We found that, in MPAL patients, there were high proportions of blast cells in bone marrow and incidence of hepatosplenomegaly, lymphadenopathy, and Philadelphia chromosome. The overall survival (OS) and relapse-free survival (RFS) in MPAL patients were significantly shorter than those in Ly+AML and Ly−AML. With regard to the patients with normal karyotype only, the OS and RFS of MPAL were significantly lower than those of the Ly+AML and Ly−AML; but there were no significant differences in OS and RFS among the patients with complex karyotype. The OS rates of 3 groups with complex karyotype were lower than those of patients with normal karyotype. In Cox multivariate analysis, complex karyotype was an independent pejorative factor for both OS and RFS. Therefore, MPAL is confirmed to be a poor-risk disease while Ly+AML does not impact prognosis. Complex karyotype is an unfavorable prognosis factor in AML patients with different immunophenotype. Mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.Mixed phenotype acute leukemia (MPAL) is a complex entity expressing both lymphoid and myeloid immunophenotyping. In the present study, 47 MPAL, 60 lymphoid antigen-positive acute myeloid leukemia (Ly However, close to 2 to 5% of cases , 2, the +AML patients have led to inconsistent conclusions and there is lack of reports comparing clinic characteristic and outcome in MPAL with Ly+AML. As the result, the therapy for this subtype of leukemia is not consistent. The goal of present study was to analyze the molecular genetic, cytogenetic, and immunophenotypic features of patients with MPAL according to WHO 2008 classification and the relationship between clinic outcomes and laboratory characteristics.Hitherto, although lack of the MPAL cases research according to the WHO 2008 classification was reported, it is well known that MPAL appears to be a complex entity with different biological characters  and low +AML), and 90 were with acute myeloid leukemia with common myeloid immunophenotype (Ly−AML). They were matched based on the subtypes, gender, and age (α-positive were excluded. Cases were classified by morphology and simultaneously by cytochemistry for myeloperoxidase (POX), sodium fluoride sensitive naphthalene acetic acid esterase (NAE/NAF), and glycogen (PAS). Study protocol underwent thorough review and approval process at the hospital's ethics committee. Informed consent was obtained from all patients included in the study.A total of 197 patients with acute myeloid leukemia diagnosed according to WHO 2008 criteria [ and age . AML aftFCM immunophenotyping was performed on isolated bone marrow mononuclear cells by flow cytometry after collection according to standard procedure. All cases were profiled by a panel of leukocyte-associated markers, including cMPO, CD117, CD13, CD33, CD14, CD15, CD64, and CD11b/c for the myeloid lineage; cyCD3, CD2, CD5, CD7, and CD8 for T-cell lineage, cyCD79a, CD22, CD19, CD10, CD22, and CD20 for B-cell linage; and CD34 and HLA-DR for the stem/progenitor cell clone. A marker was considered positive when more than 20% of the blasts showed a positive signal.β/MYHll, BCR/ABL, and MLL rearrangements as well as P53, Del(5q), Del(7q), Del(20q), and +8 were performed according to manufacturer's instructions.Conventional cytogenetic analysis was carried out on direct preparations or 24 h unstimulated culture of bone marrow cells according to standard technique of G-banding. Meanwhile, the abnormal cloning was defined based on the criteria from International System for Cytogenetic Nomenclature (ISCN). FISH studies aimed at detecting AML1/ETO, PML/RARa, CBFPatients who received at least 2 circles of chemotherapy were involved in current study in order to analyze clinic outcome. All patients received induction and maintenance treatment according to guidelines set by the Hematological Society of the Chinese Medical Association. The induction treatments were as follows: ① combined AML/ALL regimens (DOALP and DOAP), ② ALL-type induction therapy (VDLP and VDCP), and ③ AML-type induction therapy (DA/DAE/HA/TA). Meanwhile, they received postremission therapy as follows: ④ 3 circles of consolidation therapy and less than 3 circles of MD-Ara-C/HD-MTX, ⑤ equal to or more than 3 circles of MD-Ara-C/HD-MTX or more than 3 circles of Hyper-CVAD-A/B or autotransplantation after 5 median circles of standard-dose induction therapy, which ranges from 3 to 10 circles, and ⑥ allogeneic hematopoietic stem cell transplantation after 3 to 6 circles of consolidation or intensive therapy. The distribution of cases of induction regimen and consolidation therapy was summarized in χ2 tests were used for the analysis of qualitative data. All analyses were done with SPSS version 17.0, and P value of less than 0.05 was considered to be associated with statistical significance.The two independent samples nonparametric tests were used for comparison of quantitative data. Kaplan-Meier method was used to estimate the long survival of patients. Cox univariate and multivariate analyses were performed to identify risk factors related to OS and RFS. The Pearson +AML or Ly−AML patients, MPAL patients displayed a higher ratio of lymphadenopathy and hepatosplenomegaly (63.8%) and higher percentage of blasts in bone marrow (67.6%). However, there were no statistical differences between the two groups of Ly+AML and Ly−AML.Patient characteristics were summarized in +AML (81.7%) subtypes was significantly higher than in Ly−AML group (47.8%). On the contrary, a marker of differentiated cells, CD117, was significantly lower in MPAL cases (8.5%) than Ly+AML (61.7%) and Ly−AML (51.1%).Immunophenotype data showed that the positive incidence of CD34 in MPAL (76.6%) and Ly+AML  and Ly−AML . However, it seemed that MPAL patients had the lowest incidence of normal karyotype (27.0%) in the three groups (P = 0.003), and there were no statistical differences between the two groups of Ly+AML and Ly−AML. Philadelphia chromosome and fused BCR-ABL gene incidence were highest in MPAL group of patients (15 out of 37 MPAL patients with available karyotypic data (40.5%) had complex karyotype. Complex karyotype that frequently appeared in MPAL was observed in lower incidence in Lypatients . Fluores+AML, and 79 Ly−AML patients according to WHO 2008 criteria were shown in P = 0.022 and P = 0.014). By applying the results of paired comparison, we found that the OS in MPAL was significantly shorter than that in Ly+AML (P = 0.046) and Ly−AML patients (P = 0.006). Meanwhile, the RFS in MPAL was also significantly shorter than that in Ly+AML (P = 0.019) and Ly−AML group (P = 0.007). However, the OS and RFS were not significantly different between Ly+AML and Ly−AML groups  and relapse-free survival (RFS) rates for 35 MPAL, 55 LyL groups .+AML and Ly−AML groups with normal karyotype, complete remission (CR) rates were 88.5% (23/26) and 80.4% (37/46) after a second induction regimen, which was higher than the cases with complex karyotype (P = 0.013 and P = 0.029). However, in MPAL group, there was no difference in CR rate between normal karyotype and complex karyotype patients . Meanwhile, the overall final complete remission rate after two cycles of chemotherapy of AML patients with different immunophenotype  had no statistical differences in the same genetic background. Details were shown in In Ly+AML (29 months) and Ly−AML (46 months) patients. There was significant difference in overall and relapse-free survival rates among MPAL, Ly+AML, and Ly−AML patients with normal karyotype (P = 0.002 and P = 0.004). By applying the results of paired comparison, the OS rate of MPAL patients was significantly lower than that of the Ly+AML (P = 0.008) and Ly−AML (P = 0.001), but no statistical differences were found between Ly+AML and Ly−AML patients. The RFS in MPAL with normal karyotype was significantly lower than that in the Ly+AML (P = 0.02) and Ly−AML (P = 0.002), but there was no statistical difference between the latter two groups, as shown in + chromosome or BCR/ABL fusion gene), there were no statistical differences among the 3 groups in OS and RFS. The median survival time of patients with complex karyotype in three groups was 7.5 months in MPAL, 9.4 months in Ly+AML, and 15 months in Ly−AML, respectively  compared with Ly months c+AML, and Ly−AML patients with either normal or complex karyotype were shown, respectively, in Figures +AML group, the RFS rates of MPAL and Ly−AML patients with complex karyotype were lower than those in patients with normal karyotype (P = 0.05 and P = 0.04). There were statistical differences of OS between MPAL patients with normal and complex karyotype (P = 0.005). Meanwhile, MPAL patients with normal karyotype had distinctly longer median survival time than those with complex karyotype (median 18 months versus 6 months). Furthermore, statistical differences were also found in the Ly+AML and Ly−AML patients between normal and complex karyotype (P = 0.027 and P = 0.038). In the two groups (Ly+AML and Ly−AML), median survival time was 37.4 and 46 months for patients with normal karyotype versus 7.3 and 15 months for patients with complex karyotype  and complex karyotype (P < 0.01) in univariate analysis and complex karyotype (P < 0.01) in multivariate analysis were also poor prognostic factors on RFS  to attempt identifying risk factors relating to OS. Age, peripheral white blood cells, MPAL, complex karyotype, and abnormal karyotype were shown to display adverse prognostic significance on OS . HoweverS Tables  and 5. SAML is a genetically heterogeneous disease with widely different treatment outcomes and disparate survivals. Thus, it is important to propose prognostic model effectively for subtype of AML subtypes diagnosis and evaluations of outcome. Now, the most important prognostic factors for AML are age and chromosome karyotype , 10. How+AML and Ly−AML groups. And, in the current study, the median age of 34 years at diagnosis and a slight predominance of the male population (63.8%) is similar to that reported by Yan et al. [According to the WHO 2008 classification of leukemia  and the n et al. . As repon et al.  and Bachn et al. , we also−AML, and Ly+AML. However, there were no significant differences in OS and RFS between Ly−AML and Ly+AML patients. These results demonstrated that MPAL may be a poor prognostic indicator in AML. However, it needed to be taken with caution because of the high incidence of complex karyotype in MPAL patients included. Therefore, all patients with normal karyotype were considered only, and it was found that the survival of MPAL patients was also worse than that in Ly+AML and Ly−AML. Meanwhile, factors relating to survival were evaluated with the Cox proportional-hazards regression model. As a result, in multivariate analysis, only patients with complex karyotype predicted a shorter OS and RFS.The previous studies showed that survival in biphenotypic acute leukemia was shorter than in other acute leukemia patients whatever adults or children , 15–17. +AML and Ly−AML groups. These findings demonstrated that complex karyotype was strongly associated with adverse outcome in AML, which was in harmony with several early studies [+AML and Ly−AML patients with complex karyotype present similar survival time with MPAL? A possible explanation could be that complex karyotype has a more significant impact on clinical outcome in AML patients with no matter which subtype of immunophenotype. Several studies also demonstrated that complex karyotype is a poor prognosis factor [Notably, within the MPAL group, patients with complex karyotype had shorter survival compared with normal karyotype patients. Similar phenomena were also found on OS in Ly studies , 19. It s factor , 19, and+AML and Ly−AML, which was consistent with those of Casasnovas et al. [+AML patients.In multivariate analysis for OS, in addition to complex karyotype, peripheral white blood cells (WBC) were also found as risk factor as it was also established in report by Mikulic et al. . Howevers et al. . To our +AML does not impact prognosis. Complex karyotype is an unfavorable prognostic factor in AML patients with different immunophenotype. Moreover, mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.In conclusion, MPAL is confirmed to be a poor-risk acute leukemia subtype with normal chromosome karyotype, while Ly


Ketamine is an anesthetic with antidepressant properties. The rapid and lasting effect of ketamine observed in preclinical and clinical research makes it a promising therapeutic to improve current major depression (MD) treatment. Our work intended to evaluate whether the combined use of classic antidepressants (imipramine or fluoxetine) and ketamine would improve the antidepressant response. Using an animal model of depressive-like behavior, we show that the addition of ketamine to antidepressants anticipates the behavioral response and accelerates the neuroplastic events when compared with the use of antidepressants alone. In conclusion, our results suggest the need for a reappraisal of the current pharmacological treatment of MD. Therefore, herein we assessed whether the use of an acute KET treatment could improve the efficacy and/or anticipate the clinical effect of both tricyclic antidepressants and selective serotonin reuptake inhibitor agents in a rat model of depression.Currently used antidepressants target the metabolism/transport of monoaminergic neurotransmitters, such as serotonine and noradrenaline. However studies show that glutamate (GLU) deregulation is also implicated in MD.n=12 per group; Male Wistar rats , aged 3 months were housed (three per cage) under standard laboratory conditions . Eighty-four animals were used. Animals initially performed the determination of the baseline value of sucrose consumption test (SCT) and then were randomly assigned to the following experimental groups—a control group without stress exposure and six groups exposed to unpredictable chronic mild stress (uCMS). After 4 weeks used to establish a depressive-like behavior phenotype, uCMS-exposed animals were randomly assigned to six different groups: one untreated group (i) uCMS, and the other five experimental groups according to the assigned treatment: (ii) imipramine, (iii) fluoxetine, (iv) KET alone for the first 3 days and saline for the remainder period (uCMS-KET), or treated with KET daily for 3 days and either (v) FLX (KET-FLX) or (vi) IMP (KET-IMP) for the remaining treatment period  for 3 h, housing on damp bedding for 8 h, overnight illumination, 18 h food deprivation followed by exposure to inaccessible food for 1 h, water deprivation for 18 h followed by exposure to an empty bottle for 1 h, and reversed light/dark cycle for 48 h every 7 days) until establishment of the model phenotype and then through the duration of behavioral evaluation.A modified version of an uCMS protocol−1, Kemprotec, Cumbria, UK), IMP  and KET  with 0.9% saline as vehicle for 14 days and then performed behavioral evaluation. Treatment and administration scheme were chosen on the basis of their therapeutic effects in previous studies.19After establishment of depressive-like behavior, uCMS animals were treated according to the assigned group with daily intraperitoneal injections of saline, FLX .All animals (controls and uCMS-exposed) were submitted to a series of behavioral testing. Sucrose preference tests were performed weekly over the 6 weeks of exposure to uCMS. The remainder tests  were performed after 14 days of treatment .8 Anhedonia was defined as a reduction in sucrose preference.Anhedonia was assessed weekly during exposure to uCMS using the SCT. Briefly, animals were allowed to habituate to the sucrose solution for 1 week before the uCMS protocol to establish baseline preference levels. To test sucrose preference, animals that were food- and water-deprived for 18 h were presented with two pre-weighed bottles containing 1% sucrose solution or tap water for a period of 1 h. Sucrose preference was calculated according to the formula: sucrose preference=(sucrose intake/(sucrose intake+water intake)) × 100, as previously described.20 Animals were placed in the central junction facing an open arm, and allowed to explore for 5 min. Entry was defined as all four paws being positioned within one arm. The test was recorded and the ratio between time in open arms and time in closed arms was measured. Activity in the open arms was calculated as open arm entries percentage  and time spent in open arm percentage . The degree of anxiety was indirectly related to the time spent in the open arms and the number of open arm entries.To assess anxiety-like behavior, animals were tested on an elevated-plus maze  as previously described.Learned helplessness was evaluated in the forced-swim test on the last day of exposure to uCMS. Twenty-four hours after a pre-test session (10 min), rats were placed in cylinders filled with water  for a period of 5 min. Test sessions were assessed using a camera connected to a video tracking system ; the system automatically calculated immobility time and latency to immobility. Learned helplessness behavior was defined as an increase in time of immobility and a decrease in latency to immobility.To assess locomotor activity, rats were placed in an open-field apparatus (43.2 (length) × 43.2 (width) × 30.5 (height) cm, transparent acrylic walls and white floor, Med Associates) in a room illuminated by white light. Instant position was monitored over a period of 5 min by an array of two 16 beam infrared arrays. Total distance and average speed was used as a measure of locomotor activity.An additional setting of animals was used to evaluate KET safety regarding induction of PPI impairment. Startle reflexes were measured in two identical startle response systems , each consisting of a non-restrictive Plexiglas cylinder  cm, 22.2 (length) cm), mounted on a Plexiglas platform and placed in a ventilated, sound-attenuated chamber. Cylinder movements were detected and measured by a piezoelectric element mounted under each cylinder. A dynamic calibration system (San Diego Instruments) was used to ensure comparable startle magnitudes across the two devices. Startle stimuli were presented through a high frequency speaker located 33 cm above the startle chambers. Startle magnitudes were sampled each millisecond (ms) during a period of 200 ms beginning at the onset of the startle stimulus. A startle response is defined as the peak response during this 200-ms period. Animals were habituated to the apparatus 5 min daily 2 days before the testing period. After the habituation period, rats were taken from their home cage and placed in the test chamber. The chamber was then sealed and each animal allowed to acclimatize to the startle chamber for a period of 5 min. In addition, background white noise, with an intensity of 63 dB, was maintained to minimize the impact of acoustic stimuli outside the chamber environment. Following five introductory 120-dB startle trials (noise lasting 40 ms), a total of 35 test trials were pseudo-randomly delivered as follows: (a) five trials with background noise only, (b) 10 startle trials of 120 dB and (c) five pre-pulses of each of four different intensities preceding a startle trial. Pre-pulse intensities of 2, 4, 8 and 16 dB above the background noise level lasted 20 ms and preceded the 120-dB startle presentation in 100 ms. Inter-trial intervals ranged from 10 to 20 s. The average startle response was assessed in the 100-ms period following the onset of the startle stimulus presentation.M Na2S2O5 and 91.3 mM Na2-EDTA , centrifuged at 15 000 r.p.m. for 45 min at 4 °C and the supernatant stored at −80 °C until analysis.Monoamines levels were measured using high performance liquid chromatography (HPLC), combined with electrochemical detection. From each experimental group, seven rats were killed by decapitation. After decapitation, the animal heads were snap-frozen in liquid nitrogen to prevent tissue degradation. The brains were quickly extracted and placed in an icy surface; under a stereomicroscope the prefrontal cortex (PFC), dorsal hippocampus, ventral hippocampus and nucleus accumbens (NAc) were dissected. The dissected tissues were weighed and then homogenized and deproteinized in 100 μl of 0.2 N perchloric acid solution  containing 7.9 m21 The working electrode was glassy carbon, the reference electrode was Ag/AgCl and the columns used were ODS Hypersil, 250 mm × 4.6 mm, 5 μm . The voltage of the working electrode was set at +800 mV in the LC4C amperometric detector and the flow rate of the LC1150 HPLC pump was set at 1.0 ml min−1. The mobile phase consisted of an acetonitrile : 100 mM phosphate buffer (5:95) pH 4.9, containing 50 μM Na2-EDTA (Riedel-de Haën). Samples were initially diluted 1:5 with ddH2O, then further diluted 1:1 with 0.1 M Borax buffer (Sigma-Aldrich), pH 9.6. o-Phthalaldehyde (Sigma-Aldrich) was subsequently added and left to react at room temperature for 10 min before injection. Quantification of GLU and aspartate (ASP) was done by comparison of the area under the curve with that of reference external standards using HPLC software , as previously described.23The analytical measurements were performed using a GBC LC1150  HPLC pump coupled with a BAS LC4C  electrochemical detector and pre-column derivatization as described previously.After the end of behavioral evaluation, five rats from each group were perfused transcardially with saline (NaCl 0.9%) under deep pentobarbital anesthesia. Brains were removed and kept in Golgi-Cox solution for 15 days and then transferred to a 30% sucrose solution for 5 days. Sections (200 μm) were obtained using a vibratome and collected in 6% sucrose and blotted dry onto gelatin-coated microscope slides. They were alkalinized in 18.7% ammonia, developed in Dektol , fixed in Kodak Rapid Fix, dehydrated and xylene-cleared before coverslipping. Dendritic arborization and spine numbers were analyzed in layer II/III of infralimbic (IL) area of PFC, and dentate gyrus and CA3 region of hippocampus. Selected neurons had every branch of the dendritic tree reconstructed at × 1000 (oil) magnification using a motorized microscope  and Neurolucida software  and three-dimensional analysis of the reconstructed neurons was performed using NeuroExplorer software (Microbrightfield). For each animal, 20 neurons were studied and measurements from individual neurons from each animal were averaged. The following dendritic morphology parameters were examined: dendritic length and the number of primary dendrites and dendritic branching points were compared across experimental groups; dendritic spines were assessed according to their morphology mushroom-shaped, thin, thick and ramified spines and dendritic spine density (number of spines/dendritic length) and the proportion of spines in each category was calculated for each neuron in branches that were either parallel or at acute angles to the coronal surface of the section. In dentate granule cells, proximal and distal branches were analyzed for each neuron; basal branches and proximal and distal apical branches in pyramidal neurons in the CA3 region and IL area of the PFC were analyzed. Neuronal reconstructions were masked to the observer.f=0.25), a type I error α=0.05 and a statistical power (1—type II error) of 0.8, and also the performance of HPLC and Golgi analysis after behavioral analysis, with the need to establish two diferent groups of animals. In practice, we ended up using a total of 84 animals, with n=12 to each experimental group.Sample size was determined considering a medium effect size . Statistical analysis was performed using IBM SPSS statistics 20.0 .Data obtained in experiment were analyzed applying analysis of variance. Quantitative data obtained for parameters analyzed regarding elevated-plus maze, forced-swim test, open field, PPI, neuronal dendritic architecture and HPLC measurements were analyzed using one-way analysis of variance, and whenever appropriate, P<0.05). On the seventh day of treatment, animals treated with KET (P<0.05), FLX-KET (P<0.05) and IMP-KET (P<0.05) animals , but not the remaining treated groups had reversion of anhedonic phenotype (P<0.01) and FLX (P<0.05), IMP-KET (P<0.001) and FLX-KET (P<0.001)), except for those given only KET that did not display any longer the behavioral rescuing effect detected 1 week before  and increased immobility time  when compared with control animals. Comparing with uCMS, all treated animals showed a significantly increased latency to immobility: FLX, KET (P<0.05), IMP (P<0.01) and IMP-KET, FLX-KET  and IMP-KET, FLX-KET ; both IMP-KET (P<0.05) and FLX-KET (P<0.01) groups showed a significant increase in time spent in open arms when compared with uCMS animals; no other significant differences were found  and both FLX (P<0.01) and IMP-KET, FLX-KET (both P<0.001) also have significantly higher entries in open arms . Treatment with KET, in the dosage used in our experimental protocol, failed to show PPI disruption (data not shown).In MD, therapy is still largely based on the ‘monoaminergic hypothesis', which is fundamentally associated with alterations in the level of neurotransmitters. According to this and as an attempt to investigate the possible mechanism underlying the effect of giving KET together with antidepressants, the levels of neurotransmitters, more specifically GLU and ASP were analyzed by HPLC. For that, important key areas for MD such as PFC and hippocampus were selected. In addition, we have investigated the levels of these neurotransmitters in additional brain areas, which are related to the behavioral profile of recovery, such as the NAc. .6,55=2.254, P<0.01). Treatment with antidepressants produced an increase in GLU levels, but only IMP-KET and FLX-KET displayed a significant increase in GLU levels compared with untreated animals ; post hoc analysis showed control animals have higher ASP than uCMS untreated animals  and that uCMS-exposed animals treated with IMP-KET and FLX-KET have significantly higher ASP levels than untreated uCMS animals . In basal dendrites, treatment with IMP-KET (P<0.01), FLX (P<0.05) and FLX-KET (P<0.001) induced a reversion of dendritic shortening. In the apical dendrites, all treated animals (P<0.05) had a significant recovery , which was recovered by the treatment with IMP-KET (P<0.001), IMP and FLX-KET (both P<0.05). Animals treated with KET alone and FLX alone did not show significant changes in dendritic length  and apical  dendrites show a significant decrease in their length in animals that were exposed to uCMS. Treatment with KET (P<0.001), IMP-KET (P<0.001) and FLX-KET (P<0.001) recovered basal dendritic length, whereas FLX-KET was able to induce a significant increase in basal dendritic length compared with FLX alone (P<0.01). Neither IMP nor FLX alone increased CA3 basal dendritic length significantly when compared with uCMS animals. Regarding the apical dendritic tree, treatment with KET (P<0.01), IMP-KET (P<0.01), FLX alone (P<0.01) and FLX-KET (P<0.01) induced recovery, whereas treatment with IMP alone failed to increase CA3 apical dendritic length significantly. Both IMP-KET and FLX-KET promoted a higher regrowth than IMP (P>0.001) and FLX , and this feature was reversed within all treatment groups  and apical  IL dendrites of uCMS animals. Treatments with KET (P<0.001), IMP (P<0.001), IMP-KET (P<0.001), FLX (P<0.001) and FLX-KET (P<0.001) increased SD in IL basal dendrites; FLX-KET significantly increased SD in basal dendrites when compared with FLX alone . Treatment with FLX (P<0.001) and FLX-KET (P<0.001) increased SD in apical dendrites. No significant increase of SD in apical dendrites was found in animals treated with KET or IMP alone and in animals treated with IMP-KET. Regarding spine morphology, mushroom spines are decreased in uCMS animals both in apical  and basal  dendrites when compared with control animals. Treatment with KET (P<0.001), IMP-KET (P<0.001) and FLX-KET (P<0.001) reverted this pattern in basal and apical IL dendrites. No significant increase in mushroom spine population was found in animals treated with IMP or FLX alone; although the treatment with IMP-KET and FLX-KET caused a significant rise in mushroom spine population in apical and basal dendrites when compared with IMP (P<0.05) and FLX (P<0.01) alone (A significant decrease in spine density (SD) was found in basal (F1) alone .6,28=7.613, P<0.05) when compared with control animals. Treatment with FLX (P<0.01) and FLX-KET (P<0.01) significantly increased SD. There was no significant increase in SD in animals treated with KET or IMP alone or with IMP-KET. When evaluating the spine morphology, specifically the more developed mushroom spines, uCMS animals present also less number of mushroom dendritic spines in dentate gyrus neurons   but treatment with KET , IMP , IMP-KET , FLX  and FLX-KET  restored mushroom spines numbers  and basal  dendrites, which were recovered upon treatment with all the tested drugs . Addition of KET to FLX treatment statistically increases spine density  in apical dendritic tree. The population of mushroom dendritic spines is decreased in untreated uCMS animals both in apical  and basal  CA3 dendrites. In apical dendrites, treatment with KET (P<0.001), IMP (P<0.05), IMP-KET (P<0.001) and FLX-KET (P<0.001) caused an increase in mushroom spine population. Both IMP-KET (P<0.01) and FLX-KET (P<0.001) induce higher mushroom dendritic spine population. In basal dendrites, treatment with KET (P<0.01), IMP-KET (P<0.01) and FLX-KET (P<0.01) increased the amount of mushroom spines  and this feature was reversed in the treatment with IMP, IMP-KET, FLX-KET (P<0.01) and FLX (P<0.05). Regarding spine morphology, uCMS promoted a decrease in the population of mushroom spines  and all treatments significantly increase mushroom spine compared with the untreated uCMS animals (P<0.001). Importantly, the animals treated with antidepressant and ketamine (IMP-KET and FLX-KET) presented a significant increase of mushroom spine population when compared with those who were only given antidepressant .The use of KET in subanesthetic dose with antidepressant effect is described both in animal models of stress and in MD patients.The SCT was used as a behavioral hallmark for recovery from depressed phenotype. The first evaluation occurred at the seventh day of treatment and indicated that only animals treated with KET  display behavioral rescue of anhedonia. After one additional week of treatment, at the 14th day, all treated groups show reversion of anhedonia, except the one only treated with KET, which showed a reversion of the positive behavioral effect. In our results, we can see the specific effect of the interaction of KET added to antidepressant treatment on the results of the treated group KET-FLX. The effect of KET alone in anhedonia does not last until the 14th day, but when given with FLX, it enhances it.The second hallmark of this model is learned helplessness, a measure of behavior despair. There was an increase in latency to immobility time in all treated animals but the analysis of total immobility time shows that the effect of KET alone is not detected 2 weeks after being administrated. Again, when KET is given with FLX, it is evident that there is an increase in latency time compared with the antidepressant alone.The analysis of anhedonia and despair in our model showed that combining KET with other treatments seems to anticipate the increase in sucrose preference and to improve the response. KET possibly is acting as an initial booster and keeps the antidepressant response. However, when given alone it failed to show a robust result.27 and in animal models when given in multiple doses.28 However, we observed a positive effect when given with antidepressants. When given together with the antidepressants, beneficial effect of KET extends beyond the mood domain and also potentiates the anxiolytic effects of SSRIs and tricyclic antidepressant drugs.We also assessed anxiety and show that the combination of KET with FLX or IMP was able to significantly reverse the anxiety trait. Again, there seems to be no anxiolytic effect after 2 weeks of KET given alone. Some literature reports long-lasting anxyolitic effect of KET given in a single dose to patients with depressive disordersThe positive effect of initial dual therapy with KET and other antidepressant, concerning behavioral recovery in our animal model, is a faster initial increase in sucrose preference and a more robust response after 2 weeks of treatment not only in anhedonia but also in behavioral despair and anxiety.30 as a result of dysfunction occurring by several pathways. To evaluate the impact of KET in neurotransmission, we have analyzed the levels of both GLU and also we evaluated the dendritic architecture. We assessed the levels of GLU in regions relevant for depressive-like behavior. Our major finding was that NAc, a brain region implicated in anhedonia,31 was the only region where KET, in association with other antidepressants, triggered an increase in the levels of GLU and ASP suggesting that this particular effect is ascribed to GLU metabolism. The failure to observe significant changes in GLU or ASP levels in other brain regions probably relates to temporal dynamics of the experimental design. However, literature reports that both acute stressors and chronic stressors increase basal release and increased presynaptic reuptake of GLU, but most measurements were performed using mycrodialysis or synaptossomal analysis, thus reflecting extracellular contents, not the overall content of GLU. There is evidence that KET acts as a noncompetitive and nonselective high-affinity NMDA antagonist also on GABAergic neurons and may rapidly increase GLU release, for example, in the PFC.33 Although there is clinical evidence that treatment for depression increases the release of GLU in the NAc,34 other studies using KET in individuals with MD were not consistent in showing an association between therapy and changes in neurotransmitter content.36 Present data suggest changes in glutamatergic transmission, particularly in the NAc, a mechanism through which KET helps restore mood.MD disorder and stress disorders have been associated with impaired functional connectivity,37 Globally, groups treated with antidepressant and KET showed a recovery of the morphology of their dendritic trees, with a more consistent response in FLX than IMP. Yet, the most striking changes were noticed in dendritic spines; we not only found that the addition of KET to the antidepressant treatment boosted a significant recovery in spine density when compared with antidepressants alone, but also with greater spine maturation. KET is known to have effects in synaptic plasticity: the fast-acting antidepressant action of KET is known to be dependent on rapid protein synthesis  and activation of intracellular pathways such as mTOR, all of them physiologically relevant for synaptic plasticity mechanisms, neuronal growth, differentiation and synaptogenesis.41 Although the specific role of mTOR in NAc is not fully understood in depression, the effect of mTOR modulation is well studied in addiction. Therefore, we can speculate the existence of some similarity between addiction development and in antidepressant action. Indeed, the use of rapamycin not only blocks establishment of addiction, but also the antidepressant effect of KET.42 The acute and lasting effect of KET in neuronal remodeling might be one of the reasons why it improves behavioral markers of recovery in our experiment. The particular case of combined treatment in increasing more mature dendritic spine population shows that processes related to synaptic functioning and rewiring have an important role in behavioral recovery.Through the analysis of neuronal architecture, we confirmed the impact of uCMS on shrinkage of dendritic arborizations in the hippocampus and in the IL area of the PFC, and dendritic hypertrophy in NAc medium spiny neurons.In summary, the present data show that KET has the potential to improve the action of classic antidepressants in brain regions affected by uCMS, a validated animal model of depression. The addition of KET to antidepressants accelerate recovery from anhedonia. The specific addition to FLX improves not only anhedonia but also behavioral despair. Despite lack of robustness in neurochemical data, we show improvement of glutamatergic profile in an area related to anhedonic behavior. The effects in neuronal morphology show a potential mechanism for improvement in synaptic connectivity in brain areas known to be affected and responsible for behavioral hallmarks in MD. These results show that KET has potential to improve the initial response of MD treatment.


