); or pemetrexed (patients with nonsquamous histology). For further lines, we personalized the drug regimen with the highest likelihood of benefit, and toxicity was deemed acceptable to both the physician and the patient . The agents that target the epidermal growth factor receptor pathway were the initial systemic treatment for the patients in whom a driver mutation was identified . All statistical analyses were performed using spss version 20 (ibm corporation, armonk, ny, usa). Continuous variables were described by the measures of central tendency and dispersion (mean, standard deviation, median, and extreme values), by discrete variables, by frequency tables, and by the percentage over the total . The demographic and clinical variables for the patients are summarized as a description of the clinical profile . The primary analysis was based on a log rank test of the difference between the two groups with no adjustments for baseline covariates . Cox proportional hazards models were used to adjust for age, sex, tumor stage, performance status, smoking status, and gold stage . Patients with lc (number [n]=471) were consecutively recruited between january 2006 and october 2013 following referral to a specialist lc clinic at a local tertiary hospital (guadalajara, spain). These patients were older than 35 years (range: 3595 years), and the diagnosis was confirmed by histological or cytological specimens in all cases . Nonsmokers with lc were also included, and those cases of primary lc with the following pathological diagnoses were finally selected for analysis: adenocarcinoma; squamous cell carcinoma; small cell cancer; and nonsmall cell lung cancer (not otherwise specified, including large cell carcinoma). We used postbronchodilator spirometry (masterlab; jager ag, wrzburg, germany) and subjects were classified as having copd according to global initiative for chronic obstructive lung disease (gold) staging with a ratio of forced expiratory volume in 1 second (fev1) to forced vital capacity (fev1/fvc) of <0.7 . Predicted values for lung function variables are from the european community for coal and steel.15 each subject s information was recorded using a standardized database that included demographics, pulmonary function tests, image techniques, blood analysis, type of tumor, anatomical extension, treatment side effects, and survival . The patients with poor performance status (eastern cooperative oncology group score 4) for which only the best supportive care was recommended, and those who moved from our city during follow - up were excluded . All included patients gave their informed consent before entry into the database, and the study was approved by the local ethics committee (guadalajara ethics committee, guadalajara, spain). For this study, we focused our analysis on patients with advanced stages at diagnosis without surgery (stages 3b and 4). Patients with early - stage cancer and surgical treatment were not included in the study . All of the patients were treated according to gold guidelines for copd16 and according to the national comprehensive cancer network guidelines, regardless of whether they had copd.13 in most cases, first - line therapy included cisplatin or carboplatin in combination with any of the following agents: paclitaxel; gemcitabine (squamous carcinoma); etoposide (small cell carcinoma); or pemetrexed (patients with nonsquamous histology). For further lines, we personalized the drug regimen with the highest likelihood of benefit, and toxicity was deemed acceptable to both the physician and the patient . The agents that target the epidermal growth factor receptor pathway were the initial systemic treatment for the patients in whom a driver mutation was identified . All statistical analyses were performed using spss version 20 (ibm corporation, armonk, ny, usa). Continuous variables were described by the measures of central tendency and dispersion (mean, standard deviation, median, and extreme values), by discrete variables, by frequency tables, and by the percentage over the total . The demographic and clinical variables for the patients are summarized as a description of the clinical profile . The primary analysis was based on a log rank test of the difference between the two groups with no adjustments for baseline covariates . Cox proportional hazards models were used to adjust for age, sex, tumor stage, performance status, smoking status, and gold stage . From 471 evaluable patients, 324 (69%) were not treated with surgery because of the disseminated disease (stages 3b and 4). Among them 47.7% also had copd (50.4% in the overall population) with gold stage 1 (35.6%) and stage 2 (47.6%) predominance . Table 1 summarizes the clinical characteristics of the study population with lc according to copd status . At the time of cancer diagnosis, copd patients were older (7010 years versus 6613 years; p=0.002), but there were no significant differences in terms of sex distribution, performance status, or histological subtype distribution (figure 1a). In copd patients, there was a nonsignificant trend of a higher percentage of adenocarcinomas in gold 1 and squamous cell carcinoma in gold 2 (figure 1b). We looked at the correlation between the degree of airflow limitation in the entire study population; pearson s correlation coefficient between fev1, as the percent of the predicted value, and overall survival was very low (r=0.12), suggesting that collateral factors rather than the degree of airflow limitation are the main elements responsible for the prognosis of these patients . Meier curves showed no significant differences in overall survival between copd and non - copd patients (log rank, p=0.65) (figure 2). In the cox regression model, performance status (hazard ratio [hr] = 1.32, 95% confidence interval [ci]: 1.171.50; p=0.000), clinical stage (3b versus 4) (hr = 0.61, 95% ci: 0.480.78; p=0.000), and histological type (hr = 1.22, 95% ci: 1.01 1.46; p=0.04) were significantly associated with poor overall survival . In the multivariate cox proportional hazard model adjusting for the most relevant variables, the adjusted hr was statistically significant for performance status (hradj = 1.33, 95% ci: 1.111.59; p=0.002) and clinical stage (hradj = 0.67, 95% ci: 0.500.89; p=0.006), but not for copd status (hradj = 1.20, 95% ci: 0.831.50; p=0.46) (table 2). In the present study, we found that in using standard care in patients with lc and advanced disease (stages 3b and 4), the presence of copd did not worsen the prognosis . Copd also has little impact on the main characteristics of the patient at diagnosis, and it does not have a significant deleterious impact in terms of overall survival . In last few years, great advances have been made in the areas of copd and lc . Genetic studies seem to point to shared susceptibility genes that are common to copd and lc, although the reasons why some subjects develop lc and some develop copd when exposed to similar environmental challenges remain unresolved.17 despite this gap, it is clear that there is an association between these complex diseases . However, what remains unsolved is how to use this information to the benefit of individual patients . Lc in copd patients is a real problem since mortality studies of patients with copd suggest that 20%30% of patients die from lc.18 our data show that 50% of lc cases have coexisting copd, which confirms that a disproportionate number of lc cases occur in smokers with pre - existing copd when compared with those with normal lung function.19,20 in this context, one thing that should be addressed is how important copd is in patients with lc . Once the patients are diagnosed with lc, and if they are in the early stages, they are generally treated with curative intent using surgery, radiotherapy, and chemotherapy, or using a combined modality approach.21 several studies have focused on this special issue since the concomitant coexistence of copd might limit treatment with surgery . Lpez - encuentra et al22 analyzed the characteristics of copd and non - copd patients, as well as the possible prognostic value of this comorbidity over a sample of 2,994 lc nonmicrocytic cases surgically treated in hospitals participating in the bronchogenic carcinoma cooperative group of the spanish society of pneumology and thoracic surgery . This study showed that such an association may have deleterious prognostic value in patients presenting with both diseases . The effect is observed 2 years after surgical resection, and in copd, this effect is directly related to functional severity . Other authors that also investigated the comorbidity impact in nonmicrocytic resected lc found a higher mortality rate in copd patients when compared with non - copd patients.23 therefore, by and large, the prognosis for patients with copd and lc is worse than that of patients with lc without copd.14,24 certainly, there are patients in whom standard surgery is denied, offering only limited resection or nonsurgical treatment such as radiation therapy, radiofrequency ablation, stereotactic body radiotherapy, or cryotherapy because of impaired pulmonary function.2527 these options might result in poorer survival and increased rates of local recurrence when compared with surgical treatment . In common clinical practice, lc (both the small cell and nonsmall cell types) is usually diagnosed at an advanced stage, making it one of the most deadly forms of cancer . For patients with t4 extension, n2n3 disease (stage 3b), or m1 (stage 4), surgical resection is not generally recommended . In a large population with 67,725 cases of nonsmall cell lung cancer that were submitted to the staging database, only 9,137 (13%) cases were surgically managed.28 therefore, at diagnosis, most patients with lc have advanced disease that generally requires the use of systemic therapies in an effort to improve overall survival while maintaining quality of life . Although over 10% of patients can expect to be cured who survive 5 years after diagnosis with no evidence of the cancer having returned all patients can benefit from palliative treatment, which can improve the quality of survival . In fact, although very limited, the main improvement in the management of lc in recent years relates to palliative care . The patients with advanced metastatic disease may achieve improved survival and the palliation of symptoms with chemotherapy, targeted agents, and other supportive measures . Treatment options for patients are determined by histology, tumor stage, as well as by the general health and comorbidities of the patient . In this specific population that, in our study, at the moment of diagnosis, represented the 69%, we did not find significant differences in overall survival between copd and non - copd patients . In fact, overall, the presence of copd did not negatively influence the quality of life of patients, so our treatment was selected according to nccn guidelines without taking into consideration the presence of copd . In contrast with our data, abal arca et al29 found that in a large population of 996 patients with lc, the risk of death was significantly higher at stages 3b and 4, and in the absence of surgery and chemotherapy; but, astonishingly, survival was significantly higher in copd patients . To explain this result, the authors suggest some kind of diagnostic bias in this population due to the fact that copd patients may be diagnosed in earlier stages . However, in an adjusted cox regression model used for significant variables in the bivariate analysis, only stage and treatment remained in the final model . Previous series have observed a progressive tendency toward a cytohistological diagnosis of adenocarcinoma in the general population.13 in our study, there was a nonsignificant trend for a higher percentage of adenocarcinoma / large cell carcinoma in non - copd and gold 1 patients, and in squamous and small cell cancer patients in gold 24 . Tobacco smoking is a risk factor for any histological type of lc, but it has been described that this association is stronger with squamous cell carcinoma, small cell carcinoma, and large cell carcinoma than with adenocarcinoma . In a study of papi et al,30 the presence of copd increased a patient s risk for developing squamous cell carcinoma by four times . These results have been confirmed by other authors that also found a better association between poor lung function and squamous cell carcinoma or microcytic carcinoma when compared with adenocarcinoma.31 this point is particularly relevant since nowadays, chemotherapy and new targeted drugs are strongly associated to histology.13 first, most of our patients with copd were gold 1 and 2 . Although we cannot exclude some impact in patients with severe airflow deterioration, the pearson correlation coefficient between fev1, as a percent of the predicted value, and overall survival was very low, suggesting that collateral factors other than the degree of airflow limitation are mainly responsible in the prognosis of most copd patients with advanced lc . Second, at present, the overall survival of patients with stages 3b and 4 is very limited, and this could explain the absence of a clinical impact of copd; however, survival is changing with new pharmacological approaches . These results should be reassessed if significant changes occur in the survival of patients with advanced lc . Although we cannot exclude some impact in patients with severe airflow deterioration, the pearson correlation coefficient between fev1, as a percent of the predicted value, and overall survival was very low, suggesting that collateral factors other than the degree of airflow limitation are mainly responsible in the prognosis of most copd patients with advanced lc . Second, at present, the overall survival of patients with stages 3b and 4 is very limited, and this could explain the absence of a clinical impact of copd; however, survival is changing with new pharmacological approaches . These results should be reassessed if significant changes occur in the survival of patients with advanced lc . We conclude that in the current study, when using standard care, copd does not have a significant impact on the overall survival of advanced lc patients (stages 3b and 4).
Regeneration of lost periodontal structures, especially the lost alveolar bone, is a matter of prime concern in the clinical management of periodontal disease as bone destruction is primarily responsible for tooth loss . Different therapeutic modalities such as autogenous bone graft and allograft etc . Have been recommended for restoring periodontal osseous defects with their own set of advantages and inherent disadvantages such as sense of procurement of the same, patients discomfort; antigenic reactivity, danger of transmission of diseases such as hiv etc . Recently, choukroun's platelet - rich fibrin (prf) and a unique alloplastic graft material hydroxyapatite (hap)-bioactive glass (bg) composite granules (biograft habg active) because of promising regenerative potential attracted lot of attention of various researchers . The purpose of this case report is to describe the current concept of periodontal diagnosis and to evaluate clinically as well as radiographically the efficacy of choukroun's prf and biograft habg active combination in the treatment of localized advance intrabony and grade ii furcation defect in #36 . A 25-year - old female patient reported to the department of periodontics with chief complaint of teeth discoloration, mild and intermittent pain in left lower back tooth region, and bleeding gums while brushing since childhood, 12 and 2 months, respectively . On examination, dental fluorosis, local factors, and gingival inflammation were observed [figure 1]; bleeding on probing, vertical probing depth (vpd) of 45 mm with clinical attachment loss (cal) of 12 mm was present in more than 30% teeth, but distobuccal, distolingual, and mid - lingual aspect of vital tooth #36 showed vpd 11, 4 and 10 mm [figure 2] with advance cal of 9, 2, and 8 mm, respectively, along with horizontal probing depth (hpd) of 6 mm with lingual grade ii furcation involvement . The orthopantomogram [figure 3] and intraoral periapical radiograph [figure 4] revealed generalized mild bone loss with advance bone loss in furcation and on the distal surface of #36 . Generalized mild chronic periodontitis with localized advance loss of periodontal support in #36 was diagnosed . Clinical frontal aspect depicts dental fluorosis and local factors associated gingival inflammation (a) vertical probing depth of 10 mm at mid - lingual and (b) distobuccal aspect of #36, respectively orthopantomogram reveals generalized mild bone loss with advance bone loss in furcation and on the distal surface of #36 intraoral periapical radiograph showing interradicular bone loss and severe osseous defect at distal aspect of #36 after phase i therapy, plaque control was re - evaluated every 2 week and maintenance therapy was reinforced as per need . By the end of 8 weeks, gingiva was found healthy with normal sulcus depth, but no change was observed in #36 . The management of existing isolated intrabony and grade ii furcation defect in #36 utilizing different periodontal regenerative treatment modalities such as bone grafts and guided tissue regeneration was discussed in detail with the patient . Choukroun's prf and biograft habg active was selected, and duly signed consent from the patient was taken . The patient was advised routine blood, urine investigations which were within the normal limits and hiv, hepatitis c virus, and hepatitis viral markers were reported to be negative . Prf prepared just before the surgery as per protocol developed by choukroun's et al . Ten milliliters of blood sample was taken from antecubital vein of the patient in sterile glass tube without an anticoagulant and centrifuged at 24002800 rpm for 12 min . A fibrin clot was formed in the middle part of the tube whereas the upper and bottom part contained acellular plasma and red corpuscles, respectively . Under aseptic condition, after local anesthesia administration both the buccal and lingual full - thickness flaps were reflected after crevicular incision with respect to #35#37 followed by thorough subgingival scaling and root planing of both furcation and intrabony defects associated with #36 . A fibrin clot formed in the middle part of the tube was obtained and amalgamated with biograft habg active and filled in both furcation and intrabony defects of #36 followed by direct loop sutures and periodontal dressing application . The patient was instructed oral rinse twice daily with 0.2% chlorhexidine for 2 weeks and amoxicillin 500 mg and ibuprofen 400 mg was advised thrice a day for 5 days . Reduction in vpd [figure 5], hpd [figure 6], and cal was observed at 3, 9, 12 months as shown in . Postoperative radiographic evaluation of #36 at 3 months showed mild changes [figure 7a] whereas complete healing of grade ii furcation and intrabony defect up to the adjacent mesial alveolar bone margin of #37 observed at 9 and 12 months [figure 7b and c]. (a) vertical probing depth distobuccal aspect reduced to 6 mm (b) reduced to 4 mm (c) reduced to 3 mm at 3, 9 and 12 months postoperative respectively (a) horizontal probing depth 4 mm (b) 3 mm (c) 0 mm at 3, 9 and 12 months postoperatively postoperative healing of hard structures at (a) at 3 months (b) 9 months (c) 12 months respectively chronic periodontitis is characterized by loss of attachment due to destruction of periodontal ligament, loss of adjacent supporting bone; usually slow to moderate rate of progression but may have the period of rapid destruction localized, involving one area of the tooth's attachment or more generalized . The area of rapid destruction characterized by probing depth> 6 mm with attachment loss> 4 mm, furcation involvement if present will exceed class i (incipient) with radiographic evidence of same . The severity and distribution / extent of chronic periodontitis was diagnosed on the basis of 1999 international workshop for classification of periodontal diseases and condition; and american academy of periodontology task force report 2014, respectively (the task force preferred to use the percentage of affected teeth rather than the percentage of affected sites as an extent descriptor for chronic periodontitis that is generalized chronic periodontitis may be defined as periodontitis without a clear pattern of disease distribution of affected teeth or> 30% of affected teeth). Similarly, in the present case, the generalized chronic mild periodontitis was observed in> 30% of the teeth along with localized severe loss of periodontal support with respect to #36, therefore, diagnosed as generalized chronic mild periodontitis associated with localized advance loss of periodontal support in #36 . As regeneration in furcation and deep intrabony defect is difficult to attain because anatomy that impedes the accessibility for individual oral hygiene in molars, professional root debridement and due to slow and difficult integration of the grafted material into the physiological architecture as cited in reports of sharma and pradeep and rastogi et al ., respectively . In spite of different periodontal regenerative approaches, as currently, not a single regenerative material is considered gold standard in the treatment of osseous defects; therefore, amalgamation of biograft habg active and choukroun's prf was utilized because of their promising properties in the present report to obtain maximum outcome . Reported that the interleukin (il) 1, il 6, tumor necrosis factor-, il 4, vascular endothelial growth factor (vegf) in the prf clot play a crucial role in balancing the tissue homeostasis, whereas the healing cytokines il 4 and vegf inhibit inflammatory signal pathways thereby support and coordinate the neovascularization which may be the reason for uneventful healing in present case . The present case report showed reduction in pds, cal clinically and complete healing of furcation as well as intrabony defect radiographically; may be due to the property of biograft habg that bonds with host bone faster than hap ceramics and resorbs slowly but completely than bg, which is replaced and remodeled by the new bone . Prf when used as a membrane or as a grafting material creates an improved space making effect, which facilitates cell events that are favorable for periodontal regeneration leading to mineralized tissue formation, and it also induces the cell proliferation of osteoblasts, periodontal ligament cells, growth factors, but suppress the oral epithelial cell growth . The combined effect prf and biograft habg because of their distinct properties along with thorough debridement may result in excellent outcome in the present case report, which is in accordance with the reports of rastogi et al . Utilized hap particles with prf membrane in the intrabony defect, whereas salaria et al . Amalgamation of prf and biograft habg active was effective for the management of adjoin localized advance intrabony and grade ii furcation defect in the same tooth, but long - term randomized clinical trials should be undertaken before reaching final conclusion.
The online version of this article (doi:10.1007/s13300 - 015 - 0148 - 5) contains supplementary material, which is available to authorized users . In the united states (us), the goal of optimizing treatment strategies for type 2 diabetes mellitus (t2 dm) has grown in urgency with the epidemic rise of the disease . In 2012, an estimated 9.3% of the us population had diabetes, compared to 8.3% in 2010 . The number of hospitalizations each year among people with diabetes also has risen substantially, from 2.8 million in 1988 to nearly 5.5 million in 2009 . Previous research has suggested that discontinuity of care from the inpatient to the outpatient setting is common, with perhaps as many of 42% of patients discharged on medication not reporting that medication regimen to subsequent outpatient providers . Current treatment guidelines recommend the use of insulin as the preferred treatment for hyperglycemia in the hospital setting, and as a result, hospitalized patients with t2 dm often have the other antihyperglycemic agents (ahas) held and insulin initiated . However, it remains unclear how hospitalized patients transitioning to outpatient care are being treated following discharge . Whether patients receiving oral ahas / glucagon - like peptide-1 receptor agonists (glp-1s) prior to hospitalization resume their use, and whether patients who were undiagnosed or untreated before their hospitalization begin antihyperglycemic therapy after discharge, focus on linking continuity of care with adverse clinical outcomes, it is imperative to better understand which therapeutic strategies may lead to the best outcomes for patients with t2 dm . Determining current treatment patterns around transitions of care represents the first step toward optimizing therapy across treatment settings . To assess aha utilization patterns around inpatient to outpatient transitions of care, we conducted a retrospective database study among a sample of us adults hospitalized with a diagnosis of t2 dm during 20102012 . This study was a retrospective analysis of de - identified medical and pharmacy data from the 20102012 marketscan hospital drug, commercial, and medicare supplemental databases (truven health analytics). The hospital drug database is derived from hospital ordering and billing systems, and includes diagnosis and drug administration information from inpatient settings in 659 acute - care us hospitals . The commercial database includes inpatient, outpatient, and outpatient prescription drug claims for commercially insured employees and their dependents, covered under a variety of fee - for - service and managed care health plans through over 100 large employers and health plans located across the us . The medicare supplemental database contains the healthcare experience of retirees with medicare supplemental insurance paid for by a subset of employers . Over 55,000 hospital discharges in the 20102012 hospital drug database can be linked to claims in the commercial and medicare supplemental databases, and served as the basis of this study that followed patients across outpatient and inpatient settings of care . Patients selected for the study were required to meet the following criteria: (1) hospitalization recorded in the linked hospital drug database between january 1, 2010 and december 31, 2012 with a t2 dm diagnosis code [international classification of diseases, ninth revision, clinical modification (icd-9) 250.x0, 250.x2] listed in any diagnosis field, the earliest of which represented the index hospitalization; (2) available data in linked claims for a period of 90 continuous days before the index hospitalization admission date and 90 days after the index hospitalization discharge date; and (3) age 18 years or above on the index hospitalization admission date . Patients with any claims of ketoacidosis in the 90 days before the index hospitalization admission date, during the index hospitalization, or in the 90 days after the index hospitalization discharge date were excluded from the study . This exclusion was intended to help ensure patients with type 1 diabetes miscoded as type 2 on the index hospitalization were not part of the study sample . Although ketoacidosis can occur in t2 dm, aha utilization was the key outcome measured in this study and was assessed before, during, and after the index hospitalization . Pre - hospitalization aha utilization was based on retail outpatient pharmacy claims 30 days pre - admission, and mail order pharmacy claims 90 days pre - admission (including the date of admission). This time period was selected because retail pharmacy prescriptions typically cover up to 1 month of therapy while mail order prescriptions may cover up to 3 months of therapy . Therefore, aha claims in this time window were likely to represent aha therapy as of the time of admission . Retail pharmacy claims also were assessed during 60 and 90 days pre - admission to assess the sensitivity of the primary pre - index measure . Aha utilization during the index hospitalization was derived from inpatient medication administration data from the date of admission through the date of discharge . Post - hospitalization aha utilization was based on retail and mail order outpatient pharmacy claims on the date of discharge and the subsequent 30 days . This time period was selected because the intent was to capture prescriptions filled shortly after discharge, which were most likely to represent any regimen changes that occurred as a result of hospitalization . However, because some patients may have had a pre - admission supply of medication that they continued to use post - discharge, aha claims over 60 and 90 days after discharge also were measured . Within each time period, binary variables were created to record patients aha utilization at the class level (e.g., biguanides, sulfonylureas) and overall (i.e., any agent). In addition, medication utilization was categorized as insulin only (without oral ahas or glp-1), or one, two, or three oral ahas / glp-1s with or without insulin . Other information recorded for patients at the time of index admission included age, gender, health plan type, and geographic region . Additional variables measured during the 90-day pre - hospitalization period included deyo charlson comorbidity index (cci) score, and selected comorbidities and concomitant medications (displayed in table 1).table 1patient demographic profile and baseline clinical characteristicscharacteristicsstudy patients (n = 8144) n / mean%/sdage, years (mean, sd)66.213.2male (n,%) 433453.2%top 3 health plan types (n,%) ppo307637.8% comprehensive300236.9% pos plan7268.9%geographic region (n,%) northeast680.8% north central101312.4% south645979.3% west5957.3% unknown90.1%no t2 dm medical or prescription claim in 90 days pre - index (n,%) 170821.0%baseline period deyo - cci (mean, sd)1.61.8baseline presence of comorbid conditions (n,%) cardiovascular disease241929.7% diabetic peripheral neuropathy or foot ulcer6948.5% diabetic retinopathy3364.1% diabetic nephropathy2533.1%concomitant medications (n,%) antihyperlipidemic medications415351.0% antihypertensive medications513963.1% antiobesity medications100.1% deyo - cci deyo charlson comorbidity index, pos point of service, ppo preferred provider organization, sd standard deviation, t2 dm type 2 diabetes mellitus patient demographic profile and baseline clinical characteristics deyo - cci deyo charlson comorbidity index, pos point of service, ppo preferred provider organization, sd standard deviation, t2 dm type 2 diabetes mellitus the primary diagnosis on the index admission was used as the reason for admission . Diagnoses that were glycemic (hyperglycemia, non - ketotic hyperosmolar coma, hypoglycemia), microvascular (nephropathy, retinopathy, neuropathy, or foot ulcer), or macrovascular (cardiovascular, including atherosclerosis, myocardial infarction, ischemic heart disease, heart failure, stroke, or transient ischemic attack) in nature were flagged because of their potential relatedness to diabetes . Other primary diagnosis codes were grouped at the three - digit icd-9 code level to identify the top other reasons for admission among the study sample . The presence of hypoglycemia or hyperglycemia during the index admission was measured using relevant primary or secondary diagnosis codes listed on the index admission . Because hypoglycemia may be poorly coded on claims, in addition to hypoglycemia diagnosis codes, other diagnoses that may be indicative of the condition [e.g., icd-9 249.30, 250.30 (diabetic coma) and 962.3 (poisoning by insulin)] also were used to identify hypoglycemia in accordance with a published algorithm . Diagnoses of uncontrolled diabetes, diabetes with hyperosmolarity and abnormal glucose were defined as hyperglycemia . Frequencies and percentages were calculated for categorical variables and means and standard deviations were examined for continuous variables . The study described in this paper was an analysis of de - identified data and did not entail primary research with human or animal subjects . As such, institutional review board approval was not required prior to undertaking this research . A total of 9580 patients with t2 dm were identified in the data source, and 85% (n = 8144) met all inclusion criteria for the study . Study - eligible patients had a mean age of 66 years and 53% were male . Twenty - one percent had no t2 dm diagnosis or claims for ahas in the prior 90 days (table 1). The hospitals in which the index admission occurred tended to be medium (200499 beds, 57%) or large - sized (500 + beds, 30%). Most were non - teaching hospitals (97%) located in urban areas (85%). A primary or secondary diagnosis of t2 dm was required on the index hospitalization for study inclusion, but the primary reason for admission