Journal articles on the topic 'Trbd'

Abstract Objective Electroconvulsive therapy (ECT) is an effective option for treatment-resistant bipolar disorder (trBD). However, the mechanisms of its effect are unknown. Oxidative stress is thought to be involved in the underpinnings of BD. Our study is the first, to our knowledge, to report the association between notable oxidative stress parameters (superoxide dismutase [SOD], glutathione peroxidase [GSH-Px], catalase [CAT], and malondialdehyde [MDA]) levels and ECT response in trBD patients. Methods A total 28 trBD patients and 49 controls were recruited. Six-week ECT and naturalistic follow-up were conducted. SOD, GSH-Px, CAT, and MDA levels were measured by enzyme-linked immunosorbent assay, and the 17-item Hamilton Depression Rating Scale and Young Mania Rating Scale were administered at baseline and the end of the 6th week. MANCOVA, ANCOVA, 2 × 2 ANCOVA, and a multiple regression model were conducted. Results SOD levels were lower in both trBD mania and depression (P = .001; P = .001), while GSH-Px (P = .01; P = .001) and MDA (P = .001; P = .001) were higher in both trBD mania and depression compared with controls. CAT levels were positively associated with 17-item Hamilton Depression Rating Scale scores in trBD depression (radjusted = 0.83, P = .005). MDA levels in trBD decreased after 6 weeks of ECT (P = .001). Interestingly, MDA levels decreased in responders (P = .001) but not in nonresponders (P > .05). Conclusions Our study indicates that decreased SOD could be a trait rather than a state in trBD. Oxidative stress levels are associated with illness severity and ECT response. This suggests that the mechanism of oxidative stress plays a crucial role in the pathophysiology of trBD.

Objective The N-methyl-D-aspartate subtype glutamate receptor antagonist ketamine has rapid antidepressant and antisuicidal effects in treating treatment-resistant bipolar depression (TRBD). The neurocognitive effects of repeated ketamine infusions in TRBD are not known. Methods Six intravenous infusions of ketamine (0.5 mg/kg over 40 min) were administered on a Monday–Wednesday–Friday schedule during a 12-day period on 16 patients with TRBD followed by a 2-week observational period. The assessment of neurocognitive function was conducted using the MATRICS Consensus Cognitive Battery at baseline, 13 and 26 days. Tasks were designed to test speed of processing, working memory, visual learning and verbal learning. Results A significant improvement was found only in scores of speed of processing (F = 9.9, p = 0.001) after a 2-week observational period, which was accounted for by the improvement of depression symptoms. There were no significant changes over time in terms of working memory, visual learning and verbal learning. Pearson correlation analysis showed that the improvement of depression symptoms through six ketamine infusions was greater among TRBD patients with lower working memory at baseline (r = 0.54, p = 0.03). In multiple regression analysis, the significant correlation was still maintained (beta = 0.67, t = 2.2, p = 0.04). Conclusion This preliminary study indicated that six ketamine infusions were not harmful but were slightly beneficial for speed of processing in TRBD. However, this change was mainly accounted for the improvement of depression symptoms over time. Lower baseline working memory appears to be associated with greater antidepressant response after completion of six ketamine infusions in patients with TRBD.

Abstract Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by prolonged neutropenia, mainly affecting middle-age females of the HLA-DRB1*1302 type. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitors, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients, supporting an immune pathogenesis. Using Sanger sequencing, we previously reported that the T-cell receptor (TR) gene repertoire in CIN is skewed, indicating antigen selection in CIN ontogeny. However, the analytical depth afforded by Sanger sequencing is limited, hindering definitive conclusions. In order to obtain a truly comprehensive view into the role of antigen drive in CIN, using next generation sequencing (NGS) we interrogated the TR repertoire of 13 patients (8 females, 5 males) included in our previous study as well as a healthy female. TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=4) or BM (n=10) samples. PCR products were used as a substrate for paired-end sample preparation (Illumina) and subjected to NGS on the MiSeq Illumina Platform. The raw NGS data were preprocessed with a dedicated pipeline for this purpose, including: (i) quality filtering of each read; (ii) merging of paired-end reads via local alignment; (iii) preparation of fasta files for submission to the IMGT/High V-QUEST tool; and, (iv) IMGT/High V-QUEST metadata analysis, interpretation and visualization. Overall, 6,196,980 TRBV-TRBD-TRBJ gene rearrangements were analyzed (130,020-1,037,680 /case) of which 5,317,609 were productive since they used functional TRBV genes and also carried in-frame CDR3. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. For repertoire analyses, clonotypes rather than single rearrangement sequences were considered. Overall, 553,145 unique clonotypes were identified (median 39,510; range 7,732-172,253/case), of which 408,744 represented singletons. All clonotypes from the Sanger analysis were detected by NGS as well. Among the 46 functional TRBV genes identified, the most frequent were: TRBV29-1 (13.9%), TRBV19 (6.7%), TRBV12-3 (5.6%), TRBV6-5 (5.4%), TRBV27 (4.9%) and TRBV6-1 (4.0%), collectively accounting for 40,5% of the TRBV repertoire; the TRBV29-1 gene predominated in 9/13 CIN cases. All CIN cases were found to carry distinct expanded clonotypes (median 10,314; range 2,279-40,245/case). The predominant clonotype ranged in frequency from 5.25 to 20.2% of the total clonotypes observed in each case. This contrasts significantly (p<0.001) with a 0.47% frequency of the dominant clonotype in the healthy control. Cluster analysis of the sequences of all CIN cases identified 9034 different clonotypes shared by different patients and, thus, deemed as public. Notably, public clonotypes of a given CDR3 length could show high sequence similarity, further underscoring the restricted nature of the repertoire. As an example, 1632/2665 (61.2%) public clonotypes with 12 aminoacid-long CDR3 were grouped into 168 distinct communities, populated with 2-280 highly similar sequences, each linked with 1 aminoacid distance with at least another member of the community. Overall, the present study offers conclusive evidence that the TR repertoire in CIN is remarkably skewed. The finding of oligoclonal T-cell expansions and public clonotypes strongly indicates that antigen-driven immune responses are very likely implicated in the pathogenesis of CIN. Disclosures No relevant conflicts of interest to declare.

Abstract The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia (CLL) is largely unexplored, though highly relevant in light of the interactions of the CLL B cells with T cells, effectively inducing tolerance to the latter. Our recent classic subcloning/Sanger sequencing studies of the T-cell receptor beta chain (TRB) gene repertoire in CLL indicated repertoire restriction, pointing to antigenic selection. However, due to the inherent limitations of low-throughput analysis, definitive conclusions were not possible. Here, we sought to advance the analytical depth of our approach by employing high-throughput, next generation sequencing (NGS) for exploring the TRB gene repertoire in CLL. Our study included 9 untreated CLL cases assigned to two paradigmatic stereotyped subsets, namely clinically indolent subset #4 (n=7) and clinically aggressive subset #1 (n=2). RNA was isolated from peripheral blood mononuclear cells (n=7 cases) or purified CD4+ and CD8+ T cells (n=2, both subset #4). TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA according to the BIOMED2 protocol and were subjected to NGS (MiSeq Illumina Platform). The paired-end Illumina protocol allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, thus increasing the accuracy of results. Still, considering the inherent limitations of PCR-based NGS, the experimental setup included many internal controls: (i) replicate samples of the same patient at the same timepoint; (ii) samples of the same patient at sequential time points (two-timepoint longitutinal analysis for 1 case); (iii) replicate cDNA samples for PCR amplification; and, (iv) analysis of a healthy individual. A bioinformatics pipeline was developed for raw NGS data processing, performing: (i) quality filtering of reads; (ii) merging of paired-end reads via local alignment; (iii) preparation of filtered-in fasta sequences for submission to the IMGT/HighV-QUEST tool; and, (iv) IMGT/HighV-QUEST metadata clustering, analysis and interpretation. Overall, 19 samples were analyzed, producing 7,920,136 TRBV-TRBD-TRBJ reads (median 359,957 reads/sample, median Q-score 38.3). Poor quality, incomplete, out-of-frame and unproductive rearrangements were filtered out (median 2.1% of reads/sample). For repertoire analyses, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) rather than single rearrangement reads were considered, so as to avoid possible biases due to clonal expansion following antigenic stimulation (median 56194 distinct clonotypes/sample, 33619 singletons versus 13725 expanded). Among the 53 functional TRBV genes identified, the following 5 predominated: TRBV12-3/12-4 (7.5%), TRBV19 (6.1%), TRBV5-1 (5.2%), TRBV29-1 (4.9%) and TRBV27 (4.8%), collectively accounting for 28.5% of the TRBV repertoire. Comparison of the TRBV gene repertoire of CD8+ vs CD4+ cells in subset #4 CLL cases showed that TRBV19 was overrepresented in the CD4+ compartment (9.4% versus 6.9%, p<0.001). Comparison between subset #4 versus subset #1 cases revealed significant overrepresentation of TRBV12-3/12-4 in subset #4 (8.6% versus 4.1%, p<0.001). The TRB repertoire was significantly more oligoclonal in CLL compared to the healthy control (median frequency of the predominant clonotype: 7.3% versus 0.47%, respectively, p<0.001), and this skewing stemmed mainly from the CD8+ rather than the CD4+ compartment (median frequency of the predominant clonotype 10.7% versus 1.0%, respectively, p<0.001). Cluster analysis of all CLL cases identified 11281 different clonotypes (excluding singletons) shared by different patients and not present in the healthy control. Of these, 10670 and 12 were exclusively found in subset #4 and subset #1 cases, respectively. The longitudinal analysis of one case identified 14.6% of all expanded clonotypes persisting over time. Moreover, comparison of TRBV gene usage and clonotype repertoire among replicate samples revealed high reproducibility of results. Overall, our study provides large-scale, reproducible evidence of TR repertoire skewing and oligoclonality in CLL, mainly derived from the CD8+ T cell compartment, strongly supporting antigenic selection. The functional role of clonally expanded T cells remains to be elucidated. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 831 Chronic idiopathic neutropenia (CIN) is a disorder of neutrophil production usually characterized by benign and asymptomatic course and female predominance. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitor cells, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients. Recently, using flow cytometry and CDR3 spectratyping, we obtained for the first time evidence for expanded T cell populations alluding to repertore skewing in both PB and BM of patients with CIN, but more prominent in the BM CD8+ subset. Prompted by these fndings, here we significantly extended our studies of the cytotoxic T cell responses in CIN in order to obtain a comprehensive view of the role of antigen selection in CIN pathogenesis. The study included 18 patients with CIN, 17 females and 1 male, diagnosed according to the established criterial for CIN. TRBV-TRBD-TRBJ gene rearrangements were amplified on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=6) or BM (n=12) samples. PCR products were subcloned by transformation into E.Coli/TOP10F' competent bacteria and individual colonies were chosen randomly and subjected to Sanger sequencing. Sequence data were analyzed using the International imMunoGenetics information system and more particularly the IMGT/V-QUEST tool. Overall, 507 TRBV-TRBD-TRBJ gene rearrangements were analyzed (19-30/case) of which 466 were productive since they used functional TRBV genes and also carried in-frame CDR3s. A polyclonal profile was seen in only 3/18 cases (16.7%). The remaining cases were found to carry clusters of identical rearrangements corresponding to distinct immunodominant clonotypes. The frequency of each clonotype was determined by dividing the number of identical sequences by the total number of subcloned sequences analyzed. In 10/18 cases (55.5%), the dominant clonotype accounted for 12.8–22.6% of the total, whereas in 5/18 cases (27.8%) it accounted for 34.2–68.4% of the total. The TRBV28 gene was used by the dominant clonotype of three different CIN cases; the TRBV10-2, TRBV10-3, TRB19, and TRBV7-8 genes were identified in the major clonotypes of two cases each. Importantly, cluster analysis of the CDR3 sequences of all CIN cases of the present study identified 4 different rearrangements that were shared by different patients (public clonotypes). In conclusion, the present study strongly suggests that CIN may result from an autoimmune reaction directed against granulocytic progenitors triggered by a restricted range of, as yet, unidentified antigen(s). The finding of public clonotypes may indicate that public antigenic stimuli and/or shared immune processes underlie CIN development, at least for a proportion of cases. Overall, our results further support the concept that CIN may share common pathophysiologic mechanisms with other disorders characterized by T-cell and cytokine mediated suppression of hematopoiesis, perhaps representing a milder extreme within the spectrum of these acquired immune-mediated BM failure syndromes. Disclosures: No relevant conflicts of interest to declare.