P≤0.0001). TgAb  and TSH ≥1 μIU/ml (OR (95% CI)) OR: 1.49  were significant predictors of TC in multivariate analysis controlling for age and gender. TC was not associated with serum concentrations of TPOAb. In patients with TN, elevated serum concentration of TgAb and TSH ≥1 μIU/ml are independent predictors for TC. The association between HT and TC is antibody specific.This prospective study investigates the relationship between Hashimoto's thyroiditis (HT) and thyroid cancer (TC) in patients with thyroid nodules (TNs). We prospectively examined 2100 patients with 2753 TNs between January 5, 2010 and August 15, 2013. A total of 2023 patients with 2669 TNs met the inclusion criteria of TN ≥5 mm and age ≥18 years. Each patient had blood drawn before fine-needle aspiration biopsy (FNAB) for the following measurements: TSH, free thyroxine, free tri-iodothyronine, thyroid peroxidase antibody (TPOAb), and antithyroglobulin antibody (TgAb). Diagnosis of TC was based on pathology analysis of thyroidectomy tissue. The associations of TC with the independent variables were determined by univariate and multivariate logistic regression analysis and reported as adjusted odds ratio (OR) with 95% CI. A total of 248 malignant nodules were found in 233 patients. There was an association of TC with both increased serum TgAb concentration and age<45 years. An elevated serum TgAb concentration was found in 10.2% of patients (182 of 1790) with benign nodules as compared with 20.6% of patients (48 of 233) with malignant nodules ( Hashimoto's thyroiditis (HT) is the most common autoimmune thyroid disease and the most common cause of hypothyroidism  was first proposed by Heliobacter pylori-induced chronic gastritis and gastric carcinoma; iv) asbestosis and mesothelioma; v) chronic obstructive pulmonary disease and lung cancer; and vi) scleroderma and carcinoma of the breast and lung (A relationship between chronic inflammation and cancer was first proposed by Virchow (1863) and has been sustained by clinical and epidemiological evidence. The most compelling evidence is the association between: i) intestinal chronic inflammatory diseases (Crohn's disease and ulcerative rectocolitis) and adenocarcinoma of the colon; ii) chronic HBV or HCV hepatitis and liver carcinoma; iii) Two recent retrospective studies have reported a positive association between HT and TC in patients presenting with thyroid nodules (TNs). In these two studies, antithyroglobulin antibody (TgAb), but not thyroid peroxidase antibody (TPOAb), was an independent risk factor for TC .Based on these previous retrospective studies, we hypothesized that the association of HT with TC is antibody specific. Since a retrospective study's design may be subject to ascertainment bias and assay heterogeneity, we chose to further investigate the relationship between HT and TC by prospectively examining this relationship in TN patients using a multivariate approach to account for the possible influence of other variables including the levels of thyroid-stimulating hormone (TSH).The inclusion criteria were the presence of a single or multiple TNs ≥5 mm and age ≥18 years.We prospectively evaluated 2100 patients with 2753 TNs from January 5, 2010 to August 15, 2013. All TNs were evaluated with a high-resolution ultrasound (US), fine-needle aspiration biopsy (FNAB), and, when indicated, by tissue pathology. All patients were evaluated by a single endocrinologist (G A) with more than 15 years of experience in thyroid US and ultrasound-guided FNAB.The study protocol was approved by the Institutional Review Board of the New Hanover Regional Medical Center, Wilmington, NC. All patients gave written informed consent.At the time of initial US examination, the following factors were ascertained in all study subjects: gender, age, and number of US-determined TN. Treatment with any TSH-altering medication was documented. All patients had their blood drawn before FNAB for the following measurements: TSH, free thyroxine, free tri-iodothyronine, TPOAb, and TgAb. All measurements were performed by LabCorp . Both TPOAb and TgAb levels were measured using Immulite 2000 technology by Siemens Medical Solutions Diagnostics  utilizing purified antigen (TPO) immobilized to insoluble beads in a solid phase, enzyme-labeled chemiluminescent sequential immunometric assay. The reference range for TPOAb and TgAb levels was 0–34 and 0–40 IU/ml respectively. The analytical sensitivity for TPOAb and TgAb levels was 5 and 2.2 IU/ml respectively. Serum TSH level was measured at LabCorp by an electrochemiluminescence immunoassay developed by Roche Diagnostic. The reference range for TSH was 0.45–4.5 μIU/ml. Hypothyroidism is defined as TSH >4.5 μIU/ml without thyroid medication and in patients with a known history of hypothyroidism on thyroid hormone replacement. Hyperthyroidism is defined as TSH<0.45 μIU/ml without medication and in patients with prior history of hyperthyroidism on antithyroid medication.Before the FNAB, we obtained written informed consent for the procedure. The FNAB was performed under sterile conditions with US guidance to confirm accurate needle placement. Three passes were made of each lesion using 27-gauge needles. If there was no sample on visual inspection, we then used a larger needle (25-gauge). Onsite adequacy was not performed in this study. The samples were submitted for cytologic evaluation in accordance with the Bethesda system for reporting thyroid cytopathology, resulting in diagnostic Bethesda categories (BC) I (nondiagnostic or unsatisfactory), II (benign), III (atypia of undetermined significance or follicular lesion of undetermined significance), IV (follicular neoplasm or suspicious for follicular neoplasm), V , or VI  . For patSurgical resection was recommended for patients with FNAB results that were positive for malignancy, highly suspicious for PC or follicular neoplasm, or follicular cells of undetermined significance. In addition, thyroid surgery was also recommended for patients with two non-diagnostic or benign FNABs with two or more US features suggestive of TC  or increased size.s.d. were used to summarize quantitative variables, while frequencies and percentages were used to summarize qualitative data. Bivariate associations with TC were examined using non-parametric Wilcoxon tests for quantitative variables and χ2 tests for categorical variables. Univariate association of independent variables  gland, single nodule gland, thyroid medication, history of head/neck radiation, and family history of TC) on diagnosis of cancer was determined using logistic regression. Multivariable logistic regressions adjusting for age and gender were used to examine associations between TC and each of the variables and serum concentrations of TSH, TgAb, and TPOAb. A probability of <0.05 was considered statistically significant.The statistical analysis was performed by Dr M N Kuchibhatla  using SPSS/SAS program . Means and The remaining 2023 patients with 2669 TNs were included in the statistical analysis. There were 1150 TNs that were <10 mm in greatest diameter, and the remaining 1519 TNs were ≥10 mm. There were 1348 patients who had FNAB of a single TN and 675 patients who had FNAB of two or more TNs.Thyroidectomy was performed on 461 patients. Of these, 374 had abnormal FNAB  and 25 had non-diagnostic FNAB (BC I). 62 patients with benign FNAB (BC II) had surgery due to worrisome US features or a size large enough to warrant surgery. Of the 461 patients who had surgery, 233 patients with 248 malignant TNs were diagnosed with TC. TC was histologically confirmed in 233 patients: 45 patients had single nodule gland and 188 patients had MNG. Among those patients with MNG, 121 (64%) TCs were in dominant TN and 67 (36%) of TC were in non-dominant TN.Of the 248 malignant TNs, 149 were PC, 83 were follicular variant of PC, four were medullary carcinoma, and 12 were follicular carcinoma. From the 248 malignant TNs, 104 were <10 mm diameter in size and 144 were ≥10 mm diameter in size. There were 44 follicular adenomas on surgical pathology in this study.P=0.0007). The malignancy rate among males was higher than females, but did not reach statistical significance most likely due to high number of female patients (84%) in this study.As expected, young age was a risk factor for TC (n=48) of the TC patients as compared with 10.17% (n=182) of the patients with benign nodule (P<0.0001). In contrast, TC was not associated with TPOAb concentration. Serum TPOAb concentration was elevated in 31.33% (n=73) of the TC patients and 26.7% (n=478) of the benign nodule patients (P=0.1356).There is an association between TC and TgAb. Serum TgAb concentration was elevated above the normal range in 20.6% ): OR: 2.24  was a significant predictor of cancer while TPOAb (OR (95% CI)): OR: 1.19  was not . In the Our prospective study of patients with TN demonstrates that an elevated serum TgAb concentration is an independent predictor for TC. Serum TPOAb concentration was not a predictor of TC in either univariate or multivariate analyses.This prospective study was undertaken to further investigate the risk of thyroid inflammation and TC. It confirms two retrospective studies that suggested this association . For the study's assay, the normal reference range for TgAb concentration was 0 to 40 IU/ml. In the multivariate analysis, we found that patients with detectable but normal TgAb concentrations (>0 to <40 IU/ml) had an (OR (95% CI)): OR: 1.70 . This finding suggests that lowering the cutoff value for TgAb or TPOAb may reduce the false negative misclassifications, as noted by There were 114 patients with TC who had normal thyroid function and normal thyroid antibodies (TPOAb <34 IU/ml and TgAb <40 IU/ml). In this group, 17 patients (15%) had histopathologic changes in HT. This finding may indicate that the prevalence of HT in TC might be higher than expected.A recent review by  The prevalence of TC in this prospective study was 11.5% for patients and 9.2% for TNs, which is within the 5–15% TC risk expected for TNs  that had not been prespecified. As reviewed by The association of TgAb with malignancy in patients with nodule has been found in some studies , but notThe relationship between inflammation and TC is complex and still not completely understood . The mecThe present study has several strengths. The biochemical measurements and patient phenotypic data were all collected prospectively to eliminate selection bias, which is confirmed by the lack of association of surgical intervention with biochemical analysis. All measurements of TSH, TgAb and TPOAb were made at the same laboratory and using the same methodology for each patient to eliminate any interference from variability of different assay methodologies. Finally, only 3.7% of the initial 2100 patients seen were excluded from the study due to previously discussed reasons. This small fraction is unlikely to affect the outcome of this study. Since this is a cross-sectional association study, it does not directly address the mechanism that explains the association of TgAb and TC.In summary, elevated serum TgAb level was associated with an increased risk of TC in patients with TN. In single cutoff analysis, TSH ≥1 μIU/ml is a significant predictor of TC. Serum TPO was not associated with TC. We conclude that the association of HT with TC is antibody specific.


