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- Title
- POSSIBLE USE OF P20 ANTIGEN IN SERODIAGNOSIS OF INFLAMMATORY BOWEL DISEASE.
- Creator
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Yang, ShinChieh, Naser, Saleh, University of Central Florida
- Abstract / Description
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Crohn's disease (CD) is an idiopathic, chronic, relapsing inflammatory disorder, which is most commonly involved terminal ileum and colon. The incidence and prevalence of CD has dramatically increased during the last 50 years; however, the etiology and mechanism of this disorder remain unveiled. Besides genetic susceptibility, recent integrated researches investigated the role of environmental triggers such as microflora, measles viruses and mycobacteria in the pathogenesis of CD. The...
Show moreCrohn's disease (CD) is an idiopathic, chronic, relapsing inflammatory disorder, which is most commonly involved terminal ileum and colon. The incidence and prevalence of CD has dramatically increased during the last 50 years; however, the etiology and mechanism of this disorder remain unveiled. Besides genetic susceptibility, recent integrated researches investigated the role of environmental triggers such as microflora, measles viruses and mycobacteria in the pathogenesis of CD. The association between M. avium subsp paratuberculosis (MAP) and CD has been heightened because of clinical resemblance to Johne's disease (JD), a granulomatous enteritis in ruminants caused by MAP. Isolation of MAP from tissue and milk samples from CD patients and from commercial pasteurized milk and dairy products from JD-infected animals implies a possible re-classification of CD as zoonotic disorder. Clinical signs and symptoms of CD are often non-specific and a challenge to distinguish it from other disorders. Current methods for inflammatory bowel disease diagnosis, especially for CD are highly invasive, distressing and expensive. In this study, the recombinant clone pB11 containing 1.1 kb insert, identified from a MAP genomic library constructed in E. coli, expressed a 20 kDa (p20) antigen encoded on 549 bp partial MAP gene with an ORF cloned in frame within pBAD/His cloning vector. Immunoreactivity of p20 was confirmed by Immunoblot. Purified p20 antigen was then used in the development of an enzyme-linked immunosorbent assay (ELISA) for possible serodiagnosis of Inflammatory Bowel Disease (IBD) associated with MAP infection. All variables associated with ELISA test with regard to concentrations, washes and incubations were optimized using hyper immune rabbit t-anti-MAP polyclonal IgG antibodies and sera from CD and non-CD subjects. The cut-off value for positive response was established as 0.3 following the analysis of statistically formulated samples from normal and non-CD subjects. The developed ELISA test was then used to test a blindly coded 2 17 clinical sera. All sera samples were tested in duplicates and in both p20-coated and uncoated micro titer plates. Consequently, 116/134 (87%) CD sera were positive compared to 24/83 (33%) non-CD sera (P<0.05). Specifically anti-MAP IgG was detected in 8/22 (36%) Ulcerative colitis and 16/61 (26%) non-IBD sera. p20-ORF encoding sequence was recloned (pB11/B6) and the expressed protein reactivity remained consistent. Moreover, the full length of the cloned gene was also identified through blast and alignment analysis and predicted to encode 346 amino acids with unknown function and no identity with other known proteins. The latter supports the clinical data, which reflect on the unique characteristics of this antigen. The result so far suggests that the recombinant clone and its subclone derivative may have potential role in serodiagnosis of CD.
Show less - Date Issued
- 2004
- Identifier
- CFE0000089, ucf:46148
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000089
- Title
- SURVIVAL OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN THE POLYMORPHONUCLEAR LEUKOCYTES OF A CROHN'S DISEASE PATIENT.
- Creator
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Rumsey, John, Naser, Saleh, University of Central Florida
- Abstract / Description
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Mycobacterium avium subspecies paratuberculosis (map) is an intracellular pathogen that is known to parasitize macrophages and monocytes. Map infiltrates gastrointestinal tract host tissue where it is the known etiological agent of johne's disease in ruminants and implicated in the etiology of crohn's disease in humans. Map's ability to survive within macrophages enables it to disseminate throughout the rest of the host, possibly infecting other circulating blood leukocytes. In this study,...
