A quantitative three-dimensional image analysis tool for maximal acquisition of spatial heterogeneity data

Three-dimensional (3D) imaging techniques provide spatial insight into environmental and cellular interactions and are implemented in various fields, including tissue engineering, but have been restricted by limited quantification tools that misrepresent or underutilize the cellular phenomena captured. This study develops image postprocessing algorithms pairing complex Euclidean metrics with Monte Carlo simulations to quantitatively assess cell and microenvironment spatial distributions while utilizing, for the first time, the entire 3D image captured. Although current methods only analyze a central fraction of presented confocal microscopy images, the proposed algorithms can utilize 210% more cells to calculate 3D spatial distributions that can span a 23-fold longer distance. These algorithms seek to leverage the high sample cost of 3D tissue imaging techniques by extracting maximal quantitative data throughout the captured image

Missense mutations in the perforin (PRF1) gene as a cause of hereditary cancer predisposition

Perforin, a pore-forming toxin released from secretory granules of NK cells and CTLs, is essential for their cytotoxic activity against infected or cancerous target cells. Bi-allelic loss-of-function mutations in the perforin gene are invariably associated with a fatal immunoregulatory disorder, familial haemophagocytic lymphohistiocytosis type 2 (FHL2), in infants. More recently, it has also been recognized that partial loss of perforin function can cause disease in later life, including delayed onset FHL2 and haematological malignancies. Herein we report a family in which a wide range of systemic inflammatory and neoplastic manifestations have occurred across three generations. We found that disease was linked to two missense perforin gene mutations (encoding A91V, R410W) that cause protein misfolding and partial loss of activity. These cases link the partial loss of perforin function with some solid tumours that are known to be controlled by the immune system, as well as haematological cancers. Our findings also demonstrate that perforin gene mutations can contribute to hereditary cancer predisposition

Key environmental stress biomarker candidates for the optimisation of chemotherapy treatment of leukaemia

The impact of fluctuations of environmental parameters such as oxygen and starvation on the evolution of leukaemia is analysed in the current review. These fluctuations may occur within a specific patient (in different organs) or across patients (individual cases of hypoglycaemia and hyperglycaemia). They can be experienced as stress stimuli by the cancerous population, leading to an alteration of cellular growth kinetics, metabolism and further resistance to chemotherapy. Therefore, it is of high importance to elucidate key mechanisms that affect the evolution of leukaemia under stress. Potential stress response mechanisms are discussed in this review. Moreover, appropriate cell biomarker candidates related to the environmental stress response and/or further resistance to chemotherapy are proposed. Quantification of these biomarkers can enable the combination of macroscopic kinetics with microscopic information, which is specific to individual patients and leads to the construction of detailed mathematical models for the optimisation of chemotherapy. Due to their nature, these models will be more accurate and precise (in comparison to available macroscopic/black box models) in the prediction of responses of individual patients to treatment, as they will incorporate microscopic genetic and/or metabolic information which is patient-specific.peer-reviewe

Immune reconstitution and clinical recovery following anti-CD28 antibody (TGN1412)-induced cytokine storm

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud's phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm

Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design

Oxidized alginate hydrogels are appealing alternatives to natural alginate due to their favourable biodegradability profiles and capacity to self-crosslink with amine containing molecules facilitating functionalization with extracellular matrix cues, which enable modulation of stem cell fate, achieve highly viable 3-D cultures, and promote cell growth. Stem cell metabolism is at the core of cellular fate (proliferation, differentiation, death) and metabolomics provides global metabolic signatures representative of cellular status, being able to accurately identify the quality of stem cell differentiation. Herein, umbilical cord blood mesenchymal stem cells (UCB MSCs) were encapsulated in novel oxidized alginate hydrogels functionalized with the glycine-histidine-lysine (GHK) peptide and differentiated towards the osteoblastic lineage. The ADA-GHK hydrogels significantly improved osteogenic differentiation compared to gelatin-containing control hydrogels, as demonstrated by gene expression, alkaline phosphatase activity and bone extracellular matrix deposition. Metabolomics revealed the high degree of metabolic heterogeneity in the gelatin-containing control hydrogels, captured the enhanced osteogenic differentiation in the ADA-GHK hydrogels, confirmed the similar metabolism between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. Our results suggest a novel paradigm for metabolomics-guided biomaterial design and robust stem cell bioprocessing. STATEMENT OF SIGNIFICANCE: Producing high quality engineered bone grafts is important for the treatment of critical sized bone defects. Robust and sensitive techniques are required for quality assessment of tissue-engineered constructs, which result to the selection of optimal biomaterials for bone graft development. Herein, we present a new use of metabolomics signatures in guiding the development of novel oxidised alginate-based hydrogels with umbilical cord blood mesenchymal stem cells and the glycine-histidine-lysine peptide, demonstrating that GHK induces stem cell osteogenic differentiation. Metabolomics signatures captured the enhanced osteogenesis in GHK hydrogels, confirmed the metabolic similarity between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. In conclusion, our results suggest a new paradigm of metabolomics-driven design of biomaterials

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD