Bioluminescent Quantitation and Detection of Gene Expression During Infectious Disease. Pp. 273-288.
Quantitative and Bioluminescent Assay to Measure Efficacy of Conventional and DNA Vaccinations Against Helicobacter pylori. Pp. 289-302.
Ozpolat, B., Rao, X.M., Osato, M.S., Graham, D.Y. and Lachman, L.B.*
Quantitation of Chlamydia trachomatis 16S rRNA Using NASBA Amplification and a Bioluminescent Microtiter Plate Assay. Pp. 303-313.
X. Song, B. K. Coombes and J.B. Mahony*
Nucleic Acid Sequence Based Amplification (NASBA) of Chlamydia pneumoniae Major Outer Membrane Protein (ompA) mRNA with Bioluminescent Detection. Pp. 315-327.
B. K. Coombes* and J. B. Mahony
Patterns of Expression of Viral and Cytokine Gene Transcripts During Mouse Polyoma Virus Infection. Pp. 329-341.
Donald R. Drake III, Leise Knoepp, Jeffrey K.
Actor and Aron E. Lukacher*
Cytokine mRNA Expression and Serum Cortisol Evaluation During Murine Lung Inflammation Induced by Mycobacterium tuberculosis. Pp. 343-351.
Jeffrey K. Actor*, Cari D. Leonard, Virginia E. Watson, Alice Wells,
Chinnaswamy Jagannath, Robert L. Hunter Jr. and Amitava Dasgupta
[Back to top] Bioluminescent Quantitation and Detection of Gene Expression During Infectious Disease.
By combining the advantages of RT-PCR with the sensitivity of bioluminescence using the photoprotein aequorin, a bioluminescence assay has been applied to the determination of message regulation during infectious disease. The bioluminescence produced by the aequorin conjugate covers more than seven logs concentration, of which approximately five logs produces a linear relationship between product and bioluminescence signal. Aequorin - based bioluminescent detection protocols for mRNA are sensitive into the attomolar range, which obligate fewer cycles of PCR and avoid the plateau effect traditionally associated with other noncompetitive RT-PCR techniques. Additional advantages of aequorin-based bioluminescence methods are ease of automation, compatibility with microtiter plate format, low cost, and flexibility.
[Back to top] Quantitative
and Bioluminescent Assay to Measure Efficacy of Conventional and DNA
Vaccinations Against Helicobacter pylori.
Ozpolat,
B., Rao, X.M., Osato, M.S., Graham, D.Y. and Lachman, L.B.*
Vaccination
against Helicobacter pylori using DNA sequences encoding Urease A and B
subunits was compared to immunization with urease antigen and MTP-PE in a
liposome formulation. To determine the effectiveness of a vaccine against H.
pylori in a mouse model it is essential to quantify the number of H.
pylori remaining in the stomachs following challenge with an inoculum of
live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if
vaccine candidates are protective. To overcome this problem, we developed two
assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a
non-competitive direct quantitative PCR using a highly sensitive bioluminescent
readout. The competitive PCR requires coamplification of a segment of the urease
C sequence and an internal control standard in a competitive manner using a
single set of primers. PCR products were quantified colorimetrically by an
enzyme-linked immunosorbent assay and compared with known quantities of the
internal control standard added to the PCR reaction. The highly sensitive,
bioluminescent assay measures the amplified DNA directly using a flash-type
luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H.
pylori by either the bioluminescent assay or the competitive PCR was
reliable, specific and sensitive compared to quantitative growth assays which
often gave false results. The bioluminescent assay was much more sensitive and
less labor/time intensive than the competitive PCR. The bioluminescent assay was
able to quantitate as few as 100 bacteria, while the competitive assay could not
detect less than 103 bacteria per mouse stomach. Quantification of H.
pylori by bioluminescent assay was superior to the competitive assay and may
be used for research applications, such as the development of vaccines,
pathogenesis of gastric disease and monitoring of antibiotic treatment.
[Back to top] Quantitation
of Chlamydia trachomatis 16S rRNA
Using NASBA Amplification and a Bioluminescent Microtiter Plate Assay.
We developed a nucleic acid sequence based amplification (NASBA) assay which employs the recombinant photoprotein Aequorin in a microtiter plate format for detection and quantitation of C. trachomatis that may be useful in large scale epidemiological studies aimed at improving our understanding of factors affecting transmission of this sexually transmitted pathogen. The conditions for NASBA amplification of the16S rRNA target were optimized (90 mM KCl, 12 mM MgCl2, 0.2 mM P1 and P2 primers), amplified RNA was captured by a biotin-labelled capture probe immobilized onto streptavidin coated microtiter plates and detected with an FITC-labelled oligonucleotide probe and Aequorin-anti-FITC antibody conjugate. The analytical sensitivity of NASBA was 1,000 in vitro generated RNA transcripts and 1.6 IFU of C. trachomatis. The sensitivity of NASBA using the bioluminescent assay was 10 fold higher than Northern blotting. Time course amplification experiments performed with 10 fold serial dilutions of target established that amplification was linear at 75 min and extended over a range of five log units of input RNA copy number. Linear regression analysis confirmed a linear fit for the data with r2 = 0.959 (p < 0.004). A double log plot of RLU signal versus copy number was linear; analysis of residuals from a series of runs tests confirmed a fit with a linear model (number of runs = 3, p = 0.5 where p < 0.05 indicates statistical deviation from a linear model). NASBA amplification coupled with bioluminescent detection in a microtiter plate format should provide a useful tool for quantitation of C. trachomatis in clinical specimens for use in epidemiological studies.
[Back to top] Nucleic
Acid Sequence Based Amplification (NASBA) of Chlamydia pneumoniae Major Outer Membrane Protein (ompA)
mRNA with Bioluminescent Detection.
Chlamydia pneumoniae has been associated with chronic conditions such as atherosclerosis and coronary heart disease but the precise role of this intracellular bacteria in the pathogenesis of these diseases is not well defined. Several techniques have been developed for detection of C. pneumoniae in atheromatous lesions, however it remains unclear whether persistent forms of the organism and/or actively replicating bacteria contribute to associated pathology. The aim of this study was to utilize nucleic acid sequence based amplification (NASBA) technology together with a highly sensitive aequorin bioluminescent hybridization assay for the detection of C. pneumoniae ompA mRNA transcripts. A NASBA targeting the ompA gene of C. pneumoniae was developed, and the sensitivity was evaluated using both C. pneumoniae ompA RNA generated in vitro, and purified C. pneumoniae inclusion forming units (IFU). C. pneumoniae NASBA was capable of detecting between 100 and 1000 ompA RNA molecules and could detect 0.2 IFU of C. pneumoniae using the aequorin bioluminescent assay. The sensitivity of the bioluminescent assay was at least 10-fold higher than Northern blot detection. The linearity of NASBA amplification was assessed in time-course amplification experiments with different input numbers of RNA molecules. When NASBA products were analyzed during the linear phase of amplification, the dynamic range of bioluminescent detection extended over 8-log units of input RNA copy number. NASBA amplification coupled with bioluminescent detection may prove to be a useful molecular tool for the detection, quantitation and analysis of differentially expressed chlamydial genes during various stages of infection and disease pathology or for other mRNAs of interest in different disease processes.
[Back to top] Patterns of Expression of Viral and Cytokine Gene
Transcripts During Mouse Polyoma Virus Infection.
Donald
R. Drake III, Leise Knoepp, Jeffrey K. Actor and Aron E. Lukacher*
CD8+ cytotoxic T lymphocytes are critical for clearance of infection and prevention of tumors caused by mouse polyoma virus. High susceptibility to polyoma-induced tumors is manifested by neonatal inoculation of mice belonging to particular H-2k haplotype inbred strains. We previously reported that tumor-susceptible mice generate polyoma-specific CD8+ T cells, but at a frequency approximately 20-fold lower than tumor-resistant H-2k mice. To determine whether susceptibility or resistance may also be associated with a cytokine microenvironment conducive for promoting cell-mediated (i.e., type 1 cytokines) or humoral (i.e., type 2 cytokines) immune responses, we used quantitative bioluminescence RT-PCR to measure in vivo message levels for viral proteins and cytokines during infection of neonatal mice. We found that the level of polyoma viral transcripts peaked higher and fell with significantly slower kinetics in tumor-susceptible mice than in tumor-resistant mice. Interestingly, message for VP1, the major viral capsid protein, persisted in multiple organs of mice of both susceptible and resistant strains, indicating chronic productive infection regardless of tumor susceptibility. IL-1b, IL-12, IL-2, IFN-g, and IL-4 message levels were all higher in infected susceptible than resistant mice. Although both susceptible and resistant mice expressed transcripts for IFN-g and IL-4, the signature type 1 and type 2 cytokines, respectively, a dominance of IL-4 message, with concomitant drop in IFN-g message, was seen only in the susceptible mice. These results suggest that a type 2 pattern of cytokine expression may contribute to susceptibility to polyoma virus tumorigenesis.
[Back to top] Cytokine
mRNA Expression and Serum Cortisol Evaluation During Murine Lung Inflammation
Induced by Mycobacterium tuberculosis.
Jeffrey K. Actor*, Cari D. Leonard, Virginia E. Watson, Alice Wells, Chinnaswamy Jagannath, Robert L. Hunter Jr. and Amitava Dasgupta
A
model system was characterized for investigating the potential role of cortisol
in MTB induced immunopathology. Serum cortisol levels were evaluated in two
mouse strains; C57BL/6 mice develop lung granulomas following acute Mycobacterium
tuberculosis infection while A/J
mice are deficient in this process. Serum cortisol levels were examined
post infection, as well as immunoregulatory mRNA expression in the lung,
measured using bioluminescent RT-PCR techniques. Prior to infection, the A/J
mice constitutively maintain nearly 75% higher serum cortisol than C57BL/6 mice.
Both A/J and C57BL/6 mice exhibited approximately 30% reduction in relative
serum cortisol following infection. At no time did serum cortisol levels in the
A/J fall below constitutive levels in the non-infected C57BL/6. The overall
elevated cortisol in the A/J may affect pulmonary immunoresponsiveness; A/J mice
exhibited earlier induction of IL-10 and TNF-a than C57BL/6 mice, with a relative lack of IL-2
during late infection. Conversely, the C57BL/6 mice demonstrated higher
IL-12(p40) and IL-2 messages at the latter stages of disease than the A/J mice.
Both mice demonstrated high IFN-g
mRNA. The high constitutive serum cortisol in the A/J mice may therefore
contribute to establishment of an environment counter-productive to initiation
of protective Th1 cell and granulomatous responses.