Can regulatory T cells improve outcomes of sensitised patients after HLA-Ab incompatible renal transplantation: study protocol for the Phase IIa GAMECHANgER-1 trial.

BACKGROUND: Kidney transplantation is the gold-standard treatment for patients with kidney failure. However, one-third of patients awaiting a kidney transplant are highly sensitized to human leukocyte antigens (HLA), resulting in an increased waiting time for a suitable kidney, more acute and chronic rejection, and a shorter graft survival compared to non-highly sensitised patients. Current standard immunosuppression protocols do not adequately suppress memory responses, and so alternative strategies are needed. Autologous polyclonally expanded regulatory T cells (Tregs) have been demonstrated to be safe in transplant settings and could be a potential alternative to modulate memory immune alloresponses. METHODS: The aim of this trial is to determine whether adoptive transfer of autologous Tregs into HLA sensitised patients can suppress memory T and B cell responses against specific HLA antigens. This is a two-part, multi-centre, prospective clinical trial, comprising an observational phase (Part 1) aiming to identify patients with unregulated cellular memory responses to HLA (Pure HLA Proteins) followed by an interventional phase (Part 2). The first 9 patients identified as being eligible in Part 1 will undergo baseline immune monitoring for 2 months to inform statistical analysis of the primary endpoint. Part 2 is an adaptive, open labelled trial based on Simon's two-stage design, with 21 patients receiving Good Manufacturing Practice (GMP)-grade polyclonally expanded Tregs to a dose of 5-10 × 106 cells/kg body weight. The primary EP is suppression of in vitro memory responses for 2 months post-infusion. 12 patients will receive treatment in stage 1 of Part 2, and 9 patients will receive treatment in stage 2 of Part 2 if ≥ 50% patients pass the primary EP in stage 1. DISCUSSION: This is a prospective study aiming to identify patients with unregulated cellular memory responses to Pure HLA Proteins and determine baseline variation in these patterns of response. Part 2 will be an adaptive phase IIa clinical trial with 21 patients receiving a single infusion of GMP-grade polyclonally expanded Tregs in two stages. It remains to be demonstrated that modulating memory alloresponses clinically using Treg therapy is achievable. TRIAL REGISTRATION: EudraCT Number: 2021-001,664-23. REC Number: 21/SC/0253. Trial registration number ISRCTN14582152.

The Impact of the NOD2/CARD15 Variant (3020insC) and PSMA6 Polymorphism (-8C>G) on the Development and Outcome of Multiple Myeloma.

INTRODUCTION: Multiple myeloma (MM) is a hematological malignancy characterized by genetic variety. The 3020insC variant of the NOD2/CARD15 gene results in the upregulation of proinflammatory cytokines. Chronic inflammation and abnormal function of the proteasome system may lead to MM development. The polymorphism (-8C>G) in the PSMA6 gene affects proteasome activity. The aim of our study was to analyze the possible relationship of NOD/CARD15 and PSMA6 genes with the risk of development and outcome of MM, as well as the sensitivity to bortezomib (proteasome inhibitor) in cell cultures derived from MM patients. Objects and Methods. Genomic DNA from 100 newly diagnosed MM patients and 100 healthy blood donors was analyzed by methods such as PCR-RFLP (for PSMA6 genotyping) and automated DNA sequencing (for NOD2/CARD15 genotyping). In a subgroup of 50 MM patients, nucleated bone marrow cells were treated with bortezomib in vitro. RESULTS: Patients with PSMA6 CG+GG genotypes had higher chances for progressive disease (OR = 5.0, 95% CI 1.07-23.16, p = 0.05), shorter overall survival taking into account the type of treatment (p = 0.039), and increased risk of death due to MM at the level of tendency (OR = 4.74, 95% CI 1.02-21.97, p = 0.06). The presence of NOD2/CARD15 3020insC decreased the risk of renal dysfunction in MM (OR = 0.23, 95% CI 0.07-0.74, p = 0.009). The analyzed changes in NOD2/CARD15 and PSMA6 genes did not impact the MM risk. In an in vitro study, bortezomib increased the number of apoptotic cells at 8 nM and 12 nM between wild-type and 3030insC variants of NOD2/CARD15 (p = 0.018 and p = 0.03, respectively). CONCLUSION: The presented results suggest a possible impact of PSMA6 CG+GG genotypes on the MM outcome and the association of the NOD2/CARD15 variant with bortezomib in vitro sensitivity.

