Blood-borne extracellular vesicles of bacteria and intestinal cells in patients with psychotic disorders.

BACKGROUND: Human cells and bacteria secrete extracellular vesicles (EV) which play a role in intercellular communication. EV from the host intestinal epithelium are involved in the regulation of bacterial gene expression and growth. Bacterial EV (bactEV) produced in the intestine can pass to various tissues where they deliver biomolecules to many kinds of cells, including neurons. Emerging data indicate that gut microbiota is altered in patients with psychotic disorders. We hypothesized that the amount and content of blood-borne EV from intestinal cells and bactEV in psychotic patients would differ from healthy controls. METHODS: We analyzed for human intestinal proteins by proteomics, for bactEV by metaproteomic analysis, and by measuring the level of lipopolysaccharide (LPS) in blood-borne EV from patients with psychotic disorders (n = 25), tested twice, in the acute phase of psychosis and after improvement, with age- and sex-matched healthy controls (n = 25). RESULTS: Patients with psychotic disorders had lower LPS levels in their EV compared to healthy controls (p = .027). Metaproteome analyses confirmed LPS finding and identified Firmicutes and Bacteroidetes as dominating phyla. Total amounts of human intestine proteins in EV isolated from blood was lower in patients compared to controls (p = .02). CONCLUSIONS: Our results suggest that bactEV and host intestinal EV are decreased in patients with psychosis and that this topic is worthy of further investigation given potential pathophysiological implications. Possible mechanisms involve dysregulation of the gut microbiota by host EV, altered translocation of bactEV to systemic circulation where bactEV can interact with both the brain and the immune system.

High-throughput assay for effect screening of amphotericin B and bioactive components on filamentous Candida albicans.

AIMS: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB). METHODS AND RESULTS: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 µg ml-1 , and this was eightfold reduced (0.0038 µg ml-1 ) in the presence of the nutritional drinks. CONCLUSIONS: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Na+ is shifted from the extracellular to the intracellular compartment and is not inactivated by glycosaminoglycans during high salt conditions in rats.

Recently, studies have emerged suggesting that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. We investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. Na+ accumulation was induced in rats by a high salt diet (HSD) (8% NaCl and 1% saline to drink) or by implantation of a deoxycorticosterone acetate (DOCA) tablet (1% saline to drink) using rats on a low salt diet (LSD) (0.1% NaCl) on tap water as control. Na+ and K+ were assessed by ion chromatography in tissue eluates, and the extracellular volume by equilibration of 51 Cr-EDTA. By tangential sectioning of the skin, we found a low Na+ content and extracellular volume in epidermis, both parameters rising by ∼30% and 100%, respectively, in LSD and even more in HSD and DOCA when entering dermis. We found evidence for an extracellular Na+ gradient from epidermis to dermis shown by an estimated concentration in epidermis ∼2 and 4-5 times that of dermis in HSD and DOCA-salt. There was intracellular storage of Na+ in skin, muscle, and myocardium without a concomitant increase in hydration. Our data suggest that there is a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. Salt stress results in intracellular storage of Na+ in exchange with K+ in skeletal muscle and myocardium that may have electromechanical consequences. KEY POINTS: Studies have suggested that Na+ can be retained or removed without commensurate water retention or loss, and that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. In the present study, we investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. We used two common models for salt-sensitive hypertension: high salt and a deoxycorticosterone salt diet. We found a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. There was intracellular Na+ storage in muscle and myocardium without a concomitant increase in hydration, comprising storage that may have electromechanical consequences in salt stress.

Identification of novel biomarkers of inflammation in Atlantic salmon (Salmo salar L.) by a plasma proteomic approach.

Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.

The Use of Extracellular Membrane Vesicles for Immunization against Francisellosis in Nile Tilapia (Oreochromis niloticus) and Atlantic Cod (Gadus morhua L.).

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Extracellular vesicles in patients in the acute phase of psychosis and after clinical improvement: an explorative study.

