Evidence of a genetic background predisposing to complex regional pain syndrome type 1.

BACKGROUND: Complex regional pain syndrome type 1 (CRPS-1) is a rare, disabling and sometimes chronic disorder usually arising after a trauma. This exploratory study examined whether patients with chronic CRPS-1 have a different genetic profile compared with those who do not have the condition. METHODS: Exome sequencing was performed to seek altered non-synonymous SNP allele frequencies in a discovery cohort of well-characterised patients with chronic CRPS-1 (n=34) compared with population databases. Identified SNP alleles were confirmed by Sanger sequencing and sought in a replication cohort (n=50). Gene expression of peripheral blood macrophages was assessed. RESULTS: In the discovery cohort, the rare allele frequencies of four non-synonymous SNPs were statistically increased. The replication cohort confirmed this finding. In a chronic pain cohort, these alleles were not overexpressed. In total, 25 out of 84 (29.8%) patients with CRPS-1 expressed a rare allele. The SNPs were rs41289586 in ANO10, rs28360457 in P2RX7, rs1126930 in PRKAG1 and rs80308281 in SLC12A9. Males were more likely than females to have a rare SNP allele, 8 out of 14 (57.1%) vs 17 out of 70 (24.3%) (Fisher's p=0.023). ANO10, P2RX7, PRKAG1 and SLC12A9 were all expressed in macrophages from healthy human controls. CONCLUSION: A single SNP in each of the genes ANO10, P2RX7, PRKAG1 and SLC12A9 was associated with developing chronic CRPS-1, with more males than females expressing these rare alleles. Our work suggests the possibility that a permissive genetic background is an important factor in the development of CRPS-1.

Gut microbial metabolites in Parkinson's disease: Association with lifestyle, disease characteristics, and treatment status.

There is growing appreciation of the importance of the intestinal microbiota in Parkinson's disease (PD), and one potential mechanism by which the intestinal microbiota can communicate with the brain is via bacteria-derived metabolites. In this study, plasma levels of bacterial-derived metabolites including trimethylamine-N-oxide (TMAO), short chain fatty acids (SCFA), the branched chain fatty acid isovalerate, succinate, and lactate were evaluated in PD subjects (treatment naïve and treated) which were compared to (1) population controls, (2) spousal / household controls (similar lifestyle to PD subjects), and (3) subjects with multiple system atrophy (MSA). Analyses revealed an increase in the TMAO pathway in PD subjects which was independent of medication status, disease characteristics, and lifestyle. Lactic acid was decreased in treated PD subjects, succinic acid positively correlated with disease severity, and the ratio of pro-inflammatory TMAO to the putative anti-inflammatory metabolite butyric acid was significantly higher in PD subjects compared to controls indicating a pro-inflammatory shift in the metabolite profile in PD subjects. Finally, acetic and butyric acid were different between PD and MSA subjects indicating that metabolites may differentiate these synucleinopathies. In summary, (1) TMAO is elevated in PD subjects, a phenomenon independent of disease characteristics, treatment status, and lifestyle and (2) metabolites may differentiate PD and MSA subjects. Additional studies to understand the potential of TMAO and other bacterial metabolites to serve as a biomarker or therapeutic targets are warranted.

Novel role of the Mu-opioid receptor in pancreatic cancer: potential link between opioid use and cancer progression.

Opioids are the most popular drugs for both acute and chronic pain management. The G protein-coupled mu-opioid receptor (MOR) is the therapeutic target for most clinically used opioids, including morphine. A mounting number of publications suggest a relationship between the MOR and possible cancer progression and recurrence extending to managing chronic cancer pain. In this study, we studied the possible link between opioid use and pancreatic cancer (PC) progression. We found increased MOR expression in murine and human PC cell lines, human PC-derived organoids, and in the undifferentiated or poorly differentiated areas of surgically resected PC tissues. Direct stimulation of MOR by morphine (MOR agonist) caused a significant dose-dependent increase in proliferation, invasion, and levels of stemness markers in PC cells. In a co-culture system, MOR stimulation of macrophages also resulted in increased proliferation of PC cells. MOR overexpression increased proliferation and cancer stemness, whereas knock-down of MOR followed opposite results in the PC cells. Morphine induced chemoresistance to conventional chemotherapeutic agents used for PC treatment. Overall, our results suggest that MOR is expressed in pancreatic cancer and may be involved in tumor progression and chemoresistance.

Alcohol use disorder as a potential risk factor for COVID-19 severity: A narrative review.

In Dec. 2019-January 2020, a pneumonia illness originating in Wuhan, China, designated as coronavirus disease 2019 (COVID-19) was shown to be caused by a novel RNA coronavirus designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). People with advanced age, male sex, and/or underlying health conditions (obesity, type 2 diabetes, cardiovascular disease, hypertension, chronic kidney disease, and chronic lung disease) are especially vulnerable to severe COVID-19 symptoms and death. These risk factors impact the immune system and are also associated with poor health, chronic illness, and shortened longevity. However, a large percent of patients without these known risk factors also develops severe COVID-19 disease that can result in death. Thus, there must exist risk factors that promote exaggerated inflammatory and immune response to the SARS-CoV-2 virus leading to death. One such risk factor may be alcohol misuse and alcohol use disorder because these can exacerbate viral lung infections like SARS, influenza, and pneumonia. Thus, it is highly plausible that alcohol misuse is a risk factor for either increased infection rate when individuals are exposed to SARS-CoV-2 virus and/or more severe COVID-19 in infected patients. Alcohol use is a well-known risk factor for lung diseases and ARDS in SARS patients. We propose that alcohol has three key pathogenic elements in common with other COVID-19 severity risk factors: namely, inflammatory microbiota dysbiosis, leaky gut, and systemic activation of the NLRP3 inflammasome. We also propose that these three elements represent targets for therapy for severe COVID-19.

Quantitative analysis of short-chain fatty acids in human plasma and serum by GC-MS.

Short-chain fatty acids (SCFAs) are volatile fatty acids produced by gut microbial fermentation of dietary nondigestible carbohydrates. Acetate, propionate, and butyrate SCFA measures are important to clinical and nutritional studies for their established roles in promoting healthy immune and gut function. Additionally, circulating SCFAs may influence the metabolism and allied function of additional tissues and organs. The accurate quantification of SCFAs in plasma/serum is critical to understanding the biological role of SCFAs. The low concentrations of circulating SCFAs and their volatile nature present challenges for quantitative analysis. Herein, we report a sensitive method for SCFA quantification via extraction with methyl tert-butyl ether after plasma/serum acidification. The organic extract of SCFAs is injected directly with separation and detection using a polar GC column coupled to mass spectrometry. The solvent-to-sample ratio, plasma volume, and amount of HCl needed for SCFA protonation were optimized. Method validation shows good within-day and inter-day repeatability. The limit of detection was 0.3-0.6 µg/mL for acetate and 0.03-0.12 µg/mL for propionate and butyrate. Successful application of this method on clinical plasma and serum samples was demonstrated in six datasets. By simplifying the sample preparation procedure, the present method reduces the risk of contamination, lowers the cost of analysis, increases throughput, and offers the potential for automated sample preparation.

Chronic stress-induced gut dysfunction exacerbates Parkinson's disease phenotype and pathology in a rotenone-induced mouse model of Parkinson's disease.

Recent evidence provides support for involvement of the microbiota-gut-brain axis in Parkinson's disease (PD) pathogenesis. We propose that a pro-inflammatory intestinal milieu, due to intestinal hyper-permeability and/or microbial dysbiosis, initiates or exacerbates PD pathogenesis. One factor that can cause intestinal hyper-permeability and dysbiosis is chronic stress which has been shown to accelerate neuronal degeneration and motor deficits in Parkinsonism rodent models. We hypothesized that stress-induced intestinal barrier dysfunction and microbial dysbiosis lead to a pro-inflammatory milieu that exacerbates the PD phenotype in the low-dose oral rotenone PD mice model. To test this hypothesis, mice received unpredictable restraint stress (RS) for 12 weeks, and during the last six weeks mice also received a daily administration of low-dose rotenone (10 mg/kg/day) orally. The initial six weeks of RS caused significantly higher urinary cortisol, intestinal hyperpermeability, and decreased abundance of putative "anti-inflammatory" bacteria (Lactobacillus) compared to non-stressed mice. Rotenone alone (i.e., without RS) disrupted the colonic expression of the tight junction protein ZO-1, increased oxidative stress (N-tyrosine), increased myenteric plexus enteric glial cell GFAP expression and increased α-synuclein (α-syn) protein levels in the colon compared to controls. Restraint stress exacerbated these rotenone-induced changes. Specifically, RS potentiated rotenone-induced effects in the colon including: 1) intestinal hyper-permeability, 2) disruption of tight junction proteins (ZO-1, Occludin, Claudin1), 3) oxidative stress (N-tyrosine), 4) inflammation in glial cells (GFAP + enteric glia cells), 5) α-syn, 6) increased relative abundance of fecal Akkermansia (mucin-degrading Gram-negative bacteria), and 7) endotoxemia. In addition, RS promoted a number of rotenone-induced effects in the brain including: 1) reduced number of resting microglia and a higher number of dystrophic/phagocytic microglia as well as (FJ-C+) dying cells in the substantia nigra (SN), 2) increased lipopolysaccharide (LPS) reactivity in the SN, and 3) reduced dopamine (DA) and DA metabolites (DOPAC, HVA) in the striatum compared to control mice. Our findings support a model in which chronic stress-induced, gut-derived, pro-inflammatory milieu exacerbates the PD phenotype via a dysfunctional microbiota-gut-brain axis.

Microglia, inflammation and gut microbiota responses in a progressive monkey model of Parkinson's disease: A case series.

Inflammation has been linked to the development of nonmotor symptoms in Parkinson's disease (PD), which greatly impact patients' quality of life and can often precede motor symptoms. Suitable animal models are critical for our understanding of the mechanisms underlying disease and the associated prodromal disturbances. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkey model is commonly seen as a "gold standard" model that closely mimics the clinical motor symptoms and the nigrostriatal dopaminergic loss of PD, however MPTP toxicity extends to other nondopaminergic regions. Yet, there are limited reports monitoring the MPTP-induced progressive central and peripheral inflammation as well as other nonmotor symptoms such as gastrointestinal function and microbiota. We report 5 cases of progressive parkinsonism in non-human primates to gain a broader understanding of MPTP-induced central and peripheral inflammatory dysfunction to understand the potential role of inflammation in prodromal/pre-motor features of PD-like degeneration. We measured inflammatory proteins in plasma and CSF and performed [18F]FEPPA PET scans to evaluate translocator proteins (TSPO) or microglial activation. Monkeys were also evaluated for working memory and executive function using various behavior tasks and for gastrointestinal hyperpermeability and microbiota composition. Additionally, monkeys were treated with a novel TNF inhibitor XPro1595 (10 mg/kg, n = 3) or vehicle (n = 2) every three days starting 11 weeks after the initiation of MPTP to determine whether XPro1595 would alter inflammation and microglial behavior in a progressive model of PD. The case studies revealed that earlier and robust [18F]FEPPA PET signals resulted in earlier and more severe parkinsonism, which was seen in male cases compared to female cases. Potential other sex differences were observed in circulating inflammation, microbiota diversity and their metabolites. Additional studies with larger group sizes of both sexes would enable confirmation and extension of these findings. If these findings reflect potential differences in humans, these sex differences have significant implications for therapeutic development of inflammatory targets in the clinic.

