Transcription factor NF-kB in the prognosis of outcomes for patients undergoing repeat keratoplasty

Background: Obtaining a clear graft after repeat keratoplasty is still a challenging task for corneal transplant surgeons. Corneal transplant rejection rate is significantly increased in repeat keratoplasty compared with first keratoplasty. Activation of nuclear factor kappa B (NF-kB) plays a key role in the pathogenesis of rejection of transplants of various organs and tissues, primarily due to production of proinflammatory cytokines (TNF, IL-1, etc.). Purpose: To assess the role of transcription factor NF-kB in lymphocyte subsets in the prognosis of outcomes for patients undergoing repeat keratoplasty. Material and Methods: Forty-six patients (46 eyes) who underwent repeat keratoplasty were included. Of these, 30 patients (group 1) developed an opacified graft, and 16 (group 2) obtained a clear graft. Patient age ranged from 32 to 88 years. Imaging flow cytometry (ImageStream Mark II – AMNIS) and Amnis NF-kB Translocation Kits (ACS10000, Millipore, Sigma) were used to assess percentages of the cells with translocation of the NF-kB p65 subunit for lymphocyte subsets. Statistical analyses were conducted using Statistica 13.0 (StatSoft, Tulsa, OK, USA) software. The Mann–Whitney test was used to assess significance of differences. Results: Percentages of the cells with translocation of the NF-kB p65 subunit were higher in group 1 compared with group 2, particularly, for T-helpers (26.5[17; 29] vs 12.2[11;21]; p=0.003), cytotoxic T-cells (24.0[17;28] vs 11.5[7;15]; p=0.006), NK cells (45.0[34;53] vs 18.2[16;39]; p=0.014), Th17 cells (23.9[18;31] vs 14.3[12;21]; p=0.002), regulatory T cells (30.4[21;34] vs 17.4[16;22]; p=0.001) and activated T helpers (21.918;28] vs 11.4[11;17]; p=0.002). Conclusion: There were substantially increased percentages of activated cells in lymphocyte subsets (with the most pronounced increase for NK cells) of patients who developed an opacified graft compared to those who obtained a clear graft following re-keratoplasty. Assessment of percentages of the cells with translocation of the NF-kB p65 subunit for lymphocyte subsets provides valuable information for predicting graft rejection following re-keratoplasty

Dynamics of T helper subpopulations in the critical period of severe injury in children

Severe mechanical injury is one of the main reasons behind children’s disability and mortality. Severe injury induces a complex host immune response to tissue injury, a parallel pro- and anti-inflammatory state, bearing an elevated risk for infectious complications (IC) and/or multiple organ failure (MOF). This study aimed to determine the informative immunological criteria of traumatic injury severity and prognosis outcome in children (severe injury group (SInj, ISS ≥ 16), n = 87; mild/moderate injury group (MInj, ISS < 16), n = 34) based on the assessment of absolute cell count (abs) and percentage of such T helper subpopulations as regulatory T lymphocytes – CD4+CD127lowCD25high(Treg), Th17 lymphocytes – CD4+CD161+ and CD4+CD127higtCD25high T cells(T127hi) in severe injury cases grouped by the outcome (favorable, n = 47; unfavorable, n = 40) and depending on IC (n = 16) and the development of MOF (n = 11) on the 1st, 3d , 5th, 7th, 14th day after injury. The control group was comprised of 80 apparently healthy children comparable in age and sex. An inverse relationship between severity of injury, degree of blood loss and outcome of injury was revealed with the abs of all Th populations, but for Th abs and Treg abs the most significant correlation was found (Spearman’s R ≤ -0,70, p < 0.00001). For SInj group, a pronounced decrease of Th abs, Treg abs, T127hi abs and Th17 abs, in the acute post-traumatic period with an increase to 14 days was revealed. The values of in the first day for indicators of patients with MInj group correspond to the values of control group and significantly differ from SInj group. There are different kinetics of percentage Th subpopulations in peripheral blood of children with severe injuries. The Th17%CD4+ and T127hi%CD4+ significant increase in 1st-3d  and 3d -7th days after injury respectively in comparation with сontrol and MInj groups. There were no differences between groups in terms of Treg%CD4+. The lower-level Treg abs in trauma patients admitted to the ICU is significantly associated with develop the infectious complications and outcome of trauma. The Th17 abs is significantly reduced in 3-7th days after the injury in the SInj group with MOF. The results of the study indicate that in children levels of Treg, T127hi and Th17 is significantly associated with severity of injury and may be used to predict outcome of trauma and assess the risk of IC and MOF