Extracellular vesicles (EVs) are cell-derived structures that transport proteins, lipids and nucleic acids between cells, thereby affecting the phenotype of the recipient cell. As the content of EVs reflects the status of the originating cell, EVs can have potential as biomarkers. Identifying EVs, including their cells of origin and their cargo, may provide insights in the pathophysiology of psychosis. Here, we present an in-depth analysis and proteomics of EVs from peripheral blood in patients (n = 25) during and after the acute phase of psychosis. Concentration and protein content of EVs in psychotic patients were twofold higher than in 25 age- and sex-matched healthy controls (p < 0.001 for both concentration and protein content), and the diameter of EVs was larger in patients (p = 0.02). Properties of EVs did not differ significantly in blood sampled during and after the acute psychotic episode. Proteomic analyses on isolated EVs from individual patients revealed 1,853 proteins, whereof 45 were brain-elevated proteins. Of these, five proteins involved in regulation of plasticity of glutamatergic synapses were significantly different in psychotic patients compared to controls; neurogranin (NRGN), neuron-specific calcium-binding protein hippocalcin (HPCA), kalirin (KALRN), beta-adducin (ADD2) and ankyrin-2 (ANK2). To summarize, our results show that peripheral EVs in psychotic patients are different from those in healthy controls and point at alterations on the glutamatergic system. We suggest that EVs allow investigation of blood-borne brain-originating biological material and that their role as biomarkers in patients with psychotic disorders is worthy of further exploration.

Age-related changes in rat dermal extracellular matrix composition affect the distribution of plasma proteins as a function of size and charge.

Collagen and glycosaminoglycans (GAGs) constituting the ECM may limit the space available and thus exclude macromolecules from a fraction of the interstitial fluid (IF) phase. This exclusion phenomenon is of importance for transcapillary fluid and solute exchange. The purpose of the study was to examine the range of interstitial exclusion in rat skin by using probes within a span of molecular weights and electrical charge and also to test if a change in interstitial composition, occurring as a consequence of aging, affected exclusion. To this end, we used a novel approach, involving the exact determination of albumin concentration and mass in IF and tissue eluate by HPLC and thereafter, expressing the corresponding numbers relative to albumin for a set of probe proteins assessed by quantitative proteomics. Albumin was excluded from 55±4% (n=8) of the extracellular fluid phase. There was a highly significant, positive correlation between probe Stokes-Einstein (SE) radius and fractional excluded volume (VEF), described by VEF=0.078·SE radius+0.269 (P<0.001), and oppositely, a negative correlation between probe isoelectric point (pI) and exclusion for proteins with comparable size, VEF=-0.036·pI+0.719 (P=0.04). Aging resulted in a significant reduction in skin hydration and sulfated GAGs, a moderate increase in hyaluronan, and a corresponding, reduced VEF for albumin and the other macromolecular probes. Our findings suggest that the changes in the ECM in aged skin may result in delayed adjustments of fluid perturbations and reduced ability for salt storage.

Distribution volumes of macromolecules in human ovarian and endometrial cancers--effects of extracellular matrix structure.

Elements of the extracellular matrix (ECM), notably collagen and glucosaminoglycans, will restrict part of the space available for soluble macromolecules simply because the molecules cannot occupy the same space. This phenomenon may influence macromolecular drug uptake. To study the influence of steric and charge effects of the ECM on the distribution volumes of macromolecules in human healthy and malignant gynecologic tissues we used as probes 15 abundant plasma proteins quantified by high-resolution mass spectrometry. The available distribution volume (VA) of albumin was increased in ovarian carcinoma compared with healthy ovarian tissue. Furthermore, VA of plasma proteins between 40 and 190 kDa decreased with size for endometrial carcinoma and healthy ovarian tissue, but was independent of molecular weight for the ovarian carcinomas. An effect of charge on distribution volume was only found in healthy ovaries, which had lower hydration and high collagen content, indicating that a condensed interstitium increases the influence of negative charges. A number of earlier suggested biomarker candidates were detected in increased amounts in malignant tissue, e.g., stathmin and spindlin-1, showing that interstitial fluid, even when unfractionated, can be a valuable source for tissue-specific proteins. We demonstrate that the distribution of abundant plasma proteins in the interstitium can be elucidated by mass spectrometry methods and depends markedly on hydration and ECM structure. Our data can be used in modeling of drug uptake, and give indications on ECM components to be targeted to increase the uptake of macromolecular substances.

Quantification of the concentration gradient of biomarkers between ovarian carcinoma interstitial fluid and blood.