was not diabetes related for most patients in the study sample (table 2). Only 3% of patients had t2 dm (icd-9 250.x0, 250.x2) listed as the primary diagnosis on their index hospitalization . Twenty - two percent of patients had a primary diagnosis potentially related to diabetes, most of whom had a primary diagnosis of a macrovascular - related condition (21%); few patients had glycemic - related (<1%) or microvascular - related (<1%) conditions . Examination of other primary diagnoses revealed a wide variety of conditions, with no particular one predominating . The most common primary diagnoses are shown in table 2 and included osteoarthritis (6%), cardiac dysrhythmias (4%), and pneumonia (3%).table 2characteristics of the index hospitalizationcharacteristicsstudy patients (n = 8144) n / mean%/sdprimary diagnosis (n,%) t2dm2713.3% glycemic related570.7% microvascular related660.8% macrovascular related170420.9%top five other primary diagnoses (n,%) icd-9 715 osteoarthrosis4745.8% icd-9 427 cardiac dysrhythmias3224.0% icd-9 486 pneumonia, organism unspecified2132.6% icd-9 786 respiratory system / other chest symptoms1922.4% icd-9 038 septicemia1892.3%hypoglycemia at index admission (n,%) 1772.2%hyperglycemia at index admission (n,%) 89811.0%length of index hospitalization, days (mean, sd)4.24.0discharge status (n,%) discharged home694885.3% transferred to another facility84810.4% other / unknown3484.3% icd-9 international classification of diseases, ninth revision, clinical modification, sd standard deviation, t2 dm type 2 diabetes mellitus characteristics of the index hospitalization icd-9 international classification of diseases, ninth revision, clinical modification, sd standard deviation, t2 dm type 2 diabetes mellitus length of stay averaged 4.2 days (median 3 days; range 189 days). Most patients were discharged home, but 10% were transferred to another facility (e.g., long - term care, skilled nursing). Almost half (47%) of patients with t2 dm did not have any aha claims in the 30 days prior to hospitalization, and this proportion rose to about 60% in the 30 days following discharge (table 3). Seventeen percent of patients had no aha utilization while in the hospital.table 3aha utilization 30 days pre-, during, and 30 days post - hospitalizationpre - hospitalization n = 8144during hospitalization n = 8144post - hospitalization n = 8144 n% n% n% ahas biguanides210325.8199924.5140817.3 sulfonylureas152618.7178621.9105112.9 insulins130216.0573970.5120614 . Dpp-4 inhibitors5997.45937.34004.9 thiazolidinediones5857.25687.03123.8 other 1461.8420.5801.0type of aha regimen no ahas384647.2137616.9484659.5 insulin only (no oral agent or glp-1)82410.1313538.582310.1 1 oral agent / glp-1 insulin(s)213926.3239529.4174221.4 with insulin3043.7168920.72813.5 without insulin183522.57068.7146117.9 2 oral agents / glp-1s insulin(s)105312.998612.16017.4 with insulin1461.87329.0851.0 without insulin90711.12543.15166.3 3 oral agents / glp-1s insulin(s)2823.52523.11321.6 with insulin280.31832.2170.2 without insulin2543.1690.81151.4 aha antihyperglycemic agent, dpp-4 dipeptidyl peptidase-4 . Glp-1 glucagon - like peptide-1 receptor agonist patients may use more than one aha during an observation period; therefore the sum of the percentages may be greater than 100% other consists of the following aha classes; alpha - glucosidase inhibitors, amylin analogs, bile acid sequestrants, dopamine receptor agonists, glp-1 agonists, and meglitinides; all of which had little utilization (<2%) during the three observation periods aha utilization 30 days pre-, during, and 30 days post - hospitalization aha antihyperglycemic agent, dpp-4 dipeptidyl peptidase-4 . Glp-1 glucagon - like peptide-1 receptor agonist patients may use more than one aha during an observation period; therefore the sum of the percentages may be greater than 100% other consists of the following aha classes; alpha - glucosidase inhibitors, amylin analogs, bile acid sequestrants, dopamine receptor agonists, glp-1 agonists, and meglitinides; all of which had little utilization (<2%) during the three observation periods biguanides (i.e., metformin) were the most commonly filled oral ahas pre- and post - hospitalization, followed by the sulfonylureas and dipeptidyl peptidase-4 (dpp-4) inhibitors (fig . 1). Although only about 15% of patients utilized insulin pre- or post - hospitalization, insulin was utilized by the majority of patients (71%) during hospitalization.fig . 1most common antihyperglycemic agents pre-, during, and 30 days post - hospitalization in patients with pre - hospitalization utilization of any antihyperglycemic agent (n = 4298). Dpp-4 dipeptidyl peptidase-4 most common antihyperglycemic agents pre-, during, and 30 days post - hospitalization in patients with pre - hospitalization utilization of any antihyperglycemic agent (n = 4298). Dpp-4 dipeptidyl peptidase-4 patients who did not have a claim for ahas before their hospitalization were unlikely to have one afterward, and patients who had a claim for ahas before hospitalization often discontinued them following discharge . Of the patients without aha claims in the 30 days before their hospitalization, 70% continued to have no aha claims in the 30 days after leaving the hospital (fig . 2). Approximately, half of the patients who did have an aha claim before admission did not have any aha claims in the 30 days post - discharge . The majority (55%) of patients who were administered ahas during hospitalization had no aha claims after leaving the hospital (fig . 2changes in ahas from pre- to 30 days post - hospitalization all patients (n = 8144). Asterisks with or without insulins . 3changes in ahas during hospitalization to 30 days post - hospitalization all patients (n = 8144). Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist changes in ahas from pre- to 30 days post - hospitalization all patients (n = 8144). Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist changes in ahas during hospitalization to 30 days post - hospitalization all patients (n = 8144). Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist a subgroup analysis that assessed aha claims among patients with no t2 dm diagnosis or claims for ahas in the 90 days pre - admission (n = 1708) revealed that 77% of these patients had no aha claims in the 30 days after discharge . The corresponding rate among patients with evidence of either t2 dm diagnoses or aha claims pre - admission (n = 6436) was 55% . In another subgroup analysis among patients whose index admission diagnosis was t2 dm (n = 271), 61% of patients had an aha claim following discharge, compared to 40% among patients whose hospitalization had a non - t2 dm primary diagnosis (n = 7873). In the 60- and 90-day pre- and post - hospitalization analysis, the proportion of patients with no aha utilization decreased as the follow - up period increased (table 4). In the 90 days pre - admission, 35% of patients had no aha claims, compared to 47% over 30 days pre - admission . The corresponding post - discharge rates were 35% with no aha claims over 90 days and 60% over 30 days . No aha claims either pre - admission or post - discharge were observed among 24% of the sample over 90 days, compared to 35% over 30 days.table 4utilization of ahas over 30-, 60-, and 90-day periods pre- and post - hospitalization (n = 8144)pre - hospitalizationpost - hospitalization30 days (%) 60 days (%) 90 days (%) 30 days (%) 60 days (%) 90 days (%) proportion of patients, by aha regimen no ahas47.239.435.059.543.435.4 insulin only (no oral agent or glp-1)10.112.014.510.113.012.5 1 oral agent / glp-1 insulin(s)26.329.530.821.428.230.7 2 oral agents / glp-1s insulin(s)12.914.815.07.412.116.5 3 oral agents / glp-1s insulin(s)3.54.34.71.63.34.8 aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist utilization of ahas over 30-, 60-, and 90-day periods pre- and post - hospitalization (n = 8144) aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist a total of 9580 patients with t2 dm were identified in the data source, and 85% (n = 8144) met all inclusion criteria for the study . Study - eligible patients had a mean age of 66 years and 53% were male . Twenty - one percent had no t2 dm diagnosis or claims for ahas in the prior 90 days (table 1). The hospitals in which the index admission occurred tended to be medium (200499 beds, 57%) or large - sized (500 + beds, 30%). Most were non - teaching hospitals (97%) located in urban areas (85%). A primary or secondary diagnosis of t2 dm was required on the index hospitalization for study inclusion, but the primary reason for admission was not diabetes related for most patients in the study sample (table 2). Only 3% of patients had t2 dm (icd-9 250.x0, 250.x2) listed as the primary diagnosis on their index hospitalization . Twenty - two percent of patients had a primary diagnosis potentially related to diabetes, most of whom had a primary diagnosis of a macrovascular - related condition (21%); few patients had glycemic - related (<1%) or microvascular - related (<1%) conditions . Examination of other primary diagnoses revealed a wide variety of conditions, with no particular one predominating . The most common primary diagnoses are shown in table 2 and included osteoarthritis (6%), cardiac dysrhythmias (4%), and pneumonia (3%).table 2characteristics of the index hospitalizationcharacteristicsstudy patients (n = 8144) n / mean%/sdprimary diagnosis (n,%) t2dm2713.3% glycemic related570.7% microvascular related660.8% macrovascular related170420.9%top five other primary diagnoses (n,%) icd-9 715 osteoarthrosis4745.8% icd-9 427 cardiac dysrhythmias3224.0% icd-9 486 pneumonia, organism unspecified2132.6% icd-9 786 respiratory system / other chest symptoms1922.4% icd-9 038 septicemia1892.3%hypoglycemia at index admission (n,%) 1772.2%hyperglycemia at index admission (n,%) 89811.0%length of index hospitalization, days (mean, sd)4.24.0discharge status (n,%) discharged home694885.3% transferred to another facility84810.4% other / unknown3484.3% icd-9 international classification of diseases, ninth revision, clinical modification, sd standard deviation, t2 dm type 2 diabetes mellitus characteristics of the index hospitalization icd-9 international classification of diseases, ninth revision, clinical modification, sd standard deviation, t2 dm type 2 diabetes mellitus length of stay averaged 4.2 days (median 3 days; range 189 days). Most patients were discharged home, but 10% were transferred to another facility (e.g., long - term care, skilled nursing). Almost half (47%) of patients with t2 dm did not have any aha claims in the 30 days prior to hospitalization, and this proportion rose to about 60% in the 30 days following discharge (table 3). Seventeen percent of patients had no aha utilization while in the hospital.table 3aha utilization 30 days pre-, during, and 30 days post - hospitalizationpre - hospitalization n = 8144during hospitalization n = 8144post - hospitalization n = 8144 n% dpp-4 inhibitors5997.45937.34004.9 thiazolidinediones5857.25687.03123.8 other 1461.8420.5801.0type of aha regimen no ahas384647.2137616.9484659.5 insulin only (no oral agent or glp-1)82410.1313538.582310.1 1 oral agent / glp-1 insulin(s)213926.3239529.4174221.4 with insulin3043.7168920.72813.5 without insulin183522.57068.7146117.9 2 oral agents / glp-1s insulin(s)105312.998612.16017.4 with insulin1461.87329.0851.0 without insulin90711.12543.15166.3 3 oral agents / glp-1s insulin(s)2823.52523.11321.6 with insulin280.31832.2170.2 without insulin2543.1690.81151.4 aha antihyperglycemic agent, dpp-4 dipeptidyl peptidase-4 . Glp-1 glucagon - like peptide-1 receptor agonist patients may use more than one aha during an observation period; therefore the sum of the percentages may be greater than 100% other consists of the following aha classes; alpha - glucosidase inhibitors, amylin analogs, bile acid sequestrants, dopamine receptor agonists, glp-1 agonists, and meglitinides; all of which had little utilization (<2%) during the three observation periods aha utilization 30 days pre-, during, and 30 days post - hospitalization aha antihyperglycemic agent, dpp-4 dipeptidyl peptidase-4 . Glp-1 glucagon - like peptide-1 receptor agonist patients may use more than one aha during an observation period; therefore the sum of the percentages may be greater than 100% other consists of the following aha classes; alpha - glucosidase inhibitors, amylin analogs, bile acid sequestrants, dopamine receptor agonists, glp-1 agonists, and meglitinides; all of which had little utilization (<2%) during the three observation periods biguanides (i.e., metformin) were the most commonly filled oral ahas pre- and post - hospitalization, followed by the sulfonylureas and dipeptidyl peptidase-4 (dpp-4) inhibitors (fig . 1). Although only about 15% of patients utilized insulin pre- or post - hospitalization, insulin was utilized by the majority of patients (71%) during hospitalization.fig . 1most common antihyperglycemic agents pre-, during, and 30 days post - hospitalization in patients with pre - hospitalization utilization of any antihyperglycemic agent (n = 4298). Dpp-4 dipeptidyl peptidase-4 most common antihyperglycemic agents pre-, during, and 30 days post - hospitalization in patients with pre - hospitalization utilization of any antihyperglycemic agent (n = 4298). Dpp-4 dipeptidyl peptidase-4 patients who did not have a claim for ahas before their hospitalization were unlikely to have one afterward, and patients who had a claim for ahas before hospitalization often discontinued them following discharge . Of the patients without aha claims in the 30 days before their hospitalization, 70% continued to have no aha claims in the 30 days after leaving the hospital (fig . 2). Approximately, half of the patients who did have an aha claim before admission did not have any aha claims in the 30 days post - discharge . The majority (55%) of patients who were administered ahas during hospitalization had no aha claims after leaving the hospital (fig . 2changes in ahas from pre- to 30 days post - hospitalization all patients (n = 8144). Asterisks with or without insulins . 3changes in ahas during hospitalization to 30 days post - hospitalization all patients (n = 8144). Asterisks with or without insulins . Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist changes in ahas from pre- to 30 days post - hospitalization all patients (n = 8144). Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist changes in ahas during hospitalization to 30 days post - hospitalization all patients (n = 8144). Aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist a subgroup analysis that assessed aha claims among patients with no t2 dm diagnosis or claims for ahas in the 90 days pre - admission (n = 1708) revealed that 77% of these patients had no aha claims in the 30 days after discharge . The corresponding rate among patients with evidence of either t2 dm diagnoses or aha claims pre - admission (whose index admission diagnosis was t2 dm (n = 271), 61% of patients had an aha claim following discharge, compared to 40% among patients whose hospitalization had a non - t2 dm primary diagnosis (n = 7873). In the 60- and 90-day pre- and post - hospitalization analysis, the proportion of patients with no aha utilization decreased as the follow - up period increased (table 4). In the 90 days pre - admission, 35% of patients had no aha claims, compared to 47% over 30 days pre - admission . The corresponding post - discharge rates were 35% with no aha claims over 90 days and 60% over 30 days . No aha claims either pre - admission or post - discharge were observed among 24% of the sample over 90 days, compared to 35% over 30 days.table 4utilization of ahas over 30-, 60-, and 90-day periods pre- and post - hospitalization (n = 8144)pre - hospitalizationpost - hospitalization30 days (%) 60 days (%) 90 days (%) 30 days (%) 60 days (%) 90 days (%) proportion of patients, by aha regimen no ahas47.239.435.059.543.435.4 insulin only (no oral agent or glp-1)10.112.014.510.113.012.5 1 oral agent / glp-1 insulin(s)26.329.530.821.428.230.7 2 oral agents / glp-1s insulin(s)12.914.815.07.412.116.5 3 oral agents / glp-1s insulin(s)3.54.34.71.63.34.8 aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist utilization of ahas over 30-, 60-, and 90-day periods pre- and post - hospitalization (n = 8144) aha antihyperglycemic agent, glp-1 glucagon - like peptide-1 receptor agonist the results of this retrospective study of hospitalized adults with t2 dm suggest patients may not be receiving optimal aha therapy during transitions of care around the time of hospitalization . Most patients (83%) received ahas while hospitalized, but approximately half had no aha claims in the 30 days prior to hospitalization (90 days for mail order) and about 60% had no aha claims in the 30 days post - discharge . Lengthening the pre- and post - hospitalization periods reduced the proportion of patients without aha claims but one - third of patients still had no aha claim before and after the hospital stay even with a longer 90-day window . Patients who did not have ahas claims before hospitalization were unlikely to start afterward, and a large proportion of patients who utilized ahas pre - admission or during their stay often discontinued these medications upon discharge . This is concerning because lack of continuity of care has been associated with adverse clinical outcomes, especially in patients with a chronic condition . In a study of medicare beneficiaries with diabetes hospitalized for acute myocardial infarction, for example, patients discharged without aha therapy had higher mortality rates 30 days, 6 months, and 1 year post - hospitalization than patients discharged on aha therapy, even after multivariable adjustment of differences between groups . It deserves mention that although all patients in the study had a primary or secondary diagnosis of t2 dm during the index hospitalization, 21% percent of patients in this study had no healthcare claims with a t2 dm diagnosis and no pharmacy claims for ahas in 90 days prior to their index hospitalization . Specific clinical details on these patients were not available in the database used for the analysis, but these patients may represent previously undiagnosed patients whose t2 dm was initially identified during the index admission . It also is possible they were diagnosed patients treated with lifestyle interventions alone (e.g., diet and exercise but no aha) who did not incur any healthcare services carrying a t2 dm diagnosis in the 90 days prior to hospitalization . These patients had the lowest rates of post - discharge aha claims but subgroup analyses excluding them entirely did not drastically alter study results; 60% of all patients had no aha claims in the 30 days post - discharge whereas after this exclusion, 55% of the remaining patients had no aha claims in the 30 days post - discharge . Our study finding that 60% of all patients with t2 dm had no aha claims in the 30 days post - discharge (corresponding figures for 60 and 90 days post - discharge were 44, and 35%, respectively) while high, was in line with the small body of previous research examining post - discharge aha treatment patterns . Wu et al . Studied 2160 patients with t2 dm who used insulin both in the 30 days before and during hospitalization, identified through retrospective medical records review at a us health system, and found 61% discontinued insulin upon discharge . About 60% of these patients were also treated with oral ahas in the 6 months before hospitalization, but only about 2025% were treated with oral ahas in the 60 days post - discharge . Bergenstal et al . Assessed pre- and post - admission treatment patterns in a retrospective database analysis of 400 patients, and found 24% had a reduction in aha regimen after hospitalization . Lipska et al . Found that 13.4% of 8791 medicare patients on aha therapy at the time of admission for acute myocardial infarction were discharged without such therapy, according to medical record review conducted as part of the national heart care project . In a retrospective chart review of 217 diabetic patients admitted with acute myocardial infarction, lovig et al . Found 11.5% of these patients were discharged without any ahas, despite most having no clinical reason to discontinue ahas . Retrospectively examined 1359 men with poorly controlled diabetes (hba1c> 8.0%) discharged from veterans administration hospitals and found less than one quarter had a change in therapy upon discharge, and almost one - third had no change in therapy or scheduled follow - up for continuing care within 30 days . The authors suggest that this lack of treatment modification or follow - up care despite evidence of poorly controlled disease may reflect clinical inertia. An alternate explanation to clinical inertia is that there was a deliberate decision to not utilize ahas post - hospitalization . While the data used in this study did not allow examination of clinical decision making, lifestyle interventions alone may be appropriate initial treatment for t2 dm and survey data suggests about 14% of us adult diabetes patients do not use ahas, so not all patients in our sample were expected to have aha utilization post - discharge . In addition, some patients in our study may have had non - diabetes - related elevated glucose levels during hospitalization that resulted in an erroneous t2 dm diagnosis on their hospital record; any such patients may not have required aha utilization post - discharge . It is notable, though, that loving and colleagues conducted medical chart review to assess reasons for aha discontinuation among diabetic patients hospitalized for myocardial infarction and were unable to find a clear reason for discontinuation for 88% of patients . That, along with the high proportion of patients with no post - hospitalization aha in the current and previous studies, suggests hospitalization may not result in an appropriate re - evaluation of therapy . This may be problematic, as patients with t2 dm who are hospitalized tend to have poorer glycemic control than comparable patients who are not hospitalized, and the hospitalization may present an opportunity to intervene . Other literature has suggested that a hospital admission may allow an opportunity to improve long - term diabetes care . We measured aha pre- and post - hospitalization utilization based on outpatient pharmacy claims, leaving open the possibility that patients may have received ahas that do not appear in the data . For example, it is possible that some patients received samples, paid cash for low - cost generic aha prescriptions such that no claim was generated to their health plan, or had a pre - admission supply of medication that they utilized post - discharge, all of which could make it appear as if patients had no aha utilization when, in fact, they did use ahas post - discharge . In addition, inpatient medication utilization for readmitted patients (12% within 30 days; 21% within 90 days) was not captured in our post - discharge medication utilization measures . Treatment non - adherence, for example, patients receiving but not filling aha prescriptions, also could be reason for the lack of aha claims since non - adherence is a known issue in this population . The results of this study are based on patients whose data were captured in the linked marketscan hospital drug database, which may not be generalizable to all individuals with t2 dm in the us . The hospitals that contribute to the database are primarily community hospitals and are disproportionately located in the southern region of the us . Most hospitals in which an index admission occurred were non - teaching hospitals (97%) and located in urban areas (85%). Aha utilization patterns at teaching hospitals or rural facilities could be different than seen in our sample . The results of this study suggest that a large proportion of hospitalized patients with t2 dm may not receive optimal transitions of care in and out of the hospital . Most patients (83%) utilized an aha while hospitalized but one - half of all patients did not fill an aha prescription either in the 30 days before or 30 days after hospitalization, and almost one - third of patients did not fill an aha prescription in the 90 days before or after hospitalization . One - fifth of patients for whom t2 dm is listed on a claim associated with their inpatient care had no evidence of being treated for diabetes before their admission, suggesting they may have been newly diagnosed at the time of admission and would not have become aware of their t2 dm at that time if not for the hospitalization . Under - diagnosis of t2 dm and inadequate treatment of diagnosed t2 dm remains an issue in and out of the hospital; when patients are hospitalized, it is crucial to take advantage of this opportunity to appraise and optimize their care . Lien vo was an employee of janssen scientific affairs, llc at the time of the research . This article does not contain any new studies with human or animal subjects performed by any of the authors . 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Attention - deficit / hyperactivity disorder (adhd) is a behavioral disorder usually diagnosed during childhood . It is characterized by developmentally inappropriate symptoms of inattention, hyperactivity and impulsivity and significant impairment in multiple domains of functioning . Diagnostic and statistical manual of mental disorders (dsm - iv) diagnostic criteria define three adhd subtypes based on the presence of inattention and hyperactivity / impulsivity symptoms: predominantly inattentive type, predominantly hyperactive / impulsive type, and combined type . A recent us national epidemiological study of 8- to 15-year - old school children found an 8.7% prevalence of adhd, with the predominantly inattentive type most prevalent (4.4%) among the adhd subtypes (combined type, 2.2%; predominantly hyperactive - impulsive type, 2.0%). The behavioral manifestations of adhd inattentive type differ from those associated with combined and hyperactive / impulsive type . Children with combined and hyperactive / impulsive type exhibit problems with behavioral inhibition, including interrupting, blurting out, getting out of their seat at inappropriate times, and playing loudly . In contrast, the primary difficulties of children with inattentive type are non - disruptive in nature and are related to planning and organizing actions . Children with inattentive type often forget to complete or lose assignments, procrastinate, complete work carelessly, have difficulty planning for the completion of long - term projects and studying for tests, and have problems keeping materials organized [7 - 9]. Three treatments qualify as well - established interventions for children with adhd: psychopharmacological therapy, behavioral parent training, and behavioral classroom management . Psychopharmacological therapy (e.g. Stimulant medication) is the most utilized intervention modality for treating patients with adhd and produces marked improvements in sustained attention, impulse control and noisy and disruptive behaviors . While psychopharmacology ameliorates some of the core symptoms of adhd inattentive type, there is minimal evidence to suggest that medication promotes improvements in the ability to effectively plan and organize materials and/or actions . As such, stimulant medication is only minimally effective in improving important areas of functioning such as long - term academic achievement . Similarly, evidence - based behavioral treatments, such as parent training and classroom management, primarily target impulsive and disruptive behaviors and do not focus on the problematic behaviors associated with inattentive type . In the past 2 years, several advances have been made in the treatment of behaviors exhibited by children with adhd inattentive type . Interventions have been evaluated that target planning, materials organization, time - management, and homework management skills . These interventions typically focus on behavior in the school setting as demands for these skills are greatest at school and can lead to significant academic impairment . Children are taught systems for organizing school materials and managing / planning homework responsibilities . Checklists with operationalized definitions of behavior are used to monitor skills implementation . Similar to existing evidence - based interventions for adhd, these interventions incorporate behavioral therapeutic techniques, such as rehearsal, prompting, shaping and contingency management, to teach and promote skills use and their generalization . For example, point systems or token economies are often utilized to monitor and reward adherence . Interventions have been evaluated that target multiple aspects of organization and planning, including: classroom preparation, organization of bookbag, binder, and locker, and planning and tracking homework assignments and long - term projects . These interventions are associated with short - term gains on process measures of materials organization, homework management, planning and procrastination . Some of these improvements appear to be sustainable for children with adhd as assessed at 8-week and 3-month follow - ups . At this point, it is unclear if gains on process measures (e.g. Materials organization or homework management checklists) are ultimately associated with improvement in functional outcomes such as school performance . Only a subset of studies completed to date included both process measures and measures of functional impairment . Documented improvements in organizational skills, parent ratings of homework problems, and grade point average . However, the gains in grade point average were relatively small . Pfiffner et al . Documented improvements in organizational skills and a clinician completed clinical global impression, and these gains were maintained at a 3- to 5-month follow - up . Abikoff and gallagher showed improvements in both organizational skills and parent - rated homework problems, but it is unknown if improvements in homework problems resulted in overall academic improvements . Hence, alternative explanations for observed patient improvements are possible (e.g. Hawthorne effects). In addition, generalization across settings was not formally assessed in most of the studies . It would be important to determine if skills generalize across classrooms or from school to home . The behavioral difficulties of patients with adhd inattentive type may be markedly different from those of children with combined or hyperactive / impulsive type and the targets of treatment will need to be tailored accordingly . While behavioral interventions targeting organization, planning and time - management cannot be classified as a central challenge will be figuring out how to disseminate these interventions into clinical practice . The interventions evaluated to date have varied in where the intervention is delivered (e.g. School, home, or clinic), and dissemination models will need to account for these factors . Providing intervention directly in the setting where the skills are most relevant / problematic (e.g. School) should allow clinicians to achieve higher levels of skills generalization . To promote dissemination, intervention protocols should be developed that do not require a high degree of clinical specialization to implement and that can be implemented directly in the setting of interest.
Ureteral avulsion is conventionally defined as injuries of the ureter caused by blunt (non - penetrating) trauma with acute deceleration or acceleration movement mechanism due to motor vehicle accidents . With the invention of endourology tools, ureteral avulsion can also occur as a result of stretching in the weakest point of the ureter . Ureteral avulsion and perforation during ureteroscopy are the most common cause of iatrogenic ureteral injuries . The rate of ureteral injury dropped to 7% in 1990 due to increased surgical experience and development of more advanced uertroscopy tools . The incidence of ureteral perforation has been reported in 1.5% of such procedures of which 0.2% have required surgery . In spite of the prevalent use of ureteroscopy in the treatment of ureteral stones and replacement of open surgical procedures with ureteroscopic procedures, the rate major and severe complications such as avulsion and perforation commands attention [6, 7]. Ureteral avulsion from the pelvis can be managed by ureteral reimplantation, transureteroureterostomy and ureteroneocystostomy [810]. Renal autotransplantation can be an appropriate whenever a considerable length of the ureter is lost or a previous surgery has failed . Meanwhile, sometimes after such procedure renal impairment is encountered . In the present case, we report a successful autotransplantatioin in a patient who underwent urgent operation following avulsion of the ureter at a distance of 4 cm from the pelvis . Surgeons should be aware of possible complications attributed to transureteral lithotripsy (tul) and their knowledge and expertize in managing such complications . The patient was a 51-year - old lady with left flank colicky pain associated with nauseas and vomiting for a month . Study of kub (kidney, ureter, and bladder x - ray) and non - contrast abdominopelvic spiral ct scan images revealed a 1-cm proximal left ureteral stone (fig . 1). Accordingly the patient was selected to undergo tul and ureteral stenting . Figure 1:abdominopelvic spiral ct scan of the patient before surgery . Abdominopelvic spiral ct scan of the patient before surgery . Due to a narrow ureteral lumen proceeding with the procedure seemed impossible and we decided to terminate the surgery . While taking out the ureteroscope we noticed avulsion of the ureter at a point approximately 4 cm from ureteropelvic junction . The avulsed ureter was handing on the tip of the ureteroscope and the avulsed fragment came out along with the ureteroscope . After thorough evaluation of various possible methods to approach the problem, the patient was brought to a left flank position from her original lithotomy position . With a left intercostal incision nephrectomy was done . Subsequently, the position of the patient was changed to supine and a gibson incision was made over the right lower quadrant of the abdomen . Following dissection of the iliac vessels, renal vessels were anastomosed to the iliac vessels (renal artery to the internal iliac artery and renal vein to the external iliac vein). Using the lich given the fact that only a short segment of the avulsed ureter (4 cm) was left, we considered implantation of the ureter to the bladder . A drainage tube was inserted to drain any possible bleeding from the operation site and then the surgical wound was closed . The patient was discharged in a good general condition after 3 days and the drainage tube removed . An ultrasound imaging study of the genitourinary system 8 months into the patients follow up showed normal size, echo and cortical thickness in the operated kidney after renal autotransplantation . In follow up, 2 months after renal autotransplantation, the arterial and venous flow of the transplanted kidney were normal on color doppler ultrasound imaging (figs . 2 and 3). The mean parenchymal arterial vascular resistance in the transplanted kidney was 0.63 which was in the normal range . The height, anteroposterior diameter and cortical thickness of the transplanted kidney were 117.41 mm and 13 mm, respectively . The values of urea and creatinine 3 months after transplantation were 26.4 and 1.09, respectively . Size, echo and cortical thickness of the transplanted kidney were reported normal in the follow up ultrasound 8 months after autotransplantation (fig . Figure 3:color doppler ultrasound of the transplanted kidney 2 months after surgery in the zoomed area of the image . Figure 4:gray - scale ultrasonography of the patient's transplanted kidney 8 months after surgery . Color doppler ultrasound of the transplanted kidney 2 months after surgery in the zoomed area of the image . Ureteroscopy is widely used for lithotripsy procedure and resection of malignant tumors, but this method may lead to urethral mucosal trauma, hematuria, ureteral stenosis, urinary tract infection and ureteral perforation . Among all complications of ureteroscopic lithotripsy procedure, complete avulsion of the proximal ureter is one of the most challenging that happens in 0.060.45% of the cases . It is evident that appropriate decision making and a timely surgical intervention can prevent the need for nephrectomy and possible complications in the future . In general, management of ureteral avulsion depends on the location of injury, the length of the traumatized ureter, time of diagnosis, patient's age and general health . Boari flap and psoas hitch techniques are used in proximal and distal injury of the ureter . Finally, in the absence of the previous conditions, autotransplantation can be done as a vital method . It is evident that such surgical procedures require substantial expertize among all healthcare providers, a patient in a suitable physical status and proper age, and an accurate and timely decision making . Autotransplantation was done in our patient after quick evaluation and review of different options to repair a traumatized ureter and clinical evidences supported the accomplishment of a successful outcome.