Abstract Chronic lymphocytic leukemia (CLL) is characterized by remarkable skewing of the B-cell receptor immunoglobulin (BcR IG) repertoire, culminating in the existence of subsets of patients with stereotyped BcR IGs. This implies antigen selection in the natural history of CLL, ultimately affecting clonal behavior. Currently, limited information is available regarding the role of antigens in the selection and activation of cognate T cells, although this is relevant in light of B and T cell interactions inducing T cell tolerance. Our preliminary next-generation sequencing (NGS) studies in 11 patients assigned to stereotyped subset #4, a clinically indolent disease subgroup, indicated T-cell receptor beta chain (TRB) gene repertoire restriction and oligoclonality. Prompted by these observations, here we sought to obtain a comprehensive view of the T-cell repertoire in CLL by extending our analysis to 36 untreated CLL patients, either assigned to major stereotyped subsets [subset #4 (n=11), subset #1 (n=10), subset #2 (n=4), subset #16 (n=1)] or non-subset cases [with mutated-M (n=5) or unmutated-UM clonotypic BcR IGs (n=5)]. Starting material was PB mononuclear cells (n=27), purified CD4+ and CD8+ T-cell subpopulations (n=10), bone marrow (n=2) or lymph node (LN) tissue (n=1). Three patients were studied overtime. Multiple sample and PCR replicates, as well as 3 age-matched healthy controls were also included. TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA (BIOMED2 protocol) and subjected to paired-end NGS, designed to cover the complementarity determining region 3 (CDR3) twice/sequence. In order to further increase the accuracy of results, raw NGS reads were subjected to a purpose-built, bioinformatics algorithm, performing: (i) length and quality filtering of raw reads; (ii) merging of filtered-in paired reads via local alignment; (iii) length and quality filtering of stitched sequences. No base calls of Q-score<30 were allowed in the 75 nucleotide stretch ahead of the FGXG motif, thus further increasing the CDR3 sequencing reliability. Filtered-in sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline designed for clonotype computation and repertoire analysis. Overall, 66 samples were analyzed, producing 23,238,779 filtered-in sequences. Only productive, in-frame TRBV-TRBD-TRBJ rearrangements were included in the analysis (85.8% of filtered-in sequences, median 354,972/sample). For repertoire analysis, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) were considered (median 37,550 distinct clonotypes/sample; 13,134 expanded versus 22,550 singletons). The distribution of TRBV genes was asymmetric with only 5 genes [TRBV12-3/12-4 (8.4%), TRBV19 (6.8%), TRBV29-1 (6.6%), TRBV5-1 (6.1%), TRBV6-5 (5.2%)] collectively accounting for one-third of the repertoire, indicating functional constraints. No significant TRBV gene repertoire difference was identified across the different immunogenetic CLL subgroups analyzed. In contrast, significant (p<0.001) differences were seen between CD8+ versus CD4+ cells regarding certain genes (e.g. over-representation of TRBV7-2 in CD4+ and under-representation of TRBV5-1 in CD8+ cells), alluding to distinct immune selective processes. Notably, the TRB repertoire was significantly more oligoclonal in CLL compared to the healthy controls (median frequency of the predominant clonotype: 5.7% versus 1.27%, respectively, p<0.05), albeit with differences between cases with distinct BcR IG, perhaps reflecting their different immunopathogenesis. Overtime analysis showed persistence of most major clonotypes in 2/3 cases studied, likely in a context of persistent antigenic stimulation. Cluster analysis of all CLL cases identified 17,592 different clonotypes (excluding singletons) shared by different patients and not present in the healthy controls, arguing in favor of a disease-biased immunoprofile; formal proof of this claim will require analysis of a larger number of healthy controls, currently underway by our group. In conclusion, massive parallel sequencing documents the restricted nature of the TRB repertoire in CLL, supporting selection by shared antigenic elements. Whether these are the same antigens interacting with the malignant clone remains to be elucidated. Disclosures Stamatopoulos: Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.

The current psychopharmacological treatment approaches for major depression focus on monoaminergic interventions, which are ineffective in a large proportion of patients. Globally, treatment-resistant bipolar depression (TRBD) affects up to 33% of depressive patients receiving treatment. Certain needs are still unmet and require new approaches. Many studies are in favor of treatments with ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, even in single use, whose effects emerge in minutes to hours post administration. However, little data are available on ketamine performance in TRBD patients with somatic comorbidities, including highly prevalent ones, i.e., cardiovascular disease (heart failure, hypertension, post-myocardial infarct, arrhythmias, etc.) diabetes, and obesity, and depression-associated comorbidities such as stroke, epilepsy, as well as in the elderly population. The literature shows that treatment with ketamine is efficacious and safe, and the majority of adverse drug reactions are mild and tend to mostly disappear within 30 min to 2 h of ketamine administration.

Abstract B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are considered to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 36 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 100% (36/36) of patients. The most frequent rearrangements were observed in IGH genes (94%), including complete IGHV-IGHJ in 83% (30/36) and incomplete IGHD-IGHJ in 22% (8/36) of patients. Among complete IGH rearrangements 2 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 26 patients (72%) (including 64% of IGKV-IGKJ, 47% IGKV/intron-Kde, and 22% IGLV-IGLJ) what indicates active receptor editing occurring during B lymphoblasts leukemogenesis. Crosslineage TCR genes rearrangements were found in 97% (35/36) of patients. TCR beta genes rearrangements were detected in 47% (17/36) of patients (complete TRBV-TRBJ in 25% (9/36), TRBD-TRBJ in 6/36 patients - 17%). TRGV-TRGV in 58% (21/36), TRDV-TRDJ in 58% (21/36); 17 monoallelic and 4 biallelic were found. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblast undergoes rearrangement on the same IGK allele before IGL genes rearrangement occur. The data may suggest the possible of antigens in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as stable molecular marker for monitoring MRD in B-ALL.

Abstract The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. Disclosures No relevant conflicts of interest to declare.

BackgroundMost people with bipolar disorder spend a significant percentage of their lifetime experiencing either subsyndromal depressive symptoms or major depressive episodes, which contribute greatly to the high levels of disability and mortality associated with the disorder. Despite the importance of bipolar depression, there are only a small number of recognised treatment options available. Consecutive treatment failures can quickly exhaust these options leading to treatment-resistant bipolar depression (TRBD). Remarkably few studies have evaluated TRBD and those available lack a comprehensive definition of multi-therapy-resistant bipolar depression (MTRBD).AimsTo reach consensus regarding threshold definitions criteria for TRBD and MTRBD.MethodBased on the evidence of standard treatments available in the latest bipolar disorder treatment guidelines, TRBD and MTRBD criteria were agreed by a representative panel of bipolar disorder experts using a modified Delphi method.ResultsTRBD criteria in bipolar depression was defined as failure to reach sustained symptomatic remission for 8 consecutive weeks after two different treatment trials, at adequate therapeutic doses, with at least two recommended monotherapy treatments or at least one monotherapy treatment and another combination treatment. MTRBD included the same initial definition as TRBD, with the addition of failure of at least one trial with an antidepressant, a psychological treatment and a course of electroconvulsive therapy.ConclusionsThe proposed TRBD and MTRBD criteria may provide an important signpost to help clinicians, researchers and stakeholders in judging how and when to consider new non-standard treatments. However, some challenging diagnostic and therapeutic issues were identified in the consensus process that need further evaluation and research.Declaration of interestIn the past 3 years, M.B. has received grant/research support from the NIH, Cooperative Research Centre, Simons Autism Foundation, Cancer Council of Victoria, Stanley Medical Research Foundation, MBF, NHMRC, Beyond Blue, Rotary Health, Geelong Medical Research Foundation, Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Meat and Livestock Board, Organon, Novartis, Mayne Pharma, Servier, Woolworths, Avant and the Harry Windsor Foundation, has been a speaker for Astra Zeneca, Bristol Myers Squibb, Eli Lilly, Glaxo SmithKline, Janssen Cilag, Lundbeck, Merck, Pfizer, Sanofi Synthelabo, Servier, Solvay and Wyeth and served as a consultant to Allergan, Astra Zeneca, Bioadvantex, Bionomics, Collaborative Medicinal Development, Eli Lilly, Grunbiotics, Glaxo SmithKline, Janssen Cilag, LivaNova, Lundbeck, Merck, Mylan, Otsuka, Pfizer and Servier. A.J.C. has in the past 3 years received honoraria for speaking from Astra Zeneca and Lundbeck, honoraria for consulting from Allergan, Janssen, Lundbeck and LivaNova and research grant support from Lundbeck. G.M.G. holds shares in P1Vital and has served as consultant, advisor or CME speaker for Allergan, Angelini, Compass pathways, MSD, Lundbeck, Otsuka, Takeda, Medscape, Minervra, P1Vital, Pfizer, Servier, Shire and Sun Pharma. J.G. has received research funding from National Institute for Health Research, Medical Research Council, Stanley Medical Research Institute and Wellcome. H.G. received grants/research support, consulting fees or honoraria from Gedeon Richter, Genericon, Janssen Cilag, Lundbeck, Otsuka, Pfizer and Servier. R.H.M.-W. has received support for research, expenses to attend conferences and fees for lecturing and consultancy work (including attending advisory boards) from various pharmaceutical companies including Astra Zeneca, Cyberonics, Eli Lilly, Janssen, Liva Nova, Lundbeck, MyTomorrows, Otsuka, Pfizer, Roche, Servier, SPIMACO and Sunovion. R.M. has received research support from Big White Wall, Electromedical Products, Johnson and Johnson, Magstim and P1Vital. S.N. received honoraria from Lundbeck, Jensen and Otsuka. J.C.S. has received funds for research from Alkermes, Pfizer, Allergan, J&J, BMS and been a speaker or consultant for Astellas, Abbott, Sunovion, Sanofi. S.W has, within the past 3 years, attended advisory boards for Sunovion and LivaNova and has undertaken paid lectures for Lundbeck. D.J.S. has received honoraria from Lundbeck. T.S. has reported grants from Pathway Genomics, Stanley Medical Research Institute and Palo Alto Health Sciences; consulting fees from Sunovion Pharamaceuticals Inc.; honoraria from Medscape Education, Global Medical Education and CMEology; and royalties from Jones and Bartlett, UpToDate and Hogrefe Publishing. S.P. has served as a consultant or speaker for Janssen, and Sunovion. P.T. has received consultancy fees as an advisory board member from the following companies: Galen Limited, Sunovion Pharmaceuticals Europe Ltd, myTomorrows and LivaNova. E.V. received grants/ research support, consulting fees or honoraria from Abbott, AB-Biotics, Allergan, Angelini, Dainippon Sumitomo, Ferrer, Gedeon Richter, Janssen, Lundbeck, Otsuka and Sunovion. L.N.Y. has received grants/research support, consulting fees or honoraria from Allergan, Alkermes, Dainippon Sumitomo, Janssen, Lundbeck, Otsuka, Sanofi, Servier, Sunovion, Teva and Valeant. A.H.Y. has undertaken paid lectures and advisory boards for all major pharmaceutical companies with drugs used in affective and related disorders and LivaNova. He has also previously received funding for investigator-initiated studies from AstraZeneca, Eli Lilly, Lundbeck and Wyeth. P.R.A.S. has received research funding support from Corcept Therapeutics Inc. Corcept Therapeutics Inc fully funded attendance at their internal conference in California USA and all related expenses. He has received grant funding from the Medical Research Council UK for a collaborative study with Janssen Research and Development LLC. Janssen Research and Development LLC are providing non-financial contributions to support this study. P.R.A.S. has received a presentation fee from Indivior and an advisory board fee from LivaNova.

Abstract B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are postulated to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 28 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 96% (27/28) of patients. The most frequent rearrangements were observed in IGH genes (96%), including complete IGHV-IGHJ in 75% (21/28) and incomplete IGHD-IGHJ in 31% (8/28) of patients. Among complete IGH rearrangements 4 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 20 patients (71%) (including 25% of IGKV-IGKJ, 50% of IGKV/intron-Kde, and 25% of IGLV-IGLJ) indicating active receptor editing occurring during B lymphoblasts leukemogenesis. Cross-lineage TCR genes rearrangements were found in 77% (23/28) of patients. TCR beta genes rearrangements were detected in 46% (13/28) of patients (complete TRBV-TRBJ in 32% (9/28), TRBD-TRBJ in 5/28 patients - 18%). TRGV-TRGV were found in 46% (13/28), TRDV-TRDJ in 50% (14/28; 10 monoallelic and 4 biallelic). TCR beta genes rearrangements with presence of TCR gamma genes rearrangements were identified in 25% (7/28) of patients. The identified Ig and TCR rearrangements were stable in patients monitored for minimal residual disease (MRD) and patients with leukemia relapse. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblasts undergo many rearrangements on the same IGK allele before they rearrange IGL genes. The data suggest the role of antigen in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as a possible molecular marker for monitoring MRD in B-ALL.

Abstract Abstract 3908 Chronic lymphocytic leukemia (CLL) exhibits a remarkably skewed immunoglobulin (IG) gene repertoire mainly evident in the existence of subsets of patients with quasi-identical IGs in their B cell receptors (BcRs), collectively accounting for one-third of CLL patients. BcR stereotypy is strongly suggestive of clonal selection by a restricted set of antigens. However, it is not yet clear at which phase of clonal evolution these antigens act, or whether the stimulation is persistent. Furthermore, the possible role of antigens in the selection and activation of cognate T lymphocytes remains obscure yet highly relevant, given recent data about T cell interactions with CLL B cells and their tolerized behavior. Here, we analyzed the repertoire of T cell receptor β chain genes (TRB) in CLL expressing stereotyped IGHV4–34/IGKV2–30 BcR IGs (subset #4), which exhibit a series of immunogenetic features, such as pronounced intraclonal diversification of IG genes, suggestive of ongoing interactions with (auto)antigens. Furthermore, subset #4 CLL cells have distinctive functional responses to BcR and/or Toll-like receptor triggering, rendering this subset a paradigmatic example for seeking evidence of antigen selection also within the T cell population. We analyzed 18 peripheral blood samples of 12 untreated subset #4 patients (samples from different time points were analyzed in 4 cases). No case had evidence of infection at sampling. PCR amplicons for TRBV-TRBD-TRBJ gene rearrangements (BIOMED2 protocol) were subcloned by transformation into E. coli/TOP10F bacteria and randomly chosen individual colonies were subjected to Sanger sequencing. Only productive rearrangements (n=320, ranging from 14–52/case) were analyzed. All cases were found to carry clusters of identical rearrangements (≥2) corresponding to distinct clonotypes; the number of expanded clonotypes/case ranged from 1–13 (median 5). The relative frequency of each clonotype/case was determined by dividing the number of the corresponding identical sequences by the total number of subcloned sequences analyzed. The frequency of the most expanded (immunodominant) clonotype/case ranged from 8.1–70.4%. Collectively, the frequency of all expanded clonotypes/case ranged from 29.7–93.3%. In 2/4 cases that were analyzed at different time points, at least one clonotype was found to persist. Importantly, cluster analysis of the TRB CDR3 sequences of all cases identified ‘public’ clonotypes: 2 identical clonotypes (TRBV15*02/TRBD1*01/TRBJ2–2*01 and TRBV30*01/TRBD1*01/TRBJ2–2*01) each shared by a pairs of different patients and a highly similar clonotype shared by an additional pair of patients. In conclusion, the present study provides clear evidence of repertoire skewing among T cells in CLL patients belonging to subset #4, strongly supporting antigen selection. The finding of ‘public’ clonotypes raises the possibility that shared antigenic epitopes may be relevant for clonal selection of T cells in different subset #4 cases. Whether the antigens that drive T cell repertoire restriction are identical/related to those implicated in the selection of CLL progenitors of subset #4 or even the malignant cells themselves or whether they are tumor-associated antigens remains to be clarified. Disclosures: No relevant conflicts of interest to declare.