BACKGROUND: Tumor interstitial fluid (TIF) rather than plasma should be used in cancer biomarker discovery because of the anticipated higher concentration of locally produced proteins in the tumor microenvironment. Nevertheless, the actual TIF-to-plasma gradient of tumor specific proteins has not been quantified. We present the proof-of-concept for the quantification of the postulated gradient between TIF and plasma. METHODS: TIF was collected by centrifugation from serous (n = 19), endometrioid (n = 9) and clear cell (n = 3) ovarian carcinomas with early (n = 15) and late stage (n = 16) disease in grades 1 (n = 2), 2 (n = 8) and 3 (n = 17), and ELISA was used for the determination of CA-125, osteopontin and VEGF-A. RESULTS: All three markers were significantly up-regulated in TIF compared with plasma (p < 0.0001). The TIF-to-plasma ratio of the ovarian cancer biomarker CA-125 ranged from 1.4 to 24,300 (median = 194) and was inversely correlated to stage (p = 0.0006). The cancer related osteopontin and VEGF-A had TIF-to-plasma ratios ranging from 1 to 62 (median = 15) and 2 to 1040 (median = 59), respectively. The ratios were not affected by tumor stage, indicative of more widespread protein expression. CONCLUSION: We present absolute quantitative data on the TIF-to-plasma gradient of selected proteins in the tumor microenvironment, and demonstrate a substantial and stage dependent gradient for CA-125 between TIF and plasma, suggesting a relation between total tumor burden and tissue-to-plasma gradient. GENERAL SIGNIFICANCE: We present novel quantitative data on biomarker concentration in the tumor microenvironment, and a new strategy for biomarker selection, applicable in future biomarker studies.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue.

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.

Interstitial fluid-a reflection of the tumor cell microenvironment and secretome.

The interstitium or interstitial space describes the space outside the blood and lymphatic vessels. It contains two phases; the interstitial fluid (IF) and the extracellular matrix. In this review we focus on the interstitial fluid phase, which is the physical and biochemical microenvironment of the cells, and more specifically that of tumors. IF is created by transcapillary filtration and cleared by lymphatic vessels, and contains substances that are either produced and secreted locally, thus denoted secretome, or brought to the organ by the circulation. The structure of the interstitium is discussed briefly and moreover techniques for IF isolation focusing on those that are relevant for studies of the secretome. Accumulated data show that tumor IF is hypoxic and acidic compared with subcutaneous IF and plasma, and that there are gradients between IF and plasma giving information on where substances are produced and thereby reflecting the local microenvironment. We review recent data on the origin of tissue specific substances, challenges related to isolating a representative secretome and the use of this as a substrate for biomarker identification. Finally we perform a comparative analysis across human tumor types and techniques and show that there is great variation in the results obtained that may at least partially be due to the isolation method used. We conclude that when care is taken in isolation of substrate, analysis of the secretome may give valuable biological insight and result in identification of biomarker candidates. This article is part of a Special Issue entitled: An Updated Secretome.

Proteomic evaluation of inflammatory proteins in rat spleen interstitial fluid and lymph during LPS-induced systemic inflammation reveals increased levels of ADAMST1.

The spleen is a part of the immune system and is involved in the response to a systemic inflammation induced by blood borne pathogens that may induce sepsis. Knowledge about the protein composition of the spleen microenvironment in a control situation and during systemic inflammation may contribute to our understanding of the pathophysiology of sepsis. To our knowledge, the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In order to access the proximal fluid surrounding the splenic cells, we collected postnodal efferent spleen lymph from rats by cannulation, and spleen interstitial fluid (IF) by centrifugation. The origin of the isolated spleen IF was assessed by the extracellular tracer (51)Cr-EDTA and the plasma tracer (125)I-HSA. Spleen lymph, IF, and plasma samples were collected during lipopolysaccharide (LPS) induced systemic inflammation and analyzed using a cytokine multiplex assay and, for the first time, using label-free mass spectrometry based proteomics. The concentrations of TNF-α, IL-1ß, IL-6, and IL-10 increased severalfold in all fluids after LPS exposure. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were detected after LPS only. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was detected by proteomics (the pro- region) in lymph only after LPS. ADAMTS1 was assessed by ELISA (the metalloproteinase domain), and the concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. A dramatic increase in ADAMTS1 was detected in lymph, IF, and plasma after LPS exposure. In conclusion, the procedures we used to isolate IF and lymph from the spleen during LPS enabled detection of locally produced proteins. Furthermore, we have demonstrated that the inflammatory proteome is different in the spleen microenvironment when compared to that in plasma.

A new method for isolation of interstitial fluid from human solid tumors applied to proteomic analysis of ovarian carcinoma tissue.

Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample's origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na(+), two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods.