Intrinsically conducting polymers may undergo significant changes of molecular structure and material properties when exposed to highly oxidizing conditions or very positive electrode potentials, commonly called overoxidation. The type and extent of the changes depend on the experimental conditions and chemical environment. They may proceed already at much lower rates at lower electrode potentials because some of the processes associated with overoxidation are closely related to more or less reversible redox processes employed in electrochemical energy conversion and electrochromism. These changes may be welcome for some applications of these polymers in sensors, extraction, and surface functionalization, but in many cases, the change of properties affects the performance of the material negatively, contributing to material and device degradation. This report presents published examples, experimental observations, and their interpretations in terms of both structural and of material property changes. Options to limit and suppress overoxidation are presented, and useful applications are described extensively. The term “oxidation” is firmly established in chemistry with different meanings in various fields ,2. In maThe term “intrinsically conducting polymer” names a class of mostly organic oligomeric or polymeric materials that show electronic conductivity because of mobile charge carriers capable of moving along conjugated segments and hopping between such segments of the polymer. These charge carriers are created by oxidation, i.e., the formation of a radical cation by the removal of an electron. This cation is frequently called a polaron. For charge compensation, anions from the electrolyte solution move into the ICP; in the simplest case, during oxidation with iodine, the formed iodide stays in the ICP. As an alternative, cations  can move out. The ingress of anions is frequently called “doping” in a slightly stretched interpretation of the meaning of this term in solid state and semiconductor physics, where doping means the replacement of one kind of atom by atoms of another kind with a different number of valence electrons at an extremely low concentration. Consequently, the term is not used in this communication. Unfortunately and somewhat misleadingly, materials afforded also with electronic conductivity by the addition of an electronically conducting material, such as metal fibers or carbon powder, to an insulating polymer are called simply conducting polymers.N,N′-dicyanonaphthaquinonediimine (DCNNI) and tetrathiafulvalene (TTF)), resulting in some confusion   was elec3 considered as an electrochromic material, a degree of overoxidation Y depending on the nucleophile present in the electrolyte solution based on the charge Qoo was observed in a CV associated with overoxidation and the reversible redox charge Qred according to:In studies on a composite of PPy and WOY = 5 was found when water was present thiopheneoximide)  was idenThe overoxidation of PEDOT results in higher crystallinity. This was attributed to the formation of smaller oligomer units during overoxidation . In the PEDOT electropolymerized in solutions containing various supporting electrolytes, including anionic polysaccharides, was more stable vs. overoxidation and appeared smoother and denser than polymers formed in the presence of other electrolytes .PEDOT can also be formed by oxidative jet deposition, enabling inline processing less easily performed with common oxidative batch processes  . With prThe effect of the electrochemical overoxidation of poly  on chargObserved irreversible changes in the EPR spectra, particularly the steep drop of spin density, were attributed to overall degradation of the polymer and, in particular, to a decrease in the conjugation length by crosslinking or addition reactions with remaining spins confined to isolated segments of the polymer chain.Polymers formed by the electropolymerization of tetrathiafulvalene-substituted EDOT showed reduced electroactivity of the substituents after overoxidation, which was attributed to diminished activity of the polymer backbone .Bilayer systems of different ICPs were studied aiming at diode-like behavior . DetrimeInfrared spectroscopy of overoxidized polyphenylene yielded evidence of a breakdown of the polymer into shorter segments, and para-quinoid sequences were apparently destroyed .A composite of polypyrrole–polyoxyphenylene was electrosynthesized at a relatively high electrode potential needed for oxidation of the second component, 2-allylphenol . BecauseA composite of poly(3-methylthiophene) and overoxidized PPy was prepared by applying a specifically designed electrode potential time program and monomer concentrations . The aveWithin studies of the electrochemistry of ionically functionalized polyacetylene analogues, evidence of overoxidation was found .The sensitivity towards overoxidation of poly DFE and poly DTE  was compThe differences in material properties between PDFE and PDTE must be primarily related to differences between sulfur in the thiophene ring and oxygen in the furan ring, although this is nowhere addressed in the report. Oxidation of the monomers showed a behavior hardly helpful in finding an explanation: DFE was oxidized at substantially lower potentials than DTE, as seen in the presented CVs.The second oxidation step in a CV of polyindole  was assoIn a report on the preparation of nanosized polyindole by emulsion polymerization, overoxidation was addressed only as a keyword . The perThe adsorption and polymer film formation of 4-aminoindole  on platiWith a platinum electrode, already dioxygen was sufficient to start oxidative adsorption and deposition of a multilayer film on the electrode surface. Electrochemical oxidation of the monomer also resulted in polymer film formation on both metals with film properties depending on the applied electrode potential. Overoxidation may proceed both by dioxygen and high electrode potentials. It appears noteworthy to notice that linkage between monomer units proceeds only via the pyrrole entity leaving the amino group at the benzene ring available for functionalization. At too-high electrode potentials, this group may be oxidized, too, resulting in a loss in electroactivity of the film.The overoxidation of polymers of bis complexes resulted in morphology changes affecting effective diffusion coefficients and crosslinking .Poly[(tetraethyldisilanylene)oligo] derivatives showed overoxidation of the oligo(thienylene) units and Si–Si bond cleavage upon exposure to too-high electrode potentials, resulting in lower conductivity and values of the work function .Poly  can be pDegradation of this polymer during electrode potential cycling has been observed both during overoxidation  and the basically reversible redox reaction, with a much lower rate in the latter case. During overoxidation, the quinone structure is destroyed, the conjugation length decreases, the polymer chain may be broken, and chemical follow-up reactions may occur. In case of oligomeric material showing π-π stacking, conformational changes may play a role, too .At first glance, avoiding electrochemical overoxidation appears to be straightforward and simple: set a proper upper (positive) electrode potential limit. This might work in an experimental lab setup with a three-electrode arrangement. However, during potentiodynamic deposition of an ICP, overoxidation may already happen, e.g., during the initial scans where, for deposition of at least some ICPs , higher electrode potentials are needed to overcome nucleation inhibition or a very poorly conducting polymer film. In the cases of galvanostatic or current pulse deposition without real electrode potential control, the situation is even worse. Frequently, CVs of PANI prepared potentiodynamically show the middle-potential redox peak pair discussed above with the newly formed CP already. In a two-electrode arrangement, as encountered in a secondary battery  or a supE = 1.8 V. Simple PPy showed a strong peak under the same conditions already around E = 0.9 V -hyd-razono}-2,3-dihydro-benzothiazole-6-sulfonic) acid as a counter ion turned out to be particularly overoxidation-resistant . This waThe electrochemical overoxidation of polyacetylene could be suppressed by various additions, which were either oxidized themselves at too-high electrode potentials or helped to establish an equal state of polyacetylene oxidation .Contrary to the mostly negative effects of overoxidation, there are also reports on positive ones. They range from merely increased stability of a particular function of an ICP to the achievement of further capabilities of an ICP. Examples range widely across fundamental and applied electrochemistry, sensors, etc. In the following, they are briefly presented, and particular attention is paid to details revealing correlations between these changes caused by overoxidation and the obtained improvements and effects. It is noteworthy that the overoxidation of a plain carbon microfiber enhances dopamine adsorption and, consequently, sensitivity in the detection of this substance at the expense of a slower response that is possibly related to the significantly increased double layer capacitance . This apFlexible overoxidized PPy films were examined as negative electrode materials for sodium- and lithium-ion batteries with promising results .2Mo18A Pt–Co fuel cell catalyst supported on PPy carbon multiwall nanotubes employed in a direct methanol fuel cell showed enhanced catalytic activity after partial overoxidation . PPy witoxidized . A reducoxidized .Overoxidized PPy as part of a composite also containing cellulose was examined as a separator for a lithium metal battery . The welx nanoparticles showed morphological changes after overoxidation, enhancing methanol transport in the methanol oxidation reaction of a fuel cell electrode [Favorable changes of the PPy layer coating on a copper support with embedded NiOlectrode .The electrodeposition of atomic gold on PANI was complicated by polymer degradation during the procedure . “StabilSeveral overoxidized ICPs incorporated in an acrylate-based solid polymer matrix were directly electrosynthesized . OveroxiProtection against electrode fouling;Provision of permselectivity;Action as a host or cover for immobilized reactants ;Provision of the material for molecular imprinting.Overoxidation of an ICP or an ICP-containing composite can yield a material (in most cases a coating) serving various purposes:Function 1 is rather general and will be mentioned in passing whenever authors considered this property noteworthy. Functions 2–4 will be addressed in the following sections in this order, starting with the most common ICPs. Infrequently studied ICPs are collected at the end of this chapter.PANI can be