T cell receptor gene repertoire profiles in subgroups of patients with chronic lymphocytic leukemia bearing distinct genomic aberrations.

Background: Microenvironmental interactions of the malignant clone with T cells are critical throughout the natural history of chronic lymphocytic leukemia (CLL). Indeed, clonal expansions of T cells and shared clonotypes exist between different CLL patients, strongly implying clonal selection by antigens. Moreover, immunogenic neoepitopes have been isolated from the clonotypic B cell receptor immunoglobulin sequences, offering a rationale for immunotherapeutic approaches. Here, we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles aiming to identify unique signatures that would point towards an additional source of immunogenic neoepitopes for T cells. Experimental design: TR gene repertoire profiling using next generation sequencing in groups of patients with CLL carrying one of the following copy-number aberrations (CNAs): del(11q), del(17p), del(13q), trisomy 12, or gene mutations in TP53 or NOTCH1. Results: Oligoclonal expansions were found in all patients with distinct recurrent genomic aberrations; these were more pronounced in cases bearing CNAs, particularly trisomy 12, rather than gene mutations. Shared clonotypes were found both within and across groups, which appeared to be CLL-biased based on extensive comparisons against TR databases from various entities. Moreover, in silico analysis identified TR clonotypes with high binding affinity to neoepitopes predicted to arise from TP53 and NOTCH1 mutations. Conclusions: Distinct TR repertoire profiles were identified in groups of patients with CLL bearing different genomic aberrations, alluding to distinct selection processes. Abnormal protein expression and gene dosage effects associated with recurrent genomic aberrations likely represent a relevant source of CLL-specific selecting antigens.

Periodontal disease progression and gene polymorphisms: results after 3 years of active periodontal treatment.

BACKGROUND: Although non-surgical periodontal treatment is considered the gold standard, a subgroup of patients displays recurrence/progression of periodontitis after treatment. The aim of the present prospective study was to assess the effect of IL-6 -572 G/C and IL-10 -592 C/A gene polymorphisms on the risk of disease recurrence/progression at 3 years following non-surgical periodontal treatment. METHODS: Thirty-seven patients diagnosed with chronic periodontitis received oral hygiene instructions and non-surgical periodontal treatment and were monitored for 3 years. All individuals were clinically evaluated for PPD, CAL and BOP at baseline and 3 years. Based on the clinical findings at 3 years, all subjects were considered either "at risk" or "not at risk" of periodontal disease progression based on specific criteria. Blood samples were collected at baseline and genotyping of the polymorphisms in IL-6 (rs1800796) and IL-10 (rs1800872) genes were performed by PCR. RESULTS: Following DNA separation and genotyping, 70.3% of the patients were homozygous carriers of the IL-6 -572G and 45.9% were carriers of the IL-10 -592A allele. Individuals at risk of disease progression ranged from 16.2% to 56.8% based on the criteria used. IL-6 -572 G/C and IL-10 -592 C/A polymorphisms were not associated with an increased risk of further disease progression (P>0.05) when the three criteria were examined. All examined periodontal clinical measures were significantly improved (P<0.05) after treatment. Males showed a significantly higher risk of disease progression than females when full-mouth BOP ≥30% was considered (P=0.008). CONCLUSIONS: Within the limitations of this 3-year prospective study, individuals susceptible to periodontal disease as determined by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele were not associated with an increased risk of further disease progression and the potential need for further treatment following non-surgical periodontal treatment. Males were more prone to be at risk of disease progression than females.

The TΑp63/BCL2 axis represents a novel mechanism of clinical aggressiveness in chronic lymphocytic leukemia.

The TA-isoform of the p63 transcription factor (TAp63) has been reported to contribute to clinical aggressiveness in chronic lymphocytic leukemia (CLL) in a hitherto elusive way. Here, we sought to further understand and define the role of TAp63 in the pathophysiology of CLL. First, we found that elevated TAp63 expression levels are linked with adverse clinical outcomes, including disease relapse and shorter time-to-first treatment and overall survival. Next, prompted by the fact that TAp63 participates in an NF-κB/TAp63/BCL2 antiapoptotic axis in activated mature, normal B cells, we explored molecular links between TAp63 and BCL2 also in CLL. We documented a strong correlation at both the protein and the messenger RNA (mRNA) levels, alluding to the potential prosurvival role of TAp63. This claim was supported by inducible downregulation of TAp63 expression in the MEC1 CLL cell line using clustered regularly interspaced short palindromic repeats (CRISPR) system, which resulted in downregulation of BCL2 expression. Next, using chromatin immunoprecipitation (ChIP) sequencing, we examined whether BCL2 might constitute a transcriptional target of TAp63 and identified a significant binding profile of TAp63 in the BCL2 gene locus, across a genomic region previously characterized as a super enhancer in CLL. Moreover, we identified high-confidence TAp63 binding regions in genes mainly implicated in immune response and DNA-damage procedures. Finally, we found that upregulated TAp63 expression levels render CLL cells less responsive to apoptosis induction with the BCL2 inhibitor venetoclax. On these grounds, TAp63 appears to act as a positive modulator of BCL2, hence contributing to the antiapoptotic phenotype that underlies clinical aggressiveness and treatment resistance in CLL.

Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo.

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from ß-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant ß0/ß0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe ß-globin phenotype.

T-Cell Dynamics in Chronic Lymphocytic Leukemia under Different Treatment Modalities.

PURPOSE: Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens. EXPERIMENTAL DESIGN: Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays. RESULTS: T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR. CONCLUSIONS: Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.