Show moreMycobacterium avium subspecies paratuberculosis (map) is an intracellular pathogen that is known to parasitize macrophages and monocytes. Map infiltrates gastrointestinal tract host tissue where it is the known etiological agent of johne's disease in ruminants and implicated in the etiology of crohn's disease in humans. Map's ability to survive within macrophages enables it to disseminate throughout the rest of the host, possibly infecting other circulating blood leukocytes. In this study, the survival and fate of map strain atcc 43015 (human isolate) following phagocytosis was determined using in vitro murine macrophage cell line j774a.1 and polymorphonuclear cells (pmnc's) from five crohn's disease patients. Pmnc's from three healthy individuals and two ulcerative colitis patients, as well as escherichia coli (atcc 11303) and mycobacterium tuberculosis strain h37ra (atcc 25177), were included as controls (moi 10:1). Maturation of the phagosome was determined by evaluating the presence of stage specific markers on the surface of the phagosomal membrane. The endosomal protein, transferrin receptor, and the lysosomal marker, lamp-1, were then immunostained with cy-5 conjugated secondary antibodies, and colocalization of bacteria with each marker was evaluated separately using confocal scanning laser microscopy (cslm). In both tissue models, colocalization of viable map and m. Tuberculosis with the early endosomal marker, transferrin receptor occurred with an estimated five fold higher frequency than did association with the late lysosomal marker, lamp-1, as compared to live e. Coli, and all dead bacterial species. Using differential live/dead staining and fluorescent microscopy, survival of m. Tuberculosis and map was estimated at 85% and 79%, respectively compared to only 14% for e. Coli. The outcome was similar for both tissue culture and pmncs from all patients tested, suggesting that map and m. Tuberculosis can survive readily in both cell types, and regardless of the disease state of the host or the killing power of the cell. Map's survival appears to mimic m. Tuberculosis', suggesting the ability to resist phagolysosome fusion, by maintaining association with the early endosomes. Overall, the data confirms map virulence in host human blood leukocytes.
Show less - Date Issued
- 2004
- Identifier
- CFE0000184, ucf:52838
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000184
- Title
- CELLULAR IMMUNE RESPONSE AND GENE EXPRESSION PROFILING IN CROHN'S DISEASE PATIENTS ASSOCIATED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS.
- Creator
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Romero, Claudia, Naser, Saleh A., University of Central Florida
- Abstract / Description
-
Despite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious,...
Show moreDespite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious, slow growing mycobacterium avium subspecies paratuberculosis (map). Due to the similarities between cd and paratuberculosis, a mycobacterial cause in cd has been proposed. Recent discovery of a possible association between nod2/card15 mutations and risk of cd added support to microorganism-host interactions. In this study, a possible mycobacterial role in cd etiology has been evaluated by investigating the presence of map dna, the state of the cellular immune response and microarray gene expression profiling in peripheral blood and surgical tissue from cd, uc and healthy control subjects. Nested pcr detected map dna in tissue from 10/12(83%) cd patients compared to 1/6(17%) non-ibd subjects. Fluorescence in situ hybridization (fish) with the aid of confocal scanning laser microscopy (cslm) detected map dna in 8/12(67%) cd subjects compared to 0/6(0%) in non-ibd subjects. The detection of map dna by either technique in tissue from cd subjects is significant compared to non-ibd subjects (p < 0.05). Map dna was also detected in both inflamed and non-inflamed tissue from patients with cd suggesting map infiltration in human tissue. Correlation of possible map presence and the function of polymorphonuclear leukocytes (pmn) and peripheral blood mononuclear cells (pbmc) in 19 cd patients and 12 controls have been evaluated. Pmn phagocytosis of viable fitc-map was suppressed in 13/19(68%) cd patients compared to 0/12(0%) in healthy controls (p<0.05). Pbmc phagocytosis of viable fitc-map was suppressed in 5/19(26%) of cd patients compared to 0/12(0%) of healthy controls (p<0.05). The proliferative response of pbmc with t-cell majority from cd and controls subjects was evaluated against pha, candida albicans, pwm and map ppd. Dysfunctional proliferative response against pha was found in 8/19(42%) cd patients compared to 