Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Effects of Blended Diesel-Biodiesel Fuel on Emissions of a Common Rail Direct Injection Diesel Engine with Different Exhaust Gas Recirculation Rates.

The aim of the study was to investigate the exhaust gas emissions and fuel consumption of a common rail direct injection (CRDI) diesel engine using mixed diesel (B10) and biodiesel (B20-B100) fuels. The study's primary objective was to determine the effects of blended diesel-biodiesel fuel on CRDI emissions based on different exhaust gas recirculation (EGR) rates, including carbon monoxide (CO), carbon dioxide (CO2), oxygen (O2), nitrogen oxide (NOx), and hydrocarbon (HC) emissions, smoke opacity, exhaust gas temperature, and fuel consumption. The CRDI experiments involved adjusting the different engine speeds (1400-3000 rpm) and EGR rates (0 and 12.5%), which were analyzed to determine their impact on these parameters for both blended diesel and biodiesel fuels. The results showed that under the conditions of no EGR (0%), the CO and HC emissions and the smoke opacity were lower than those with a 12.5% EGR rate for all fuel types and all cases. With 12.5% EGR rate, the O2 emissions and the EGT of the CRDI diesel engine decreased, which resulted in significantly lower NOx emissions because of EGR into the combustion chamber. For the maximum engine speed of 3000 rpm and with no EGR, the CO and HC emissions and the smoke opacity were lower than those with a 12.5% EGR rate for all fuel types. With a 12.5% EGR rate at 3000 rpm, the O2 emissions and the exhaust gas temperature were reduced by 0.07% and 2.27%, respectively, and the NOx emissions were reduced by 2.54%. However, with EGR, the CO and HC emissions and the smoke opacity increased by 7.70%, 18.61%, and 0.4%, respectively. Furthermore, the fuel consumption of pure biodiesel (B100) at 3000 rpm with a 12.5% EGR rate was reduced by 2.81% compared to that with a 0% EGR rate. Because the temperature in the combustion chamber is high enough for the engine to run, the EGR reuses a portion of the exhaust gases and can help to minimize the quantity of fuel in the combustion chamber. As a suggestion based on these observations, biodiesel fuel should not exceed B80 because the viscosity and density of fuel that are too high may affect the fuel injection system, both the injectors, and the pressure pump, causing the injectors to be unable to work correctly. These findings can contribute to the development of strategies and technologies for reducing emissions and improving fuel efficiency in CRDI diesel engines.

Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells.

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

Thyroid hormone receptor alpha modulates fibrogenesis in hepatic stellate cells.

OBJECTIVE: Progressive hepatic fibrosis can be considered the final stage of chronic liver disease. Hepatic stellate cells (HSC) play a central role in liver fibrogenesis. Thyroid hormones (TH, e.g. thyroxine; T4 and triiodothyronine; T3) significantly affect development, growth, cell differentiation and metabolism through activation of TH receptor α and/or ß (TRα/ß). Here, we evaluated the influence of TH in hepatic fibrogenesis. DESIGN: Human liver tissue was obtained from explanted livers following transplantation. TRα-deficient (TRα-KO) and wild-type (WT) mice were fed a control or a profibrogenic methionine-choline deficient (MCD) diet. Liver tissue was assessed by qRT-PCR for fibrogenic gene expression. In vitro, HSC were treated with TGFß in the presence or absence of T3. HSC with stable TRα knockdown and TRα deficient mouse embryonic fibroblasts (MEF) were used to determine receptor-specific function. Activation of HSC and MEF was assessed using the wound healing assay, Western blotting, and qRT-PCR. RESULTS: TRα and TRß expression is downregulated in the liver during hepatic fibrogenesis in humans and mice. TRα represents the dominant isoform in HSC. In vitro, T3 blunted TGFß-induced expression of fibrogenic genes in HSC and abrogated wound healing by modulating TGFß signalling, which depended on TRα presence. In vivo, TRα-KO enhanced MCD diet-induced liver fibrogenesis. CONCLUSION: These observations indicate that TH action in non-parenchymal cells is highly relevant. The interaction of TRα with TH regulates the phenotype of HSC via the TGFß signalling pathway. Thus, the TH-TR axis may be a valuable target for future therapy of liver fibrosis.

Upgrading Pyrolysis Bio-Oil through Esterification Process and Assessing the Performance and Emissions of Diesel-Biodiesel-Esterified Pyrolysis Bio-Oil Blends in Direct Injection Diesel Engines.

