To evaluate the fracture resistance of simulated immature teeth with an apical plug of biodentine followed by composite resin vs total obturation with biodentine tested immediately and after 3 months of aging and also to find out the chemical composition of dentin in contact with these materials.Extracted human maxillary central incisors with simulated immature apex with radicular dentin thickness (RDT) of 1 to 1.5 mm selected and divided into three groups of 20 each. Group I (control)—4 mm biodentine apically and thermoplasticized gutta-percha. Group II—4 mm biodentine apically and composite resin. Group III—complete obturation with biodentine. About 10 samples from each group were tested immediately and remaining 10 stored in phosphate buffered solution (PBS) and tested after 3 months for fracture resistance and chemical analysis of dentin.No significant difference in fracture resistance between the groups was observed when tested immediately. After 3 months of aging, only biodentine group showed a significant reduction in fracture resistance with increased Ca/P ratio of root dentine.Biodentine group has shown drastic reduction in fracture resistance after 3 months of aging, and hence, cannot be recommended as a reinforcement material in immature teeth with thin dentin walls.How to cite this article: Zhabuawala MS, Nadig RR, Pai VS, Gowda Y, Aswathanarayana RM. Reinforcing Effects of Calcium Silicate-based Cement and Dual Cure Composite Resin in Simulated Immature Teeth with an Open Apex: An in vitro Study. Int J Clin Pediatr Dent 2017;10(4):351-357. Garcia-Godoy and Murray11 Mineral trioxide aggregate has a good sealing ability, high degree of biocompatibility, low cytotoxicity, antimicrobial properties, ability to set in the presence of moisture, shorter treatment time, and induction of hard tissue deposition periradicularly.12 However, White et al13 reported that tooth strength is weakened after exposure to MTA for more than 5 weeks.Apical placement of mineral trioxide aggregate (MTA) and completion of a procedure in single appointment have been advocated as an promising alternative treatment option with high clinical success rate.14 Inspite of their favorable characteristics as an apical plug, the fragility of roots due to thin dentinal walls can compromise tooth strength, hence, reinforcement of radicular dentin is of utmost importance in such cases.15Recently, a new calcium silicate-based cement bio-dentine has been introduced which contains tricalcium silicate, calcium carbonate, and zirconium oxide and water-based liquid that has calcium chlorite and water reducing agent. It sets in 12 minutes and is recommended for use as dentine substitute. Similar to MTA, biodentine is biocompatible and when in contact with vital tissues it is said to be biologically active. The compressive strength and elasticity modulus are comparable with dentine and handling characteristics of biodentine is superior to MTA.19 Composite resin has the ability to bond to root dentine walls, increasing the strength of the roots. However, it has certain disadvantages like polymerization shrinkage stresses, difficulty in complete curing, and increased c-factor in the root canal system.20 An alternative is the use of fiber post with an elastic modulus similar to dentine shown to have “Monoblock” effect.2122 A mismatch between the diameter of post space and the fiber post is a problem, particularly in immature teeth with large root canals.23Structural strengthening of non-vital immature teeth has received a great deal of attention and different materials and techniques have been examined.24 However, in all of these above studies, testing was done immediately that too with canal wall thickness of >2.5 mm. Stuart et al25 suggested that the canal wall reinforcement of teeth with canal wall thickness of 2 mm or more may not be necessary. But most of the studies available in the literature has been done with the canal wall thickness of 2 mm or more. Therefore, evaluation of fracture resistance of rehabilitated teeth with canal wall thickness less than 1.5 mm is crucial and more relevant. Reinforcement of tooth root depends not only on the inherent strength of the obturating material but also on the quality of bond of the material with the root canal dentin and structural alteration that can happen in the material as well as root dentin over a period of time. Hence, this study is undertaken to evaluate the reinforcing effects of dual cure composite resin and biodentine on immature root dentin and also to find out if these materials can bring about any alteration in root canal dentin that can influence the tooth strength with time.Further, placing an apical plug of MTA or biodentine followed by placement of composite resin for radicular reinforcement has certain practical difficulties, such as many interfaces between the materials used, multiple treatment visits, removal of extra MTA/biodentine that has adhered to the root dentine may interfere with bonding and an attempt to remove the adhered material might result in unnecessary removal of already weakened radicular dentin. To overcome the above practical difficulties, there have been attempts to fill the entire canal with these materials. A study conducted to evaluate the fracture resistance of simulated immature teeth with biodentine apical plug and backfilling with various materials concluded that backfilling with guttapercha, fiber post, or biodentine does increase the force required to fracture immature teeth.Sixty extracted human maxillary central incisors measuring 5 mm faciolingually and 6 mm mesiodistally were selected for the study, disinfected with 5.2% sodium hypochlorite for 2 hours, and stored in distilled water until the beginning of the experiment. Preoperative radiographs were taken to rule out any aberrations in the anatomy. The roots of teeth were standardized to a length of 10 mm as measured from the apex to the facial cementoenamel junction (CEJ) by cutting the root tip to simulate incomplete root formation. Endodontic access cavities were made using round bur and Endo Z bur in a high speed hand piece and root canals were prepared using the Protaper rotary instruments. To achieve a simulation of teeth with immature apices, Peeso reamers between #1 and #6 were introduced into the root canal until #6 pass freely out of the apex. After instrumenting with Peeso reamers, the radicular dentin thickness was 2.5 mm. Further 703 carbide bur was used to obtain the remaining dentin thickness around 1.5 mm . The samGroup I (n = 20): AH Plus sealer was applied to the canal walls, backfilled with gutta-percha using Obtura II. Excess sealer was removed from the chamber with dry cotton pellet.Group II (n = 20): Dual-cured composite resin para-core was inserted into the canal and the excess resin was removed and light activation was performed for 40 seconds.Group III (n = 20): Biodentine was mixed and placed to a level just below the facial CEJ.In all the groups after backfilling procedures, the access openings 2 mm below CEJ were filled with nano-composite . Immediately after filling, 10 samples from each group were randomly divided into two subgroups. In subgroup A: specimens were stored for 1 week in PBS. In subgroup B: specimens were stored in PBS for 3 months until fracture resistance testing. To simulate periodontal membrane, the root surfaces were coated with 0.2 to 0.3 mm thick layer of polyvinyl-siloxane impression material and then embedded in the acrylic resin leaving the gap of 2 mm between the top of acrylic and to the CEJ. A custom-made jig was used to fix the cylinders at an angle of 45° and a compressive load was applied from the palatal surface at crosshead speed of 1 mm/minute until fracture using universal testing machine. The maximum force required to fracture each specimens was recorded in Newtons. The data were statistically analyzed using one-way analysis of variance. The level of significance was set at p = 0.05.After testing fracture resistance, all the specimens were subjected to energy dispersive x-ray analysis to find out the chemical composition of the dentin. The root surfaces were split longitudinally with diamond disk and surfaces were etched with 37% phosphoric acid for 5 second and rinsed thoroughly with water and subjected to elemental analysis under high vacuum at 1,500x magnification using energy dispersive system, which is attached to an environmental scanning electron microscope. The recorded values represented the average mineral weight percentage.The mean values and their respective standard deviations of the force required to fracture the roots are presented in  and Tab. In immeThe mean values and their respective standard deviations of the Ca/P ratio of dentin are presented in  and Tab. In imme25 Reinforcing capability of biodentine was evaluated in this study because in addition to being bioactive and biocompatible material, it has improved handling characteristics and less time consuming than MTA. Han and Okiji26 suggested that BD may have a remarkable biomineralization capability than MTA.Reinforcement of endodontically treated immature teeth assumes great importance as these teeth are more susceptible to external and masticatory forces. The selected material for reinforcement must be applied easily in clinical practice, bond successfully to dentine, and serve as a good barrier against microleakage.25 Therefore, preparation of the canal was further enlarged with 702 carbide burs to approximate Cvek’s stage 3 root development. Cvek’s stage 3 root development was selected for the present model; because at this stage, the root-to-canal ratio in a mesiodistal dimension at the CEJ is nearly 1:1.1527 In the present study, after the simulation of all the samples, CBCT was used to find out the accurate thickness of the remaining radicular dentin.In most of the studies, found in the literature, to simulate immature teeth, the canals were instrumented with Peeso reamers (1-6) until a size 6 Peeso can be passed 1 mm beyond the apex to simulate open apex, this would only result in dentin thickness of 2 to 2.5 mm. Investigations have revealed that the reinforcement of a canal with a diameter less than 1.5 mm is not necessary while wall thickness of 2.63 mm is insufficient to weaken tooth struc-ture.2425 On evaluating the results, in immediate tested specimens, teeth restored with paracore group showed the highest fracture resistance followed by biodentine and then gutta-percha. However, there was no statistical significant difference between the groups tested in the study. A preliminary study by Yasir28 evaluated the fracture