A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing

The variable domain resurfacing and CDR-grafting approachesto antibody humanization were compared directly on the two murinemonoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19).Resurfacing replaces the set of surface residues of a rodentvariable region with a human set of surface residues. The methodof CDR-grafting conceptually consists of transferring the CDRsfrom a rodent antibody onto the Fv framework of a human antibody.Computer-aided molecular modeling was used to design the initialCDR-grafted and resurfaced versions of these two antibodies.The initial versions of resurfaced N901 and resurfaced anti-B4maintained the full binding affinity of the original murineparent antibodies and further refinements to these versionsdescribed herein generated five new resurfaced antibodies thatcontain fewer murine residues at surface positions, four ofwhich also have the full parental binding affinity. A mutationalstudy of three surface positions within 5 Å of the CDRsof resurfaced anti-B4 revealed a remarkable ability of the resurfacedantibodies to maintain binding affinity despite dramatic changesof charges near their antigen recognition surfaces, suggestingthat the resurfacing approach can be used with a high degreeof confidence to design humanized antibodies that maintain thefull parental binding affinity. By comparison CDR-grafted anti-B4antibodies with parental affinity were produced only after seventeenversions were attempted using two different strategies for selectingthe human acceptor frameworks. For both the CDR-grafted anti-B4and N901 antibodies, full restoration of antigen binding affinitywas achieved when the most identical human acceptor frameworkswere selected. The CDR-grafted anti-B4 antibodies that maintainedhigh affinity binding for CD19 had more murine residues at surfacepositions than any of the three versions of the resurfaced anti-B4antibody. This observation suggests that the resurfacing approachcan be used to produce humanized antibodies with reduced antigenicpotential relative to their corresponding CDR-grafted versions.     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.     

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059–152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.     

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding

The crystallographic study of chimeric B72.3 antibody illustratedthat there are three FR side-chain interactions with eitherCDR residue's side chain or main chain. For example, hydrogenbonds are formed between the hydroxyl group of threonine atL5 in FR1 and the guanidinal nitrogen group of arginine at L24in CDR1, between the hydroxyl group of tyrosine at L36 in FR2and the amide nitrogen group of glutamine at L89 in CDR3 andbetween the hydroxyl group of tyrosine at L71 in FR3 and thecarbonyl group of isoleucine at L29 as well as the amide nitrogengroup of serine at L31 in CDR1. Elimination of these hydrogenbonds at these FR positions may affect CDR loop conformations.To confirm these assumptions, we altered these FR residues bysite-directed mutagenesis and determined binding affinitiesof these mutant chimeric antibodies for the TAG72 antigen. Wefound that the substitution of tyrosine by phenylalanine atL71, altering main-chain hydrogen bonds, significantly reducedthe binding affinity for the TAG72 antigen by 23-fold, whereasthe substitution of threonine and tyrosine by alanine and phenylalanineat L5 and L36, eliminating hydrogen bonds to side-chain atoms,did not affect the binding affinity for the TAG72 antigen. Ourresults indicate that the light-chain FR residue tyrosine atL71 of chimeric B72.3 antibody plays an important role in influencingthe TAG72 antigen binding. Our results will thus be of importancewhen the humanized B72.3 antibody is constructed, since thisimportant mouse FR residue tyrosine at L71 must be maintained.     

Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin

Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A-G) which differ from each other by 34-64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30- fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.      

Brain Disposition of Antibody-Based Therapeutics: Dogma,Approaches and Perspectives

Due to their high specificity, monoclonal antibodies have been widely investigated for their application in drug delivery to the central nervous system (CNS) for the treatment of neurological diseases such as stroke, Alzheimer’s, and Parkinson’s disease. Research in the past few decades has revealed that one of the biggest challenges in the development of antibodies for drug delivery to the CNS is the presence of blood–brain barrier (BBB), which acts to restrict drug delivery and contributes to the limited uptake (0.1–0.2% of injected dose) of circulating antibodies into the brain. This article reviews the various methods currently used for antibody delivery to the CNS at the preclinical stage of development and the underlying mechanisms of BBB penetration. It also describes efforts to improve or modulate the physicochemical and biochemical properties of antibodies (e.g., charge, Fc receptor binding affinity, and target affinity), to adapt their pharmacokinetics (PK), and to influence their distribution and disposition into the brain. Finally, a distinction is made between approaches that seek to modify BBB permeability and those that use a physiological approach or antibody engineering to increase uptake in the CNS. Although there are currently inherent difficulties in developing safe and efficacious antibodies that will cross the BBB, the future prospects of brain-targeted delivery of antibody-based agents are believed to be excellent.     

Epitope‐Resolved Detection of Peanut‐Specific IgE Antibodies by Surface Plasmon Resonance Imaging

Peanut allergy can be life‐threatening and is mediated by allergen‐specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope‐resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ?‐chain‐specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross‐reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 μL each) in a 45 min assay. Excellent correlation of Ara h2‐specific IgE values was found between ImmunoCAP assays and the new SPRi method.     

TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.     

Isolation of anti-T cell receptor scFv mutants by yeast surface display

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.      

Changes in Non-Deamidated versus Deamidated Epitope Targeting and Disease Prediction during the Antibody Response to Gliadin and Transglutaminase of Infants at Risk for Celiac Disease

Celiac disease (CeD) is a conditional autoimmune disorder with T cell-mediated immune response to gluten coupled with antibody production to gliadin and the self-protein tissue transglutaminase (TG2). TG2 contributes to the CeD pathomechanism by deamidating gliadin, thereby generating more immunogenic peptides. Anti-gliadin antibodies may appear before the autoantibody production. The scope of this study was to dissect these early antibody responses by investigating serum samples collected during the PreventCD prospective double-blind study, where infants with high CeD risk were randomized to 200 mg daily gluten intake or placebo from 4 to 6 months of age, followed by frequent blood testing on regular gluten consumption in both groups. After primary gluten intake, children with or without later CeD produced IgA and IgG antibodies which preferentially recognized non-deamidated gliadin peptides. At CeD development with anti-TG2 seroconversion, there was a significant increase in the antibody reaction toward deamidated gliadin peptides (DGP), with maturation in the binding strength for the PEQPFP gamma-gliadin core peptide. The earliest produced autoantibodies targeted TG2’s celiac epitope 2. Our results reveal a qualitative change in the gliadin-directed humoral immune response at the time when anti-TG2 antibodies appear, but anti-DGP antibodies in the absence of anti-TG2 antibodies are not disease-predictive.     

Stepwise transformation of a cholera toxin and a p24 (HIV-1) epitope into D-peptide analogs

We have transformed two peptide epitopes into D-peptide analogs: VPGSQHIDS derived from cholera toxin recognized by the antibody TE33, and GATPQDLNTML from the HIV-1 capsid protein p24 recognized by the antibody CB4-1. The transformation process was performed by stepwise substitution of each single epitope position by all 19 D-amino acids and glycine followed by antibody binding studies and selection of one D- analog for further transformation. Thus, each transformation step introduced one novel D-position into the peptide. For both epitopes complete D-analogs were obtained. The cholera toxin-derived variant dwGsqhydp binds to the antibody TE33 with higher affinity than its original epitope, whereas in the case of the p24-derived analog saGdwwGkssl lower affinity was detected. Both D-peptides are completely stable in serum for several days. Antibody interaction models for both D-molecules were generated by computer-assisted modelling based on the crystal structures of the starting complexes. Compared with the L- peptides, the binding conformation of dwGsqhydp is very similar, whereas saGdwwGkssl displays a completely different interaction mode.      

Identification of Biomarkers in Cerebrospinal Fluid and Serum of Multiple Sclerosis Patients by Immunoproteomics Approach

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. At present, the molecular mechanisms causing the initiation, development and progression of MS are poorly understood, and no reliable proteinaceous disease markers are available. In this study, we used an immunoproteomics approach to identify autoreactive antibodies in the cerebrospinal fluid of MS patients to use as candidate markers with potential diagnostic value. We identified an autoreactive anti-transferrin antibody that may have a potential link with the development and progression of MS. We found this antibody at high levels also in the serum of MS patients and created an immunoenzymatic assay to detect it. Because of the complexity and heterogeneity of multiple sclerosis, it is difficult to find a single marker for all of the processes involved in the origin and progression of the disease, so the development of a panel of biomarkers is desirable, and anti-transferrin antibody could be one of these.     

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.     

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.     

A comparative study on antibody immobilization strategies onto solid surface

Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering real-to-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels) had the following order: secondary antibody-mediated immobilization>protein A/G-mediated immobilization>direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies, which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.     

Two Epitope Regions Revealed in the Complex of IL-17A and Anti-IL-17A VHH Domain

Interleukin-17 (IL-17) is a cytokine produced by the Th17 cells. It is involved in chronic inflammation in patients with autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and psoriasis. The antibodies targeting IL-17 and/or IL-17R are therapy tools for these diseases. Netakimab is an IL-17A-specific antibody containing a Lama glama V_HH derivative domain and a VL variable domain. We have determined the crystal structure of the IL-17A-specific V_HH domain in complex with IL-17A at 2.85 Å resolution. Certain amino acid residues of the three complementary-determining regions of the V_HH domain form a network of solvent-inaccessible hydrogen bonds with two epitope regions of IL-17A. The β-turn of IL-17A, which forms the so-called epitope-1, appears to be the main region of IL-17A interaction with the antibody. Contacts formed by the IL-17A mobile C-terminal region residues (epitope-2) further stabilize the antibody–antigen complex.     

Intestinal Permeability Is a Mechanical Rheostat in the Pathogenesis of Liver Cirrhosis

Recent studies have suggested that an alteration in the gut microbiota and their products, particularly endotoxins derived from Gram-negative bacteria, may play a major role in the pathogenesis of liver diseases. Gut dysbiosis caused by a high-fat diet and alcohol consumption induces increased intestinal permeability, which means higher translocation of bacteria and their products and components, including endotoxins, the so-called “leaky gut”. Clinical studies have found that plasma endotoxin levels are elevated in patients with chronic liver diseases, including alcoholic liver disease and nonalcoholic liver disease. A decrease in commensal nonpathogenic bacteria including Ruminococaceae and Lactobacillus and an overgrowth of pathogenic bacteria such as Bacteroidaceae and Enterobacteriaceae are observed in cirrhotic patients. The decreased diversity of the gut microbiota in cirrhotic patients before liver transplantation is also related to a higher incidence of post-transplant infections and cognitive impairment. The exposure to endotoxins activates macrophages via Toll-like receptor 4 (TLR4), leading to a greater production of proinflammatory cytokines and chemokines including tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, which play key roles in the progression of liver diseases. TLR4 is a major receptor activated by the binding of endotoxins in macrophages, and its downstream signal induces proinflammatory cytokines. The expression of TLR4 is also observed in nonimmune cells in the liver, such as hepatic stellate cells, which play a crucial role in the progression of liver fibrosis that develops into hepatocarcinogenesis, suggesting the importance of the interaction between endotoxemia and TLR4 signaling as a target for preventing liver disease progression. In this review, we summarize the findings for the role of gut-derived endotoxemia underlying the progression of liver pathogenesis.