John Hermon-Taylor

Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis

Trypsinogen activation can be quantified by measurement of released activation peptides (TAP assay). TAP assay in urine was performed on admission for 55 patients with acute pancreatitis. TAP concentration correlated with subsequent disease severity in 87%, whereas C-reactive protein concentration, and multifactorial scoring at 48 h, were correct in 55% and 84%. Sensitivity and specificity for TAP assay were 80% and 90%, for C-reactive protein 53% and 55%, and for multifactorial scoring at 48 h, 60% and 93%. Urine TAP assay distinguishes acute pancreatitis without trypsinogen activation from acute pancreatitis with trypsinogen activation, and helps to identify patients who will progress to the severe acute disease. Use of the assay should allow early intensive treatment of those who need it.

Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohns disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohns disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohns disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.

Characterization of IS900 loci in mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohns disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/Bst:EII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johnes disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacterium avium subsp. paratuberculosis infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohns disease and healthy control groups were also included. Mycobacterium avium subsp. paratuberculosis was detected by IS900 PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained. Mycobacterium avium subsp. paratuberculosis was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohns disease (P = 0.0003 for irritable bowel syndrome patients versus healthy controls and P = 0.0000 for Crohns disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS900 that was also found in isolates from seven of eight dairy sheep. There was a significant association (P = 0.0018) between Mycobacterium avium subsp. paratuberculosis infection and the consumption of hand-made cheese. Mycobacterium avium subsp. paratuberculosis is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohns disease.

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

SummaryConclusionsUrinary TAP obtained within the first 48 h of the onset of symptoms can distinguish patients with severe acute pancreatitis.BackgroundUrinary trypsinogen activation peptide (TAP) has recently been described as an early marker of severity in acute pancreatitis.MethodsIn 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.ResultsMedian 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 48h 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 48h 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≥10 for severe pancreatitis was 100 and 85%, respectively. Given a cutoff of 10 ng/mL for an admission urinary TAP obtained within 48h of the onset of symptoms, the negative predictive value was 100% for mild pancreatitis.

Development of radioimmunoassays for free tetra-L-aspartyl-L-lysine trypsinogen activation peptides (TAP)

Tetra-L-aspartyl-L-lysine (D4K) containing trypsinogen activation peptides were synthesised on solid-phase supports. Synthetic D4K peptides were N-terminally haptenised and used to generate specific C-terminally directed anti-D4K antibodies. Affinity purification of antisera using Sepharose-immobilised synthetic D4K segregated two highly purified populations of anti-D4K antibodies, one eluting with EDTA recognising the calcium chelate and the other eluting with propionic acid recognising an alternative epitope on the anionic oligopeptide. Both specific anti-D4K antibodies were C-terminally directed and did not bind trypsinogen. Specific antisera and calcium-independent antibodies were used to develop and characterise solution and solid-phase immunoassays specific for free trypsinogen activation peptides (TAP assay), with a detection limit of 10(-11) M and between assay CV of 10.7% for the solution-phase system. The release of D4K peptides by enteropeptidase activation of trypsinogen and dog pancreatic secretion is demonstrated. TAP assays specifically indicate trypsinogen activation and may contribute to the recognition and understanding of disease states such as pancreatitis.

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.

Mycobacterium avium subspecies paratuberculosis , Crohn's disease and the Doomsday scenario

Johnes disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johnes is a new disease which emerged at the turn of the 19th and 20th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohns disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohns disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohns disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease result from the perturbed neuroimmune response to penetration into the gut wall of secondary pathogens from the lumen. These include both normal gut organisms and educated members of the enteric microbiome such as more aggressive E. coli. More new diseases may arise from failure to apply a range of remedial measures to this longstanding zoonotic problem.

DNA PROBES DEMONSTRATE A SINGLE HIGHLY CONSERVED STRAIN OF MYCOBACTERIUM AVIUM INFECTING AIDS PATIENTS

Strains of the Mycobacterium avium intracellulare complex (MAIC) have become important colonisers of patients with acquired immunodeficiency syndrome (AIDS). Restriction fragment length polymorphisms were used to study the DNA from 88 MAIC isolates, including 51 derived from 47 AIDS patients. MAIC isolates from 33 of 45 AIDS patients were identical at the molecular level and distinct from the mycobacteria isolated from the stools of healthy subjects. The study also showed that serotyping correlates poorly with the genetic identity of these organisms. Mycobacterium paratuberculosis, which has been implicated in Crohns disease, was not identified in any of the cultures studied.

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

ABSTRACT Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johnes disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohns disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.