transformed by electrochemical overoxidation into a permselective membrane . Permsel−1) for dopamine determination with high rejection of ascorbate and dihydroxyphenylacetic acid [®. A carbon ceramic electrode modified with overoxidized PPy showed high sensitivity for the electro-oxidation of folic acid; selectivity was not addressed [Permselective properties of overoxidized and, thus, electronically insulating PPy were reported first in 1992 . Presumatic acid . This elddressed . A graphddressed .Highly boron-doped diamond microfibers coated with overoxidized PPy served as amperometric sensor electrodes for dopamine with high rejection of ascorbic and 3,4-dihydroxyphenylacetic acid . Compare2O2 and surface coverage of overoxidized PPy films on a platinum substrate was determined simultaneously with a newly developed method enabling better insight into the influence of preparation conditions [m-phenylenediamine) films as permselective coatings for H2O2-sensing electrodes was compared, and the latter film performed better [m-phenylenediamine) subsequently coated with a film of bismuth on a glassy carbon electrode was used successfully for the simultaneous detection of Cd2+ and Pb2+ ions [2+ and Pb2+ ions were determined with a bismuth-covered glassy carbon electrode coated with Nafion® and overoxidized 2-mercaptoethanesulfonate-tethered polypyrrole [o-phenylenediamine) ) prepared electrochemically with dodecyl sulfate as a counter anion [Vanillin could be determined selectively in commercial samples with a glassy carbon electrode coated with overoxidized PPy . Electronditions . The pred better . Amperomd better . Thiolatb2+ ions . A glassb2+ ions . Preaccub2+ ions . With thypyrrole . Dopaminer anion . Ascorbier anion .N-pentylphenylboronic acid as a counter anion increased affinity towards diols [The overoxidation of a PPy layer in the presence of various counter anions resulted in enhanced sensitivity towards the amperometric detection of dopamine . Microscds diols . This wads diols .2+ ions with high selectivity and sensitivity [2+ ions suitable for subsequent stripping voltammetry was prepared [PPy prepared with sulfosalicylic acid as a counter anion and selective ligand was used after overoxidation for the determination of Cusitivity . The samsitivity . A furthsitivity . With thprepared . Overoxiprepared . A “memoA glassy carbon electrode modified with a composite of overoxidized polyimidazole/graphene oxide enabled the simultaneous determination of ascorbic acid, dopamine, uric acid, guanine, and adenine . A similN-acetylaniline) with β-cyclodextrin chemically attached to it formed an inclusion complex of cyclodextrin with cocaine determined with impedance measurements [Electropolymerized and overoxidized poly was achieved by the overoxidation of PPy . L-lysinGlucose oxidase was immobilized on gold nanoparticle-decorated overoxidized PPy for use as an amperometric glucose biosensor . Using CGlassy carbon coated with several layers of biotin-loaded overoxidized PPy and nanosheets of reduced graphene oxide was studied as an enantioselective electrochemical sensor for R-mandelic acid . No inteThe electrochemistry of antibody-modified PPy and its interaction with an antigen with the ICP in neutral, oxidized, and overoxidized states was studied . OveroxiGraphite modified with overoxidized PTh was examined as a sensor material for the amperometric determination of propham, showing high selectivity . A similOveroxidized PEDOT on a screen-printed carbon electrode was successfully applied in the determination of submicromolar dopamine concentration without interference from a 1000-fold excess of ascorbic acid .Several ionophores immobilized in overoxidized PPy enabled the joint quantification of potassium, ammonium, and sodium by impedance measurements .The enantioselective uptake of glutamic acids by carbon fibers coated with overoxidized PPy packed into a column and the subsequent release by applying a suitable electrode potential were realized . The con® with attached glutamate oxidase was used as a microelectrode for the detection of alanine aminotransferase [o-phenylenediamine) was tested in the determination of L-lactate [o-phenylendiamine instead of PPy resulted in lower selectivity. Simultaneous determination of ethanol and glucose with a dual-amperometric biosensor with a dual gold electrode coated with overoxidized PPy, glucose oxidase, and alcohol oxidase immobilized with bovine serum albumin and glutaraldehyde was developed and tested successfully [2O2 reduction at EAg/AgCl = 0.15 V was tested [EAg/AgCl = 0.7 V operates based on H2O2, oxidation had lower sensitivity. The stability of the electrode in both modes of operation was not examined. The electropolymerization of pyrrole–glucose oxidase mixtures on a platinum electrode yielded a highly sensitive glucose biosensor [® for better selectivity. The latter inner layer resulted in a better selectivity performance.A glassy carbon electrode modified with an overoxidized PPy–gold nanoparticles composite subsequently covered with electrostatically attached transglutaminase antigen and, finally, capped with bovine serum albumin was developed as an immunosensor for the electrochemical determination of antitransglutaminase antibodies . A platinsferase . A cholensferase . An ampe-lactate . In an e-lactate . The useessfully . A glucos tested . At EAg/iosensor . Noticeao-phenylenediamine) was assessed and compared with a single layer system  [The overoxidation of PPy as a part of a bioactive and electrically conducting hydrogel used as a biotransducer for glucose eliminated the background current, similar to observations noticed above . The statopcoat) . The pertopcoat) . Other iPPy was proposed as sensor material for the detection of OH radicals . The oveAn electrochemically oxidized graphite electrode modified with overoxidized poly(pyrrole-3-carboxylic acid) was studied as a disposable dopamine sensor, showing sufficient selectivity against common interferents .M NaOH (effecting overoxidation) [Electrochemical overoxidation by controlled application of higher electrode potentials to a molecularly imprinted ICP layer may be used to remove the molecular template ,333,334.idation)  removed idation) . This pridation) ,339. Molidation) . Ultra-tidation) .The selectivity of molecularly imprinted overoxidized PPy was applied in the microextraction of ibuprofen . SimilarMolecularly imprinted overoxidized PPy was used in an electrode for nicotinamide analysis . PreparaInstead of using PPy films, colloids of PPy were suggested  and reviThe recognition of Gram-negative and -positive bacteria was achieved with overoxidized PPy imprinted during potentiostatic PPy deposition in a solution also containing the monomer and bacteria . BacteriThe electrode-potential-triggered release of L-glutamate from molecularly imprinted overoxidized PPy was realized .The incorporation of chelating ions with metal specificity into an ICP film may be an option for trace metal determination . OveroxiOveroxidized PEDOT:PSS was incorporated into an organic bioelectronic ion pump .Molecularly imprinted and overoxidized poly(indole-3-acetic acid) deposited on multi-walled carbon nanotubes immobilized on graphite enabled the enantioselective detection of D- and L-aspartic acid .p-nitrophenol [Overoxidized polydopamine used for embedding silver nanoparticles provided capabilities similar to those of overoxidized PPy for sensing rophenol . Such ovrophenol . Overoxirophenol .The overoxidation of electropolymerized poly(1-naphthylamine) yielded a sensor material for the selective determination of dopamine in the presence of a large excess of ascorbic acid .p-aminophenol) on a glassy carbon support enabled the simultaneous determination of dopamine, uric acid, and tryptophan [Longitudinally unzipped, multi-walled carbon nanotubes incorporating overoxidized poly voltage  has been applied without the control of a particular electrode potential ,377,378.Overoxidation of the ICP employed as channel material in an organic electrochemical transistor was used to afford permanent changes of material properties .The overoxidation of PEDOT used in an all-polymer electrochemical transistor with associated conductivity loss was employed in a “write-once read-many-times” application . The preOveroxidation by air (presumably dioxygen) has been noticed as a problem with ICP-based heterojunctions, diodes, and capacitors prepared using a lithographic approach ,383Powders of PPy and PEDOT have been prepared by chemical oxidation with ammonium persulfate for use as radical scavengers ,385. ScaTo avoid issues with PPy overoxidation in a polymer battery, the authors switched to PEDOT as a positive electrode material .Because of the apparently significant importance of overoxidation, sometimes the high stability of an ICP versus overoxidation is claimed without providing any evidence .Overoxidation of electrochemically active  polymers, such as 2,5-dimercapt-l,3,4-thiadiazole , were reIn many applications of ICPs, overoxidation mostly by electrode potential excursions results in performance losses for the ICP in its particular function, such as irreversibly diminished electronic conductivity, reduced charge storage capability, and changed morphology. Various options to avoid this are either obvious  or have been suggested ; in any case, poorer performances of electrochemical systems based on ICPs may be attributed to overoxidation as a likely cause. According to the rich evidence surveyed above, it can be stated that overoxidation is inseparably connected with the redox activity of ICPs because the oxidized form of every ICP is basically susceptible to chemical, particularly nucleophilic, attacks from the chemical environment. Thus, from the oxidized state, an ICP may—as desired—return by reduction to the neutral form or—as not desired—by chemical reactions into overoxidation or degradation products. Consequently, the definition of a clear-cut overoxidation electrode potential is basically impossible. All reported data may be interpreted in relative terms, in which potential range reversible redox activity dominates and overoxidation takes over.There is, quite to the contrary, a large amount of scientific reports wherein ICPs overoxidized in a controlled manner have been applied in sensors, electrocatalysts, and further functions. A better understanding of the beneficial effects of overoxidation will be helpful in the development and optimization of sensors and further applications. It might also help in controlling the unwelcome effects of overoxidation in other applications better. The occasionally noticed expulsion of counter anions during overoxidation sheds some doubt on the role of ionic species  incorporated in overoxidized ICPs and, apparently, still present after overoxidation.