resistance between biodentine and gutta-percha in immature teeth and reported that there was no statistical significant difference between groups. A similar study by Topc.uoglu et al24 evaluated the fracture resistance between bioden-tine, gutta-percha, and fiber post group in immature teeth and suggested that there is no significant difference between biodentine and gutta-percha group. In the present study, access composite is extended 2 mm apical to CEJ to provide an additional root strength necessary to minimize cervical root fracture. Seto et al29 reported that composite resin to a depth of 3 mm would significantly strengthen the roots in immature teeth. So this could be one possible reason, why there was no statistical significant difference in the fracture strength between the control group and the experimental groups in the current study.While testing for fracture resistance, the load was applied at an angle of 45° to the long axis of the tooth to simulate the average angle of contact between maxillary and mandibular incisors in a class I occlusion.30 reported that the degradation patterns of resin-dentin interface inside the root canal created by self-etching adhesive systems due to water sorption and induced collagenolytic activity adversely affects the longevity of adhesively bonded post. Tay and Pashley31 reported that the self-etching systems due to their hydro-philic characteristics tend to present potential degradation in the course of time, as water plays a key role in polymer degradation and concluded that the tested etch-and-rinse adhesive systems had a better performance in terms of bond durability over time than the self-etching adhesive systems and thus monoblock effect is questionable over a period of time. However, our study was of 3 months duration and the longevity of the bond with extended time periods needs further evaluation. The difference between immediate and delayed biodentine group, there is a drastic reduction in the tooth strength after 3 months of storage. Bowes32 and Leiendecker et al33 reported that highly alkaline calcium hydroxide induces a caustic degradation effect on exposed collagen and this is mediated by the breakdown of intermolecular bonds in collagen fibrils, increasing their water absorption leading to swelling. A recent study by Sawyer et al34 reported that dentin flexural strength for dentin exposed to biodentine and MTA decreased significantly after 2 and 3 months. Atmeh et al35 examined the dentin cement interfacial interaction with biodentine and reported that in confocal images a layer of mineral infiltration zone is associated with an altered intertubular microstructure leading to change in optical properties of interfacial dentin. SEM micrographs showed the same band of structurally altered dentin immediately beneath the biodentine which is due to high alkalinity of hydrated biodentine, which has induced a caustic denaturing and permeability of organic collagen component of interfacial dentin. Therefore, it is possible that the above reaction might have lead to decreased tooth strength of the biodentine samples after 3 months. Another reason for the decreased tooth strength with biodentine after 3 months of aging could be the solubility of biodentine. Singh et al36 have reported that the solubility of biodentine after 30 days and 60 days immersion period was significantly higher as compared with MTA. Vivan et al37 theorized that a material releasing calcium ions to exert biologic effect to some extent solubilize and dissociate from fully hardened cement thus resulting in disintegration. In the present study, radiographic examination of the samples observed showed reduction in the radioopacity of biodentine after 3 months of aging which could suggest the disintegration of this material over a period of time. Hence, further evaluation is required for biodentine to be used in apexi-fication procedure. In the present study, radicular dentin of biodentine group has shown a significant increase in the Ca/P ratios from the mean value of 2.33 to 3.02, thus connoting a gain in the mineral content. Thus, these results are in accordance with the previous mentioned study by Han and Okiji26 who stated that both biodentine and MTA caused the uptake of Ca and Si in the adjacent root canal dentine in the presence of PBS who reported that Ca and Si uptake in dentin areas. The dentin element uptake was more prominent for biodentine than MTA. Therefore, in the present study, solubility of biodentine could be due to higher uptake of ions by the root canal dentin, which could have altered the physical properties of biodentine and therefore, strength of biodentine itself is reduced over a period of time. It is interesting to note that Hatibovic-Kofman et al38 compared the influence of MTA and CH on fracture resistance in sheep teeth and stated that over 1 year period, MTA and CH have showed a 2% and 26% decrease in fracture resistance. Authors reported that after initial decrease in the fracture resistance of MTA treated teeth, the process is reversed, and fracture resistance increases for a period between 2 months and 1 year. It can be explained by the fact that MTA induces the expression of TIMP-2 in dentin matrix and suppress the degenerative activities of MMP-2 and -14. Whether similar phenomenon can be observed in biodentine needs further evaluation.After 3 months of storage, teeth restored with para-core have shown the highest fracture resistance followed by gutta-percha and biodentine showed the least fracture resistance. The difference between immediate and delayed paracore groups, there was a reduction in fracture resistance although it was not statistical significant. Tay et alin vitro and in vivo investigations are necessary to validate the above findings.There was no statistically significant difference in the fracture resistance of an immature tooth with an apical plug of biodentine followed by obturation with gutta-percha, paracore, or biodentine. After 3 months of aging, biodentine group has shown a drastic reduction in the fracture resistance whereas there is no significant reduction in gutta-percha and paracore groups. EDX analysis shows increased Ca/P ratio of root canal dentin of bio-dentine group compared to gutta-percha and paracore after 3 months of aging, whether this uptake of minerals is responsible for decreased tooth strength needs further evaluation. Therefore, biodentine cannot be recommended as a reinforcement material in immature teeth with thin dentinal walls. Further  Dental impact injuries involve children at school age between 8 and 12 years can lead to pulpal necrosis with incomplete root formation. Management of such cases is a significant challenge endodontically as well as restoratively. Structural strengthening of immature teeth has received a great deal of attention and therefore, different materials and techniques have been examined.


Identification of the infarct-related artery (IRA) in patients with STEMI using coronary angiography (CA) is often based on the ECG and can be challenging in patients with severe multi-vessel disease. The current study aimed to determine how often percutaneous intervention (PCI) is performed in a coronary artery different from the artery supplying the territory of acute infarction on cardiac magnetic resonance imaging (CMR).We evaluated 113 patients from the Reduction of infarct Expansion and Ventricular remodeling with Erythropoetin After Large myocardial infarction (REVEAL) trial, who underwent CMR within 4±2 days of revascularization. Blinded reviewers interpreted CA to determine the IRA and CMR to determine the location of infarction on a 17-segment model. In patients with multiple infarcts on CMR, acuity was determined with T2-weighted imaging and/or evidence of microvascular obstruction.A total of 5 (4%) patients were found to have a mismatch between the IRA identified on CMR and CA. In 4/5 cases, there were multiple infarcts noted on CMR. Thirteen patients (11.5%) had multiple infarcts in separate territories on CMR with 4 patients (3.5%) having multiple acute infarcts and 9 patients (8%) having both acute and chronic infarcts.In this select population of patients, the identification of the IRA by CA was incorrect in 4% of patients presenting with STEMI. Four patients with a mismatch had an acute infarction in more than one coronary artery territory on CMR. The role of CMR in patients presenting with STEMI with multi-vessel disease on CA deserves further investigation. The ACC/AHA and ESC/EACTS guidelines currently give a class I recommendation to intervene on the infarct related artery (IRA) in patients presenting with ST-Elevation Myocardial Infarction (STEMI)., 2 In a The Reduction of infarct Expansion and Ventricular remodeling with Erythropoetin After Large myocardial infarction (REVEAL) trial was a randomized placebo-controlled trial assessing the efficacy of intravenous erythropoietin on infarct size in patients with acute STEMI. The triaThe design of REVEAL has been published previously., 6 BriefAn independent interventional cardiologist, blinded to clinical data and CMR results, reviewed coronary angiograms from the REVEAL trial. The IRA was determined based on angiographic parameters including visually significant stenosis ≥70%), presence of thrombus and TIMI flow.0%, prese The LVEF2, matrix 192×132 pixels, slice thickness 6 mm, parallel imaging factor 2, acquisition in late diastole on every fourth heartbeat, T2 preparations; 0 ms, 24 ms, 55 ms, 90 ms, size 1.9×1.4×6.0 mm, temporal resolution 239 ms.CMR scans were performed at 2–6 days post presentation. Patients were placed supine in a 1.5-T clinical scanner , and a phased-array receiver coil was placed on the chest for imaging. Cine images were acquired in six to ten short-axis views and two long-axis views. Short-axis views were obtained just below the level of the mitral-valve insertion plane and then every 1 cm throughout the left ventricle. A gadolinium-based contrast agent was then administered intravenously, and contrast-enhanced images were acquired in the same views as those for cine-CMR. DE images were acquired with a segmented inversion-recovery gradient-echo pulse sequence., 9 The tThe CMR scans were analyzed by one blinded observer blinded to clinical data and CA results using the identical 17-segment model as for CA. Cine-CMRFor the determination of a match between the IRA by CA and the infarct territory on CMR, the segments affected by the IRA as coded according to the 17-segment model on CA were compared to the segments of hyperenhancement noted on DE-CMR. A match was defined as