Balanced Crystalloids versus Saline in Critically Ill Adults — A Systematic Review with Meta-Analysis

BACKGROUND: The comparative efficacy and safety of balanced crystalloid solutions and saline for fluid therapy in critically ill adults remain uncertain. METHODS: We systematically reviewed randomized clinical trials (RCTs) comparing the use of balanced crystalloids with saline in critically ill adults. The primary outcome was 90-day mortality after pooling data from low-risk-of-bias trials using a random-effects model. We also performed a Bayesian meta-analysis to describe the primary treatment effect in probability terms. Secondary outcomes included the incidence of acute kidney injury (AKI), new treatment with renal replacement therapy (RRT), and ventilator-free and vasopressor-free days to day 28. RESULTS: We identified 13 RCTs, comprising 35,884 participants. From six trials (34,450 participants) with a low risk of bias, the risk ratio (RR) for 90-day mortality with balanced crystalloids versus saline was 0.96 (95% confidence interval [CI], 0.91 to 1.01; I2 = 12.1%); using vague priors, the posterior probability that balanced crystalloids reduce mortality was 89.5%. The RRs of developing AKI and of being treated with RRT with balanced crystalloids versus saline were 0.96 (95% CI, 0.89 to 1.02) and 0.95 (95% CI, 0.81 to 1.11), respectively. Ventilator-free days (mean difference, 0.18 days; 95% CI, −0.45 to 0.81) and vasopressor-free days (mean difference, 0.19 days; 95% CI, −0.14 to 0.51) were similar between groups. CONCLUSIONS: The estimated effect of using balanced crystalloids versus saline in critically ill adults ranges from a 9% relative reduction to a 1% relative increase in the risk of death, with a high probability that the average effect of using balanced crystalloids is to reduce mortality

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1–0.2 μg ml−1) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6–28%). Hypericin (0.1–0.2 μM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy

An investigation of regulatory T cell therapy in advanced human skin experimental transplantation models

Regulatory T cells (Tregs) are powerful suppressors of immune responses and help preserve immune homeostasis and self-tolerance. Specific Treg subsets—including those expressing the chemokine receptors CCR4 and CCR8—demonstrate high suppressive potency in certain clinical contexts. Several studies have revealed the promise of Treg cellular therapies in counteracting immune-mediated pathologies including transplant rejection. Clinical translation of Treg therapies must be supported by ongoing pre-clinical and clinical research to both evidence and enhance its safety and efficacy. Humanised experimental mice provide a cost-effective and practical approach to create in vivo models for pre-clinical transplant research. However, issues including high technical demand and variable capacities to represent human biology can limit their use. The two aims of this study were to develop an advanced humanised mouse model that could support multilineage human haematopoeisis and accurately represent human allotransplantation, and to investigate whether specific skin-homing human Treg subsets can provide effective anti-rejection therapy for skin allografts. In the first part of this study, HSPC-NBSGW mice—non-irradiated NOD,B6.SCID IL-2rγ-/-KitW41/W41 (NBSGW) mice engrafted with human CD133+ hematopoietic stem and progenitor cells (HSPCs)—were assessed for function capability in the context of allotransplantation. In depth characterisation of durable human haematopoeitic cell engraftment was followed by quantitative transcriptomic and immunophenotypic analyses of complete skin allograft rejection, which this model can effect through functional human innate and adaptive immune cells. In the second part, the suppressive capabilities of chemokine receptor-expressing Tregs were evaluated phenotypically and functionally. In addition to potent immune suppression mediated by CCR4-expressing Tregs, subcutaneous CCL22 administration in the presence of Treg therapy prevented skin allograft rejection. As the potential of Treg therapy has become clearer, pre-clinical research is becoming an increasingly essential tool for uncovering features of Treg behaviour and function that may help optimise safety and efficacy. The findings presented in this thesis advance the in vivo research toolkit and describe an additional method by which Tregs may be manipulated to enhance their therapeutic application.</p

Development of LT-HSC-reconstituted non-irradiated NBSGW mice for the study of human hematopoiesis in vivo

Humanized immune system (HIS) mouse models are useful tools for the in vivo investigation of human hematopoiesis. However, the majority of HIS models currently in use are biased towards lymphocyte development and fail to support long-term multilineage leucocytes and erythrocytes. Those that achieve successful multilineage reconstitution often require preconditioning steps which are expensive, cause animal morbidity, are technically demanding, and poorly reproducible. In this study, we address this challenge by using HSPC-NBSGW mice, in which NOD,B6.SCID IL-2r&#x3B3; Kit (NBSGW) mice are engrafted with human CD133 hematopoietic stem and progenitor cells (HSPCs) without the need for preconditioning by sublethal irradiation. These HSPCs are enriched in long-term hematopoietic stem cells (LT-HSCs), while NBSGW mice are permissive to human hematopoietic stem cell (HSC) engraftment, thus reducing the cell number required for successful HIS development. B cells reconstitute with the greatest efficiency, including mature B cells capable of class-switching following allogeneic stimulation and, within lymphoid organs and peripheral blood, T cells at a spectrum of stages of maturation. In the thymus, human thymocytes are identified at all major stages of development. Phenotypically distinct subsets of myeloid cells, including dendritic cells and mature monocytes, engraft to a variable degree in the bone marrow and spleen, and circulate in peripheral blood. Finally, we observe human erythrocytes which persist in the periphery at high levels following macrophage clearance. The HSPC-NBSGW model therefore provides a useful platform for the study of human hematological and immunological processes and pathologies. -/- W41/W41

Humanization of immunodeficient animals for the modeling of transplantation, graft versus host disease and regenerative medicine

The humanization of animals is a powerful tool for the exploration of human disease pathogenesis in biomedical research, as well as for the development of therapeutic interventions with enhanced translational potential. Humanized models enable us to overcome biological differences that exist between humans and other species, whilst giving us a platform to study human processes in vivo. To become humanized, an immune deficient recipient is engrafted with cells, tissues or organoids. The mouse is the most well studied of these hosts, with a variety of immunodeficient strains available for various specific uses. More recently, efforts have turned to the humanization of other animal species such as the rat, which offers some technical and immunological advantages over mice. These advances, together with ongoing developments in the incorporation of human transgenes and additional mutations in humanized mouse models, have expanded our opportunities to replicate aspects of human allotransplantation and to assist in the development of immunotherapies. In this review, the immune and tissue humanization of various species is presented with an emphasis on their potential for use as models for allotransplantation, graft versus host disease and regenerative medicine

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability thereby limiting potential therapeutic applications. As Bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the Bromodomain and Extra-terminal Motif (BET) inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells (HSPCs). This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy

Chimeric antigen receptor-modified human regulatory T cells that constitutively express interleukin-10 maintain their phenotype and are potently suppressive

Clinical trials of regulatory T cell therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations which harbour a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CAR), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2 and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation