Uptake Transporters at the Blood-Brain Barrier and Their Role in Brain Drug Disposition.

Uptake drug transporters play a significant role in the pharmacokinetic of drugs within the brain, facilitating their entry into the central nervous system (CNS). Understanding brain drug disposition is always challenging, especially with respect to preclinical to clinical translation. These transporters are members of the solute carrier (SLC) superfamily, which includes organic anion transporter polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), and amino acid transporters. In this systematic review, we provide an overview of the current knowledge of uptake drug transporters in the brain and their contribution to drug disposition. Here, we also assemble currently available proteomics-based expression levels of uptake transporters in the human brain and their application in translational drug development. Proteomics data suggest that in association with efflux transporters, uptake drug transporters present at the BBB play a significant role in brain drug disposition. It is noteworthy that a significant level of species differences in uptake drug transporters activity exists, and this may contribute toward a disconnect in inter-species scaling. Taken together, uptake drug transporters at the BBB could play a significant role in pharmacokinetics (PK) and pharmacodynamics (PD). Continuous research is crucial for advancing our understanding of active uptake across the BBB.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Commercializing Sonic Seasoning in Multisensory Offline Experiential Events and Online Tasting Experiences.

The term "sonic seasoning" refers to the deliberate pairing of sound/music with taste/flavour in order to enhance, or modify, the multisensory tasting experience. Although the recognition that people experience a multitude of crossmodal correspondences between stimuli in the auditory and chemical senses originally emerged from the psychophysics laboratory, the last decade has seen an explosion of interest in the use and application of sonic seasoning research findings, in a range of multisensory experiential events and online offerings. These marketing-led activations have included a variety of different approaches, from curating pre-composed music selections that have the appropriate sonic qualities (such as pitch or timbre), to the composition of bespoke music/soundscapes that match the specific taste/flavour of particular food or beverage products. Moreover, given that our experience of flavour often changes over time and frequently contains multiple distinct elements, there is also scope to more closely match the sonic seasoning to the temporal evolution of the various components (or notes) of the flavour experience. We review a number of case studies of the use of sonic seasoning, highlighting some of the challenges and opportunities associated with the various approaches, and consider the intriguing interplay between physical and digital (online) experiences. Taken together, the various examples reviewed here help to illustrate the growing commercial relevance of sonic seasoning research.

Metacognition and Crossmodal Correspondences Between Auditory Attributes and Saltiness in a Large Sample Study.

Mounting evidence demonstrates that people make surprisingly consistent associations between auditory attributes and a number of the commonly-agreed basic tastes. However, the sonic representation of (association with) saltiness has remained rather elusive. In the present study, a crowd-sourced online study ( n = 1819 participants) was conducted to determine the acoustical/musical attributes that best match saltiness, as well as participants' confidence levels in their choices. Based on previous literature on crossmodal correspondences involving saltiness, thirteen attributes were selected to cover a variety of temporal, tactile, and emotional associations. The results revealed that saltiness was associated most strongly with a long decay time, high auditory roughness, and a regular rhythm. In terms of emotional associations, saltiness was matched with negative valence, high arousal, and minor mode. Moreover, significantly higher average confidence ratings were observed for those saltiness-matching choices for which there was majority agreement, suggesting that individuals were more confident about their own judgments when it matched with the group response, therefore providing support for the so-called 'consensuality principle'. Taken together, these results help to uncover the complex interplay of mechanisms behind seemingly surprising crossmodal correspondences between sound attributes and taste.

COVID-19 patient bridged to recovery with veno-venous extracorporeal membrane oxygenation.

BACKGROUND: In severe cases, the coronavirus disease 2019 (COVID-19) viral pathogen produces hypoxic respiratory failure unable to be adequately supported by mechanical ventilation. The role of extracorporeal membrane oxygenation (ECMO) remains unknown, with the few publications to date lacking detailed patient information or management algorithms all while reporting excessive mortality. METHODS: Case report from a prospectively maintained institutional ECMO database for COVID-19. RESULTS: We describe veno-venous (VV) ECMO in a COVID-19-positive woman with hypoxic respiratory dysfunction failing mechanical ventilation support while prone and receiving inhaled pulmonary vasodilator therapy. After 9 days of complex management secondary to her hyperdynamic circulation, ECMO support was successfully weaned to supine mechanical ventilation and the patient was ultimately discharged from the hospital. CONCLUSIONS: With proper patient selection and careful attention to hemodynamic management, ECMO remains a reasonable treatment option for patients with COVID-19.

Hyaluronan and LYVE-1 and allograft function in lung transplantation recipients.

Hyaluronan (HA) is associated with innate immune response activation and may be a marker of allograft dysfunction in lung transplant recipients. This was a prospective, single center study comparing levels of bronchioalveolar lavage (BAL) and serum HA and the HA immobilizer LYVE-1 in lung transplant recipients with and without acute cellular rejection (ACR). Chronic lung allograft dysfunction (CLAD)-free survival was also evaluated based on HA and LYVE-1 levels. 78 recipients were enrolled with a total of 115 diagnostic biopsies and 1.5 years of median follow-up. Serum HA was correlated with BAL HA (r = 0.25, p = 0.01) and with serum LYVE-1 (r = 0.32, p = 0.002). There was significant variation in HA and LYVE-1 over time, regardless of ACR status. Levels of serum HA (median 74.7 vs 82.7, p = 0.69), BAL HA (median 149.4 vs 134.5, p = 0.39), and LYVE-1 (mean 190.2 vs 183.8, p = 0.72) were not associated with ACR. CLAD-free survival was not different in recipients with any episode of elevated serum HA (HR = 1.5, 95% CI = 0.3-7.7, p = 0.61) or BAL HA (HR = 0.94, 95% CI = 0.2-3.6, p = 0.93). These results did not differ when stratified by bilateral transplant status. In this small cohort, serum HA, BAL HA, and LYVE-1 levels are not associated with ACR or CLAD-free survival in lung transplant recipients.

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development.

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

Activated protein C restores hepatic microcirculation during sepsis by modulating vasoregulator expression.

Activated protein C (aPC) promotes fibrinolysis while inhibiting coagulation and inflammation. In septic patients, aPC levels are depleted, and aPC treatment has emerged as a therapeutic option. To better understand the mechanism(s) by which aPC improves survival in sepsis, we sought to determine the effect of aPC treatment on hepatic vasoactive gene and protein expression, leading to changes in hepatic vascular responsiveness in a septic animal model. Under anesthesia, rats underwent sham or cecal ligation and puncture followed by aPC treatment (1 mg/kg, twice daily, i.v.). Treatment with aPC significantly decreased hepatic endothelin 1 (ET-1)/ET A receptor mRNA and protein expression. To determine the effect of aPC on hepatic microvasculature, ET-1-induced changes in liver microcirculation were assessed by intravital microscopy. This approach demonstrated aPC significantly improved hepatic perfusion index in the animals that underwent cecal ligation and puncture in the absence of significant changes in portal venous pressure. Furthermore, although aPC did not affect ET-1-dependent sinusoidal vasoconstriction, aPC induced hepatoprotective effects via enhanced red blood cell velocity. Collectively, these data demonstrate aPC ameliorates ET-1-dependent changes in hepatic microcirculation and improves hepatic function in the setting of sepsis.

A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension.

BACKGROUND: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs. METHODS AND RESULTS: Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, -361 (A/T), -254(C/G), and -218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the -361 and -218 SNPs were not different between the groups, the allele frequency of the -254(C-->G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of -254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the -254(C-->G) SNP creates a binding sequence for nuclear factor-kappaB. Functional analyses revealed that the -254(C-->G) SNP enhanced nuclear factor-kappaB-mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-kappaB activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the -254G allele. CONCLUSIONS: These results suggest that the -254(C-->G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-kappaB, an inflammatory transcription factor.

Segmentation of vessels cluttered with cells using a physics based model.

Segmentation of vessels in biomedical images is important as it can provide insight into analysis of vascular morphology, topology and is required for kinetic analysis of flow velocity and vessel permeability. Intravital microscopy is a powerful tool as it enables in vivo imaging of both vasculature and circulating cells. However, the analysis of vasculature in those images is difficult due to the presence of cells and their image gradient. In this paper, we provide a novel method of segmenting vessels with a high level of cell related clutter. A set of virtual point pairs ("vessel probes") are moved reacting to forces including Vessel Vector Flow (VVF) and Vessel Boundary Vector Flow (VBVF) forces. Incorporating the cell detection, the VVF force attracts the probes toward the vessel, while the VBVF force attracts the virtual points of the probes to localize the vessel boundary without being distracted by the image features of the cells. The vessel probes are moved according to Newtonian Physics reacting to the net of forces applied on them. We demonstrate the results on a set of five real in vivo images of liver vasculature cluttered by white blood cells. When compared against the ground truth prepared by the technician, the Root Mean Squared Error (RMSE) of segmentation with VVF and VBVF was 55% lower than the method without VVF and VBVF.

Dynamics of alpha-synuclein aggregation and inhibition of pore-like oligomer development by beta-synuclein.

Accumulation of alpha-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, beta-synuclein (beta-syn), a close homologue, does not aggregate and reduces alpha-synuclein (alpha-syn)-related pathology. Although considerable information is available about the conformation of alpha-syn at the initial and end stages of fibrillation, less is known about the dynamic process of alpha-syn conversion to oligomers and how interactions with antiaggregation chaperones such as beta-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of alpha-syn showed that alpha-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating alpha-syn dimers on the membrane incorporate additional alpha-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, beta-syn dimers do not propagate and block the aggregation of alpha-syn into ring-like oligomers. Under in vitro cell-free conditions, alpha-syn aggregates formed ring-like structures that were disrupted by beta-syn. Similarly, cells expressing alpha-syn displayed increased ion current activity consistent with the formation of Zn(2+)-sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, alpha-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of beta-synuclein might be related to its ability to block the formation of pore-like structures.

Thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels.

Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC50 was 335 and 58 microM for 40 mM K+ - and 2 microM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3'-5'-cyclic monophosphate (50 microM) and HA-1004 (30 microM). A rise in extracellular K+ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 microM) and active store depletion by phenylephrine (in the absence of external Ca2+ both induced PA contraction due to capacitative Ca2+ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca2+ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels, which subsequently attenuate Ca2+ influx and decrease cytosolic free Ca2+ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K+ channels and inhibition of store-operated Ca2+ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway.

Confirming human antibody responses to a therapeutic monoclonal antibody using a statistical approach.

Confirmatory assays for immunogenicity testing typically involve testing a sample in the presence or absence of excess drug. A decrease in assay signal in the presence of drug is taken to indicate the presence of anti-drug antibodies (ADAb) and the sample is confirmed positive. While there is widespread acceptance of the principle, there are currently no published guidelines for determining how much the signal should be reduced for a sample to be confirmed positive. In this report we address this issue using a novel approach employing a Student's t-test. The basic premise is to assess if an observed decrease in signal in the presence of drug is greater than what might be expected to occur as a result of the normal variation in the system. A key component of the method involves being able to capture and measure all of the normal variation. This requires a modification of commonly employed methods of sample preparation. We validated the method and tested samples from a clinical study. In addition, we reanalyzed the data to see what would have been the outcome had we used two other common approaches for confirmatory assays, one based on a minimum percent decrease in signal to confirm positivity (arbitrarily set at 25%), and one requiring a minimum drop in signal, set by a low quality control (QC) sample. The t-test approach proved superior over a wide range of assay signals and appeared to result in fewer false negatives.

Inhibition of endothelin-1-mediated up-regulation of iNOS by bosentan ameliorates endotoxin-induced liver injury in cirrhosis.

Endothelin-1 (ET-1) has been shown to regulate the expression of various genes in addition to its vasoconstrictor role in the liver. Elevated levels of ET-1 during cirrhosis play an important role in the observed microcirculatory dysfunction; however, its role as a transcription regulator remains unclear. This study aimed to determine the role of ET-1 in the hepatic gene expression of vasomediators after cirrhosis in response to LPS. Cirrhosis was induced by bile duct ligation (BDL) for 1 or 3 weeks in male Sprague-Dawley rats. Following 1 or 3 weeks of BDL or sham operation (sham), rats received an intravenous (i.v.) injection of bosentan, a dual-selective ETA/B receptor antagonist (30 mg/kg bw) or saline, and an intraperitoneal (i.p.) injection of LPS (1 mg/kg bw). Plasma alanine aminotransferase (ALT) levels were significantly elevated in 1- and 3-week BDL animals. Six hours following LPS, the elevated ALT levels were markedly exacerbated in 3-week BDL animals, which were significantly ameliorated with bosentan treatment. LPS resulted in increased ET-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 mRNA expressions in both sham and BDL rats. Bosentan significantly inhibited the up-regulations of ET-1, iNOS, and COX-2 mRNA. Our data strongly suggest that ET-1 plays an important role in up-regulating the expression of iNOS, COX-2, and ET-1 itself in hepatic tissue following LPS challenge, which may contribute to the observed hepatocellular injury during endotoxemia in cirrhosis. Thus, due to significant increases in ET-1 levels during cirrhosis, ET-1 receptor blockade may prove to be of great therapeutic value in the treatment of cirrhotic patients exposed to secondary injuries such as endotoxemia.

Induction of biphasic changes in perfusion heterogeneity of rat liver after sequential stress in vivo.

Trauma and subsequent sepsis lead to hepatic microcirculation disruption through various molecular mechanisms in which endothelin-1 (ET-1) plays a pivotal role. These stresses are thought to alter hepatic perfusion, heterogeneously leading to a mismatch of oxygen supply and demand. We hypothesize that mild remote stresses prime the liver to sequential sepsis through direct effects on the hepatic lobular flow distribution. We also propose to investigate the extent and the localization of the stress-induced microcirculation disruption. Sprague-Dawley rats were randomly divided into four experimental groups: sham, femur fracture (FFX), cecal ligation and puncture (CLP), and sequential stress (SS). Hepatic intravital microscopy was performed for in vivo assessment of the liver microcirculation flow distribution under baseline and after ET-1 infusion. Red blood cell motion distribution was used to quantify intralobular and interlobular heterogeneity of perfusion (HoP). Intralobular HoP, which reflects lobular regulation sites, was significantly increased in the FFX and CLP groups, but was not changed or decreased in the SS group compared with control. ET-1 infusion exerted opposite effects depending on the pathological condition, further increasing the difference between groups. SS induced decrease in intralobular HoP, contrasted with a significant increase in interlobular HoP, suggesting multiple disruption sites. Our data suggest that increased intralobular HoP may be indicative of a compensatory response to moderate stress; its decrease under sequential stress conditions corresponds with a total breakdown of hepatic lobular flow regulation. This may be another instance of the rich variability characteristic of normal physiology that "decomplexifies" under critical decompensated conditions.

Kupffer cell ablation improves hepatic microcirculation after trauma and sepsis.

BACKGROUND: Macrophages undergo maladaptive alterations after trauma. In this study, we assessed the role of Kupffer cells in hepatic microcirculatory response to endothelin-1 (ET-1) after femur fracture (FFx) and cecal ligation and puncture (CLP). METHODS: Sprague-Dawley rats (200-300 g) underwent sham, FFx, CLP, or FFx + CLP. To ablate Kupffer cells, group 1 animals were treated with gadolinium chloride, and group 2 animals received saline. Hepatic microcirculation was assessed by intravital microscopy. Liver mitochondrial redox state and tissue oxygen (tPo2) were determined by NADH and ruthenium fluorescence, respectively. Liver damage was estimated by alanine aminotransferase levels. Differences were assessed using analysis of variance followed by Student-Newman-Keuls post hoc test. RESULTS: After 10 minutes of ET-1, CLP and FFx + CLP caused significant reduction in hepatic perfusion index (2.5-fold and 5-fold vs. sham, p < 0.05, respectively), redox state (36% and 45% vs. sham, p < 0.01, respectively), tPo2 (10% and 12% vs. sham, p < 0.05, respectively), and more liver damage compared with sham and FFx-treated animals. Kupffer cell depletion restored microcirculation, redox state, and tPo2 and abrogated hepatocellular damage. CONCLUSION: Kupffer cells contribute directly to hepatic microcirculatory dysfunction and liver injury after inflammatory stress. Furthermore, Kupffer cell depletion ameliorates the microcirculatory perturbations of trauma and sepsis. Thus, modulation of Kupffer cell response may prove beneficial.

Kupffer cell ablation attenuates cyclooxygenase-2 expression after trauma and sepsis.

BACKGROUND: Prostaglandins, synthesized by cyclooxygenase (COX), play an important role in the pathophysiology of inflammation. Severe injuries result in immunosuppression, mediated, in part, by maladaptive changes in macrophages. Herein, we assessed Kupffer cell-mediated cyclooxygenase-2 (COX-2) expression on liver function and damage after trauma and sepsis. MATERIALS AND METHODS: To ablate Kupffer cells, Sprague Dawley rats were treated with gadolinium chloride (GdCl3) 48 and 24 h before experimentation. Animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). Controls received sham operations. After 24 h, liver samples were obtained, and mRNA and protein expression were determined by PCR, Western blot, and immunohistochemistry. Indocyanine-Green (ICG) clearance and plasma alanine aminotransferase (ALT) levels were determined to assess liver function and damage, respectively. One-way analysis of variance (ANOVA) with Student-Newman-Keuls test was used to assess statistical significance. RESULTS: After CLP alone, FFx+CLP, and GdCl3+FFx+CLP, clearance of ICG decreased. Plasma ALT levels increased in parallel with severity of injury. Kupffer cell depletion attenuated the increased ALT levels after FFx+CLP. Femur fracture alone did not alter COX-2 protein compared with sham. By contrast, COX-2 protein increased after CLP and was potentiated by sequential stress. Again, Kupffer cell depletion abrogated the increase in COX-2 after sequential stress. Immunohistochemical data confirmed COX-2 positive cells to be Kupffer cells. CONCLUSIONS: In this study, sequential stress increased hepatic COX-2 protein. Depletion of Kupffer cells reduced COX-2 and attenuated hepatocellular injuries. Our data suggest that Kupffer cell-dependent pathways may contribute to the inflammatory response leading to increased mortality after sequential stress.