Evaluation of the functional activity of CD39 ectonucleotidase in regulatory T cells in children with inflammatory bowel diseases

In connection with the increasing incidence and prevalence of inflammatory bowel disease (IBD), the search for prognostic markers of the effectiveness of therapy is an urgent problem. An imbalance between Th17 lymphocytes and regulatory T cells (Treg) is a major defect in the immune system leading to IBD. Extracellular ATP produced during tissue damage, rebound pro-inflammatory effects, and activates Th17 cell differentiation. Ectonucleotidase CD39 catalyzes the dephosphorylation of ATP to AMP, followed by conversion to adenosine by CD73. CD39 is expressed in various cell types, including Treg. Aim – evaluate the functional activity of CD39+ in Treg in children with IBD using the luciferin-luciferase method.68 children with IBD were examined. Of these, 28 children were in remission, 40 were in exacerbation. The number of Tregs (CD4+CD25highCD127low) expressing CD39 was estimated by flow cytometry. The ATP concentration in supernatants and cells was determined using the luciferin-luciferase test. Results are presented as median (Me) and quartiles (Q0.25-Q0.75). The significance of differences between groups was assessed using the nonparametric Mann–Whitney U test.The relative number of CD39+Treg in patients in remission of IBD was significantly higher than in patients in a state of exacerbation. A decrease in ATP concentration under the influence of CD39+Treg in patients with IBD occurred immediately upon the addition of exogenous ATP. ATP in patients in remission decreased by 44.5% (Me 54.5 (41.5-65.9)), in patients in exacerbation – by 32.5% (Me 67.5 (59.7-71.3)). At the same time, in patients in remission, the decrease in the ATP content after 5 minutes of the reaction was significantly higher than in patients in the state of exacerbation (p = 0.01), after 30 minutes of the reaction, no significant difference was found. It was shown that samples with a smaller number of cells and a lower intensity of CD39 expression in Treg had a higher activity of CD39 ectonucleotidase.For efficient ATP hydrolysis, in addition to the amount of CD39 in Treg, their functional activity is important. The assessment of the catalytic activity of CD39 in Treg in patients with IBD is most informative in the first minutes after the addition of exogenous ATP. In patients in remission, the catalytic activity of CD39 in Treg was higher than in patients in a state of exacerbation

Moxetumomab pasudotox in heavily pre-treated patients with relapsed/refractory hairy cell leukemia (HCL): long-term follow-up from the pivotal trial

Background: Moxetumomab pasudotox is a recombinant CD22-targeting immunotoxin. Here, we present the long-term follow-up analysis of the pivotal, multicenter, open-label trial (NCT01829711) of moxetumomab pasudotox in patients with relapsed/refractory (R/R) hairy cell leukemia (HCL). Methods: Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 purine nucleoside analogs (PNAs), or ≥ 1 PNA followed by rituximab or a BRAF inhibitor. Patients received 40 µg/kg moxetumomab pasudotox intravenously on Days 1, 3, and 5 of each 28-day cycle for up to six cycles. Disease response and minimal residual disease (MRD) status were determined by blinded independent central review. The primary endpoint was durable complete response (CR), defined as achieving CR with hematologic remission (HR, blood counts for CR) lasting > 180 days. Results: Eighty adult patients were treated with moxetumomab pasudotox and 63% completed six cycles. Patients had received a median of three lines of prior systemic therapy; 49% were PNA-refractory, and 38% were unfit for PNA retreatment. At a median follow-up of 24.6 months, the durable CR rate (CR with HR > 180 days) was 36% (29 patients; 95% confidence interval: 26–48%); CR with HR ≥ 360 days was 33%, and overall CR was 41%. Twenty-seven complete responders (82%) were MRD-negative (34% of all patients). CR lasting ≥ 60 months was 61%, and the median progression-free survival without the loss of HR was 71.7 months. Hemolytic uremic and capillary leak syndromes were each reported in ≤ 10% of patients, and ≤ 5% had grade 3–4 events; these events were generally reversible. No treatment-related deaths were reported. Conclusions: Moxetumomab pasudotox resulted in a high rate of durable responses and MRD negativity in heavily pre-treated patients with HCL, with a manageable safety profile. Thus, it represents a new and viable treatment option for patients with R/R HCL, who currently lack adequate therapy. Trial registration: ClinicalTrials.gov identifier: NCT01829711; first submitted: April 9, 2013. https://clinicaltrials.gov/ct2/show/NCT0182971