Role of TLR4 in the gut-brain axis in Parkinson's disease: a translational study from men to mice.

OBJECTIVE: Recent evidence suggesting an important role of gut-derived inflammation in brain disorders has opened up new directions to explore the possible role of the gut-brain axis in neurodegenerative diseases. Given the prominence of dysbiosis and colonic dysfunction in patients with Parkinson's disease (PD), we propose that toll-like receptor 4 (TLR4)-mediated intestinal dysfunction could contribute to intestinal and central inflammation in PD-related neurodegeneration. DESIGN: To test this hypothesis we performed studies in both human tissue and a murine model of PD. Inflammation, immune activation and microbiota composition were measured in colonic samples from subjects with PD and healthy controls subjects and rotenone or vehicle-treated mice. To further assess the role of the TLR4 signalling in PD-induced neuroinflammation, we used TLR4-knockout (KO) mice in conjunction with oral rotenone administration to model PD. RESULTS: Patients with PD have intestinal barrier disruption, enhanced markers of microbial translocation and higher pro-inflammatory gene profiles in the colonic biopsy samples compared with controls. In this regard, we found increased expression of the bacterial endotoxin-specific ligand TLR4, CD3+ T cells, cytokine expression in colonic biopsies, dysbiosis characterised by a decrease abundance of SCFA-producing colonic bacteria in subjects with PD. Rotenone treatment in TLR4-KO mice revealed less intestinal inflammation, intestinal and motor dysfunction, neuroinflammation and neurodegeneration, relative to rotenone-treated wild-type animals despite the presence of dysbiotic microbiota in TLR4-KO mice. CONCLUSION: Taken together, these studies suggest that TLR4-mediated inflammation plays an important role in intestinal and/or brain inflammation, which may be one of the key factors leading to neurodegeneration in PD.

Alcohol Effects on Colon Epithelium are Time-Dependent.

BACKGROUND: Alcohol intake increases the risk of developing colon cancer. Circadian disruption promotes alcohol's effect on colon carcinogenesis through unknown mechanisms. Alcohol's metabolites induce DNA damage, an early step in carcinogenesis. We assessed the effect of time of alcohol consumption on markers of tissue damage in the colonic epithelium. METHODS: Mice were treated by alcohol or phosphate-buffered saline (PBS), at 4-hour intervals for 3 days, and their colons were analyzed for (i) proliferation (Ki67) and antiapoptosis (Bcl-2) markers, (ii) DNA damage (γ-H2AX), and (iii) the major acetaldehyde (AcH)-DNA adduct, N2 -ethylidene-dG. To model circadian disruption, mice were shifted once weekly for 12 h and then were sacrificed at 4-hour intervals. Samples of mice with a dysfunctional molecular clock were analyzed. The dynamics of DNA damage repair from AcH treatment as well as role of xeroderma pigmentosum, complementation group A (XPA) in their repair were studied in vitro. RESULTS: Proliferation and survival of colonic epithelium have daily rhythmicity. Alcohol induced colonic epithelium proliferation in a time-dependent manner, with a stronger effect during the light/rest period. Alcohol-associated DNA damage also occurred more when alcohol was given at light. Levels of DNA adduct did not vary by time, suggesting rather lower repair efficiency during the light versus dark. XPA gene expression, a key excision repair gene, was time-dependent, peaking at the beginning of the dark. XPA knockout colon epithelial cells were inefficient in repair of the DNA damage induced by alcohol's metabolite. CONCLUSIONS: Time of day of alcohol intake may be an important determinant of colon tissue damage and carcinogenicity.

Diurnal variations in intestinal barrier integrity and liver pathology in mice: implications for alcohol binge.

Recent studies suggest that circadian rhythms regulate intestinal barrier integrity, but it is not clear whether there are daily variations in barrier integrity. This study investigated daily variations in intestinal barrier integrity, including whether there are differences in alcohol-induced intestinal barrier dysfunction after an alcohol binge at different times of day and whether this is associated with concurrent liver injury. C57BL6/J male mice were fed a standard chow diet, an alcohol-containing liquid diet, or an alcohol control diet for 4 wk. During week 5 (i.e., on days 43-45), mice received three once-daily gavages of alcohol (6 g/kg) or the control (phosphate-buffered saline) at the same time each day. Immediately after the binge on the second day, intestinal permeability was assessed. Four hours after the third and final binge, mice were euthanized and tissue samples collected. The results demonstrated diet-specific and outcome-specific effects of time, alcohol, and/or time by alcohol interaction. Specifically, the alcohol binge robustly influenced markers of intestinal barrier integrity, and liver markers were robustly influenced by time of day. Only intestinal permeability (i.e., sucralose) demonstrated a significant effect of time and also showed a binge by time interaction, suggesting that the time of the alcohol binge influences colonic permeability. NEW & NOTEWORTHY This study investigated daily variations in intestinal barrier integrity, including whether there are differences in alcohol-induced intestinal barrier dysfunction after an alcohol binge at different times of day and whether this is associated with concurrent liver injury. We conclude that 1) alcohol binge significantly impacted markers of intestinal permeability, 2) time of day significantly affected liver outcomes, and 3) the time of day influenced colonic permeability.

Circadian Mechanisms in Alcohol Use Disorder and Tissue Injury.

Heavy use of alcohol can lead to addictive behaviors and to eventual alcohol-related tissue damage. While increased consumption of alcohol has been attributed to various factors including level of alcohol exposure and environmental factors such as stress, data from behavioral scientists and physiological researchers are revealing roles for the circadian rhythm in mediating the development of behaviors associated with alcohol use disorder as well as the tissue damage that drives physiological disease. In this work, we compile recent work on the complex mutually influential relationship that exists between the core circadian rhythm and the pharmacodynamics of alcohol. As we do so, we highlight implications of the relationship between alcohol and common circadian mechanisms of effected organs on alcohol consumption, metabolism, toxicity, and pathology.

CREB Protein Mediates Alcohol-Induced Circadian Disruption and Intestinal Permeability.

BACKGROUND: Alcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol-induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 in vitro blocks alcohol-induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol-induced increase in PER2 and suggesting that oxidative stress may mediate alcohol-induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol-derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability. METHODS: Caco-2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real-time polymerase chain reaction. Wild-type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue. RESULTS: Alcohol increased oxidative stress, caused Caco-2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco-2 cells with an antioxidant scavenger. Alcohol-derived oxidative stress activated cAMP response element-binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco-2 cell monolayer hyperpermeability. CONCLUSIONS: Taken together, these data suggest that strategies to reduce alcohol-induced oxidative stress may alleviate alcohol-mediated circadian disruption and intestinal leakiness, critical drivers of ALD.

Alcohol Feeding in Mice Promotes Colonic Hyperpermeability and Changes in Colonic Organoid Stem Cell Fate.