PICRUSt2 was used to predict microbial metabolic potential in axSpA patients and healthy controls (HCs). IgA-SEQ analyses revealed enrichment of several microbes in the fecal  and salivary  microbiome in axSpA patients as compared with HCs. Fecal microbiome from axSpA patients showed a tendency towards increased alpha diversity in IgA+ fraction and decreased diversity in IgA- fraction in comparison with HCs, while the salivary microbiome exhibits a significant decrease in alpha diversity in both IgA+ and IgA- fractions. Increased IgA coating of Clostridiales Family XIII in feces correlated with disease severity. Inferred metagenomic analysis suggests perturbation of metabolites and metabolic pathways for inflammation  and metabolism (propanoate and butanoate) in axSpA patients. Analyses of fecal and salivary microbes from axSpA patients reveal distinct populations of immunoreactive microbes compared to HCs using the IgA-SEQ approach. These bacteria were not identified by comparing their relative abundance alone. Predictive metagenomic analysis revealed perturbation of metabolites/metabolic pathways in axSpA patients. Future studies on these immunoreactive microbes may lead to better understanding of the functional role of IgA in maintaining microbial structure and human health.Axial spondyloarthritis (axSpA) is an inflammatory arthritis involving the spine and the sacroiliac joint with extra-articular manifestations in the eye, gut, and skin. The intestinal microbiota has been implicated as a central environmental component in the pathogenesis of various types of spondyloarthritis including axSpA. Additionally, alterations in the oral microbiota have been shown in various rheumatological conditions, such as rheumatoid arthritis (RA). Therefore, the aim of this study was to investigate whether axSpA patients have an altered immunoglobulin A (IgA) response in the gut and oral microbial communities. We performed 16S rRNA gene (16S) sequencing on IgA positive (IgA Axial spondyloarthritis (axSpA) is an immune-mediated inflammatory arthritis, which affects the sacroiliac and spinal joints, and is associated with microscopic lesions or inflammation in the gut . It inclE. coli (AIEC) pathotype, induced Th17 immunity through microbial metabolic enzyme propanediol dehydratase  . In thisydratase . These fydratase . HoweverThe gut is the largest immune organ, facilitating interaction between the gut microbiota and local mucosal immune response. We hypothesize that other mucosal sites that are home to microbes contribute significantly to systemic rheumatic disease, particularly the oral cavity. It has been shown that patients with rheumatoid arthritis (RA) have a higher prevalence of severe periodontitis as compared to healthy controls . Disease+ fraction in the fecal and salivary samples showed perturbation of many predictive metabolites and metabolic pathways in axSpA patients. These potentially immune reactive microbes may reveal novel host-microbial interactions in health and disease. Taken together, this study adds to our understanding of HLA-B27-associated host immune response and its interaction with the gut microbes in axSpA.In this study, we evaluated IgA coating (IgA-SEQ) and traditional 16S sequencing of microbes in the saliva and feces from axSpA patients and healthy controls (HCs). IgA-SEQ analyses identified potentially immune-targeted microbes that were not observed to be differentially abundant using traditional 16S sequencing alone. In addition, differential IgA coating on some fecal and salivary microbes was associated with disease activity. Using a computational approach to predict the microbial function, we found that the IgAWe performed a prospective cohort study at the Oregon Health and Science University (OHSU). Subjects were excluded from all cohorts if they were younger than age 18 years, pregnant, had a history of prior intestinal surgery or colon cancer, or had antibiotic use 6 months prior to sample collection. This study was approved by the Institutional Review Board at OHSU and written informed consent was obtained from all participants.AxSpA cohort: Subjects with a diagnosis of axSpA were recruited from either the OHSU rheumatology clinic or via the Spondylitis Association of America (SAA). The SAA is a patient advocacy group with physicians on its board, and members with AS are highly engaged and informed. Samples: All subjects enrolled at OHSU provided a blood sample for genomic DNA extraction and subsequent HLA-B typing. Subjects recruited through the SAA shipped fecal and/or salivary samples to our lab using next-day shipping packets. For microbiome analysis, fecal and salivary samples were snap frozen upon collection and stored at -80°C until further analyses. All processing thereafter was performed blinded to patient phenotype. HLA-B typing: Genomic DNA was extracted from anti-coagulated blood by the salting out method performed in a shared core resource at the Casey Eye Institute, OHSU. The LABType XR HLA-B SSO typing kit from One Lambda Thermo Fisher (RSSOX1B) was used according to manufacturer’s instructions in the OHSU Laboratory of Immunogenetics and Transplantation.https://www.earthmicrobiome.org/). Sequencing of the V4 region was accomplished with Illumina MiSeq using barcoded primers (+ fraction was stained with phycoerythrin (PE) labeled mouse anti-human IgA (Miltenyi 130-093-128), and enriched using anti-PE magnetic activated cell sorting (MACS) beads, and the negative fraction was collected for IgA- microbes. IgA+ fraction was further purified through a FACS Aria (BD Biosciences). Genomic DNA from IgA+ and IgA- microbial fractions was isolated and the V4 region of the16S rRNA gene was sequenced as described above.To determine the microbial community structure, genomic DNA from the salivary and fecal samples was isolated using the methods described previously . Briefly primers . For IgAhttps://github.com/KarstensLab/igaseq). Samples that had 7,500 reads per sample in both the IgA- and IgA+ fractions were used. The IgA index was calculated as the log ratio of the difference between IgA coated and IgA uncoated bacteria over the sum of IgA coated and IgA uncoated bacteria (IgA index= - (log(IgA+ taxon)- log(IgA- taxon))/(log(IgA+ taxon) + log(IgA- taxon))) (Sequencing data were demultiplexed and processed using the DADA2 package in R . This intaxon))) . Alpha dtaxon))) . Beta ditaxon)))  and visuvia alignments and hidden state prediction models  . PICRUStn models , using dn models  and indin models .+ and IgA- fractions from salivary and fecal microbiome data from axSpA patients and HCs.All statistical analyses were performed in R. For 16S sequencing and IgA-SEQ analyses, non-parametric tests (Wilcoxon Rank Sum) were used to assess within and between group differences for the alpha diversity measures. Differences in individual microbial relative abundances at the genus and ASV level were assessed with non-parametric tests (Wilcoxon Rank Sum) and corrected for multiple tests using the Benjamini-Hochberg method . Differe+ and IgA- sorted fractions from the fecal and salivary samples. The fecal cohort had 31 subjects (17 axSpA patients and 14 HCs), and the salivary cohort had 26 subjects (14 axSpA patients and 12 HCs). While there were no significant differences in age or gender (P> 0.2 and 0.6 respectively) between the axSpA patients and HCs for the fecal samples, the HCs were younger (P= 0.018), and with less female representation (P= 0.038) than the axSpA patients in the salivary cohort. However, both axSpA patients and HCs had similar BMI in the fecal (P = 0.5) and salivary (P = 0.4) cohort. AxSpA patients had a mean disease activity (BASDAI) of 3.5 in the fecal cohort, and 2.0 in the salivary cohort. The fecal cohort was comprised of 47% axSpA patients on biologics such as adalimumab, etanercept or secukinumab, while 36% of the salivary axSpA cohort were on biologics  measures of alpha diversity  , however microbial composition as assessed by beta diversity was not significantly different between axSpA subjects and HCs   in the axSpA patients as compared with HCs  between axSpA and HCs did not yield any significant differences (data not shown).Although we did not observe differences in the overall microbial composition in fecal samples as measured by 16S sequencing, we suspected that there may be differences in how the host immune system interacted with these microbes. To determine whether microbiota from the fecal and saliva samples can elicit an immune response, we performed IgA-SEQ on IgA+ fraction as compared with the IgA- fraction) and depleted (decreased relative abundance of that microbe in IgA+ fraction as compared with IgA- fraction) bacteria from patients with axSpA identified a greater number of unique IgA-coated microbial genera in fecal . Interestingly, some of the IgA enriched microbes overlapped between axSpA patients and HCs, such as those belonging to genus Escherichia/Shigella, Ruminococcaceae-NK4A214, Intestinibacter  and Ruminococcaceae (UCG-002) trended towards a negative correlation . At the ASV level ].To determine whether IgA enriched microbes in feces from axSpA patients were associated with disease, we correlated genus and ASV level microbes with the disease activity score (measured by BASDAI). The analysis showed a significant negative correlation between + or IgA- fractions in the perturbation of microbial metabolic function. We found multiple metabolic pathways (MetaCyc) significantly increased  in the fecal IgA+ fraction of axSpA patients  in both fecal and salivary fractions , acetyl CoA fermentation to butanoate II, purine nucleobases degradation I (anaerobic), guanosine nucleotides degradation  to predict microbial metabolic potential. Previous inferred microbial metabolic profiling of SpA patients  and HLA-+ fraction and 28 genes in the IgA- fraction of axSpA fecal samples, with a minimal overlap of 1 gene (+ and IgA- fractions had higher numbers of predictive genes (191 and 154 respectively) perturbed in axSpA patients, with 36 shared genes between the IgA+ and IgA- fractions , glutathione metabolism , oxidative phosphorylation (K00239), and biofilm formation  were perturbed. The IgA- fraction showed a decreased abundance of predicted genes from metabolic pathways such as propanoate and , and butanoate metabolism  in axSpA patients. The salivary IgA+ fraction from axSpA patients had decreased abundance of genes from butanoate , and propanoate  metabolism. Multiple genes for other inflammatory pathways such as tryptophan metabolism , oxidative phosphorylation , flagellar assembly , and glutathione metabolism (K07232) had altered abundances in the IgA+ fraction of the saliva samples. We also performed additional exploratory analyses , which revealed additional KOs belonging to pathways mentioned above  level for predictive genes/metabolite revealed perturbation of 72 predictive genes in the IgAIn this study, we examined the IgA coating of microbes in the fecal and salivary microbiome from axSpA patients and HCs. To our knowledge, this is the first study to focus on IgA-enriched and depleted microbes and their predictive metabolic contributions in axSpA patients. We found an enrichment of IgA coated microbes in the fecal samples from axSpA patients. Also, the salivary microbiome from axSpA patients had a lower number of IgA coated microbes despite showing significant microbial dysbiosis. Some of these IgA coated microbes showed an inverse correlation with the BASDAI. In addition, predictive microbial function revealed various inflammatory and metabolic pathways perturbed in fecal and salivary samples from axSpA patients. Taken together, our data suggest that IgA selectively marks inflammatory or immune reactive microbes, resulting in an altered microbial community structure and function in axSpA patients.Akkermansia, and members of family Ruminococcaceae and Lachnospiraceae. These microbes have been shown to be associated with clinical studies in SpA and IBD  and salivary  samples from axSpA patients, which have been shown to be associated with various spondyloarthropathies . While IgA coated microbes have shown to exacerbate gut inflammation  with increased relative abundance of  studies , 13. Oth studies , 46, sugammation , 17, IgAammation  and reguammation . Additiohobionts . These ohobionts . Furtherhobionts . Togethe+ and IgA- fractions from saliva samples of axSpA patients. A possible explanation is that perturbation of oral mucosa may be a result of disease instead of driving the disease in axSpA patients.Our results demonstrate alterations in the