This research aimed to evaluate the performance and emissions of direct injection diesel engines using blends of diesel-biodiesel-esterified pyrolysis bio-oil (D-B-EPB). The pyrolysis process was employed to produce pyrolysis bio-oil (PBO) from solid biomass obtained from fresh palm fruits. Furthermore, a simple and effective esterification process was used to upgrade the PBO. The methyl ester (ME) purity of EPB production was studied to optimize three independent variables: methanol (14.8-65.2 wt %), sulfuric acid (1.6-18.4 wt %), and reaction time (16-84 min) using the response surface methodology. The actual experiment yielded a ME purity of 72.73 wt % under the recommended conditions of 40.3 wt % methanol, 13.0 wt % sulfuric acid, 50 min reaction time, 60 °C reaction temperature, and 300 rpm stirrer speed. Additionally, the stability and phase behaviors of D-B-EPB blends were analyzed by using a ternary phase diagram to determine the potential blending proportion. The results revealed that a fuel blend consisting of 30 wt % diesel, 60 wt % biodiesel, and 10 wt % EPB (D30B60EPB10) met the density and viscosity requirements of diesel standards. This D30B60EPB10 blend was subjected to performance and emission tests in diesel engines at various speeds ranging from 1100 to 2300 rpm and different engine loads of 25, 50, and 75%. In terms of performance analysis, the brake thermal efficiencies of biodiesel and D30B60EPB10 were 7.19 and 3.88% higher than that of diesel, respectively. However, the brake-specific fuel consumption of the D30B60EPB10 blend was 6.60% higher than that of diesel due to its higher density and viscosity and lower heating value compared with that of diesel. In the emission analysis, the D30B60EPB10 blend exhibited performance comparable to diesel while being more environmentally friendly, reducing carbon monoxide, carbon dioxide, nitrogen oxide, and smoke opacity by 8.73, 30.13, 37.55, and 59.75%, respectively. The results of this study suggest that the D-B-EPB blend has the potential to serve as a viable biofuel option, reducing the proportion of diesel in blended fuel and benefiting farmers and rural communities..

Addressing extreme size mismatch in pediatric intestinal transplantation: Outcomes of intestinal length reduction.

BACKGROUND: Bench liver reduction, with or without intestinal length reduction (LR) (coupled with delayed closure and abdominal wall prostheses), has been a strategy adopted by our program for small children due to the limited availability of size-matched donors. This report describes the short, medium, and long-term outcomes of this graft reduction strategy. METHODS: A single-center, retrospective analysis of children that underwent intestinal transplantation (April 1993 to December 2020) was performed. Patients were grouped according to whether they received an intestinal graft of full length (FL) or following LR. RESULTS: Overall, 105 intestinal transplants were performed. The LR group (n = 10) was younger (14.5 months vs. 40.0 months, p = .012) and smaller (8.7 kg vs. 13.0 kg, p = .032) compared to the FL group (n = 95). Similar abdominal closure rates were achieved after LR, without any increase in abdominal compartment syndrome (1/10 vs. 7/95, p = .806). The 90-day graft and patient survival were similar (9/10, 90% vs. 83/95, 86%; p = .810). Medium and long-term graft survival at 1 year (8/10, 80% vs. 65/90, 71%; p = .599), and 5 years (5/10, 50% vs. 42/84, 50%; p = 1.00) was similar. CONCLUSION: LR of intestinal grafts appears to be a safe strategy for infants and small children requiring intestinal transplantation. This technique should be considered in the situation of significant size mismatch of intestine containing grafts.

Investigation of free fatty acid reduction from mixed crude palm oil using 3D-printed rotor-stator hydrodynamic cavitation: An experimental study of geometric characteristics of the inner hole.

A continuous esterification process is employed to decrease the free fatty acid (FFA) concentration of FFA-rich mixed crude palm oil. Both optimal and recommended conditions are determined for the esterification reaction conditions and the geometry of the 3D-printed rotor design in the rotor-stator hydrodynamic cavitation reactor. This study is primarily concerned with the effect of the cavitation device configuration, especially the rotor design, on FFA reduction. Instead of conventional spherical or cylindrical drilled holes, a point angle cone-shaped hole is used to create cavities over the rotor surface. These point angles are adjusted to clarify their effect on FFA reduction. The response surface methodology is applied to determine the optimal concentrations of methanol and sulfuric acid, rotor speed, hole diameter and depth, and cone point angle. The recommended conditions are 20.8 wt% methanol, 2.6 wt% sulfuric acid, 3000 rpm, 5 mm hole diameter, 5 mm hole depth, and 110°, respectively. Under this configuration, the FFA content is reduced from 12.014 wt% to around 1 wt%. A maximum yield of 97.34 vol% esterified oil is obtained through a completed phase separation step, and 93.31 vol% pure oil is collected after the cleansing step. The recommended conditions result in reduced chemical usage, cheaper FFA reduction, and lower environmental impact. This creative rotor design effectively improves our understanding of the geometry of the cavitation device, thus enhancing the cavitation effect in industrial operations.