overlap of at least one segment of any of the 17-segments. To account for possible misregistration two independent research investigators re-analyzed both the CA and CMR side by side to confirm a mismatch.As this manuscript is purely descriptive in nature, no formal statistical analysis was performed.The present analysis included 113 patients with a median age of 57.3 years and 21 (19%) women. A total of 17 (15%) had a history of diabetes, 54 (48%) had hypertension and hyperlipidemia. Analysis of angiographic images revealed that in patients presenting with STEMI, 63% experienced an acute infarct to the RCA, 27% to the LAD, and 10% to the LCx. The average ejection fraction measured on CMR was 47% (±11) and 43% (±14) by angiography. Analysis of the agreement between the IRA identified by CA and the infarct territories by CMR found a total of 5 (4%) patients to have a mismatch. In four of the five cases, there were multiple infarcts noted on CMR. With the use of T2-weighted edema imaging and microvascular obstruction on DE-CMR, the acuity of each infarct could be determined. Interestingly, the IRA identified by CA in these four cases were in an area of chronic infarct. In the fifth case, the coronary artery supplying the infarcted myocardium by CMR did not show any significant stenosis, and the IRA selected by CA was in a different territory that had no infarct on CMR.In the first identified mismatch, the IRA was determined to be the RCA, which underwent PCI with stent placement. On initial blinded analysis, the IRA was indeterminate. CMR showed an infarct in the inferolateral wall. It was only in hindsight, after reviewing the CMR and looking again at angiography, that a flush occlusion with retrograde filling was identified in the obtuse marginal (OM) branch of the LCx . In the In this trial cohort of patients without prior known MI (n = 113), 13 patients (11.5%) had multiple infarcts in separate territories on CMR with 4 patients (3.5%) having multiple acute infarcts and 9 patients (8%) having both acute and chronic infarcts. Of the multiple acute and chronic infarcts 3 involved all three vessels  and 6 involved the RCA and LCx. Twenty-three patients (20%) with STEMI exhibited right ventricular infarction. Among the 62 patients with the IRA as the RCA, 21 (34%) experienced right ventricular infarcts (RVI). Additionally, there were two patients who had an infarct in the right ventricle from an IRA that was identified from the LAD and one from the LCx, however, both were left dominant coronary anatomy. Among patients in the present study, 9 (8%) were found to have aneurysms and 10 (9%) were found to have the presence of left ventricular thrombi. There were a total of 44 (39%) patients with microvascular obstruction (MVO) on CMR images of which, 22 (50%) were RCA infarcts, 17 (39%) were LAD infarcts, and 13 (30%) were LCx infarcts. In the analysis of coronary dominance, 78% of patients were found to be right dominant, 12% were left dominant, and 10% were co-dominant.The present study examined patients presenting with STEMI who had TIMI flow grade 0 or 1 and no prior history of PCI to the IRA and no prior MI. We found a mismatch between the IRA identified by CA and CMR in five patients. Additionally, we found a relatively high incidence of RVI and multiple infarcts. The lack of ECG data in the present analysis is a potential major limitation, however, in all five mismatch cases, the clinical team who had access to the ECG intervened on an artery that was not identified as the IRA as defined by CMR.In one case, we could not identify the IRA on CA even with the information provided by CMR. A few potential explanations for this finding are: a) vasospasm, b) spontaneous thrombolysis prior to angiography being performed, or c) flush occlusion of a branching vessel, which could not be visualized by angiography. This patient did not have multiple territories of infarction by CMR and thus misinterpretation of the acute lesion could not explain this finding.An interesting observation from this study is that MVD with at least one vessel being completely occluded was seen in 4 of the 5 cases and could be a potential avenue for when the IRA is missed. It has been shown that up to half of patients with STEMI present with MVD, associated with increased risk of morbidity and mortality.–14 Our sIn reviewing the CMR images, we determined that 4 patients had more than one acute territory of infarct as determined by T2-weighted imaging and/or MVO. Plaque instability associated with acute myocardial infarction is not a local event but may in fact be associated with a diffuse process, occurring throughout the coronary anatomy., 19 AmonRight ventricular infarction in acute myocardial infarction is a poor prognostic indicator, associated with cardiogenic shock, ventricular arrhythmias, and advanced atrioventricular block. ContrastThe present study looked at a very specific subset of patients, as noted by the fewer anterior infarcts compared to the general population, with TIMI flow grade 0 or 1 and no prior history of MI or revascularization to the IRA. Therefore, it is likely that with a broader STEMI population, there may be an even higher incidence of mismatch. Another limitation to this study is the challenges of mapping the myocardial territory supplied by the IRA on a 17-segment model and the potential for misregistration. To limit this potential methodological challenge, we only classified a mismatch after meticulously reviewing each mismatch case with angiography and CMR images side by side. An additional major limitation is the qualitative as opposed to quantitative analysis of CMR images. An important consideration is that in patients with a chronic occlusion of a vessel within the identified infarct region, supplied from collaterals from a neighboring territory, a sudden occlusion of the neighboring, collateral-supplying vessel, may in fact be the culprit, infarct related artery, and an appropriate intervention was in fact performed clinically. As mentioned previously, a potential major limitation to this study is that the blinded interventional cardiologist did not have access to the ECG, however, the clinical team who did have access to the ECG, intervened on the vessel that was not determined by CMR to be the IRA in all five of the cases, thus suggesting that this did not impact the results of our study.In this select population of patients who met the strict entry criteria of the REVEAL study, the identification of the IRA by CA was incorrect in 4% of patients presenting with STEMI. Four patients with a mismatch had an acute infarction in more than one coronary artery territory on CMR. The role of CMR in patients presenting with STEMI with MVD on CA deserves further investigation.S1 Appendix(XLSX)Click here for additional data file.


Despite technological advances, the prognosis and survival of acute myeloid leukemia (AML) adult patients remain low, compared with other hematologic malignancies. Some antigens detected by immunophenotyping may soon play a significant role in the pathophysiologic, prognostic, and overall survival (OS) rate of AML patients. Therefore, we conducted a systematic review and meta-analysis of PubMed, Scopus, Science Direct, Web of Science, and the Cochrane Library (using PRISMA guidelines). We analyzed 11 studies and 13 antigens, detected through the immunophenotyping of 639 patients. From them, twelve exhibited a negative impact with AML prognosis. The meta-analysis demonstrated a high expression of AML markers, which have been associated with a decrease in survival over 10 months  and over 20 months . Knowing that the expression of immunophenotypic markers, which are not used on a routine basis, might be able to influence disease behavior, looks promising. However, they have been associated with a poor prognosis as well as a decrease in survival. This may allow for different chemotherapeutical protocols, including future studies for new therapeutic targets. The genetic heterogeneity of this group of hematological malignancies makes it impractical to perform initial analyses that can encompass different genes involved in AML. This makes diagnoses difficult, which can negatively influence therapeutic strategy4. Even with major improvements in the understanding and treatment of AML over the past several years, few advances have been made in the outcomes and survival of AML patients6. Complete remission is expected for approximately 60‒70% of adults with AML after the induction phase of treatment, but only about 25% survive three or more years with the possibility of being cured7. The 5-year relative survival rate for patients from birth to 19 years has been reported to be 62.8%, but declines to 5.4%, for patients older than 65 years8.Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by clonal proliferation of immature myeloid cells at various stages of maturation10. AML presents highly heterogeneous immunophenotypic profiles, which is probably due to genetic diversity. Leukemia-associated phenotypic markers (LAIPS) are useful to discriminate between normal/reactive immature myeloid precursors from leukemic cells and is commonly used in minimal residual disease (MRD) studies. In addition, LAIPS are also associated with molecular alterations with well-recognized prognostic implications (such as CD19 expression in AML with RUNX1-RUNX1T1)12.Multiparameter flow cytometry (MFC) immunophenotyping provides relevant information for AML diagnosis, classification, and monitoring. MFC allows identification, quantification, and lineage assessment of abnormal blast cells and disease classification according to the maturation stage to be made13. Improving the accuracy of prognostic assessment of AML may allow the treatment to be more specific and risk-adapted, increase the probability of cure, and minimize treatment-related morbidity and mortality14.Previous studies show that a great number of distinct antigens affect AML prognosis and prediction. Nevertheless, difficulties are still found in performing accurate risk stratification for diagnoses based on immunophenotypic featuresSince there are no clear recommendations and no consistent approaches to the use of new markers in the immunophenotypic panels for AML evaluations as well as for the markers’ influence on determining prognosis and survival in clinical practice, a systemic review was done. In this review, we aimed to identify relevant publications about the influence of these new monoclonal antibodies used as immunophenotypic markers in the prognosis and survival of AML patients.15.A systematic review was performed based