salivary microbial community associated with axSpA patients. While the salivary microbes in axSpA patients showed significant alterations in the microbial community and structure, we observed a smaller number of IgA coated microbes in comparison with the fecal samples. However, there are significant changes in the predictive microbial function in both IgA+ and IgA- fractions in feces and saliva. Despite secretory IgA being the most abundant immunoglobulin produced in our body , degradation of various sugars ], nucleotide biosynthesis pathways  and adjusted p-values (FDRP). Similarly, for the inferred microbial metabolic pathway analysis, we performed a stringent and relaxed cutoff as done in our previous microbiome studies , accession number under bioproject PRJNA873789.This study was reviewed and approved by OHSU Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.MA (†) was involved in the study design and data generation for the current manuscript. TG, TM, JR and LK had access to all the data and patient records and share the responsibly for data integrity and its accurate analysis. All authors contributed to the article and approved the submitted version.This study was supported by NIH R01EY029266 (JR-PI), NIH P30EY010572 (Casey Eye Institute), Research to Prevent Blindness (Casey Eye Institute), the Rheumatology Research Foundation, the Spondylitis Association of America (Jane Bruckel ECI Award to TG), NIH K01DK116706 (LK), NIH T32HL083808 (IL) and NIH National Library of Medicine Training Grant T15-LM007088 (JN). JR receives support from the Grandmaison Fund for Autoimmunity Research, the Stan and Madelle Rosenfeld Family Trust, and the William and Mary Bauman Foundation.The authors wish to thank Lindsey Watson and Puthyda Keath for human subjects’ recruitment and sample processing, Manny Rodriguez for laboratory assistance, Erin Dahl for help with data visualization, and the Spondylitis Association of America for allowing us to advertise the study to its members.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We present a novel case of severe hyperosmolar hyperglycemic derangement in an elderly patient - without a known history of diabetes mellitus - after the first injection of leuprolide for the treatment of metastatic prostate adenocarcinoma. Whilst the available literature provided accumulative evidence of an association between insulin resistance and the use of gonadotropin-releasing hormone (GnRH) agonists, the initial presentation of leuprolide-induced impaired glycemic tolerance with a hyperosmolar hyperglycemic state (HHS) represents a clinical rarity that was seldom reported. A literature review was conducted to explore the underlying mechanisms of leuprolide-associated glucose intolerance. Screening for diabetes is recommended for patients receiving leuprolide therapy to identify at-risk patients and close glycemic monitoring is warranted in diabetic patients to minimize serious complications from poor glycemic control induced by leuprolide. The use of gonadotropin-releasing hormone (GnRH) agonists in metastatic prostate cancer has increased in the last two decades . The sysAfrican American male patient was brought to our emergency department with generalized weakness, nausea, and anorexia for four days associated with an altered mental status for one day as per the patient’s son. Past medical history was significant for essential hypertension, gout, and prostate adenocarcinoma status post-prostatectomy and radiotherapy in 2016 with a recent metastatic recurrence to the liver, which was diagnosed three months prior to his index presentation. Regular medications included amlodipine 10 mg daily, allopurinol 100 mg daily, and bicalutamide 50 mg daily. The patient received the first injection of leuprolide two months prior to his present admission. He was scheduled for a three-monthly leuprolide 22.5 mg depot intramuscular therapy. There was no history of chest pain, shortness of breath, or cough. The rest of the systemic review was non-contributory. There was neither known personal nor family history of diabetes mellitus.A 75-year-old The initial evaluation revealed an ill-looking afebrile elderly patient with a pulse rate of 115 beats per minute and blood pressure of 77/55 mmHg. The patient was confused, drowsy, and difficult to arouse. The patient’s body mass index (BMI) was 19.9 kg/m². Physical examination revealed dry mucous membranes with generalized abdominal tenderness on deep palpation without focal guarding. Neurological examination demonstrated no focal weakness. No acanthosis was identified on the examination. The rest of the systemic examination was unremarkable. Initial laboratory results showed a blood glucose of 996 mg/dL (reference range 90-110 mg/dL) with normal serum ketones level, normal serum bicarbonate , and a normal anion gap. Serum osmolality was 330 mOsm/Kg (reference limits 270-300 mOsm/Kg). Table Hemoglobin A1c was 13.5% (reference < 5.7%). Computed tomography of the abdomen revealed no acute intra-abdominal process apart from the metastatic hepatic nodules. Notably, the patient had a recent admission to our facility three months prior to index presentation, when the recurrent prostate cancer was diagnosed, and serial fasting glucose readings ranged between 90-100 mg/dL. There were no previous hemoglobin A1c results in our institution or the available outside records.A diagnosis of hyperosmolar hyperglycemic state (HHS) was made. The patient was admitted to the intensive care unit for aggressive fluids resuscitation, continuous insulin infusion, and optimization of electrolytes disturbances as per our institution-based protocol. The patient’s glucose levels normalized after 12 hours of insulin infusion, and mentation improved remarkably. The following day, the patient was alert, returned to baseline mental status per family, and resumed oral feeding. Endocrinology consultation recommended fast-acting insulin (lispro) six units three times with meals and basal insulin (levemir) 12 units twice daily alongside intensive diabetic education.Leuprolide is a gonadotropin-releasing hormone (GnRH) agonist that has been authorized to be used in a range of benign and neoplastic conditions  -3. The rIn fact, in the year 2010, the Food and Drug Administration (FDA) warned patients with prostate cancer treatment who are using GnRH agonists about an increased risk of diabetes while they are receiving these medications . NeverthA large nation-based Canadian study was conducted in a cohort of men aged above 66 years who received at least 6 months of ADT and reported an increased risk of type 2 diabetes mellitus (T2DM) , but the latter cohort did not assess the effects of GnRH and AA separately as it combined all modalities of ADT as a single exposure variable [Early observational studies have shown more tendency of hypogonadotropic males to develop diabetes mellitus than their eugonadotropic counterparts . The avaAn early Japanese cohort conducted by Suzuki K et al  enrolledThe authors postulated leuprolide therapy as the culprit of poor glycemic control and onset of HHS in this reported patient given a robust temporal association between a recent leuprolide exposure and the new onset of severe hyperglycemia. Although our patient was not known to be diabetic and had normal fasting glucose levels prior to leuprolide introduction, the possibility of undiagnosed diabetes that might have been progressively worsened by leuprolide use cannot be entirely ruled out. Nevertheless, this case is considered the first - to the best of the authors' knowledge - to report HHS as a presenting feature of leuprolide-induced impaired glycemic control in a patient without a previous history of diabetes within a short-term interval following the first injection of leuprolide. Diabetes screening with close glycemic monitoring is recommended for patients receiving leuprolide to minimize the occurrence of serious diabetic complications such as the HSS described in this patient .We described a novel case of hyperosmolar hyperglycemic state (HHS) in an elderly patient with no previous history of diabetes mellitus that was induced by leuprolide therapy, in keeping with the current literature that reported an association between the use of GnRH agonists and the development of diabetes. Screening for diabetes is recommended for patients receiving GnRH agonists to minimize the risk of complications like HSS reported in this presented case.


Prenatal exposure to multiple phthalates is ubiquitous, and yet few studies have evaluated these exposures as a mixture in relation to child autistic traits and behavioral problems.To assess cumulative associations between prenatal phthalate mixtures and child behaviors, including effect modification by exposure timing and child sex.Analyses included 501 mother/child pairs from the multicenter pregnancy cohort The Infant Development and Environment Study (TIDES). Nine maternal urinary phthalate metabolites were measured in early and late pregnancy, and behavior was assessed at ages 4–5 years using composite T scores for the Behavioral Assessment System for Children (BASC-2), which measures several dimensions of child behavior, and the Social Responsiveness Scale (SRS-2), which measures social impairment consistent with autistic traits. We utilized weighted quantile sum (WQS) regressions to examine pregnancy period-specific associations between phthalate mixtures and behavioral outcomes. Full-sample 95% WQS confidence intervals are known to be anticonservative, so we calculated a confirmatory p-value using a permutation test. Effect modification by sex was examined with stratified analyses.A one-quintile increase in the early pregnancy phthalate mixture was associated with increased SRS-2 total score  and worse adaptive skills  in both sexes. In sex-stratified analyses, the early pregnancy phthalate mixture was associated with increased SRS-2 total score in boys  and girls  and worse BASC-2 adaptive skills score in girls , while the late pregnancy phthalate mixture was associated with increased BASC-2 externalizing score in boys .Our results suggest cumulative adverse associations between prenatal phthalate mixtures and multiple facets of childhood behavior. They arStudies of prenatal phthalate metabolites and child neurodevelopment generally show adverse associations, though these findings are somewhat mixed in terms of specific associations for each phthalate and difficult to interpret due to variations in the metabolite exposures studied, the timing of assessment, and the outcomes assessed . A recenIn this study, we address these gaps in the literature by using weighted quantile sum (WQS) regression to examine associations between maternal prenatal phthalates measured in early pregnancy and late pregnancy and child neurobehavioral outcomes at ages 4–5 years. WQS regression estimates the cumulative association of the exposure mixture with outcomes, as well as the weighted contribution of each individual exposure in the mixture to the combined association , a signi2.2.1.The Infant Development and Environment Study (TIDES) is a prospective cohort of pregnant women recruited in their first trimester from 2010 to 2012 at the University of California, San Francisco (UCSF); University of Minnesota (UMN); University of Rochester Medical Center (URMC); and Seattle Children’s Hospital/University of Washington (UW). Study design and methods have been previously published . Informe2.2.