Nomenclature, diagnosis and management of drug-induced autoimmune-like hepatitis (DI-ALH): An expert opinion meeting report.

Drug-induced liver injury (DILI) can mimic almost all other liver disorders. A phenotype increasingly ascribed to drugs is autoimmune-like hepatitis (ALH). This article summarises the major topics discussed at a joint International Conference held between the Drug-Induced Liver Injury consortium and the International Autoimmune Hepatitis Group. DI-ALH is a liver injury with laboratory and/or histological features that may be indistinguishable from those of autoimmune hepatitis (AIH). Previous studies have revealed that patients with DI-ALH and those with idiopathic AIH have very similar clinical, biochemical, immunological and histological features. Differentiating DI-ALH from AIH is important as patients with DI-ALH rarely require long-term immunosuppression and the condition often resolves spontaneously after withdrawal of the implicated drug, whereas patients with AIH mostly require long-term immunosuppression. Therefore, revision of the diagnosis on long-term follow-up may be necessary in some cases. More than 40 different drugs including nitrofurantoin, methyldopa, hydralazine, minocycline, infliximab, herbal and dietary supplements (such as Khat and Tinospora cordifolia) have been implicated in DI-ALH. Understanding of DI-ALH is limited by the lack of specific markers of the disease that could allow for a precise diagnosis, while there is similarly no single feature which is diagnostic of AIH. We propose a management algorithm for patients with liver injury and an autoimmune phenotype. There is an urgent need to prospectively evaluate patients with DI-ALH systematically to enable definitive characterisation of this condition.

Research gaps and opportunities in autoimmune hepatitis-Results of the international autoimmune hepatitis group research workshop 2022.

Autoimmune hepatitis (AIH) is a rare autoimmune liver disease that is characterised by a chronic inflammatory immune reaction directed against hepatocytes. The disease results in a substantial reduction in quality of life and potentially leads to liver-related complications or death. The International Autoimmune Hepatitis Group (IAIHG) initiated a series of research workshops to uncover the scientific gaps and opportunities in AIH. This review summarises the results of the latest workshop in Maastricht in 2022 and reviews the current challenges in adult AIH, particularly in relation to four important aspects of AIH: diagnostics; new immunomodulatory therapies; clinical trial design; and unmet clinical needs. This review also summarises the progress made since the AIH workshop in 2017. Patients and patient representatives were actively involved in the parallel working groups alongside clinicians and researchers. Despite 40 years of experience with diagnosing and treating AIH, false diagnoses occur and treatment is still based on nonselective immunosuppression. In addition to the need for more specific diagnostic tests, prognostic markers and tailor-based treatments, a major unmet clinical need was identified in areas of care delivery and health-related quality of life.

Breakdown in hepatic tolerance and its relation to autoimmune liver diseases.

The liver is a complex immunological organ. It has both immunogenic and tolerogenic capacity. Tolerogenic potential of human liver with its protective firewalls is required to guard the body against the continuous influx of microbial product from the gut via the sinusoids and biliary tree. Immunotolerance and anergic state is maintained by a combined effort of both immune cells, parenchyma cells, epithelial and endothelial cells. Despite this, an unknown trigger can ignite the pathway towards breakdown in hepatic tolerance leading to autoimmune liver diseases. Understanding the initial stimulus which causes the hepatic immune system to switch from the regulatory arm towards self-reactive effector arm remains challenging. Dissecting this pathology using the current technological advances is crucial to develop curative immune based therapy in autoimmune liver diseases. We discuss the hepatic immune cells and non-immune cells which maintain liver tolerance and the evidence of immune system barrier breach which leads to autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis.

Beyond FOXP3: a 20-year journey unravelling human regulatory T-cell heterogeneity.

