Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution.

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

Toxicokinetic Modeling of Per- and Polyfluoroalkyl Substance Concentrations within Developing Zebrafish (Danio rerio) Populations.

Per- and polyfluoroalkyl substances (PFAS) are pervasive environmental contaminants, and their relative stability and high bioaccumulation potential create a challenging risk assessment problem. Zebrafish (Danio rerio) data, in principle, can be synthesized within a quantitative adverse outcome pathway (qAOP) framework to link molecular activity with individual or population level hazards. However, even as qAOP models are still in their infancy, there is a need to link internal dose and toxicity endpoints in a more rigorous way to further not only qAOP models but adverse outcome pathway frameworks in general. We address this problem by suggesting refinements to the current state of toxicokinetic modeling for the early development zebrafish exposed to PFAS up to 120 h post-fertilization. Our approach describes two key physiological transformation phenomena of the developing zebrafish: dynamic volume of an individual and dynamic hatching of a population. We then explore two different modeling strategies to describe the mass transfer, with one strategy relying on classical kinetic rates and the other incorporating mechanisms of membrane transport and adsorption/binding potential. Moving forward, we discuss the challenges of extending this model in both timeframe and chemical class, in conjunction with providing a conceptual framework for its integration with ongoing qAOP modeling efforts.

Robot-Assisted vs Laparoscopic Right Hemicolectomy in Octogenarians.

OBJECTIVE: With increasing age, there is greater need for right-sided colonic resections than its left-sided counterparts. Older age is associated with limited physical and functional status, which carries greater operative risk. Improvements in robotic surgery questions its role, especially in older adults, compared with laparoscopy. The objective is to investigate whether robotic right hemicolectomy (RRH) is as safe and effective as laparoscopic right hemicolectomy (LHR) in octogenarians (age >80 years). DESIGN: Retrospective cross-sectional analysis. SETTINGS AND PARTICIPANTS: Octogenarians who underwent elective RRH and LRH by the Tweed Colorectal Group over 5 years. METHODS: Complications within 30 days, age, gender, smoking status, immunocompromised status, presence of diabetes, American Society of Anesthesiologists (ASA) physical status score, preoperative Eastern Cooperative Oncology Group (ECOG) performance status, mFI-5 (modified frailty index), operative time, method of anastomosis, postoperative length of stay (LOS), need for rehabilitation, and short-term oncologic data using the TNM criteria were compared using univariate and multivariate analysis. RESULTS: Seventy-eight elective patients were included. LRH and RRH groups had similar median ages, gender distribution, and comorbidities. Across the entire cohort, 61.5% had no 30-day complications. RRH had nonsignificantly shorter operative time but significantly shorter LOS (5 vs 8 days) and fewer minor complications (24.5% vs 34.5%). Major complications and overall complications were not significantly different between the groups. Lower ASA and ECOG status were associated with lower complication rates across both groups. Oncologic resection outcomes were similar for both approaches. CONCLUSIONS AND IMPLICATIONS: RRH does not confer an increased risk of complications compared to LRH in the octogenarians and may be a viable alternative in the field of minimally invasive surgery for older patients. Future research should focus on intracorporeal anastomoses, as it is a potential confounder leading to the shorter inpatient LOS shown in our robotic group.

Comparison of outcome and costs of robotic and laparoscopic right hemicolectomies.

To compare the outcomes of patients undergoing right hemicolectomy using laparoscopic or robotic approaches and perform a cost analysis. Retrospective review of all patients undergoing elective laparoscopic and robotic right hemicolectomies at a public and private hospital in NSW/QLD from January 2015 to June 2018. Cost analysis was calculated using actual and estimated costs by the local health district. A total of 101 patients were identified. 59 (58%) had Robotic resection, of which 44 (75%) had an intra-corporeal anastomosis. There were no demographic or oncological differences between the two groups. The robotic group had a significantly earlier time to bowels opening (2 vs 4 days, p < 0.001) and shorter length of stay (3 vs 5 days, p < 0.001). The robotic group had a lower rate of ileus (2% vs 14%, p = 0.02) and complications (5% vs 33%, p = 0.006). The mean lymph node harvest was higher in the robotic group (18 vs 14, p = 0.001). The operative time was longer in the robotic group (110 vs 97 min, p = 0.021). The total instrument costs of robotic surgery were A$2565.37 compared with $1507.50 for laparoscopic surgery. The cost of bed days was A$1167.00/day. The average difference in cost of care was calculated as A$1276.13 and A$464.43 less in the robotic with intra-corporeal and extra-corporeal anastomosis, respectively. Patients have significantly faster return to bowel function and shorter length of stay after Robotic vs laparoscopic right hemicolectomy and experience fewer complications. This difference in length of stay may make robotic right hemicolectomies more cost effective.