on a scientific research protocol describing the aims and methods used. Within the limitations of the research in this area, this synthesis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statementAre there new immunophenotypic markers that may aid in the prognosis and survival of acute myeloid leukemia”?The question of this systematic literature review was: “The literature search was conducted using Pubmed, Science Direct, Web of Science, Scopus and Cochrane Library databases for articles published from 2012 to 2015. In addition, the reference lists of relevant papers were searched for additional AML studies.The following search terms consisted of a range of pertinent terms: 1) antigens CD (MeSH); 2) antigens, differentiation (MeSH); 3) biological Markers (MeSH); 4) tumor markers, 5) biological (MeSH) and prognosis (MeSH); 6) survival rate (MeSH); 7) survival analysis (MeSH) and leukemia myeloid, acute (MeSH); or 8) acute leukemia.16); (5) articles assessing AML cases; and (6) articles with available abstract and full text.The articles found in the search were compared with the previously defined inclusion criteria to determine the relevance of the study: (1) papers published from 2012 to 2015; (2) articles published in English, Spanish, and Portuguese; (3) articles that used immunophenotyping in their methodologies; (4) articles assessing monoclonal antibodies not included in the consensus diagnostic panels for AML  assessment scale, which consisted of 22 itemsFrom the included studies, information regarding several parameters was obtained: (1) journal of publication; (2) The Journal Citation Reports (JCR) impact factor; (3) location; (4) study design; (5) aim of the study; (6) number of samples analyzed; (7) AML classification; (8) most incident subtype; (9) immunophenotypic marker; (10) prognostic value; (11) first induction treatment protocol (12) follow up; (13) survival; (14) limitations; and (15) STROBE scores. For inaccessible or incomplete full texts, authors were contacted for additional information.A meta-analysis of the relative risks related to the probability of survival at 10 to 20 months was performed. For analysis purposes, low expression of the immunophenotypic marker was considered as absent, and high expression was considered as present.18. A contingency table was constructed  classification for AML, six used the French-American-British (FAB) classification, and three used both FAB and WHO to classify the AML subtypes30. The most reported subtype in the articles was FAB M2 followed by FAB M4.An overview of the characteristics of the 11 studies included in the final analysis is summarized in Tables 27. The Journal Citation Reports (JCR) impact factor found in the search ranged from 1.826 to 22.26825. There were only two pediatric studies, and the others focused on adults30. The expression of 13 different antigens was evaluated in all articles (Table 30. The most common chemotherapy regimen was anthracycline-based induction therapy (3 + 7), although other alternative treatments were chosen by some groups.Most studies were performed in countries with high scientific and technologic development, including the United States (US), Germany, and Japanes Table . All 11 30.The methodological quality of observational studies conducted with the STROBE tool showed that eight articles scored >90% revealing a high methodological quality of the included studies. One article scored 19 points, which was equivalent to 86.4%, four scored 20 points, which was equivalent to 91%, and four scored 21 points, which was equivalent to 95.4%27. All five cohort articles showed a decreased survival related to the high expression of the immunophenotypic markers.Most articles showed that the expression of the evaluated antigens has a negative impact on prognosis of AML. Only one article showed that CD263 has a positive value on prognosis, and two articles showed that the values of CD90 and ILT3 were not determined+/CD38− cells) and the CD34+/CD38+ compartments of AML cells. They showed that LSC expressed a higher amount of CD82 than CD34+/CD38+ AML cells; these findings suggested that overexpression of CD82 may render LSC able to adhere to the bone marrow (BM) niche where it appears to regulate maintenance of leukemia stem cells within the BM niche. In addition, they showed that down-regulation of CD82 in LSC may stimulate mobilization of these cells from the BM niche to PB and sensitize them to chemotherapeutic agents24.Two articles by the same authors discussed CD82, a member of the tetraspanin superfamily that was originally identified as an accessory molecule in T-cell activation and in nonimmune cells in integrin-mediated cell adhesion to the extracellular matrix. Both articles evaluated the expression of CD82 in the self-renewing leukemia stem cell (LSC) compartments  and showed that CD135 was expressed in 82% of all cases. There was no association of CD135 expression with the FLT3 internal tandem duplication mutation, a molecular abnormality associated with unfavorable AML prognosis. Nevertheless, co-expression of CD135 and CD177 was associated with a decrease in event free survival (EFS) and overall survival (OS) in multivariable analysis in both age groups30.CD135 (FLT3) is a tyrosine kinase receptor that has a significant role in leukemogenesis. Sharawat et al. investigated the expression of CXCR4 (CD184) in AML. CXCR4 is a receptor for stromal-derived factor 1 (SDF1) that plays a very important role in hematopoiesis development and organization of immune system. After analyzing whole blasts and CD34+ cells in 142 adult non-M3 AML cases, the authors used mean fluorescence intensity (MFI) and showed a correlation between high CXCR4 expression and decrease in EFS and OS in addition to an unfavorable prognosis. In addition, CXCR4 expression was associated with high leukemic burden, as estimated by DHL level and white blood cell and peripheral blast cell counts29.Mannelli et al. evaluated the expression of this antigen in 30 AML cases and 30 acute lymphoblastic leukemia patients and observed that CD133 was expressed in 56.6% of AML (n = 17). The authors observed an important correlation between the expression of CD133 and the survival of AML patients and demonstrated a decrease in OS with an increase in CD133 expression. As for the prognostic value, it was concluded that CD133 expression was highly associated with poor prognosis in AML patients22.CD133 is a novel five transmembrane molecule expressed on primitive normal hematopoietic progenitors. Tolba et al. assessed the expression of the death receptors, including TRAILR1, 2, and 3 , TNFR1 (CD120a), and FAS (CD95). CD262 is involved in induction of apoptosis in lymphoid cells. CD263 is a marker of neutrophilic granulocytes that participates in apoptosis regulation and inhibition of cell death through competitive binding activity. TNFR1 is expressed on monocytes, lymphocytes, and granulocytes, and is involved in cytotoxicity mediation. The authors concluded that CD262 and TNFR1 expressions showed significantly shorter OS, earlier disease onset, and higher probability of relapse in AML patients. Conversely, CD263 expression exhibited an enhanced OS. As for prognostic value, high expressions of CD262 and TNFR1 were found to be associated with unfavorable prognostic groups, and CD263 was found in cases with favorable risk19.In 46 AML patients, Schmohl et al. evaluated one member of the immunoglobulin-like transcripts (ILT3 expression) in normal and leukemic myeloid precursors in 20 healthy individuals and 37 AML cases. ILT3 is a member of the large family of ILT molecules, leukocyte Ig-like receptors (LIRs), and monocyte/macrophage Ig-like receptors (MIRs). The authors showed that ILT3 was expressed in all cases of AML displaying monocytic differentiation but not in the AML subtypes M1/M2 and M3. It was shown that expression of ILT3 by leukemic cells contribute to the inhibition of tumor specific T cell responses. As for the prognostic impact, frequent cytogenetic abnormalities observed in AML patients with ILT3+ were those associated with intermediate prognosis, but it’s potential value as a prognostic marker, particularly in cytogenetically normal AML, remains to be determined27.Dobrowolska et al. evaluated hMICL expression in one article in 93 AML patients. hMICL is a heavily glycosylated transmembrane C-type lectin with an unknown function. Cryopreserved mononuclear cells and bone marrow samples were used in the analysis. They showed that hMICL was found to be restricted to the CD45low/SSClow population of AML cells. The authors suggest that no loss of hMICL expression in AML patients may be an early indicator of relapse. In addition, hMICL preserved fluorescence intensity at relapse, suggesting that this antigen could be a tool for minimal residual disease quantification by flow cytometry in AML28.Larsen et al. evaluated the expression of these two antigens in two distinct primitive cell population compartments from bone marrow. CD34+ CD38− cells (enriched for HSC) and CD34+ CD38+ cells (enriched for HPC) obtained from 12 pediatric AML patients were used for the analysis. The results showed that CD90 showed slight incremental increases in patients who reached remission and did not relapse. As for CD96, the authors describe greater expression in relapse and higher levels in AML cells than in normal bone marrow cells before and after chemotherapy26.CD90, a cell-surface glycoprotein, seems to be involved in proliferation and expansion processes, and CD96 is a member of the immunoglobulin superfamily. In one article, Chaves-Gonzales 30. The expression of these markers are considered independent events, since their pathogenic significance are not necessarily interconnected within certain pathways. The survival analysis over 10 months showed a great heterogeneity with a significant Cochran Q test  and the Higgins I² test showed a result of 83.33%, indicating high heterogeneity and suggesting an impediment to achieving the meta-analysis. Despite that impediment, the tests were still performed showing a meta-analytical estimate of no significant risk (1.49). The funnel plot and regression testing for evaluation of publication bias showed a large asymmetry   = 1.0512; p = 0.7889), and the Higgins I² test showed a result of 0% indicating no heterogeneity. The same results were found in relation to the markers collected at 20 months, both in relation to Q Cochran test (Q (df = 4) = 2.6083; p = 0.6254) and the Higgins I² test (0%). It was decided then, to perform the two meta-analyses using the fixed effects model.The meta-analytical estimate