Nevertheless, completely laparoscopic colorectal procedures have been adopted in clinical practice by a limited number of surgeons . This is possibly due to increased operating time and costs, to the fact that their relative benefit is as yet unclear, and that these procedures are technically demanding . The latter reason prompted this experimental study, which was carried out to evaluate whether a previously described intracorporeal approach to sigmoid resection could be facilitated and applied to the rectum in a porcine model . Ten domestic pigs with a median weight of 40.7 kg (range 38.5 - 43 kg) were anesthetized with intubation and ventilation with halothane . Prior to surgery, the animals were fasted for 48 hours and kept on fluids ad libitum and received a 2-liter enema . Pneumoperitoneum was induced using carbon dioxide insufflated to a pressure of 10 mm hg by placement of a trocar in the infraumbilical skin using a cut - down technique . Two 12 mm ports were then inserted in the left hypocondrium and in the midline cephalad to the umbilicus . Due to porcine anatomy, a 33 mm cannula (endopath, ethicon endosurgery, cincinnati, oh) was inserted in the right iliac fossa rather than suprapubically . All subjects were turned into a steep head - down position in addition to a right lateral tilt until no small bowel was seen in the field . A surgical technique previously described for laparoscopic sigmoid resection with intracorporeal colorectal anastomosis and specimen removal via a suprapubic incision once the proximal site of bowel division was identified, a laparoscopic pursestring clamp (ethicon endosurgery, cincinnati, oh) (figure 1) was applied to the colon, and two straight needles (placed at opposite ends of a monofilament suture and held in position against the clamp by an endoloop) were passed through the clamp and retrieved through a port, as previously described . The colon was divided with scissors against the pursestring clamp after having been ligated with an intracorporeally knotted suture . The lower third of the rectum was transected with an articulating endoscopic stapler (ets flex 35, ethicon endosurgery, cincinnati, oh). A plastic bag allowed temporary intra - abdominal storage of the specimen . The rectal stump was irrigated by 1uminal wash - out in order to verify that the linear staple row was tight . The anvil of a circular stapler (ecs 25 ethicon endosurgery, cincinnati, oh) held by a modified allis - clamp was inserted through the 33 mm port into the colon and the pursestring was tied intracorporeally around the anvil notch . The same circular gun with a spike fixed to its shaft was introduced per anum (figure 2), and a doublestapled colorectal anastomosis was performed according to a previously - described technique . The integrity of the anastomosis was checked by irrigation with methylene blue after the application of a noncrushing intestinal clamp just proximal to the circular staple line . The specimen, in a plastic bag, was delivered through the 33 mm port . Circular stapler with spike fixed to its shaft is advanced behind the staple line of the rectal stump . Complete proximal and distal doughnuts were obtained in all cases and anastomoses were all methylene blue tight . The mean anastomotic level from the anal verge was 5.2 cm (range 4 - 6 cm). All porcine subjects had an uneventful 5-week postoperative course at the end of which both laparoscopy and rigid proctoscopy were carried out . At celioscopy there were no adhesions or other abnormalities at the anastomotic site, while endoscopy revealed normal suture lines . Histology of colorectal anastomoses revealed healing mucosa with neither foreign body reactions nor thickening of bowel wall at anastomotic site . Transection of the rectum at a lower level can be achieved inserting an open - surgery articulating stapler through a suprapubic incision . However, this approach requires a third stapler (in addition to endoscopic linear and circular staplers), re - establishing pneumoperitoneum twice, and manipulation of an open - surgery stapling device with laparoscopic graspers . The results of the present experimental study show that the use of a laparoscopic articulating stapling device helps to overcome these downsides and allows secure division of the low rectum . Intracorporeally hand - sewn pursestring sutures are usually fashioned with curved or ski - shaped needles on the proximal colon, but this is a time - consuming procedure . The introduction of the t - needle technique (endo - stitch, ussc, norwalk, ct) failed to facilitate this procedure . In fact, since the t - needle should not be detached from the instrument's jaws (unless for reloading), the anvil shaft must, at a certain point, be loosened from the allis - clamp to allow 360 suturing and tying around the pursestring notch of the anvil ., that the use of a laparoscopic pursestring suture clamp seems to facilitate the construction of intracorporeal pursestring sutures and results in a safe colorectal anastomosis . According to previously described techniques, once the circular stapling gun's central spike (auto suture, pceeatm, ussc, norwalk, connecticut) is advanced behind the staple line of the rectal stump, the spike must be disconnected and removed through a port (figure 3). The use of a gun adapted for laparoscopy (with a spike fixed to its shaft) (figure 2) eliminates the need for retrieval of the spike . The present study shows that a previously described intracorporeal approach to sigmoid resection can be facilitated and applied safely to the rectum in a porcine model . However, further experimental and clinical comparative studies are needed with regard to safety, time - frame and costs of the construction of intracorporeal pursestring sutures.
Transversus abdominis plane (tap) block is a widely practiced peripheral nerve block, utilized to anesthetize the somatic nerves supplying the anterior abdominal wall by depositing local anesthetic in the neurovascular plane between internal oblique and transversus abdominis muscle layers . It was introduced in anesthesia practice in 2001 by rafi utilizing the traditional anatomical land marks . Tap block has subsequently been used as a component of multimodal analgesia for postoperative pain relief following various surgical procedures such as large bowel resection, open appendectomy, retropubic prostatectomy, nephrectomy, hernia repair, laparoscopic cholecystectomy and cesarean section . Although carney et al . And atim et al . Have observed analgesic benefit of tap block in total abdominal hysterectomy by landmark based approach and ultrasound guided (usg) approach respectively, griffith et al . Found that tap block does not confer any definite analgesic benefit in major gynecological procedures over a multimodal analgesic regimen . Furthermore, the effect of preincisional tap block on intraoperative as well as postoperative analgesia in patients undergoing total abdominal hysterectomy remains yet to be elucidated . Based on these observations, this study was conceptualized to elucidate the efficacy of bilateral preincisional tap block as a component of multimodal analgesia for providing perioperative pain relief in patients undergoing total abdominal hysterectomy . After obtaining approval by the institute ethics committee and written informed consent, 90 adult female patients of american society of anesthesiologists (asa) physical status (ps) i or ii, scheduled for total abdominal hysterectomy by a lower abdominal transverse incision were recruited in this randomized double - blind controlled clinical trial . Unwilling patients and patients with body mass index> 30 kg / m, compromised renal and liver function, uncontrolled diabetes, severe cardiovascular, respiratory disease, having a history of allergy to any of the study drug, and history of abdominal surgery were excluded from the study . Secondary outcomes were intraoperative fentanyl requirement and hemodynamic changes, postoperative hemodynamic changes and time to request first postoperative analgesic . A thorough review of related literature was performed from standard textbooks and an internet search of related articles was performed pubmed . Sample size was calculated on the basis that a 20 mm difference in the mean vas between the two groups would be clinically useful . We used ps power and sample size calculations software, version 3.0 [department of biostatistics, vanderbilt school of medicine, nashville, tn]. We assumed a standard deviation of 30 mm as a standard deviation of vas score in the population . Forty one patients in each group would be needed, assuming the probability of alpha () error is 5% and a power of the study is 85% . Assuming a probable drop out of 10%, 90 patients were recruited . Patients were randomly allocated into two equal groups of 45 patients in each group using a random number generators in microsoft excel 2003 [microsoft corporation, redmond, wa] and allocation concealment were maintained by using an opaque sealed envelope technique . Baseline parameters such as heart rate, continuous electrocardiogram, noninvasive blood pressure, spo2 were noted down . Patients were randomly allocated into two groups, group b or group n to receive one of the following solutions for bilateral tap block . Group b: injection bupivacaine, 0.25% (0.5 ml / kg body weight on each side).group n: injection normal saline (0.5 ml / kg body weight on each side). Group b: injection bupivacaine, 0.25% (0.5 ml / kg body weight on each side). Group n: injection normal saline (0.5 ml / kg body weight on each side). The anesthesiologist, who prepared the solution in identical syringes, remained unaware of the nature of the study and was not involved in further data collection . The lumbar triangle of petit, located just anterior to the latissimus dorsi muscle was identified by palpating the iliac crest in an anterior to posterior direction, until the edge of the latissimus dorsi was felt . The skin was pierced just cephalic to the iliac crest over the triangle of petit with a blunt 18 gauge tuhoy needle [smiths medical international limited, hythe, kent, uk] after infiltration with 2% lignocaine . The needle was advanced perpendicular to the skin in the coronal plane until the first resistance of external oblique muscle was encountered . Gentle advancement of the needle resulted in a pop sensation as the needle entered the plane between the external and internal oblique fascial layers . A second loss of resistance was encountered when the needle reaches the transversus abdominis fascial plane between the internal oblique and transversus abdominis muscle . A test dose of 1 ml was injected to determine resistance to flow and confirm the needle tip placement within the neurovascular plane . After this one of the study solutions was injected on each side following careful aspiration to exclude vascular puncture . Ten min after tap block, all patients received a standardized general anesthesia with fentanyl 1 mcg / kg, propofol and atracurium . They were monitored for any signs of inadequate analgesia in the intraoperative period such as sweating, lacrimation, tachycardia (> 100/min) and hypertension (> 20% elevation of baseline mean arterial pressure) and supplemental doses of injection fentanyl 0.5 mcg / kg were given as needed . An anesthesiologist who was unaware about the patient allocation did recording of intraoperative hemodynamic data and other anesthesia management . Intravenous infusion of paracetamol (1 g to patients with body weight> 40 kg and 750 mg to patients with body weight <40 kg) was given 30 min prior to completion of surgery . Fluid deficit arising from preoperative fasting was corrected by maintenance fluid . Blood loss and other plasma losses blood losses up to the transfusion threshold were replaced with 3 ml of ringer's lactate for each ml of blood loss . After the patient had adequately recovered from anesthesia, and was able to assess pain, postoperative analgesia was assessed with vas 0 - 100 mm in the immediate postoperative period (when the patient was able to communicate in the postanesthesia care unit), at 1, 2, 3, 4, 5, 6 and 24 h and whenever the patient complained of pain . The time of administration of rescue analgesic in the form of injection tramadol 2 mg / kg intravenous (iv) was noted when vas> 40 mm . Vas score was assessed in both rest and movement (knee flexion) by an independent observer who was unaware about the allocation . After administration of rescue analgesic, patients were shifted to a postoperative analgesic regimen of injection tramadol iv 2 mg / kg 8 hourly and injection paracetamol 6 hourly up to 24 h. the incidence of postoperative nausea and vomiting was noted during the first 24 h. rescue antiemetics were given to any patient who complained of nausea or vomiting . Any signs of adverse effects of the technique like local site infection, hematoma formation, local anesthetic toxicity due to intravascular injection of anesthetic (like dizziness, tinnitus, perioral numbness and tingling, lethargy, seizures, signs of cardiac toxicity like atrio - ventricular conduction block, arrhythmias, myocardial depression and cardiac arrest), peritoneal perforation, bowel perforation, difficulty ambulating or fall and injury secondary to spread of local anesthetic to nerves of the buttock, lateral thigh or leg in the distribution of the femoral nerve were sought for . All raw data were entered into a microsoft excel spreadsheet and analyzed using standard statistical software . Continuous numerical data were expressed as mean and standard deviation (for normally distributed data), or median and inter - quartile range (for data that are not normally distributed). Normally distributed numerical data between groups were analyzed using the student's t - test . Skewed data between groups were analyzed using the mann categorical variables were analyzed using the fisher's exact test or the pearson's chi - square test as applicable . Ninety patients were recruited for the trial and data from all of them has been analyzed . A consort flow diagram depicting the passage of participants through the trial has been provided in figure 1 . The two groups were comparable in terms of baseline demographic parameters (age, sex and body weight), duration of surgery and anesthesia and preoperative