Recent evidence suggests immunomodulatory effects of daratumumab in heavily pre-treated Multiple Myeloma (MM) patients (pts); however, the precise effects remain under-characterized, particularly at the molecular level. REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study that evaluates the effects of daratumumab monotherapy on bone metabolism of pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Secondary endpoint of the study included the evaluation of T cell dynamics by comprehensive analysis of the T cell receptor (TR) repertoire employing next generation sequencing (NGS) and multi-color flow cytometry. Herein we report the results of this secondary endpoint for the first 14 pts who completed 3 cycles of daratumumab monotherapy. In total, we analyzed 28 paired samples collected at screening (n=14) and on Day 1 of Cycle 4 (C4D1, n=14) of treatment in order to assess potential changes in relation to treatment and clinical response. Patients were grouped based on best responses into responders (i.e. patients with partial response [PR, n=1] and very good PR [VGPR, n=6]), and non-responders (i.e. patients with minimal response [MR, n=2], stable disease [SD, n=4], or progressive disease [PD, n=1]). Starting material was peripheral blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads (n=6,715,406 | median 221,145/sample) were processed through a previously published, purpose-built bioinformatics pipeline. Only productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=3,097,565 | median 101,670/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 151,153 distinct clonotypes (median 5,084 clonotypes/sample) were assessed. Both groups (responders/non-responders) displayed clonal T cell expansions both pre- and post-treatment. Clonality was found to be increased after treatment for both responders and non-responders, with statistical significance in the former (median cumulative frequency of the 10 most expanded T cell clonotypes/sample: 31% pre-treatment versus 40% post-treatment, respectively | p=0.04). In both groups, the clonotype repertoire appeared to be renewed with only a small fraction of pre-treatment clonotypes remaining after treatment (1% for non-responders; 0.6% for responders). Interestingly, in the responders' group we noticed a significant shift in the major clonotype repertoire at screening vs C4D1. In particular, in the responders' group the 10 most expanded clonotypes/sample at C4D1 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment, suggesting that daratumumab treatment led to the emergence of anti-myeloma T cell clones which contributed to clinical response. On the contrary, the 10 most expanded pre-treatment clonotypes in the non-responders' group tended to dominate also the post-treatment repertoire. Of note, 13 shared clonotypes were identified amongst the post-treatment repertoires of different patients (responders/non-responders); shared clonotypes were not found in other entities in public databases, raising the possibility that they may be "disease-specific" and selected by common tumor-associated antigens. With a single exception, shared clonotypes were detected in cases with relevant HLA restrictions, which is noteworthy given the random HLA background of our cohort. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at screening 60% versus 83% at C4D1 | p=0.003), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at screening 30.8% versus 48.9% at C4D1 | p=0.03) in both groups. In conclusion, TR clonality increases post-treatment through a renewal mechanism; however, pre-treatment clones significantly expanded post-treatment in responders, alluding to the existence of clonotypes with anti-MM properties that may be activated after treatment with daratumumab, arguably contributing to clinical response. Disclosures Kastritis: Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria. Katodritou:Genesis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Amgen: Honoraria. Delimpasi:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant. Symeonidis:Sanofi: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding. Chatzidimitriou:Janssen: Honoraria.

ABSTRACT Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.

In 2000, investigators discovered Tribbles, a Drosophila protein that coordinates morphogenesis by inhibiting mitosis. Further work has delineated Xenopus (Xtrb2), Nematode (Nipi-3), and mammalian homologs of Drosophila tribbles, which include TRB1, TRB2, and TRB3. The sequences of tribbles homologs are highly conserved, and despite their protein kinase structure, to date they have not been shown to have kinase activity. TRB family members play a role in the differentiation of macrophages, lymphocytes, muscle cells, adipocytes, and osteoblasts. TRB isoforms also coordinate a number of critical cellular processes including glucose and lipid metabolism, inflammation, cellular stress, survival, apoptosis, and tumorigenesis. TRB family members modulate multiple complex signaling networks including mitogen activated protein kinase cascades, protein kinase B/AKT signaling, mammalian target of rapamycin, and inflammatory pathways. The following review will discuss metazoan homologs of Drosophila tribbles, their structure, expression patterns, and functions. In particular, we will focus on TRB3 function in the kidney in podocytes. This review will also discuss the key signaling pathways with which tribbles proteins interact and provide a rationale for developing novel therapeutics that exploit these interactions to provide better treatment options for both acute and chronic kidney disease.

Abstract Non-neoplastic lymphadenopathy (NNL) associated with the human immunodeficiency virus (HIV) infection may develop concurrently with the onset of HIV viremia (acute retroviral syndrome) that can persist beyond the acute phase. Histopathological findings at this early phase mainly pertain to hyperplastic changes with large lymphoid follicles; with time, the number of lymphoid follicles diminishes, while plasma cells increase; at the extreme is a pattern characterized by sclerosis of the germinal centers in the residual follicles. HIV-specific CD8+ T cell responses have been reported and certain viral protein epitopes have been identified e.g. the p24 protein, a component of the HIV particle capsid. Overall, these findings reflect an ongoing immune response that is still incompletely characterized at the molecular level, particularly as it concerns the composition of the T cell receptor (TR) gene repertoire. In order to obtain a comprehensive view into the role of antigen selection in shaping T cell responses in HIV-associated NNL [HIV(+) NNL], we studied in-depth the TR repertoire in: (i) lymph node biopsy samples from 12 patients with HIV(+) NNL, (ii) lymph node samples from 5 non-HIV patients with reactive lymphadenopathy [HIV(-) RL]; and, (iii) peripheral blood samples from 4 healthy, HIV-seronegative individuals without lymphadenopathy [healthy controls, HIV(-) HC]. Genomic DNA was isolated from either paraffin-embedded lymph nodes (for patients with lymphadenopathy) or blood mononuclear cells (for healthy individuals). TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol. PCR products were subjected to next generation sequencing (NGS) on the MiSeq Illumina Platform. NGS data analysis, interpretation and visualization was performed by a validated, in-house bioinformatics pipeline. Overall, we obtained: (i) 1,440,305 (mean: 120,025) productive rearrangement sequences in the HIV(+) NNL group; (ii) 702,533 (mean: 140,506) productive sequences in the HIV(-) RL group; and, (iii) 539,981 (mean: 134,995) productive sequences in HIV(-) HC cases. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. In total, we identified 15,553 unique clonotypes in patients with HIV(+) NNL (mean: 1,296, range: 337-6,212), 53,874 in HIV(-) RL (mean: 10,774, range: 3,336-16,304) and 220,069 clonotypes in HIV(-) HC cases (mean; 55,017, range: 35,430-68,916), indicating significant repertoire restriction in the former group. Indeed, this group was characterized by an increased level of oligoclonality compared to the other two groups: the mean values of the sum of relative frequencies for the 10 most frequent clonotypes were 80%, 19.6% and 16.5%, respectively. Seven of 12 HIV(+) NNL cases carried the same dominant clonotype (TRBV29-1, SVDPSGTGGEGYT) that was also found in the remaining 5 patients of this group, albeit at lower frequencies; in contrast, it was completely absent in the HIV(-) RL and HIV(-) HC groups. Regarding the TRBV gene repertoire, the TRBV29-1 gene was overrepresented (p<0.005) in the HIV(+) NNL group, whereas the TRBV6-5 and TRBV19 genes were frequent in both groups of patients with lymphadenopathy (HIV-associated or not); finally, the TRBV5-1 was underrepresented (p<0.005) in patients with lymphadenopathy (HIV-associated or not) compared to HIV(-) HC cases. Comparison of the present TR gene sequence dataset against public databases identified 2 clonotypes with an established reactivity against the p24 protein that were present in 2 different patients with HIV(+) NNL of the present cohort. In conclusion, the TR gene repertoire of patients with HIV(+) NNL displays increased level of clonality, distinct TRBV gene repertoire as well as a widely shared, specific dominant clonotype compared to HIV(-) RL cases or HIV(-) healthy controls. These findings allude to an antigen-driven, HIV-specific immune process, a claim also supported by the detection of clonotypes with established anti-HIVp24 reactivity in at least a fraction of the analyzed patients. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding.

Preliminary evidence for T cell receptor (TR) repertoire renewal and increased TR clonality has been reported by our group (Vlachonikola et al., ASH 2019) in multiple myeloma (MM) patients (pts) receiving daratumumab monotherapy within the context of the REBUILD study, an ongoing prospective, multicenter, non-comparative, open-label, phase II study in pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Herein, we report the results from the longitudinal analysis of the TR repertoire employing next generation sequencing (NGS) and multi-color flow cytometry in 24 pts who completed 3 cycles (n=24) and 6 cycles (n=11/24) of daratumumab monotherapy, in order to assess the immunomodulatory effects of daratumumab. We assessed 59 peripheral blood samples collected at screening (SCR, n=24), on Day 1 of Cycle 4 (C4, n=24) and Day 1 of Cycle 7 (C7, n=11). Patients were grouped based on best responses at C4 into responders (i.e. pts with partial response [PR, n=7] and very good PR [VGPR, n=8]), and non-responders (i.e. pts with minimal response [MR, n=2], stable disease [SD, n=5], or progressive disease [PD, n=2]). TRBV-TRBD-TRBJ gene rearrangements were subjected to paired-end NGS and raw reads (n=13,886,646 | median 239,969/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=6,324,986 | median 100,738/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 325,789 distinct clonotypes (median 4,535 clonotypes/sample) were analyzed. The TR repertoire displayed clonal T cell expansions in both groups (responders/non-responders) in all pre/post-treatment timepoints. Clonality increased after treatment for both responders and non-responders in all assessed timepoints, with statistical significance at C4 in both groups (median cumulative frequency of the 10 most expanded T cell clonotypes/sample in responders: 31.6% pre-treatment vs 43% C4 post-treatment, p=0.009; and, in non-responders: 19.8% pre-treatment vs 39.6% C4 post-treatment, p=0.009). In both groups, the clonotype repertoire appeared to be renewed. Interestingly, in the responders' group a significant shift was noticed in the major clonotype repertoire at screening vs C4. In particular, the 10 most expanded clonotypes/sample at C4 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment. Additionally, although the major post-treatment clonotype at C4 also dominated at C7 in most cases, certain lower frequency clonotypes at C4 emerged among the top-10 at C7. Thirteen shared clonotypes were identified amongst the post-treatment repertoires of different patients but not in other entities in public databases, raising the possibility that they may be "MM-specific" and selected by common MM-associated antigens. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at SCR 63% vs 78.7% at C4 | p=0.0045 and 87.4% at C7 | p=0.0009), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at SCR 31.4% vs 45.3% at C4 | p=0.0045 and 53.8% at C7 | p=0.001) in both groups. In conclusion, we document T cell clonal expansions and clonal drift after daratumumab treatment in MM. Our results suggest that daratumumab acts through renewing the greatest part of the pre-treatment TR clonotype repertoire, suggesting dynamic changes of the T cell compartment under treatment, a claim also supported by the significant increase in CD8+ cytotoxic T cell numbers overtime. The significant post-treatment expansion of certain low-frequency pre-treatment clones in responders raises the intriguing hypothesis that daratumumab treatment may have led to the outgrowth of anti-MM T cell clones, arguably contributing to clinical response. Disclosures Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hatjiharissi:Abbvie: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria. Katodritou:Theagenion Cancer Hospital: Current Employment; Takeda: Honoraria, Other: Expenses, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Delimpasi:GENESIS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Symeonidis:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding. Stamatopoulos:AstraZeneca: Honoraria; Janssen, Gilead, Abbvie: Honoraria, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau. Terpos:Amgen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Sanofi: Honoraria; BMS: Honoraria. Chatzidimitriou:Janssen: Research Funding.

Abstract INTRODUCTION: T-prolymphocytic leukemia (T-PLL) is a rare T cell malignancy with an aggressive clinical course. Preliminary clinical data suggest that allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in a proportion of patients. However, direct evidence that graft-versus-leukemia (GVL) activity is indeed effective in T-PLL is lacking. We sought to investigate GVL in T-PLL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions (immunosuppression tapering, donor lymphocyte infusions (DLI), chronic graft-versus-host disease (cGvHD)), and T cell receptor (TCR) repertoire diversity alterations after alloSCT. METHODS: The study sample consisted of 10 consecutive patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma (TRG) gene rearrangements. Data interpretation followed EuroMRD guidelines. In selected patients, TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS). TRBV-TRBD-TRBJ gene rearrangements were amplified according to BIOMED2 protocol on genomic DNA. PCR products were sequenced on Illumina's MiSeq platform. NGS data were analyzed through a purpose-built bioinformatics immunoprofiler (EuroClonality-NGS consortium). Rearrangements with very similar junctional amino acid sequences and identical TRBV and TRBJ gene usage were defined as clonotypes. RESULTS: Patients underwent alloSCT in remission after first-line (8) or salvage (2) alemtuzumab-based therapy. 5 patients were allografted with an unrelated donor, 4 with a related donor, and one received haploidentical alloSCT. Conditioning was fludarabine with cyclophosphamide and/or total body irradiation-based. All patients had a cytological complete response (CR) after alloSCT. 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 81+ and 12+ months post transplant), whilst MRD re-increased in 3 patients after 5-28 months despite ongoing cGvHD in one of them. NGS of the TCR repertoire was performed longitudinally in 3 patients with the longest follow-up. A total of 104 samples (blood (n=91) and BM (n=10) plus 3 donor blood samples) were sequenced. The sample at diagnosis showed one or two major clonotypes in all 3 sequenced T-PLLs, predominantly reflecting a mono- or biallelic leukemic TRB gene rearrangement. Kinetics of this leukemic clonotype followed kinetics of RQ-PCR MRD measurement, demonstrating that NGS can be used to quantify MRD in T cell lymphoma. Immediately after transplantation, the TRB repertoire was heavily skewed in all 3 patients, but recovered over time. Also in all 3 patients, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. This corresponded to the donor clonotype repertoire and disappeared with increasing MRD levels. In each of the samples after alloSCT, several expanded non-leukemic clonotypes were observed at a maximum frequency from 3% to 54% of total TRB sequences. However, during MRD response, novel dominant clonotypes that could explain a clonal GVL effect did not emerge. Figure 1 shows an example of a patient repeatedly showing MRD response to immune interventions. Currently, 5 patients are alive and in cytological CR (4-81 months after alloSCT), translating into a median relapse-free survival of 40 months. CONCLUSIONS: The MRD responses to immune interventions observed here provide the first direct evidence for the efficacy of GVL in T-PLL. However, the GVL effect in T-PLL appears to be often only limited or transient. It also does not seem to be caused by the emergence of novel dominant T cell clones but is rather relying on a poly-/oligoclonal T cell response. Figure 1. Example for MRD kinetics in relation to immune interventions. Figure 1. Example for MRD kinetics in relation to immune interventions. Disclosures No relevant conflicts of interest to declare.

ABSTRACT The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production ofAgrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58ΔaccR mutations intrbB, -C, -D, -E, -L, -F, -G, and -Habolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbImutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation intraI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of thetrb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Abstract Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.

ABSTRACT Using yeast two-hybrid screens, we have defined an interaction group of six Tra proteins encoded by the F plasmid and required by F+ cells to elaborate F pili. The six proteins are TraH, TraF, TraW, TraU, TrbI, and TrbB. Except for TrbI, these proteins were all identified as hallmarks of F-like type IV secretion systems (TFSSs), with no homologues among TFSS genes of P-type or I-type systems (T. Lawley, W. Klimke, M. Gubbins, and L. Frost, FEMS Microbiol. Lett. 224:1-15, 2003). Also with the exception of TrbI, which is an inner membrane protein, the remaining proteins are or are predicted to be periplasmic. TrbI consists of one membrane-spanning segment near its N terminus and an 88-residue, hydrophilic domain that extends into the periplasm. Hence, the proteins of this group probably form a periplasmic cluster in Escherichia coli. The interaction network identifies TraH as the most highly connected node, with two-hybrid links to TrbI, TraU, and TraF. As measured by transcriptional activation of lacZ, the TrbI-TraH interaction in Saccharomyces cerevisiae requires the TraH amino acid segment from residues 193 to 225. The TraU and TraF interactions are localized to C-terminal segments of TraH (amino acids 315 to 458 for TraF and amino acids 341 to 458 for TraU). The TrbI-TraH interaction with full-length (less the signal peptide) TraH is weak but increases 40-fold with N-terminal TraH deletions; the first 50 amino acids appear to be critical for inhibiting TrbI binding in yeast. Previous studies by others have shown that, with the exception of trbB mutations, which do not affect the elaboration or function of F pili under laboratory conditions, a mutation in any of the other genes in this interaction group alters the number or length distribution of F pili. We propose a model whereby one function of the TraH interaction group is to control F-pilus extension and retraction.

Aktualisierung der Technischen Regel zur Vermeidung von Brand-, Explosions- und Druckgefährdungen an Tankstellen und Gasfüllanlagen zur Befüllung von Landfahrzeugen: Im Oktober 2012 erschien als Ersatz für die frühere TRbF 40 [1] die TRBS 3151/TRGS 751 [2], die Anforderungen zur Vermeidung von Brand-, Explosions- und Druckgefährdungen an Tankstellen und Gasfüllanlagen zur Befüllung von Landfahrzeugen enthält. Bereits in 2015 erschien eine Neufassung dieser Regel [3], in der neben Änderungen in der Struktur der Regel auch eine Anpassung der TRBS 3151/TRGS 751 an die Novelle der Betriebssicherheitsverordnung und die Änderung der Gefahrstoffverordnung vom 3. Februar 2015 [4, 5] erfolgte.

