Joseph Macri

The Intestinal Microbiota Affect Central Levels of Brain-Derived Neurotropic Factor and Behavior in Mice

BACKGROUND & AIMS Alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to inflammatory and functional bowel disorders and psychiatric comorbidities. We examined whether the intestinal microbiota affects behavior and brain biochemistry in mice. METHODS Specific pathogen-free (SPF) BALB/c mice, with or without subdiaphragmatic vagotomy or chemical sympathectomy, or germ-free BALB/c mice received a mixture of nonabsorbable antimicrobials (neomycin, bacitracin, and pimaricin) in their drinking water for 7 days. Germ-free BALB/c and NIH Swiss mice were colonized with microbiota from SPF NIH Swiss or BALB/c mice. Behavior was evaluated using step-down and light preference tests. Gastrointestinal microbiota were assessed using denaturing gradient gel electrophoresis and sequencing. Gut samples were analyzed by histologic, myeloperoxidase, and cytokine analyses; levels of serotonin, noradrenaline, dopamine, and brain-derived neurotropic factor (BDNF) were assessed by enzyme-linked immunosorbent assay. RESULTS Administration of oral antimicrobials to SPF mice transiently altered the composition of the microbiota and increased exploratory behavior and hippocampal expression of BDNF. These changes were independent of inflammatory activity, changes in levels of gastrointestinal neurotransmitters, and vagal or sympathetic integrity. Intraperitoneal administration of antimicrobials to SPF mice or oral administration to germ-free mice did not affect behavior. Colonization of germ-free BALB/c mice with microbiota from NIH Swiss mice increased exploratory behavior and hippocampal levels of BDNF, whereas colonization of germ-free NIH Swiss mice with BALB/c microbiota reduced exploratory behavior. CONCLUSIONS The intestinal microbiota influences brain chemistry and behavior independently of the autonomic nervous system, gastrointestinal-specific neurotransmitters, or inflammation. Intestinal dysbiosis might contribute to psychiatric disorders in patients with bowel disorders.

Chronic Gastrointestinal Inflammation Induces Anxiety-Like Behavior and Alters Central Nervous System Biochemistry in Mice

BACKGROUND & AIMS Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. METHODS AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. RESULTS T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. CONCLUSIONS Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve.

Cardiac remodeling in fish: Strategies to maintain heart function during temperature change

Rainbow trout remain active in waters that seasonally change between 4°C and 20°C. To explore how these fish are able to maintain cardiac function over this temperature range we characterized changes in cardiac morphology, contractile function, and the expression of contractile proteins in trout following acclimation to 4°C (cold), 12°C (control), and 17°C (warm). The relative ventricular mass (RVM) of the cold acclimated male fish was significantly greater than that of males in the control group. In addition, the compact myocardium of the cold acclimated male hearts was thinner compared to controls while the amount of spongy myocardium was found to have increased. Cold acclimation also caused an increase in connective tissue content, as well as muscle bundle area in the spongy myocardium of the male fish. Conversely, warm acclimation of male fish caused an increase in the thickness of the compact myocardium and a decrease in the amount of spongy myocardium. There was also a decrease in connective tissue content in both myocardial layers. In contrast, there was no change in the RVM or connective tissue content in the hearts of female trout with warm or cold acclimation. Cold acclimation also caused a 50% increase in the maximal rate of cardiac AM Mg2+-ATPase but did not influence the Ca2+ sensitivity of this enzyme. To identify a mechanism for this change we utilized two-dimensional difference gel electrophoresis to characterize changes in the cardiac contractile proteins. Cold acclimation caused subtle changes in the phosphorylation state of the slow skeletal isoform of troponin T found in the heart, as well as of myosin binding protein C. These results demonstrate that acclimation of trout to warm and cold temperatures has opposing effects on cardiac morphology and tissue composition and that this results in distinct warm and cold cardiac phenotypes.

Proliferating brain cells are a target of neurotoxic CSF in systemic autoimmune disease.

Brain atrophy, neurologic and psychiatric (NP) manifestations are common complications in the systemic autoimmune disease, lupus erythematosus (SLE). Here we show that the cerebrospinal fluid (CSF) from autoimmune MRL-lpr mice and a deceased NP-SLE patient reduce the viability of brain cells which proliferate in vitro. This detrimental effect was accompanied by periventricular neurodegeneration in the brains of autoimmune mice and profound in vivo neurotoxicity when their CSF was administered to the CNS of a rat. Multiple ionic responses with microfluorometry and protein peaks on electropherograms suggest more than one mechanism of cellular demise. Similar to the CSF from diseased MRL-lpr mice, the CSF from a deceased SLE patient with a history of psychosis, memory impairment, and seizures, reduced viability of the C17.2 neural stem cell line. Proposed mechanisms of cytotoxicity involve binding of intrathecally synthesized IgG autoantibodies to target(s) common to different mammalian species and neuronal populations. More importantly, these results indicate that the viability of proliferative neural cells can be compromised in systemic autoimmune disease. Antibody-mediated lesions of germinal layers may impair the regenerative capacity of the brain in NP-SLE and possibly, brain development and function in some forms of CNS disorders in which autoimmune phenomena have been documented.

Associations between bacterial communities of house dust and infant gut

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Proteomic analysis of plasma from Holstein cows testing positive for Mycobacterium avium subsp. paratuberculosis (MAP).

Johnes disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johnes antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.

The Canadian Healthy Infant Longitudinal Development Birth Cohort Study: Biological Samples and Biobanking

BACKGROUND It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors. METHODS CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected. RESULTS A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses. CONCLUSIONS The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

Strong anion determination in biological fluids by capillary electrophoresis for clinical diagnostics.

New methods for quantitative analysis of strong anions are required for diagnostic testing of human diseases. Current techniques suffer from poor selectivity and/or long analysis times that are not amenable for labile anions in high-saline or volume-restricted samples. We introduce a rapid assay (<5 min) based on capillary electrophoresis (CE) with indirect UV detection for simultaneous analysis of sulfate, sulfite, and chloride in human urine, plasma, and sweat specimens. Remarkable selectivity for strong anions is achieved by using an acidic background electrolyte under reversed polarity that results in electrokinetic rejection of matrix interferences at the capillary inlet. A dual co-ion probe system consisting of 5 mM naphthalene disulfonate (NDS) and 5 mM naphthalene trisulfonate (NTS) in 0.4 M formic acid, pH 2.0 is developed for detection of UV transparent anions (S/N ≈ 3, 60 μM with a 25 μm inner diameter fused-silica capillary) with good peak symmetry and baseline stability. Due to the chemical reactivity of sulfite, dilute formaldehyde is used as a reagent to form an acid-stable hydroxymethylsulfonate adduct. Method validation confirmed excellent linearity (R(2) > 0.999), good accuracy (mean bias ≈7%), and acceptable long-term reproducibility (CV < 10%) over 20 days. The assay allows for artifact-free determination of sulfate and sulfite with consistent results for chloride when compared to standard electrochemical methods (R(2) > 0.975). Preliminary data suggest that kidney-stone formers have lower urinary sulfate excretion relative to non-kidney-stone patient controls (p = 0.0261). CE offers a selective yet robust platform for routine analysis of strong anions that is needed for confirmatory testing of cystic fibrosis, sulfite oxidase deficiency, urolithiasis, and other disorders of sulfur metabolism and/or anion transport.

Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char).

Intestinal overproduction of apolipoprotein B (apoB)-48-containing chylomicrons is increasingly recognized as an underlying factor in metabolic dyslipidemia commonly observed in insulin-resistant states. Enhanced chylomicron assembly and secretion has been documented in animal models of insulin resistance, but the underlying mechanistic factors are unknown. Chylomicron assembly occurs through a series of complex vesicular interactions involving prechylomicron transport vesicles (PCTVs), which transport lipids from the endoplasmic reticulum (ER) to the Golgi. We report proteomic profiles of PCTVs isolated from the enteric ER in the small intestine of the fructose-fed hamster, an established model of diet-induced insulin resistance. Using 2D gel electrophoresis and tandem mass spectrometry, PCTVs were characterized and proteomic profiles of PCTV-associated proteins from insulin-resistant and control enterocytes were developed, with the intention of identifying proteins involved in insulin signaling attenuation and lipoprotein overproduction. A number of PCTV-associated proteins were found to be differentially expressed including microsomal triglyceride transfer protein (MTP), apoB-48, Sar1 and VAMP7. We postulate that altered expression of Sar1 and MTP may contribute to increased chylomicron assembly in the fructose-fed hamster. These findings have increased our understanding of the intracellular assembly and transport of nascent chylomicrons and potential cellular factors responsible for lipoprotein overproduction in insulin-resistant states.

Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2‐DE and MS. Proteomic profiles of PCTV‐associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B‐48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle‐associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid‐binding proteins, and lipid‐related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.