The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation.

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.

Macrophage regulation of the "second brain": CD163 intestinal macrophages interact with inhibitory interneurons to regulate colonic motility - evidence from the Cx3cr1-Dtr rat model.

Intestinal macrophages are well-studied for their conventional roles in the immune response against pathogens and protecting the gut from chronic inflammation. However, these macrophages may also have additional functional roles in gastrointestinal motility under typical conditions. This is likely to occur via both direct and indirect influences on gastrointestinal motility through interaction with myenteric neurons that contribute to the gut-brain axis, but this mechanism is yet to be properly characterised. The CX3CR1 chemokine receptor is expressed in the majority of intestinal macrophages, so we used a conditional knockout Cx3cr1-Dtr (diphtheria toxin receptor) rat model to transiently ablate these cells. We then utilized ex vivo video imaging to evaluate colonic motility. Our previous studies in brain suggested that Cx3cr1-expressing cells repopulate by 7 days after depletion in this model, so we performed our experiments at both the 48 hr (macrophage depletion) and 7-day (macrophage repopulation) time points. We also investigated whether inhibitory neuronal input driven by nitric oxide from the enteric nervous system is required for the regulation of colonic motility by intestinal macrophages. Our results demonstrated that CD163-positive resident intestinal macrophages are important in regulating colonic motility in the absence of this major inhibitory neuronal input. In addition, we show that intestinal macrophages are indispensable in maintaining a healthy intestinal structure. Our study provides a novel understanding of the interplay between the enteric nervous system and intestinal macrophages in colonic motility. We highlight intestinal macrophages as a potential therapeutic target for gastrointestinal motility disorders when inhibitory neuronal input is suppressed.

Helicobacter pylori Chronic Infection Selects for Effective Colonizers of Metaplastic Glands.

Chronic gastric infection with Helicobacter pylori can lead to progressive tissue changes that culminate in cancer, but how H. pylori adapts to the changing tissue environment during disease development is not fully understood. In a transgenic mouse gastric metaplasia model, we found that strains from unrelated individuals differed in their ability to infect the stomach, to colonize metaplastic glands, and to alter the expression of the metaplasia-associated protein TFF3. H. pylori isolates from different stages of disease from a single individual had differential ability to colonize healthy and metaplastic gastric glands. Exposure to the metaplastic environment selected for high gastric colonization by one of these strains. Complete genome sequencing revealed a unique alteration in the frequency of a variant allele of the putative adhesin sabB, arising from a recombination event with the related sialic acid binding adhesin (SabA) gene. Mutation of sabB in multiple H. pylori strain backgrounds strongly reduced adherence to both normal and metaplastic gastric tissue, and highly attenuated stomach colonization in mice. Thus, the changing gastric environment during disease development promotes bacterial adhesin gene variation associated with enhanced gastric colonization. IMPORTANCE Chronic infection with Helicobacter pylori is the primary risk factor for developing stomach cancer. As disease progresses H. pylori must adapt to a changing host tissue environment that includes induction of new cell fates in the cells that line the stomach. We tested representative H. pylori isolates collected from the same patient during early and later stages of disease in a mouse model where we can rapidly induce disease-associated tissue changes. Only the later-stage H. pylori strains could robustly colonize the diseased stomach environment. We also found that the ability to colonize the diseased stomach was associated with genetic variation in a putative cell surface adhesin gene called sabB. Additional experiments revealed that SabB promotes binding to stomach tissue and is critical for stomach colonization by the late-stage strains. Thus, H. pylori diversifies its genome during disease progression and these genomic changes highlight critical factors for bacterial persistence.

Examining enteric nervous system function in rat and mouse: an interspecies comparison of colonic motility.

Gastrointestinal motility is crucial to gut health and has been associated with different disorders such as inflammatory bowel diseases and postoperative ileus. Despite rat and mouse being the two animal models most widely used in gastrointestinal research, minimal studies in rats have investigated gastrointestinal motility. Therefore, our study provides a comparison of colonic motility in the mouse and rat to clarify species differences and assess the relative effectiveness of each animal model for colonic motility research. We describe the protocol modifications and optimization undertaken to enable video imaging of colonic motility in the rat. Apart from the broad difference in terms of gastrointestinal diameter and length, we identified differences in the fundamental histology of the proximal colon such that the rat had larger villus height-to-width and villus height-to-crypt depth ratios compared with mouse. Since gut motility is tightly regulated by the enteric nervous system (ENS), we investigated how colonic contractile activity within each rodent species responds to modulation of the ENS inhibitory neuronal network. Here we used Nω-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide synthase (NOS) to assess proximal colon responses to the stimulatory effect of blocking the major inhibitory neurotransmitter, nitric oxide (NO). In rats, the frequency of proximal colonic contractions increased in the presence of l-NNA (vs. control levels) to a greater extent than in mice. This is despite a similar number of NOS-expressing neurons in the myenteric plexus across species. Given this increase in colonic contraction frequency, the rat represents another relevant animal model for investigating how gastrointestinal motility is regulated by the inhibitory neuronal network of the ENS.NEW & NOTEWORTHY Mice and rats are widely used in gastrointestinal research but have fundamental differences that make them important as different models for different questions. We found that mice have a higher villi length-to-width and villi length-to-crypt depth ratio than rat in proximal colon. Using the ex vivo video imaging technique, we observed that rat colon has more prominent response to blockade of major inhibitory neurotransmitter (nitric oxide) in myenteric plexus than mouse colon.