The initial idea of a distinct group of T-cells responsible for suppressing immune responses was first postulated half a century ago. However, it is only in the last three decades that we have identified what we now term regulatory T-cells (Tregs), and subsequently elucidated and crystallized our understanding of them. Human Tregs have emerged as essential to immune tolerance and the prevention of autoimmune diseases and are typically contemporaneously characterized by their CD3+CD4+CD25high CD127lowFOXP3+ phenotype. It is important to note that FOXP3+ Tregs exhibit substantial diversity in their origin, phenotypic characteristics, and function. Identifying reliable markers is crucial to the accurate identification, quantification, and assessment of Tregs in health and disease, as well as the enrichment and expansion of viable cells for adoptive cell therapy. In our comprehensive review, we address the contributions of various markers identified in the last two decades since the master transcriptional factor FOXP3 was identified in establishing and enriching purity, lineage stability, tissue homing and suppressive proficiency in CD4+ Tregs. Additionally, our review delves into recent breakthroughs in innovative Treg-based therapies, underscoring the significance of distinct markers in their therapeutic utilization. Understanding Treg subsets holds the key to effectively harnessing human Tregs for immunotherapeutic approaches.

Diesel-Biodiesel-Water Fuel Nanoemulsions for Direct Injection and Indirect Injection Diesel Engines: Performance and Emission Characteristics.

An experimental research is assessed to examine the engine performance and exhaust emissions of direct injection (DI) and indirect injection diesel (IDI) engines fueled with petroleum diesel, biodiesel, and nanoemulsion fuel. The nanoemulsion fuel was produced using a hydrodynamic cavitation reactor. These three fuels were used to study the exhaust emissions, brake power, brake specific fuel consumption (BSFC), brake thermal efficiency (BTE), and exhaust gas temperature at engine speeds ranging from 1100, 1400, 1700, 2000, and 2300 rpm with engine loads of 25, 50, and 75%. Furthermore, three fuels were burned in two types of combustion engines such as DI and IDI diesel engines under identical conditions. The finding showed that using DI and IDI engines influenced the magnitude of emissions as well as the performance with different speeds and loads. By comparing the performance of DI and IDI engines at a maximum engine load of 75%, the most concerning parameter among the efficiency of an engine of BTE of diesel, biodiesel, and nanoemulsion fuel from the DI engine was higher at 24.19, 24.83, and 20.76%, respectively, than that of the IDI engine at 2300 rpm engine speed. At the maximum load and speed of engines, the BSFC of diesel, biodiesel, and nanoemulsion fuel in the DI engine were 4.44, 23.73, and 20% lower than in the IDI engine, respectively. Emission results of the DI and IDI engines were analyzed at 75% load and 2300 rpm speed. The results demonstrated that emissions of NO x from nanoemulsion fuel from the IDI engine was significantly reduced by 82.46% when the values were compared to the DI engine. In terms of CO emissions, the IDI engine emits significantly less than the DI diesel engine. The CO emissions of diesel, biodiesel, and nanoemulsion fuel in the IDI engine were 69.02, 28.95, and 48.75% lower than those in the DI engine, respectively. The studies conclude that the emissions from IDI engines clearly show that pollution from exhaust emissions can be reduced to a low level compared to the DI engine. However, when high-performance engines are considered, the DI engine is recommended rather than the IDI engine.

Normothermic Machine Perfusion-Improving the Supply of Transplantable Livers for High-Risk Recipients.

The effectiveness of liver transplantation to cure numerous diseases, alleviate suffering, and improve patient survival has led to an ever increasing demand. Improvements in preoperative management, surgical technique, and postoperative care have allowed increasingly complicated and high-risk patients to be safely transplanted. As a result, many patients are safely transplanted in the modern era that would have been considered untransplantable in times gone by. Despite this, more gains are possible as the science behind transplantation is increasingly understood. Normothermic machine perfusion of liver grafts builds on these gains further by increasing the safe use of grafts with suboptimal features, through objective assessment of both hepatocyte and cholangiocyte function. This technology can minimize cold ischemia, but prolong total preservation time, with particular benefits for suboptimal grafts and surgically challenging recipients. In addition to more physiological and favorable preservation conditions for grafts with risk factors for poor outcome, the extended preservation time benefits operative logistics by allowing a careful explant and complicated vascular reconstruction when presented with challenging surgical scenarios. This technology represents a significant advancement in graft preservation techniques and the transplant community must continue to incorporate this technology to ensure the benefits of liver transplant are maximized.

Interplay between Mast Cells and Regulatory T Cells in Immune-Mediated Cholangiopathies.