hemodynamic parameters (pulse rate, systolic and diastolic blood pressure, respiratory rate) and the volume of the study drug required in tap block . A summary of base line characteristics of the patients has been furnished in table 1 . Consort flow diagram for patient selection baseline characteristics of the patients in each group from the analysis of the intraoperative hemodynamic parameters it was found that pulse rate was significantly higher in patients receiving placebo (95.9 11.2 bpm vs. 102.9 8.8 bpm, mean the difference 7.0 s, p = 0.001) after surgical skin incision . Both systolic and diastolic blood pressure after surgical skin incision was also significantly higher in patients receiving placebo, but similar at all other time points . Comparison of mean preoperative and intraoperative pulse rate, b = group b, n = group n, pr1 = 10 min after tap block, pr2 = before induction, pr3 = after induction, pr4 = after incision, pr5 = 15 min intraoperative, pr6 = 30 min intraoperative, pr7 = 60 min intraoperative, pr8 = 90 min intraoperative, pr9 = 120 min intraoperative comparison of mean preoperative and intraoperative systolic blood pressure and diastolic blood pressure . Bsbp = systolic blood pressure of group b, nsbp = systolic blood pressure of group n, bdbp = diastolic blood pressure of group b, ndbp = diastolic blood pressure of group n, bp1 = 10 min after tap block, bp2 = before induction, bp3=after induction, bp4 = after incision, bp5 = 15 min intraoperative, bp6 = 30 min intraoperative, bp7 = 60 min intraoperative, bp8 = 90 min intraoperative, bp9 = 120 min intraoperative median requirement of intraoperative fentanyl was significantly higher in patients receiving placebo in comparison to bupivacaine in tap block (81 mcg vs. 114 mcg, p = 0.000, mann the difference between the medians is 32.0 mcg with a 95% confidence interval (ci) of 22.0, 42.5 (hodges lehman median difference). There was no statistically significant difference between the two groups in terms of median values of immediate postoperative pulse rate, systolic blood pressure and diastolic blood pressure (mann whitney u - test). Postoperative oxygen saturation varied from 97% to 100% in all patients of both groups b and n. vas scores in the immediate postoperative period both at rest (median vas 3 mm vs. 27 mm) and with activity (median 8 mm vs. 35 mm) were significantly lower in patients who received tap block . Median duration of analgesia was significantly higher in patients belonging to group b (290 min vs. 16 min, p = 0.000, mann the difference in median duration of analgesia is 275 min with 95% ci of 250307 min (hodges lehman median difference). A kaplan meier survival analysis for the cumulative duration of analgesia in first 24 h shows a significantly longer duration of analgesia in patients receiving tap block [figure 4]. Comparison of quality of analgesia duration of analgesia in either group has been depicted in by kaplan meyer survival analysis . The survival graph shows significant cumulative analgesia in patients receiving transversus abdominis plane block incidence of postoperative nausea - vomiting was also similar in both groups . No opioid related side effects such as respiratory depression, pruritus or urinary retention was noted in any of the patients . None of the patients in either group had any complication that can be attributed to tap block . Median value of pulse rate, systolic blood pressure, diastolic blood pressure, vas scores at 1, 2, 3, 4, 5, 6 and 24 h were not compared as> 30% patients in group n received rescue analgesic during the immediate postoperative period . The principal finding of our study is that bupivacaine in tap block provides effective intraoperative and immediate postoperative analgesia in patients undergoing total abdominal hysterectomy . Our finding of preincisional tap block reducing intraoperative fentanyl requirement was consistent with those of mukhtar and khattak who reported a significant reduction in intraoperative morphine consumption in patients receiving tap block with 0.5% bupivacaine in renal transplant recipients (0.4 1.2 mg vs. 9.3 1.4 mg; p <0.0001). El - dawlatly et al . Reported a similar significant reduction in intraoperative sufentanil consumption in patients undergoing laparoscopic cholecystectomy (8.6 3.5 mcg vs. 23.0 4.8 mcg, p <0.01). Similar findings were reported in a study by ra et al . In patients undergoing laparoscopic cholecystectomy where intraoperative remifentanil use was significantly lower in patients receiving either 0.5% or 0.25% bupivacaine in comparison to placebo . However, no other rct, to the best of our knowledge has addressed the efficacy of preincisional tap block in preventing hemodynamic response to surgical stimuli . We have found the superiority of tap block in providing immediate postoperative analgesia reflected by a lower vas score both at rest and with activity . The current literature on tap block is not unanimous in the matter that whether it improves postoperative pain score or not . Carney et al . In open appendicectomy . In 2008, carney et al . Found that anatomical tap block in total abdominal hysterectomy patients significantly reduces postoperative pain scores up to 48 h period . Postoperative morphine consumption also decreased at 12 h, 36 h and 48 h time period . Recently, sharma et al . Also found that tap block by landmark technique improves vas score in first 24 h in patients undergoing major abdominal surgery . Petersen et al . In 2012 also found that us guided bilateral tap block in patients undergoing laparoscopic cholecystectomy provides superior postoperative pain scores . Petersen et al . In 2013 found that tap block does not provide superior analgesia in comparison to placebo after inguinal hernia repair . A previous cochrane review and a meta - analysis in 2012 failed to demonstrate the beneficial effect of tap block on postoperative pain scores . In this context, it is worth mentioning that the meta - analysis found that tap block decreases postoperative opioid consumption, which may be a more important parameter to decide an analgesic regimen . The median duration of effective postoperative analgesia from our study was 290 min in patients receiving tap block, and we did not use any additive in tap block . Clonidine in peripheral nerve block has been shown to significantly increase the duration and may be considered here also . Time to the requirement of first postoperative analgesic is also significantly increased in patients received tap block (290 min vs. 16 min). This was consistent with mcdonnell et al ., who demonstrated in their anatomic study that tap block with 0.5% lignocaine may provide analgesia for 4 - 6 h. only three patients in bupivacaine tap group required rescue analgesic in first two postoperative hours whereas 43 patients in the saline group required the same . Four patients in bupivacaine group did not require rescue analgesia in first 24 h postoperative period . The cause of prolonged duration of analgesic effect following single shot tap block is not entirely clear . This may be explained by the fact that the tap is relatively poorly vascularized, and therefore drug clearance may be slowed . Inadequate analgesia even after tap block may be either due to technical failure or due to visceral pain component, which is not addressed by tap block . As such, until now, all local anesthetic techniques carry an inherent failure rate of 5 - 20%, depending on the skill of the operator . The most important clinical implication of our findings is the significant opioid sparing effects of tap block both in the intraoperative as well as the postoperative period . Opioids, though very effective in perioperative pain management, may be associated with nausea - vomiting, pruritus and respiratory depression . Moreover, some patients who are morbidly obese or having obstructive sleep apnea will be maximally benefitted from tap block as it provides opioid sparing effects . Patients having ischemic heart disease or stenotic valvular lesion like mitral or aortic stenosis, where tachycardia is undesirable, will also be benefitted from preincisional tap block . It may be a relatively safer alternative to neuraxial block for intra and postoperative analgesia in patients having coagulopathy . Though we controlled the depth of anesthesia by bis monitoring, ensured adequate muscle relaxation, prevented hypovolemia, indirect assessment of intraoperative pain by hemodynamic parameters may be unreliable . Second use of real time usg for tap block is increasing; we used a landmark based anatomical approach . However, as real time us guidance may increase the efficacy of tap block, it wo nt change the primary finding of our study . Third, use of patient controlled analgesia in the postoperative period could have accurately delineated postoperative opioid consumption . Preincisional tap block decreases intraoperative fentanyl requirements, prevents hemodynamic responses to surgical stimuli and also provides effective postoperative analgesia.
Under innate immune conditions, the inflammatory responses mediated by macrophages, mast cells, and neutrophils comprise an important barrier against infectious pathogens, such as viruses, fungi, and bacteria, as well as chemical toxins [1, 2]. Among the cellular components of innate immunity, macrophages are regarded as central inflammatory cells, as they identify external pathogens using special surface receptors (e.g., toll - like receptors (tlrs)) and are widely distributed in the human body . The inflammatory responses mediated by macrophages and their role in pathophysiology have been previously studied . Activated macrophages induce various intracellular signaling cascades, including src, syk, phosphatidylinositide 3-kinase (pi3k), akt, inhibitor of b (ib) kinase (ikk), and ib [46]. The signaling pathway also stimulates the nuclear translocation of nuclear factor- (nf-) b and activator protein ap-1, triggering the expression of inflammatory genes that lead to secretion of inflammatory mediators (e.g., nitric oxide (no), reactive oxygen species (ros), prostaglandin e2 (pge2), chemokines, and cytokines (e.g., tumor necrosis factor- (tnf-))) [79]. Recently, ample evidence has suggested that unchecked, prolonged inflammatory responses can cause serious immunological diseases, including diabetes, septic shock, cancer, arthritis, and cardiovascular disease . The understanding of inflammatory responses and exploration of strategies for suppressing inflammation are thus considered appropriate approaches to reducing disease incidence [1013]. The cordyceps genus including cordyceps sinensis, cordyceps militaris, cordyceps pruinosa, and cordyceps bassiana grow in korea, japan, china, and the congo . The cordyceps genus can be administered through traditional routes and is known to ameliorate various inflammatory diseases, including chronicle bronchitis, asthma, and eczema . The biological and pharmacological activities of cordyceps genus are antioxidative, antiviral, antifibrotic, anti - inflammatory, antinociceptive, antiangiogenic, antiplatelet aggregation, and antidiabetic [14, 15]. Studies have also demonstrated the anti - inflammatory mechanisms of butanol (bf) and hexane (hf) fractions of cordyceps bassiana . However, the specific chemical compounds responsible for the plant's anti - inflammatory properties have not yet been elucidated . Recently, we isolated a promising novel compound [kth-13: 4-isopropyl-2,6-bis(1-phenylethyl)phenol] with anticancer activity from cordyceps bassiana . Despite the novel chemical structure of this compound, we have established a method for its total synthesis and derivatization to develop more effective molecules . So far, almost 60 compounds were newly synthesized and tested to check their activities by employing no assay and antiproliferative activity . Of them, interestingly, kth-13-amine - diastereomer 1 [4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 (kth-13-ad1)] has been reported to have stronger activity than that of the original compound in terms of anticancer activity (data not shown). In this study, therefore, we further aimed to demonstrate the anti - inflammatory potential of kth-13-ad1, a derivative of kth-13, and to explore its mechanism of action using activated macrophages . Sodium nitroprusside (snp), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt), dihydrorhodamine 123 (dhr123), fluorescein isothiocyanate- (fitc-) dextran, ascorbic acid, and lps (e. coli 0111:b4) were purchased from sigma chemical co. (st . Louis, mo, usa). Fetal bovine serum and rpmi 1640 the murine macrophage cell line raw264.7 and human embryonic kidney (hek) 293 cells were purchased from the american type culture collection (rockville, md, usa). Luciferase constructs containing binding promoters for nf-b and ap-1 were gifts from professors chung, hae young (pusan national university, pusan, korea) and rhee, man hee (kyungpook national university, daegu, korea). Phospho- and total protein - specific antibodies to p65, p50, c - fos, c - jun, ib, ikk, akt, p85, src, syk, lamin a / c, and -actin were obtained from cell signaling technology (beverly, ma, usa). Primers (table 1) designed in our laboratory were synthesized by bioneer (daejeon, korea). To synthesize kth-13-ad1 (figure 1(a)), a solution of 4-isopropylaniline (4.00 g, 29.6 mmol) in xylene (14 ml) was mixed with styrene (9.48 g, 91.1 mmol) and cf3so3h (1.0 ml, 11.4 mmol). The reaction mixture was allowed to heat to 160c and stirred for 24 h. at that time, the reaction was allowed to cool to room temperature and the volatiles were removed under vacuo . The resulting residue was purified by silica gel column chromatography (hexanes: etoac = 9: 1) to afford the desired kth-13-ad1 (2.60 g, 7.57 mmol, and 1: 1 diastereomers) in 26% yield as a brown oil . Infrared (ir) spectra of kth-13-ad1 were recorded on a bruker vertex 70 spectrophotometer, max in cm . Bands are characterized as strong (s), medium (m), and weak (w). H nuclear magnetic resonance (nmr) spectra of this compound were recorded on a jeol jnm - al400 (400 mhz) spectrometer . Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard (cdcl3: 7.27 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), coupling constants (hz), and integration . C nmr spectra were recorded on a jeol jnm - al400 (100 mhz) spectrometer with complete proton decoupling . Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard (cdcl3: 77.16 ppm). Low - resolution mass spectrometry was performed on an agilent 6890n gc (hewlett - packard co., palo alto, california, usa). H nmr (400 mhz, cdcl3, diastereomer 1): 7.407.12 (m, 12h), 4.07 (q, j = 6.9 hz, 2h), 3.29 (br s, 2h), 3.00 (sept, j = 6.9 hz, 1h), 1.67 (d, j = 7.1 hz, 6h), 1.37 (d, j = 7.1 hz, 6h); c nmr (100 mhz, cdcl3): 145.8, 139.4, 138.9, 130.2, 128.7, 127.5, 126.3, 123.5, 40.2, 33.7, 24.4, 22,2; ir (neat): 3471 (s), 3384 (s), 3060 (m), 3024 (m), 2982 (s), 2869 (s), 2293 (w), 1947 (w), 1878 (w), 1803 (w), 1623 (s), 1600 (s), 1471 (s), 1450 (s), 1373 (m), 1318 (m), 1257 (m), 1172 (m), 1027 (m), 881 (s), 761 (s), 700 (s) cm; lr - ms (esi): m / z calculated for c25h30n ([m + h]) 344.2, and found 344.2 . Raw264.7 and hek293 cells were cultured with rpmi1640 medium supplemented with 10% heat - inactivated fbs, glutamine, and antibiotics (penicillin and streptomycin) at 37c in a 5% co2 atmosphere . In each experiment, cells were detached with a scraper . Examination of cell densities at 2 10 cells / ml revealed that the proportion of dead cells was consistently <1% according to trypan blue dye exclusion as the criterion for viability . Raw264.7 macrophage cells (1 10 cells / ml) were cultured for 18 h, pretreated with kth-13-ad1 (0 to 200 m) for 30 min, and further incubated with lps (1 g / ml) for 24 h. the inhibitory effect of kth-13-ad1 on lps - induced no production was determined by analyzing no level using griess reagent, as previously described [18, 19]. The od at 550 nm (od550) was measured using a spectramax 250 microplate reader (molecular devices, sunnyvale, ca, usa). The level of intracellular ros was determined by recording the change in fluorescence resulting from the oxidation of the fluorescent probe dhr123 . Briefly, 5 10 raw264.7 cells were exposed to kth-13-ad1 (0 to 150 m) for 30 min and then incubated with snp (0.25 mm) at 37c for 20 min to induce ros production . The cells were further incubated with 20 m of the fluorescent probe dhr123 for 30 min at 37c . The degree of fluorescence, which corresponded to the level of intracellular ros, was determined using a facscan flow cytometer (becton - dickinson), as reported previously . To measure the phagocytic activity of raw264.7 cells, we modified a previously reported method . Raw264.7 cells (5 10) were pretreated with kth-13-ad1 (0 to 150 m) for 1 h, resuspended in 100 l phosphate buffered saline (pbs) containing 1% human ab serum, and then incubated with fitc - dextran (1 mg / ml) at 37c for 20 min . The reactions were stopped by adding 2 ml ice - cold pbs containing 1% human serum and 0.02% sodium azide . The cells were then washed three times with cold pbs - azide and analyzed on a facscan flow cytometer (becton - dickinson, san jose, ca, usa), as reported previously . The level of fitc - dextran or ros in raw264.7 cells was determined through flow cytometric analysis [22, 23]. 10 cells / ml) treated with kth-13-ad1 in the presence or absence of fitc - dextran (1 mg / ml) or dhr123 (20 m) were washed with staining buffer containing 2% rabbit serum and 1% sodium azide in pbs and then incubated with direct - labeled antibodies for an additional 45 min on ice . After washing three times with staining buffer, stained cells were analyzed on a facscan flow cytometer (becton - dickinson). Raw264.7 cells (1 10 cells / ml) were cultured for 18 h, after which kth-13-ad1 (0 to 150 m) was added for the final 24 or 8 h of culture . The cytotoxic effects of ats - e3 kth-13-ad1 were then evaluated using a conventional mtt assay, as reported previously [24, 25]. For the final 3 h of culture, 10 l mtt solution (10 mg / ml in pbs, ph 7.4) was added to each well . The incubation was stopped by the addition of 15% sodium dodecyl sulfate (sds) to each well, which solubilized the formazan . The absorbance at 570 nm (od570630) was measured using a spectramax 250 microplate reader (biotek, bad friedrichshall, germany). Raw264.7 cells (1 10 cells / ml) were cultured for 18 h, pretreated with kth-13-ad1 (0 to 150 m) for 30 min, and further cultured with lps (1 g / ml) for 6 h. the inhibitory effect of kth-13-ad1 on the expression of inos and tnf- was determined using semiquantitative rt - pcr and real - time quantitative reverse transcription - polymerase chain reaction (qrt - pcr), as reported previously [18, 27]. The primers (bioneer, daejeon, korea) used in these reactions are listed in table 1 . Hek293 cells (1 10 cells / ml) were transfected with 1 g plasmids to drive the expression of -galactosidase and either nf-b - luc or ap-1-luc in the presence or absence of an inducing molecule (myd88, trif, or ha - src). Transfections were performed using the pei method in 12-well plates, as previously outlined [28, 29]. Luciferase assays were performed using the luciferase assay system (promega, madison, wi, usa), as previously reported . Raw264.7 cells (5 10 cells / ml) were washed three times in cold pbs supplemented with 1 mm sodium orthovanadate, resuspended in lysis buffer (20 mm tris - hcl, ph 7.4, 2 mm edta, 2 mm ethyleneglycotetraacetic acid, 50 mm -glycerophosphate, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 1% triton x-100, 10% glycerol, 10 g / ml aprotinin, 10 g / ml pepstatin, 1 mm benzamide, and 2 mm pmsf), lysed by sonication, and rotated for 30 min at 4c . The lysates were clarified by centrifugation at 16,000 g for 10 min at 4c and stored at 20c until use . The soluble fractions of the cell lysates were immunoblotted, and total and phosphoprotein levels of c - fos, c - jun, p50, p65, ib, ikk, akt, p85, src, syk, lamin a / c, and -actin were visualized, as previously reported . Data are expressed as the mean standard deviation (sd), as calculated from one (n = 6) of two independent experiments . Other data are representative of three different experiments with similar results . For statistical comparisons, the results were analyzed using analysis of variance / scheffe's post hoc test and the kruskal - wallis / mann - whitney test . A p value <0.05 was considered to be statistically significant . All statistical tests were conducted using spss (spss inc ., chicago, il, usa). In previous studies, we and other investigators have reported on the anti - inflammatory, anticancer, and immunomodulatory activities of cordyceps bassiana [3235]. Unlike other cordyceps species, such as cordyceps militaris and cordyceps sinensis, recently, we identified a promising new compound called 4-isopropyl-2,6-bis(1-phenylethyl)phenol with anticancer activity against several cancer cell lines, such as c6 glioma and mda - mb-231 . We further established a method for total synthesis of this compound in order to facilitate its mass production and derivatization . In this study, we tested one (kth-13-ad1 (figure 1(a))) of its derivatives, specifically to see whether this compound is able to suppress macrophage - mediated inflammatory responses . As shown in figure 1(b), left panel, the production of no was reduced by up to 70% in a dose - dependent manner by kth-13-ad1 in lps - stimulated macrophage - like raw264.6 cells at 150 m, though it did not downregulate no released from snp, a drug that directly releases no in vitro (figure 1(b), right panel). These results indicate that kth-13-ad1 does not act as a chemically directed neutralizing agent but rather as a modulator of no biosynthesis during tlr4 stimulation following lps treatment . In similar fashion, the radical scavenging activity of kth-13-ad1 was observed only at 100 and 150 m when applied to snp - treated cells (figure 1(c), left panel) but not in the dpph assay (figure 1(c), right panel). Since kth-13-ad1 did not suppress the viability of raw264.7 cells (figure 1(d)), the results demonstrate that though this compound does not have direct antioxidative activity, it displays anti - inflammatory properties that indirectly suppress the production of inflammatory mediators, such as no, similarly to ginsenosides that have only anti - inflammatory properties [37, 38]. However, unlike these compounds, flavonoids (e.g., quercetin, luteolin, and kaempferol) and anthraquinones (e.g., rhodoptilometrin), are active components of numerous medicinal plants and have been reported to demonstrate both antioxidative and anti - inflammatory activities [3942]. To determine the mechanism by which kth-13-ad1 suppresses no production, we investigated tlr4-mediated transcriptional activation levels in lps - treated raw264.7 cells pretreated with kth-13-ad1 . First, we evaluated mrna levels in proinflammatory genes, including inos and tnf-, using quantitative real - time and semiquantitative pcr . As shown in figures 2(a) and 2(b), kth-13-ad1 (150 m) significantly decreased the mrna levels of inos and tnf-, suggesting that kth-13-ad1-driven suppression of inflammatory responses occur at the transcriptional level . Since nf-b and ap-1 are representative of transcription factors regulating inflammatory responses [43, 44], we determined the ability of this compound to suppress the activation of nf-b and ap-1 using a luciferase reporter gene assay with promoter sites for nf-b and ap-1 . To determine how kth-13-ad1 regulates promoter activity in inflammatory genes, we pretreated the kth-13-ad1 for 30 min before activating hek293 cells with pma, an activator of pkc that induces the inflammatory signaling pathway . As shown in figure 2(c), luciferase activity mediated by nf-b and ap-1 was greatly enhanced by pma, up to 500- and 40-fold, respectively . Interestingly, kth-13-ad1 (100 and 150 m) strongly suppressed nf-b - mediated luciferase activity (figure 2(c), left panel) but not ap-1 (figure 2(c), right panel). It has also been reported that myd88 and trif, adaptor molecules regulating the tlr4-mediated intracellular signaling pathway, are good inducers of luciferase activity in hek293 cells [47, 48]. The results in figure 2(d) support this conclusion, as cotransfection of these molecules increased both nf-b- and ap-1-mediated luciferase activity up to 4,000- and 250-fold, respectively, as seen in previous studies . Comparing these two conditions, myd88-induced nf-b activation was strongly suppressed by kth-13-ad1, with 100 m of the compound inhibiting activation by 45% (figure 2(d)). In addition, kth-13-ad1 reduced the nuclear translocation of the nf-b subunits p65 and p50 at 30 and 60 min, although there was no inhibition at 15 min (figure 2(e)). These results suggest that the nf-b signaling pathway affecting the activation of nf-b dimerization at 30 to 60 min might be a potential target for kth-13-ad1 . These inhibitory patterns observed by kth-13-ad1 treatment seem to also imply that nf-b activation signals might be timely regulated by some upstream signaling units . To prove such raised possibility, we subsequently focused on identifying the nf-b regulatory pathways that are recognized by this compound . First, we determined the phosphorylation level of ib, an essential step in the nuclear translocation of nf-b . The phosphorylation of ib was blocked by kth-13-ad1 at 15, 30, and 60 min, while the total form of ib was greatly increased at 30 and 60 min, compared to lps treatment alone (figure 3(a)), implying that this compound is able to suppress both phosphorylation and degradation of ib between 30 min and 1 h. in addition, kth-13-ad1 was found to reduce the phosphorylation of ikk/, upstream of ib, at 5 min (figure 3(a)). Similarly, the phosphorylation of akt and p85/pi3k, upstream enzymes for ikk activation, was blocked by kth-13-ad1 at 3 to 5 min (figure 3(b)). Additionally, src phosphorylation occurring at 1 and 3 min was also suppressed by this compound (figure 3(b)). To confirm our results, we evaluated the dose - responsive activity of kth-13-ad1 on the phosphorylation levels of putative target proteins, p85/pi3k and src . As seen in figure 3(c), the phosphorylation of p85 and src was continuously diminished by kth-13-ad1 (100 and 150 m) under the same conditions . As these results strongly suggest that the putative target of kth-13-ad1 is src, we next analyzed whether this compound is able to regulate the autophosphorylation of src, using an overexpression strategy, as reported previously [50, 51]. As shown in figure 3(d), kth-13-ad1 (100 and 150 m) clearly decreased the level of src phosphorylation, implying that src is directly modulated by this compound . This is further supported by the fact that the promoter activity of nf-b, as stimulated by src overexpression, was also blocked by kth-13-ad1 (figure 3(e)). Based on these results, we propose that src serves as a potential target protein in kth-13-ad1-mediated anti - inflammatory actions . Many anti - inflammatory compounds, both naturally occurring and chemically synthesized, have been found to possess direct inhibitory activity against src kinase [5052]. Additionally, previous studies have reported on src activity under nf-b activation and inflammatory gene expression [51, 53, 54], suggesting an important role of src in inflammatory responses and in oncogenic activity . Finally, to demonstrate whether kth-13-ad1 modulates macrophage phagocytic uptake, fitc - dextran was applied to raw264.7 cells during exposure to this compound . As shown in figure 4, there was no inhibition of phagocytosis by kth-13-ad1, as measured by fitc - derived fluorescence [55, 56]; however, significant enhancement was observed at 100 and 150 m concentrations, implying that the compound stimulates macrophage phagocytosis, an important step in the innate immune response . This result suggests that innate immune responses might be regulated by kth-13-ad1 via different cellular mechanisms . In summary, kth-13-ad1 demonstrated impressive suppression of inflammatory mediator production, including ros and no, without inducing cytotoxicity, while this compound strongly enhanced the phagocytotic uptake, suggesting that kth-13-ad1 is able to regulate cellular responses related to innate immunity . In addition, the transcriptional activation of nf-b and its upstream signaling pathway, composed of src, p85/pi3k, and akt, was blocked by kth-13-ad1, as summarized in figure 5 . Thus, the present study proposes that kth-13-ad1, a chemical analog of kth-13 extracted from cordyceps bassiana, has clinical utility as an anti - inflammatory drug.