Features of parameters of cellular immune depending on the activity of foci of demyelination in children with multiple sclerosis

MS is a common disease of the central nervous system that leads to disability and reduced quality of life. The debut of disease in 3-5% of patients occurs in childhood and has a less favorable course compared to adults. MS is caused by the activation of autoreactive T cells in the breakdown of peripheral tolerance, which is normally controlled by regulatory T cells (Tregs). It is promising to study expression of CD39 and CD73 in Treg and Th17 populations to assess their suppressive activity. Aim is to evaluate content of major and minor lymphocyte populations and expression of CD39 and CD73 in CD4+ lymphocyte population in children with MS. 111 children with MS were examined, 66 with contrast-negative lesions on MRI (Group 1), 45 with contrast-positive lesions (Group 2). The comparison group consisted of 46 healthy children (Group 3). Content of T, B, NK lymphocytes, Treg (CD4+CD25highCD127low), Thact (CD4+CD25highCD127high), Th17 cells (CD3+CD4+CD161+); expression of CD39 and CD73 in Treg, Th17 and Thact was performed by flow cytometry. An increase in content of T helpers, a decrease in NK cells in patients in group 2 was revealed. An increase in number of Thact and Th17 lymphocytes was obtained in patients of both groups with MS. Number of Tregs in group 1 was significantly higher than in group 3. Ratio of cells expressing CD39 and CD73 in MS patients depended on lymphocyte population as well as in the group 3. The highest content of CD39+ cells was observed in Treg population, and the lowest in Thact population. For CD73 expression, on the contrary, the highest expression of CD73 was observed in Thact cells, the lowest in Treg. When comparing groups of patients, it was found that in patients of group 1, number of cells expressing CD39 ectonucleotidase was significantly increased, and number of supTh17 was comparable with group 3. In both groups of MS patients, an increase in CD73 counts in Treg, Thact and Th17 was observed. Thus, informative populations of lymphocytes (CD4+ cells, Treg, CD39+Treg, supTh17) have been identified, which can be used to monitor condition of children with multiple sclerosis

Nuclear transcription factor kB (NF-kB) activity in lymphocyte populations in children with Wilson-Konovalov disease

Wilson's disease (WD) is a rare hereditary disease caused by a deficiency of the ATF7B transporter. The accumulation of copper can cause damage to organs and cells, mainly the liver. Copper exposure can modulate cytokine synthesis through molecular and cellular signaling pathways, including the nuclear transcription factor NF-kB pathway. NF-kB is the main regulator of inflammation and cell death, acts as a central link between liver damage, fibrosis and hepatocellular carcinoma. An excess of NF-kB-dependent cytokine response stimulates inflammatory reactions, but excessive inhibition of NF-kB can negatively affect the viability of hepatocytes. Method of flow cytometry with visualization — Amnis ImageStreamX allows to evaluate the activity of NF-kB (% of activated cells in cell populations). The aim: to evaluate the activity of NF-kB in lymphocyte populations in children with WD disease. Immunophenotyping of lymphocytes and assessment of the level of translocation of NF-kB were performed in 52 children with WD and in 25 children of comparison group. The mass concentration of copper in daily urine was determined by atomic absorption method using the AAnalyst 800 spectrometer. In children with WD, the content of cells with NF-kB translocation varied from 5 to 90% depending on the lymphocyte population; the highest level was detected in B cells — 57.5 (37-68) %. A significant difference in distributions of the number of cells with NF-kB translocation between WD and healthy children was shown (F-criterion, p < 0.01). In most cases, children with WD are characterized by a decrease in the activity of NF-kB in populations of B cells (in 43% of cases), T helper cells (48%), T cytotoxic (44%) and Th17 lymphocytes (41%). In children with WD, the concentration of copper varied from 9.7 to 2582 mcg/day, Me = 616 (210-1173). A direct relationship was obtained between the copper content in urine and the level of translocation of NF-kB in B lymphocytes, r = 0.34, p = 0.016. The activity of the NF-kB correlates with biochemical markers of the severity of liver damage (ALT, AST, GGT) and with copper content in urine. The study of the NF-kB signaling pathway seems promising for a better understanding of the pathogenetic mechanisms of the formation of inflammation and liver fibrosis in children with WD

