Defective macrophage handling of Escherichia coli in Crohn's disease.

BACKGROUND AND AIM: Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. METHODS: Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). RESULTS: Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CONCLUSIONS: CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure.

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.

Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff.

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.

Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex.

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.

Further studies on the GS element. A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp. silvaticum but not in Mycobacterium avium subsp. paratuberculosis.

We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.

Cerebral trypsinogen expression in human and rat cerebrospinal fluid.

Trypsinogen was identified in cerebrospinal fluid (CSF), where it has not previously been reported and its activation state in experimental subarachnoid haemorrhage (SAH) in rats and in neurosurgical patients was determined. Trypsinogen activation peptide (TAP) release provided an equimolar marker for trypsinogen. Total TAP was significantly reduced to 26% of the baseline level (P<0.02) following experimental SAH in 15 rats but not in ten sham operated controls (P=0.3). TAP was also measured in patients with ruptured (n=11) and unruptured (n=9) aneurysms who underwent craniotomy to clip an aneurysm. Postoperatively there was a significant fall in TAP concentration (P<0.005) in both groups. Trypsinogen, as identified by CSF levels of TAP, is activated by SAH in rats and by craniotomy for aneurysmal clipping in patients.

Hyperenterostatinemia in premenopausal obese women.

Enterostatins [Val-Pro-Asp-Pro-Arg (VPDPR), Val-Pro-Gly-Pro-Arg (VPGPR), and Ala-Pro-Gly-Pro-Arg (APGPR)] are pentapeptides derived from the NH2-terminus of procolipase after tryptic cleavage and belong to the family of gut-brain peptides. Although enterostatin-like immunoreactivities exist in blood, brain, and gut, and exogenous enterostatins decrease fat appetite and insulin secretion in rats, the roles of these peptides in human obesity remain to be examined. To determine whether VPDPR and APGPR secretion is altered in obesity, serum VPDPR and APGPR levels were measured in 38 overnight-fasted subjects (body mass index, 17.9-54.7 kg/m2) before and after a meal. The mean fasting VPDPR in the serum of lean subjects was significantly lower than that in obese subjects [lean = 603 +/- 86 nmol/L (n = 17); obese, 1516 +/- 227 nmol/L (n = 21); P = 0.0023]. In addition, the rise in serum APGPR after a meal (postmeal/fasting ratio) was significantly higher in lean than in obese subjects [lean, 1.71 +/- 0.24 (n = 17); obese, 1.05 +/- 0.14 (n = 21); P = 0.0332]. The results of these studies show hyperenterostatinemia in obesity and a diminution in enterostatin secretion after satiety.

Distribution and characterization of enterostatin-like immunoreactivity in human cerebrospinal fluid.

Enterostatins belong to a family of peptides (e.g., Val-Pro-Asp-Pro-Arg, VPDPR; Ala-Pro-Gly-Pro-Arg, APGPR; and Val-Pro-Gly-Pro-Arg, VPGPR) derived from the tryptic cleavage of amino-terminal pentapeptide from procolipase. Pharmacologic studies have suggested a role for these peptides in appetite regulation and insulin secretion. Studies into the distribution of enterostatins or the role of endogenous peptides have not been possible until now due to the lack of a suitable method for assay. Using two polyclonal antibodies raised against VPDPR and APGPR and different chromatographic methods, we have examined the nature and distribution of enterostatin-like immunoreactivity in human cerebrospinal fluid. The results reported here show for the first time the presence of enterostatin-like immunoreactivity in the human cerebrospinal fluid. Further characterization of cerebrospinal fluid enterostatin-like immunoreactivity revealed that it is not due to APGPR, VPGPR, or VPDPR but to another peptide similar to VPDPR.

Urinary trypsinogen activation peptide (TAP) predicts severity in patients with acute pancreatitis.

CONCLUSIONS: Urinary TAP obtained within the first 48 h of the onset of symptoms can distinguish patients with severe acute pancreatitis. BACKGROUND: Urinary trypsinogen activation peptide (TAP) has recently been described as an early marker of severity in acute pancreatitis. METHODS: In a multicenter study, urine samples were collected for TAP concentration at 6-12, 24, and 48 h after admission from 139 patients with acute pancreatitis (99 with mild disease, 40 with severe disease) and from 50 control patients. Severity of acute pancreatitis was defined by the presence of organ failure and/ or pancreatic necrosis on dynamic contrast-enhanced computed tomography. RESULTS: Median urinary TAP in the 139 patients with acute pancreatitis compared to the 50 control patients was significantly higher at admission, 4.6 vs 0.8 ng/mL (p < 0.001), and 6-12 h, 1.9 vs 0.55 ng/mL (p = 0.04). Among patients who presented within 48 h of the onset of symptoms, the median urinary TAP for severe pancreatitis (9 patients) compared to mild pancreatitis (40 patients) was significantly higher at admission, 29.6 vs. 3.6 ng/mL (p = 0.001). Also, when obtained within 48 h of the onset of symptoms, all patients with severe pancreatitis had an admission urinary TAP level > 10 ng/mL. The sensitivity and specificity of an admission urinary TAP > or = 10 for severe pancreatitis was 100 and 85%, respectively. Given a cutoff of 10 ng/mL for an admission urinary TAP obtained within 48 h of the onset of symptoms, the negative predictive value was 100% for mild pancreatitis.

Two-year-outcomes analysis of Crohn's disease treated with rifabutin and macrolide antibiotics.

Fifty-two patients with severe Crohn's disease were enrolled in this study. Six (11.5%) were intolerant of the medication and had to be excluded. The remaining 46 patients were treated with rifabutin in combination with a macrolide antibiotic (clarithromycin or azithromycin). Patients were treated for a mean of 18.7 (range 6-35) months and followed up for 25.1 (range 7-41) months. Of the 19 patients who were steroid dependent at the start of this study, only two continued to require steroids when treatment was established. A reduction in the Harvey-Bradshaw Crohn's disease activity index occurred after 6 months' treatment (P = 0.004, paired Wilcoxon test) and was maintained at 24 months (P < 0.001). An improvement in inflammatory parameters was observed as measured by a reduction in erythrocyte sedimentation rate (P = 0.009) and C-reactive protein (P = 0.03) at 18 months compared with pretreatment levels, and an increase in serum albumin at 12 months (P = 0.04). When subsets of the study population were analysed, patients with pan-intestinal disease achieved better remission at 2 years than did those with less extensive involvement (P = 0.04, Mann-Whitney U-test). No difference in treatment response by age, disease duration, the presence of granulomas on histology, or the occurrence of drug-induced side-effects, was observed. These data suggest that treatment with rifabutin and clarithromycin or azithromycin may result in a substantial clinical improvement in Crohn's disease and justify the conduct of a randomized controlled trial.

IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales.

IS900 PCR for Mycobacterium paratuberculosis was applied to cream, whey, and pellet fractions of centrifuged whole cows' milk. The test and simultaneous control reactions gave correct results for spiked milk and for native milk samples obtained directly from M. paratuberculosis-free, subclinically infected, and clinically infected cows. The test was then applied to units of whole pasteurized cows' milk widely obtained from retail outlets throughout central and southern England from September 1991 to March 1993. With peak periods in January to March and in September to November, when up to 25% of units were affected, an overall 22 of 312 samples (7%) tested positive for M. paratuberculosis. In 18 of the 22 positive samples (81%), the PCR signal segregated to the cream or pellet fractions or both, consistent with the presence of intact mycobacteria. Nine of 18 PCR-positive milk samples (50%) and 6 of 36 PCR-negative milk samples (16%) yielded long-term liquid cultures which tested positive for M. paratuberculosis after 13 to 40 months of incubation, despite overgrowth by other organisms. Taken together with data on the prevalence of M. paratuberculosis infection in herds in the United Kingdom, the known secretion of M. paratuberculosis in milk from subclinically infected animals, and the inability of laboratory conditions simulating pasteurization to ensure the killing of all these slowly growing or unculturable organisms, there is a high risk, particularly at peak times, that residual M. paratuberculosis will be present in retail pasteurized cows' milk in England.

Activation of human granulocyte type 1-prophospholipase A2.

Using an assay for measurement of released type 1-prophospholipase A2 (type 1-proPLA2) propeptides (PROP assay), we have shown that human granulocytes, but not lymphocytes or macrophages, abundantly express this 'pancreatic' type 1-proPLA2 zymogen. Stimulation with tumour necrosis factor-alpha (TNF-alpha) and other cytokines results in the immediate release from granulocytes of a mixture of free propeptides and type 1-proPLA2 precursor. We also found that granulocytes contain an approximately 29 kDa trypsin-like endogenous type 1-proPLA2 activator. PROP assay and TAP (trypsinogen activation peptide) assay of plasma samples accurately predicts the segregation of acute pancreatitis into three clearly defined categories of severity--mild, intermediate and severe--at the time of first hospital admission and over the next few hours of observation. Mild and intermediate pancreatitis are associated with a degree of granulocyte stimulation limited to the release of the unactivated type 1-proPLA2 precursor. Progression to severe disease is accompanied by the activation of granulocyte type 1-proPLA2, apparently carried to completion. This identifies the approximately 29 kDa endogenous activator of type 1-proPLA2 in granulocytes as a critical mediator at a threshold stage in acute pancreatitis, which marks the transition from uncomplicated pancreatitis to the potentially lethal disease. Specific inhibitors of this key regulatory enzyme modelled on the P3-P1 domain of the type 1-proPLA2 activation peptide would seem to be promising candidates for a new class of chemotherapeutic agents.

Cholecystokinin-8 induces edematous pancreatitis in dogs associated with short burst of trypsinogen activation.

To study the early pathogenesis of acute edematous pancreatitis in dogs, we examined the relationship of pancreatic hyperstimulation with cholecystokinin-8 (10 micrograms/kg/hr intravenously for 6 hr) to alterations in circulating pancreatic enzymes and pancreatic morphology with special reference to trypsinogen activation. Cholecystokinin-8 infusion was associated with increases in plasma amylase, lipase, trypsin-like immunoreactivity, and plasma and urine trypsinogen activation peptide. Pancreatic parenchymal swelling and interlobular and subcapsular fluid accumulations were detected ultrasonographically within 2 hr of cholecystokinin-8. Circulating trypsin-like immunoreactivity and trypsinogen activation peptide in urine reached a peak at 2 and 4 hr, respectively, then declined despite progressive increases in circulating amylase and lipase and intrapancreatic fluid. No significant changes were observed in dogs receiving a saline infusion. This study illustrates that cholecystokinin-8 induces edematous pancreatitis in dogs that is associated with a short-lived burst of trypsinogen activation.

Solid-phase hybridization capture of low-abundance target DNA sequences: application to the polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum.

Polymerase chain reaction (PCR) has been widely applied to the detection of microorganisms. Overall sensitivity of PCR tests may be substantially reduced due to a large excess of nontarget DNA and inhibitory substances in the sample. We used a 5'-biotinylated 513-bp probe from the 3' region of the IS 900 element specific for Mycobacterium paratuberculosis (Mptb) to capture target Mptb DNA from crude sample DNA extracts. Captured target DNA was separated using streptavidin-coated magnetic particles (Dynal). Since the IS 900 element shares homology over this region with IS 902 in Mycobacterium avium subsp. silvaticum (Mavs), target DNA from this other pathogen was also retained. Highly specific PCR for the detection of either organism directed to the 5' regions of IS 900 or IS 902 was then performed directly on the solid phase. Hybridization capture of target DNA using sequence adjacent to the desired specific PCR site applied to Mptb increased overall sensitivity of detection in tissue and fecal extracts 10- to 100-fold. False positives due to contamination artifact were substantially excluded since the capture probe did not retain amplicons from the detection PCR. Development of the method to involve covalent 5' immobilization of capture probes on heat-resistant polymers should, in the future, provide a simple system with broad potential applications.