11beta-hydroxysteroid dehydrogenases are regulated during the pulmonary granulomatous response to the mycobacterial glycolipid trehalose-6,6'-dimycolate.

OBJECTIVE: Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown. METHODS: One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6'-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology. RESULTS AND CONCLUSION: IL-6, IL-1alpha and TNF-alpha mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11beta-hydroxysteroid dehydrogenases (11betaHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11betaHSD type 1 mRNA decreased 4-fold and 11betaHSD type 2 (11betaHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11betaHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

Requisite role for complement C5 and the C5a receptor in granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate.

The development of pulmonary granulomatous lesions during mycobacterial infection is a complex phenomenon, in part caused by responses elicited towards the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor). The molecular mechanisms underlying granuloma formation following challenge with TDM are not yet completely understood. The present study defines pathologic differences in acute response to Mycobacterium tuberculosis TDM in C57BL/6 mice and mice lacking the C5a receptor (C5aR-/-). Mice were intravenously injected with TDM prepared in water-in-oil-in-water emulsion and examined for histologic response and changes in proinflammatory cytokines and chemokines in lung tissue. Control C5a receptor-sufficient mice demonstrated a granulomatous response that peaked between days 4 and 7. Increased production of macrophage inflammatory protein-1 alpha (MIP-1alpha), interleukin-1beta (IL-1beta) and CXC chemokine KC (CXCL1) correlated with development of granulomas, along with modest change in tumor necrosis factor-alpha (TNF-alpha). In contrast, the C5aR-/- mice revealed markedly exacerbated inflammatory response. The receptor-deficient mice also demonstrated a lack of coherent granulomatous response, with severe oedema present and instances of lymphocytic cuffing around pulmonary vessels. Lung weight index was increased in the C5aR-/- mice, correlating with increased MIP-1alpha, KC, IL-1beta and TNF-alpha over that identified in the congenic C5aR-sufficient controls. Correlate experiments performed in C5-deficient (B10.D2-H2d H2-T18c Hco/oSnJ) mice revealed similar results, leading to the conclusion that C5 plays a significant role in mediation of chemotactic and activation events that are the basis for maturation of granulomatous responses to TDM.

Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice.

Mice injected with endotoxin develop endotoxaemia and endotoxin-induced death, accompanied by the oxidative burst and overproduction of inflammatory mediators. Lactoferrin, an iron binding protein, provides a natural feedback mechanism to control the development of such metabolic imbalance and protects against deleterious effects of endotoxin. We investigated the effects of intraperitoneal administration of human lactoferrin on lipopolysaccharide (LPS)-induced release of tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 10 (IL-10) and nitric oxide (NO) in vivo. Lactoferrin was administered as a prophylactic, concurrent or therapeutic event relative to endotoxic shock by intravenous injection of LPS. Inflammatory mediators were measured in serum at 2, 6 and 18 h post-shock induction. Administration of lactoferrin 1 h before LPS resulted in a rather uniform inhibition of all mediators; TNF by 82%, IL-6 by 43%, IL-10 by 47% at 2 h following LPS injection,and reduction in NO (80%) at 6 h post-shock. Prophylactic administration of lactoferrin at 18 h prior to LPS injection resulted in similar decreases in TNF-alpha (95%) and in NO (62%), but no statistical reduction in IL-6 or IL-10. Similarly, when lactoferrin was administered as a therapeutic post-induction of endotoxic shock, significant reductions were apparent in TNF-alpha and NO in serum, but no significant effect was seen on IL-6 and IL-10. These results suggest that the mechanism of action for lactoferrin contains a component for differential regulation of cellular immune responses during in vivo models of sepsis.

A role for complement C5 in organism containment and granulomatous response during murine tuberculosis.