Nonviral gene vectors have many advantages such as mass production, easier transportation, less immunogenicity, and being easily targeted to organs [1, 2]. Among the nonviral vectors, chitosan is known to possess efficient properties owing to their ability to condense nucleic acid into stable complexes, which protects dna from degradation by nuclease . The dna / polymer complexes are taken up into the cells via endocytosis into the endosomes, following with burst release of complexes fraction in endosomes and the dna translocates into the nucleus . It is soluble at acidic ph value, and the amino groups carry positive charge in acidic mediums; it can combine with negatively charged dna . Previous studies have revealed that chitosan, like other cationic polymers, displayed concentration - dependent toxicity toward cells in vitro, although it had many advantages as a gene vector . Magnetic ferriferous oxide nanoparticles possess prominent advantages that might correct the defects of traditional drugs and gene carriers . Whereby numerous dna strands attached to the surface of these ferriferous oxides could reach the desired position with the help of static magnetic field . In order to improve the properties of nanoparticles such as biocompatibility, transfection efficiency, and controlled release, we embedded the biodegradable polymers on the surface of ferriferous oxide to form a core shell structure . Therefore, the focus of our research was on how to improve the target property and remove the application barriers of nonviral gene vectors in vivo . The use of a static magnetic field has been shown to result in dramatic increase in transfection efficiency of gene delivery when compared with the conventional transfection system [9, 10]. Magnet - assisted transfection is a new, easy - to - handle, very highly efficient technology . It is a very gentle method with almost no toxicity and has been successfully used on many and also critical cell lines . All types of nucleic acids from plasmid dna or oligonucleotides to sirna can be used with this approach . In this research, the synthesized magnetic nanoparticles have an approximately size of 100 nm and are additionally coated with biodegradable polymers . We used both of the advantages of magnetic nanoparticles and biodegradable polymers, and the application of the novel polymer - fe3o4 complexes as gene vectors in vitro was then described at length . These iron oxides can be generated by precipitation from acidic iron - salt solutions upon addition of appropriate bases . A cts (mws 45 kda, 20% w / w, ph6.9) solution carrying a positive charge or peg (mws 6 kda, 20% w / w) solution was prepared . 0.2 ml of this solution was added to 0.8 ml of iron oxide dispersion (10% w / w) for 8 h incubation . After filtration sterilization with a 0.45 m filter, the nanoparticles were used for the next transfection experiments . Nanoparticles and dna form complexes by ionic interaction of the negatively charged nucleic acid and the positively charged surface of the cts - fe3o4 nanoparticle (n / p ratio 4: 1). The polymer - fe3o4 was analyzed by means of a transmission electron microscope (tem, hitachi h-700h), x - ray diffraction (xrd, philips x'pert pro). The size and zeta potential of the polymer - fe3o4 were both assessed using the zetasizer nano instrument . The plasmid pegfp - c1 was propagated in escherichia coli and was purified using an endotoxin - free plasmid maxiprep kit (qiagen). At the ph level of 7.4 the polymer - fe3o4 complexes were mixed with dna at different volume ratios in a 50 l reaction system . The final concentration (fc) of plasmid dna and polymer fe3o4 was 4 g/l and 1 mm (concentrations related to fe) diluted with double - distilled water (ddh2o). After 1 h incubation at 37c the concentration of dna in the supernatant was measured by uv spectrophotometric absorption at 260 nm . The encapsulation efficiency (e.e .) Of the process indicates the percentage of dna encapsulated used for the preparation of polymer - fe3o4 complexes . To observe the target distribution of polymer - fe3o4 nanoparticles in different organs of mice, 40 pathogen - free balb / c female mice were purchased from the sichuan industrial institute of antibiotic for the in vivo studies . The polymer fe3o4 was redispersed as described previously and injected through the caudal vein on the dosage of 1 mm iron oxide in 0.8 ml . A neodymium - iron - boron (ndfeb) permanent magnet (br 1/4 1.5 t) the mice were sacrificed at different times after the injection (2 h, 6 h, 12, and 24 h), and the liver, spleen, lungs, heart, and brain were taken out and made into tissue slices . The target distribution of polymer fe3o4 was observed by prussian blue and neutral red staining . Preweighed polymer - fe3o4 complexes containing dna were incubated in a test tube with phosphate - buffered saline (pbs, ph 7.4), for 30 min under moderate stirring at 37c . Dna was reacted with polymer - fe3o4 nanoparticles at three different volume ratios (1: 3, 1: 1, and 3: 1). At predetermined time intervals (12, 24, 48, 72, and 96 h), 50 l of the released medium was collected by centrifugation (3,000 g, 1 min), and 50 l of fresh pbs was added back into the test tube . Dna release was monitored by uv spectroscopy at 260 nm, and dna integrity was evaluated on a 1% agarose gel . The amount of released dna was calculated from the free dna concentration in the supernatants, and the curve of dna release in vitro was described . At last, to confirm the functionality of released dna, the discharged dna was applied to the assay of transfection in vitro . The polymer - fe3o4 complexes (1 mm) were mixed with plasmid dna (4 g/l) according to the optimal e.e . Naked plasmid dna and dna / polymer - fe3o4 complexes were incubated with or without dnasei (0.5 u) in the 30 l reaction system for 1 hour at ph 7.4 . The digestion was stopped by addition of 0.5 m edta . The product of enzymatic digestion was analyzed by 1% agarose gel electrophoresis, and dna in the gel was visualized by ethidium bromide staining . Naked plasmid dna after being digested by dnasei and naked plasmid dna without digestion were used as controls . Human embryonic kidney 293 cells (hek-293), human liver carcinoma cells (hepg2), and mouse myeloma cell line (sp2/0) were maintained in dmem or rpmi-1640 medium (gibco - brl), supplemented with 10% fetal calf serum (fcs, gibco - brl) and 1% penicillin / streptomycin . For the transfection and cytotoxicity test, the cells were grown under standard conditions for 24 hours until 70% to 80% confluency in 96-well flat - bottomed microassay plates before the addition of either the plasmid dna / polymer - fe3o4 complex or only the polymer fe3o4 . Firstly, the precipitate polymer - fe3o4 complexes were resuspended under conditions of ultrasonic agitation for 10 min . Subsequently, the complexes were added into the cell - culture fluid at a different concentration (0.2 ~ 1.0 mm, 2 ~ 20 mm), diluted with a serum - free medium . At the end of each predetermined time (6 h, 12 h, 24 h, and 48 h), the polymer - fe3o4 complexes were replaced with 200 l of fresh dmem medium . Then, 20 l of mtt (5 g/l) in dmem was added to each well and incubated for an additional 4 hours . All mediums were then removed, and 150 l of dmso was added to dissolve the crystals formed by the live cells . The cell viability was calculated, and the viability of nontreated control cells was arbitrarily defined as 100% . 24-well plates were seeded with 2 10 cells (hepg2 and sp2/0 cells) and grown for 24 hours to obtain 7080% confluence . Prior to transfection, the medium was removed, and the cells were rinsed once with pbs (ph 7.4), then supplied with serum - free medium . The plasmid dna was mixed with cts - fe3o4 and peg - fe3o4 as described previously and incubated for 30 minutes at 37c . Dna / polymer - fe3o4 complexes were suspended in a serum - free medium to get the final concentrations of 2 g/l and 1.5 mm, respectively . To verify the short exposure to a static magnetic field would improve transfection efficiency; the cells were placed on a (ndfeb) magnet for 30 min at a distance of 3 mm from the magnet surface, which leads to a magnetic flux density of 340 mt and a magnetic field gradient perpendicular to the well plate of 14 t / m . After a further incubation of 4 h the cells were incubated with plasmid dna alone and dna / polymer - fe3o4 complexes under standard conditions and grown in culture medium for 24 hours to allow for egfp expression . Chitosan (mws 45 kda), lipofectamine (bestbio), and pbs were added to an equal volume of dna as controls . Tem images showed that most of the iron oxide complexes were approximately spherical (unpublished data). The xrd measurements also indicated that the samples had a cubic crystal system and magnetite fe3o4 was the dominant body of the polymer - fe3o4 complexes . The size and zeta potential showed the two samples to have a uniform size of 100 nm (figure 1(a)) and almost the same distribution . The sizes of 10100 nm in diameter are desirable since they are too small not to be eliminated by the reticuloendothelial system (res) but too large to be filtered out by the kidneys . Cts - fe3o4 had a positive charge of about 20 mv (figure 1(b)), and the zeta potential of peg - fe3o4 was 0 mv . It has been reported that surface charge plays an important role in determining the efficiency and mechanism of cellular uptake . It is also an important factor to improve stability of polymer - fe3o4 complexes and to prevent from further aggregation in aqueous solution via electrostatic repulsion . The polymer - fe3o4 complexes were mixed with plasmid dna according to different volume ratios (1: 3, 1: 2, 1: 1, 2: 1, and 3: 1) in a 50 l reaction system . Increased along with the proportion of the magnetic materials mainly because of the electrostatic interactions, surface energy of nanoparticles, and branched structures of polymers . The optimal e.e emerged when the iron oxide complexes were mixed with dna at 3: 1 volume ratio, and the final concentration of dna and iron oxide was 2 g/l and 1.5 mm respectively . Of peg - fe3o4 was inferior to cts - fe3o4 notably for the lack of electrostatic attraction . The different organs from the mice injected with polymer - fe3o4 were taken out and made into tissue slices . Target distribution of polymer fe3o4 in vivo was demonstrated with the help of outer static magnetic field . Figure 2(b) shows a large number of iron particles scattered in the hepatic tissue; many of them were distributed along the hepatic sinusoid 2 h after injection . The iron particles decreased gradually over time and disappeared 24 h after injection (data not shown). The shape of the liver cells was seen under a high - power microscope to be integrated . There was no iron staining in the other organs, such as the lungs (figure 2(d)), the spleen, and the heart . Naked pegfp - c1 without digestion and naked pegfp - c1 following digestion by dnasei were used as controls . We could evidence partial protection of dna coated by polymer fe3o4 from nuclease - mediated dna degradation (unpublished data). It was assumed that dna degradation occurs in several layers; external layers will be degraded easily but not internal layers . Furthermore, cts - fe3o4 nanoparticles offered higher protection for dna than peg - fe3o4, as the dna chains could be attached more strongly to the former . In addition, dnasei digestion resulted in a shift in the most distribution of the dna isoforms: supercoiled plasmid in nontreated samples was replaced by the open loop form in treated samples . The in vitro release rates of dna from polymer - fe3o4 complexes were studied at different volume ratios . A significant proportion (30%) of the adsorbed dna was released very rapidly from the cts - fe3o4 nanoparticles in the initial 12 hours . After 48 h, the amount of released dna reached 55% at the optimal e.e . And the remainder of the adsorbed dna was released slowly, reaching 70% at 96 h (figure 3(a)). Compared to dna release from cts - fe3o4, a burst release phase of more than 61% from peg - fe3o4 was observed . The release curve showed that the dna was released more rapidly; more than 80% of dna was discharged from peg - fe3o4 after 24 h at the optimal e.e ., and the entire release was mostly completed at 72 h (figure 3(b)). The dna integrity test at predetermined time points was assessed by agarose gel electrophoresis (data not shown). No differences were observed between egfp expression from the released dna and the controlled plasmid pegfp - c1, indicating that adsorption and release from the polymer - fe3o4 do not alter the functionality of plasmid dna . Overall, the controlled release effect of cts - fe3o4 complexes was relatively obvious compared with peg - fe3o4 . The speed of dna release was inversely proportional to the volume ratios of nanoparticles . The n / p ratio (the ratio of negatively charged dna to positively charged chitosan) is a key factor to determine the optimal complexation conditions . The difference ph and counterions in the medium might directly affect the binding between cts and dna . It could be inferred that the burst release was induced by the dna degradation in the external layers . The results showed that the controlled - release effect of cts - fe3o4 was more obvious, and the unsteady binding power made the efficient binding with dna and peg - fe3o4 impossible . In addition, the small proportion of chitosan in the polymer - fe3o4 complexes actually hindered the effect of controlled release . Increasing the proportion of chitosan would slow down the dna release but augment the particle size and positive charge of the complexes . It has been reported that positively charged nanoparticles exhibited dose - dependent hemolytic activities and cytotoxicities . In addition, most of the larger nanoparticles (> 150 nm) are trapped by the liver and lung where many macrophages are located . For the drug and gene target delivery application, the nonspecific uptake of nanoparticles by macrophages in the res should be minimized . The contradictory issue of controlled - release and particle size needs to be resolved urgently by carrying out a further study . It has been reported that chitosan derivatives are less toxic than other cationic polymers such as pei in vitro and in vivo . Evaluation of cell viability was conducted on hek-293 and hepg2 cells using a 0.220 mm concentration gradient of polymer - fe3o4 complexes for different incubation periods . More than 90% cell viability of both polymer - fe3o4 complexes was obtained after 24 h of incubation with a concentration of 2 mm or less, and apparent cytotoxicity emerged when the concentration of polymer fe3o4 was more than 10 mm (data not shown). This result showed that both cts - fe3o4 and peg - fe3o4 had low cytotoxicity . The security application could therefore be deduced according to the previously mentioned data and the optimal e.e . Hepg2 and sp2/0 cells were transfected as described previously with either dna / cts - fe3o4 or dna / peg - fe3o4, with dna / chitosan, dna / lipofectamine, and naked plasmid as controls . Exposure to a permanent magnetic field (magnet) for 30 min was followed by 4 h incubation . The highest transfection rates were achieved in hepg2 cells corresponding to 67.2% and 45.8% after transfected with cts - fe3o4 and peg - fe3o4 complexes . Significantly lower transfection rates of 14.3%, 8.7%, and 0.4% resulted from transfection with lipofectamine, chitosan, and naked plasmid, respectively . In addition, the transfection rates were significantly increased by 4.1- and 3.2-fold in hepg2 and sp2/0 cells, when compared to cells not exposed to the magnetic field . Similar transfection results were also obtained with sp2/0 cells, and lower rates of 43.7% and 32.5% treated with cts - fe3o4 and peg - fe3o4 complexes were achieved . Compared with conventional transfected methods, the results were still statistically significant (figure 4). Thus, the transfections rates enhanced by the assistance of magnetic field were verified again in hepg2 and sp2/0 cells . It seems that the use of a static magnetic field can improve the translocation of the particles across the cell membrane . It has been reported that the higher transfection rates with magnetic nanoparticles were mainly attributed to their size surface charge, since the larger nanoparticles faster sedimentated on the surfaces of the cells, and this resulted in higher endocytic uptake, and positively charged nanoparticles were more easily taken up by cells . The chosen cells used in our study were malignant cells from human and mice, and these cells differed in characteristics used as models for different human diseases . Thus, they were good representative samples for enhancement of delivery and effective targeting of gene expression . Furthermore, the egfp expression was strong in transfected cells indicating that the function of dna was kept and no fragmentation occurred . Magnetic materials modified by biodegradable polymers as gene carriers possess many merits . For examples, simple manufacturing operation, arriving at the target point with the help of an outer magnetic field; a powerful surface energy effect and a small size effect are their outstanding characters . Moreover, it is easy to modify all kinds of multifunctional groups or targeting molecules to form the structure of the core shell, such as cts, pei, specific ligands, and monoclonal antibodies, since the complexes have multiple binding sites on their surface, and dna attaches itself to them in sizeable amounts either through an electrostatic effect or by chemical bond coupling . In order to improve the e.e . Of the polymer - fe3o4 complexes and realize the controlled release of the dna, we modified the fe3o4 with multifunctional groups cts and peg . In addition, the process of linking polymeric groups did not utilize organic solvent extraction, and the iron content used does not surpass the acceptable daily intake . Furthermore, some of the novel nanoparticles could improve the antigen presentation effect, show a better adjuvant effect, and make a long - term, single - immunization vaccine possible . There are likely to be further applicative studies of polymer - fe3o4 complexes as gene delivery systems . Preliminary data from our studies suggest that fe3o4 nanoparticles when decorating with positive - charged polymer cts exhibit preferential gene delivery . Dna encapsulation efficiency increased with the proportion of polymer - fe3o4 nanoparticles, and the optimal e.e . Simultaneously, the attachment of dna to polymer - fe3o4 complexes did provide protection against cleavage by nuclease . The target distribution of polymer - fe3o4 complexes with an outer magnetic field was demonstrated in vivo . The controlled - release effect of cts - fe3o4 complexes was more apparent than peg - fe3o4, and the dna binding and release from the polymer - fe3o4 do not alter its functionality . Both cts - fe3o4 and peg - fe3o4 had low cytotoxicity to hek-293 and hepg2 cells . The concentration of 2 mm or less in an in vitro application was shown to be absolutely safe . In addition, the magnetic forces lead to an accelerated sedimentation of polymer - fe3o4 complexes on the cell surface and do directly enhance the transfection efficiency in hepg2 and sp2/0 cells compared with conventional transfection methods . The novel gene delivery system proved to be an effective tool for future, and it is promising in targeting expression and delivery of therapeutic genes in in vivo studies . We will continue to do research in this field to provide a more detailed evaluation about the transfer of dna.

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