°C until analysis. Samples were analyzed over time in subsets, the first being the early pregnancy samples, which were initially analyzed by the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention (CDC) and the University of Washington (UW) as previously described using a method involving enzymatic deconjugation of phthalates from glucuronidated forms, solid phase extraction, separation with high performance liquid chromatography (HPLC), and detection by either isotope-dilution tandem mass spectrometry (CDC) or electrospray ionization-tandem mass spectrometry  and late pregnancy between gestational weeks 26 and 42 (mean = 33 weeks gestation) using previously described methods . Measuretrometry . For latmetry  and the Social Responsiveness Scale, 2nd Edition (SRS-2) were completed by mothers of TIDES children between 4 and less than 6 years of age. The BASC-2 provides four composite scores: externalizing, internalizing, behavioral symptoms index, and adaptive skills composite scores. The BASC-2 externalizing composite score is comprised of hyperactivity and aggression subscale scores; the internalizing composite is comprised of anxiety, depression, and somatization subscale scores; the behavioral symptoms index (BSI) is comprised of attention problems, atypicality, and withdrawal subscale scores; and the adaptive skills composite is comprised of activities of daily living, adaptability, functional communication, and social skills subscale scores. Higher scores indicate more problematic behaviors for all BASC-2 scores except for the adaptive skills scores. The SRS-2 provides a total composite score indicating social communication deficits and restrictive behaviors that can be used to screen for autism  or to ca2.4.10-transformed in all models due to right skew. We chose to include the highly correlated DEHP metabolites MEHP, MEOHP, MEHHP, and MECPP separately in mixture models rather than as one sum measure of DEHP metabolites to avoid the potential loss of information on differential toxicity of individual metabolites.Associations between maternal urinary phthalate mixtures and child behavioral outcomes were analyzed separately between early and late pregnancy. Phthalate measurements with concentrations above the LOD in at least 50% of the samples or for which no more than 20% of the samples were missing for either pregnancy period were included in this analysis and listed in 10-transformed, specific gravity-adjusted phthalates were transformed into quintiles because quintiles provided a balance between having quantiles large enough to include all < LOD values for most metabolites but not being so large as to lose resolution. Median levels and variability differed for certain metabolites across study centers, and so to address this heterogeneity we used study center-specific quintiles. WQS regressions iteratively select weights that optimize the likelihood of the model when the coefficient for the sum mixture is positive or negative, and so we ran each model twice, once for each direction, in order to characterize potential positive and negative mixture associations. To ensure estimate stability, we excluded results for a particular direction if fewer than 100 bootstrapped weights out of 1000 total bootstraps were associated with sum mixture coefficients in that direction. The full-sample confidence intervals from the WQS regression were calculated using the “HC0” Huber-White heteroskedasticity-consistent standard error sandwich estimator (Weighted quantile sum (WQS) regression was used to examine associations. Logstimator . To incrstimator  to obtaistimator . To examAll models adjusted for the a priori-selected covariates maternal and child age, pre-pregnancy BMI, study center, race, income, maternal education, child sex, parity (primiparous vs. multiparous), time of the day and gestational age at urine collection, self-reported alcohol and cigarette consumption during pregnancy, and maternal depression and stress as measured by mean PHQ-9 and PSS-10 score, respectively. Covariates were chosen a priori because of their potential confounding effect on one or more outcome measures  or for p3.3.1.t-test p = 0.2).501 participants had complete data for phthalates in at least one pregnancy period, at least one behavioral outcome, and all covariates. Demographic characteristics for these participants included in the final analysis are shown in 3.2.10-transformed concentrations were moderately to highly correlated within each pregnancy period  increase in BASC-2 externalizing score associated with a phthalate mixture heavily weighted for MCPP and MBzP. Other coefficients such as those for the suggestive mixture associations with worse adaptive skills did not appear to differ by sex. IPW had little effect on any model estimates see .4.4.1.Phthalate mixtures were significantly associated with adverse outcomes for many behavioral measures in the original models, and after applying the permutation tests, we have the highest confidence in the early pregnancy associations with autistic traits in both sexes, as well as the male-specific late pregnancy association with externalizing behaviors. The magnitudes of observed associations were all around 1 point per 1 quintile simultaneous increase in each component of the mixture, which is a substantial effect size considering that each T score unit can be interpreted as 0.1 SD of the population distribution. MBzP and MCPP were important metabolites in almost all observed associations, while sex-stratified analyses suggested that early pregnancy phthalate mixture associations specific to girls were predominantly driven by low molecular weight phthalates (LMWP) like DEP and DBP metabolites, while higher molecular weight phthalates like DEHP metabolites were only important in some male-specific associations. The mixture associations we evaluated more accurately model real-world, ubiquitous phthalate co-exposures than the conventional approach of evaluating individual phthalates.Our results suggest significant relationships between everyday ambient exposures and adverse child behavior and therefore have substantial implications for neurobehavioral development. Although associations were relatively modest, and most children in this low-risk cohort scored within the average range relative to BASC-2 and SRS-2 test norms, the clinical significance of our findings is underscored by the widespread exposure to phthalates in the environment. From a public health perspective, even a small association between phthalates and neurodevelopment may have profound implications for the general population . While l4.2.This study did not assess potential mechanisms underlying the associations between phthalate mixtures and neurobehavioral outcomes. However, research in animal models and a few human studies provide mechanistic hypotheses to inform future research. Phthalates are known to inhibit the synthesis of testosterone , and ourAlternatively, multiple phthalates have been shown in vitro to competitively inhibit the thyroid receptor . In anot4.3.Our data suggest that the associations between phthalates and neurobehavioral outcomes may be sex and time-specific. DEHP metabolites were moderately or highly weighted only for significant mixture associations in boys; MEP, MBP, and MiBP weights were only highly weighted for significant associations in girls; and MBzP and MCPP were highly weighted for most associations. A recent study also used WQS regressions to examine associations between age 7 Conner’s Parent Rating Scale (CPRS) scores and mixtures of 8 phthalate metabolites measured in the third trimester and at child ages 3 and 5, finding no significant mixture associations when evaluating all measured phthalates . HoweverRegarding studies that have analyzed phthalate metabolites individually, a recent study observed early pregnancy MBP and MCPP positive associations with age 3–4 SRS-2 total score, similar to our findings, though only in boys with inadequate folic acid supplementation, which we did not measure . Further4.4.Strengths of this study include the measurement of several phthalate metabolites across multiple time points during pregnancy; a large sample of healthy pregnancies; the assessment of both behavioral problems and adaptive behaviors using standardized, norm-referenced measures; consideration of effect modification by sex; adjustment for important predictors of child behavior outcomes; and a modern statistical approach to analyze mixture exposure associations with outcomes. This study has several key limitations. Spot urine samples may be influenced by random variation in daily concentrations due to acute exposures. We attempted to reduce that variation by curvilinearly controlling for the time of day of urine collection, which should improve estimate precision. If this variation is similarly random across pregnancy periods, this should not bias our estimates. Our reliance on spot urine samples at only two different time points will increase the chances of results being affected by exposure misclassification, and so our timing-specific results should be interpreted with caution until further verification. It is possible that differences in WQS mixture weights for individual metabolites across time may be in part due to differences in concentrations over time, but this is unlikely to be a major determinant of the observed timing-specific mixture weights because weights do not consistently change in the same direction as concentrations between the time periods. The TIDES cohort is also limited in that it is mainly comprised of higher income, predominantly white, well-educated families, which limits the generalizability of these findings. In addition, participants without complete data excluded from this analysis were more likely to be black, less well-educated, and have lower incomes, though IPW analyses suggest that this had minimal influence on effect estimates. The WQS regression method is limited to evaluating additive, monotonic associations between mixture exposures and outcomes, and so we did not examine synergistic, antagonistic, or non-monotonic associations between phthalate mixtures and behavioral outcomes. Mixture association effect sizes should be interpreted keeping in mind that within each time period, correlations for some phthalates were relatively low in our population, so a simultaneous quantile increase in each phthalate metabolite may be uncommon. However, this may not be the case in other populations, and the mixture weights combined with the mixture association effect size provide information on estimating effect sizes for increases of one or more components out of the total mixture.5.After analyzing the mixture effects of maternal phthalate concentrations on child behavioral outcomes, we observed adverse mixture associations with many measured behavior scores, in particular significant associations with autistic traits and externalizing behaviors. These results suggest cumulative, additive impacts of phthalate mixtures, with the most important phthalates in that mixture differing by outcome, sex, and timing of exposure.Supplementary Material


Metabolic syndrome is associated with an increased risk of vascular cognitive impairment or, in the more extreme, vascular dementia. Animal models are used to investigate the relationship between pathology and behaviour. This review summarizes the latest understanding of the role of the hippocampus and prefrontal cortex in vascular cognitive impairment, the influence of inflammation in this association while also commenting on some of the latest interventions proposed.Models of vascular cognitive impairment and vascular dementia, whether they develop from an infarct or non-infarct base, demonstrate increased neuroinflammation, reduced neuronal function and deficits in prefrontal and hippocampal-associated cognitive domains. Promising new research shows agents and environmental interventions that inhibit central oxidative stress and inflammation can reverse both pathology and cognitive dysfunction.While preclinical studies suggest that reversal of deficits in vascular cognitive impairment models is possible, replication in patients still needs to be demonstrated. Metabolic syndrome (MetSyn) is a cluster of metabolic disturbances—abdominal obesity, hypertension, glucose intolerance and atherogenic dyslipidemia (increased plasma triglycerides and decreased high-density lipoprotein cholesterol concentration)—which is associated with increased risk for heart disease, type 2 diabetes (T2D) and stroke , 2. CurrThe association between MetSyn and cardiovascular risk is well studied; however, the identification of how MetSyn metabolic changes can lead to functionally debilitating changes in cognition is less understood. Vascular cognitive impairment (VCI) is a form of cognitive deficit caused by vascular abnormalities . The mosThere are currently no medications that successfully treat VCI. Interventions such as antihypertensives and statins focus on managing risk factors  while boPathological analysis of brain changes in VCI and VD includes both neuroimaging and pathology-confirmed diagnosis . CerebroThe hippocampus and prefrontal cortex play an essential role in cognitive functioning. The hippocampus is important in spatial memory and episodic memory, information formation and processing and associated behavioural regulation , while tFrom our understanding of cognition and cognitive deficits in its many forms, it is clear that a ‘one area fits all’ concept cannot address why cognitive deficits occur after metabolic syndrome and its associated diseases. The use of preclinical models with controlled parameters is advantageous to mimic certain