Implementation and Evaluation of Quality Improvement Training in Surgery: A Systematic Review.

OBJECTIVE: The aim of this study was to review and appraise how quality improvement (QI) skills are taught to surgeons and surgical residents. BACKGROUND: There is a global drive to deliver capacity in undertaking QI within surgical services. However, there are currently no specifications regarding optimal QI content or delivery. METHODS: We reviewed QI educational intervention studies targeting surgeons or surgical trainees/residents published until 2017. Primary outcomes included teaching methods and training materials. Secondary outcomes were implementation frameworks and strategies used to deliver QI training successfully. RESULTS: There were 20,590 hits across 10 databases, of which 11,563 were screened following de-duplication. Seventeen studies were included in the final synthesis. Variable QI techniques (eg, combined QI models, process mapping, and "lean" principles) and assessment methods were found. Delivery was more consistent, typically combining didactic teaching blended with QI project delivery. Implementation of QI training was poorly reported and appears supported by collaborative approaches (including building learning collaboratives, and coalitions). Study designs were typically pre-/post-training without controls. Studies generally lacked clarity on the underpinning framework (59%), setting description (59%), content (47%), and conclusions (47%), whereas 88% scored low on psychometrics reporting. CONCLUSIONS: The evidence suggests that surgical QI training can focus on any well-established QI technique, provided it is done through a combination of didactic teaching and practical application. True effectiveness and extent of impact of QI training remain unclear, due to methodological weaknesses and inconsistent reporting. Conduct of larger-scale educational QI studies across multiple institutions can advance the field.

General tissue mass transfer model for cryopreservation applications.

Successful cryopreservation of complex specimens, such as tissues and organs, would greatly benefit both the medical and scientific research fields. Vitrification is one of the most promising techniques for complex specimen cryopreservation, but toxicity remains a major challenge because of the high concentration of cryoprotectants (CPAs) needed to vitrify. Our group has approached this problem using mathematical optimization to design less toxic CPA equilibration methods for cells. To extend this approach to tissues, an appropriate mass transfer model is required. Fick's law is commonly used, but this simple modeling framework does not account for the complexity of mass transfer in tissues, such as the effects of fixed charges, tissue size changes, and the interplay between cell membrane transport and transport through the extracellular fluid. Here, we propose a general model for mass transfer in tissues that accounts for all of these phenomena. To create this model, we augmented a previously published acellular model of mass transfer in articular cartilage to account for the effects of cells. We show that the model can accurately predict changes in CPA concentration and tissue size for both articular cartilage and pancreatic islets, tissue types with vastly different properties.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

Mathematical Modeling of Protectant Transport in Tissues.

Mass transfer of protectant chemicals is a fundamental aspect of cryopreservation and freeze-drying protocols. As such, mass transfer modeling is useful for design of preservation methods. Cell membrane transport modeling has been successfully used to guide design of preservation methods for isolated cells. For tissues, though, there are several mass transfer modeling challenges that arise from phenomena associated with cells being embedded in a tissue matrix. Both cells and the tissue matrix form a barrier to the free diffusion of water and protective chemicals. Notably, the extracellular space becomes important to model. The response of cells embedded in the tissue is dependent on the state of the extracellular space which varies both spatially and temporally. Transport in the extracellular space can also lead to changes in tissue size. In this chapter, we describe various mass transfer models that can be used to describe transport phenomena occurring during loading of tissues with protective molecules for cryopreservation applications. Assumptions and simplifications that limit the applicability of each of these models are discussed.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

The cost effectiveness of vaginal versus abdominal repair of vesicovaginal fistulae.