The molecular mechanisms underlying protective granuloma formation and control of bacterial growth during infection with Mycobacterium tuberculosis (MTB) are not yet completely understood. MTB-infected mice with natural deficiency in complement component C5 are unable to develop productive granulomatous responses, and are impaired in limiting organism growth within the lung. To address the molecular basis for this histologic dysfunction, congenic complement C5-sufficient (B10.D2-H2d H2-T18c Hcl/nSnJ) and complement C5-deficient strains (B10.D2-H2d H2-T18c Hco/oSnJ) congenic mice were infected with Mycobacterium tuberculosis, and cytokine and chemokine responses were examined. Twelve and 28 days after infection, lungs showed elevated messages for multiple inflammatory cytokines in both congenic strains. Interleukin (IL)-12(p40) mRNA was also induced during infection in C5-deficient mice, although levels were significantly decreased compared to C5-sufficient congenics. C5-deficient mice also demonstrated reduced KC, MIP-2, IP-10, and MCP-1 mRNA. The defect may directly involve C5-mediated effects on macrophage responses; C5-deficient bone marrow derived macrophages had significantly reduced secretion of KC, MIP-1 alpha and MIP-2 compared to C5-sufficient macrophages following in vitro infection. These findings indicate a role for C5 in mediation of chemotactic and activation events that are the basis for granulomatous responses during murine tuberculosis.

Digitoxin-like immunoreactivity in sera of mice after feeding with chinese medicine Chan Su: study of protein binding of Chan Su in normal sera, uremic sera and sera from patients with liver disease.

Traditional Chinese medicines are readily available without prescription from herbal drug stores. One such Chinese medicine, Chan Su, which is prepared from the skin gland of toads, has cardiotonic effect due to bufadienolides. Here we report rapid detection of the presence of Chan Su in blood using the fluorescence polarization immunoassay for digitoxin. In our study mice were fed with a dose of 75 mg/kg of Chan Su and blood was drawn before, and 1 and 2 h after feeding. We observed significant digitoxin-like immunoreactivity in the sera. For example in one mouse the digitoxin-like immunoreactivity was undetectable before feeding with Chan Su, but was 19.7 ng/ml 1 h and 8.8 ng/ml 2 h afterwards. The apparent half-life of Chan Su is approximately 1 h in mice. In another experiment, we studied protein binding of Chan Su by measuring total and free Chan Su concentrations (ultrafiltrate prepared by using Centrifree Micropartition Filter, molecular weight cutoff: 30000 Da). Chan Su was strongly bound to serum proteins. We observed higher free fraction in uremic sera and sera from patients with liver disease. We identified albumin as one of the proteins that bind Chan Su in serum.

Dysregulated response to mycobacterial cord factor trehalose-6,6'-dimycolate in CD1D-/- mice.

The biologic effects of the mycobacterial glycolipid trehalose-6,6'-dimycolate (TDM) include granuloma formation and macrophage activation and are dependent on physical conformation. In mice, the group II CD1 surface molecule CD1d has been implicated in glycolipid presentation. The importance of CD1d interactions in pathology has yet to be established. We hypothesized that mice lacking CD1d (CD1D(-/-)) would demonstrate dysregulated granulomatous response to TDM, compared with CD1D(+/-) heterozygous controls. Mice were intravenously injected with TDM-coated polystyrene-divinylbenzene beads and examined for histologic response and for changes in inflammatory cytokine and chemokine mRNA. Control CD1D heterozygous mice demonstrated a granulomatous response, which peaked at day 5. Increased mRNA for tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha) correlated with development of granulomas, with very little change in interleukin-1beta (IL-1beta) and monocyte chemoattractant protein-1 (MCP-1). In contrast, the CD1D(-/-) mice revealed markedly different responses. Five days after administration, severe pulmonary hemorrhage was induced. The relative size of inflammation surrounding coated bead in the CD1D(-/-) mice was nearly double that induced in the CD1D(+/-) mice. CD1D(-/-) mice also demonstrated elevated mRNA for both inflammatory cytokines and chemokines by day 1 after administration, significantly earlier than responses seen in the heterozygous controls.

Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

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.

Patterns of expression of viral and cytokine gene transcripts during mouse polyoma virus infection.

