Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism.

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Risk assessment of the asthma-induction potential of substances in spray products for car cabin detailing - based on EU's Chemical Agents Directive, using harmonised classifications and quantitative structure-activity relationship (QSAR).

Exposure to spray-formulated products for car cabin detailing is a potential risk for asthma induction. With a focus on the asthma-related endpoints sensitisation and irritation of the lungs, we performed an occupational risk assessment based on requirements in the EU Chemical Agents Directive. We identified 71 such spray products available in Denmark. We identified ingredient substances in safety data sheets and screened for harmonised classifications of respiratory sensitisation and airway irritation. For respiratory sensitisation, we also applied quantitative structure-activity relationship (QSAR). We modelled the exposure during 15 min of work inside a car cabin, and determined the risk ratio of the products by further applying occupational exposure limits - mainly derived no-effect levels (DNELs) from the European Chemicals Agency (ECHA) set on respiratory irritation. Four substances had a harmonised classification for respiratory irritation (bronopol, 2-phenoxyethanol, 2-methoxypropanol, and butan-1-ol). Seven substances were positive in the QSAR model for respiratory sensitisation (monoethanolamine, bronopol, glycerol, methyl salicylate, benzoic acid, ammonium benzoate, and sodium benzoate). Two vinyl treatment products had a risk ratio > 1 based on the level of sodium benzoate and its DNEL set on respiratory irritation. Two products had risk ratios of 0.69 and 0.73, respectively, based on 2-methyl-2 H-isothiazol-3-one and its acute DNEL set on respiratory irritation. In conclusion, 10 substances that may pose a risk for asthma induction were identified in the products. Two of the 71 products had a risk ratio > 1, meaning they may pose an asthma-induction risk in the modelled exposure scenario and using respiratory irritation DNELs from ECHA.

An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&5), conferring ß-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other ß-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.

Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms.

Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

Adaptive laboratory evolution and independent component analysis disentangle complex vancomycin adaptation trajectories.

Human infections with methicillin-resistant Staphylococcus aureus (MRSA) are commonly treated with vancomycin, and strains with decreased susceptibility, designated as vancomycin-intermediate S. aureus (VISA), are associated with treatment failure. Here, we profiled the phenotypic, mutational, and transcriptional landscape of 10 VISA strains adapted by laboratory evolution from one common MRSA ancestor, the USA300 strain JE2. Using functional and independent component analysis, we found that: 1) despite the common genetic background and environmental conditions, the mutational landscape diverged between evolved strains and included mutations previously associated with vancomycin resistance (in vraT, graS, vraFG, walKR, and rpoBCD) as well as novel adaptive mutations (SAUSA300_RS04225, ssaA, pitAR, and sagB); 2) the first wave of mutations affected transcriptional regulators and the second affected genes involved in membrane biosynthesis; 3) expression profiles were predominantly strain-specific except for sceD and lukG, which were the only two genes significantly differentially expressed in all clones; 4) three independent virulence systems (φSa3, SaeR, and T7SS) featured as the most transcriptionally perturbed gene sets across clones; 5) there was a striking variation in oxacillin susceptibility across the evolved lineages (from a 10-fold increase to a 63-fold decrease) that also arose in clinical MRSA isolates exposed to vancomycin and correlated with susceptibility to teichoic acid inhibitors; and 6) constitutive expression of the VraR regulon explained cross-susceptibility, while mutations in walK were associated with cross-resistance. Our results show that adaptation to vancomycin involves a surprising breadth of mutational and transcriptional pathways that affect antibiotic susceptibility and possibly the clinical outcome of infections.

Functional Characterization of Type III-A CRISPR-Cas in a Clinical Human Methicillin-R Staphylococcus aureus Strain.

CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.

The Novel Membrane-Associated Auxiliary Factors AuxA and AuxB Modulate ß-lactam Resistance in MRSA by stabilizing Lipoteichoic Acids.

A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.

Carcinoembryonic Antigen (CEA)-Specific 4-1BB-Costimulation Induced by CEA-Targeted 4-1BB-Agonistic Trimerbodies.

4-1BB (CD137) is an inducible costimulatory receptor that promotes expansion and survival of activated T cells; and IgG-based 4-1BB-agonistic monoclonal antibodies exhibited potent antitumor activity in clinical trials. However, the clinical development of those antibodies is restricted by major off-tumor toxicities associated with FcγR interactions. We have recently generated an EGFR-targeted 4-1BB-agonistic trimerbody that demonstrated strong antitumor activity and did not induce systemic inflammatory cytokine secretion and hepatotoxicity associated with first-generation 4-1BB agonists. Here, we generate a bispecific 4-1BB-agonistic trimerbody targeting the carcinoembryonic antigen (CEA) that is highly expressed in cancers of diverse origins. The CEA-targeted anti-4-1BB-agonistic trimerbody consists of three 4-1BB-specific single-chain fragment variable antibodies and three anti-CEA single-domain antibodies positioned around a murine collagen XVIII-derived homotrimerization domain. The trimerbody was produced as a homogenous, non-aggregating, soluble protein purifiable by standard affinity chromatographic methods. The purified trimerbody was found to be trimeric in solution, very efficient at recognizing 4-1BB and CEA, and potently costimulating T cells in vitro in the presence of CEA. Therefore, trimerbody-based tumor-targeted 4-1BB costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions.

Cochlear implantation in a 10-year old boy with Pendred syndrome and extremely enlarged endolymphatic sacs.

A 10-year-old boy with fluctuating sensorineural hearing loss (SNHL) and biallelic mutations in the SLC26A4 gene and with inner ear anomalies received a cochlear implantation. SLC26A4 mutations are associated with variable degrees of SNHL and enlarged vestibular aqueducts (EVA), identified either as non-syndromic EVA or classic Pendred syndrome; the latter also associated with thyroid dysfunction. The inner ear malformations in this group of patients have been considered a relative contraindication against cochlear implantation because of the potential per- and postoperative complications such as peroperative cerebrospinal fluid leak or postoperative vestibular symptoms. In the current case there were no surgical or postoperative complications, indicating that extremely enlarged endolymphatic sacs are not as such a contraindication for cochlear implantation. This case also illustrates the management dilemma of an appropriate timing for cochlear implantation.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies.

The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells has shown satisfactory results in preclinical cancer models. Even so, the approach's translation into the clinic will require incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE), consisting of an EGFR-specific single-domain VHH antibody fused to a CD3-specific scFv. We generated two LiTEs with the anti-EGFR VHH and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers are, therefore, suitable for future applications in gene-based immunotherapy approaches.

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy.

The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies.

The lumped constant for the galactose analog 2-18F-fluoro-2-deoxy-D-galactose is increased in patients with parenchymal liver disease.

UNLABELLED: The galactose analog 2-(18)F-fluoro-2-deoxy-d-galactose ((18)F-FDGal) is a suitable PET tracer for measuring hepatic galactokinase capacity in vivo, which provides estimates of hepatic metabolic function. As a result of a higher affinity of galactokinase toward galactose, the lumped constant (LC) for (18)F-FDGal was 0.13 in healthy subjects. The aim of the present study was to test the hypothesis of a significantly different LC for (18)F-FDGal in patients with parenchymal liver disease. METHODS: Nine patients with liver cirrhosis were studied in connection with a previous study with determination of hepatic intrinsic clearance of ¹8F-FDGal (V*(max/K*(m)). The present study determined the hepatic removal kinetics of galactose, including hepatic intrinsic clearance of galactose (V(max)/K(m)) from measurements of hepatic blood flow and arterial and liver vein blood galactose concentrations at increasing galactose infusions. LC for ¹8F-FDGal was calculated as (V*(max)/K*(m))/(V(max)/K(m)). On a second day, a dynamic ¹8-FDGal PET study with simultaneous infusion of galactose (mean arterial galactose concentration, 6.1 mmol/L of blood) and blood samples from a radial artery was performed, with determination of hepatic systemic clearance of ¹8F-FDGal (K*(+gal) from linear analysis of data (Gjedde-Patlak method). The maximum hepatic removal rate of galactose was estimated from ¹8F-FDGal PET data (V(max)(PET)) using the estimated LC. RESULTS: The mean hepatic V(max) of galactose was 1.18 mmol/min, the mean K(m) was 0.91 mmol/L of blood and the mean V(max)/K(m) was 1.18 L of blood/min. When compared with values of healthy subjects, K(m) did not differ (P = 0.77), whereas both V(max) and V(max)/K(m) were significantly lower in patients (both P < 0.01). Mean LC for ¹8LF-FDGal was 0.24, which was significantly higher than the mean LC of 0.13 in healthy subjects (P < 0.0001). Mean K*(+gal) determined from the PET study was 0.019 L of blood/min/L of liver tissue, which was not significantly different from that in healthy subjects (P = 0.85). Mean hepatic V(max)(PET) was 0.57 mmol/min/L of liver tissue, which was significantly lower than the value in healthy subjects (1.41 mmol/min/L of liver tissue (P < 0.0001). CONCLUSION: Disease may change the LC for a pet tracer, and this study demonstrated the importance of using the correct LC.

Regional metabolic liver function measured in patients with cirrhosis by 2-[¹8F]fluoro-2-deoxy-D-galactose PET/CT.

BACKGROUND & AIMS: There is a clinical need for methods that can quantify regional hepatic function non-invasively in patients with cirrhosis. Here we validate the use of 2-[(18)F]fluoro-2-deoxy-d-galactose (FDGal) PET/CT for measuring regional metabolic function to this purpose, and apply the method to test the hypothesis of increased intrahepatic metabolic heterogeneity in cirrhosis. METHODS: Nine cirrhotic patients underwent dynamic liver FDGal PET/CT with blood samples from a radial artery and a liver vein. Hepatic blood flow was measured by indocyanine green infusion/Fick's principle. From blood measurements, hepatic systemic clearance (Ksyst, Lblood/min) and hepatic intrinsic clearance (Vmax/Km, Lblood/min) of FDGal were calculated. From PET data, hepatic systemic clearance of FDGal in liver parenchyma (Kmet, mL blood/mL liver tissue/min) was calculated. Intrahepatic metabolic heterogeneity was evaluated in terms of coefficient-of-variation (CoV, %) using parametric images of Kmet. RESULTS: Mean approximation of Ksyst to Vmax/Km was 86% which validates the use of FDGal as PET tracer of hepatic metabolic function. Mean Kmet was 0.157 mL blood/mL liver tissue/min, which was lower than 0.274 mL blood/mL liver tissue/min, previously found in healthy subjects (p<0.001), in accordance with decreased metabolic function in cirrhotic livers. Mean CoV for Kmet in liver tissue was 24.4% in patients and 14.4% in healthy subjects (p<0.0001). The degree of intrahepatic metabolic heterogeneity correlated positively with HVPG (p<0.05). CONCLUSIONS: A 20-min dynamic FDGal PET/CT with arterial sampling provides an accurate measure of regional hepatic metabolic function in patients with cirrhosis. This is likely to have clinical implications for the assessment of patients with liver disease as well as treatment planning and monitoring.

Hepatic galactose metabolism quantified in humans using 2-18F-fluoro-2-deoxy-D-galactose PET/CT.

UNLABELLED: Accurate quantification of regional liver function is needed, and PET of specific hepatic metabolic pathways offers a unique method for this purpose. Here, we quantify hepatic galactose elimination in humans using PET and the galactose analog 2-(18)F-fluoro-2-deoxy-d-galactose ((18)F-FDGal) as the PET tracer. METHODS: Eight healthy human subjects underwent (18)F-FDGal PET/CT of the liver with and without a simultaneous infusion of galactose. Hepatic systemic clearance of (18)F-FDGal was determined from linear representation of the PET data. Hepatic galactose removal kinetics were determined using measurements of hepatic blood flow and arterial and liver vein galactose concentrations at increasing galactose infusions. The hepatic removal kinetics of (18)F-FDGal and galactose and the lumped constant (LC) were determined. RESULTS: The mean hepatic systemic clearance of (18)F-FDGal was significantly higher in the absence than in the presence of galactose (0.274 ± 0.001 vs. 0.019 ± 0.001 L blood/min/L liver tissue; P < 0.01), showing competitive substrate inhibition of galactokinase. The LC was 0.13 ± 0.01, and the (18)F-FDGal PET with galactose infusion provided an accurate measure of the local maximum removal rate of galactose (V(max)) in liver tissue compared with the V(max) estimated from arterio-liver venous (A-V) differences (1.41 ± 0.24 vs. 1.76 ± 0.08 mmol/min/L liver tissue; P = 0.60). The first-order hepatic systemic clearance of (18)F-FDGal was enzyme-determined and can thus be used as an indirect estimate of galactokinase capacity without the need for galactose infusion or knowledge of the LC. CONCLUSION: (18)F-FDGal PET/CT provides an accurate in vivo measurement of human galactose metabolism, which enables the quantification of regional hepatic metabolic function.