Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells

While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments

Varijabilnost gibanja konja u hodu tijekom hipoterapijskog jahanja

The impulses emitted from the back of a horse during hippotherapy stimulate the rider’s postural reflex mechanisms, resulting in balance and coordination training. The objectives of this study were to evaluate the movement variability of the horse’s back and limbs and to determine significant relationships between the movement of the selected body points on the horse. Two English thoroughbreds and twelve female riders participated in six sessions of hippotherapy. Three-dimensional (3-D) videography was used to assess movement of the selected points on the horse’s back and limbs. The spatiotemporal parameters of the horse’s walk showed no significant changes throughout the entire measuring process. Horse movement within a given session was stable, and overall, inter-individual (between-horse) variability was greater than intra-individual variability. The maximum differences in the vertical displacement of the horse’s back across individual sessions were significant. With respect to the range of movement of the caudal part of the horse’s back, it is necessary to consider the instability of movements during longitudinally repeated sessions.Impulsi s leđa konja za vrijeme hipoterapijskog jahanja podražuju jahačeve posturalne refleksne mehanizme, što rezultira treningom ravnoteže i koordinacije. Ciljevi ovog istraživanja bili su vrednovati varijabilnosti gibanja leđa i udova konja i utvrditi značajnost veza između gibanja odabranih točaka na tijelu konja. Dva čistokrvna engleska konja i dvanaest jahačica sudjelovalo je u šest hipoterapijskih sesija. Trodimenzionalna videografija se koristila za utvrđivanje gibanja izabranih točaka na konjskim leđima i udovima. Kinematički parametri konjskog hoda nisu se značajno mijenjali tijekom cijelog procesa mjerenja. Gibanje konja tijekom jedne sesije bilo je stabilno i, generalno, inter-individualna varijabilnost kinematičkih parametara između konja bila je veća nego intra-individualna varijabilnost između mjerenja u svih 6 sesija. Maksimalne razlike u okomitim pomacima leđa konja između pojedinačnih sesija bile su značajne. Promatra li se donji dio leđa konja, potrebno je u obzir uzeti nestabilnost gibanja tijekom ponovljenih jahačkih sesija

Evaluation of miRNA detection methods for the analytical characteristics necessary for clinical utilization

miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/mu l) and methods evading the RT process (amol/mu l). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement. METHOD SUMMARY Three miRNA detection technologies were compared: 1) RT-qPCR where the RT step was performed with either a specific (TaqMan miRNA assay) or universal (TaqMan Advanced assay) priming strategy; 2) miREIA technology, using hybridization and specific antibody to DNA/RNA hybrids and 3) SplintR-qPCR, which utilizes a hybridization and ligation step followed by qPCR

Insulin sensitivity and secretion in obese Type 2 diabetic women after various bariatric operations

Objective: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity

PHD2/3 deletion in CD8 T cells enhances their effector functions and improves tumor response to adoptive cell therapy

Adoptive cell therapy, a promising treatment for hematological malignancies, faces limitations in solid tumors due to the hypoxic and immunosuppressive tumor microenvironment. The transcription factor Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in adaptative response to hypoxia. Here, we aimed to understand the impact of hypoxia and HIF-1α on CD8 T cells with the aim to find new immunotherapy strategies. Using genetic modulations, we deleted or stabilized HIF-1α in CD8 T cells. We observed that HIF-1α stabilization in CD8 T cells led to increased glycolysis, expression of activation markers, and enhanced cytolytic capacity. Furthermore, adoptive transfer of CD8 T cells expressing high levels of HIF-1α resulted in improved tumor control in multiple preclinical models. Finally, stabilization of HIF-1α with the pharmacological inhibitor IOX4 demonstrated potential to enhance the function of human CD8 T cell, offering a promising avenue for improving cancer immunotherapies.(MED - Sciences médicales) -- UCL, 202

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes

Regulation of Prepro-NeuropeptideW/B and Its Receptor in the Heart of ZDF Rats: An Animal Model of Type II DM

Neuropeptide B (NPB) and neuropeptide W (NPW) are neuropeptides, which constitute NPB/W signaling systems together with G-protein coupled receptors NPBWR1. The location and function of NPB/W signaling systems have been predominantly detected and mapped within the CNS, including their role in the modulation of inflammatory pain, neuroendocrine functions, and autonomic nervous systems. The aim of the study is to investigate the impact of diabetes on the neuropeptide B/W signaling system in different heart compartments and neurons which innervates it. In the RT-qPCR analysis, we observed the upregulation of mRNA for preproNPB in RV, for preproNPW in LA, and for NPBWR1 in DRG in diabetic rats. On the contrary, the expression of mRNA for NPBWR1 was downregulated in LV in diabetic rats. In the WB analysis, significant downregulation of NPBWR1 in LV (0.54-fold, p = 0.046) in diabetic rats was observed at the proteomic level. The presence of NPBWR1 was also confirmed in a dissected LCM section of cardiomyocytes and coronary arteries. The positive inotropic effect of NPW described on the diabetic cardiomyocytes in vitro could point to a possible therapeutic target for compensation of the contractile dysfunction in the diabetic heart. In conclusion, the NPB/W signaling system is involved in the regulation of heart functions and long-term diabetes leads to changes in the expression of individual members of this signaling system differently in each cardiac compartment, which is related to the different morphology and function of these cardiac chambers