BACKGROUND: Alcohol increases intestinal permeability to proinflammatory microbial products that promote liver disease, even after a period of sobriety. We sought to test the hypothesis that alcohol affects intestinal stem cells using an in vivo model and ex vivo organoids generated from jejunum and colon from mice fed chronic alcohol. METHODS: Mice were fed a control or an alcohol diet. Intestinal permeability, liver steatosis-inflammation, and stool short-chain fatty acids (SCFAs) were measured. Jejunum and colonic organoids and tissue were stained for stem cell, cell lineage, and apical junction markers with assessment of mRNA by PCR and RNA-seq. ChIP-PCR analysis was carried out for Notch1 using an antibody specific for acetylated histone 3. RESULTS: Alcohol-fed mice exhibited colonic (but not small intestinal) hyperpermeability, steatohepatitis, and decreased butyrate/total SCFA ratio in stool. Stem cell, cell lineage, and apical junction marker staining in tissue or organoids from jejunum tissue were not impacted by alcohol. Only chromogranin A (Chga) was increased in jejunum organoids by qPCR. However, colonic tissue and organoid staining exhibited an alcohol-induced significant decrease in cytokeratin 20+  (Krt20+) absorptive lineage enterocytes, a decrease in occludin and E-cadherin apical junction proteins, an increase in Chga, and an increase in the Lgr5 stem cell marker. qPCR revealed an alcohol-induced decrease in colonic organoid and tissue Notch1, Hes1, and Krt20 and increased Chga, supporting an alteration in stem cell fate due to decreased Notch1 expression. Colonic tissue ChIP-PCR revealed alcohol feeding suppressed Notch1 mRNA expression (via deacetylation of histone H3) and decreased Notch1 tissue staining. CONCLUSIONS: Data support a model for alcohol-induced colonic hyperpermeability via epigenetic effects on Notch1, and thus Hes1, suppression through a mechanism involving histone H3 deacetylation at the Notch1 locus. This decreased enterocyte and increased enteroendocrine cell colonic stem cell fate and decreased apical junctional proteins leading to hyperpermeability.

Night workers with circadian misalignment are susceptible to alcohol-induced intestinal hyperpermeability with social drinking.

Alcohol-induced intestinal hyperpermeability (AIHP) is a known risk factor for alcoholic liver disease (ALD), but only 20-30% of heavy alcoholics develop AIHP and ALD. The hypothesis of this study is that circadian misalignment would promote AIHP. We studied two groups of healthy subjects on a stable work schedule for 3 mo [day workers (DW) and night workers (NW)]. Subjects underwent two circadian phase assessments with sugar challenge to access intestinal permeability between which they drank 0.5 g/kg alcohol daily for 7 days. Sleep architecture by actigraphy did not differ at baseline or after alcohol between either group. After alcohol, the dim light melatonin onset (DLMO) in the DW group did not change significantly, but in the NW group there was a significant 2-h phase delay. Both the NW and DW groups had no change in small bowel permeability with alcohol, but only in the NW group was there an increase in colonic and whole gut permeability. A lower area under the curve of melatonin inversely correlated with increased colonic permeability. Alcohol also altered peripheral clock gene amplitude of peripheral blood mononuclear cells in CLOCK, BMAL, PER1, CRY1, and CRY2 in both groups, and inflammatory markers lipopolysaccharide-binding protein, LPS, and IL-6 had an elevated mesor at baseline in NW vs. DW and became arrhythmic with alcohol consumption. Together, our data suggest that central circadian misalignment is a previously unappreciated risk factor for AIHP and that night workers may be at increased risk for developing liver injury with alcohol consumption.

Light/Dark Shifting Promotes Alcohol-Induced Colon Carcinogenesis: Possible Role of Intestinal Inflammatory Milieu and Microbiota.

BACKGROUND: Colorectal cancer (CRC) is associated with the modern lifestyle. Chronic alcohol consumption-a frequent habit of majority of modern societies-increases the risk of CRC. Our group showed that chronic alcohol consumption increases polyposis in a mouse mode of CRC. Here we assess the effect of circadian disruption-another modern life style habit-in promoting alcohol-associated CRC. METHOD: TS4Cre × adenomatous polyposis coli (APC)lox468 mice underwent (a) an alcohol-containing diet while maintained on a normal 12 h light:12 h dark cycle; or (b) an alcohol-containing diet in conjunction with circadian disruption by once-weekly 12 h phase reversals of the light:dark (LD) cycle. Mice were sacrificed after eight weeks of full alcohol and/or LD shift to collect intestine samples. Tumor number, size, and histologic grades were compared between animal groups. Mast cell protease 2 (MCP2) and 6 (MCP6) histology score were analyzed and compared. Stool collected at baseline and after four weeks of experimental manipulations was used for microbiota analysis. RESULTS: The combination of alcohol and LD shifting accelerated intestinal polyposis, with a significant increase in polyp size, and caused advanced neoplasia. Consistent with a pathogenic role of stromal tryptase-positive mast cells in colon carcinogenesis, the ratio of mMCP6 (stromal)/mMCP2 (intraepithelial) mast cells increased upon LD shifting. Baseline microbiota was similar between groups, and experimental manipulations resulted in a significant difference in the microbiota composition between groups. CONCLUSIONS: Circadian disruption by Light:dark shifting exacerbates alcohol-induced polyposis and CRC. Effect of circadian disruption could, at least partly, be mediated by promoting a pro-tumorigenic inflammatory milieu via changes in microbiota.

Decreased melatonin secretion is associated with increased intestinal permeability and marker of endotoxemia in alcoholics.

Chronic heavy alcohol use is known to cause gut leakiness and alcoholic liver disease (ALD), but only 30% of heavy drinkers develop increased intestinal permeability and ALD. The hypothesis of this study was that disruption of circadian rhythms is a potential risk factor in actively drinking alcoholics for gut leakiness and endotoxemia. We studied 20 subjects with alcohol use disorder (AD) and 17 healthy controls (HC, 6 day workers, 11 night workers). Subjects wore a wrist actiwatch for 7 days and underwent a 24-h dim light phase assessment and urine collection for intestinal permeability. The AD group had significantly less total sleep time and increased fragmentation of sleep (P < 0.05). AD also had significantly lower plasma melatonin levels compared with the HC [mean area under the curve (AUC) 322.78 ± 228.21 vs. 568.75 ± 304.26 pg/ml, P = 0.03]. In the AD group, AUC of melatonin was inversely correlated with small bowel and colonic intestinal permeability (lactulose-to-mannitol ratio, r = -0.39, P = 0.03; urinary sucralose, r = -0.47, P = 0.01). Cosinor analysis of lipopolysaccharide-binding protein (marker of endotoxemia) and lipopolysaccharide every 4 h for 24 h in HC and AD subjects had a midline estimating statistic of rhythm of 5,026.15 ± 409.56 vs. 6,818.02 ± 628.78 ng/ml (P < 0.01) and 0.09 ± 0.03 vs. 0.15 ± 0.19 EU/ml (P < 0.05), respectively. We found plasma melatonin was significantly lower in the AD group, and lower melatonin levels correlated with increased intestinal permeability and a marker of endotoxemia. Our study suggests the suppression of melatonin in AD may promote gut leakiness and endotoxemia.

The Role of miR-212 and iNOS in Alcohol-Induced Intestinal Barrier Dysfunction and Steatohepatitis.

BACKGROUND: Alcoholic liver disease is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of intestinal tight junction proteins, such as Zonula Occludens-1 (ZO-1), plays an important role in alcohol-induced gut leakiness. However, the mechanism of alcohol-induced disruption of tight junction proteins is not well established. The goal of this study was to elucidate this mechanism by studying the role of microRNA 212 (miR-212) and inducible nitric oxide synthase (iNOS) in alcohol-induced gut leakiness. METHODS: The permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance and flux of fluorescein sulfonic acid. miR-212 was measured by real-time polymerase chain reaction. The wild-type, iNOS knockout, and miR-212 knockdown mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. The LNA-anti-miR-212 was used to inhibit miR-212 expression in mice. The alcohol-induced intestinal permeability, miR-212 expression, and liver injuries in mice were measured. RESULTS: Our in vitro monolayer and in vivo mice studies showed that: (i) alcohol-induced overexpression of the intestinal miR-212 and intestinal hyperpermeability is prevented using miR-212 knockdown techniques; and (ii) iNOS is up-regulated in the intestine by alcohol and that iNOS signaling is required for alcohol-induced miR-212 overexpression, ZO-1 disruption, gut leakiness, and steatohepatitis. CONCLUSIONS: These studies thus support a novel miR-212 mechanism for alcohol-induced gut leakiness and a potential target that could be exploited for therapeutic intervention to prevent leaky gut and liver injury in alcoholics.