aspects of MetSyn and VCI in animals and explore the relationship between brain pathology and the cognitive deficits associated with the disorders.VCI animal models are largely based on modelling the cerebrovascular pathology observed in patients  Table 11. Transidb/db mice which are a model of T2D. A third group are MetSyn traits induced by environmental stressors such as being fed high-fat and/or sugar diet [streptozotocin (STZ), a widely used chemical for the induction of experimental diabetes in rodents [Metabolic disturbance can be used to model systemic cause or as an adjunct to the aforementioned models with a number of rodent models that encompass features of MetSyn. Some models are inbred strains selected for one or more traits underlying MetSyn—most commonly the SHR (spontaneous hypertensive rat) modelling hypertension. Others are population models with genetic risk for MetSyn traits, such as Zucker rats which model obesity and gar diet  or admin rodents .With regard to neuroinflammation, both infarct and non-infarct models of VCI demonstrate upregulation of inflammatory cells and markers. Astrocytes and microglia are activated in the brain  and pro-Cognitive tests in VCI models demonstrate impaired memory. Spatial reference memory and working memory is deficient in VCI models , 46 whicCarotid artery occlusion and MCAO models produce a loss of hippocampal neurons , impaire-α [.Utilizing a STZ/MCAO MetSyn-VCI model, researchers observed that diabetes induced hippocampal-dependant impaired cognitive function in the Y-maze, social recognition and novel object recognition tasks in concert with upregulation of NLRP3  inflammasome expression in the hippocampus •. These -α  while mi-α .db/db model hippocampal long-term potentiation is inhibited and memory impaired [Looking at non-surgical models, a decrease in neuron number and vitality is observed in the frontal cortex and hippocampus of Zucker rats, along with a reduction in synaptic markers and a memory retention deficit in the passive avoidance task , 59. Conimpaired , 61 whilimpaired . High-faimpaired –65.Based on these recent studies models of VCI and VD, whether they develop from a surgical or metabolic base, deficits in prefrontal and hippocampal-associated cognitive domains, decreased neuronal function and increased neuroinflammation are observed. However, we are little closer to identifying the cellular and molecular mechanisms underlying VCI and VD. Working backwards from interventions that show attenuation of deficits is providing a potential alternative path to identifying potential targets for treatment of VCI and VD.Several new treatments have been proposed to reverse the cognitive and pathological deficits in animal models of VCI. These include anti-inflammatory, antihypertensive or antioxidant pharmacological interventions that may guide researchers towards appropriate mechanisms of action for human treatments. Other potential interventions are environmental, where the mechanisms are often poorly understood.Injection of MCC950, a selective inhibitor of the NLRP3 inflammasome, after reperfusion in the STZ/MCAO MetSyn-VCI model ameliorated the diabetes-mediated deficits in hippocampal-dependant memory, lowered cell death of the neurons in the CA1 and dentate gyrus regions of the hippocampus and reduced levels of IL-1β and NLRP3 after MCAO •, sugges2+-channel blocker in BPH/2 mice prevented hypertension-related damage to functional hyperemia through the diminished activity of the capillary endothelial cell inward-rectifier potassium channel, Kir2.1 [Chronic 3-month treatment with the antihypertensive amlodipine, a voltage-dependent Ca, Kir2.1 . Interes, Kir2.1 , alignin, Kir2.1 .Recent studies suggested that antioxidants also have promising effects in VCI and VD models. The Chinese herb mix Chitosan reduces hippocampal-memory impairment and reduces neuron cell loss induced by CCAO while also significantly reversing reactive oxygen species production, neuronal apoptosis and microglia overactivation in the hippocampus through activation of the antioxidative response element Nrf2 (NF-E2 p45-related factor 2) causing the subsequent upregulation in the expression of many cytoprotective enzymes . MeanwhiEvidence suggests that there are effects of physical exercise on neuroplasticity, learning and memory and investigations in preclinical models of MetSyn-VCI have produced very positive results , 79. In Using models of MetSyn, VCI and VD researchers show cognitive and pathological deficits that model what is observed in humans. Promising reversal of these deficits is observed with environmental interventions, anti-inflammatory agents and antioxidants, although whether this can be replicated in patients is still to be seen. What is evident from the latest studies is that an expansion of focus is required. Interestingly, Ward and colleagues suggest that when investigating the hippocampus’s role in VCI, we should consider it as a conceptual neurovascular unit which is composed of neurons, endothelial cells and glial cells, assessing the interaction between vasculature and the surrounding brain cells rather than neurons alone •. This c


Varicella zoster virus (VZV) is one of the eight known human herpesviruses. Initial VZV infection results in chickenpox, while viral reactivation following a period of latency manifests as shingles. Separate vaccines exist to protect against both initial infection and subsequent reactivation. Controversy regarding chickenpox vaccination is contentious with most countries not including the vaccine in their childhood immunization schedule due to the hypothesized negative impact on immune-boosting, where VZV reactivation is suppressed through exogenous boosting of VZV antibodies from exposure to natural chickenpox infections.Population-level chickenpox and shingles notifications from Thailand, a country that does not vaccinate against either disease, were previously fitted with mathematical models to estimate rates of VZV transmission and reactivation. Here, multiple chickenpox and shingles vaccination scenarios were simulated and compared to a model lacking any vaccination to analyze the long-term impacts of VZV vaccination.As expected, simulations suggested that an introduction of the chickenpox vaccine, at any coverage level, would reduce chickenpox incidence. However, chickenpox vaccine coverage levels above 35% would increase shingles incidence under realistic estimates of shingles coverage with the current length of protective immunity from the vaccine. A trade-off between chickenpox and shingles vaccination coverage was discovered, where mid-level chickenpox coverage levels were identified as the optimal target to minimize total zoster burden. Only in scenarios where shingles vaccine provided lifelong immunity or coverage exceeded current levels could large reductions in both chickenpox and shingles be achieved.The complicated nature of VZV makes it impossible to select a single vaccination scenario as universal policy. Strategies focused on reducing both chickenpox and shingles incidence, but prioritizing the latter should maximize efforts towards shingles vaccination, while slowly incorporating chickenpox vaccination. Alternatively, countries may wish to minimize VZV complications of both chickenpox and shingles, which would lead to maximizing vaccine coverage levels across both diseases. Balancing the consequences of vaccination to overall health impacts, including understanding the impact of an altered mean age of infection for both chickenpox and shingles, would need to be considered prior to any vaccine introduction.The online version contains supplementary material available at 10.1186/s12916-022-02534-7. Varicella zoster, commonly referred to as chickenpox, is a respiratory infectious disease that causes a characteristic red rash and pox on the skin surface . It is cThe chickenpox vaccine was first approved for use in the USA in 1995. Despite its successful use for a quarter century, global VZV vaccination policy remains a topic of much debate. It is a live-attenuated vaccine administered in two doses during childhood. The shingles vaccine is either a single (Zostovax) or double (Shingrix) dose recombinant vaccine given later-in-life to suppresses reactivation , 8, 9. CThe primary reason the VZV vaccine has not been implemented worldwide is the presumed reduction of immune-boosting in adults. Theoretical models have predicted an increase in shingles incidence with the inclusion of the chickenpox vaccine on a countries childhood immunizations schedule. , 24. ImpIt is difficult to discern whether increases in shingles are due to the vaccine or improved reporting. Prior to the introduction of the chickenpox vaccine, cases of shingles had been increasing in Canada, , 31, theTo evaluate the impact of chickenpox vaccination, we estimated the number of cases that would have been averted had the chickenpox vaccine had been introduced in Thailand’s routine immunization program in 1996 under various roll-out scenarios  . With a By the end of the 100-year simulation period, chickenpox cases were near zero under any of the vaccination coverage and uptake combinations Tables S and S3. Shingles dynamics were more complex due to chickenpox vaccination increasing the number of individuals susceptible to VZV reactivation after the initial 20 year protective period from chickenpox vaccination. Vaccine simulations were evaluated against a null simulation which lacked both chickenpox and shingles vaccination. To examine the impact of vaccination at different points of the 100-year simulation, we separated the results into four 25-year time periods Fig. S. Over thUnsurprisingly, most scenarios that included shingles vaccination decreased shingles cases in the first period years 1–25) of our simulation. Only when chickenpox coverage was high and shingles coverage was low did shingles cases increase in this period. This is because the length of immunity from chickenpox vaccination lasted 20 years, so the increase in cases was driven by those losing immunity in the last few years of this period. In the second and third periods (years 25-75) all scenarios saw in increase in shingles cases compared to the first period. In the final period, all scenarios saw a drop in shingles cases compared to the second and third periods  reveal that any introduction of a chickenpox vaccine would drastically reduce chickenpox incidence; (ii) identify a non-linear relationship in chickenpox coverage and reduction of chickenpox cases; (iii) demonstrate that any introduction of a shingles vaccine with realistic coverage levels , simulations that included slow, moderate, or aggressive chickenpox vaccine roll-out increased shingles incidence Fig. S, though Under realistic vaccination scenarios observed in countries that currently vaccinate against both chickenpox and shingles, where the shingles vaccine provides 5 years of immunity and high chickenpox coverage  would likely increase the number of shingles cases, particularly at higher chickenpox vaccination levels seen in Figs. The complicated nature of VZV makes it impossible to select a single vaccination scenario as universal policy. Some countries may wish to minimize total VZV cases, while others may prefer to focus on chickenpox or shingles individually. Strategies focused on reducing both chickenpox and shingles incidence, but prioritizing the latter, should concentrate on raising awareness for shingles vaccination  and maxiMonthly clinical case reports for chickenpox and shingles were obtained from the Thailand Ministry of Health . PopulatChickenpox and shingles data from Thailand were previously fit using a mechanistic model . The modTo model the introduction of a chickenpox vaccine in Thailand, three vaccine roll-out scenarios were used: (i) slow — chickenpox vaccine uptake matching measles vaccine uptake in Thailand, which was introduced in 1984, (ii) moderate — chickenpox vaccine coverage matching hepatitis B vaccine coverage in Thailand, which began in 1992, and (iii) aggressive — a chickenpox vaccine uptake matching Japanese Encephalitis vaccine coverage in Thailand, which was introduced in 2006 Fig. 5-92.6%) –57. The 5-92.6%) . To keep-92.6% –55-92.6%) . We alsopomp [Shingles coverage was modeled as either non-existent (no shingles vaccination) or similar to the estimated coverage values from the USA (ent}33%)  or the Uent}50%) –46. Init 5 years  or lifelig. pomp .Though most of the results and discussion are focused on the vaccination scenarios listed in Fig. Additional file 1: Supplementary Materials.