1/12(8.3%) in controls suggesting possible t-cell anergy. Pbmc from 11 cd subjects reacted normally to pha, 7/11(64%) reacted strongly to map ppd suggesting previous exposure to mycobacteria, and 3/11(27%) did not react with map ppd suggesting lack of pre-exposure to mycobacteria. From the seven mycobacterial pre-exposed samples, 6/7(86%) showed a normal ability to recall antigens by activated macrophages when exposed to c. Albicans, and all 7 samples had a normal pwm response. Finally, microarray-chip technology was employed to identify the expression profile of genes that have a role in the immune response of cd patients. Rna was isolated from fresh buffy coats from 8 healthy controls, 2 cd, and 1 uc patients. Chips with an estimated of 30,000 human genes were hybridized to cdna from these samples. We found that 17% of the total number of genes was differentially expressed. Over 200 genes were involved in the immune response, 7 genes where common to both forms of ibd (uc and cd), and 8 genes were found to be either downregulated in cd and upregulated in uc or viceversa. The ifngr1 gene, which encodes the ligand-binding chain of the ifn-gamma receptor, was found to be downregulated in 2/2(100%) of cd patients, but not in uc patients. It is known that defects in ifngr1 are a cause of atypical mycobacterial infection and bcg infection. Patients suffering from this deficiency have an immunologic defect predisposing them to infection with mycobacteria. This correlates with the proposed theory as map being the causative agent of cd. Furthermore, the results indicate a host susceptibility requirement for the establishment of mycobacterial infection in cd patients. Further characterization of ifngr1 using real-time pcr is underway. Collectively, detection of map dna in the majority of cd tissue and the alteration in pmn and pbmc to respond efficiently to map may be related to the fact that mycobacterial pathogens infect phagocytic cells of susceptible hosts and consequently the immune response is dysregulated. Furthermore, the fact that a gene linked to mycobacterial susceptibility was found to be downregulated in cd patients only, strengthens the mycobacterial etiology of cd. In general, the data suggest a possible role for a bacterial pathogen in cd pathogenesis.
Show less - Date Issued
- 2004
- Identifier
- CFE0000170, ucf:46170
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000170
- Title
- CORRELATION OF RPOB GENE MUTATION WITH CLINICAL RIFABUTIN AND RIFAMPICIN RESISTANCE FOR TREATMENT OF CROHN'S DISEASE.
- Creator
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Beckler, Daniel, Naser, Saleh, University of Central Florida
- Abstract / Description
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Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located...
Show moreEmerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain 18 had an MIC > 30 ug/ml and > 5 ug/ml for RIF and RFB respectively, and a significant rpoB mutation C1367T, compared to an MIC of < 1.0 ug/ml for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site; the beta subunit of RNA polymerase. Our in vitro induced mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC > 30 ug/ml and > 10 ug/ml for RIF and RFB respectively. The T1442C mutation resulted in a Leu481 to Pro481 amino acid change, consequently altering the beta subunit sequence. Sequencing the entire 3.5 kb rpoB in strains 18 and UCF5-RIF16r revealed no additional expressed nucleotide mutation. Of the 10 MAP strains analyzed, an additional one strain (UCF4) exhibited a slight increase in the MIC against RIF and RFB compared to the wild-type. Nucleotide sequencing of the rpoB gene revealed an A2284C mutation in strain UCF4 that occurred further downstream of the expected probable rpoB region and resulted in an amino acid alteration Asn762 to His762. The location of this mutation outside the binding site and its correlation with the minor increase in MIC suggests a possible secondary interaction between the drug and the beta subunit. We have provided three dimensional images through the utilization of PyMol Molecular-based Graphics to display a clear comparison of the mutations observed in the beta subunit for MAP strains 18, UCF5-RIF16r, and UCF4. We propose that these alterations may have caused a less stable interaction between RIF and the beta subunit, resulting in the observed increased in MIC. Furthermore, the change in amino acid sequence did not affect the viability for our RIF resistant strains. The data clearly illustrates that clinical and in vitro-induced MAP mutants with rpoB mutations result in resistance to RIF and RFB. Consequently, unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.