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-2(k) 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-2(k) 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-1beta, IL-12, IL-2, IFN-&gama; and IL-4 message levels were all higher in infected susceptible than resistant mice. Although both susceptible and resistant mice expressed transcripts for IFN-&gama; and IL-4, the signature type 1 and type 2 cytokines, respectively, a dominance of IL-4 message, with concomitant drop in IFN-&gama; 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.

Cytokine mRNA expression and serum cortisol evaluation during murine lung inflammation induced by Mycobacterium tuberculosis.

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&percent; higher serum cortisol than C57BL/6 mice. Both A/J and C57BL/6 mice exhibited approximately 30&percent; 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-alpha 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-&gama; 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.

Hypersusceptibility of A/J mice to tuberculosis is in part due to a deficiency of the fifth complement component (C5).

Mycobacterium tuberculosis (MTB) causes tuberculosis in man, which occurs as an acute, chronic or dormant disease reactivating over several years. The mechanisms of persistence and reactivation are not well understood and there is a need for animal models. Moderate-dose, aerosol infection killed A/J mice earlier than partially resistant C57Bl/6 mice, whereas a low-dose, aerosol-induced chronic infection exacerbated earlier in A/J mice. A/J mice lethally infected with MTB but drug cured of disease underwent reactivation of tuberculosis at least 100 days before similarly infected C57Bl/6 mice. Because A/J mice were C5 deficient, congenic B10 mice sufficient and deficient for C5 were infected intravenously with MTB to define the role of C5. C5-deficient mice again showed enhanced growth of MTB in the lungs. MTB-infected macrophages from C5-deficient mice showed enhanced growth of MTB coinciding with a reduced secretion of both cytokines (TNF-alpha, IL-1beta, IL-6, IL-12) and chemokines (KC, MIP-2 and MIP-1alpha) in A/J and TNF-alpha and chemokines in C5-deficient mice. Because C5-deficient macrophages could be activated from extraneous C5 and TNF-alpha we suggest that both play a role in the macrophage-mediated killing as well as containment mechanisms in tuberculosis.

Effect of poloxamer CRL-1072 on drug uptake and nitric-oxide-mediated killing of Mycobacterium avium by macrophages.

Mycobacterium avium-intracellulare complex (MAI) are common pathogens of opportunistic infections that are naturally resistant to most antibiotics and develop acquired resistance rapidly. An experimental drug, poloxamer CRL-1072, was found to have two unusual properties: it synergistically enhanced the activity of several antibiotics against MAI even though it had little activity as a single agent and it had greater activity against MAI in macrophage culture or in mice than in broth culture. Studies were undertaken to investigate the mechanisms of these effects. CRL-1072 was taken up by MAI and enhanced the uptake of fluorescent-labeled streptomycin and erythromycin in broth culture. The labeled antibiotics had reduced activity so the relevance for naive antibiotics must be inferred. In culture with human U937 monocytoid cells, CRL-1072 became localized in phagosomes and promoted uptake of streptomycin. Finally, CRL-1072 was found to induce production of mRNA for inducible nitric oxide synthase (iNOS) and nitric oxide (NO) by U937 cells. The antimycobacterial effect in macrophages was reversed by the iNOS inhibitor N-monomethyl L-arginine (NMMA), suggesting that CRL-1072 promotes killing of MAI by inducing NO. These effects were induced by noncytotoxic concentrations of CRL-1072. These data suggest that the antimycobacterial mechanisms of CRL-1072 include enhancing the delivery of antibiotic to targets within MAI and enhancement of the ability of macrophages to kill ingested organisms.

Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA.

Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organisms in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of MTB that can distinguish between viable and non-viable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensitivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homogenate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finally, in a mouse model of dormancy, direct RT-PCR detected a signal among multiple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Thus the method to identify its mRNA message may be useful to detect viable but dormant bacteria.

Quantitation of Helicobacter pylori in the stomach using quantitative polymerase chain reaction assays.