Immune-mediated cholangiopathies are characterised by the destruction of small and large bile ducts causing bile acid stasis, which leads to subsequent inflammation, fibrosis, and eventual cirrhosis of the liver tissue. A breakdown of peripheral hepatic immune tolerance is a key feature of these diseases. Regulatory T cells (Tregs) are a major anti-inflammatory immune cell subset, and their quantities and functional capacity are impaired in autoimmune liver diseases. Tregs can undergo phenotypic reprogramming towards pro-inflammatory Th1 and Th17 profiles. The inflamed hepatic microenvironment influences and can impede normal Treg suppressive functions. Mast cell (MC) infiltration increases during liver inflammation, and active MCs have been shown to be an important source of pro-inflammatory mediators, thus driving pathogenesis. By influencing the microenvironment, MCs can indirectly manipulate Treg functions and inhibit their suppressive and proliferative activity. In addition, direct cell-to-cell interactions have been identified between MCs and Tregs. It is critical to consider the effects of MCs on the inflammatory milieu of the liver and their influence on Treg functions. This review will focus on the roles and crosstalk of Tregs and MCs during autoimmune cholangiopathy pathogenesis progression.

Continuous Double-Step Esterification Production of Palm Fatty Acid Distillate Methyl Ester Using Ultrasonic Tubular Reactor.

Double-step esterification to produce biodiesel from palm fatty acid distillate (PFAD) was performed by utilizing an ultrasound clamp reactor. Six pairs of ultrasonic clamps were attached to the left and right sides of the stainless-steel tube, and each pair was separated 100 mm apart from each other. Therefore, a total of 12 units of ultrasound clamps distributed 4800 W maximum power (12 × 400 W) throughout the continuous reactor by an ultrasonic generator. To optimize each step of the continuous esterification process for producing methyl ester from PFAD, a response surface methodology was used. The final 93.32 wt % methyl ester purity was attained under a double-step esterification process. For the first step, a 3.75:1 molar ratio of methanol to PFAD (46.4 vol % methanol), 6.6 vol % sulfuric acid, and 400 mm length of ultrasound clamp at 25 L/h PFAD flow rate for converting the PFAD to 60.24 wt % methyl ester were recommended. For the second step, the esterification was repeated under a molar ratio of methanol to the first esterified oil of 2.87:1 (61.6 vol % methanol), 5.6 vol % of sulfuric acid, and 400 mm length of ultrasound clamp at 25 L/h esterified oil flow rate. The ultrasonic clamp reactor achieved high yields of esterified oil and the crude biodiesel in a relatively short residence period of 32 s. To determine the product yields of a double-step esterification process, the maximum yields were 103.9 wt % first esterified oil, 107.6% crude biodiesel, and 98 wt % purified biodiesel when calculated on the basis of 100 vol % initial PFAD. The average energy consumed in the production of double-step esterification biodiesel was 0.05796 kWh/L. Therefore, this current approach has a high potential for producing biodiesel with less energy and requires less time to convert the PFAD to a high purity of methyl ester.

Continuous production of nanoemulsion for skincare product using a 3D-printed rotor-stator hydrodynamic cavitation reactor.

In this study, nanoemulsions for skincare products were continuously produced using a hydrodynamic cavitation reactor (HCR) designed with a rotor and stator. The key component of this research is the utilization of a 3D-printed rotor in a HCR for the production of an oil-in-water nanoemulsion. Response surface methodology was used to determine the process conditions, such as speed of the rotor, flow rate, as well as, Span60, Tween60, and mineral oil concentrations, for generating the optimal droplet size in the nanoemulsion. The results showed that a droplet size of 366.4 nm was achieved under the recommended conditions of rotor speed of 3500 rpm, flow rate of 3.3 L/h, Span60 concentration of 2.36 wt%, Tween60 concentration of 3.00 wt%, and mineral oil concentration of 1.76 wt%. Moreover, the important characteristics for consideration in skincare products, such as polydispersity index, pH, zeta potential, viscosity, stability, and niacin released from formulations, were also assessed. For the niacin release profile of emulsion and nanoemulsion formulations, different methods, such as magnetic stirring, ultrasound, and hydrodynamic cavitation, were compared. The nanoemulsion formulations provided a greater cumulative release from the formulation than the emulsion. Particularly, the nanoemulsion generated using the HCR provided the largest cumulative release from the formulation after 12 h. Therefore, the present study suggests that nanoemulsions can be created by means of hydrodynamic cavitation, which reduces the droplet size, as compared to that generated using other techniques. The satisfactory results of this study indicate that the rotor-stator-type HCR is a potentially cost-effective technology for nanoemulsion production.