Show less - Date Issued
- 2007
- Identifier
- CFE0001729, ucf:47310
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001729
- Title
- Malondialdehyde (MDA) and Glutathione Peroxidase (GPx) are elevated in Crohns disease-associated with Mycobacterium avium subspecies paratuberculosis (MAP).
- Creator
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Qasem, Ahmad, Naser, Saleh, Masternak, Michal, Parthasarathy, Sampath, Andl, Claudia, University of Central Florida
- Abstract / Description
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Inflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The...
Show moreInflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 healthy controls), 27 CD subjects, 27 of CD-healthy relatives, 66 subjects with various diseases and 34 non-related healthy subjects were investigated. Overall, GPx activity was significantly higher in MAP infected humans (0.80941(&)#177;0.521) versus MAP (-ve) samples (0.42367(&)#177;0.229 units/ml), P(<)0.01. Similarly, the average of GPx activity in cattle infected with MAP was 1.59(&)#177;0.65 units/ml compared to 0.46907(&)#177;0.28 units/ml in healthy cattle (P(<)0.01). Although it was not statistically significant, MDA average level was higher in MAP infected human samples versus MAP (-ve) controls (1.11(&)#177;0.185 nmol/ml versus 0.805(&)#177;0.151 nmol/ml, respectively). Similarly, MDA average level in CD samples that are MAP+ (1.703(&)#177;0.231 nmol/ml) was higher than CD samples that are MAP (-ve) (1.429(&)#177;0.187 nmol/ml). In cattle, MDA average level in MAP infected samples was significantly higher at 3.818(&)#177;0.45 nmol/ml compared to 0.538(&)#177;0.18 nmol/ml in healthy cattle (P(<)0.01). Clearly, the data demonstrated that MAP infection is associated with oxidative stress and resulting in the pathophysiology of worsening of the condition of CD patients.
Show less - Date Issued
- 2016
- Identifier
- CFE0006699, ucf:51906
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006699
- Title
- EXPRESSION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS 19KDA WITH POSTTRANSLATIONAL MODIFICATION.
- Creator
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Safavi-Khasraghi, Mitra, Naser, Saleh, University of Central Florida
- Abstract / Description
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Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant...
Show moreDespite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.
Show less - Date Issued
- 2006
- Identifier
- CFE0001170, ucf:52851
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001170
- Title
- Downregulation in IFNGR1 increases suspectiblity to Mycobacterium avium subspecies paratuberculosis infection in Crohn's disease.
- Creator
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Htun, Zin Mar, Naser, Saleh, Andl, Claudia, Tigno-Aranjuez, Justine, University of Central Florida
- Abstract / Description
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BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in...
Show moreBACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (?-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in association with IFNGR1 mutation in CD patients have not been investigated.METHODS: In this study, we investigated blood samples of 79 human subjects for MAP infection in association with IFNGR1 gene dysfunction. Samples were divided into 22 CD, 6 Ulcerative colitis (UC), 32 normal healthy and 19 non-inflammatory bowel disease (NIBD). Five variants of IFNGR1 single nucleotide polymorphisms (SNP) were investigated using Taqman Genotyping assay, then IFNGR1 expression measured by RT-PCR and serum IFNGR1 and ?-IFN levels were measured using ELISA. MAP infection was detected using nested PCR. RESULTS: Among 28 IBD patients, 4/6 (66.67%) of UC and 18/22 (81.82%) of CD are tested positive for at least one SNP homozygous minor form compared to 21.88% and 47.37%% in 32 healthy and 19 NIBD (P (<)0.05). IFNGR1 gene expression was downregulated 1.4-fold in IBD patients (P =0.07) and 1.7-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients (P=0.06). Serum IFNGR1 protein levels were downregulated 1.53-fold in IBD patients compared to normal, and 1.4-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients. MAP infection is more common in rs2234711 SNP positive patients (5/7 =71.42%) (P(<)0.05). Serum ?-IFN levels were not elevated in both groups.CONCLUSION: IFNGR1 SNP's, MAP infection and IFNGR1 downregulation were found in higher incidence in IBD, suggesting role of IFNGR1 in susceptibility of MAP infection in IBD patients.
