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Biomedical subjects

S A Naser

Publications and source records attributed to S A Naser.

17 recordsLinked to original sources
PubMed2006

A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens.

A novel fluorescence imaging technique based on deconvolution microscopy and spectral analysis is presented here as an alternative to confocal laser scanning microscopy. It allowed rapid, specific and simultaneous identification of five major opportunistic pathogens, relevant for public health, in suspension and provided quantitative results.

Bacteria↗
PubMed2002

In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy.

The diversity in the methodology employed to investigate Crohn's disease (CD) etiology has added significantly to the controversy of the mycobacterial role in this chronic inflammatory bowel disease. Mycobacterium avium subsp paratuberculosis (MAP), a proposed and suspected agent in many CD patients, is a fastidious and very slow grower bacillus, which causes Johne's disease (JD) in cattle. The methodology that has been widely and successfully used for isolation and identification of MAP from and in JD animals is not reliable and has proven to be unsuccessful in achieving the same objectives for CD diagnosis. In this study, a Confocal Scanning Laser Microscopy (CSLM) system has been employed in an attempt to detect MAP in CD patient. In situ hybridization was performed on full thickness tissue using rabbit anti-MAP polyclonal antibody that was adsorbed with E. coli protein extracts. Consequently, MAP was detected in the microvilli region in tissue specimens from CD patient and not in the controls. In the same CD tissue specimen, MAP was not detected when isotype normal rabbit sera was employed. The polyclonal antibody marker may be replaced with monoclonal antibodies, if available, or with MAP-specific-DNA or RNA probes. This technique adds an additional approach to investigate MAP role in CD etiology especially when the culture approach is long and inconsistent.

Animals↗
PubMed2002

Open clinical trial of rifabutin and clarithromycin therapy in Crohn's disease.

BACKGROUND: Crohn's disease, an inflammatory bowel disease in humans, has a suspected aetiology of Mycobacterium avium subsp. Paratuberculosis. AIMS: To evaluate the role of rifabutin and clarithromycin anti-Mycobacterium avium subsp. Paratuberculosis treatment in Crohn's disease patients using an open clinical trial. METHODS: . A total of 36 patients with acute presentations of Crohn's disease, whose sera tested positive against p35 and p36 antigens (two recombinant proteins of Mycobacterium avium subsp. Paratuberculosis), were selected for treatment with rifabutin and macrolide antibiotic therapy Rifabutin and macrolide antibiotic therapy medications included 250 mg 1 po bid clarithromycin and 150 mg 1 po bid Ri-fabutin accompanied with a probiotic. Crohn's disease patients' response to rifabutin and macrolide antibiotic therapy was monitored over a period ranging from 4 to 17 months. RESULTS: Seven patients (19.4%) withdrew from the study since they were unable to tolerate medications. Of the remaining 29 patients, 21 (58.3%) reached a sustained state of improvement, traditionally defined as a decrease of 70 points between their entrance and exit Crohn's disease activity index scores together with the absence of the need of all other Crohn's medications, such as immunosuppressants and corticosteroids. Three Crohn's disease patients [8. 3%) noticed significant improvements, but required other Crohn's medications, concurrently with rifabutin and macrolide antibiotic therapy, to achieve and sustain improvement. Only 5 Crohn's disease patients (13.8%) were non-responders, noticing no marked improvement while on rifabutin and macrolide antibiotic therapy. CONCLUSION: The data add further evidence to support the role of rifabutin and macrolide antibiotic therapy in the treatment of Crohn's disease specifically in those patients with evidence of Mycobacterium avium subsp. Paratuberculosis infection. A large multi-centre clinical trial is needed to further explore these findings.

Adolescent↗
PubMed2000

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients. Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.

Adolescent↗
PubMed2000

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples.

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.

Animals↗
PubMed2000

Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization.

M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn's disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis.

Animals↗
PubMed2000

Use of short-term culture for identification of Mycobacterium avium subsp. paratuberculosis in tissue from Crohn's disease patients.

OBJECTIVE: To investigate the role of Mycobacterium avium subsp. paratuberculosis (MAP) in Crohn's disease (CD), using short-term mycobacterial culture media. METHODS: Sixty-three tissue specimens from 27 CD patients and 36 controls were processed and inoculated into a modified 7H9 broth base medium and incubated at 37 degrees C and 5% CO2 for up to 1 year. Acid-fast staining, determination of mycobactin dependency, PCR analysis using two IS900-derived oligonucleotides and hybridization with an internal probe were performed. RESULTS: MAP was present in six of seven (86%) surgically resected tissue samples and in four of 20 (20%) biopsies, with an overall 37% from CD patients, as compared to two of 36 (5.6%) of control specimens. The presence of MAP in Mycobacterial Growth Indicator Tube (MGIT) cultures was detected within 10-12 weeks for surgically resected tissue and after 40 weeks for biopsy specimens, with no MAP growth detected in 12B* Bactec cultures. CONCLUSIONS: Because MAP was present in 86% of resected tissue compared to 20% of biopsy specimens from CD patients, we speculate that MAP resides in the submucosal layer closer to the active part of the ulcer rather than on the surface of the mucosal cells. Thus, surgically resected tissue cultured in MGIT medium is a favorable protocol for rapid cultivation of MAP and for investigating its role in CD pathogenesis. The data support the mycobacterial role in CD pathogenesis.

Crohn Disease↗
PubMed1999

Occurrence of the IS900 gene in Mycobacterium avium complex derived from HIV patients.

The occurrence of the insertion sequence IS900 in Mycobacterium avium subsp. avium strains isolated from HIV infected patients has been investigated. In this study, genomic DNA from 62 mycobacterial isolates [31 strains of M. avium complex (MAC) consisting of 26 M. avium subsp. avium HIV-isolates and five non-HIV isolates and 31 additional Mycobacterium species] were analysed by an IS900 -based polymerase chain reaction (PCR) assay and Southern hybridization using a non-radioactive-labelled 251 bp DNA fragment located at the 5'-region of the IS900 sequence. As expected, none of the 28 Mycobacterium species contained the IS900 in their genomic DNA. Of the 26 M. avium subsp. avium HIV-isolates, 15 (57.6%) were strongly positive for the IS900 or IS900 related sequence. The five pulmonary non-HIV MAC isolates were negative for the IS900. As expected, the three strains of M. avium subsp. paratuberculosis were positive. This PCR guided study suggest that the IS900 gene is very common in clinical strains of M. avium subsp. avium especially those isolated from HIV patients. Ultimately the IS900 PCR-based assay may provide a useful tool for diagnostic and epidemiological studies related to MAC infections in HIV patients.

Animals↗
PubMed1999

Characterization of Mycobacterium paratuberculosis p36 antigen and its seroreactivities in Crohn's disease.

Recent data using improved cultural, molecular, and serological techniques have strengthened the association of Mycobacterium paratuberculosis with Crohn's disease, an inflammatory bowel disease (IBD) with unknown etiology. To provide more evidence of an etiological association, antibody reactivities of Crohn's disease patients were tested by immunoblotting against M. paratuberculosis-recombinant antigens. A clone containing a 1,402-bp insert and expressing a 36K-antigen (p36) was analyzed. No homology was found between the deduced amino acid sequence of p36 and any protein sequences compiled in the GenBank indicating that p36 is a novel mycobacterial protein. The reactivity of 199 serum samples was tested against the p36 by immunoblotting technique. Sera from 77 of 89 (86.5%) Crohn's disease patients and 16 of 18 (89%) sera from patients with tuberculosis and leprosy reacted with p36 compared to 5 of 42 (12%) ulcerative colitis and non-IBD control sera (p < 0.0001). In addition, p36 reacted to all sera from 10 normal controls that were Bacillus Calmette-Guerin (BCG)-immunized and only to 10% of 40 normal controls that were not BCG-immunized. The fact that sera from Crohn's disease patients reacted to p36 with the same high frequency as the sera from patients that were exposed to mycobacterial antigens further supports the hypothesis of the mycobacterial etiology in Crohn's disease.

Antibodies, Bacterial↗
PubMed1998

Effect of IS900 gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis.

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 microM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33. 5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis.

Bacterial Proteins↗
PubMed1997

Characterization of a specific Mycobacterium paratuberculosis recombinant clone expressing 35,000-molecular-weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease.