Interleukin-6 and interleukin-10 gene polymorphisms and the risk of further periodontal disease progression.

Susceptible genotypes to periodontal disease are associated with disease onset and progression. The aim of this study was to examine the effect of gene polymorphisms on the risk of further disease progression and the need for further treatment among adults with chronic periodontal disease. Sixty-seven patients diagnosed with chronic periodontitis were grouped according to genotype status and risk of further progression of disease and tooth loss. All individuals were clinically evaluated for probing pocket depth, clinical attachment loss and bleeding on probing at baseline and 45 days after treatment. Blood samples were collected at baseline and genotyping of the polymorphisms in IL-6 (rs1800796) and IL-10 (rs1800872) genes were performed by PCR. Following DNA separation and genotyping, 65.7% of the patients were homozygous carriers of the IL-6 -572G and 49.3% were carriers of the IL-10 -592A allele. Individuals at risk of disease progression ranged from 7.5% to 62.7% based on the criteria used. Carriers of the IL-10 -592A allele were significantly associated with BOP ≥ 30% and therefore exhibited a higher risk of further periodontal breakdown (p = 0.018) with an odds ratio of 1.18. None of the other definitions of disease progression were significantly associated with the examined IL-6 and IL-10 genotypes (p > 0.05). IL-10 polymorphism was associated with an increased risk of further disease progression and the potential need for further treatment following non-surgical periodontal treatment. Susceptible IL-6 genotypes were not associated with the risk of persisting or recurrent disease activity.

Interleukin-6 and interleukin-10 gene polymorphisms and the risk of further periodontal disease progression

Abstract: Susceptible genotypes to periodontal disease are associated with disease onset and progression. The aim of this study was to examine the effect of gene polymorphisms on the risk of further disease progression and the need for further treatment among adults with chronic periodontal disease. Sixty-seven patients diagnosed with chronic periodontitis were grouped according to genotype status and risk of further progression of disease and tooth loss. All individuals were clinically evaluated for probing pocket depth, clinical attachment loss and bleeding on probing at baseline and 45 days after treatment. Blood samples were collected at baseline and genotyping of the polymorphisms in IL-6 (rs1800796) and IL-10 (rs1800872) genes were performed by PCR. Following DNA separation and genotyping, 65.7% of the patients were homozygous carriers of the IL-6 −572G and 49.3% were carriers of the IL-10 −592A allele. Individuals at risk of disease progression ranged from 7.5% to 62.7% based on the criteria used. Carriers of the IL-10 −592A allele were significantly associated with BOP ≥ 30% and therefore exhibited a higher risk of further periodontal breakdown (p = 0.018) with an odds ratio of 1.18. None of the other definitions of disease progression were significantly associated with the examined IL-6 and IL-10 genotypes (p > 0.05). IL-10 polymorphism was associated with an increased risk of further disease progression and the potential need for further treatment following non-surgical periodontal treatment. Susceptible IL-6 genotypes were not associated with the risk of persisting or recurrent disease activity.

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human.

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.

Tumour necrosis factor gene polymorphisms and migraine in Greek children.

INTRODUCTION: Migraine is considered to be a multifactorial, complex disease. Various genetic and environmental factors contribute to the manifestation of this disease. The aim of this study was to determine whether polymorphisms in the tumour necrosis factor (TNF) region are associated with the risk of migraine. We examined the association between 6 single nucleotide polymorphisms in the coding regions of TNF-α and TNF-ß genes and migraine. MATERIAL AND METHODS: The study included two groups of children (group A and group B). Group A consisted of 103 unrelated children with typical migraine without aura 5-14 years of age. Group B (control group) consisted of 178 unrelated healthy children. The diagnosis of migraine was, in all patients, made according to the International Classification of Headache Disorders (ICHD II). RESULTS: According to our results positive family history was present in 62.2% of patients of group A. No significant differences were found in the frequencies of genotypes or alleles between patients and controls. The non-parametric analyses of variance showed no significant differences in the age at onset between genotype groups of the TNF-α and TNF-ß gene polymorphisms. Comparison of genotype frequencies between boys and girls in affected patients and control individuals were not significantly different (p = 0.089, p =0.073 respectively). The distribution of TNF polymorphisms was not associated with the presence of family history of migraine in patients. CONCLUSIONS: Our data indicate that TNF-α and TNF-ß gene polymorphisms are not a significant risk factor for migraine without aura in Greek children.

Chronic lymphocytic leukaemia: an immunobiology approach.

B cell chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia that follows an extremely variable clinical course. Several important prognostic parameters defining pathogenic and clinical subgroups of CLL have been identified and validated recently. The biological significance of immunoglobulin (Ig) heavy chain variable region gene (IgHV) mutational status and associated ZAP-70 over-expression, CD38 and chromosomal aberrations have enabled to identify patients at high risk for early disease progression and inferior survival. Moreover, studies of the B cell antigen receptor (BCR) structure and receptor signalling have been most helpful in revealing some new aspects of the biology of this disease. In particular, the analysis of IG genes has revealed that the expressed IgHV/IgKV/IgLV gene repertoires of CLL cells differ from those of normal B cells. A further unique feature of the CLL IG repertoire is the existence of subsets of cases with "stereotyped" BCRs. Accumulating molecular and phenotypic data support the notion that CLL development and evolution is not a simple scholastic event and strongly indicates a role for antigen in driving the cell of origin for at least some subsets of CLL cases.