Show less - Date Issued
- 2017
- Identifier
- CFE0007121, ucf:51951
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007121
- Title
- REAL TIME REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR DIRECT DETECTION OF VIABLE MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN CROHN'S DISEASE PATIENTSANDASSOCIATION OF MAP INFECTION WITH DOWNREGUALTION IN INTERFERON-GAMMA RECEPTOR (INFG1) GENE IN CROHN'S DISEASE PATIENTS.
- Creator
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Chehtane, Mounir, Naser, Saleh, University of Central Florida
- Abstract / Description
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Association of Mycobacterium avium subspecies paratuberculosis (MAP) with Crohn's disease (CD) and not with ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), has been vigorously debated in recent years. This theory has been strengthened by recent culture of MAP from breast milk, intestinal tissue and Blood from patients with active Crohn's disease. Culture of MAP from clinical samples remained challenging due to the fastidious nature of MAP including its lack of cell...
Show moreAssociation of Mycobacterium avium subspecies paratuberculosis (MAP) with Crohn's disease (CD) and not with ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), has been vigorously debated in recent years. This theory has been strengthened by recent culture of MAP from breast milk, intestinal tissue and Blood from patients with active Crohn's disease. Culture of MAP from clinical samples remained challenging due to the fastidious nature of MAP including its lack of cell wall in infected patients. The advent of real time PCR has proven to be significant in infectious disease diagnostics. In this study, real time reverse transcriptase PCR (RT-PCR) assay based on targeting mRNA of the IS900 gene unique to MAP has been developed. All variables included in RNA isolation, cDNA synthesis and real time PCR amplification have been optimized. Oligonucleotide primers were designed to amplify 165 bp specific to MAP and the assay demonstrated sensitivity of 4 genomes per sample. In hope this real time RT-PCR may aid in the detection of viable MAP cells in Crohn's disease patients, a total of 45 clinical samples were analyzed. Portion of each sample was also subjected to 12 weeks culture followed by standard nested PCR analysis. The samples consisted of 17 cultures (originated from 13 CD, 1 UC and 3 NIBD subjects), 24 buffy coat blood (originated from 7 CD, 2 UC, 11 NIBD and 4 healthy subjects) and 4 intestinal biopsies from 2 CD patients. Real time RT-PCR detected viable MAP in 11/17 (65%) of iii suspected cultures compared to 12/17 (70%) by nested PCR including 77% and 84% from CD samples by both methods, respectively. Real time RT-PCR detected MAP RNA directly from 3/7 (42%) CD, 2/2 (100%) UC and 0/4 healthy controls similar to results following long term culture incubation and nested PCR analysis. Interestingly, real time RT-PCR detected viable MAP in 2/11 (13%) compared to 4/11 (26%) by culture and nested PCR in NIBD patients. For tissue samples, real time RT-PCR detected viable MAP in one CD patient with the culture outcome remains pending. This study clearly indicates that a 12-hr real time RT-PCR assay provided data that are similar to those from 12 weeks culture and nested PCR analysis. Consequently, use of real time In our laboratory, we previously demonstrated a possible downregulation in the Interferon-gamma receptor gene (IFNGR1) in patients with active Crohn's disease using microarray chip analysis. In this study, measurement of RNA by real time qRT-PCR indicated a possible downregulation in 5/6 CD patients compared to 0/12 controls. The preliminary data suggest that downregulation in INFGR1 gene, and the detection of viable MAP in CD patients provides yet the strongest evidence toward the linkage between MAP and CD etiology.
Show less - Date Issued
- 2005
- Identifier
- CFE0000629, ucf:46504
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000629
- Title
- SYSTEMATIC REVIEW AND META-ANALYSIS: TUBERCULOSIS, TNFΑ INHIBITORS, AND CROHN'S DISEASE.
- Creator
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Cao, Brent L, Naser, Saleh A., University of Central Florida
- Abstract / Description
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Inflammation is often a protective reaction against harmful foreign agents. However, in many disease conditions, the mechanisms behind the inflammatory response are poorly understood. Often times, the inflammation causes adverse effects, such as joint pain, abdominal pain, fever, fatigue, and loss of appetite. Thus, many treatments aim to inhibit the inflammatory response in order to control adverse symptoms. Such treatments include TNFα inhibitors. However, a major risk associated with drugs...
Show moreInflammation is often a protective reaction against harmful foreign agents. However, in many disease conditions, the mechanisms behind the inflammatory response are poorly understood. Often times, the inflammation causes adverse effects, such as joint pain, abdominal pain, fever, fatigue, and loss of appetite. Thus, many treatments aim to inhibit the inflammatory response in order to control adverse symptoms. Such treatments include TNFα inhibitors. However, a major risk associated with drugs inhibiting tumor necrosis factor alpha (TNFα) is serious infection, including tuberculosis (TB). Anti-TNFα therapy is used to treat patients with Crohn's disease, for which the risk of tuberculosis may be even more concerning. Recent literature suggests Crohn's might involve Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular TB-like bacterium. This study seeks to investigate the risk of developing TB in patients with Crohn's disease treated with TNFα inhibitors. A meta-analysis synthesized existing evidence. Evidence came from published randomized, double-masked, placebo-controlled trials of TNFα inhibitors for treatment of adult Crohn's disease. Twenty-three trials were identified, including 5,669 patients. The risk of tuberculosis was significantly increased in anti-TNFα treated patients, with a risk difference of 0.028 (95% confidence interval [CI], 0.0011-0.055). The odds ratio was 4.85 (95% CI, 1.02-22.99) when all studies were included and 5.85 (95% CI, 1.13-30.38) when studies reporting zero tuberculosis cases were excluded. The risk of tuberculosis is increased in patients with Crohn's disease treated with TNFα inhibitors. The medical community should be alerted about this risk and the potential for TNFα inhibitor usage favoring granulomatous infections and worsening the patient condition.
Show less - Date Issued
- 2018
- Identifier
- CFH2000360, ucf:45909
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000360
- Title
- Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients.
- Creator
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Alcedo, Karel, Naser, Saleh, Cheng, Zixi, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
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Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three...
Show moreCrohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs (-) Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
Show less - Date Issued
- 2015
- Identifier
- CFE0005917, ucf:50831
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005917
- Title
- MOLECULAR TYPING OF MYCOBACTERIAL ISOLATES CULTURED FROM THE TISSUE OF INFLAMMATORY BOWEL DISEASE (CROHN'S DISEASE) PATIENTS.
- Creator
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Adams, Leanne M, Naser, Saleh, University of Central Florida
- Abstract / Description
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The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900...
Show moreThe role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.
Show less - Date Issued
- 2004
- Identifier
- CFE0000031, ucf:46125
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000031
- Title
- Role of Single Nucleotide Polymorphisms (SNPs) in PTPN2/22 and Mycobacterium avium subspecies paratuberculosis (MAP) in Rheumatoid Arthritis and Crohn's Disease.
- Creator
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Sharp, Robert, Naser, Saleh, Parks, Griffith, Roy, Herve, Singla, Dinender, University of Central Florida
- Abstract / Description
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Both genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs...