Johne's disease is a chronic enteritis of ruminants associated with enormous worldwide economic losses for the dairy cow- and goat-rearing industries. Management limitations and eradication programs for this disease have been hampered by the lack of a simple and specific diagnostic test for the detection of subclinical cases. We used a recombinant clone expressing a 35,000-molecular-weight Mycobacterium paratuberculosis antigen (p35 antigen) from a previously constructed expression library of M. paratuberculosis in Escherichia coli. The DNA fragment encoding the p35 gene hybridized only to DNA from Mycobacterium avium complex, but not to DNAs from other mycobacteria and nonmycobacterial organisms. The seroreactivity of p35 was evaluated by immunoblotting against 57 reference serum samples obtained from infected and uninfected animals. p35 was recognized by sera from 100% of animals with advanced Johne's disease (clinical stage) (12 cattle, 2 goats, and 2 sheep) and by sera from 75% of 20 cattle with early infection (subclinical stage). None of the sera from 15 M. paratuberculosis-free cows, 3 Mycobacterium bovis BCG-infected tuberculous cattle, or 3 cows artificially inoculated with multiple doses of viable M. paratuberculosis reacted with p35. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 86, 100, 100, and 75%, respectively. The accuracy of p35 immunoblotting was superior to those of commercially available diagnostic tests for Johne's disease. These results suggest that the p35 recombinant protein has potential for use in the serodiagnosis of animals with Johne's disease at all stages of infection. The DNA fragment encoding p35 may also serve as a probe for identification of M. avium complex infection.

Animals↗
PubMed1996

Identification of Mycobacterium avium complex in sarcoidosis.

Cell wall-defective bacteria which later reverted to acid-fast bacilli have been isolated from sarcoid tissue. These have not been conclusively shown to be mycobacteria. Specific PCR assays were applied to identify mycobacterial nucleic acids in these cultured isolates and in fresh specimens obtained from patients with sarcoidosis. Positive amplification and hybridization were observed with Mycobacterium avium complex- and/or Mycobacterium paratuberculosis-specific probes in five of the six cultured isolates and two fresh skin biopsy samples and one cerebrospinal fluid specimen. There was no amplification or hybridization with Mycobacterium tuberculosis or M. avium subsp. silvaticum probes, respectively. Patients' sera were also tested for antibody reactivities by immunoblotting with M. paratuberculosis recombinant clones expressing the 36,000-molecular-weight antigen (36K antigen) (p36) and the 65K heat shock protein (PTB65K). All seven sarcoidosis, four of six tuberculosis, and all six leprosy patient serum specimens showed strong reactivity with p36 antigen. In contrast, 13 of 38 controls showed only weak reactivity with p36 (P = 0.002 for controls versus sarcoidosis samples). Similarly, PTB65K reacted with high intensity with sera from 5 of 5 sarcoidosis, 5 of 6 tuberculosis, and 5 of 6 leprosy patients, compared with its low-intensity reaction with 5 of 22 controls (P = 0.001 for controls versus sarcoidosis samples). This study demonstrates the isolation and/or identification of M. paratuberculosis or a closely related M. avium complex strain from sarcoid skin lesions and cerebrospinal fluid. Furthermore, the reactivity of antibodies in sarcoid patient sera against p36 and PTB65K antigens was comparable to the reactivity of sera obtained from patients with known mycobacterial disease. Collectively, these data provide further support for the theory of the mycobacterial etiology of sarcoidosis.

DNA, Bacterial↗
PubMed1995

Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.

Animals↗
PubMed1994

Identification and characterization of Mycobacterium paratuberculosis recombinant proteins expressed in E. coli.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants, and it has also been isolated and identified from patients with Crohn's disease, an inflammatory bowel disease. The control of Johne's disease has been hampered by the lack of a reliable diagnostic test because of the large degree of antigenic cross-reactivity between mycobacterial and non-mycobacterial species. To help identify specific antigen(s) or epitope(s), an M. paratuberculosis expression library was screened with antibodies and DNA probes. In total, 54 clones were randomly picked, purified, and characterized by DNA probes and monoclonal antibodies with known specificity to individual mycobacterial antigens. Four clones carrying the heat shock protein 65K-, two representing the secreted protein 32K-, three representing the 21K-, and 20 clones representing the specific insertion element of M. paratuberculosis (IS900)-encoding genes and their gene products were identified and characterized. Well-defined recombinant antigens and/or epitopes representing M. paratuberculosis may facilitate the development of specific diagnostic tests and the investigation of their role in these chronic diseases.

Antibodies, Monoclonal↗
PubMed1993

Low temperature protocol for efficient transformation of Mycobacterium smegmatis spheroplasts.

Spheroplasts of Mycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0 x 10(8) to 1.0 x 10(9) spheroplasts to saturating DNA (1 microgram) for 15 min at 5 degrees C resulted in a transfection efficiency of approximately 0.009% . The transfer of the beta-lactamase marker with DNA purified from strain LM15 to spheroplasts of a beta-lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillin-resistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for beta-lactamase Cell-free extracts of penicillin-resistant transformants contained beta-lactamase activity that ranged from 0.046 to 0.134 micromol of benzylpenicillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation of M. smegmatis.

Mycobacteriophages↗