Monte Carlo (MC) methods have been applied for studying interactions between polarized light and biological tissues, but most existing MC codes supporting polarization modeling can only simulate homogeneous or multi-layered domains, resulting in approximations when handling realistic tissue structures.Over the past decade, the speed of MC simulations has seen dramatic improvement with massively parallel computing techniques. Developing hardware-accelerated MC simulation algorithms that can accurately model polarized light inside three-dimensional (3D) heterogeneous tissues can greatly expand the utility of polarization in biophotonics applications.Here, we report a highly efficient polarized MC algorithm capable of modeling arbitrarily complex media defined over a voxelated domain. Each voxel of the domain can be associated with spherical scatters of various radii and densities. The Stokes vector of each simulated photon packet is updated through photon propagation, creating spatially resolved polarization measurements over the detectors or domain surface.We have implemented this algorithm in our widely disseminated MC simulator, Monte Carlo eXtreme (MCX). It is validated by comparing with a reference central-processing-unit-based simulator in both homogeneous and layered domains, showing excellent agreement and a 931-fold speedup.http://mcx.space/.The polarization-enabled MCX offers biophotonics community an efficient tool to explore polarized light in bio-tissues, and is freely available at  Due to the rapid emergence of massively parallel computing techniques, benefit largely from the fast advances in many core processors such as graphics processing units (GPUs), MC simulations of polarized light have seen significant speed improvement over the past decade. Several groups have reported parallel EMC implementations.Although these studies have demonstrated significantly improved simulation speed, most of these simulators only support layered domains and can not address the needs in modeling increasingly complex media.In this work, we present an open-source and GPU-accelerated MC simulator to model polarized light inside 3D heterogeneous media. This MC algorithm utilizes a 3D voxelated grid to represent spatially varying distributions of spherical scatterers, characterized by their radii and densities. We use Muller calculus to update the Stokes vectors of simulated photon packets, from which we can compute various polarimetry related measurements along the surface of the domain. In the remainder of this paper, we first briefly review the steps of the meridian-plane MC algorithm.22.1The meridian-plane polarized light MC algorithmAfter the “launch” step, the photon packet starts propagating inside the simulation domain. In lossy media, the packet weight is monotonically reduced along the photon’s paths and the weight loss is accumulated into the local grid element  in a two-layer domain. The slab-shaped simulation domain has a size of In Finally, we show simulation of a slab-shaped medium embedded with a spherical inclusion, showcasing pMCX’s capability of modeling heterogeneous domains. In this benchmark , the sim4In summary, we report a massively parallel implementation of polarized MC algorithm in our MCX simulator for modeling the propagation of polarized light inside complex media filled with spherical scatterers. Enabled by its built-in voxel-based geometric representation, the pMCX can handle arbitrarily heterogeneous media. We have described the preprocessing steps to encode the scattering properties of spherical particles with various radii and volume densities into a 3D voxel-based data structure. We provide validation and speed benchmarks ranging from simple homogeneous to complex heterogeneous domains. In all benchmarks, our pMCX solver reports excellent match with the widely used reference solver mcMeridian, while providing a speedup nearly three orders of magnitude. In addition, we observed different GPU memory utilization efficiency among global, constant, and shared memories, with the global memory implementation yielding the highest speed and least restriction. It is noteworthy that a limitation in both mcMeridian and pMCX simulations is that all media boundaries are assumed to have matched refractive indices. We plan to further extend this work to update the Stokes vector across mismatched boundaries.


Malignant pleural mesothelioma (MPM) is a deadly cancer primarily of the lung pleura, strongly associated with asbestos exposure. According to the ESMO Clinical Practice Guidelines, MPM patients may be suitable for best supportive care only, active  or multimodality treatments. Surgery, although controversial, is an option in some centres, but the recognised standard of care is systemic chemotherapy (cisplatin-pemetrexed) while other therapies, including immunotherapy  or combination therapies  are being investigated . DespiteRobinson et al. review their use of different mouse models to investigate therapeutic approaches to treat MPM. They developed some of the first human and mouse mesothelioma cell lines using human tumours in immunocompromised mice and intraperitoneal injection of asbestos respectively. They also developed the MexTAg mouse, an asbestos-induced mesothelioma mouse model in which expression of the Simian Virus 40 large T antigen (SV40 TAg) is controlled by a mesothelin tissue-specific promoter. The advantage with this model is it generates 100% mesotheliomas in response to asbestos. More recently, they also developed a bilateral tumour model which was developed to analyse the difference between immune checkpoint blockade responsive and non-responsive tumours against a homogeneous background.The development of novel treatments for MPM requires substantial preclinical data before drugs can enter a clinical trial. Mesothelioma cell lines are extensively used to screen individual or combinations of drugs to determine cytotoxicity, anti-cancer mechanisms and drug resistance, and to identify potentially clinically relevant biomarkers. However, 2D cell culture has limitations as lack of heterogeneity and a tumour microenvironment and acquisition of mutations limit their usefulness . Three-dPezzicoli et al. discussed current and potentially upcoming standards of care for MPM and reviewed several novel therapeutic agents recently in clinical trial. Lurbinectedin is a transcriptional inhibitor and blocks the function of tumour associated macrophages. Mesothelioma cell lines were highly sensitive to lurbinectedin and in the SAKK 17/16 phase II clinical trial, lurbinectedin demonstrated activity following immune checkpoint inhibitor therapy, indicating a phase III trial is warranted. Pezzicoli et al. also discussed the outcome pf several tyrosine kinase inhibitor trials. Of particular interest, were results from preclinical studies using CDK4/6 inhibitors, that demonstrated cell toxicity and inhibition of tumour growth in mouse models. The MiST2 study  was recently announced. The median progression free survival and overall survival was 128 and 217 days respectively. The authors plan to further investigate CDK4/6 inhibition in another randomised study  in MPM which have not yet entered clinical trial. STAT3 is important for cell survival, proliferation, self-renewal and chemoresistance. KDM4A demethylates the tumor suppressor SETD2 products H3K36me3 and H3K9me3, which are essential in cell growth and DNA damage repair. Heparanase promotes tumor growth, angiogenesis, metastasis and chemoresistance. All molecules reviewed impact some of the hallmarks of cancers but their role in MPM and the effect of targeting these molecules on MPM tumour growth require considerably more preclinical studies.Oien et al. to have anti-cancer effects in cisplatin-resistant and pemetrexed-resistant MPM cell lines. Interestingly, NF2-mutated cell lines were more sensitive to QC. Inhibition of the hippo pathway was identified using RNA-seq and closely-associated transcription factors YAP1, CDK4, and KRAS were downregulated. However, the effect of QC in vivo has not been investigated.Repurposing of existing drugs has also been proposed for MPM. Quinacrine (QC), which is an antimalarial drug and an antibiotic, was shown by Precision medicine approaches to treat MPM have been proposed  and the in vitro and in vivo models to test gene and variant function. Together, scientists and clinicians can utilise their specific expertise to collaborate and plan better animal studies and clinical trials to maximise effective drug design to treat diseases like MPM which have shown limited therapeutic improvement.Developing new approaches for treating MPM are urgently needed and it has been suggested that a circular pathway of bench to bedside and back again approach may be key to clinical breakthroughs . Many pr


After entry into the lymphatic initials, the lymph drained retrograde to the portal blood flow towards the exit at the liver hilum. Perinatally, the liver undergoes complex changes transforming from the main hematopoietic to the largest metabolic organ. We investigated the time course of lymphatic vessel development and identified the hepatic lymphatics to emerge postnatally in a process that relies on input from the VEGF-C/VERGFR-3 growth factor—receptor pair for formation of the fully articulate hepatic lymph vessel bed.The liver is a major biosynthetic and detoxifying organ in vertebrates, but also generates 25%–50% of the lymph passing through the thoracic duct and is thereby the organ with the highest contribution to lymph flow. In contrast to its metabolic function, the role of the liver for lymph generation and composition is presently severely understudied. We took a rigorous, volume imaging-based approach to describe the microarchitecture and spatial composition of the hepatic lymphatic vasculature with cellular resolution in whole mount immune stained specimen ranging from thick sections up to entire mouse liver lobes. Here, we describe that in healthy adult livers, lymphatic vessels were exclusively located within the portal tracts, where they formed a unique, highly ramified tree. Ragged, spiky initials enmeshed the portal veins along their entire length and communicated with long lymphatic vessels that followed the path of the portal vein in close association with bile ducts. Together these lymphatic vessels formed a uniquely shaped vascular bed with a delicate architecture highly adapted to the histological structure of the liver. Unexpectedly, with the exception of short collector stretches at the porta hepatis, which we identified as exit point of the liver lymph vessels, the entire hepatic lymph vessel system was comprised of capillary lymphatic endothelial cells only. Functional experiments confirmed the space of Disse as the origin of the hepatic lymph and flow  The lymph vasculature is connected to the blood vasculature by the thoracic and the right lymphatic duct. While capillary lymphatic vessels are specialized for uptake of fluid and immune cells, collectors are designated to lymph transport , pericentral (zone 3) and intermediary area (zone 2).via three pathways; the first running alongside the portal vessels to the porta, the second following the central vein and the third reaching and following the hepatic capsule  description of the hepatic lymph vessels in mice and the details of their development are still missing. The dense and pigmented hepatic tissue challenges volumetric optical imaging and molecular lymphatic markers lack specificity in the liver.Here, we used light sheet and confocal microscopy-based volume imaging to visualize the lymphatic vasculature in specimens ranging from thick sections to complete liver lobes. We identified the VEGF-C/VEGFR-3 axis as a crucial player in the formation of the hepatic lymphatic vascular network.+/LacZ mice  with a step size of 2 µm. 3D reconstruction was performed on high end commodity hardware using the volume rendering software package Voreen (voreen.uni-muenster.de) ®20, in PBS). Immunostaining was performed freely floating in antibody solution at 37°C. After each staining step the specimens were extensively washed in PBS-T (0.1% Tween®20 in PBS).Liver specimens were permeabilized using 5% Triton-X 100/PBS at 37°C and blocked using Permblock solution , confocal images were captured with a Zeiss LSM880 confocal microscope .Liver specimens were optically cleared in BABB . After whole mount immunostaining, the specimens were embedded in low melting point agarose and dehydrated using escalating methanol concentrations . Methanol was then exchanged by a 1:1 mixture of methanol and BABB and finally replaced by BABB.via the portal vein using a peristaltic pump and afterwards explanted from the mouse.Mice were anaesthetized by intraperitoneal injection of Ketamine (125 mg/kg body weight) and Xylazine (12.5 mg/kg body weight). Surgical depth of the anesthesia was reached after a few minutes, indicated by the absence of the paw withdrawal reflex. The abdomen was opened and the intestine was moved to the left side allowing free access to the portal vein. The portal vein was cannulated with a 27 G ¾ needle and the antibody solution (0.75 mg/kg body weight) was injected. After 3 min the thoracic cavity was opened and the lung was cut. The liver was then perfused with heparin/PBS (10 U/ml) and 4% PFA The following antibodies were used: goat anti-LYVE-1 , rat anti-PECAM-1 , rabbit anti-LYVE-1 , goat anti-LDLR , rabbit anti-GS , rabbit anti-CK19 , mouse anti-smooth muscle actin (SMA)-Cy3 , rat anti-Lyve-AlexaFluor594 , secondary antibodies coupled to AlexaFluor488, 568 and 647 (Invitrogen).The acquired image volumes were converted into the easy-to-use HDF5 format and the relevant inner volume was extracted by using a global erosion operation on the slice-wise concave hull of the imaged liver tissue sample. For segmentation, straightforward hysteresis thresholding was utilized, of which the upper and lower limit were set manually for each volume. Subsequently, connected component analysis and size thresholding was used to remove small and erroneous artifacts. To ensure segmentations of lymphatic vessels only, all resultant volumes were manually inspected and incorrectly extracted blood vessels were removed by an expert. The resultant 3D segmentation volume was further processed using a graph and feature extraction pipeline (VIPAR) adapted from Several marker proteins are well established for the identification of lymph vessels, including the transcription factor PROX-1, the transmembrane tyrosine kinase receptor VEGFR-3, the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and the surface glycoprotein podoplanin (PDPN) [summarized in ]. While Although LYVE-1 is expressed on sinusoids, good differentiation of lymphatic structures in the proximity of larger vessels was still possible as LYVE-1 expression was most pronounced in zone 2 sinusoidal endothelial cells, but weaker in zone 1 and 3 . BesidesBased on the pronounced staining of PROX-1, VEGFR3 and PDPN on non-lymphatic structures, we elected to focus on LYVE-1 as lymphatic marker. However, since LYVE-1 expression is generally restricted to lymphatic capillaries, probing of the other markers was still required to identify lymphatic collectors.Hepatic lymph vessels have been detected in the portal tracts, subcapsular and alongside the central veins . We reviLYVE-1-positive vascular structures were also found in sections close to the hepatic venous confluence in the median lobe, an anatomical position that made it unlikely that they arose from portal lymph vessels . The conThe observation of lymphatic vessels exclusively near portal tracts was confirmed in whole mount stainings of the complete caudate lobe for VEGFR-3. Despite the VEGFR-3-positive sinusoids, lymphatic vessels were successfully identified by following vessel branches, which ultimately derived from the proximity of the portal vein. .While the confocal analysis of thick vibratome sections allowed a detailed study of lymphatic morphology with subcellular resolution, the mesoscopic light sheet microscopy enabled us to visualize the lymphatic network on a larger scale in the context of whole hepatic lobes or the entire liver . Whole mLYVE-1 expression, oak-leaved shaped lymphatic endothelial cells and button junctions are hallmarks of lymphatic capillaries . The widvia the interstitial lymph flow the dermal lymphatics. Such lymph vessel staining was observed with far longer incubation periods. In contrast, liver lymphatics in the portal tracts showed clear LYVE-1 staining (While immune staining of sections and whole mounts revealed the localization and, more importantly, 3D architecture of lymphatics in the portal tracts, we aimed to further determine the route of fluid flow towards the initial lymph vessels. To this end, we infused primary-labeled antibodies targeting PECAM-1, LYVE-1 and LDLR in the form of a bolus injection into the portal vein . While Pstaining . Furtherstaining  while upstaining .porta hepatis as well as the portal vein itself to check for efferent collecting lymphatic vessels characterized by the absence of LYVE-1. Indeed, PROX-1-positive, but LYVE-1-negative vessels were present at the porta hinting at collecting lymphatic vessels (porta in various instances suggests a lymph flow following this hierarchical organization towards the porta hepatis.The clear spatial restriction of lymphatic vessels to the portal tracts directly raises the question which route the lymphatic vasculature takes to exit the liver. For this purpose, we examined the large portal tracts, which run close to the entrance  vessels . In orde vessels . Taken tDevelopmental studies in the mouse identified cellular contributions from the foregut epithelium and the septum transversum mesenchyme to the nascent liver bud. Between E10.5 and E15.5, the fetal liver undergoes rapid expansion and becomes the major fetal hematopoietic organ. Perinatally, functional differentiation of hepatocytes and biliary epithelium commences preparing the liver for its future primarily metabolic role .Whole mount staining of midgestation E12.5 fetuses for PROX-1 and LYVE-1 identified besides embryonic macrophages and the known sites of lymph vessel formation also the expanding fetal liver as an organ highly expressing both proteins . DigitalWe followed the further maturation of lymphatic vessels after birth in vibratome sections of the right hepatic lobe and whole mount preparations of the caudate lobe. At P1, rather compact, LYVE-1-positive lymph vessels were found associated with large superficial portal tracts , P1. ThiIn summary LYVE-1-positive lymphatic vessels appeared to form after birth with their maturation going along with the development of deep portal tracts.Vegfc+/LacZ livers revealed no obvious deficiencies, as branched LYVE-1-positive lymph vessels associated with the portal tracts were readily detected (Vegfr3+/LacZ heterozygous livers, even whole mount staining captured an intact, continuous lymphatic vascular network as apparent by simple visual inspection (Vegfr3+/LacZ and Vegfc+/LacZ livers showed a significant lengthening of the initial lymphatic vessel segments between branchpoints with a higher average cross section area (Motivated by the crucial role of the VEGF-C/VEGFR-3 signaling axis for other lymphatic vessel beds forming postnatally, we probed for a potential role of this growth factor, growth factor receptor pair in hepatic lymph vessel formation. Immune staining of vibratome sections from WT and detected . As the detected . When wespection . In ordeion area . Taken tThe liver is the organ with the largest contribution to lymph production. Although lymphatic vessels are known to be important in the context of disease and tissue homeostasis, a detailed morphological description of the 3D structure of the hepatic lymph vessel bed was lacking. This study now describes the spatial arrangement of the murine liver lymphatic vasculature with cellular resolution by analyzing immunostained thick sections or whole mount preparations covering analysis of liver lobes from micro-to mesoscale.via optical sectioning, which avoids tissue loss and artefacts associated with the mechanical sectioning process. Since, in general, expression of lymph vessel markers is not homogeneous throughout the entire lymphatic vasculature, we stained for several markers including LYVE-1 to identify lymph capillaries, in conjunction with VEGFR-3, which is found more widely distributed on the lymphatic vasculature. In addition, we put a particular emphasis on highlighting non-lymphatic liver structures that also expressed molecular markers found on lymphatic vessels.This comprehensive approach showed that lymphatic vessels in the healthy murine liver are exclusively localized at portal tracts. Blind-ending initials were found starting near small bile ducts, covering the portal vein branches and finally growing into a tree of lymphatics including straight, larger vessels running alongside the portal vein branches and larger bile ducts. The evidence for this description is based on large multi-tile scans of vibratome sections, and staining of whole liver lobes. Light sheet microscopy allowed investigation of whole liver lobes The current model of lymph flow in the liver includes three routes including the portal tracts, central veins and the capsule . While oporta hepatis most likely transport the lymph to abdominal lymph nodes (Unexpectedly, LYVE-1-positive lymphatic vessels were present at the liver surface near the confluence of hepatic veins in the median. Since we found no evidence for similar structures in other lobes and the median lobe is directly facing the diaphragm, we speculate that these lymphatic vessels extend from the lymphatic network covering the diaphragm. Connections between hepatic and diaphragmatic lymphatic vessels were described earlier and until now they were classified as anastomoses emerging from the central venous lymphatics . The lymph nodes .via the portal vein, the lymph flow could provide the liver with leaked bile ingredients by carrying them back to the systemic circulation. Lymphatic uptake of components destined for excretion may in this way prevent the liver from buildup of potentially toxic local deposits. This mechanism could be important in case of cholestasis upon biliary obstruction, when the pressure within the bile ducts increases. Supporting this hypothesis, bile components were found in the lymph upon biliary obstruction (Immunostaining revealed a strong association of lymph vessels and bile ducts. Lymphatic vessels not only started next to small bile ducts, but also ran in very close contact to larger ducts. While it is widely accepted that the capillary plexus surrounding larger bile ducts contributes to hepatic lymph flow , these etruction .The majority of hepatic lymphatic vessels started as spiky blind ending initials and was composed of oak leaf-shaped, LYVE-1-expressing cells and thereby qualifies as lymphatic capillaries. Despite its overall composition of the ubiquitous capillary LECs the spatial structure of the hepatic lymph vessels differs from other vessel beds like the skin, diaphragm, meninges or mesentery . Since lVegfc+/LacZ and less noticeable of Vegfr3+/LacZ mice suggesting an indispensable role of the VEGF-C/VEGFR-3 signaling axis. Similar postnatal development and dependence on VEGF-C has recently been described for meningeal lymphatic vessels (Perinatally the liver undergoes a complex maturation accompanied by a functional switch from the major fetal hematopoietic to an adult metabolic organ. In accordance with these structural changes, we detected the formation of hepatic lymph vessels only after birth tightly associated with the formation of deep portal tracts. Not unexpected, we noted structural defects in the hepatic lymph vessels of  vessels .We also aimed to identify fluid flow resulting in lymph generation. From antibody bolus injection into the portal vein, we suggest that plasma and molecules from the portal inflow leave the sinusoids in zone 1 and continue from the space of Disse to the space of Mall, where they enter lymphatic capillaries. Sinusoidal fenestrations with a diameter of 50–200 nm allow almost free circulation of fluid and antibodies between the vessel lumen and the space of Disse, which we visualized by detection of LDLR antibodies on the basal membrane of hepatocytes. The concentration of this LDLR staining, which was pronounced near portal tracts, indicated a preferential exit of the sinusoids proximal to the portal tracts. Since the anti-PECAM-1 antibody homogenously stained the whole sinusoidal vasculature, it is unlikely that this distribution pattern was due to insufficient access of antibodies to the sinusoids near central veins.porta closely associated with the portal vein. Although not functionally demonstrated by our experiments, we propose that these collectors provide the outflow route for hepatic lymph. In close proximity to the porta, we identified lymph nodes, which should correspond to those identified as the first functional nodes filtering hepatic lymph (Collecting vessels characterized as PROX-1 or VEGFR-3-positive, but LYVE-1-negative vessels, were found on sections of the ic lymph .The route for hepatic lymph generation we propose here is in agreement with the described traditional portal route . Howevervia portal lymphatics rather than the bloodstream and the central veins. From this perspective hepatic lymphatic vessels would not only act as “vasa privata” in order to maintain tissue homeostasis of the liver, but in addition also act as “vasa publica” providing the body with hepatic products. This assumption is underscored by previous observations where nascent proteins were observed in hepatic lymph (The liver not only metabolizes blood components, but also synthesizes large amounts of biomolecules. We have not addressed contribution of liver synthetic activity to the lymph. However, since there is a substantial fluid flow from the sinusoids towards the space of Disse and further to the space of Mall, we suggest that molecules secreted into the space of Disse by hepatocytes will exit ic lymph . Liver fic lymph . Both scic lymph . While t