BACKGROUND: The density of Helicobacter pylori is thought to correlate with the degree of inflammation and thus indirectly with the outcome of the infection. Rapid quantitative assays of H. pylori in gastric or duodenal mucosa are lacking. The aim was to develop quantitative assays using the polymerase chain reaction to assess the quantity of H. pylori in the gastric mucosa. METHODS: Competitive PCR was based on coamplification of a segment of the ureC sequence and an internal control 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, noncompetitive PCR assay does not use coamplification and measures the amplified DNA sequence using a flash-type luminescent tag and a specific probe. The mouse infected model using H. pylori strain SS-1 was used to develop the assays. RESULTS: Quantification of H. pylori using either the competitive or noncompetitive PCR was reliable, highly sensitive and specific. CONCLUSIONS: The ability to rapidly quantitate H. pylori from gastric mucosa should be useful to investigate the role of H. pylori density and infection outcome, as well as to monitor the effectiveness of antibiotic treatment or vaccines against H. pylori.

Apoptosis in mycobacterium tuberculosis infection in mice exhibiting varied immunopathology.

This study examined mechanisms contributing to pulmonary immunopathology following acute Mycobacterium tuberculosis (MTB) infection in vivo in a murine model. A/J and C57BL/6 mice were intravenously infected with MTB (Erdman). Pathological differences were found between strains, unrelated to pulmonary load of bacilli. A/J mice developed progressive interstitial pneumonitis, while C57BL/6 mice maintained granuloma formation. The contribution of FAS and FAS ligand-mediated apoptosis was assessed via bioluminescent reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemical staining, and TUNEL assessment of DNA fragmentation. Cytokine messages for pulmonary tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as well as for the lytic molecules perforin and granzyme B, were quantified. Immunohistochemical staining for CD3 receptor was performed to monitor lymphocytic lung infiltration. Soon after infection, A/J mice exhibited increased pulmonary IFN-gamma message, concurrent with the appearance of CD3+ lymphocytes distributed throughout the lung. C57BL/6 mice exhibited perivascular cuffing, with no accompanying increase in IFN-gamma message. A/J mice also had elevated levels of FAS and FAS ligand message and protein early after infection, while the C57BL/6 mice had no increased expression of these molecules. Both strains exhibited qualitatively similar numbers of TUNEL-positive cells throughout infection, with a marked increase on day 7. Apoptotic cells appeared to co-localize with acid fast bacilli. It is therefore proposed that apoptosis during initial granuloma formation following MTB infection may occur through a FAS/FAS ligand-independent pathway. Moreover, a failure of completion of the FAS/FAS ligand-mediated apoptosis pathway in the A/J mice may contribute to inefficient elimination of lymphocytes, thus further aggravating pulmonary pathology.

Relationship of survival, organism containment, and granuloma formation in acute murine tuberculosis.

The relationship among organism growth, immunopathology, and survival was studied in C57BL/6 and A/J mice acutely infected with Mycobacterium tuberculosis (MTB) (Erdman). Although organisms grew at similar rates in the lungs of both mouse strains, A/J mice died prior to 14 days after infection, whereas C57BL/6 mice survived twice as long. The lungs of A/J mice exhibited necrotizing interstitial inflammation and widely distributed acid-fast bacilli without granuloma formation. In contrast, the lungs of C57BL/6 mice had relatively mild interstitial inflammation, which was replaced by focal granulomas, and acid-fast bacilli were primarily within granulomas. MTB induced similar granulomas for A/J and C57BL/6 mice in spleen and liver. In the lung, the A/J mice produced only transient messages for interferon-y (IFN-y), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-10, and inducible nitric oxide synthase (iNOS). The C57BL/6 mice, in contrast, produced a delayed but sustained response in the lung correlating with granuloma onset and characterized by high induction of IL-6, IFN-gamma, IL-1beta, IL-10, and TNF-alpha. Responses in the liver and spleen were also evaluated. These results demonstrate that histopathology and cytokine response to MTB infection varies among organs in mice. Increased survival during acute infection may, therefore, depend on the ability to contain organisms within granulomas in the lung.

A flexible bioluminescent-quantitative polymerase chain reaction assay for analysis of competitive PCR amplicons.