Abstract In patients with B lymphoid malignancies, depletion of B cells by Rituximab, an anti-CD20 humanized monoclonal antibody, can induce changes in the subset composition, activation and function of T cells. The spectrum of resultant immune-mediated sequelae encompasses organ-specific manifestations (e.g. pneumonitis, gastritis) as well as Rituximab-associated late onset neutropenia (R-LON). Although the pathogenesis of these clinical syndromes is not fully elucidated, evidence suggests that at least a fraction of cases may develop in a setting of expanded cytotoxic T cell populations with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Similar cytotoxic T cell expansions can be observed after Rituximab administration in other clinical settings e.g. allogeneic transplantation (allo-HCT), where selective restriction of the T-cell receptor (TR) gene repertoire is probably driven by multifactorial mechanisms. Here, we sought to obtain more insight into this phenomenon by molecular immunoprofiling of the TR gene repertoire in two groups of patients who received Rituximab: (i) Group A: patients (n=10) with chronic lymphocytic leukemia (CLL) treated with fludarabine-cyclophosphamide-rituximab (FCR); and, Group B: patients (n=14) who underwent allo-HCT for hematologic malignancies and received Rituximab either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. Each patient included in the study received a mean of 6 cycles of Rituximab (range, 1-14). TR repertoire analysis was performed 11-88 (median, 36) and 5-24 months (median, 5) after the first Rituximab administration in Group A and Group B, respectively. TR beta gene rearrangements were PCR amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data were analyzed using the IMGT/V-QUEST tool. A total of 579 productive TRBV-TRBD-TRBJ rearrangements were analysed, 291 for Group A, 288 for Group B (6-91/case, median=20). Among the 46 TRBV functional genes identified, 3 accounted for >25% of cases in both groups: (i) TRBV27 (13% in both Groups A and B); ii) TRBV19-1 (13% in group A, 7% in group B); and, (iii) TRBV6-1 (7% and 6%, respectively). Clusters of identical (>=2) rearrangements corresponding to clonotypes were identified in all patients. Oligoclonality with immunodominant clonotypes (>12% of the repertoire) accounting for over 30% of the analyzed sequences was more frequent in Group A (7/10 cases) versus Group B (5/10 cases); however, larger clonotype expansions were seen in group B. Longitudinal analysis was performed in 3 patients with oligoclonality, 1 from group A and 2 from Group B: in the Group A patient, immunodominant clonotypes disappeared, while both patients in Group B retained the oligoclonal repertoire. Lymphocyte subpopulation analysis by flow cytometry was performed in 6 patients of each group. T-LGL proliferations (defined as CD4/CD8 abnormal ratio and CD3+CD8+CD57+ >30%) were found more often in Group B (3/6 cases in Group A versus 6/6 in Group B). They were related to oligoclonal TR gene repertoire in Group A (3/3) but not in Group B patients (3/6 cases). However, true expansions could be considered only in group B patients, since CD8+ lymphocytes >1.0*109/l were seen in all 6 Group B versus only 1/6 group A cases. Self-limiting R-LON was observed in 8 patients (4 in each group), but no association of oligoclonality to R-LON could be found. In conclusion, we report frequent development of oligoclonal T cell populations after Rituximab treatment in two different clinical contexts. The Groups analyzed differed with respect to the extent of oligoclonality, suggesting that the precise clinical setting determines the amplitude of TR repertoire skewing after Ritximab. Sustained oligoclonal cytotoxic expansions were recognised more often among allo-HCT patients, presenting with a highly restricted TR gene repertoire and likely reflecting strong antigenic stimulation by viruses and/or cGVHD aggravated by T cell imbalances induced by Rituximab. Disclosures Stamatopoulos: Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

ABSTRACT Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the cag pathogenicity island of Helicobacter pylori (HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.

Abstract Using next-generation sequencing (NGS), we recently documented the clonal architecture of the T cell repertoire in treatment-naive chronic lymphocytic leukemia (CLL), with ample immunogenetic evidence indicating selection by restricted antigens. Our preliminary NGS study in 16 patients pre- and 3-month post-treatment indicated a differential impact of standard chemoimmunotherapy (FCR) versus B cell receptor signaling inhibitors (BcRi) on CLL T cells. Prompted by these observations, here we sought to comprehensively assess CLL T cell repertoire changes over treatment in relation to both treatment type and clinical response by combining NGS immunoprofiling, flow cytometry and functional assays. NGS profiling of the T cell receptor (TR) gene repertoire was performed in 28 CLL patients who received FCR (n=9), ibrutinib (IB, n=15) and/or rituximab-idelalisib (R-ID, n=10) at successive timepoints (pre, +3mo, +9mo and at deepest clinical response, total samples: n=113). TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw reads were processed through a purpose-built, validated bioinformatics pipeline, culminating to 20,347,768 productive, filtered-in TRB sequences (median 155,479/sample). For repertoire analysis, clonotypes (i.e. rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,420 distinct clonotypes/sample). All cases displayed significant clonal T cell expansions both pre- and post-treatment [median clonality, measured as the cumulative frequency of the 10 most expanded (major) clonotypes/sample: 30.3% and 39.6%, respectively]. Median clonality significantly increased at +3mo in the FCR (29.0% to 46.9%, p<.001) and R-ID group (33.0% to 39.1%, p<.001), but not in the IB group (33.3% to 31.2%, p>.05). Overtime analysis revealed a gradual increase of clonality over deepening clinical response (pre-, +3mo, +9mo, deepest response) in the R-ID group (33.0% to 39.1% to 46.0% to 46.1%, respectively; p<.001), but only a trend in this respect for IB (33.3% to 31.2% to 33.8% to 42.0%; p>.05). Considering that FCR resulted in T cell repertoire reconstitution whereas BcRis retained pre-treatment clones, we then focused on major clones persisting over treatment and found that they significantly expanded in the R-ID group, peaking at +3mo (p<.01). Cross-comparison across all CLL patients and against 767,438 unique TRB sequences retrieved from multiple public databases (HSV infections, T-cell lymphoproliferations, autoimmune disorders, healthy individuals), revealed 23/563 major clonotypes shared exclusively among CLL patients, alluding to selection by conserved CLL-related antigens. We then sought to test the functional effect of treatments on T cells. To this end, we evaluated activation markers on CLL T cell subpopulations for 8 CLL patients (R-ID, n=4; IB, n=4) pre- and +3mo post-treatment by flow cytometry and found statistically significant upregulation of T cell activation markers for R-ID compared to IB, particularly for: (i) CD69 in CD4+ effector memory T cells (p<.01); (ii) CD25 in CD8+ TEMRA T cells (p=0.006); and, (iii) CD38 in CD8+ effector memory T cells (p<.05) and CD8+ TEMRA T cells (p<.05). We also investigated the ability of CD3+ T cells, purified from 13 patients pre- and +3mo post-treatment (FCR, n=3; R-ID, n=5; IB, n=5), to form immune synapses with autologous pre-treatment CD19+ tumor cells. Quantitative relative recruitment index (RRI) analysis for F-actin showed that both R-ID (p<.01) and IB (p<.05) treated T cells form polarized immune synapses in contrast to FCR (p>.05). Taken together, NGS immunoprofiling suggests that BcRis retain T cell clones that may have developed in response to CLL-related antigens, which in the case of R-ID expand and peak at +3mo. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response. Overall, this data provides rationale for designing combination strategies, e.g. of R-ID with immunomodulating drugs, aiming to boost cytotoxic anti-tumor responses. Moreover, identifying the relevant neoepitopes may eventually pave the way for stratified treatments by means of engineered T cells or peptide vaccines, especially if these epitopes are conserved among CLL. Disclosures Vardi: Janssen: Honoraria; Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

464 Background: VEGF targeted therapy (VEGF-TT) are standard in advanced metastatic renal cell carcinoma (mRCC), however, toxicities that can lead to drug discontinuation can have a significant impact on patient (pt) outcomes. We aimed to identify risk factors for toxicity and develop the first model to predict toxicity-related treatment discontinuation (TrRD) in mRCC pts treated with VEGF-TT. Methods: Baseline characteristics and treatment outcome data were collected on 936 mRCC pts on first-line VEGF-TT from 7 IMDC institutions. TrTD was analyzed using a competing risk regression model for treatment discontinuation. Results: Median follow up was 23 months. Treatment discontinuation occurred in 833 pts (89%), of which 198 (23.8%) were related to drug toxicity. Sunitinib was the most common VEGF-TT (77%) in our series followed by sorafenib (18.4%). Median time on therapy was 7.1 months in all pts and 4.4 months for pts with TrTD. Most common toxicities leading to TrTD included fatigue, diarrhea and mucositis. On multivariate analysis, significant adverse predictors for TrTD (p<0.05) were: age (≥60 years), baseline glomerular filtration rate (GFR) <30 cc/min, number of metastatic sites (>1), and baseline sodium level (<LLN). A model was developed using the number of patient risk factors to predict the risk of TrTD (Table). Conclusions: In the largest series reported to date, age, GFR, number of metastatic sites, and baseline sodium level were found to be independent risk factors that predict toxicity-related treatment discontinuation in mRCC pts treated with VEGF-TT. Based on the number of risk factors present, we built the first model to predict treatment-related drug discontinuation. This model can be used for treatment and frequency of monitoring considerations in clinical practice. [Table: see text]

Abstract Epo cytoprotection of developing erythroblasts mechanistically is thought to involve 1] PI3K and Akt pathways (eg. mTOR, Foxo3A inhibition) as engaged via a distal EpoR PY479 site, and 2] anti-apoptotic effects as exerted by EpoR proximal box1 and PY343 signals (eg. Bcl-xl transcription). To globally assess Epo-regulation of survival- associated genes, a maximally Epo-responsive cohort of bone-marrow derived KitposCD71high erythroblasts was prepared, purified and used in Affymetrix 430–2.0 array based profiling of Epo responsive genes. Quadruplicate array analyses and RT-QPCR revealed selective modulation of PI3K pathway associated factors Irs2, p85-alpha, Trb3, and Foxo3a as well as the Bcl2-related factor Bim1. Irs2, p85-alpha and Trb3 were induced three- to five-fold, while Foxo3a was rapidly down-modulated. As studied using knocked-in minimal EpoR alleles, induction of Irs2 and Trb3 (a pseudo-kinase Akt inhibitor) depended sharply upon an EpoR PY343 Stat5 binding site. Epo down-modulation of Foxo3a (as well as Trb2 and Bim1), in contrast, occurred via EpoR PY-independent routes. Unexpectedly, the EpoR allele, EpoR-H-Y343 also protected erythroblasts from LY294002 and wortmanin induced apoptosis, while an EpoR-HM-F343 allele was hyper-sensitive. For EpoRH, this proved to correlate specifically with Y343 dependent phosphorylation of GAB2. PY343 couples specifically to Stat5, and phospho-Stat5 therefore is proposed to recruit GAB2 and enable its PY452 phosphorylation. GAB2 then provides a shunt to PI3K activation. Findings provide new insight into Epo-regulation of a select set of PI3K-associated genes, and core EpoR cytoprotective mechanisms.