Environmental stimuli repeatedly paired with drugs of abuse can elicit conditioned responses that are thought to promote future drug seeking. We recently showed that healthy volunteers acquired conditioned responses to auditory and visual stimuli after just two pairings with methamphetamine . This study extended these findings by systematically varying the number of drug-stimuli pairings. We expected that more pairings would result in stronger conditioning. Three groups of healthy adults were randomly assigned to receive 1, 2 or 4 pairings  of an auditory-visual stimulus with MA, and another stimulus with placebo (PBO). Drug-cue pairings were administered in an alternating, counterbalanced order, under double-blind conditions, during 4 hr sessions. MA produced prototypic subjective effects  and alterations in physiology . Although subjects did not exhibit increased behavioral preference for, or emotional reactivity to, the MA-paired cue after conditioning, they did exhibit an increase in attentional bias  toward the drug-paired stimulus. Further, subjects who had four pairings reported “liking” the MA-paired cue more than the PBO cue after conditioning. Thus, the number of drug-stimulus pairings, varying from one to four, had only modest effects on the strength of conditioned responses. Further studies investigating the parameters under which drug conditioning occurs will help to identify risk factors for developing drug abuse, and provide new treatment strategies. Environmental cues that are repeatedly paired with drugs acquire the ability to produce conditioned responses through the process of associative learning  and placebo, each with distinctive stimuli. Group P1 received one pairing of MA and placebo , Group P2 received two pairings (4 sessions) and Group P4 received four pairings . The study consisted of four phases: i) a pre-test assessing responses to the cues before conditioning, ii) a conditioning phase consisting of one, two, or four presentations of MA, and placebo paired with distinctive audio/visual stimuli, iii) a post-conditioning test, and iv) three online follow-up tests. The primary outcome measures were four indices of conditioning of the MA-paired stimuli: behavioral preference, attentional bias, emotional reactivity, and ‘liking’ of the stimuli. The primary goal was to determine whether the strength or persistence of conditioned responses was related to the number of pairings. The procedure was based on previous studies in our laboratory , 41, witHealthy volunteers (N = 45) aged 18–35 were recruited from the university and surrounding community using flyers and online advertisements approved by the University of Chicago Institutional Review Board . They weMethamphetamine  dose and dosage procedures were identical to those used previously , 41. MetDuring an initial orientation visit, study procedures were explained to participants and written informed consent was obtained. Participants were told that they could be given a placebo, stimulant, sedative or alcohol in order to minimize expectancy effects. Participants were asked to abstain from recreational drug use for 48 h before any session (7 d for marijuana) as well as 6 h after any session, and to consume their normal amounts of caffeine and nicotine before all sessions. Participants practiced the study tasks and questionnaires.Sessions were held in comfortably furnished rooms that contained a television, VHS player, and computer. At each session, participants first completed drug screening and pregnancy tests, subjective drug effects ratings , and cardiovascular measures (blood pressure [BP] and heart rate [HR]). Compliance was assessed at each session using breathalyzers , urine drug tests , and pregnancy tests for women . When not completing experimental tasks, participants could watch selected movies, read, or relax.This 2-hr session was conducted between 9 am and 5 pm. The primary goal was to assess subjects’ baseline responses toward the two to-be-conditioned visual/auditory cues as in . After iThese 4-hr sessions were conducted from 9 am to 1 pm, 2–10 d apart. Subjective drug effects and cardiovascular measures were taken six times throughout the session . At 9:30 am, the drug (MA) or placebo (PBO) was administered orally under double blind conditions. Subjects received MA and PBO on alternating sessions, with the order (MA or PBO first) counterbalanced between subjects. Thirty min after MA/PBO administration, timed to coincide with peak drug effects, see , particiThe four ‘distractor’ tasks included the Balloon Analogue Risk Taking (BART) task , a simplThis 2-hr session was conducted at the same time of day as session 1, 2–10 d after the last conditioning session, to assess the change in responses to the conditioning stimuli. Subjects completed the same four tasks conducted during the pre-test: forced choice, visual probe, VAS, and EMG.Participants also completed online versions of three study tasks  at 2, 7, and 20 d following the post-test session to assess the persistence of the conditioned responses. Participants were asked to use a personal computer to access the tasks on a website using a web application . Participants were instructed to complete the follow-up tasks in a quiet location with the computer audio turned on. The tasks were presented similarly to the pre- and post-test sessions, e.g., stimulus duration, inter-trial interval, etc.A forced choice task was used to assess behavioral preference for the two cues. Participants viewed composite images of the background scenes with images of distractor tasks superimposed . These were presented first individually and then in pairs. Thus, in each trial, two composite pictures were presented individually for 3 s (with audio corresponding to the presented background image), and then the two images were presented side by side (no audio) for the preference test. Participants indicated their preference by pressing the corresponding mouse button (left or right). A total of 28 pairs of background (2 types) plus task images (4 types) were presented using a full-factorial design, and order presentation was randomized. The primary outcome measure was choice preference for the drug-paired cue, which was calculated as the number of trials on which subjects chose the MA-paired background cue when both cues were present on-screen , minus the number of choices for the PBO-paired backgrounds. This left a total of 16 comparisons of interest. Individual choice behavior was used to calculate the change in preference for the drug-paired cue before and after conditioning. For the purposes of comparison, the prior study using this protocol  utilizedA VAS scale was used as a self-reported measure of subjective “liking” for each cue, and was given during the pretest, post-test, and follow-ups. In this task, the two conditioned stimuli were presented individually, twice for 5 s each. Participants were asked to move a small vertical bar on a horizontal line indicating how much they liked each cue on a scale of 0 (“dislike very much”) to 100 (“like very much”).Bias in attention towards the two conditioned stimuli was examined using a modified visual-probe task in combination with EOG , 50. TheEmotional reactivity was assessed by measuring corrugator and zygomatic reactivity in response to each cue , 13, 52.Subjective drug effects were assessed during conditioning sessions using three measures. The Drug Effects Questionnaire (DEQ) measuresHR and BP were monitored during the conditioning sessions at 6 regular time points (TPs) during the 4 h sessions. Mean arterial pressure (MAP) was calculated using the formula: [systolic BP + 2 x diastolic BP]/3.All data were analyzed using the SPSS statistical software package . Physiological data (HR and MAP) were analyzed using raw data. Subjective drug effects  were analyzed using change scores, i.e., the difference between scores at each TP and the pre-drug score . Session differences were determined for groups P2 and P4 using a repeated measures analysis of variance (rmANOVA) with Treatment , Session (2 or 4), and Time  as within subjects factors. Group differences for change scores were assessed using average session data with Treatment and Time as within subjects factors and Group  as a between subjects factor. Peak change scores (PCS) were calculated using the maximum change score over the course of the 6 TPs during conditioning sessions and were analyzed using a Group by Treatment by Session rmANOVA.For analyses of behavioral preference, choices for the MA-paired cue were assessed at five sessions . A rmANOVA compared behavioral choice scores using a Group by Time analysis. VAS scores of cue “liking” were calculated using averaged scores for the two types of cue presentations (2 for MA and PBO each) at each of five sessions. Behavioral preference scores were also calculated by subtracting the PBO scores from MA scores at each TP. Analysis of VAS scores for the MA and PBO cues, and preference scores, were conducted using Group by Time rmANOVA analysis.For attention measures, first-gaze proportions towards the MA-paired cue were calculated as the total number of initial gazes towards the MA-paired cue immediately after cue onset, as a fraction of total gazes (MA and PBO). A rmANOVA compared scores using Group by Time (before and after conditioning) analysis. Mean gaze times for each cue were averaged across 20 MA and PBO trials, before and after conditioning, and analyzed using a rmANOVA Group by Cue Type (MA/PBO) by Time analysis. Average RT data from the dot probe task were calculated for each probe type at the three follow-up sessions and a rmANOVA compared Group by Cue Type by Time. EMG data were analyzed for zygomatic and corrugator channels separately, using