represented by the relative risks of survival rate at 10 months Fig.  was 2.55After resetting the final models, especially the 10-month survival, the funnel plot and the regression test for analysis of the asymmetry were used to assess the potential for publication bias. Both tests showed that publication bias was not significantly associated with both the 10 and 20 month survival   and new analytical strategies. However, the discovery of new immunophenotypic markers for AML diagnosis was limited, and immunophenotypic panels have remained similar over the last twenty years. Therefore, there is still a need for new markers for leukemic myeloid cells to be included in clinical routines to increase the value of MFC not only as a diagnostic but also prognostic tool for monitoring of MRD and for development of drugs for targeted therapy5. The inhibitory receptor ILT3 is a highly sensitive and specific marker for both diagnosis and monitoring of AML with monocytic differentiation; ILT3 as a marker is particularly useful in the differential diagnosis of AML with monocytic differentiation and microgranular acute promyelocytic leukemia, two leukemia subtypes that require different treatment strategies27. Another marker, hMICL, expressed significantly higher fluorescence intensity compared to normal bone marrow, suggesting suitability of this antigen as a pan-AML marker28. Regarding cell adhesion, CD82 has been shown to have a high expression in AML cells suggesting an increase of adhesion to the BM niche. On the other hand, down-regulation of CD82 in AML cells may stimulate circulation of these cells into peripheral blood23.In this systematic review, we observed that some markers could be used for AML diagnosis and during the follow up. For example, the high expression of CD87 is an indicator of morphologically and antigenically poorly differentiated disease, especially in the M4 and M5 subtypes5. CXCR4 could be also included due to its high expression on the surface of the entire leukemic population and demonstration of its influence on the overall CR rate and as poor prognostic factor for DFS and OS29. Another interesting study described the association between high expression of CD93 and loss of CDKN2B (p15Ink4b) expression; a cell-cycle inhibitor gene that has been shown to play a prominent role in leukemia pathogenesis and correlate with poor prognosis25.Many prognostic factors have been established in the past few decades in AML, including age, cytogenetic abnormalities, white blood cell count, serum lactate dehydrogenase, and the presence of antecedent hematologic disorder. This systematic review has shown that the high expression of CD87 on peripheral blood blasts was associated with relapse and poor prognosis and could be incorporated into the initial diagnostic work-up of AML patients29. Another study showed that FLT3 receptor (CD135) was associated with the FLT3 Internal tandem duplication (ITD) mutation, which confers a poor prognosis30. Since gene mutations are of utmost importance in deciding the patients’ risk-stratification, the knowledge of this association can bring advancement on treatment decision and give even more precision to it, raising the chances of cure. From this preliminary information, it is possible to envision future studies involving the connection of these two types of markers to predict prognosis and survival of AML.The demonstration that the high expression of these immunophenotypic markers influence not only the survival of AML patients but also the prognosis, brings up some questions. Since immunomarkers are signaling those important prognostic data and it is known that molecular mutation can also have influence on the disease pathways regarding both survival and prognostic features, how these two information can be combined on determine better accurate prognostic factors, and what association could be found between them? We could observe that this data are rarely combined on the articles included on this systematic review and meta-analysis. It was evidenced that majority of patients that expressed high CXCR4 also presented the NPM1 mutated gene, what gives it a worst prognosis5. One method to attempt to increase these patients’ survival could be a more specific chemotherapeutic protocol for this heterogeneous group of diseases. It is also necessary to identify in advance those patients who have greater resistance to treatment, higher relapse rates, and lower survival. We performed a meta-analysis using the fixed effects model. Four new immunophenotypic markers, CD133, CD135, TRAIL2 (CD262), and TNFR1 (CD120a) were demonstrated and showed correlation with lower survival at 10 and 20 months. Thus, these antigens can be used as early markers in combination with other prognostic factors for risk stratification of relapse. CD133 antigen appears to be expressed restrictively in the more immature cell population, and examination of the articles has made it possible to observe that expression of the CD133 in acute leukemia could be correlated with an immature phenotype of the myeloid blasts and highly associated with poor prognosis5. Proliferation regulators (such as tyrosine kinase receptors) play an important role in the pathogenesis of acute myeloid leukemia. The high expression of CD135 was associated with lower EFS and OS30. As verified by this study, the expression of death receptors is typically associated with the apoptotic regulation of leukemic blasts that demonstrated a significant association of TRAILR2 expression on blasts from patients in adverse risk groups and showed a negative impact on overall survival. Results concerning TNFR1 showed that this receptor is for the immune-modulating cytokine TNF. TNFR1 may play a role in initiation and proliferation of AML blasts due to its high expression, which appears to be related to a lower survival in AML patients19. Technically, this meta-analysis demonstrated that meta-analytical estimates, represented by the relative survival risk at 10 and 20 months, were both significant when the risk of survival in the absence and presence of the markers was analyzed. Notably, in our analysis, there was no heterogeneity among the cohort studies, which implied that the present method to combine the results from these studies was reasonable. Thus, the conclusions from this analysis should be credible. However, due to the relatively limited number of cases included in these studies, further analyses of larger series of patients are needed to confirm these preliminary observations.Despite the advancement of therapeutic progress, the overall survival of AML patients remains low27. CD93, an AML with MLL rearrangements marker expressed on leukemia stem cells (LSC), may also be a useful therapeutic target candidate for anti-LSC therapy and prognostic marker for quantitation of minimal residual disease25. hMICL is a pan-AML marker uniformly present on cells. It can be poorly immunophenotypically lineage-characterized and difficult to monitor for residual disease and development of drugs for targeted therapy28. The glycoprotein CD82 could be an attractive target for LSC eradication, due to its important role in regulation of the AML cell survival and their adhesion to bone marrow microenvironment23.Minimal residual disease is a term used to describe detection of subclinical levels of leukemia using multiparameter flow cytometry or molecular-based approaches. Employing the MFC for minimal residual disease detection appears to be a reliable method for obtaining rapid and objective patient remission status, provide early end points in clinical trials, and to inform patient management of a patient’s status. Emerging evidence indicates that MRD detection in patients with AML is also associated with poor prognosis, and early therapeutic interventions may be of clinical benefit. In addition, other studies have reported on the progression of new drug development that target specific areas of the leukemia cells; this is important since the current treatment can help, but does not always cure, AML patients. In this review, we observed that some antigens can be useful both as a marker for MRD and potential therapeutic target. ILT3 is an antigen expressed in AML displaying monocytic differentiation that supports differentiation and subsequent AML diagnosis of AML. It may also be a candidate marker for MRD detection in AML patients due to its high sensitivity, specificity, and stable expression. This antigen also can be a target for therapy in AML with monocytic differentiation as a result of inhibition of ILT3 signaling with specific antibodies or antagonists, which may render ILT3+ AML cells more susceptible to differentiation agents and anti-tumor T cell responses8. It was notable that some data discrepancies were found as in the case with hMICL, in which the author associated its high hMICL expression with relapse, but its role in the prognosis was not clear. More data are necessary to establish the extent that hMICL-based immunophenotyping can detect treatment failure, which is common in most AML patients. The same results were observed for CD90 and CD96, in which the author also shows an association of its high expression with relapse, but does not associate either CD90 or 96 with prognosis. CD96, especially, does not demonstrate high fluorescence in the first diagnosed sample, only in the normal bone marrow sample.There are some limitations that should be noted in our systematic review and meta-analysis. One of the most important is the lack of actual patient data included in the analysis. Despite that, it is known that AML is a disease with low incidence. In the US, the estimate for 2024 predicts that leukemia, including AML, will represent 3% of all cancers in male and femalesRegarding the limitations of our systematic review and meta-analysis, survival information was extracted from survival curves and not from a mortality table. Secondly, positive and negative expression were considering basing on the MFI of each study Third, the criteria to determine the positive or negative antigen expression varied across the included studies. Fourth, from the six immunophenotypic markers included in the meta-analysis, two were removed as they caused heterogeneity between the studies, which may have been due to some methodological differences. Fifth, we only searched for articles published in English, Spanish, and Portuguese and may have missed relevant publications in other languages. Finally, some studies included in the qualitative synthesis also evaluated M3 AML patients, a subgroup recognized for its excellent prognosis. However, only two studies included in the meta-analysis contained M3 AML cases, both with a low number of patients (<10%, Table Despite the limitations listed above, the present analysis revealed the prognostic value of new antigen expression in AML. Although the high expression of CD133, CD135, TRAIL2 and TNFR1 was not significant in most of the individual studies regarding the occurrence of the outcome, when they were collected in the meta-analysis a high correlation was observed with poor prognosis and low DFS and OS. However, further prospective studies with larger sample sizes are required to include these new immunomarkers in MFC routine of acute myeloid leukemia.Supplementary Informationpdf