The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the ‘pseudocatalytic’ domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an ‘analogue-sensitive’ (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.

<p class="Body1" style="text-align: justify;"><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">Presentamos el caso de un paciente de 23 meses de edad quien desarrollo par</span><span style="font-size: 12.0pt; font-family: ";Arial Unicode MS";,";sans-serif";; mso-ascii-font-family: Arial; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">á</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">lisis facial unilateral secundaria a Enfermedad de Lyme sin s</span><span style="font-size: 12.0pt; font-family: ";Arial Unicode MS";,";sans-serif";; mso-ascii-font-family: Arial; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">í</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">ntomas otol</span><span style="font-size: 12.0pt; font-family: ";Arial Unicode MS";,";sans-serif";; mso-ascii-font-family: Arial; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">ó</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">gicos previos y en Colombia que es considerada </span><span style="font-size: 12.0pt; font-family: ";Arial Unicode MS";,";sans-serif";; mso-ascii-font-family: Arial; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">á</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">rea no end</span><span style="font-size: 12.0pt; font-family: ";Arial Unicode MS";,";sans-serif";; mso-ascii-font-family: Arial; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">é</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-hansi-font-family: ";Arial Unicode MS";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">mica.</span><span style="font-size: 12.0pt; font-family: ";Arial";,";sans-serif";; mso-bidi-font-family: ";Times New Roman";; color: windowtext; mso-ansi-language: ES-TRAD;" lang="ES-TRAD"></span></p>

<p style="margin: 0cm 0cm 0pt;"><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">La</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">competencia</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">digital</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">desempeña</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">un</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">papel</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">fundamental</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">en</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">formación</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">de</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">los</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">estudiantes</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">actualmente.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Sólo</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tenemos</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">considerar</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tecnología</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">es</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">ubicua</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">en</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">vida</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">de</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">los</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">estudiantes</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">también</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">en</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">sus</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">rutinas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">diarias.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Las</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">escuelas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">deben</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">plantearse</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">cómo</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">implementar</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">e</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">integrar</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">en</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">el</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">currículo</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tecnología</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">educativa.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">La</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">alfabetización</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">mediática</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">se</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">refiere</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">al</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">conocimiento</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">destrezas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">el</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">alumnado</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">debe</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">adquirir</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">para</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">poder</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">analizar,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">evaluar</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">crear</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">mensajes</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">mediáticos.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Este</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">artículo</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">trata</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">sobre</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">alumnado</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">entre</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">8</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">11</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">años</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">no</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tiene</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">formación</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">multimedia</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">previa,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">aprende</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">lo</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">básico</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">sobre</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">competencia</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">digital,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">así</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">como</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">el</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">impacto</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">esa</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">formación</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tiene</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">en</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">su</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">progreso</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">académico.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Dada</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">edad</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">de</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">la</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">muestra,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">las</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">destrezas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">mediáticas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">trabajadas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">no</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">han</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">ido</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">más</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">allá</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">de</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">las</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tecnológicas,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">aunque</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">algunas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">habilidades</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">evaluadoras</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">críticas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">se</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">han</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">practicado</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">oralmente.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Los</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">resultados</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">muestran</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">los</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">estudiantes</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">han</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">mejorado</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">significativamente</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">sus</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">destrezas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">básicas</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">también</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">su</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">conocimiento</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">sobre</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">los</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">media.</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">Sin</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">embargo,</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">no</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">podemos</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">concluir</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">que</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">una</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">formación</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">tan</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">corta</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">y</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">básica</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">impacte</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">su</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">progreso</span><span style="font-family: 'Century Gothic',sans-serif; font-size: 11pt; mso-bidi-font-family: 'Century Gothic';" lang="ES-TRAD">académico.</span></p>

<p class="Formatolibre" style="margin-bottom: 10.0pt; text-align: justify;"><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">Memorias de un periodista musical </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">es una novela gráfica que realizo con Santi Carrillo, periodista musical y director de </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">Rockdelux</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">. Las memorias tienden a la evocación abstracta de recuerdos, sensaciones y emociones; por eso, su texto literario me ha planteado una serie de desafíos formales a la hora de transformarlo en una narración en imágenes, particularmente en pasajes ensayísticos y de memoria sobre su infancia o la muerte temprana de su padre. La obra, aún en proceso, se estructura en capítulos asociados a canciones concretas que forman parte de su biografía y memoria. </span></p><p class="Formatolibre" style="margin-bottom: 10.0pt; text-align: justify;"><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">El cómic tradicional ha sido durante la mayor parte de su historia una forma / medio enfocado a la acción concreta y </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">externa</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">: la comedia de </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">slapstick</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">, ya desde sus comienzos como medio de masas a finales del siglo XIX, y la ficción de aventuras (exóticas, ciencia ficción, superheroicas, etc.) han nutrido la mayoría del cómic del siglo XX, juvenil. No ha sido hasta décadas recientes cuando un cómic artístico para adultos, la llamada novela gráfica, ha abordado otras temáticas: la memoria, la historia, la biografía, lo ensayístico; en resumen, la </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">realidad</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">. Estas nuevas temáticas exigen la elaboración de </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">nuevos recursos formales</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD"> para representar el mundo </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">interior</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD"> de ideas, emociones y sensaciones. En </span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri Italic','serif'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD">Memorias de un periodista musical</span><span style="font-size: 9.0pt; mso-bidi-font-size: 10.0pt; font-family: 'Calibri','sans-serif'; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: ES-TRAD;" lang="ES-TRAD"> he acudido al uso de diseños alegóricos, metáforas visuales y la combinación de diversos lenguajes (contrastes entre dibujo y fotografía) y estilos representacionales (de la caricatura al realismo).</span></p>

<p class="CuerpoAA"><span lang="ES-TRAD">En este artículo se </span><span class="Ninguno"><span lang="ES-TRAD">plantea la presencia </span></span><span lang="ES-TRAD">y función </span><span class="Ninguno"><span lang="ES-TRAD">semántica</span></span><span lang="ES-TRAD">del paisaje en el cartel cinematográfico contemporáneo. Se realiza inicialmente un breve recorrido histórico </span><span class="Ninguno"><span lang="ES-TRAD">sobre el </span></span><span lang="ES-TRAD">cartel de cine, se repasan los diversos estilos en los que se inscriben; además de establecerse una definición, tanto conceptual como gráfica. A tal efecto, se </span><span class="Ninguno"><span lang="ES-TRAD">enuncian</span></span><span lang="ES-TRAD"> los elementos gráficos y tipográficos que lo conforman y se estudian siguiendo las corrientes </span><span class="Ninguno"><span lang="ES-TRAD">nortea</span></span><span lang="ES-TRAD">mericanas y europeas. </span><span lang="ES-TRAD">El cartel de cine se presenta como una síntesis visual del argumento fílmico y de la identidad visual de la película. Debido a esta vinculación entre la imagen representacional y el contenido </span><span class="Ninguno"><span lang="ES-TRAD">del film </span></span><span lang="ES-TRAD">se analizan varios carteles, examinando sus aspectos gráficos en relación a la representación del paisaje en los mismos. La ausencia o presencia de esta representación paisajística, y su interacción con el resto de elementos que conforman el cartel, revelan la importancia que est</span><span class="Ninguno"><span lang="ES-TRAD">a</span></span><span lang="ES-TRAD"> ha tenido en su uso fílmico.</span></p>

In this paper we present the research and implementation of TRFD, a Trusted Removable USB Flash Device which can resist various attacks. TRFD is an independent system which would authenticate the user and host before data access. All the data secure processing of TRFD, such as encrypting, signing, are performed inside the device, so TRFD can protect the data against host hacking. The evaluation of our prototype shows that TRFD can solve some security issues of removable storages indeed.

ObjectivesTribbles homologue 3 (TRB3) is a pseudokinase that modifies the activation of various intracellular signalling pathways to control fundamental processes extending from mitosis and cell activation to apoptosis and modulation of gene expression. Here, we aimed to analyse the role of TRB3 in fibroblast activation in systemic sclerosis (SSc).MethodsThe expression of TRB3 was quantified by quantitative PCR, western blot and immunohistochemistry. The role of TRB3 was analysed in cultured fibroblasts and in experimental fibrosis using small interfering RNA (siRNA)-mediated knockdown and overexpression of TRB3.ResultsTRB3 expression was increased in fibroblasts of patients with SSc and in murine models of SSc in a transforming growth factor-β (TGF-β)/Smad-dependent manner. Overexpression of TRB3 stimulated canonical TGF-β signalling and induced an activated phenotype in resting fibroblasts. In contrast, knockdown of TRB3 reduced the profibrotic effects of TGF-β and decreased the collagen synthesis. Moreover, siRNA-mediated knockdown of TRB3 exerted potent antifibrotic effects and ameliorated bleomycin as well as constitutively active TGF-β receptor I-induced fibrosis with reduced dermal thickening, decreased hydroxyproline content and impaired myofibroblast differentiation.ConclusionsThe present study characterises TRB3 as a novel profibrotic mediator in SSc. TGF-β induces TRB3, which in turn activates canonical TGF-β/Smad signalling and stimulates the release of collagen, thereby inducing a positive feedback loop that may contribute to aberrant TGF-β signalling in SSc.

