The dietary supplementation of yeast cell wall extracts (YCW) has been found to reduce pathogenic bacteria load, promote immunoglobulin production, prevent diseases by pro-inflammatory responses, and alter gut microbiota composition. This study evaluated growth and slaughter results, health, gut morphology, immune status and gut transcriptome of 576 male chickens fed two diets, i.e. C (control) or Y (with 250–500 g/t of YCW fractions according to the growth period). At 21 and 42 d the jejunum of 12 chickens per diet were sampled and stained with hematoxylin/eosin for morphometric evaluation, with Alcian-PAS for goblet cells, and antibodies against CD3+ intraepithelial T-cells and CD45+ intraepithelial leukocytes. The jejunum sampled at 42 d were also used for whole-transcriptome profiling.P ≤ 0.10) and improved feed conversion . Regarding the gut, YCW supplementation tended to increase villi height (P = 0.07); it also increased the number of goblet cells and reduced the density of CD45+ cells compared to diet C (P < 0.001). In the gut transcriptome, four genes were expressed more in broilers fed diet Y compared to diet C, i.e. cytochrome P450, family 2, subfamily C, polypeptide 23b (CYP2C23B), tetratricopeptide repeat domain 9 (TTC9), basic helix-loop-helix family member e41 (BHLHE41), and the metalloreductase STEAP4. Only one gene set (HES_PATHWAY) was significantly enriched among the transcripts more expressed in broilers fed diet Y. However, a total of 41 gene sets were significantly over-represented among genes up-regulated in control broilers. Notably, several enriched gene sets are implicated in immune functions and related to NF-κB signaling, apoptosis, and interferon signals.Dietary YCW supplementation did not affect final live weight, whereas it decreased feed intake (114 to 111 g/d; The dietary YCW supplementation improved broiler growth performance, increased gut glycoconjugate secretion and reduced the inflammatory status together with differences in the gut transcriptome, which can be considered useful to improve animal welfare and health under the challenging conditions of intensive rearing systems in broiler chickens. In a context moving towards antibiotic-free livestock production, research in feed additives has largely increased to evaluate different products and their effects on animal health and immune response, in addition to animal productivity . Among tSalmonella typhimurium and Clostridium perfringens), MOS positively affected immune and metabolic pathways in the gut and reduced its colonization [Dietary supplementation with MOS has been found to improve gut morphology in terms of longer villi, shorter crypts, and a higher number of goblet cells . In infenization , 4, 5.C. perfringens [Dietary supplementation with β-glucans increased villi height in the gut and antibody levels against fringens . In chicfringens , while dfringens . NotablyThus, in broiler chickens, dietary supplementation with YCW extracts containing different rates of MOS and β-glucans might improve growth performance and gut morphology –10; promSaccharomyces cerevisiae  on growth performance and slaughter results, health, gut morphology, immune status and gut transcriptome in broiler chickens.Nevertheless, to our knowledge, comprehensive data about the effects of dietary supplementation with YCW extracts on the response of broiler chickens in terms of gut inflammatory pattern , gut whole-transcriptome profiling, animal performance, and gut morphology are still lacking. Therefore, the present study aimed at evaluating the effect of dietary supplementation with YCW extracts (mainly mannan-oligosaccharides and β-glucans) from The trial was performed at the poultry house of the Experimental Farm of the University of Padova , after a long period of downtime (about 6 months). The study was approved by the Ethical Committee for Animal Experimentation of the University of Padova. All animals were handled according to the principles stated by the EC Directive 86/609/EEC  about th2; 120 cm wide × 250 cm large × 120 cm height) were used, each equipped with 5 automatic nipple drinkers and a circular feeder (diameter: 37 cm) for manual distribution of feed. The pens had a concrete floor bedded by wood shavings-wheat straw litter . Twenty-four hours of light were provided during the first 2 d after the chickens arrived at the poultry house. After the first 2 d, hours of lights were progressively reduced until a 18L:6D light program was reached, which was maintained from the 13th day onwards.The poultry house was equipped with a cooling system, forced ventilation, radiant heating and controlled light system. A total of 24 wire-net pens  were transported by appropriate and authorized transport means to the experimental facilities of the University on the hatching day. All chicks had been vaccinated against Marek’s disease, Infectious Bronchitis (H120 + 793B) and Newcastle disease at the hatchery. On their arrival, 24 chicks per pen were placed in 24 pens, randomly allocated to two experimental groups (12 pens per group), i.e. two dietary treatments: C, control, and Y, supplemented with YCW extracts (Safmannan®).Chicks were individually weighed on their arrival, identified by a leg ring, and weighed for live weight once a week until slaughtering at 44 d. Pen feed consumption was measured daily during the trial.For each treatment, three commercial diets in crumble form were administered during the trial as usual, i.e. diet C1 and diet Y1 from 1 to 14 d; diet C2 and diet Y2 from 15 to 28 d; and diet C3 and diet Y3 from 29 d until slaughtering (on d 44) , ash (967.05), and crude protein (2001.11) by AOAC  methods.At both 21 d and 42 d of age, 24 chickens  were selected on treatment-wise average live weight and slaughtered by cervical dislocation to sample gut tissue. One sample of about 2 cm was taken from jejunum at the midpoint between the end of the duodenal loop and the location of the Meckel’s diverticulum  and wash2).Two serial sections of 4 μm per sample were obtained using a microtome and stained with haematoxylin/eosin for morphometric evaluation and with Alcian blue (pH 2.5)-PAS method for a quantitative and qualitative analysis of goblet cells, respectively. Moreover, two serial sections were used for CD3+ and CD45+ immunohistochemical analysis for the sampling at 42 d of age. The villi length and the depth of the crypts were measured with image-analysis software  according to the procedure described by Hampson , with thAfter RNAlater solution removal, tissues were ground using a steel pestle and total RNA was extracted using the RNAeasy Mini Kit , following the manufacturer’s instructions. The RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer , and its quality assessed with a 2100 Bioanalyzer . All samples had an RNA Integrity Number (RIN) value > 7.Equal amounts of RNA  of three different chicks from the same experimental group were pooled together, thus obtaining a total of four RNA pools (i.e. replicates) for each of the two dietary treatments. Therefore, RNA-Seq data were representative of a total of 24 animals (12 per group). A total of 8 tagged RNA-Seq libraries were prepared from RNA pools using Agilent’s SureSelect Strand Specific RNA Library Preparation Kit , following the manufacturer’s instructions. Briefly, poly(A) mRNA was purified from 1.5 μg of total extracted RNA and fragmented using an RNA-Seq Fragmentation Mix. First-strand and second-strand cDNA were synthetized and end-repaired. Adenylation of cDNA 3′ ends and adaptor ligation were performed. Twelve cycles of PCR were used to amplify and index the adaptor-ligated cDNA library, and the PCR products were purified and size selected using the SPRIselect reagent kit . Library concentrations were measured by both a Qubit RNA Assay kit in a Qubit 2.0 Fluorometer  and a PCR-based method by using the NEBNext Library Quant Kit for Illumina . Individual libraries were monitored for insert size using the Agilent DNA 1000 assay on the Agilent Bioanalyzer 2100 system (Agilent Technologies). Multiplexed single-end sequencing 50 bp was carried out on an Illumina Hi-Seq 4000 .GeneCounts quantification while running STAR.Initial quality control was carried out with the FastQC software version 0.11.5 . Reads wAt 44 d of age, all chickens were slaughtered in a commercial slaughterhouse. Feed and water were removed at the same exact time for all chickens, corresponding to approximately 7 h feed and 4 h water withdrawal, respectively, before slaughtering. Chickens were individually weighed before crating. All chickens of a pen were loaded in a transport cage. Chickens were slaughtered according to the standard practice of the commercial slaughterhouse . Ready-to-cook carcasses were recovered after 2 h of refrigeration at 2 °C.A total of 120 carcasses , which had been previously selected on the basis of the slaughter live weight to be representative within a pen, were individually weighed to measure slaughter dressing percentage and transported to the Department laboratories to be stored at 2 °C. Twenty-four hours after slaughter, carcasses were dissected for the main cuts, i.e. breast and hind legs .t test. Differences between means with P ≤ 0.05 were accepted as statistically significant. Differences between means with P ≤ 0.10 were accepted as representative of a statistically significant trend.Individual data related to live weight and daily growth was analysed with a generalized linear mixed model considering the diet as the main factor of variability and the pen as a random effect using PROC MIXED . Cage daP ≤ 0.10, log2 fold change (log2FC) > 1) [P ≤ 0.05) indicates that the biological activity  characterized by the gene set is correlated with the supplied ranking. As GSEA is typically used with gene sets from the Molecular Signatures Database (MSigDB), consisting of HUGO (Human Genome Organization) gene symbols, chicken gene IDs were used to obtain human orthologues and related HUGO gene symbols in BioMart. The input was prepared as follows: the raw P-values obtained through the DE analysis  were used to rank the list of genes by significance. When multiple genes with the same gene symbol were detected, only the most significant one  was retained. To specify the direction of the gene expression variation, the P-values (P) were replaced by 1−P or −(1−P) when a gene was over-expressed or under-expressed in chickens fed diet Y, respectively. The analysis was carried out by using a classic enrichment statistic and the MSigDB curated gene sets belonging to the BIOCARTA collection.Regarding whole-transcriptome profiling, a pairwise Differential Expression (DE) analysis was performed using Deseq2  > 1) . Using tFC) > 1)  was emplP = 0.08; and − 2.9%, P = 0.03, respectively). At 44 d of age, the average live weight was 2,964 g without significant differences between the two dietary treatments.On the hatching day, the weight of chicks averaged 46.2 ± 3.2 g without significant differences between experimental groups (Table P = 0.06) without effects on feed intake, which tended to impair feed conversion (P = 0.10) compared to diet C (Table P = 0.04) and feed intake (P ≤ 0.01), without effects on feed conversion. Conversely, during the third period (28–44 d), the YCW supplementation tended to promote daily weight gain (P = 0.06) without a difference in feed intake and, thus, significantly improved feed conversion (P ≤ 0.001). In the whole trial, YCW supplementation did not affect daily weight gain, whereas it tended to decrease feed intake (P = 0.09) and improved feed conversion (P = 0.01) (Table During the first period of growth (1–14 d), dietary supplementation with YCW extracts tended to decrease daily weight gain  (data not reported in tables).Total losses due to mortality during the trial ranged from 9.85% to 6.82% in animals fed diets C vs. those receiving diets Y (P = 0.07), whereas the proportions of other main carcass cuts of carcass did not differ between dietary treatments.Slaughter results and carcass quality did not change according to the dietary treatment Table . The weiP = 0.07), which was higher in chickens fed diets Y compared to those fed diets C (Table P ≤ 0.001) higher (47.0 vs. 21.7 cells/500 μm) and the area of the same cells lower (14.4 vs. 39.2 μm2) in chickens fed diet Y compared to chickens fed diet C (Table P ≤ 0.001) and, at a lower extent, CD3+ (P = 0.08) were significantly lower in birds fed diet Y compared to those fed diet C.The effect of the dietary treatment on gut morphology approached statistical significance only in the case of the villi height  and crypt depth decreased  from 21 to 42 d. As age increased, the density of goblet cells decreased   are reported in Table CYP2C23B), tetratricopeptide repeat domain 9 (TTC9), basic helix-loop-helix family member e41 (BHLHE41), and the metalloreductase STEAP4.The DE analysis showed only slight differences in the gut transcriptome of broilers according to the dietary treatment. A total of 24,145 genes were assessed. Genes whose mRNA levels were significantly different  was significantly enriched among the genes more expressed in broilers fed diet Y. However, a total of 41 gene sets were significantly over-represented among genes up-regulated in control broilers. Notably, several enriched gene sets are implicated in immune functions and related to NF-κB signaling, apoptosis, and interferon signals . The complete list of enriched BIOCARTA gene sets is provided in Supplementary Table SS. cerevisiae groups and their substrates  [Among the different feed additives, YCW extracts have been widely used in animal feeding and, specifically, in broiler chickens. Nevertheless, their effects on performance and health are not always consistent among studies, because several conditions may differ. Firstly, the contents of the different YCW fractions  can vary in the different products according to fermentation conditions and cultivation methods of the yeast as well as of d bread) , 28. AlsBased on the literature, when no specific challenge was applied, the dietary supplementation with YCW extracts in the range tested in our trial (i.e. 250–500 ppm) did not produce significant improvements in growth performance of broilers in the starter period (21 d) , 29. ConC. perfringens challenge, the dietary supplementation tested in the present trial (Safmannan®) had a positive impact on final body weight, weight gain and feed conversion ratio [Salmonella typhimurium [In broilers at 21 d of age previously submitted to an immune stress and a on ratio . A positon ratio  and for himurium  as well himurium . YCW suphimurium .Under our conditions, the reduction in daily growth observed during the first and, especially, the second period of growth 15 to 28 d of age) might be related to the higher YCW supplementation rate used in this period (i.e. 500 vs. 250 ppm). In fact, Fowler et al.  estimate d of ageOn the other hand, according to Reisinger et al. , a high Interestingly, the increased density of the goblet cells in animals fed diet Y went about with decreased cells size compared to chickens fed the control diet. It is possible that in the control group, cells were larger because they were storing and accumulating mucin and the stimulus to release the mucin was lower, whereas in the case of chickens fed diet Y several cells were continuously producing and releasing mucus .The increase in the number of goblet cells can be positively considered in view of the protective effect of mucus production by goblet cells under any challenging conditions , but undOnly a few studies are available on local gut associated immune reactions in poultry and they refer to the effect of different dietary protein sources (soybean vs. differently processed peas) . Our stuCYP2C23B, whose expression was 2.3 fold higher in broilers supplemented with YCW. This drug-metabolizing enzyme (an epoxygenase) catalyzes the conversion of arachidonic acid in epoxyeicosatrienoic acids (EETs), short-lived signaling eicosanoids. Previous studies showed EETs possess anti-inflammatory and antiapoptotic activity in kidney [TTC9 is concerned, little information about its role in the gastrointestinal tract is available. Nevertheless, we cannot exclude this gene might contribute to chicken gut homeostasis, as the human isoform TTC7A is crucial in maintaining intestinal epithelial cell integrity [BHLHE41 regulates the mammalian molecular clock; interestingly, in humans it controls the B-cells receptor repertoire, the self-renewal of B-1 cells, and innate-like B-lymphocytes, thus providing a first line of defence against pathogens [In our trial, further insights in the effects of YCW supplementation on the immune status of animals were obtained by whole transcriptome analysis (i.e. RNA-Seq). Based on the literature, the four genes whose expression was significantly increased by YCW supplementation might play pivotal roles in immune responses, but these traits have not yet been extensively investigated in chickens. Notably, the most significant differentially expressed gene (DEG) was n kidney  as well n kidney . As far ntegrity . The traathogens . Finallyathogens . Thus, iHES1 was enriched among the up-regulated genes in birds fed diet Y. This is of particular interest, since HES1 is a transcription factor proposed to be one of the primary mediators of intestinal Notch signals, which in turn contribute to epithelial cells choice of an absorptive versus a secretory lineage [HES1, through the repression of a secretory lineage transcription factor (i.e. HATH1), drives cells to become absorptive enterocytes [HES1 pathway in animals fed diet Y seems to be consistent with the observed increase of villi height after yeast-supplementation, giving both these mechanisms a role in nutrient absorption. Nonetheless, in mice HES1 has been identified as a homeostatic suppressor of inflammatory responses both in vivo (i.e. serum) and in vitro(i.e. primary macrophages) [The GSEA analysis pointed out additional results elucidating the processes triggered by YCW-supplemented diets in chickens. The pathway mediated by the epithelial transcription factor  lineage . In parterocytes . Thus, tophages) .Interesting results were also noticed for downregulated genes in chickens fed diet Y. The GSEA analysis pointed out a significant enrichment of several genes sets, most of them being related to viral infection response , antigen recognition , stress response (e.g. NF-κB activation), apoptosis induction , and inflammation . Overall, this demonstrates that immune, pro-inflammatory and pro-apoptotic processes are activated to a higher extent in chicks fed the control diet compared to those fed with the supplemented diet. The activation of these pathways might be a consequence of higher stress levels due to any challenging condition  in control animals, and is in agreement with the higher density of intraepithelial leukocytes and the reduced amount of goblet cells observed in this experimental group.Considering the limited number of DEGs, we can reasonably conclude that the yeast-supplemented diet tested in this study induce small changes in the gut transcriptome of broiler chickens. Indeed, results provided by the GSEA, which is powerful in detecting even small but coordinated transcriptional changes, are of particular interest, as they are highly consistent with the hypothesis that yeast supplementation might exert some important benefits on the intestinal mucosa. Overall, based on the biological functions of the DEGs and the significantly enriched gene sets, we hypothesize that molecular pathways mostly impacted by YCW extract supplementation are those related to immunity. This suggests that yeast supplementation might play a key role in triggering anti-inflammatory mechanisms and an animal’s response to pathogens. Despite that, additional studies are needed to draw conclusive remarks about the molecular mechanisms modulated by YCW extract supplementation in this species (i.e. whole gene expression at different time points and/or in other target tissues).The dietary addition of yeast cell wall extracts improved broiler feed conversion. Moreover, despite the absence of any specific challenging issue, it improved gut conditions in terms of increased glycoconjugate secretion at the gut level, which would provide chickens with more defences against any pathogens. The reduction of the gut inflammatory status was associated with differences in the gut transcriptome, which highlights that YCW extracts might act in different ways, at the level of intestinal integrity or at different stages of the immune response. Based on these multiple effects and action mechanisms, the inclusion of yeast cell wall extracts can be considered useful to improve animal welfare and health under the challenging conditions of intensive rearing systems in broiler chickens.Additional file 1: Table S1. Sequencing and mapping results. The table reports the RNA-Seq libraries sequenced including for each of them i) the number of raw reads obtained, ii) the number of reads after trimming and rRNAs removal iii) the number of mapped reads (and the percentage of mapped reads). Table S2. GSEA results. Enriched BIOCARTA Gene Sets (GS) at both the highest (up-regulation in “diet Y” condition) and lowest (up-regulation in “diet C” condition) part of the genes ranking are reported. ES: enrichment score; NES: normalized enrichment score; NOM p-val: nominal P-value.


Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma and may occur with lymph node and/or extranodal involvement. Recurrence in patients with diffuse large B-cell lymphoma usually occurs within the first few years after treatment and may occur in a different area outside the initial localization.A female Turkish patient who was diagnosed with nodular sclerosing Hodgkin lymphoma through lymphadenopathy examination reached remission after chemotherapy and radiotherapy. In the 11th year of follow-up and at the age of 45, newly developed multiple lymphadenopathies were diagnosed with a pathological result of diffuse large B-cell lymphoma in her advanced examination. Due to massive splenomegaly and cystic necrotic splenic residues, splenectomy was performed after eight cycles of a first-line chemotherapy regimen and two cycles of high-dose methotrexate treatment for central nervous system prophylaxis. A pericardial mass , which was not present at the time of diagnosis and interim evaluation of positron emission tomography/computed tomography, was detected through chest pain in the third month after the last screening, although a complete response had been obtained. Pathological examination of the pericardial area revealed the pathological result was a recurrence.Patients with diffuse large B-cell lymphoma have an aggressive clinical course, but cardiac involvement is very rare. In our patient’s case, pericardial involvement was observed after treatment and scanning revealed that recurrence took place in an area different from the pericardium. Cooperation of clinicians and pathologists and rapid evaluation are very important in cases of diffuse large B-cell lymphoma relapse. Although a tumoral invasion of the pericardium mostly suggests secondary malignancies, it should be kept in mind that recurrence of lymphoma is also possible. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype among non-Hodgkin lymphomas (NHLs). It accounts for about 30–40% of NHL cases and more than 80% of aggressive lymphomas . Like ma3, neutrophil count 8540/mm3, platelet count 456,000/mm3, urea 22 mg/dl, creatinine 0.64 mg/dl, uric acid 7.1 mg/dl, aspartate transaminase 25 U/L, alanine transaminase 13 U/L, lactate dehydrogenase 848 U/L, total protein 6.4 g/dl, albumin 3.2 g/dl, and C-reactive protein 68 mg/dl. Positron emission tomography/computed tomography (PET/CT) showed hypermetabolic fluorodeoxyglucose (FDG) involvement in the bilateral jugular chain in the neck , spleen sizes in the abdominopelvic sections (270 mm), hypermetabolic FDG in places of heterogeneous character in this cystic lesion area, the largest of which reaches a diameter of 70 mm (SUVmax 30.7), hypermetabolic FDG involvement (SUVmax 14.0) in the para-aortocaval region, hepatogastric area, bilateral inguinal fossa at the portal hilus level, and sclerotic lesion in the midline in the sacrum (SUVmax 15.8). The patient was diagnosed with DLBCL. Immunohistochemistry results were as follows; Ki-67 80%, CD20 (+), BCL2 (+), BCL6 (+), MUM1 (+), C-MYC (−), nongerminal center type DLBCL, and fluorescence in situ hybridization negative for c-myc by cervical lymph node biopsy. In addition to eight cycles of R-CHOP  treatment, the patient received central nervous system prophylaxis with two cycles of high-dose methotrexate because of her high central nervous system international prognostic index score . Doxorubicin was also removed from the protocol after reaching the cumulative dose of 450 mg/m2. The pathology of the splenectomy specimen did not include tumoral invasion; only a cystic necrotic area was observed. At the end of treatment, PET/CT was performed 1 month after surgery and was compatible with complete remission.A female Turkish patient who was diagnosed with nodular sclerosing HL by being examined with lymphadenopathy reached remission after receiving an ABVD  chemotherapy regimen for three cycles and 30-Gy radiotherapy. In the 11th year of follow-up and at the age of 45, newly developed multiple lymphadenopathies were diagnosed with a pathological result of DLBCL in her advanced examination. Laboratory test results of the patient were as follows: hemoglobin 8.8 g/dl, white blood cell 11,620/mmThree months after the last PET/CT control, she was admitted to our outpatient clinic due to chest pain and B symptoms. A hypermetabolic mass, which was evaluated in favor of the involvement of primary disease, was observed isolated in the widest place in contact with the pericardium at the left precardial distance 2.9 × 2.2 cm (SUVmax 34.8) Figs.  and 2, aPrimary tumors of the pericardium are extremely rare. The first malignancies that come to mind in the presence of pericardial effusion and especially tamponade are lung cancer with 30% and breast cancer with 20% , 12. TheCardiac and pericardial involvement of lymphoma is extremely rare, accounting for 0.5% of cardiac involvement and 1% of extranodal NHL. The most common lymphoma subtype is known to be DLBCL, but Burkitt lymphoma, T-cell lymphomas, small lymphocytic lymphoma, and plasmablastic lymphoma also occur . The forPrimary mediastinal large B-cell lymphoma accounts for 7% of DLBCLs and represents 2.4% of NHL cases. The disease is observed especially in the third and fourth decades and predominantly in women. Tumor compression or invasion, especially of anterior mediastinal origin, is observed. Isolated pericardial mass or effusion is not expected .Involvement-related arrhythmia may occur with many clinical findings such as shortness of breath and syncope. It usually indicates a patient with lower chance of survival. It is more common in high-grade lymphomas, especially double-hit/triple-hit subtypes, and shows a poor prognosis. Although it is mostly seen with cardiac symptoms, it can also occur asymptomatically. Generally, clinical manifestation occurs with right ventricular dysfunction and tamponade, which also carry important clues for central nervous system involvement .Our present case report describes a very rare condition because it is about a patient with DLBCL long after HL-specific treatment and recurrence with an isolated pericardial mass after DLBCL treatment. Although the literature indicates that lymphomas with isolated cardiac recurrence or cardiac involvement were particularly associated with acquired immunodeficiency syndrome, our patient’s case does not have such comorbidity. Although isolated pericardial mass suggested secondary malignancies, pathology revealed recurrence of isolated DLBCL recurrence.Pericardial involvement is rarely seen among lymphomas, and DLBCL is the most common subtype of them. Although nonlymphoma tumors first come to mind in patients with a history of cytotoxic therapy anamnesis and in cases that are at risk for secondary malignancy, it should be kept in mind especially in high-grade lymphomas in terms of recurrence.


Bile acid diarrhoea (BAD) is a common disorder resulting from increased loss of bile acids (BAs), overlapping irritable bowel syndrome with diarrhoea (IBS-D). The gut microbiota metabolises primary BAs to secondary BAs, with differing impacts on metabolism and homeostasis. The aim of this study was to profile the microbiome, metabolic products and bile acids in BAD. Patients with BAD diagnosed by SeHCAT testing, were compared with other IBS-D patients, and healthy controls. Faecal 16S ribosomal RNA gene analysis was undertaken. Faecal short chain fatty acid (SCFA) and urinary volatile organic compounds (VOCs) were measured. BAs were quantified in serum and faeces. Faecal bacterial diversity was significantly reduced in patients with BAD. Several taxa were enriched compared to IBS-D. SCFA amounts differed in BAD, controls and IBS-D, with significantly more propionate in BAD. Separation of VOC profiles was evident, but the greatest discrimination was between IBS-D and controls. Unconjugated and primary BA in serum and faeces were significantly higher in BAD. The faecal percentage primary BA was inversely related to SeHCAT. BAD produces dysbiosis, with metabolite differences, including VOC, SCFA and primary BAs when compared to IBS-D. These findings provide new mechanistic insights into the pathophysiology of BAD. Functional bowel disorders such as IBS form the largest group of patients seen in a general gastroenterology clinic, but clinicians often neglect the opportunity to diagnose BAD. The established gold standard diagnostic tests for BAD, faecal bile acid measurements, 75SeHCAT scan or serum 7α-hydroxy-4-cholesten-3-one (C4), are limited in their availability3.Bile acid diarrhoea (BAD) is a commonly missed cause of chronic diarrhoea and has been demonstrated in excess of a quarter of patients who were previously diagnosed with IBS-D4. Bile acids undergo an enterohepatic circulation. Less than 5% of the glycine- and taurine-conjugated primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), escape active absorption in the terminal ileum5. Unabsorbed primary BAs can undergo biotransformation by the microbiota in the colon, to form the secondary BAs, deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA). These are partially absorbed passively in the colon, or excreted in the faeces5. Biotransformation enzymatic reactions comprise, first, deconjugation, via bile salt hydrolase (BSH), which catalyses the hydrolysis of glycine or taurine from the C24 N-acyl amide bond of conjugated BAs6. Epimerization, oxidation, dehydroxylation and hydroxylation by hydroxysteroid dehydrogenase (HSDHs) enzymes can then occur. 7α-dehydroxylation of primary BAs, forming secondary BAs, is the most quantitatively important and complex microbial bile salt transformation8.BAD may be due to malabsorption or overproduction of BAs. The ileal hormone fibroblast growth factor 19 (FGF19) regulates hepatic BA synthesis and is low in BAD5.BA act as signalling molecules through activation of receptors: farnesoid X receptor (FXR) is most potently stimulated by CDCA, and the secondary BAs stimulate TGR5 . BA regulate intestinal homeostasis by inhibiting inflammation, preventing pathogen invasion and maintaining cell integrity, and furthermore stimulate production of hormones including FGF19, GLP-1 and PYY5. The dihydroxy-BAs, DCA and CDCA, stimulate water secretion9 and CDCA accelerates colonic transit, increases stool frequency and decreases stool consistency. Increased faecal primary BAs have been demonstrated in IBS-D and have been suggested as a diagnostic biomarker for BAD13.Increased colonic exposure to BAs influences stool volume, colonic transit time and bowel habit18. Only recently have studies including IBS-D patients taken into account the possibility that some will have BAD20. Dysbiosis occurs when imbalances in gut bacteria precipitate disease and has been linked to the changes in metabolism of BAs in the gut21. Evidence suggests that dysbiosis may be secondary to a reduction in bacteria bearing BSH activity22.Data on the faecal or colonic microbial composition in IBS subjects are inconsistent and sometimes contradictory23. We expect patients with IBS-D and BAD to exhibit altered VOC profiles compared to healthy controls (HCs)24, reflecting underlying dysbiosis with altered metabolic output. Furthermore, rapid intestinal transit could reduce the time for bacteria to metabolise and absorb nutrients and their products, in particular, the short-chain fatty acids (SCFAs). The three primary SCFAs produced by the microbiome are acetate, propionate and butyrate and are integral to health, including providing 5–10% of human basal energy requirements26.Volatile organic compounds (VOCs) are the resultant gas by-products of colonic fermentation by gut bacteria, which derive energy through oxidation of organic compounds. Changes in VOC, measured in breath, faeces or urine, indirectly reflect changes of the metabolome. A reduction in the number of faecal VOCs in certain diarrhoeal conditions has been observed, suggesting reduced overall biodiversity of the gut flora and decreased synthesis of compounds, perhaps due to more rapid intestinal transitThe objective of this study was to define mechanisms associated with the pathophysiology of BAD, through characterisation of the colonic microbiome, SCFA, VOC metabolites, and quantification of serum and faecal BAs.In this exploratory mechanistic study, 156 subjects participated, including 62 with BAD, 55 with IBS-D and 39 healthy controls (HC). Different subsets of patients were involved in the various parts of the study; the demographic and clinical characteristics are outlined in Supplemental Table p = 0.01), and Shannon’s diversity box plot (p = 0.014) shown in Fig. 668 operational taxonomic units (OTUs) were identified from the faecal microbiota in patients from the BAD and IBS-D groups. There was statistically significant difference in the alpha-diversity of OTUs with reduced bacterial diversity in BAD compared to IBS-D. This is demonstrated in the rarefaction curve p = 0.0, and Shap values were adjusted for multiple comparisons. The 10 OTUs that were most significantly enriched in BAD  were two unidentified members of the Lachnospiraceae family {OTU_136 and 268}, another from the Ruminococcaceae family {OTU_356}, a member of the Ruminococcus genus {OTU_519}, Bifidobacterium longum {OTU_283}, Prevotella copri {OTU_17 and OTU_127}, Akkermansia