Responding of the test rats was then extinguished over 7 days under isolated conditions. Tests of reinstatement were then conducted in the presence of the cocaine partner and abstinent partner under extinction conditions. Neither social partner reinstated responding relative to that observed on the final day of extinction; however, responding was greater in the presence of the cocaine partner (S+) than the abstinent partner (S−) during the reinstatement test. These data fail to demonstrate that a social partner reinstates cocaine-seeking behavior after a period of abstinence, but they do indicate that social partners can serve as either excitatory or inhibitory discriminative stimuli to influence drug-seeking responses.Social partners influence the likelihood of using drugs, developing a substance use disorder and relapse to drug use after a period of abstinence. Preclinical studies report that social cues influence the acquisition of cocaine use, the escalation of cocaine use over time, and the compulsive patterns of cocaine use that emerge during an extended binge. The purpose of this study was to examine the effects of social cues on the reinstatement of cocaine-seeking behavior after a period of abstinence. Male rats were obtained at weaning, assigned to triads (three rats/cage), reared to adulthood and implanted with intravenous catheters. Rats from each triad were then assigned to one of three conditions: (1) test rats were trained to self-administer cocaine and were tested for reinstatement; (2) cocaine partners were trained to self-administer cocaine and were predictive of response-contingent cocaine delivery; and (3) abstinent partners were not given access to cocaine and were predictive of extinction. The test rats alternated social partners every 5 days for 20 days such that responding was reinforced with cocaine in the presence of the cocaine partner (S Social peers influence the likelihood of using drugs  that predict cocaine availability reinstate responding after a period of extinction, whereas inhibitory contextual cues that predict extinction do not ; however, an individual that practices abstinence and discourages drug use in others may serve as a discriminative stimulus predicting that drug-seeking behaviors will not be reinforced (S−). As noted previously, cues that predict cocaine availability reliably reinstate drug-seeking behavior, whereas cues that predict extinction do not  reinstates responding to a greater extent than an abstinent partner that does not predict response-contingent drug availability (S−). To this end, male rats were implanted with intravenous catheters and trained to self-administer cocaine on a fixed ratio (FR1) schedule of reinforcement. The test rats alternated social partners every 5 days over 20 consecutive days during which they were tested with response-contingent cocaine in the presence of a cocaine partner and tested under extinction conditions in the presence of an abstinent partner. Next, responding was extinguished in test rats under isolated conditions over 7 days. Reinstatement tests were then conducted in the presence of the cocaine partner and the abstinent partner. We predicted that the reinstatement of drug-seeking behavior would be greater in the presence of the cocaine partner than in the presence of the abstinent partner.The purpose of the present study was to determine whether a drug-using social partner that predicts response-contingent drug availability  in polycarbonate cages (interior dimensions: 50 cm × 28 cm × 20 cm) until surgery and catheter implantation. All subjects were kept in a temperature- and humidity-controlled vivarium on a 12-h light/dark cycle (lights on: 05:00). Except during lever-press training, food was continuously available in the home cage. Fresh tap water was continuously available in the home cage throughout the study. All subjects were maintained in accordance with the Institutional Animal Care and Use Committee of Davidson College and the Guide for the Care and Use of Laboratory Animals , Male, Long-Evans rats were obtained at weaning (~21 days) and housed in triads . These chambers were equipped with a house light, two response levers and a food hopper. Software and interfacing for these chambers were supplied by Med Associates, Inc.Drug self-administration sessions were conducted in custom-built, operant conditioning chambers  described previously Smith, . BrieflyFive weeks after arrival and 1 week prior to catheter implantation, rats were food restricted to no less than 85% of their free-feeding body weights and trained to press a response lever on an FR schedule of food reinforcement during daily training sessions. During these sessions, each response was reinforced with a single 45-mg grain pellet . Sessions terminated automatically once 40 pellets were delivered or 2 h elapsed, whichever occurred first. Training continued in this manner until a rat earned 40 pellets during any four individual sessions.Six weeks after arrival, rats were anesthetized with a combination of ketamine  and xylazine  and surgically implanted with intravenous catheters into the right jugular vein. Ketoprofen  was given immediately after surgery and again 24 h later for analgesia. Beginning on the day of surgery, a solution of heparinized saline and ticarcillin  was infused through the catheter daily to prevent infection and maintain patency. After 7 days, ticarcillin was discontinued and only heparinized saline was used to maintain catheter patency.+); and (3) abstinent partners never had access to cocaine and served as discriminative stimuli for extinction (S−).Immediately after surgery, the three rats of each triad were randomly assigned to one of three conditions: (1) test rats were trained to self-administer cocaine and examined during tests of reinstatement; all primary hypotheses were tested using data from these rats; (2) cocaine partners were trained to self-administer cocaine and served as discriminative stimuli for response-contingent cocaine delivery  and the insertion of the retractable lever into the cage. Each response on the response lever produced an infusion of cocaine on an FR1 schedule of reinforcement. Each infusion delivered 0.5 mg/kg cocaine at an 0.05 mg/ml concentration over a duration of 2.0–2.4 s (based on body weight), representing infusion volumes of 0.036–0.043 ml/infusion. Coincident with the infusion, the lever retracted for 20 s to signal a timeout period in which cocaine was not available. After 20 s, the lever was reinserted and cocaine was again available on the FR1 schedule of reinforcement. This initial acquisition session was terminated automatically once 21 infusions were obtained.+) and the abstinent partner served as a discriminative stimulus for extinction (S−). During sessions with the cocaine partner, both rats had access to cocaine on an FR1 schedule and no limit was placed on the number of infusions that could be earned. During sessions with the abstinent partner, neither rat had access to cocaine. For the test rat, responses on the lever produced response-contingent infusions of saline on an FR1 schedule and no limit was placed on the number of infusions that could be earned. The order of exposure to the cocaine and abstinent partners  was counter-balanced across test rats. All sessions were 2 h in duration and began promptly at the beginning of the dark cycle (lights off: 1700).Over the next 20 consecutive days, test rats alternated social partners every 5 days. During this period, test rats were tested with cocaine in the presence of their cocaine partner for 10 days (2 cycles × 5 days) and under extinction conditions in the presence of their abstinent partner for 10 days (2 cycles × 5 days). Consequently, the cocaine partner served as a discriminative stimulus for response-contingent cocaine delivery  and in the presence of the abstinent partner on the other day. The order of partner presentation was counter-balanced across test rats. Regardless of partner, responses on the lever by the test rat produced response-contingent infusions of saline .On days in which cocaine partners were not tested in the presence of a test rat, they self-administered cocaine in isolated chambers under identical experimental conditions . On days in which abstinent partners were not used for testing, they remained isolated in polycarbonate cages.t-tests using partner/session as the factor . The effect size was calculated via the Cohen’s d statistic. All tests were two-tailed and the alpha level was set at 0.05.Data from the first 20 days of testing were examined via three-way (partner × cycle × day), repeated-measures analysis of variance (ANOVA). Data from the following 7 days  were examined via one-way, repeated-measures ANOVA using day as the factor. Data from the reinstatement tests were examined via dependent-samples n = 21 infusions; data not shown).All test rats responded on the first day of cocaine self-administration when they were tested in isolation, and 11 out of 12 test rats received the maximum number of infusions available  = 4.939, p = 0.048), but no other significant main effect or interaction was observed. As shown in Figure The test rats alternated partners every 5 days over 20 consecutive days in which they were tested with cocaine in the presence of a cocaine partner for 10 days (2 cycles × 5 days) and tested under extinction conditions in the presence of an abstinent partner for 10 days (2 cycles × 5 days). A three-way ANOVA using the factors of partner, cycle and day revealed a significant main effect of partner  = 4.360, p = 0.001). Following extinction, neither the presence of the cocaine partner nor the presence of the abstinent partner significantly reinstated responding relative to the final day of extinction . During the reinstatement tests, responding was significantly greater in the presence of the cocaine partner than in the presence of the abstinent partner (t(12) = 2.558, p = 0.025), and this difference was characterized by a large effect size (d = 1.01).Prior to reinstatement testing, responding in test rats decreased in a monotonic fashion when they were tested under extinction conditions in isolation over seven consecutive days  were not sufficient to reinstate responding to levels above those observed at the end of extinction. Consequently, the hypothesis that a social partner predictive of response-contingent cocaine would reinstate responding was not supported.The principal findings of this study are that: (1) test rats responded more when tested with response-contingent cocaine in the presence of a cocaine partner than when tested under extinction conditions in the presence of an abstinent partner; (2) the presence of a social partner previously paired with response-contingent cocaine failed to reinstate responding after a period of abstinence; and (3) non-reinforced responding during the reinstatement test was greater in the presence of a cocaine partner than in the presence of an abstinent partner. The observation that responding was significantly greater during the reinstatement test in the presence of a cocaine partner suggests that test rats were able to discriminate between the two partners and responding was under stimulus control; however, the excitatory effects contributed by the cocaine partner  and one partner was explicitly paired with extinction . Previous studies using both discrete and contextual stimuli as excitatory/inhibitory discriminative stimuli have reported that excitatory discriminative stimuli reliably and robustly reinstate drug seeking but inhibitory stimuli do not. For instance, passive contextual cues  that predict cocaine availability reinstate cocaine seeking, whereas passive contextual cues that predict extinction do not  is critically important in determining the effects of social contact on measures of drug-seeking behavior. Specifically, cocaine intake is increased in the presence of a partner self-administering cocaine and decreased in the presence of a partner without access to cocaine  is a moderate dose of cocaine that engenders high rates of responding in rats  than the abstinent partner (S−), indicating that social partners can serve as discriminative stimuli to produce either excitatory or inhibitory effects on drug-seeking behavior.In conclusion, we were not successful at demonstrating that a social partner self-administering cocaine and predictive of response-contingent cocaine could reinstate cocaine-seeking behavior after a period of abstinence. On the other hand, we were able to demonstrate that a social partner self-administering cocaine could serve as a discriminative stimulus. This latter observation was evidenced by the differential responding observed during the reinstatement tests when the response-reinforcer contingencies were identical in the presence of the two partners. During this test, responding was significantly greater in the presence of the cocaine partner (SMAS conceived of the study and was responsible for the experimental design. HZ and AMR conducted the study and collected the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has been observed that the efficacy of dipeptidyl peptidase-4 (DPP-4) inhibitors as compared to the placebo groups in some clinical trials conducted in China is weaker than that in trials conducted outside China, leading to the suspicion that this may be caused by differential Glycosylated Hemoglobin (HbA1c) response in the placebo arm of DPP-4 inhibitor clinical trials conducted in China compared to other countries.We searched published articles and other documents related to phase III placebo-control trials of DPP-4 inhibitors in Type 2 diabetes mellitus (T2DM). We included studies from different countries and compared those conducted in China to those conducted in other countries. Meta-regression analysis was used to analyze the HbA1c response in the placebo arms.p-value < 0.001), compared to 0.015 %  for 56 trials conducted outside of China. The difference of placebo effect between trials conducted in and outside China is -0.273 %  while after excluding trials conducted in Japan, the difference is -0.203 % . They are both statistically significant.A total of 66 studies met the inclusion criteria and 10 were conducted within China. There were a total of 8303 participants  in placebo groups. The pooled change in HbA1c for the placebo groups of 10 trials conducted in patients with T2DM in China was 0.26 % (95 % CI , The meta-analysis in the article demonstrates that there is statistically significant difference in the HbA1c response in the placebo arm of DPP-4 inhibitor clinical trials conducted in China compared to other countries. This differential HbA1c response in the placebo arm should be taken into consideration by both experimenters and medical decision makers when future DPP-4 studies are conducted in China. Guideline of Prevention and Treatment for T2DM in China  and There may be various reasons for this high placebo effect. However, what these reasons exactly are is unknown. Although the investigation of these reasons is not the purpose in this article, we have the following two major guesses. First, the practical process of these trials could cause a bias. Most of the trials provided the participants the significant benefit to obtain more resource of medical care, esp. in China. Because of no established PCP system, low awareness of diabetes management, China has most diabetes patients in the world  but muchThe results on the placebo effect on other countries may also provide values for future trials and medical interpretation. For example, clinical trials conducted in Japan had a statistically significant reverse placebo effect, which may be important information for conducting future DDP-4 trials as well as for interpreting trial results in Japan.The meta-analysis in the article demonstrates that there are significant differences in response in the placebo group of DPP-4 trials conducted in China compared to those conducted outside of China. This difference may give some clue to why the efficacy of these drugs is less statistically significant in trials conducted in China. More importantly, this difference in response in the placebo group should be taken into account for future DDP-4 trials conducted in China. In addition, the difference in placebo should be carefully considered by medical decision makers when future DPP-4 studies are conducted in China.


A novel on-line microfluidic assay for the chemotherapy agent carboplatin will allow sensitive detection of the drug directly applied to the tumour as it emerges from the tumour into healthy tissue. A microfluidic sensor system based on a carbon nanotube-epoxy composite electrode was fabricated to allow detection of the presence of the anti-cancer drug carboplatin in healthy tissue in real time during chemotherapy. Detection of carboplatin was carried out by observing the effects of the drug on the differential pulse voltammetry of free purine bases using a novel carbon nanotube-epoxy composite electrode. In free solution these electrodes performed better than glassy carbon electrodes for oxidation of the free purine bases AMP and GMP, and than DNA-modified carbon nanotube-epoxy composite sensors for detection of carboplatin. On-line carboplatin detection was performed using a computer-controlled microfluidic platform. The methodology for on-line carboplatin detection was optimised in terms of the analysis time and to allow repeated carboplatin measurement using the same electrode. Microdialysis sampling and our microfluidic platform were combined to give a proof-of-concept system for real-time carboplatin detection with a limit of detection of 0.014 μM carboplatin in the sampled media. This paper is dedicated to Craig Lunte's pioneering work in analysis and microdialysis. Carboplatin has proven activity against a variety of solid tumours including ovarian, testicular, head and neck and lung cancers .6 However, although carboplatin is an effective anti-cancer drug, it causes many side effects. This is because, in common with all anti-cancer drugs, carboplatin works by binding to the DNA of active cells that are growing and dividing and as a result it cannot distinguish between cancer cells and healthy cells such as blood cells, skin cells, hair follicles and the cells inside the digestive system. Consequently, carboplatin affects not only the cancer cell growth but also blocks the growth of healthy rapidly dividing cells, leading to side effects such as mouth and throat sores, diarrhea, nausea and vomiting, blood disorders, nervous system effects, appetite loss and hair loss.8Chemotherapy is central to most cancer treatment regimes. It usually involves systemic administration of one or more anti-cancer drugs, which are designed to kill cancer cells and hence inhibit the growth of tumours in order to prevent their spread to other organs. Platinum-based anti-cancer drugs such as oxaliplatin, nedaplatin, lobaplatin, heptaplatin and carboplatin are used for chemotherapy.13 These methods involve large instruments with high costs, require highly-trained personnel for testing and as they are sample-based give poor time resolution. There is a need for rapid, continuous, low-cost techniques for analysis of drug–DNA interaction and high time resolution. Electrochemical techniques have high sensitivity, low cost, and rapid response. Typically once the method is established they do not require a great deal of user expertise. This has made electrochemical devices an attractive choice for point-of-care applications with high time resolution.Several analytical techniques have been used to detect the interaction of carboplatin with the structure of deoxyribonucleic acid (DNA). These include high-performance liquid chromatography, inductively coupled plasma-mass spectrometry and capillary electrophoresis.17 Carboplatin has been detected electrochemically through its effects on DNA electrochemistry by the Brett-Oliveira group using glassy carbon electrode (GCE).17 Initially, the GCE was coated with DNA to detect the oxidation peaks due to four DNA bases ,18 of these only guanine and adenine respond to carboplatin. It was also necessary to condition the DNA sensors in single-stranded (ss) DNA (forming a triplex structure) to stabilise DNA-base oxidation peaks.19Carboplatin is not electroactive and therefore cannot be detected directly using electrochemistry, however, it can be detected through its effect on DNA bases, which are electrochemically active. These interactions have been