mean EMG responses for each of the 10 MA- and PBO-paired cue trials. Averaged data were analyzed using rmANOVA Cue Type by Time analysis. All planned post-hoc pairwise tests used the Bonferroni correction.In separate analyses, we examined the relationship between the four outcome measures using Pearson’s product-moment correlations on change scores (before vs after conditioning) for each outcome measure. Using similar correlation analysis, acute responses to MA (vs PBO) and conditioned response measures were compared using averaged PCS for MA and PBO sessions. Similar correlation analyses compared the four outcome measures with the average inter-session interval for conditioning sessions for each individual.The characteristics of the participants are summarized in Methamphetamine  produced prototypic subjective effects  and alterations in physiology . The groups did not differ in these effects except on HR, where the P1 group exhibited a later increase than groups P2 and P4 (see below). Responses to MA compared to PBO did not differ across successive sessions in groups P2 and P4. = 56.40, p < 0.001, ηp2 = 0.573, corrected for sphericity]. HR increased more quickly for groups P2 and P4 compared to P1. That is, at TP 3 HR was higher in the MA condition for groups P2 and P4 individually, but not group P1 . At other time points the groups did not differ. Analysis of peak change scores (PCS) found similar results and post-hoc comparisons between treatments indicated treatment effects for all three groups (p’s < 0.001) and are listed in  = 46.49, p < 0.001, ηp2 = 0.525], at TPs 3–6 for all three groups [all p’s ≤ 0.017], but the groups did not differ. Analyses of MAP PCS values yielded similar results , and three subscales of the POMS , but no significant group differences were found. PCS analyses of treatment effects for each group are listed in  = 31.94, 26.99, p’s < 0.001, ηp2’s = 0.437, 0.395, respectively, corrected for sphericity], starting at TP 3 [all p’s ≤ 0.019]. No group differences were observed. Note that two subjects were excluded from analyses (one from P1 and P4 groups) due to missing data.ARCI. MA increased mean ARCI Arousal (A) and Euphoria (MBG) subscale scores across 6 TPs , with significant differences starting at TP 3 [all p’s ≤ 0.045]. No significant effects were observed for measures of Anger, Confusion, or Depression. No group differences for any measure were found.POMS. MA increased mean POMS subscale scores of Friendliness, Elation, Vigor, Anxiety, and decreased Fatigue across 6 TPs , with effects beginning at TP 2 or 3 and lasting until the end of the session [all p’s ≤ 0.043]. For “Dislike,” MA increased scores at TP 6 for group P1 [p = 0.013], had no significant treatment effects for group P2, and showed a marginally significant reduction compared to PBO for group P4 at TP 4 [p = 0.058]. No group differences were found for any measure.DEQ. MA increased mean scores on DEQ measures of “Feel,” “Like,” “Dislike,” “High,” and want “More” drug across 6 TPs .Relative to PBO ratings, subjects in group P4 (but not P1 or P2) reported “liking” the MA-paired cue (VAS scores) more after conditioning . For all, 0.365) . However = 2.15, p = 0.034, ηp2 = 0.097] (p = 0.05), and a trend elevation compared to group P2 (p = 0.084). No group differences were detected at the 7 and 20 d follow-up. Note that for all VAS analyses, data from two participants (1 each from groups P2 and P4) were excluded from analyses due to outlier status  compared to groups P1  and P2   . Group c = 4.99, p = 0.032, ηp2 = 0.131]  towards the MA-paired cues increased from before to after conditioning to a similar extent in all three groups, means ± SEM of all three groups combined were 0.49 ± 0.02 and 0.54 ± 0.02, respectively (N = 36)  . Individ= 0.131] . Note thEMG activity for the zygomatic and corrugator muscles in response to the MA- and PBO-paired cues did not change after conditioning see .r = 0.588, p < 0.001, N = 39). Within-group analyses indicated that this relationship was significant for group P2 , marginally significant for group P4 , but not for group P1. This suggests that individuals who displayed greater changes in behavioral preference for the MA-paired cue also exhibited stronger preference “liking” ratings for the MA cue, but this relationship only emerged after at least two MA-cue pairings. No significant correlations were observed for the changes in attentional or emotional reactivity measures.Changes in VAS preference “liking” ratings for the MA-paired cue (from before to after conditioning) were positively correlated with changes in behavioral preference scores on the forced choice task . That is, when all 41 participants were examined together, those who reported greater MA liking showed the greatest changes in VAS preferences rating for the MA cue. Separate group analyses revealed no group differences. Ratings of wanting “More” MA  during conditioning were also positively correlated with changes in VAS preference rating for the MA cue  , but not for groups P1 and P2. This suggests that participants who reported wanting more MA exhibited greater changes in rating preference for the drug-paired cue, but this relationship only existed for those individuals who had received four drug-cue pairings. No other significant relationships were identified.Ratings of MA “Liking” (vs PBO) during conditioning (average PCS) were positively correlated with the change in VAS preference rating for the MA cue (Pearson’s  N = 41) . When anThe interval between conditioning sessions was unrelated to the conditioned responses on any of the four outcome measures.The purpose of this study was to replicate and extend previous findings , 41 showThe conditioned responses to the MA-paired cue were modest, especially relative to our previous studies using this procedure. For example, in the previous studies, we detected conditioned responses on behavioral preference and emotional reactivity after two pairings, but these measures did not increase in this study. The reasons for the differential results are not clear. It is possible that the apparently minor methodological changes, especially the different and increased number of distractor tasks, could have influenced the results. For example, one of the tasks used previously  was seleConditioning studies with laboratory animals indicate that the magnitude and persistence of conditioned responses are directly related to the number of pairings between the conditioned stimulus (CS) and unconditioned stimulus (US) . NotablyThe relative increase in ratings of “liking” of the MA-paired cue in the P4 group followed an interesting pattern. That is, whereas subjects’ absolute ratings of the MA cue “liking” did not increase from before to after conditioning, their ratings of the PBO cue (but not the MA cue) declined after conditioning. This is similar to our previous study , in whicDespite the absence of a significant increase in behavioral preference (forced choice) after conditioning, subjects’ rated “liking” for the drug-related stimulus was positively correlated with behavioral cue preference within the P4 group. This suggests that with a larger sample it may be possible to demonstrate conditioned effects with the behavioral preference measure. Alternatively, it is also possible that behavioral indices of “seeking”  are separate from measures of “liking” of a drug or stimulus  towards the drug-paired cue, whereas here we observed only increased attentional bias as measured by initial gaze and cue “liking.” The reasons for the differences are not known, but could be related to differences in sample size (N = 90 vs N = 42). On the measure of behavioral preference, Mayo and de Wit (2015) found that preference increased in 50/90 participants (55.5%) compared to the 19/42 (45.2%) participants in the current study. As noted above, subject sampling and methodological differences might also have contributed in ways that are difficult to detect. For example, when alcohol was used as the drug reinforcer, Mayo and de Wit (2016) observed increases in initial gazes towards the drug-paired cue, but no changes in emotional reactivity, possibly indicating that participant sampling influences the specific expression of conditioned drug-cue responses.Limitations of this study include a small sample size and the use of healthy participants. As noted above, the failure to replicate some of our previous findings may be related to the small number of subjects, and relatedly, more subjects may be needed to detect the effect of number of pairings. The participants in this study were healthy adults with no history of problem drug use. It is possible that this approach under-sampled individuals with a predisposition for drug addiction or who were sensitive to drug-cue conditioning.de novo conditioning paradigm with discrete neutral cues paired with methamphetamine. Our findings provide some evidence that conditioned responses  are greater with more pairings. The findings also provide preliminary evidence on individual differences in susceptibility to developing conditioned drug-cue responses based on the relation between individual subjective responses to a drug. New treatment strategies might utilize this information and target drug users who are particularly sensitive to developing conditioned drug responses, and targeting the cue-induced response using extinction or exposure therapy [In conclusion, results from laboratory models of drug conditioning, such as this study, advance our basic understanding of how conditioned drug-cue responses are acquired , 38, 61. therapy . Future S1 TableNote that cue types indicate whether the cue was paired with methamphetamine (MA) or placebo (PBO).(DOCX)Click here for additional data file.


Background. Tumor necrosis factor-alpha (TNF-α) is an important proinflammatory cytokine which plays an important role in the immunopathogenesis of Behcet's disease (BD). B cell activating factor (BAFF) and its homolog A proliferation inducing ligand (APRIL) are members of the tumor necrosis factor family. BAFF binds to 3 receptors, B cell activating factor receptor (BAFF-R), transmembrane activator and calcium modulator ligand interactor (TACI), and B cell maturation antigen (BCMA) that are expressed by B cells. Objective. Estimation of the serum levels of TNF-α, APRIL, BAFF, and BCMA in patients with BD in an effort to evaluate their degree of involvement in the pathogenesis and development of BD. Patients and Methods. This study included 30 male patients fulfilling the international study group criteria for the diagnosis of BD. Twenty age-matched healthy male volunteers served as control. Serum samples were used for quantification of TNF-α, APRIL, BCMA, BAFF, and hsCRP using ELISA techniques. Results. The mean serum levels of TNF-α, APRIL, BCMA, and BAFF were more elevated in cases than in controls in a statistically significant manner (P < 0.001). Positive correlation was observed between hs-CRP and BDCAF (Behcet's disease current activity forum) index . None of the TNF family members tested was affected by a positive pathergy test. Conclusions. Patients have significantly higher levels of TNF family members'  compared to controls which might contribute to the pathogenesis of BD. Behcet's disease (BD) is a multisystem inflammatory disorder, currently classified as a vasculopathy. Its etiopathogenesis is unclear, but environmental, genetic, and autoimmune factors have been considered. There have been ongoing efforts to elucidate the aetiology of BD .α) is an important proinflammatory cytokine which plays an important role in the immunopathogenesis of Behcet's disease. The serum level of TNF-α is elevated in patients with BD, and a dramatic response to anti-TNF-alpha antibody treatment further supports the role of TNF in BD [Tumor necrosis factor-alpha  and its homolog A proliferation inducing ligand (APRIL) are members of the tumor necrosis factor family and are expressed by monocytes, dendritic cells (DC)s, neutrophils, basophils, stromal cells, activated T cells, activated and malignant B cells, and epithelial cells . BAFF biThis study included thirty Egyptian male patients with BD fulfilling the international study group criteria for the diagnosis of BD . PatientPatients with other associated disorders unrelated to BD including hepatic or renal diseases, diabetes, or essential hypertension were not included in this study. The nature of the study was explained to all participants. The study was approved by the Ethical Committee of Faculty of Medicine, Cairo University, and an oral and written consent was obtained from all participants. The ethical committees approve all the consent procedures.We selected 30 patients fulfilling ISG criteria for the diagnosis of BD from outpatient clinic of rheumatology and dermatology and those admitted to internal medicine departments. We excluded patients with any disorder as hepatic, renal disease, diabetes, other autoimmune diseases, or essential hypertension. The patients were grouped as active or inactive at the time of the study, and the patients who met at least one criterion of the ISG for BD were considered to be in the active stage of the disease. Patients who had been free of lesions for the previous 30 days or more were accepted as having inactive BD.Twenty patients received immunosuppressives and the other 10 patients were controlled on colchicines. So, at the time of sampling 20 patients were active and 10 were in remission.Patients were subjected to the following: complete medical assessment including medical history and complete physical examination with special emphasis on Behcet's disease symptoms and signs including vascular involvement either arterial or venous. Fundus examination assessment was done by a slit lamp examination for detection of any ocular manifestations reported in Behcet's disease, for example, anterior uveitis, posterior uveitis, iridocyclitis, keratitis, retinal vasculitis, retinal vein occlusion, and optic neuritis . AssessmTwenty age-matched healthy male volunteers were recruited and served as a control group. Control subjects with history or signs of recent acute infection or chronic disease  were not included.Control subjects are age-matched and sex-matched healthy male volunteers from similar ethnic background and we excluded those with recent acute infection.α, BAFF, BCMA, APRIL, and hsCRP.Peripheral blood was obtained by venipuncture from patients and healthy adult controls using an Institutional Review Board approved protocol and informed consent. Serum was separated to be used for measurements of TNF-Serum BAFF levels were measured using a specific ELISA kit  according to the manufacturer's protocol. This assay employs the quantitative sandwich enzyme immunoassay technique. Serum BCMA (TNFRSF17) was quantitated in serum by ELISA using 96 well plates . This assay also employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for BCMA (TNFRSF17) has been precoated onto a microplate. APRIL was determined in the serum, using a kit from Bender MedSystems . An anti-APRIL polyclonal coating antibody is adsorbed onto microwells.α was done by using TNF-α ELISA kits provided by AviBon, Helsinki, Finland. The TNF-α ELISA is an enzyme-linked immunosorbent assay for the quantitative detection of human TNF-α in cell culture supernatants and plasma. High sensitive C-reactive protein (hs-CRP) in serum was performed using ELISA technique and interpreted according to the American Heart Association  [TNF-gh risk) .t-test was used for analysis of two pieces of quantitative data. One way ANOVA test was used for comparison of more than 2 pieces of quantitative data followed by post-HOCC test for detection of significance. Pearson's and Spearman correlation was also done. r was consider weak if <0.25, mild if ≥0.25–<0.5, moderate if ≥0.5–<0.75, and strong if ≥0.75. P value was considered significant if <0.05*.Statistical package of social science (SPSS) version 15.0 was used for analysis of data. Data was summarized as mean and SD, percentage.  ranged from 0.08 to 21 yrs, and median was 1. For the control group, ages of participants ranged from 23.0 to 50.0 years with mean of 33.1 ± 8.1 years. No significant age difference was found between patients and controls (P 0.53).Thirty male patients and 20 healthy male controls were recruited for this study. Patients' ages ranged from 16 to 54 years with a mean of 34.3 ± 9.9 years. Their disease durationOnly two patients (6.7%) of the thirty studied had positive family history and 17 patients (56.7%) had positive pathergy test. The patients' BDCAF index ranged from 1 to 7 with mean of 3.4 ± 1.4. The clinical findings and results of both opthalmological and vascular assessment are represented . Twenty α, APRIL, BCMA, and BAFF were higher in patients than in controls in a statistically significant manner (P < 0.001) (P < 0.001 (The mean serum levels of TNF-< 0.001) . hs-CRP  < 0.001 .r 0.68, P < 0.001). However no correlation was detected between BDCAF and any of the TNF family members investigated  .Positive significant correlation was observed between BDCAF and hs-CRP (α level in patients with ocular lesions (P 0.007) was observed. There was no statistical difference in serum levels of APRIL, BCMA, or BAFF in patients with or without clinical manifestation. None of the TNF family members tested was affected by a positive pathergy test.A significantly higher TNF-r 0.34, P 0.047).Only serum APRIL was correlated significantly with hsCRP , while with the TNF-α the correlation was nonsignificant .An inverse significant correlation existed between the duration of the disease and the serum levels of APRIL, BCMA, and BAFF . Also APRIL correlated significantly with TNF-α   .α, BAFF, and its homolog APRIL together with their receptor BCMA in patients with Behcet's disease (BD) in comparison to controls. Similarly, other investigators reported an increased TNF-α level in BD patients, especially in the exacerbation period [In the present study we detected significantly higher serum levels of TNF-n period –15. Revin period . It has α, the serum levels of BCMA, APRIL, and BAFF correlated negatively with disease duration. Of the TNF family members we examined, TNF-α may be the most crucial member for disease maintenance and it seems that TNF-α downregulation in response to disease improvement in patients under treatment may be delayed. This implication is supported by the finding that BDCAF of our patients, who were all under treatment, did not correlate significantly with serum levels of TNF-α, while Durmazlar et al. [α correlates with BDCAF in untreated BD patients. Treatment may suppress inflammation and activity, as measured by BDCAF index and hsCRP, with minimal or delayed effect on TNF-α.Apart from TNF-r et al.  reportedThe ESR and CRP have been recognized as crude markers of disease activity in BD . Indeed,γ and TNF-α, are present in the peripheral blood in significantly higher numbers in patients with BD than in those with recurrent aphthous stomatitis (RAS) and in healthy controls [+ and CD8+ T cells can be involved in the recruitment of neutrophils to the site of inflammation [α, among others. Neutrophils have enhanced chemotaxis and phagocytosis and induce superoxide generation and myeloperoxidase expression. Neutrophils accumulate in inflammatory lesions where they induce neutrophilic vasculitis [The precise mechanisms of tissue destruction in BD have not been fully elucidated. Nevertheless, a growing number of reports have involved T and B lymphocytes mediated immune responses , 21 thatcontrols . These aammation . The neusculitis .κB activation and IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF) expression through functional BCMA receptors, an activation inhibited by anti-BCMA short hairpin RNA [BAFF and its homolog APRIL, which are expressed by neutrophils, may be indirectly involved in regulation of neutrophils, through upregulating IL-6 and GMCSF. BAFF and its homolog APRIL are members of the tumor necrosis factor family and are expressed by monocytes, DCs, neutrophils, basophils, stromal cells, activated T cells, activated and malignant B cells, and epithelial cells . APRIL arpin RNA . This carpin RNA , 27.BCMA stimulation in turn stimulates B cell and, possibly, T cell responses locally. It has been reported that B cell abnormalities could be involved in the pathogenesis of BD where increased levels of activated and memory B cell subsets had been previously demonstrated suggesting a modified B cell function in BD . Increasα is involved in the blood retinal barrier breakdown by opening tight junctions of retinal vascular endothelial cells and retinal pigmented epithelial (RPE) cells [α levels in the serum of patients withactive ocular manifestations were significantly higher than inactive ocular BD patients. Our results were in agreement with Santos Lacomba et al. [α than those of healthy controls. On the other hand, we did not find any statistically significant difference between patients with or without ocular manifestations regarding the serum levels of BAFF, APRIL, and BCMA indicating a minor role of these TNF family members in the pathogenesis of ocular manifestations of BD in comparison to TNF-α.TNF-E) cells . Our stua et al.  who repoα, APRIL, BCMA, and BAFF) we investigated showed any significant difference in expression between patients with or without other active clinical manifestations . Bozoglu et al. [α when patient subgroups were compared to each other. However, Durmazlar et al. [α have strong association with oral ulcer, genital ulcer, the presence of positive pathergy test, and vascular lesion, but no association was found between TNF-α levels and erythema nodosum or arthritis. Moreover, Hamzaoui et al. [None of the TNF family members  as compared to controls. Our findings suggest that TNF-α, BAFF/APRIL, and BCMA might contribute to the pathogenesis of BD possibly through augmentation of both innate and adaptive immune responses as well as by collaborating with other inflammatory cytokines to promote the activation and differentiation of effecter immune cells involved in the pathogenesis of BD.In conclusion, patients with BD have significantly higher inflammatory markers (ESR and hs-CRP) as well as several of the TNF family members' (TNF-The limitation of this study is the small sample size.