Survivin antiapoptotic gene expression as a prognostic factor in non-small cell lung cancer: in situ hybridization study.

Survivin is an inhibitor of apoptosis that plays a significant role in cell cycle regulation and is important for survival prognosis in many neoplasms. Survivin expression was assessed by in situ hybridization (ISH) in 60 consecutive patients (54 males and 4 females) with NSCLC treated between 1993 and 1997. The examined patients had IIB and IIIA stage according to TNM system. In all cases the chemotherapy with cisplatin and etoposide (2 cycles) was administered prior the surgery; in patients responding to the therapy one more cycle was applied. Survivin gene overexpression was observed in 35 patients (58.3%). There was no correlation between survivin mRNA level and histological type of tumor, stage of cell differentiation, stage of disease according to TNM classification, performance status according to WHO and number of chemotherapy regimens administered (p > 0.05). However, the correlation between survivin gene expression and response to the chemotherapy was statistically significant (p = 0.04). Statistical analysis showed that median survival in patients with survivin gene overexpression was shorter (14.0 months) as compared to patients with no expression (60.0 months; p = 0.00002). In survival assessment by means of Kaplan-Meier test, 14.3% of five-year survival was achieved in the former group versus 60% in the latter (p = 0.00003). Univariate analysis (log-rank test) showed that significant independent prognostic factors in NSCLC included: stage of the disease according to TNM classification (p = 0.006), response to chemotherapy (p = 0.005) and pattern of survivin gene expression (p = 0.00003). Multivariate analysis utilizing Cox's model showed that for survival assessment the stage according to TNM, response to the chemotherapy and survivin expression estimated by means of ISH are of statistical significance (p=0.00001). The calculated predictive values showed that ISH technique was quite accurate in assessment of five-year survival. Our data show that survivin expression may be used as a prognostic factor and a target for therapy.

Effect of tyrphostins on programmed cell death in colon adenocarcinoma cell line LS-180.

Programmed cell death is an important process in the regulation of cellular proliferation, rest, differentiation and death. It is a genetically controlled process with characteristic biochemical and morphological features. Apoptosis directly regulates tumorigenesis and its induction could be a useful method of cancer therapy. Cancer cells could be influenced by some factors which induce apoptosis. We investigated the influence of tyrphostins, that specifically inhibits protein tyrosine kinases and stops the cell cycle in apoptosis of the colon adenocarcinoma cell line LS180. We used them at the concentration of 1-10 microM for 24 and 48 hours. We detected apoptosis using techniques that monitor either biochemical and morphological features of this process, such as staining with 7-amino-actinomycin D, staining with Grünwald-Giemsa, TUNEL reaction, in situ hybridization and with immunoperoxidase staining procedures. We examined the expression of genes and proteins connected with programmed cell death (p53, c-myc, p21, bcl-2). We estimated the results by cytophotometry and documented them by colour photography. We found that tyrphostin rapidly inhibits the cell cycle, particularly at the concentration of 5 microM. The expression of genes and proteins was strongly correlated with the increased apoptotic cell death conforming to the results of TUNEL and staining methods.

[Results of surgery after intraocular disc lens implantation]. / Wyniki operacji po wszczepach soczewek dyskowych.

PURPOSE: To evaluate the results of Medi Contur IOLs implantation, which are a slightly modified Galand disc lens. MATERIAL AND METHODS: In the years 1994-1995, 31 eyes were operated on using modified method with horizontal, linear anterior capsule incision and intercapsular disc lens implantation. The follow-up was 6 months. RESULTS: Mean visual acuity was 0.7-0.8. There were no complications different from those occurring in other types of IOLs. The residual anterior capsule has no negative influence on visual acuity. The shape, size and localization of the IOL in physiological place, between anterior and posterior capsule, cause the good position and stability of the lens. The risk of development of the posterior capsule folds and opacity was decreased on account of a uniform capsular tension and contact of the IOL with the posterior capsule. CONCLUSION: The presented method of cataract extraction and IOL implantation, with all manipulations in intercapsular space is safer in comparison with others methods and residual anterior capsule decreases the risk of lens dislocation.