studied with a range of electrodes including hanging mercury drop electrodes, screen printed electrodes, graphite electrodes and glassy carbon electrodes.20 At present these approaches use histology to determine drug effectiveness followed potentially by systemic administration of the drugs.22 An alternative approach is to consider treatment by delivering the drug directly to a targeted specific area (the tumour) at a high dose and to monitor the surrounding healthy tissue for the first arrival of the drug. This could be achieved either by detecting carboplatin or DNA damage caused by carboplatin in real time. Such an approach could enhance the effectiveness of chemotherapy, would reduce side effects and would also reduce the cost of drug administration as the drug would be injected directly into the cancer cells.The problem with carboplatin is that its therapeutic index (the ratio between effective dose and toxic dose) is low. Consequently, avoiding toxicity by controlling the systemic dose is difficult. Recently, there has been a move to bioengineer devices that allow personalisation of cancer treatment by applying the drugs either singly or in combination directly to the tissue.in vivo and in vitro. MD sampling has been used extensively for monitoring amino acids, neurotransmitters and energy metabolites such as glucose, lactate and pyruvate in the brain for both fundamental and clinical research studies.25 Microfluidics has attracted a great deal of attention for miniaturisation of diagnostic devices due to their low analysis volume, short time for analysis, compact size and low cost26 and is particularly well suited for analysis of the low-volume flow streams produced with microdialysis.Microdialysis (MD) is a powerful sampling technique that is capable of continuously monitoring the extracellular space both 28 Retrodialysis using a microdialysis probe could allow clinicians to deliver carboplatin directly to the tumour site.In addition to monitoring analytes, sampling probes have also been used to deliver drugs or other substances for pharmacokinetic and drug metabolism studies in the blood and various tissues of both animals and humans, and have also been used to investigate drug distribution to specific regions.In this study we present a novel microfluidic sensing system that we envisage being used alongside retrodialysis drug delivery into the tumour to detect in real time when carboplatin disperses into surrounding healthy tissue. The concept is shown in 22.1in vitro using an autocalibration system, 1.5 mM AMP was dissolved in 0.1 M ACB and T1 solution mixed in the ratio of 3 : 1.Carboplatin (Sigma-Aldrich) was prepared in 0.1 M acetate buffer . T1 perfusate solution was prepared with 147 mM sodium chloride, 4 mM potassium chloride, and 2.3 mM calcium chloride in distilled water and 400 μl of 5-chloro-2-methyl-4-isothiazoline-3-one  was added as an antibacterial agent. All chemicals were of analytical grade and obtained from Sigma-Aldrich. Guanosine 5-monophosphate disodium salt hydrate  and adenosine 5-monophosphate  were obtained from Sigma-Aldrich. Stock solutions of both 1 mM GMP and 1 mM AMP were dissolved in ACB pH 4.5 as a supporting electrolyte for use in a beaker experiment. In the case of testing 2.229 although in the present work we use a different ratio of MWCNT : epoxy. Briefly, plastic pipette tips (10 μl size) were employed to contain the carbon-epoxy composite. Epoxy resin was prepared by mixing resin and hardener in the ratio of 3 : 1. The MWCNT were thoroughly combined with the epoxy resin in the proportion of 1 : 4 by weight and stirred well. A pipette tip was filled densely with the MWCNT-epoxy composite paste by a tapping and dragging method. Then an electrical wire was inserted into the filled pipette and it was allowed to dry at room temperature for two days. The epoxy resin with the same ratio as mentioned above was used to fill up the internal volume of the pipette tip using a 18G hypodermic needle, and it was allowed to cure for two days. After this process, a scalpel was used to cut the end of the electrode to expose a surface area of the MWCNT-epoxy composite that was approximately 0.50 mm in diameter. In the microfluidic platform experiment, MWCNT-epoxy composite electrodes were fabricated using Radel™ tubing  instead of the pipette tip.Multiwalled carbon nanotubes  were purchased from Sigma-Aldrich. Epoxy resin and hardener were obtained from Robnor . The method of MWCNT-epoxy composite fabrication is reported previously by the Patel group,2.3Preliminary experiments were conducted in a small glass beaker using the MWCNT-epoxy composite electrode as a working electrode. A coiled Pt wire and a commercial Ag|AgCl in 3 M NaCl solution (BASi) were used as a counter and a reference electrode, respectively.In this work, differential pulse voltammetry (DPV) was carried out using Echem software  and a PowerLab 8SP (ADinstruments). Data was processed using Igor Pro software.A novel on-line analysis chip was fabricated based on a microfludic interconnect TEE . A new channel was drilled to make a cross shape for the outlet channel and a piece of tube was glued to connect the channel using Araldite glue. This is shown schematically in 33.13.1.1The electrochemical response of MWCNT-epoxy composite pipette tip to 1 mM GMP and 1 mM AMP using differential pulse voltammetry (DPV) is shown in 19 and is the reason why Oliveira-Brett's group modified the glassy carbon electrode with DNA followed by conditioning with ssDNA.As seen in 19Surprisingly, stable oxidation currents were observed within a few DPV scans at the bare MWCNT-epoxy composite electrode for both AMP and GMP solutions. These DPV voltammograms show prominent oxidation peaks at +1.0 V for GMP  and at +30 There is much in the recent literature on the advantages of graphene, boron-doped diamond and carbon nanotube-based electrodes including their fast electrochemistry, chemical stability and resistance to fouling.31 Indeed we have recently built an ultra-sensitive flow-cell for dopamine detection based on metallic single-walled carbon nanotubes.32 Our choice of a MWCNT-epoxy composite electrode was a compromise between excellent electrochemical performance and ease of fabrication into an electrode small enough to be incorporated into a robust microfluidic cell.The MWCNT-epoxy composite electrodes gave more stable oxidation peaks than at GCEs, probably because composite electrodes provide improved mass transfer and enhanced electrochemical behaviour.3.1.2As bare MWCNT-epoxy composite electrodes showed such stable electrochemistry for the free nucleotides we investigated the effect of different carboplatin concentrations directly on the signals of free GMP and AMP. The results are shown in Kd, which is the dissociation constant. A reversible binding equilibrium is shown in eqn (1). In this case, the binding reaction of carboplatin (CP) to AMP is shown below:To model this behaviour we can consider Kd is given by eqn (2).For the above reaction, By balancing the mass of AMP:free by [AMP]0 – [AMP·CP], the equation for this case is given in eqn (4)Substituting [AMP]0 – [AMP]free term corresponds to the loss of peak height (reduction of oxidation current). The fitted lines for the GMP and AMP results are shown in Kd for carboplatin interacting with AMP was 124.9 ± 17.3 μM. For GMP, saturation was not reached with the carboplatin concentrations used, so it is more difficult to accurately determine Kd. Nevertheless we can estimate Kd of GMP to be approximately 398 ± 20 μM. It is quite useful here to plot the data on a semi-log graph to give the curves shown in Kd is when log[carboplatin] is a half. The data in Kd (lower affinity for carboplatin) compared to AMP. Broadly similar results were found by Oliveira-Brett et al., although they found the response saturated at 80 μM carboplatin in the same concentration of AMP using a DNA-modified GCE.17This [AMP]We chose AMP for the on-line experiments mainly due to its greater sensitivity in the 0–100 μM range . If carb3.2To realise the on-line analysis capabilities required for the schematic shown in Whilst the DNA-base electrochemistry is stable  and addi–1 using a reaction coil before feeding into the analysis chip. In Kd. We can clearly resolve 10 μM carboplatin, and based on the SD of the AMP oxidation peaks we estimate the limit of detection is 0.49 μM.To carry out initial testing on the board, AMP was supplied to the analysis chip directly from a syringe pump (a*). Carboplatin dissolved in T1 solution at any concentration between zero and the syringe concentration of 160 μM is provided by the careful variation of flow rates of syringes and control of valves shown in b*. The two solutions mixed at point ** and were allowed to react for 2 min at 2 μl min3.3The microfluidic system was coupled with an MD probe in order to validate the capability of detecting carboplatin physiologically during cancer chemotherapy. In terms of the schematic shown in –1 flow rate using a portable syringe pump . The T1 solution from the syringe pump flowed continuously during the experiment in order to allow carboplatin molecules to diffuse through the membrane and to travel in the liquid outlet stream towards the analysis system. The dialysate interacted with AMP in the microfluidic system and flowed towards the analysis chip for detection at the WE.A photograph of the complete system is shown in For the dialysate analysis, DPV voltammograms were obtained when the MD probe was placed in a well-stirred beaker containing T1 solution. Aliquots of 1 mM carboplatin standard solution were added to the beaker to give final concentrations of 0, 10, 20 and 25 μM.The dotted line in 4in vivo carboplatin cytotoxicity measurements. This fabricated system has potential for development of a clinical device for the detection of carboplatin in real time during chemotherapy.The fabricated microfluidic platform coupled with an MD probe was successfully used to detect the interaction of carboplatin and AMP on-line. A decrease in the oxidation current of AMP was clearly observed when the dialysate containing carboplatin interacted with the AMP solution on the microfluidic board. We have chosen AMP as it shows greatest sensitivity to carboplatin. The detection limit of our device is in the appropriate range for TP carried out the experiments, wrote and revised the manuscript, CLL assisted with experiment and revised the manuscript, SG assisted with experiments and revised the manuscript, BAP assisted with experiments and revised the manuscript, MGB directed the experiments, wrote and revised the manuscript.