muciniphila {OTU_319} and two members of the Bacteroides genus {OTU_72 and OTU_553} . The median water content was higher in the BAD group (75.6%) and IBS-D (71.7%) compared to the HC . The total amount of SCFA varied between individuals, particularly in the IBS-D group, but was not significantly different between the three groups (Table p < 0.05) in the concentration of isocaproic, and in the proportions of isobutyric, valeric and caproic.Faecal water content differed between the three groups  and isocaproic (p < 0.01) in the BAD cohort. The increase in proportion of propionate was less significant (p = 0.12). The proportion of isobutyrate was significantly lower (p = 0.04). Concentrations of caproic and heptanoic were significantly lower, as were their proportions and that of valeric. The proportion of isocaproic was higher. Comparison of the BAD and IBS-D groups gave broadly similar differences to those found in the comparison of BAD and HC, although the significance was usually weaker. There were no significant differences between the IBS-D and HC cohorts.Direct comparison of the BAD and HC groups showed significantly greater concentrations of propionate  with total SCFA in the separate groups, with stronger negative correlations in the BAD group  and between BAD and IBS-D . However, there was a clearly significant separation of VOC profiles between the IBS-D and HC groups .To analyse urinary VOCs, five different classification algorithms were assessed. The Support Vector Machine algorithm produced the best results to predict the probability of a sample being from one of the three groups of HC, IBS-D or BAD. Predictive values comparing pairs of groups are shown in Table −1 (1.33–3.11). Total secondary BA were lower in the BAD group at 0.56 (0–1.34) versus 1.04 μmol L−1 (0.49–1.51), p = 0.22, and were very low or absent in three subjects with severe BAD. The amounts of DCA, LCA, tauro- and sulfo-conjugates were reduced to 40–70% with p values between 0.13 and 0.28.Comparing the BAD and IBS-D patients with known SeHCAT tests, there was considerable individual variability, and total serum BA were similar in both groups: medians 2.08 (IQR 1.13–4.15) and 1.94 μmol Lp = 0.09). The median percentages for total glyco-, tauro- and sulfo-conjugates were all reduced in BAD to around half of the values in IBS-D . The proportions of GCDCA and UDCA were not increased.Individual serum BA when expressed as a proportion of the total showed broadly similar findings. The median percentage of LCA was lower in BAD, at 2.9% vs. 9.8% in IBS-D (p = 0.08). The unconjugated primary BAs were specifically increased , with a similar ninefold difference found for unconjugated cholic acid (p = 0.15), but not for DCA (p = 0.96).Along with the reduction in conjugated BA, there were increases in the proportion of unconjugated (free) BAs, to a median of 55.6% 25.6–72.4) in BAD vs. 21.5% (7.0–55.5) in IBS-D (5.6–72.4 −1 (2.26–6.17), p = 0.01, Table p < 0.05, , sulfated BA , and the sum of all unconjugated BA excluding UDCA .The individual faecal BA composition on a single sample showed considerable variation, but several differences were apparent between the two groups. The median total faecal BA in the BAD group was about twofold higher than in the IBS-D group, at 9.17 (IQR 7.79–14.12) vs. 4.72 µmol gThe percentage proportion of primary BA (%PBA) in the total was double (median 14.3% BAD vs. 7.1% IBS-D), with both cholic acid and CDCA increased . The individual values for %PBA and SeHCAT, shown in Fig. p = 0.19). The percentages of the individual primary BA acids (CA or CDCA) were also negatively associated with SeHCAT, whereas the secondary BA, except UDCA, were positively related  and the Ruminococcus family, (including former C. leptum)8. Reduction in their abundance in BAD will contribute to the increases in faecal primary BAs and ratio of primary/secondary BAs.The dissimilar faecal BA composition of BAD and IBS-D cohorts is are not unexpected given the differences in bacterial diversity observed between the two groups. The increase in unconjugated, primary BAs in BAD in faeces and in serum is likely due to reduced biotransformation in the conversion of primary to secondary BAsClostridia) with HSDH responsible for 7α/β epimerisation were maintained8. We have only limited species-level data to address this. An increase in faecal sulfated BAs was also observed in BAD. The liver is the predominant site of 3-sulfation, which decreases BA toxicity and enhances elimination. In the colon, various bacteria, including Clostridia species, can desulfate BA, allowing reabsorption31. The increase may be a further adaptive change to alleviate BA accumulation in BAD and a protective mechanism in intestinal dysbiosis32.Faecal ursodeoxycholic acid was higher in BAD, which suggest that the several species  of IBS-D patients with higher total BA on a single stool sample (> 90th-centile of controls) and compared them to control and IBS-D subjects with lower BA19. Their cut-off was 10.61 µmol∙g−1 stool, slightly higher than our median of 9.17 µmol g−1. These BA + IBS-D patients had not undergone SeHCAT testing, but had fasting C4 and FGF19 values similar to other cohorts with BAD4. As in our current study, faecal primary BA (CDCA and CA) and UDCA were higher in their BAD group. Faecal bacterial α-diversity did not differ between their two groups although the β-diversity (instability) was higher in BAD. Their taxonomic analysis showed their BAD group had increased Firmicutes, and an abundance of Clostridia, including Ruminococcus species and C. scindens. These were then related to BA production (C4), feedback (FGF19) and to BA-transforming enzymes (BSH and HSDH). It should be noted that their group definition was BA in a single stool sample, rather from 7-day SeHCAT test. Potentially, adaptation of the microbiota and BA synthesis may occur with more prolonged BA loss.Two recently published studies have expanded these findings significantly.20 Principal component analysis of the microbiota was able to separate those with severe BAD (SeHCAT < 5%) from those with less severe BAD or normal SeHCAT. Analysis of the metabolome could separate groups with BAD, identifying glycerophospholipids and oligopeptides among the main predictive metabolites, although BA were less clear.Faecal microbiomes and metabolomes in IBS patients, including a subset with SeHCAT tests, were investigated by Jeffery et al33. . We used a single stool sample, which is more variable and affected by diet, but is more user friendly than a 48 h collection on a defined diet. The Mayo group have recently extended their findings to also use a single stool sample33. We found that predictive values improved, to a diagnostic odds ratio of 11, using 15% PBA to detect moderate BAD (SeHCAT < 10%). However, we demonstrate  study. Scientific and ethical approval was acquired from the local Research and Development Office as well as Warwickshire Ethical committee ref: 09/H1211/38. Written informed consent was obtained from all participants in the study. In line with good medical practice and research governance framework, the study was undertaken in accordance with relevant guidelines and regulations.https://www.bad-uk.org) meeting led by patients. The aims of the study were presented and outline especially of recruitment process was discussed. Specific advice was sought in that regard and changes made to improve recruitment pathway. The study received support from the group and upon publication, results will be disseminated on the charity website and Facebook patient group. Study results were also presented at the UK Bile Acid Diarrhoea Network annual meeting.Patient and public involvement–This study was reviewed and presented at the Bile Acid Diarrhoea charity  or a previous cholecystectomy were only included if 75SeHCAT was < 15% (type 1 and type 3 BAD respectively). Participants were excluded if they suffered from coeliac disease, active IBD , colorectal cancer, antibiotics/probiotics use in the last three months, or recent bile acid sequestrants. HCs had no evidence of chronic disease, were not on any regular medications, pregnant or had taken antibiotics/probiotics in at least three months. Urine, stool and serum samples were collected in standard specimen collection bottles at 9am and stored at − 80 °C within 2 h.Participants with chronic diarrhoea were included in the BAD or IBS-D groups based on 34. To amplify genes for coding and sequencing, polymerase chain reaction (PCR) was used. V3-V4 primers and extensor ready mix (Thermo scientific) were used to amplify the 16S rRNA gene V3-V4 gene fragment from isolated metagenomic DNA using a PCR thermal cycler program. After PCR, the DNA samples were separated and sequenced using the ILLUMINA MISEQ V2 2 × 300 bp paired end protocol.Stool DNA was isolated from 200 mg of stool samples using the QIAamp Fast DNA stool extraction kit , following the manufacturer’s protocol for pathogen detection. Methods were similar to those previously publishedDefault Illumina software trimmed sequences to remove adapter sequences, primers, barcodes, low-quality sequences and those with < 1000 reads. Using a custom Java program, formation of contigs was performed by joining together forward and reverse reads with a quality filtering step to remove contigs that had more than three mismatches. The contigs were de-replicated and then clustered at 97% identity to form operational taxonomic units (OTUs) using the UPARSE pipeline. Singleton contigs from the dataset were discarded and chimeras were removed. The UPARSE pipeline calculated the abundance of each OTU by mapping the de-replicated contigs against the OTUs sequences. Taxonomy was assigned to 16S RNA gene OTU sequences using QIIME  and the RDP classifier. Using the QIIME pipeline, the level of alpha diversity in our samples was determined by generation of rarefied OTU tables, computing measures of alpha diversity for each rarefied OTU table, collating the rarefied OTU tables and then generating rarefaction curves. The depth of rarefaction was defined by either the lowest number or median number of sequences assigned to a sample within a group that was analysed. The Shannon index was used to calculate alpha diversity indexes from rarefied samples. Rarefaction was performed on OTU tables to remove sample heterogeneity.35. Moisture content  was measured the day prior to SCFA analysis with the samples being freeze dried for 24 h in an Edwards apparatus (Freezer Dryer Micro Modulyo). Each sample was measured in duplicate to improve accuracy of results. Data are presented per mass of dry faecal material (µmol g−1) and as the proportion (%) of total SCFAs.Absolute and relative quantification of SCFAs (C2-C8) and branched-chain fatty acids  was undertaken using gas chromatography in diethyl ether extracts as described previously24.The samples were analysed using a commercial field asymmetric ion mobility spectrometry (FAIMS) instrument , fitted with an ATLAS headspace sampling system. This instrument operates at room temperature and pressure, undertaking separation of complex chemical mixtures by measuring ionised molecular movement in high-electric fields. By scanning through a range of electric field strengths and compensation voltages, the instrument is able to produce a mobility map of a sample24.Frozen urine samples were defrosted; 5 ml was aliquoted into a 20 ml vial and placed in the ATLAS sampling system (no cap is fitted), set to 40 °C and left for 10 min to equilibrate and generate an VOC headspace. Clean synthetic air is the passed over the sample and into the Lonestar instrument. The instrument was set to sweep the dispersion field between 0 to 90% in 51 steps, the compensation voltage from − 6 to + 6 V in 512 steps and both positive and negative ion measurement undertaken, resulting in 52,254 data points being generated per sampleSerum and faecal BA profiles were generated using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Faecal samples were prepared by the addition of ammonium carbonate to release the BA from the binding protein, followed by centrifugation and solid-phase extraction using reversed-phase silica Chromabond C18 cartridges  for pre-analysis clean up.An analytical column  with a 5 µm silica particle (RESTEK) fitted on an HPLC binary pump  was used for the chromatographic separation of BAs. Mass spectra were obtained using an API 2000 Q-Trap  equipped with a turbo ion-spray (ESI) source. Analyst software  was used to acquire the data.The total primary BAs is the sum of CA and CDCA and their respective glyco-, tauro- and sulfo-derivatives. The total secondary BAs is the sum of LCA and DCA and their respective glyco-, tauro- and sulfo-derivatives, as well as hyodeoxycholic acid and its tauro-derivate.24. Kruskal–Wallis analyses were used to compare 3 groups and the Mann–Whitney U test was used for two groups. Spearman rank correlations were used to identify associations. P values < 0.05 were considered statistically significant.Data for patient demographics are presented as means and standard deviations (SD). The data for SCFA and BA are presented as medians with interquartile ranges (IQR). VOC data were analysed using a previously developed processing pipelineSupplementary Information.