Evaluating the patient with low back pain.

In the UK, low back pain is the most common cause of disability in young adults and every year 6-9% of adults consult their GP about back pain. A thorough history and examination is required to exclude an alternative diagnosis, such as pain arising from the hip or trochanteric bursa and to categorise patients as having: serious spinal pathology, nerve root/radicular pain or non-specific back pain. Inflammatory back pain is often missed, particularly in the early stages when examination may be normal. The primary features are pain arising in patients under 40, thoracolumbar or sacroiliac pain and alternating buttock pain. Stiffness in the early morning and after rest is a hallmark of inflammatory back pain. There may also be peripheral joint involvement with evidence of inflammatory arthritis as well as extra-articular manifestations such as iritis, psoriasis and colitis. Sphincter disturbance leading to loss of bladder or bowel control should also be explored as it is a sign of spinal cord compression or cauda equina syndrome. Both of these are neurosurgical emergencies and need urgent referral for further investigation and possible intervention. The majority of patients with low back pain can be managed in primary care as the pain will usually be self-limiting. Patients with suspected inflammatory back pain should be referred to rheumatology as soon as possible in order to institute early management and prevent long-term deformity and disability. Patients with suspected serious spinal pathology should be referred urgently for further investigation. Red flag symptoms should raise concerns regarding a possible sinister cause such as malignancy and more than one red flag mandates urgent further investigation.

Progression of intestinal permeability changes and alpha-synuclein expression in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a multifocal degenerative disorder for which there is no cure. The majority of cases are sporadic with unknown etiology. Recent data indicate that untreated patients with de novo PD have increased colonic permeability and that both de novo and premotor patients have pathological expression of α-synuclein (α-syn) in their colon. Both endpoints potentially can serve as disease biomarkers and even may initiate PD events through gut-derived, lipopolysaccharide (LPS)-induced neuronal injury. Animal models could be ideal for interrogating the potential role of the intestines in the pathogenesis of PD; however, few current animal models of PD encompass these nonmotor features. We sought to establish a progressive model of PD that includes the gastrointestinal (GI) dysfunction present in human patients. C57/BL6 mice were systemically administered one dose of either LPS (2.5 mg/kg) or saline and were sacrificed in monthly intervals (n = 5 mice for 5 months) to create a time-course. Small and large intestinal permeability was assessed by analyzing the urinary output of orally ingested sugar probes through capillary column gas chromatography. α-Syn expression was assessed by counting the number of mildly, moderately, and severely affected myenteric ganglia neurons throughout the GI tract, and the counts were validated by quantitative optical density measurements. Nigrostriatal integrity was assessed by tyrosine hydroxylase immunohistochemistry stereology and densitometry. LPS caused an immediate and progressive increase in α-syn expression in the large intestine but not in the small intestine. Intestinal permeability of the whole gut (large and small intestines) progressively increased between months 2 and 4 after LPS administration but returned to baseline levels at month 5. Selective measurements demonstrated that intestinal permeability in the small intestine remained largely intact, suggesting that gut leakiness was predominately in the large intestine. Phosphorylated serine 129-α-syn was identified in a subset of colonic myenteric neurons at months 4 and 5. Although these changes were observed in the absence of nigrostriatal degeneration, an abrupt but insignificant increase in brainstem α-syn was observed that paralleled the restoration of permeability. No changes were observed over time in controls. LPS, an endotoxin used to model PD, causes sequential increases in α-syn immunoreactivity, intestinal permeability, and pathological α-syn accumulation in the colon in a manner similar to that observed in patients with PD. These features are observed without nigrostriatal degeneration and incorporate PD features before the motor syndrome. This allows for the potential use of this model in testing neuroprotective and disease-modifying therapies, including intestinal-directed therapies to fortify intestinal barrier integrity.