Increasing evidence suggests that TRB3, a mammalian homolog of Drosophila tribbles, plays an important role in cell growth, differentiation, and metabolism. In the liver, TRB3 binds and inhibits Akt activity, whereas in adipocytes, TRB3 upregulates fatty acid oxidation. In cultured muscle cells, TRB3 has been identified as a potential regulator of insulin signaling. However, little is known about the function and regulation of TRB3 in skeletal muscle in vivo. In the current study, we found that 4 wk of voluntary wheel running (6.6 ± 0.4 km/day) increased TRB3 mRNA by 1.6-fold and protein by 2.5-fold in the triceps muscle. Consistent with this finding, muscle-specific transgenic mice that overexpress TRB3 (TG) had a pronounced increase in exercise capacity compared with wild-type (WT) littermates (TG: 1,535 ± 283; WT: 644 ± 67 joules). The increase in exercise capacity in TRB3 TG mice was not associated with changes in glucose uptake or glycogen levels; however, these mice displayed a dramatic shift toward a more oxidative/fatigue-resistant (type I/IIA) muscle fiber type, including threefold more type I fibers in soleus muscles. Skeletal muscle from TRB3 TG mice had significantly decreased PPARα expression, twofold higher levels of miR208b and miR499, and corresponding increases in the myosin heavy chain isoforms Myh7 and Myb7b, which encode these microRNAs. These findings suggest that TRB3 regulates muscle fiber type via a peroxisome proliferator-activated receptor-α (PPAR-α)-regulated miR499/miR208b pathway, revealing a novel function for TRB3 in the regulation of skeletal muscle fiber type and exercise capacity.

The article presents a simulation model of a thyristor- reactor switching device (TRSD) created in the Matlab Simulink program. The model of a TRSD single- phase connected to a single-phase transformer has been created, and transients that occur during voltage regulation in a single-phase circuit are considered and analyzed. Supplement was introduced to the method for calculating TRSD thyristors. The developed model of a single phase of TRSD can be used to create models of converter transformers on traction substations with TRSD.

ABSTRACT TRB3 has been implicated in the regulation of several biological processes in mammalian cells through its ability to influence Akt and other signaling pathways. In this study, we investigated the role of TRB3 in regulating adipogenesis and the activity of adipogenic transcription factors. We find that TRB3 is expressed in 3T3-L1 preadipocytes, and this expression is transiently suppressed during the initial days of differentiation concomitant with induction of C/EBPβ. This event appears to be a prerequisite for adipogenesis. Overexpression of TRB3 blocks differentiation of 3T3-L1 cells at a step downstream of C/EBPβ. Ectopic expression of TRB3 in mouse fibroblasts also inhibits the C/EBPβ-dependent induction of PPARγ2 and blocks their differentiation into adipocytes. This inhibition of preadipocyte differentiation by TRB3 appears to be the result of two complementary effects. First, TRB3 inhibits extracellular signal-regulated kinase activity, which prevents the phosphorylation of regulatory sites on C/EBPβ. Second, TRB3 directly interacts with the DR1 domain of C/EBPβ in the nucleus, further inhibiting both its ability to bind its response element and its ability to transactivate the C/EBPα and a-FABP promoters. Thus, TRB3 is an important negative regulator of adipogenesis that acts at an early step in the differentiation cascade to block the C/EBPβ proadipogenic function.

Tribbles 3 (TRB3) is a recently recognized atypical inactive kinase that negatively regulates Akt activity in hepatocytes, resulting in insulin resistance. Recent reports link TRB3 to nutrient sensing and regulation of cell survival under stressful conditions. We studied the regulation of TRB3 by glucose, insulin, dexamethasone (Dex), and the unfolded protein response (UPR) in 3T3-L1 adipocytes and in L6 myotubes. In 3T3-L1 adipocytes, incubation in high glucose with insulin did not increase TRB3 mRNA expression. Rather, TRB3 mRNA increased fourfold with glucose deprivation and two- to threefold after incubation with tunicamcyin (an inducer of the UPR). Incubation of cells in no glucose or in tunicamcyin stimulated the expression of CCAAT/enhancer-binding protein homologous protein. In L6 myotubes, absent or low glucose induced TRB3 mRNA expression by six- and twofold, respectively. The addition of Dex to 5 mM glucose increased TRB3 mRNA expression twofold in 3T3-L1 adipocytes but decreased it 16% in L6 cells. In conclusion, TRB3 is not the mediator of high glucose or glucocorticoid-induced insulin resistance in 3T3-L1 adipocytes or L6 myotubes. TRB3 is induced by glucose deprivation in both cell types as a part of the UPR, where it may be involved in regulation of cell survival in response to glucose depletion.

The prevalence of diabetic nephropathy continues to rise, highlighting the importance of investigating and discovering novel treatment strategies. TRB3 is a kinase-like molecule that modifies cellular survival and metabolism and interferes with signal transduction pathways. Herein, we report that TRB3 expression is increased in the kidneys of type 1 and type 2 diabetic mice. TRB3 is expressed in conditionally immortalized podocytes; however, it is not stimulated by elevated glucose. The diabetic milieu is associated with increased oxidative stress and circulating free fatty acids (FFA). We show that reactive oxygen species (ROS) such as H2O2 and superoxide anion (via the xanthine/xanthine oxidase reaction) as well as the FFA palmitate augment TRB3 expression in podocytes. C/EBP homologous protein (CHOP) is a transcription factor that is associated with the endoplasmic reticulum stress response. CHOP expression increases in diabetic mouse kidneys and in podocytes treated with ROS and FFA. In podocytes, transfection of CHOP increases TRB3 expression, and ROS augment recruitment of CHOP to the proximal TRB3 promoter. MCP-1/CCL2 is a chemokine that contributes to the inflammatory injury associated with diabetic nephropathy. In these studies, we demonstrate that TRB3 can inhibit basal and stimulated podocyte production of MCP-1. In summary, enhanced ROS and/or FFA associated with the diabetic milieu induce podocyte CHOP and TRB3 expression. Because TRB3 inhibits MCP-1, manipulation of TRB3 expression could provide a novel therapeutic approach in diabetic kidney disease.

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, inflammation, and angiogenesis. Overexpression of tribbles homolog3 (TRB3), which belongs to the tribbles family of pseudokinases, has been found in several human tumors and metabolic diseases, but its role in psoriasis has not been fully clarified. The aim of this study is to investigate the expression of TRB3 in psoriasis and explore its roles in the proliferation of keratinocytes. Twenty-four patients with psoriasis vulgaris were recruited for the study. Diagnosis of psoriasis was based on clinical and histologic examinations. Immunohistochemistry and real-time reverse transcription PCR (RT-PCR) were performed to determine protein and messenger RNA (mRNA) expression of TRB3 in psoriasis lesions. 5-Bromo-2-deoxyUridine (BrdU) incorporation assay were performed for cell proliferation. Cell cycle distribution was assessed by flow cytometry analysis. The levels of TRB3 is elevated in psoriatic lesions compared with psoriatic non-lesions. The HaCat cells expressed the TRB3 gene. We found TRB3 silencing to significantly inhibit HaCat cell proliferation. Furthermore, the specific knockdown of TRB3 slowed down the cell cycle at the gap 0/first gap phase. In conclusion, our data suggest that TRB3 is overexpressed in lesions of patients with psoriasis and may be involved in the abnormal proliferation of keratinocytes. Therefore, TRB3 may be a potential therapeutic target for psoriasis.

Thyroid hormones (THs) induce pleiotropic effects in vertebrates, mainly through the activation or repression of gene expression. These mechanisms involve thyroid hormone binding to thyroid hormone receptors, an event that is followed by the sequential recruitment of coactivator or corepressor proteins, which in turn modify the rate of transcription. In the present study, we looked for specific coregulators recruited by the long isoform of the teleostean thyroid hormone receptor beta 1 (L-Trb1) when bound to the bioactive TH, 3,5-T2 (T2). We found that jun activation domain-binding protein1 (Jab1) interacts with L-Trb1 + T2 complex. Using both the teleostean and human TRB1 isoforms, we characterized the Jab1–TRB1 by yeast two-hybrid, pull-down and transactivation assays. Our results showed that the TRB1–Jab1 interaction was ligand dependent and involved the single Jab1 nuclear receptor box, as well as the ligand-binding and N-terminal domains of TRB1. We also provide evidence of ligand-dependent, dual coregulatory properties of Jab1. Indeed, when T2 is bound to L-Trb1 or hTRB1, Jab1 acts as a coactivator of transcription, whereas it has corepressor activity when interacting with the T3-bound S-Trb1 or hTRB1. These mechanisms could explain some of the pleiotropic actions exerted by THs to regulate diverse biological processes.