Limited data suggest serum chloride levels associate with mortality in heart failure, chronic kidney disease (CKD), and pulmonary arterial hypertension. Randomized trials have also shown that administration of crystalloid intravenous fluids with lower chloride concentration may have better renal outcomes. However, chloride has not been studied longitudinally for CKD progression.We used a prospective cohort of subjects with stage 3 and 4 CKD recruited from a nephrology clinic at a single medical center. Linear regression, linear regression with generalized estimating equations, and Cox proportional hazards models were created for outcomes of overall change in estimated glomerular filtration rate (eGFR), longitudinal changes in eGFR, and time to > 30% decline in eGFR, respectively. Baseline chloride was modeled continuously and categorically, and models were adjusted for potential confounders.2. In multivariable analysis, higher serum chloride associated with worsened eGFR decline. Every 1 mEq/L increase in chloride associated with an overall eGFR decline of 0.32 mL/min/1.73m2 (p = 0.003), while the difference in eGFR decline in the highest quartile of chloride was 3.4 mL/min/1.73m2 compared to the lowest quartile (p = 0.004). No association between serum chloride and time to 30% decline in eGFR was observed in multivariable analysis .Median follow-up was 1.7 years. Baseline median age was 72 years and median eGFR was 35.7 mL/min/1.73mIn CKD patients, higher serum chloride associated with a modestly steeper rate of eGFR decline, and may be a useful biomarker to predict CKD progression. Further studies are needed to determine causality. Chronic kidney disease (CKD) is highly prevalent globally  and assoSerum chloride is a frequently neglected laboratory value typically only considered during states of metabolic acidosis. However, it has become increasingly clear that chloride is an important driver of numerous homeostatic mechanisms including regulation of renin secretion, tubuloglomerular feedback, blood pressure response, and renal sodium handling –7.Clinically, serum chloride has been gaining traction as a predictor of adverse outcomes in various clinical settings. In two observational studies of patients with acute decompensated heart failure, low serum chloride associated with diuretic resistance and increased mortality , 9. In aIn this study, we used a prospective cohort of mostly CKD stage 3 and 4 patients to determine if there was an association between serum chloride levels and CKD progression. Given recent data that higher concentration of chloride in intravenous fluids were detrimental to short term kidney outcomes, we hypothesized that higher serum chloride levels at baseline would associate with increased risk of long-term CKD progression.N = 1340). Of these, 717 patients were excluded either because 1) there was no actual in-person visit, 2) requisite laboratory data were not available, incomplete, or were outside the study enrollment window, 3) there were multiple ICD-9 codes present with at least one of them being for stage 5 CKD or end stage renal disease, 4) the MDRD eGFR did not place them into CKD stage 3 or 4, 5) they were kidney transplant recipients, or 6) they had incomplete data sets. There were 623 participants ultimately enrolled in the cohort. For this study, participants that only had one visit were eliminated (N = 32), thus the sample size was 591. The institutional review board of New York University Winthrop Hospital approved this study.Participants were enrolled into this observational, prospective, clinic-based cohort study using the electronic health records (EHR) of the patients registered for a visit in the outpatient general nephrology clinic of New York University Winthrop Hospital within the two-year period of October 1, 2011 to September 30, 2013 , medical comorbidities , and smoking history (never smoked or current/former smoker) were collected at baseline through medical chart review. Insurance status was classified as Medicare, private insurance, or Medicaid/self-insured. Smoking status was classified as current, former, or never smoker based on self-report. Medications including angiotensin converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and diuretics (either loop or thiazide) were recorded at baseline. Laboratory data including serum creatinine, chloride, bicarbonate, phosphate, albumin, calcium, and urine albumin-to-creatinine ratio (ACR) were measured at baseline, with additional data obtained at multiple follow-up visits at the discretion of each patient’s treating physicians. Laboratory tests were run at either NYU Winthrop or an accredited commercial laboratory. The regression equation developed by vailable . Enrollmvailable , but wasvailable , 21, whiThere were three primary outcomes: 1) time to > 30% decline in eGFR; 2) change in eGFR from beginning to the end of the study; 3) change in eGFR longitudinally. Participants were censored for death, dialysis initiation, or kidney transplantation.The distribution of baseline characteristics was summarized within each quartile of baseline serum chloride using medians with interquartile ranges (IQRs) for continuous variables and frequencies for levels of categorical variables. Spearman’s rank correlation and Cochran-Armitage trend tests were used to test for increasing and decreasing trends across quartiles for values of continuous variables and frequencies of categorical variables, respectively. Kaplan-Meier (KM) survival curves for the time to eGFR decline of at least 30% from baseline were estimated for each quartile of baseline chloride to visualize differences in survival distributions. We presented KM estimates for survival times where at least 10 subjects remained in all four quartile groups. The survival distributions between quartiles were also compared with a logrank test.We examined the association between serum chloride and CKD progression in three ways: 1) Cox proportional hazards models for the time to first decline of 30% or greater in eGFR from baseline; 2) linear regression models for overall change in eGFR from the baseline visit to the last follow-up visit; and 3) generalized estimated equations (GEE) for eGFR change between visits. For Cox and linear regression models, baseline chloride was modeled both continuously and categorically as quartiles. We also examined the effect of chloride in unadjusted, demographic-adjusted , and fully-adjusted  models, where covariates were all measured at baseline. Linear models additionally controlled for study length for all models. Finally, we tested whether there were linear trends between chloride quartile groups and the responses of interest by modelling quartile group as an ordinal variable.We examined the effect of chloride on eGFR changes between visits in unadjusted, demographic-adjusted and fully-adjusted models as well, but used chloride as a continuous variable only. For each visit, the covariates were those belonging to that visit, and the outcome was eGFR at the next visit. For all models, including unadjusted models, we additionally controlled for the time difference between visits, as well as eGFR of the previous visit. Thus, our models measured the effect of chloride on the rate of eGFR change by the next visit. Covariates that were not laboratory variables, including demographics, medications and comorbidities, were only measured at baseline. For the purposes of this analysis, we assumed that these variables remained the same as baseline for each visit. We used an independent working correlation structure with sandwich estimates for the standard errors. Exchangeable and autoregressive correlation structures did not lead to an improvement in model fit as measured by the quasi-Akaike Information Criterion (QIC), and covariance estimates for visits within the same subject were very close to zero and statistically insignificant. For all GEE, linear and Cox models, we tested for interactions between chloride and bicarbonate as well as between chloride and diuretic usage.p-values less than 0.05 were considered statistically significant. All analyses except for drawing the survival curves and at-risk table, including generating the KM estimates and the number of persons at risk at each time point, were done in SAS version 9.4 . KM curves and the at-risk table under them were displayed using R version 3.5.1  with the help of the following packages: ggplot2 v3.1.1 [Two-sided 2 v3.1.1 , gridExt2 v3.1.1  and cowp2 v3.1.1 .2. The median change in eGFR from baseline to the last follow-up visit was − 2.5 mL/min/1.73m2 (IQR -7.1 – 2.4 mL/min/1.73m2), with a median of 4 data points (IQR 3–5) for each patient. Baseline characteristics stratified by quartiles of chloride are shown in Table The final sample size included 591 participants with a median follow-up of 1.7 years (IQR 1–2.4 years) and mean follow-up of 1.7 years (SD 1.0 years). The median number of data points per participant during the entire study was 4 (IQR 3–5), and the median frequency of data points was 2.6 per year. At baseline, median age was 72 years, 62% were male, 53% were diabetic, 92% were hypertensive, and median eGFR was 35.7 mL/min/1.73mp = 0.013). A total of 121 participants (20%) reached this threshold of decline by the end of follow-up. While there was a significant association between hazard of > 30% eGFR decline and continuous serum chloride in unadjusted  and demographic-adjusted models , this result became insignificant in fully-adjusted models . Similarly, there was no significant association between quartiles of serum chloride at baseline and > 30% decline in eGFR in fully-adjusted models  -0.538 to − 0.109 mL/min/1.73m2, p-value = 0.003) overall reduction in eGFR  compared to the first (lowest) quartile. In fully adjusted GEE models based on repeated eGFR measurements, we found that a 1 mEq/L increase in serum chloride was significantly associated with a 0.208 mL/min/1.73m2 reduction  in eGFR at the next visit . Finally, interactions between serum chloride and diuretic use and serum bicarbonate level were insignificant for all models.When tested categorically, the association with eGFR decline was stronger with higher quartiles of chloride, with the fourth quartile of chloride associated with an overall eGFR decline of 3.378 mL/min/1.73mIn this observational, prospective cohort we found that higher serum chloride associated with modestly worsened eGFR decline during the course of 1.7 years of median follow-up, independent of other well-established risk factors for CKD progression. Serum chloride was associated with > 30% decline in eGFR in demographic adjusted models, but the results were attenuated and insignificant in fully adjusted models. There was no clear threshold effect, with continuous models of serum chloride demonstrating an association with eGFR decline for every 1 mEq/L increase.Our study adds to the growing body of evidence implicating a role for chloride in human disease. Several recent observational studies have focused on heart failure. In one analysis of patients admitted for acute decompensated heart failure, lower levels of serum chloride were independently associated with long-term mortality . In anot2 vs 36.3 mL/min/1.73m2, in the highest versus the lower quartile of serum chloride, respectively), which correlates with our findings, although this study did not examined changes in eGFR longitudinally [While these observational studies yielded adverse outcomes associated with hypochloremia, ours showed possible worsened outcomes with higher serum chloride levels. This discordance may be due to differences in study design and outcomes, as prior studies were conducted in different populations where there may be more hemodynamic and volume issues, and a stronger role for diuretics which may have impacted chloride levels and outcomes. These studies also did not specifically focus on kidney function, where chloride has important physiologic roles. For instance, previous observational studies found associations between hyperchloremia, chloride-liberal intravenous fluids, and acute kidney injury (AKI) , 24. Theudinally .Whether or not a possible association seen in this study between chloride and long-term change in eGFR is causal or translates into meaningful clinical outcomes requires further investigation and replication, however there are possible physiologic mechanisms that may link these findings. For instance, plasma chloride, not sodium, experimentally reduced renal blood flow in denervated canine kidneys . In the Another possible explanation for the observed association involves metabolic acidosis, since higher serum chloride levels are often seen concurrently with metabolic acidosis, which is associated with increased risk of CKD progression , 31. MetThere are several strengths to this study including rigorous collection of data, a cohort comprised of a real-world population with chronic kidney disease, meticulous follow-up, several statistical models each adjusted for a broad range of well-established CKD risk factors, and several data points on average for each participant to limit chance fluctuations in eGFR. However, our study also has limitations. First, the median follow-up was only 1.7 years with an overall relatively small change in eGFR, which may have reduced the power to detect > 30% decline in eGFR. Power was not calculated a priori to conducting this analysis. Our study was also too short to determine hard outcomes including end stage renal disease. However, two different methods to test the association with continuous eGFR change both yielded similar results, making the findings more robust. Second, participants were all from a nephrology clinic with established CKD, which may limit generalizability to the general population either with or without CKD. The study population also was skewed older and towards whites with Medicare and private insurance. Finally, causality cannot be determined from an observational study and we cannot exclude the possibility that our findings may be related to unmeasured bias and confounding. It is entirely possible that associations between serum chloride and long-term changes in eGFR are hemodynamic, and may not reflect underlying kidney disease progression. However, we adjusted for diuretics, ACE inhibitors/ARBs, and heart disease in our final models, which are most likely to cause hemodynamic changes, and there was still a significant association between serum chloride and change in eGFR.In conclusion, in a prospective cohort of men and women with CKD, we demonstrated that higher serum chloride is significantly associated with a modest decrease in eGFR independent of other well-established CKD risk factors. Future studies should examine and confirm this relationship over longer periods of follow-up, and in more diverse populations including those without preexisting CKD. Further research is also needed to determine causality, and possible underlying pathophysiology.Additional file 1: Table S1. Baseline Characteristics. Results are presented as mean (standard deviation).