Objective. The aim of the present study was an analysis of heteroplasmy level in mitochondrial mutations 652delG, A1555G, C3256T, T3336C, 652insG, C5178A, G12315A, G13513A, G14459A, G14846A, and G15059A in normal and affected by atherosclerosis segments of morphologically mapped aortic walls. Methods. We investigated the 265 normal and atherosclerotic tissue sections of 5 human aortas. Intima of every aorta was divided according to morphological characteristics into segments with different types of atherosclerotic lesions: fibrous plaque, lipofibrous plaque, primary atherosclerotic lesion (fatty streak and fatty infiltration), and normal intima from human aorta. PCR-fragments were analyzed by a new original method developed in our laboratory on the basis of pyrosequence technology. Results. According to the obtained data, mutations G12315A and G14459A are significantly associated with total and primary atherosclerotic lesions of intimal segments and lipofibrous plaques . Mutation C5178A is significantly associated with fibrous plaques and total atherosclerotic lesions (P ≤ 0.01). A1555G mutation shows an antiatherosclerotic effect in primary lesion in lipofibrous plaques (P ≤ 0.05). Meanwhile, G14846A mutation is antiatherogenic for lipofibrous plaques (P ≤ 0.05). Conclusion. Therefore, mutations C5178A, G14459A, G12315A, A1555G, and G14846A were found to be associated with atherosclerotic lesions. Atherosclerosis of great vessels in the overwhelming majority of cases is predominantly a morphological basis of cardiovascular mortality , 2. In tA specific feature of atherosclerosis is a complicacy of this disease detection at early stages. Molecular-genetic markers associated with atherosclerosis can help early diagnosis of this pathology. In recent times, a great number of studies were dedicated to searching genetic biomarkers of atherosclerosis and CVD in human nuclear genome –5. HowevAccording to literature data, various pathologies are associated with certain mitochondrial mutations –9. MitocInvestigations of researchers all over the world are mainly dedicated to autosomal mutations, associated with atherosclerosis –12. OnlyIt is necessary to mention that because of mitochondrial genome instability, somatic mutations often occur in mitochondrial genome. While analyzing the association of mitochondrial genome with pathologies, it is necessary to carry out a quantitative assessment of mitochondrial genome heteroplasmy level –21.In the present study, an association of eleven mitochondrial genome mutations with atherosclerosis was analyzed in individuals, aortic intima of which was morphologically divided into segments with atherosclerotic lesions of varying severity.265 normal and atherosclerotic segments of morphologically mapped aortic walls of varying severity of five individuals were taken as a material for investigation.Samples of autopsy material were taken from thoracic section of aortic intima of men and women, who died at the age of 30–65 years as a result of an accident or a sudden death .To determine a type of a lesion, normal segments of aortas and segments with atherosclerotic lesions were identified macroscopically and then microscopically in accordance with classification of Atherosclerosis Board of American Heart Association .Exteriorly unchanged segments of aorta had a smooth luminal surface. On vertical section in intima, it was possible to detect 2 layers: adjacent to aorta opening proteoglycan layer and an adjoining media myoelastic layer.Segments of intima with primary lesions (lesion type I) were macroscopically segments with smooth yellowish surface, sometimes with small yellow spots. Microscopical changes were minimal. Small accumulations of extracellular lipidic drops in connective tissue matrix were observed. Along with tissue-fixed cells, in segments of preliminary lesions, there were more mononuclear cells compared to normal intima. There were no changes in tissue structure detected.Fatty streaks (lesion type II) macroscopically were stripes and spots of yellow colour, slightly standing out over the vessel surface. Fatty streaks often merge together forming larger structures (clusters). In tissue sections, lipids were detected mainly intracellularly. Intracellular lipids were also detected in connective tissue matrix. Sometimes there can be observed an excessive overgrowth of extracellular matrix in fatty streaks.During macroscopic investigation, lipofibrous plaques (lesion type Va) looked like yellowish or pearl round or ellipsoidal formations standing far out over luminal surface. Microscopically in these lesions, all the changes were detected, which are characteristic for fatty streaks: accumulation of intracellular lipids and overgrowth of extracellular matrix. Furthermore, there were a massive necrotic nucleus and a connective tissue cap found in lipofibrous plaques. In lipofibrous plaques, there were such segments, which are morphologically similar to segments of lipidic lesions, arms of lipofibrous plaques.Fibrous plaques (lesion type Vc) are macroscopically highly elevated round and oval formations of pearl colour, microscopically consisting mainly of coarse connective tissue matrix with cells “mured” in the matrix.Aortic intima was morphologically mapped according to the presence of atherosclerotic lesions of varying severity or healthy vascular tissue.Morphological Mapping of Aortas. In this study, 5 aortic samples were used. Each sample was a segment of a vascular wall with a size approximately 7 × 9 cm2, divided according to morphological characteristics into regions with atherosclerotic lesions of varying severity . In each aortic sample, 38 to 70 of such regions were identified , and late lesions (combination 4+5).μg) was carried out using the phenol-chloroform extraction with the proteinase K lysis. The concentration of the DNA solution was measured by nanospectrophotometer IMPLEN NanoPhotometer at a wavelength of 260 nm.Isolation of total DNA from aortic tissue samples .For PCR, we used DNA with concentration 0.1  2O)-4.6 μL;MQ 2SO4, 67 mM Tris-HCl (pH 8.8)-4 μL);10X buffer (16.6  2: 25 mМMgCl(i) μL (the required concentration is 2.5 mM);4 (ii) μL (the required concentration is 1.5 mM);2.4  μLTaq- polymerase -1.33  μL;DNA template-4  μL;primer F (+)-2.7  μL.primer R (−)-2.7 A reaction mixture and PCR conditions were as follows:μL volume.The reaction was carried out in 40 Conditions for PCR are presented in Supplemental Table 2.An electrophoresis of extracted DNA samples and PCR-fragments was performed in horizontal apparatus  in agarose gel with the use of 0.5Х ТBЕ buffer. The concentration of agarose (≪Fluka≫) was 0.8% (for DNA samples) and 1.5–2.0% (for PCR-fragments).One of the primers was biotinylated with the aim of further pyrosequencing of PCR-fragment. The investigation was performed with the use of an amplifier ≪PTC DNA Engine 200≫.The examples of gel electrophoresis of PCR-fragments of investigated mitochondrial mutations are presented in Supplemental Figures 1–4.After carrying out PCR, the PCR-fragments were pyrosequenced to detect point substitutions, microinsertions, or microdeletions of human mitochondrial genome. The investigation was performed with an automatic pyrosequencing system PSQ HS96MA. During the experiment, a scheme of sample conditioning, described in a manual attached to the pyrosequenator, was realized (sepharose particles were used). Sequences of primers for sequencing are listed in Supplemental Table 3.A visualization of results was performed using a software attached to the pyrosequencing system. The statistical evaluation of the results was performed by using SPSS version 21.0.For assessment of 11 mitochondrial mutations, identified as potential atherosclerosis markers, an investigation on autopsy material with morphological and mutational mapping was made.P ≤ 0.1 level of significance. Meanwhile, mutation G13513A had a significantly negative association with total atherosclerotic lesion of mapped aortas  and mutation 652delG is at d aortas .P ≤ 0.05 level of significance. During analyzing a total of segments of lipofibrous plaques, a significantly negative correlation of mutation G14846A with this type of lesion was found.According to Wilcoxon Matched-Pairs Signed-Ranks test , it was found  that mitP ≤ 0.1 level of significance. However, this mutation is absent in primary total atherosclerotic lesion.At the same time, there was found a positive correlation between a total of segments of lipofibrous and fibrous plaques and mitochondrial genome mutation 652delG at P ≤ 0.05 level of significance with mutations C3256T and C5178A (positively) and G12315A (negatively).Moreover, a total of fibrous plaque segments correlated at To detect the presence of mutational burden interrelation with the degree of atherosclerotic lesion, a linear regression analysis was carried out. Because of a high individual variability of the characteristic, these values were normalized as quartiles. In every aorta for each mutation, an investigation of heteroplasmy index distribution was carried out and interquartile boundaries were detected. Individual scalar indexes of heteroplasmy were transformed into single values 1, 2, 3, or 4, characterizing a belonging of an index to a certain quartile within the limits of a given autopsy sample. The results are presented in Tables P < 0.001 level of significance. Taking into consideration a sufficient quantity of degrees of freedom (11), it can therefore be said that the degree of atherosclerotic lesion is associated with cumulative burden for these mutations with 99.9% probability of error-free prognosis.Therefore, during the assessment of cumulative burden for 11 mutations, the model of linear regression reached For each mutation associated with atherosclerosis, sensitivity and specificity indexes were studied. The analysis was carried out by a method of ROC-curve constructing with a further evaluation of the area under the curve. This enabled us to describe explanatory characteristics of genotypic markers .For this analysis, rank values (quartile numbers) of heteroplasmy were summed according to a sign of beta coefficient, obtained during regression analysis . The obtained parameter was named “mutational burden.”P ≤ 0.05, because a 95% confidence interval lies in the range from 74.6 to 95.3). Specificity index was 77.1 .During the model using, the sensitivity index was 88.2  and also 1, 2, and 6, NADH-dehydrogenase subunit, and cytochrome B.Conspicuous is the fact that the mutation spectrum in different types of atherosclerotic lesions of the aortic intima may slightly differ. For example, mutation A1555G showed an antiatherogenic effect in early atherosclerotic lesions (lipidic spots and fatty streaks) and lipofibrous plaques. At the same time, antiatherogenic mutation G14846A turned out to be typical only for lipofibrous plaques.In fibrous plaques, mutation spectrum significantly differed from other types of atherosclerotic lesions. Proatherogenic mutations were C5178A and C3256T; antiatherogenic mutation was G12315A. Apparently, the defective parts in G12315A transport RNA-Leu (codon recognized CUN) play an important role in pathophysiological process of the origin and development of atherosclerotic lesions before the fibrous plaque stage begins, when the percentage of heteroplasmy for this mutation decreases to such an extent that it is higher in normal intima than in the type of atherosclerotic lesion. The heteroplasmy level of some other mitochondrial genome mutations in fibrous plaques apparently decreases and does not vary from this parameter in normal aortic intima.In the present study, a focality of atherosclerotic lesions in human aortic intima was confirmed. The basis for this confirmation was differences in the heteroplasmy level of 11 mitochondrial mutations for different segments of both normal and atherosclerotic morphologically mapped human aortic intima. It was found that a certain spectrum of pro- and antiatherogenic mitochondrial genome mutations is characteristic for different types of atherosclerotic lesions of aortic intima. In total atherosclerotic lesion, lipofibrous plaques, and primary atherosclerotic lesions of the aortic intima, six proatherogenic mutations  and one antiatherogenic mutation (G13513A) were identified. Differences in the spectrum of mutations between primary atherosclerotic lesions, lipofibrous plaques, and total atherosclerotic lesions lie in the fact that, for the first two types, antiatherogenic mutation A1555G is representative. In lipofibrous plaques, mutation G14846A is also antiatherogenic. The mutation spectrum in fibrous plaques is different from other types of atherosclerotic lesions. Proatherogenic mutations in fibrous plaques are C5178A and C3256T. Mutation G12315A is antiatherogenic.The findings of this study may be useful for practitioners and medical geneticists for early detection and family analysis of atherosclerosis.Supplemental material provides the data about gel/electrophoresis of PCR-fragments, containing mutation sites (Supplementary FIGURES 1-4), primers for PCR (Supplementary TABLE 1), the conditions for the PCR of the mitochondrial genome fragments (Supplementary TABLE 2), and primers for pyrosequencing (Supplementary TABLE 3).