Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.     

Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.     

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.     

The influence of different cements on the fracture resistance and marginal adaptation of all-ceramic and fiber-reinforced crowns

PURPOSE: This in vitro study investigated the marginal adaptation and fracture resistance of heat-pressed glass-ceramic and fiber-reinforced composite molar crowns luted with resin, resin-modified glass-ionomer, or zinc-oxide-eugenol-free cements. MATERIALS AND METHODS: A total of 24 heat-pressed all-ceramic and 24 glass fiber-reinforced composite crowns were constructed and cemented using the above-mentioned luting agents (eight crowns per cement). The restorations were thermocycled and mechanically stressed, and fracture resistance was determined. Marginal adaptation was evaluated before and after stress application using semiquantitative analysis in a scanning electron microscope. RESULTS: All-ceramic and fiber-reinforced composite crowns reached the highest fracture resistance after stress application in combination with the resin cement. When luted with resin-modified glass-ionomer or zinc-oxide-eugenol-free cements, the fracture resistance of all-ceramics decreased significantly, while the fiber-reinforced composite crowns maintained their fracture resistance level; the lowest values were found for zinc-oxide-eugenol-free cements. The marginal adaptation remained unchanged after stress for all-ceramics and fiber-reinforced composite restorations if they were luted with resin cements. Luting with resin-modified glass-ionomers significantly deteriorated the marginal adaptation after stress application, with the exception of the crown-cement interface of all-ceramics. CONCLUSION: The highest fracture resistance and marginal adaptation were found for all-ceramic and glass fiber-reinforced composite molar crowns if they were luted with resin cement.     

Cholecystokinin-B/Gastrin receptor-targeting peptides for staging and therapy of medullary thyroid cancer and other cholecystokinin-B receptor-expressing malignancies

The high sensitivity of the pentagastrin stimulation test in detecting primary or metastatic medullary thyroid cancer (MTC) suggests a widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in more than 90% of MTCs, but also in a high percentage of small-cell lung cancers, stromal ovarian tumors, and potentially a variety of other tumors, including gastrointestinal adenocarcinomas, neuroendocrine tumors, and malignant glioma. The aim of our work was to develop and systematically optimize suitable radioligands for targeting CCK-B receptors in vivo and to investigate their role in the staging and therapy of MTC and other CCK-B receptor expressing malignancies. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the C-terminal CCK-receptor binding tetrapeptide sequence-Trp-Met-Asp-PheNH(2) or derivatives thereof, were investigated. They were members of the gastrin or cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety. Their stability and affinity were studied and optimized in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in preclinical models. Best tumor uptake and tumor to nontumor ratios were obtained with members of the gastrin family, because of their superior selectivity and affinity for the CCK-B receptor subtype. Radiometal-labeled derivates of minigastrin showed excellent targeting of CCK-B receptor expressing tissues in animals and healthy human volunteers. Preclinical therapy experiments in MTC-bearing animals showed significant antitumor efficacy. In a subsequent clinical study, 45 MTC patients with metastatic MTC were investigated; 23 had known and 22 had occult disease. CCK-B receptor scintigraphy was performed with (111)In-diethylenetriamine pentaacetic acid-d-Glu(1)-minigastrin. The normal organ uptake was essentially confined to the stomach (and, to a lesser extent, to the gallbladder and, in premenopausal women, to normal breast tissue) as a result of CCK-B receptor specific binding and to the kidneys, as excretory organs. All tumor manifestations known from conventional imaging were visualized as early as 1 hour postinjection, with increasing tumor to background ratios over time; at least 1 lesion was detected in 20 of 22 patients with occult disease (patient-based sensitivity, 91%). Among them were local recurrences and lymph node, pulmonary, hepatic, splenic, and bone (marrow) metastases. Eight patients with advanced metastatic disease were injected in a dose-escalation study with potentially therapeutic activities of a (90)Y-labeled minigastrin derivative at 4 to 6-week intervals (30-50 mCi/m(2) per injection for a maximum of 4 injections). Hematologic and renal toxicities were identified as the dose-limiting toxicities at the 40 and 50 mCi/m(2) levels. Two patients experienced partial remissions, and 4 experienced stabilization of their previously rapidly progressing disease. These data suggest that CCK-B receptor ligands may be a useful new class of receptor-binding peptides for diagnosis and therapy of a variety of (CCK-B receptor expressing) tumor types. They allow for sensitive and reliable staging of patients with metastatic MTC. Initial therapeutic results are promising, but nephrotoxicity is a major concern to be solved.