The Role of Intestinal Macrophages in Gastrointestinal Homeostasis: Heterogeneity and Implications in Disease.

Intestinal macrophages play a key role in the gut immune system and the regulation of gastrointestinal physiology, including gut motility and secretion. Their ability to keep the gut from chronic inflammation despite constantly facing foreign antigens has been an important focus in gastrointestinal research. However, the heterogeneity of intestinal macrophages has impeded our understanding of their specific roles. It is now becoming clear that subsets of intestinal macrophages play diverse roles in various gastrointestinal diseases. This occurs through a complex interplay between cytokine production and enteric nervous system activation that differs for each pathologic condition. Key diseases and disorders in which intestinal macrophages play a role include postoperative ileus, inflammatory bowel disease, necrotizing enterocolitis, as well as gastrointestinal disorders associated with human immunodeficiency virus and Parkinson's disease. Here, we review the identification of intestinal macrophage subsets based on their origins and functions, how specific subsets regulate gut physiology, and the potential for these heterogeneous subpopulations to contribute to disease states. Furthermore, we outline the potential for these subpopulations to provide unique targets for the development of novel therapies for these disorders.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Engineering orthogonal ligand-receptor pairs from "near drugs".

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.

A mouse model of familial porphyria cutanea tarda.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

Altering the reaction specificity of eukaryotic ornithine decarboxylase.

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.

Determination of trace level bromate in drinking water by direct injection ion chromatography.

Disinfection byproduct anions such as bromate, chlorite and chlorate pose significant health risks, even at low microgram/l levels in drinking water. A direct injection, ion chromatographic method was developed using a Dionex AS9-HC anion-exchange column with a carbonate eluent and suppressed conductivity detection for the determination of these disinfection byproduct anions, and bromide, at low microgram/l levels in drinking water. No additional sample pretreatment, other than filtration, is required. The method is linear for the oxyhalides and bromide over the typical concentration range expected for these analytes in drinking water; and quantitative recoveries were obtained for drinking water samples spiked at 10 micrograms/l. This ion chromatographic method, based on the recently developed AS9-HC column, is applicable for the quantitation of bromate in finished drinking water at the 10 micrograms/l maximum contaminant level currently being proposed by the US EPA.

Seasonal activity and relative abundance of Amblyomma americanum in Mississippi.

Ecological investigations of the lone star tick, Amblyomma americanum (L.), were conducted in 3 adjacent 60-m2 plots, located in Noxubee National Wildlife Refuge, Noxubee County, Mississippi. Ticks were collected weekly from July 1992 to July 1993 by flagging randomly selected lanes. During the year, larval ticks were collected first in early July, with peak numbers in September, and they were collected no later than late October. Nymphal ticks were collected from mid-March to late October, with peak numbers occurring in mid-May and early August. Adult ticks were found initially in early March, with peak numbers from mid-May to mid-June and were no longer collected by late August. Analyses of meteorological data indicated that the most influential parameter on tick activity was humidity.

Amiodarone hepatotoxicity: prevalence and clinicopathologic correlations among 104 patients.

The prevalence of apparent amiodarone-related hepatic injury in 104 patients followed prospectively is compared to that reported in the literature. Asymptomatic elevation of serum aminotransferase levels was detected in approximately one-fourth of the patients, a figure similar to the average of reported cases. The frequency of extrahepatic organ toxicity was increased in patients with elevated levels. Symptomatic "hepatitis" developed in 3% of this series and in less than 1% of cases in the literature. Evidence of hepatic phospholipidosis and the development of pseudoalcoholic liver injury is most likely due to the biochemical effects of the drug and to possible metabolic idiosyncrasy, respectively. Serial blood enzyme measurements, as recommended by the manufacturer, may offer some protection against the development of more serious liver injury. However, levels of amiodarone may persist in various tissues for weeks to months following withdrawal, and stopping the drug does not guarantee the prompt reversal of any organ toxicity. Accordingly, the risks posed and benefits offered by amiodarone should be carefully weighed prior to discontinuing the drug, as the risk of sudden cardiac death may outweigh the hazards of ongoing hepatic, pulmonary or other toxicity.

Giant cell carcinoma of the lung. Clinical and roentgenographic manifestations.

Giant cell carcinoma of the lung is an unusual form of pulmonary malignancy that follows an extremely aggressive clinical course. We report the clinical and roentgenographic manifestations of 14 patients with pathologically proven giant cell carcinoma of the lung, and compare our data to other reports in the literature. Our patients often presented with or developed constitutional or nonthoracic symptoms. This neoplasm was characterized by early evidence of widespread metastases. However, extension of tumor to the chest wall was not as frequent in our series as has been previously described. The survival from the time of diagnosis was extremely short. Any hope of successful treatment of this neoplasm depends on prompt, early diagnosis. Pulmonary giant cell carcinoma should be included in the differential diagnosis of large, round or oval, sharply outlined peripheral lung masses.

Sustained and nonsustained ventricular tachycardia: genesis, significance, and management.

Patients with sustained and nonsustained ventricular tachycardia may present the clinician with difficult management problems in the assessment of risk, decision for long-term treatment, and selection of appropriate therapy. Many therapeutic modalities are available, and combinations may be required in a comprehensive treatment program. The development of safer and more effective means for VT control is progressing and is needed. Nonetheless, the results of therapy with many of the treatment programs described are encouraging, as exemplified by the report from Graboys and coworkers. Long-term survival was studied in a group of 123 patients with malignant ventricular arrhythmia treated with antiarrhythmic drugs chosen primarily with noninvasive testing methods. In 98 patients in whom complex ventricular ectopy was controlled, there were 6 sudden deaths, that is, an annual mortality of 2.3 percent. In 25 patients in whom ectopy persisted despite drug therapy, there were 17 sudden deaths. Continued study of the specific mechanisms leading to ventricular tachycardia and sudden death are needed in order to control this increasingly prevalent clinical problem.

Functional aspects of asthma.

Asthma is characterized by reversible abnormalities of maximum airflow rate, lung volumes, and gas exchange. Many different pulmonary function tests can be used to help establish the diagnosis and severity of asthma. Certain methodologic considerations and factors which modify test results are discussed in this article. Reversibility of airways disease can be established by pulmonary function tests before and after bronchodilator treatment. Bronchoprovocation tests are becoming increasingly important both in diagnosis of asthma and as a tool in clinical investigation.

Biodegradation of Xanthan Gum by Bacillus sp.

Strains tentatively identified as Bacillus sp. were isolated from sewage sludge and soil and shown to elaborate extracellular enzymes that degrade the extracellular polysaccharide (xanthan gum, polysaccharide B-1459) of Xanthomonas campestris NRRL B-1459. Enzyme production by one strain was greatly enhanced when the strain was incubated in a mixed culture. Products of degradation were identified as d-glucuronic acid, d-mannose, pyruvylated mannose, 6-O-acetyl d-mannose, and a (1-->4)-linked glucan. These products correlate with the known structure of the gum. The complexity of the product mixture indicated that the xanthanase was a mixture of carbohydrases. The xanthanase complexes were similar to one another in temperature stability, pH and temperature optima, degree of substrate degradation, and enzymolysis products. Differences in pH stability, salt tolerance, recoverability, and yields of enzyme were observed.

Total and hindlimb O2 uptake and blood flow in hypoxic dogs given dopamine.

Cardiovascular interactions between dopamine and hypoxia were examined in 8 anesthetized, paralyzed dogs ventilated at constant rates. Total and hindlimb (less paw) O2 uptake, blood flow, and vascular resistance were measured with and without dopamine infusion of 10 micrograms/kg . min during both normoxic and hypoxic ventilation. Another 8 dogs were similarly treated after beta-blockade with propranolol infusion (1 mg/kg). During the baseline period, normoxic dopamine significantly increased total O2 uptake, cardiac output, and stroke volume, and significantly decreased total vascular resistance in the control group. Hypoxia decreased total O2 uptake, cardiac output, and heart rate but increased total vascular resistance. Dopamine reversed each of these hypoxic changes and restored total O2 uptake to normoxic levels. Hindlimb measurements were not significantly changed by dopamine or hypoxia in the control group. During hypoxia, beta-blockade abolished dopamine's effects except for the decrease in total vascular resistance. The improvement in cardiac output and O2 transport by dopamine infusion resulted from increased stroke volume during normoxia and from increased heart rate during hypoxia.