Tumor inflating lymphocytes. Purification, expanding and cytotoxicity analisys on primary tumor cultures

Background. Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. However, the isolation of TILs from solid tumors is technically difficult. A suppressive tumor microenvironment, in particular, a high level of expression of check-point inhibitors PD-1 CTLA4, tissue hypoxia and other factors cause that T cells isolated from the tumor do not proliferate well and do not exhibit cytotoxic properties. Aims. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4+, CD8+ cytotoxic antitumor lymphocytes. Methods. An urgent task is the activation of TILs, turning off immunosuppressive mechanisms and increasing their antitumor cytotoxic activity. Various approaches are used for this: activation by a cocktail of cytokines and antibodies, editing the lymphocyte genome by knocking out suppressor genes or, conversely, transduction of activating genes, coincubation with feeder cells, etc. Cells were obtained from samples of resected tumors in 16 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture. Results. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2, IL-15, IL-21, IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4+, CD8+ cells containing populations of CD3+CD8+CD45+(CTL) CD3+CD4+CD45+ (T-helpers), CD4+CD25+CD127- (Т-regulatory cells), CD3-CD56+CD45+ (NK-cells), CD3+CD56+CD45+ (Т-NK-cells). The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD-1- activated TILs in an amount sufficient for adoptive therapy (500106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death. Conclusion. Our developed protocol for the production and activation of TILs can be recommended for the phase III clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donor's, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity

Genetic polymorphisms in DNA repair and damage response genes and late normal tissue complications of radiotherapy for breast cancer

Breast-conserving surgery followed by radiotherapy is effective in reducing recurrence; however, telangiectasia and fibrosis can occur as late skin side effects. As radiotherapy acts through producing DNA damage, we investigated whether genetic variation in DNA repair and damage response confers increased susceptibility to develop late normal skin complications. Breast cancer patients who received radiotherapy after breast-conserving surgery were examined for late complications of radiotherapy after a median follow-up time of 51 months. Polymorphisms in genes involved in DNA repair (APEX1, XRCC1, XRCC2, XRCC3, XPD) and damage response (TP53, P21) were determined. Associations between telangiectasia and genotypes were assessed among 409 patients, using multivariate logistic regression. A total of 131 patients presented with telangiectasia and 28 patients with fibrosis. Patients with variant TP53 genotypes either for the Arg72Pro or the PIN3 polymorphism were at increased risk of telangiectasia. The odds ratios (OR) were 1.66 (95% confidence interval (CI): 1.02–2.72) for 72Pro carriers and 1.95 (95% CI: 1.13–3.35) for PIN3 A2 allele carriers compared with non-carriers. The TP53 haplotype containing both variant alleles was associated with almost a two-fold increase in risk (OR 1.97, 95% CI: 1.11–3.52) for telangiectasia. Variants in the TP53 gene may therefore modify the risk of late skin toxicity after radiotherapy

A Bioinformatics Filtering Strategy for Identifying Radiation Response Biomarker Candidates

The number of biomarker candidates is often much larger than the number of clinical patient data points available, which motivates the use of a rational candidate variable filtering methodology. The goal of this paper is to apply such a bioinformatics filtering process to isolate a modest number (<10) of key interacting genes and their associated single nucleotide polymorphisms involved in radiation response, and to ultimately serve as a basis for using clinical datasets to identify new biomarkers. In step 1, we surveyed the literature on genetic and protein correlates to radiation response, in vivo or in vitro, across cellular, animal, and human studies. In step 2, we analyzed two publicly available microarray datasets and identified genes in which mRNA expression changed in response to radiation. Combining results from Step 1 and Step 2, we identified 20 genes that were common to all three sources. As a final step, a curated database of protein interactions was used to generate the most statistically reliable protein interaction network among any subset of the 20 genes resulting from Steps 1 and 2, resulting in identification of a small, tightly interacting network with 7 out of 20 input genes. We further ranked the genes in terms of likely importance, based on their location within the network using a graph-based scoring function. The resulting core interacting network provides an attractive set of genes likely to be important to radiation response