INTRODUCTION AND HYPOTHESIS: The objective was to assess the comparative provider costs of vaginal and open abdominal repair of vesicovaginal fistula (VVF) and to determine the most cost-effective means of managing VVF. METHODS: A prospectively acquired database of all women undergoing VVF repair by a single surgeon between 2007 and 2015 was retrospectively reviewed to determine operating time, perioperative complications, inpatient stay and 30-day readmissions. The success and cost of the VVF repair were identified. Statistical analysis was by unpaired t test, Chi-squared test and Mann-Whitney U test. RESULTS: Forty-seven consecutive women of mean age 51 years (range 21-88) undergoing a first attempt at VVF repair at our institution were included; 32(68%) had vaginal repair with Martius fat pad interposition and 15 (32%) had open abdominal repair with omental interposition. There were no perioperative complications or 30-day readmissions in either group. Mean operative time was longer for open abdominal (223.4 min) than vaginal repair (196.9 min). Median inpatient stay was longer for an open abdominal (8 days) than for a vaginal approach (4 days). Successful anatomical closure was achieved in 91% of vaginal and 86% of open abdominal repairs at first attempt, and in 100% after second repair, where required. Mean/median costs for an abdominal repair were significantly higher, at £4,608.69/£4,169.20 than for vaginal repair at £3,381.50/£3,009.24 (P<0.05). CONCLUSIONS: Vesicovaginal fistulae were successfully repaired in 89% of cases at first attempt. The success rate did not differ between approaches. Vaginal repair is significantly more cost-effective than abdominal repair owing to the shorter operative time and length of stay.

Migratory DCs activate TGF-ß to precondition naïve CD8+ T cells for tissue-resident memory fate.

Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.

Improving the effectiveness of cancer multidisciplinary team meetings: analysis of a national survey of MDT members' opinions about streamlining patient discussions.

Background: Cancer is diagnosed and managed by multidisciplinary teams (MDTs) in the UK and worldwide, these teams meet regularly in MDT meetings (MDMs) to discuss individual patient treatment options. Rising cancer incidence and increasing case complexity have increased pressure on MDMs. Streamlining discussions has been suggested as a way to enhance efficiency and to ensure high-quality discussion of complex cases. Methods: Secondary analysis of quantitative and qualitative data from a national survey of 1220 MDT members regarding their views about streamlining MDM discussions. Results: The majority of participants agreed that streamlining discussions may be beneficial although variable interpretations of 'streamlining' were apparent. Agreement levels varied significantly by tumour type and occupational group. The main reason for opposing streamlining were concerns about the possible impact on the quality and safety of patient care. Participants suggested a range of alternative approaches for improving efficiency in MDMs in addition to the use of treatment protocols and pre-MDT meetings. Conclusions: This work complements previous analyses in supporting the development of tumour-specific guidance for streamlining MDM discussions considering a range of approaches. The information provided about the variation in opinions between MDT for different tumour types will inform the development of these guidelines. The evidence for variation in opinions between those in different occupational groups and the reasons underlying these opinions will facilitate their implementation. The impact of any changes in MDM practices on the quality and safety of patient care requires evaluation.

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

Tumor Tolerance-Promoting Function of Regulatory T Cells Is Optimized by CD28, but Strictly Dependent on Calcineurin.

Regulatory T cells (Treg) restrain immune responses against malignant tumors, but their global depletion in cancer patients will likely be limited by systemic autoimmune toxicity. Instead, approaches to "tune" their activities may allow for preferential targeting of tumor-reactive Treg. Although Ag recognition regulates Treg function, the roles of individual TCR-dependent signaling pathways in enabling Treg to promote tumor tolerance are not well characterized. In this study, we examined in mouse tumor models the role of calcineurin, a key mediator of TCR signaling, and the role of the costimulatory receptor CD28 in the differentiation of resting central Treg into effector Treg endowed with tumor tropism. We find that calcineurin, although largely dispensable for suppressive activity in vitro, is essential for upregulation of ICOS and CTLA-4 in Treg, as well as for expression of chemokine receptors driving their accumulation in tumors. In contrast, CD28 is not critical, but optimizes the formation of tumor-homing Treg and their fitness in tumor tissue. Accordingly, although deletion of either CnB or CD28 strongly impairs Treg-mediated tumor tolerance, lack of CnB has an even more pronounced impact than lack of CD28. Hence, our studies reveal distinct roles for what has classically been defined as signal 1 and signal 2 of conventional T cell activation in the context of Treg-mediated tumor tolerance.

Cystitis cystica and glandularis producing large bladder masses in a 16-year-old boy.

The macroscopic appearances of florid cystitis cystica et glandularis can be mistaken for malignancy, and it is therefore important to perform a prompt resection to confirm the histological diagnosis and exclude sinister pathology.