The human gut microbiome influences depression. Differences between the microbiomes of HIV-infected and uninfected individuals have been described, but it is not known whether these are due to HIV itself, or to common HIV comorbidities such as HCV coinfection. Limited research has explored the influence of the microbiome on depression within these groups. Here, we characterized the microbial community and metabolome in the stools from 373 people, noting the presence of current or lifetime depression as well as their HIV and HCV infection status. Our findings provide additional evidence that individuals with HIV have different microbiomes which are further altered by HCV coinfection. In individuals coinfected with both HIV and HCV, we identified microbes and molecules that were associated with depression. These results suggest that the interplay of HIV and HCV and the gut microbiome may contribute to the HIV-associated neuropsychiatric problems. Depression is influenced by the structure, diversity, and composition of the gut microbiome. Although depression has been described previously in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) monoinfections, and to a lesser extent in HIV-HCV coinfection, research on the interplay between depression and the gut microbiome in these disease states is limited. Here, we characterized the gut microbiome using 16S rRNA amplicon sequencing of fecal samples from 373 participants who underwent a comprehensive neuropsychiatric assessment and the gut metabolome on a subset of these participants using untargeted metabolomics with liquid chromatography-mass spectrometry. We observed that the gut microbiome and metabolome were distinct between HIV-positive and -negative individuals. HCV infection had a large association with the microbiome that was not confounded by drug use. Therefore, we classified the participants by HIV and HCV infection status . The three groups significantly differed in their gut microbiome (unweighted UniFrac distances) and metabolome (Bray-Curtis distances). Coinfected individuals also had lower alpha diversity. Within each of the three groups, we evaluated lifetime major depressive disorder (MDD) and current Beck Depression Inventory-II. We found that the gut microbiome differed between depression states only in coinfected individuals. Coinfected individuals with a lifetime history of MDD were enriched in primary and secondary bile acids, as well as taxa previously identified in people with MDD. Collectively, we observe persistent signatures associated with depression only in coinfected individuals, suggesting that HCV itself, or interactions between HCV and HIV, may drive HIV-related neuropsychiatric differences.IMPORTANCE The human gut microbiome influences depression. Differences between the microbiomes of HIV-infected and uninfected individuals have been described, but it is not known whether these are due to HIV itself, or to common HIV comorbidities such as HCV coinfection. Limited research has explored the influence of the microbiome on depression within these groups. Here, we characterized the microbial community and metabolome in the stools from 373 people, noting the presence of current or lifetime depression as well as their HIV and HCV infection status. Our findings provide additional evidence that individuals with HIV have different microbiomes which are further altered by HCV coinfection. In individuals coinfected with both HIV and HCV, we identified microbes and molecules that were associated with depression. These results suggest that the interplay of HIV and HCV and the gut microbiome may contribute to the HIV-associated neuropsychiatric problems. Deple–ky gut”) , 5, whicky gut”) . Leaky gky gut”) , 5, 7, 8ky gut”) , 9–11. Gteriales , enrichmbacteria , depletiteroidia  and incrichaceae . Some ofvotella) –18. Togeotella) –, 20.Bacteroides fragilis or Bacteroides thetaiotaomicron polysaccharide A [PSA]  , butyrat A [PSA] , and try A [PSA] ) with thBacteroidetes and Firmicutes compared to healthy subjects  expression in the ventral hippocampus and higher depression-like behaviors relative to rats receiving microbiota from rats resistant to social stress, suggesting that the gut microbiome contributes to the depression-like behavior and inflammatory processes in the brain . In HIV-Bacteroidetes, Proteobacteria, and Actinobacteria and reduced Firmicutes  and -negative individuals (n = 106) (unweighted UniFrac distances [–P = 0.001). However, we found that alpha diversity  did not differ between HIV-positive and -negative individuals. There was also a significant difference in the gut metabolome between HIV-positive and -negative individuals .We first evaluated the gut microbiome and metabolome in the context of HIV infection status. As in previous studies –51, we f–istances –54: permTo characterize the impact of covariates on the microbiome, we performed regularized discriminant analysis (RDA)  to calcuDue to the large relative effect size of HCV status, we classified the participants by the presence or absence of both HIV and HCV infection  with both HIV and HCV, “HIV monoinfected” (n = 219) with HIV but not HCV, and “uninfected” individuals (n = 106) with neither virus. A subset of these participants  were additionally assessed using untargeted metabolomics by liquid chromatography-mass spectrometry.To explore the relationship between the gut microbiome and depression in people with HIV monoinfection, HIV and HCV coinfection, or neither, we analyzed 16S rRNA gene amplicon sequencing data from a total of 571 fecal samples , 398 of 2 = 76.9, P < 0.0001) than the other infection groups, but the three groups were similar in terms of age. The uninfected group had a higher estimated verbal IQ than the coinfected group. The uninfected group also had lower current depressive symptoms and fewer problems with activities of daily living than the HIV-monoinfected and coinfected groups and a higher rate of employment than the coinfected group. Lifetime substance use disorders were lower in the uninfected group, while lifetime major depression showed a stair step pattern with the uninfected group at 33%, the HIV-monoinfected group at 52%, and the coinfected group at 71% . In terms of HIV disease, the HIV-monoinfected and coinfected groups did not differ by AIDS status, current or nadir CD4, or plasma viral load detectability. While the coinfected group had been HIV positive an average of 4 years longer than the HIV-monoinfected group, they were less likely to be on ART at their study visit . The coinfected group was also more likely to be composed of minorities , but in all other respects (including history of substance use disorders), the individuals in the coinfected group were comparable to the individuals in the HIV-monoinfected group.Sample characteristics of each infection group are included in 56–n = 7; HIV monoinfected, n = 39; uninfected, n = 34) completed a dietary intake survey .A subset of participants with a history of MDD were taking antidepressants at the time of evaluation, which may have the capacity to alter gut microbiome and metabolome composition. Of the participants with a lifetime history of MDD, 26% of the coinfected, 34% of the HIV monoinfected, and 15% of the uninfected were taking antidepressants, which included selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs) . In addi10.1128/mSystems.00465-20.1TABLE S1P < 0.05) and in which direction. Download Table S1, CSV file, 0.00 MB.Dietary intake comparison using chi-square tests between the full infection groups. The final column indicates if there is a significant difference between groups  are known to have nfection –66. To a2 = 7.7, P = 0.02): in the coinfected subgroup, 24% reported sex with men and women and 76% reported sex with only men; for the HIV-monoinfected subgroup, 10% reported sex with men and women and 90% reported sex with only men. In the uninfected subgroup, 20% reported sex with men and women and 80% reported sex with only men. As in the full group, the coinfected individuals in the MSM subgroup had longer estimated duration of HIV infection and a smaller percentage were on antiretroviral therapy (ART) in comparison to the HIV-monoinfected MSM subgroup. Additional cohort descriptors are included in When limited to MSMs, the uninfected group had somewhat higher education levels than the coinfected group, and higher premorbid IQ estimates than both infected groups; otherwise, demographic characteristics did not differ between the three subgroups . Coinfected individuals also had lower alpha diversity than uninfected individuals (P = 0.0006). Coinfected and uninfected individuals were also significantly different in their overall gut metabolome (P = 0.002). However, between coinfected and uninfected MSM subgroups, there were no differences in the overall composition of the gut microbiome and metabolome .First, we compared coinfected to uninfected groups. In the full cohort, we observed a statistically significant difference in the overall gut microbial communities in unweighted UniFrac beta diversity distances between coinfected and uninfected individuals (P = 0.001), and in their overall gut metabolome (P = 0.002). Unlike coinfected individuals, however, there was no difference in alpha diversity between the HIV-monoinfected and uninfected groups . Between the MSM subgroups of HIV-monoinfected and uninfected groups, there were no differences in the overall composition of the gut microbiome and metabolome .Next, we compared HIV-monoinfected to uninfected groups. HIV-monoinfected individuals were also significantly different from uninfected individuals in unweighted UniFrac beta diversity distances (P = 0.001). Coinfected individuals also had lower alpha diversity than HIV-monoinfected individuals (P = 0.0006). Furthermore, coinfected and HIV-monoinfected individuals were significantly different in their overall gut metabolome (P = 0.004). In the MSM subgroups, the unweighted UniFrac beta diversity distances between the coinfected and HIV-monoinfected subgroups remained statistically significantly different (P = 0.05). Again, the coinfected individuals had a lower alpha diversity than HIV-monoinfected individuals (P = 0.04). The differences in the overall gut metabolomes of the coinfected and HIV-monoinfected individuals also remained significant in the MSM cohort (p = 0.03).Finally, we compared coinfected to HIV-monoinfected groups. In the full cohorts, we observed a statistically significant difference in unweighted UniFrac beta diversity distances between coinfected and HIV-monoinfected individuals  in any of the infection groups  diversity correlations with continuous BDI-II and continuous biomarkers in the infection groups and MSM subgroups. Download Copyright © 2020 Taylor et al.2020Taylor et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Elevated levels of the proinflammatory cytokine interleukin-6 (IL-6), even in the context of viral suppression on ART, are associated with adverse outcomes such as myocardial infarction and death –72. Ther–Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV)  using (DSM-IV)  criteriaWe first tested for association between the gut microbiome and BDI-II in any of the three groups. Individuals were considered currently depressed if they reported at least mild depressive symptoms; otherwise they were considered not depressed. In no infection cohort was there a significant difference in alpha or beta diversity between individuals stratified by current depressive symptoms . Consist10.1128/mSystems.00465-20.3TABLE S3Table S3, CSV file, 0.00 MB.Alpha and beta diversity between categorical BDI-II and lifetime (LT) MDD groups in all infection groups. Beta (unweighted UniFrac) and alpha (Shannon) diversity in the 16S data, and beta (Bray-Curtis) diversity in the metabolomics data. Download Copyright © 2020 Taylor et al.2020Taylor et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.044). We found no significant differences in the HIV-monoinfected full group or MSM subgroup in unweighted UniFrac distances or Shannon diversity between MDD states  in an individual’s life would be associated with gut microbiome differences, separately within the three infection groups. Only in the full coinfected group did we observe a statistically significant difference between those who met lifetime diagnostic criteria for MDD versus those who did not . Multiple compounds of interest were found to be annotated as bile acids. Further analysis of all annotated bile acids revealed that in both the full coinfected group and the coinfected MSM subgroup, a cluster of primary and secondary bile acids were significantly increased  in individuals with a lifetime history of MDD  for the bile acid networks shown in all figures. The annotation indicates the closest spectral match found in the GNPS libraries for the feature. Download Copyright © 2020 Taylor et al.2020Taylor et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00465-20.8TABLE S8Table S8, CSV file, 0.00 MB.Sets of taxa identified using Songbird and used in log ratio calculations. Download Copyright © 2020 Taylor et al.2020Taylor et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bile acids and the gut microbiome exist in a dynamic equilibrium . PrimaryBile acid imbalances are known to be associated with pathological states such as liver disease, gastrointestinal cancers, and gallstones . Shifts t test, P = 9.055e−06, t = −5.210, df = 34.183, Cohen’s D = 1.434), suggesting that they were associated with set 1. Several microbes that were associated with coinfected individuals with a lifetime history of MDD    , 85, 86,teroides , 85, andtasonis) . Likewistasonis)  that werter spp. , 87, Laccus spp. .This is to our knowledge the first study of the association between infection with HIV and HCV, depression, and the gut microbiome and metabolome. We performed 16S rRNA sequencing and liquid chromatography-mass spectrometry using stool samples from nearly 400 individuals and evaluated the data with state-of-the-art tools. We observed that although the gut microbiome of HIV-positive and -negative individuals differed, HCV had a large effect on the microbiome which warranted consideration in our study. The infection groups differed from each other in terms of both alpha and beta diversity in the full cohort as well as the MSM subgroups. Furthermore, we found that depression was associated with differences in the gut microbiome and metabolome only in HIV-HCV coinfected individuals. Coinfected individuals with a lifetime history of MDD were enriched in primary and secondary bile acids, as well as particular depression-related taxa. Importantly, our results suggest that microbiome and metabolome investigations in HIV-infected cohorts should carefully consider possible effects of HCV coinfections, which are not uncommon among people living with HIV.Bacteroides, which we found to be associated with coinfected individuals with a history of MDD, can be decreased in the human gut by reducing consumption of animal protein and/or increasing carbohydrate intake . e intake . We alsoed foods , again shttps://hnrp.hivresearch.ucsd.edu/) including the HIV Neurobehavioral Research Center (HNRC) at the University of California San Diego (UCSD). Study details can be found in references This was a cross-sectional prospective observational cohort study of persons with or without HIV infection recruited from community sources, who agreed to undergo comprehensive neuromedical and neurobehavioral evaluations for NIH-funded studies at the HIV Neurobehavioral Research Program . HIV and HCV infections were confirmed by a point-of-care vertical flow test . Participants were designated as follows: (i) “HIV monoinfected” if they tested positive for HIV but not HCV, (ii) “coinfected” if they tested positive for both HIV and HCV, or (iii) “uninfected” if they tested positive for neither HIV or HCV. Group characteristics were compared using + T cells (flow cytometry) were performed at a Clinical Laboratory Improvement Amendments (CLIA)-certified medical center laboratory. HIV RNA was measured in plasma using reverse transcriptase PCR  with a lower limit of quantitation of 40 copies/ml.All participants underwent a comprehensive neuromedical assessment, including a medical history that collected antiretroviral therapy (ART) and other medications, data to determine Centers for Disease Control (CDC) HIV disease staging, and specimen collection . Routine clinical chemistry panels, complete blood counts, rapid plasma reagin, and CD4DSM-IV diagnosis of lifetime major depressive disorder was evaluated using the computer-assisted Composite International Diagnostic Interview (CIDI) , a struchttp://www.earthmicrobiome.org/protocols-and-standards/16s). DNA was extracted with the Qiagen MagAttract PowerSoil DNA kit as previously described (DNA extraction and 16S rRNA amplicon sequencing were done using Earth Microbiome Project (EMP) standard protocols (escribed . Amplicot tests in QIIME 2. Alpha diversity (Shannon diversity [QIIME 2 v2020.2  was usediversity ) was comt tests and Cohen’s D were calculated to assess the significance  and effect size of the log ratios.Songbird v1.0.1  in QIIMEMetabolomics sample processing and data acquisition protocols followed the standard Center for Microbiome Innovation’s seed grant project protocol to allow for comparison of this data set to many reference data sets and standards. Human fecal samples were transferred to clean 2-ml sample tubes , and the weights were recorded. The samples were then extracted in a solution of 1:1 methanol to water spiked with an internal standard of 1 μM sulfamethazine, using a 1:10 sample weight (in milligrams) to solvent volume (microliter) ratio. Using a Tissuelyser II (Qiagen), the samples were homogenized for 5 min at 25 Hz. This was followed by a 15-min centrifugation at 14,000 rpm. From the supernatant, 400 μl was transferred to a prelabeled 96-Well DeepWell plate, and the plates were concentrated using a CentriVap Benchtop Vacuum Concentrator (Labconco) for approximately 4 h. The dry plates were placed into a −80°C freezer until time for analysis.18; 1.7 μm, 2.1 mm × 50 mm) was used, as this column has demonstrated robust separation of a large variety of the compounds within the parameters used. The mobile phase consisted of solvent A (100% LC-MS grade water with 0.1% formic acid) and solvent B (100% acetonitrile with 0.1% formic acid). Each sample was injected at a volume of 5 μl into a flow rate of 0.5 ml for the entire analysis. The 12-min chromatographic gradient began at 5% solvent B for the first minute, an increase to 100% solvent B from min 1 to min 11, a hold at 100% B until min 11.5, and back down to 5% solvent B reached at min 11.5. All data were collected using electrospray ionization in positive mode. Positive mode was selected in order to allow for spectral matches to be found using the GNPS spectral libraries, a majority of which were collected in positive ionization mode. Data-dependent acquisition was set to a scan range of 100 to 2,000 m/z.The plates were resuspended in 150 μl of a 1:1 methanol-to-water solution with a 1 μM sulfadimethoxine internal standard solution. For metabolomics analysis, an ultrahigh performance liquid chromatography system (Thermo Dionex Ultimate 3000 UHPLC) coupled to an ultrahigh resolution quadrupole time of flight (qToF) mass spectrometer . For chromatographic separation, a Phenomenex Kinetex column (Chttps://massive.ucsd.edu/ProteoSAFe/static/massive.jsp). Feature detection was completed using MZmine version 2.37 software (The raw data in Bruker (.d) format were lock mass corrected using hexakis phosphazene  and were exported as .mzXML files using the Bruker Data Analysis software. Both the raw.d and the.mzXML files were uploaded to the UC San Diego mass spectrometry data repository MassIVE  including matches to drug and drug metabolite standards, bile acids, food-related compounds, and dipeptide molecules. The results reflect MSI level 2 or 3 annotations , and sequence data associated with this study have been deposited at EBI/ENA under accession number ERP122366. The raw experimental data are available at MassIVE (https://massive.ucsd.edu/), data set MSV000083664.The data generated in this study are available publicly in Qiita under the study ID 11135 (


Fibrin formation and histidine-rich glycoprotein (HRG) are involved in primary hemostasis and wound healing. Little is known regarding the relationship of clot characteristics, bleeding time, and wound healing. Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower p < 0.001). After adjusting for potential confounders, Ks), clot lysis time (CLT), and histidine-rich glycoprotein (HRG). Compared with controls, BT was 45% shorter in CAD cases. CAD patients had 32% lower n = 79, 25.6%) was independently predicted by both short and prolonged BT in CAD cases  and controls .  We studied 154 patients with coronary artery disease (CAD) and 154 subjects free of CAD matched for age, obesity, and current smoking. We evaluated bleeding time (BT) using standardized skin incisions on a forearm, along with plasma clot permeability (Translational Perspective. Elucidation of the complex relationships between plasma fibrin clot phenotype and wound healing might have important practical implications. The study shows that plasma fibrin clot density and HRG may influence BT and that appropriate skin wound healing is associated with medium BT.  Fibrin formation is the final step of blood coagulation involved in hemostasis following vascular injury and the maintenance of vessel patency under pathological conditions. There is evidence that plasma fibrin clots composed of compact networks that are less susceptible to lysis, the so-called “prothrombotic clot phenotype,” can be observed in patients with myocardial infarction (MI), ischemic stroke, and peripheral artery disease , 2. UnfaμM concentration in human plasma [Fibrin is essential for wound healing as it provides a scaffold for macrophages and fibroblasts migrating into the wound . Fibrin n plasma . HRG binn plasma –19.Standardized skin incisions performed with a single-use device  have been long employed to measure BT in patients suspected of bleeding disorders , 21. BloTo our knowledge, there have been no studies exploring plasma clot features in relation to BT and skin wound healing in healthy subjects and patients with coronary artery disease (CAD) known to form denser fibrin clot structures , 7, 9. WWe assessed 154 consecutive patients (cases) with stable CAD (Canadian Cardiovascular Society classes II or III) and, as controls, 154 unrelated subjects free of CAD, who were recruited from the hospital personnel or family members. All study participants were enrolled between 1999 and 2007 and were partly described previously . Patient2. Diabetes was defined as a history of diabetes, the use of hypoglycemic agents, or fasting plasma glucose ≥126 mg/dL (7 mM) on two separate occasions. Arterial hypertension was defined as a systolic and/or a diastolic blood pressure ≥ 140 mmHg or ≥90 mmHg, respectively, or antihypertensive therapy. Heart failure was defined according to the European guidelines [Obesity was defined as body mass index (BMI) ≥ 30 kg/mThe study was performed in accordance with the Declaration of Helsinki and its protocol was approved by the Bioethical Committee of the Jagiellonian University. All subjects enrolled provided written informed consent.Fasting blood was drawn from an antecubital vein with minimal stasis between 8 and11 AM. Lipid profiles, blood cell counts, glucose, creatinine, D-dimer, international normalized ratio (INR), and activated partial thromboplastin time (APTT) were assayed by routine laboratory techniques. Factor VIII activity (FVIII) was measured by one-stage clotting assay using factor-deficient plasma . Fibrinogen was determined using the von Clauss method. High-sensitivity CRP was measured by nephelometry . Fasting plasma tHcy levels were measured using HPLC. Histidine-rich glycoprotein (HRG) and VEGF levels were measured using ELISAs . The two parameters were assessed in once thawed samples frozen at -80 degrees Celsius. The interassay and intra-assay coefficients of variation for the ELISAs were <8%.BT assessment was performed by 2 experienced investigators as described previously . In briePlasma samples (9 : 1 of 3.2% trisodium citrate) for fibrin assays were centrifuged  within 30 minutes of collection to obtain platelet-poor plasma, immediately frozen, and stored in aliquots at -80°C. All measurements were performed by technicians blinded to the sample status in the material stored at -80 degrees Celsius and thawed prior to analysis.Ks), indicating the pore size, was calculated from the equation: Ks = Q × L × η/t × A × Δp, where Qis the flow rate in time t, L is the length of a fibrin gel, η is the viscosity of liquid (in poise), t is percolating time, A is the cross-sectional area (in cm2), and Δp is a differential pressure (in dyne/cm2). The intraindividual variability of results was 8%.Permeation of plasma fibrin clots was determined as described . BrieflyμM phospholipid vesicles , and 60 ng/ml recombinant t-PA . The mixture was transferred to a microtiter plate and its turbidity was measured at 405 nm at 37°C. Clot lysis time was defined as the time from the midpoint of the clear-to-maximum-turbid transition, which represents clot formation, to the midpoint of the maximum-turbid-to-clear transition. The coefficients of intra- and interassay variations were 6%.CLT was measured as described , 29. BriFormation of the scar at the site of prior BT test was assessed macroscopically by the independent investigators two weeks after BT measurement. The scar was defined as a linear mark at the site of skin incision different in colour from the surrounding skin and >1 mm wide. The interobserver variability was estimated at 10%, based on evaluation of 50 randomly selected patients performed independently by 2 investigators.All methods were performed in accordance with the relevant guidelines and regulations.Ks using a p value of 0.05. To demonstrate such a difference or greater in Ks, at least 31 patients were required in each group [The study was powered to have a 90% chance of detecting a 10% difference in χ2 exact test. Continuous variables were tested for the normality of the distribution by the Shapiro-Wilk W test. Those normally distributed are given as the mean ± standard deviation and otherwise as median (interquartile range). Comparisons were performed using unpaired (Student's) t-test or Mann–Whitney U test, respectively. Associations between continuous variables were calculated using Spearman's rank correlation coefficient. Uni- and multivariate logistic regression models were used to determine predictors of scar formation. We determined predictors of long and short BT, defined as the upper and the lower BT quartile, respectively, along with predictors of a very long BT with its 90th percentile as a cutoff value. Multivariate analyses adjusted for age, diabetes, ASA use, and statin treatment. Models for BT prediction involved also current smoking, platelet count, FVIII, CLT, Ks, and HRG. A p value of less than 0.05 was considered significant. Statistical analyses were conducted using the Statistica 64 v.13  or MedCalc 64-bit v.13.1.2.0  program.Categorical variables were compared using The CAD group had a lower proportion of females and a higher prevalence of hypertension, diabetes, higher BMI, total cholesterol, and LDL cholesterol as well as a more frequent statin use, compared with controls . FibrinoR = −0.54, p < 0.001). As expected, BT was longer in CAD patients treated by ASA , while those on statins had shorter BT .BT was 45% shorter in CAD patients than controls . An inveR = −0.30, p < 0.001 and R = −0.52, p < 0.001, respectively) and platelet count . There was an inverse weak correlation between BT and FVIII  in controls who all had FVIII above 80%.In the CAD and control groups, there were inverse correlations between BT and fibrinogen  and fibrinogen  in controls. In both CAD patients and controls, HRG correlated negatively with platelet count  and CRP . Interestingly, there was a positive correlation of HRG with BT  in both groups. There was no difference in HRG and VEGF between subjects whose plasma samples were collected within the first 2 years and those from the remaining years (data not shown).Plasma HRG levels were 50% lower in CAD patients than in controls . HRG invR = 0.44, p < 0.001) and fibrinogen  only in controls. We observed inverse correlation between VEGF and BT  in controls, but not in CAD patients.VEGF levels were 147% higher in CAD patients compared with controls . VEGF coKs) and more resistant to fibrinolysis (17% longer CLT) in CAD patients compared with controls . Of note, positive correlations between Ks and BT were observed in both CAD  in controls but not in CAD patients. HRG or VEGF showed no associations with CLT in either group. The duration of storage at -80 degrees Celsius showed association with neither Ks nor CLT (data not shown).As expected, plasma fibrin clots were denser  was associated in CAD patients with statin treatment as well as platelets, uartiles . In contte model .Ks together with 36% higher homocysteine and 41% higher CRP  were observed in controls who developed scars following skin incisions (Supplementary Scar formation at sites of BT measurement was observed in 41 (26.6%) CAD patients and 38 (24.7%) controls. CAD patients with scars were more frequently affected by diabetes and treated with ASA or statins and had shorter BT (by 18%) compared with the remainder (Supplementary th percentile) BT  or mostly (controls) in subjects with a short (in the lower quartile) or a very long (above 90tile) BT . Not surtile) BT . Multivatile) BT . A long Ks is an independent predictor of longer BT in both studied groups suggesting that fibrin network density affects bleeding time and has the impact on the primary hemostasis. Another novel finding is that elevated HRG in circulating blood correlates with longer BT in humans, which suggests that this abundant protein may affect primary hemostasis at least in part through its impact on fibrin clot structure. We showed an interesting U-shaped relationship between BT and scar formation following skin incisions, suggesting that appropriate skin wound healing is associated with medium BT. The current study expands our knowledge on the role of fibrin clot properties in primary hemostasis and wound healing in healthy subjects and cardiovascular patients, independently of fibrinogen and platelet count.To our knowledge, this study is the first to show that increased permeability of plasma fibrin clots is correlated with longer BT in human subjects. We demonstrated that Previously, it has been shown that plasma fibrin clot characteristics may influence bleeding risk as observed in women with heavy menstrual bleeding  and patiin vitro on BT.In our study, BT was shorter in CAD patients compared to controls, which corroborates with earlier findings , but alsin vivo in humans. HRG interacts with fibrinogen while assessed in vitro which leads to the formation of less compact fibrin clots [γ′-fibrinogen, known to increase fibrin clot density [γ′-fibrinogen levels. The present study demonstrated that higher HRG levels are associated with looser plasma clot networks in human subjects, suggesting additional anticoagulant effects of HRG reported in vitro [in vitro experiments. Of note, the presence of HRG in plasma clots in patients with deep vein thrombosis has been shown recently [in vivo.The current study is the first to show that plasma levels of HRG are involved in primary hemostasis in both CAD patients and controls. We found that HRG correlated positively with BT, which provides evidence for a role of this anticoagulant and antifibrinolytic protein in clots . In animin clots . It is ein clots , 35. Morin clots  demonstr density . On the  density  have shoin vitro , 34, 35.in vitro . The prerecently . Taken tUnexpectedly, a very long as well as a short BT correlated with skin scar formation. We hypothesized that more compact and less permeable clots would be associated with impaired wound healing due to attenuated fibroblast invasion into the wound space filled with dense fibrin network . As longKs in controls, but not in CAD patients. Most likely, these associations reflect the impact of endothelial damage, along with platelets as an important source of circulating VEGF, a major growth factor stimulating angiogenesis during wound healing [Interestingly, we observed inverse correlations between VEGF and BT as well as between VEGF and  healing , 45. Des healing . This isγ′-fibrinogen, which is known to alter clot structure [γ′-fibrinogen explains all differences and associations observed by us. The same holds true for factor XIII levels, a potent modulator of fibrin clot structure [in vitro experiments, especially elucidating the impact of HRG and VEGF on clots, are needed to get more mechanistic insights into the phenomena presented; however, this study is hypothesis-generating and strongly supports further research in this field given observations made in patients and control subjects.The current study has several limitations. The number of subjects in the study was limited; however, the study was adequately powered to detect significant differences in fibrin clot characteristics. It should be noted that the observed associations do not necessarily mean the cause-effect relationship. Several other factors that may modulate clot properties have not been assessed, for example, complement C3, fibronectin, and some genetic determinants , 7. We atructure , 46, whitructure . MoreoveTo conclude, this study indicates that fibrin clot structure may affect BT in humans, suggesting a role of fibrin properties in primary hemostasis. The current study demonstrated a potential role of circulating HRG in primary hemostasis and its possible impact on fibrin clot structural properties. Further studies are warranted to elucidate complex relationships between plasma fibrin clot phenotype and wound healing, which might have important practical implications.