Show moreBoth genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs effect on PTPN2/22 and IFN-y expression was determined using RT-PCR. Blood samples were analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. T-cell proliferation and response to phytohematoagglutonin (PHA) mitogen and MAP cell lysate were determined by BrdU proliferation assay. Out of 9 SNPs, SNP alleles of PTPN2:rs478582 occurred in 79% RA compared to 60% control (p-values ? 0.05). SNP alleles of PTPN22:rs2476601 occurred in 29% RA compared to 6% control (p-values ? 0.05). For the haplotype combination of PTPN2:rs478582/PTPN22rs2476601, 21.4% RA had both SNPs (C-A) compared to 2.4% control (p-values ? 0.05). PTPN2/22 expression in RA was decreased by an average of 1.2 fold. PTPN2:rs478582 upregulated IFN-y in RA by an average of 1.5 fold. Combined PTPN2:rs478582/PTPN22:rs2476601 increased T-cell proliferation by an average of 2.7 fold when treated with PHA. MAP DNA was detected in 34% RA compared to 8% controls (p-values ? 0.05), where samples with PTPN2:rs478582 and/or PTPN22:rs2476601 were more MAP positive. PTPN2:rs478582/PTPN22:rs2476601 together with MAP infection significantly increased T-cell response and IFN-y expression in RA samples. The same experimental approach was followed on blood samples from CD patients. Both PTPN2:rs478582/PTPN22:rs2476601 affected PTPN2/22 and IFN-y expression along with T-cell proliferation significantly more than in RA. MAP DNA was detected in 64% of CD. This is the first study to report the correlation between SNPs in PTPN2/22, IFN-y expression and MAP in autoimmune disease.
Show less - Date Issued
- 2018
- Identifier
- CFE0007371, ucf:52094
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007371
- Title
- Determining differential effects of Interleukin-2 on innate and adaptive immune cells in lymphoid organs and the gastrointestinal Tract.
- Creator
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Singh, Ayushi, McKinstry, Karl, Naser, Saleh, Copik, Alicja, University of Central Florida
- Abstract / Description
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Interleukin-2 (IL-2) is a pleiotropic cytokine demonstrated to be effective in treating cancer. However, the clinical use of IL-2 can be associated with severe side effects including gastrointestinal toxicity (GT). Similar GT symptoms are observed in inflammatory diseases such as CD (CD). Interestingly mounting evidence indicates a role for IL-2 in CD, but the underlying mechanisms are unknown. Indeed, studies on the in-vivo activities of IL-2 have mostly focused on secondary lymphoid organs...
Show moreInterleukin-2 (IL-2) is a pleiotropic cytokine demonstrated to be effective in treating cancer. However, the clinical use of IL-2 can be associated with severe side effects including gastrointestinal toxicity (GT). Similar GT symptoms are observed in inflammatory diseases such as CD (CD). Interestingly mounting evidence indicates a role for IL-2 in CD, but the underlying mechanisms are unknown. Indeed, studies on the in-vivo activities of IL-2 have mostly focused on secondary lymphoid organs and immune cells associated with them. Very few studies have addressed how IL-2 signals impact populations of immune cells in the gut. Here, we aim to identify and compare the effects of systemic IL-2 administration on six major leukocyte population and their subsets in mice using multicolor flow cytometry. While we confirmed previously observed changes in specific immune cell populations in the spleen, very few changes were seen in the gut and gut associated lymphoid tissues. Unexpectedly, a sharp decline was seen in B cells, most notably in Peyer's Patches, in mice treated with IL-2. Our data furthermore indicates that B cells in IL-2 treated mice undergo enhanced apoptosis in Peyer's Patches. Some studies suggest that changes in B cells may contribute to development of CD. Thus, this study may aid in defining ways in which IL-2 can contribute to disease etiology, and lead to novel treatments for CD.
Show less - Date Issued
- 2019
- Identifier
- CFE0007865, ucf:52777
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007865