Aequorin-based flash-type bioluminescent methods can detect nucleic acid molecules in the attomolar range (10(-18)) enabling improved monitoring of the polymerase chain reaction (PCR) at cycles previously considered too low for product detection. The high sensitivity of bioluminescence (BL) was used to examine the efficiency of the PCR and to assess the effect of substrate variation during the linear phase of amplification. Primer efficiency was dependent on initial template concentration, in a manner indicative of a two-component reaction. However, the rate of amplicon formation was significantly impaired at low template levels and could not be overcome by excess primer. The PCR was directly dependent upon nucleotide concentration, which was independent of template concentration. Conditions were identified for optimal linear amplification and detection using BL. Accurate quantitative analysis was performed using competitive coamplification of a specific target standard sequence containing identical target primer recognition sites and novel internal sequences. Quantitation was most accurate when target molecule was similar in concentration to the internal standard. The Bioluminescent Quantitative-PCR (BLQ-PCR) assay has the potential to eliminate processing variability. We demonstrated high quantitative potential with a broad dynamic range. Overall, the BLQ-PCR assay is flexible and a viable alternative to contemporary Q-PCR techniques.

CRL-1072 enhances antimycobacterial activity of human macrophages through interleukin-8.

CRL-1072 is a poloxamer surfactant that kills mycobacteria more effectively within macrophages than in broth cultures. Human macrophages treated with CRL-1072 synthesized interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in a dose-dependent manner. About 3000 pg of IL-8 per million human macrophages accumulated in cultures treated with 100-1500 ng of poloxamer, with mRNA message for IL-8 induced as early as 2 h. As macrophages do not have IL-RA receptors, a transwell culture was used to study the chemotactic and activating effects of IL-8 between CRL-1072-treated human macrophage effectors and polymorphonuclear neutrophil (PMN) targets. PMN were activated by IL-8 and secreted hydrogen peroxide and myeloperoxidase (MPO). MPO derived from PMN, in turn, activated monocytes for an enhanced killing of intracellular Mycobacterium avium. The ability of CRL-1072 to modulate macrophage-mediated activation of neutrophils and receive a feedback activation signal may form one mechanism by which its antimycobacterial activity is achieved in vivo.

Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis.

The induction of nitric oxide in human monocytes during mycobacterial infection has been a controversial issue. This study describes a comparative evaluation of the colorimetric and fluorometric methods for the detection of NO in response to Mycobacterium tuberculosis (MTB) infection in human peripheral blood-derived monocytes (PBM) and in U937, a human monocyte-derived cell line. MTB was grown in monocyte cultures in vitro for 7 or 10 days. RPMI 1640 medium, without antibiotics and supplemented with L-arginine, Hepes, 5% human AB serum, and tetrahydrobiopterin was used to support monocyte growth. As early as 72 h after infection, soluble nitrite was detectable in the medium using the fluorometric assay with diaminonaphthalene (DAN). Early induction of NO correlated with an increase in the levels of iNOS mRNA as quantitated by RT-PCR. NO levels increased progressively up to day 10 (PBM) or day 7 (U937), when 150-200 nM/10(6) cells of soluble nitrite accumulated in cultures, as measured by DAN. Furthermore, monocytes stained positively for human iNOS protein and peroxynitrite after infection with MTB. The induction of NO by MTB was inhibited by four different inhibitors of iNOS enzyme including N-monomethylarginine. Inhibition of NO resulted in the enhancement of the intracellular growth of two of five clinical isolates of MTB. NO released from a donor (S-nitroso-N-penicillamine) also had a direct bacteriostatic effect on the same isolates in broth cultures. MTB strains thus showed a differential susceptibility to intracellular and extracellular NO. In most of these assays, the Greiss reagent was limited by its sensitivity and remained negative for soluble nitrite throughout the 7-10 days of incubation. Thus, the colorimetric method, which is widely used, may give false-negative results in NO assays. This report also demonstrates for the first time that MTB induces mRNA for iNOS, iNOS protein, NO, and peroxynitrite in human monocyte/macrophage cultures.

A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: quantitative detection of cytokine cDNA in activated and resting human cells.

Because of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10(-18) M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3delta amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3delta, TNF alpha or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.