Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitry... more Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare-induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs Bai et al. AAT and NTM in Macrophages macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.
American Journal of Respiratory and Critical Care Medicine, 1995
Recent studies indicate that chronic asthma is associated with a spectrum of glucocorticoid recep... more Recent studies indicate that chronic asthma is associated with a spectrum of glucocorticoid receptor (GCR) binding abnormalities that are cytokine-inducible. These GCR abnormalities may contribute to poor asthma control and failure to respond to glucocorticoid (GC) therapy. The purpose of this study was to determine whether GCR defects are associated with poorly controlled asthma, and whether diminished GCR binding is reversible following a course of GC therapy. We enrolled 12 patients with poorly controlled asthma characterized by nocturnal awakening with cough or wheezing, AM FEV1 < 70%, or FEV1 variability of > 25% requiring a short course of high dose GC therapy. GCR binding affinity was measured in peripheral blood mononuclear cells using a radioligand binding assay before and after the GC course. Spirometry, serum cortisol, eosinophil cationic protein (ECP), and soluble IL-2 receptor (sIL-2R) levels were also performed before and after the GC course. At baseline, all subjects had airflow obstruction that significantly improved (median FEV1 increased from 65.0% to 89.5% of predicted, median FEV1/FVC ratio increased from 0.60 to 0.72) with therapy. A diminished GCR binding affinity at baseline was noted with an elevated median dissociation constant (Kd) of 29.0 nM (interquartile range at the 25th and 75th percentile [IQ] of 22.3 and 44.5 nM) compared with normal controls (Kd 8.0 nM [IQ 7.0, 9.2]). Following the GC course, a significant decrease in the Kd was seen. Serum ECP and sIL-2R levels at baseline were elevated, with serum ECP demonstrating a significant reduction following the GC course.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, Sep 1, 2009
A new analytical method suitable for high throughput measurements of LTE(4) in human urine is des... more A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.
We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidi... more We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidia. Rat SP-D but not rat SP-A bound the conidia, and the binding was inhibited by EDTA, mannose, glucose, maltose, and inositol. Binding studies using a mutant recombinant rat SP-D with altered carbohydrate recognition but normal structural organization clearly established a role for the carbohydrate recognition domain in binding to conidia. However, neither rat SP-A nor SP-D increased the association of fluorescein isothiocyanate-labeled conidia with rat alveolar macrophages as determined by flow cytometry. Both human SP-A (isolated from normal and alveolar proteinosis lungs) and SP-D (recombinant protein and protein isolated from alveolar proteinosis lungs) bound the conidia. These data indicate that important differences exist between rat and human SP-A in binding to certain fungi. Human SP-A and SP-D binding to conidia was also examined in the presence of hydrophobic surfactant components (HSC), containing both the phospholipid and hydrophobic proteins of surfactant. We found that HSC inhibited but did not eliminate human SP-A binding to Aspergillus conidia. In contrast, the SP-D binding to conidia was unaffected by HSC. These findings indicate that SP-D plays a major role in the recognition of Aspergillus conidia in alveolar fluid.
Employing a hydroxyapatite double-stranded (ds) DNA column for affinity chromatography, anti-ds-D... more Employing a hydroxyapatite double-stranded (ds) DNA column for affinity chromatography, anti-ds-DNA antibodies were isolated from the sera of aging NZB/W mice. These antibodies were radiolabeled and injected intravenously into NZB/W mice at various ages. Significant deposition of radiolabeled antibodies were shown to occur in NZBlW mice 4 months of age and older. While the injected antibodies displayed heterogeneity in their isoelectric points (PI'S), those antibodies which bound to the kidney were restricted to a more alkaline pI range. Glomerular cultures were prepared from NZB/W mice at various ages and incubated with radiolabeled anti-ds-DNA antibodies with restricted pl's. Antibodies having pi's between 7 and 9 bound in vitro to glomeruli obtained from 9-month-old NZBlW mice but not to glomeruli obtained from 2-monthold mice. In contrast, antibodies with pi's between 5 and 7 were unable to bind to glomeruli obtained from either age group. An exchange between free antibodies and antibodies bound to glomeruli was also demonstrated to occur in vitro. These findings suggest the presence of subpopulations of anti-ds-DNA antibodies which preferentially bind to glomeruli containing immune complexes.
Adult mice can be depleted of essentially all mature alpha beta T lymphocytes by chronic treatmen... more Adult mice can be depleted of essentially all mature alpha beta T lymphocytes by chronic treatment with the framework-recognizing, pan-specific anti-TCR alpha beta mAb, H57-597. Similar findings have been reported in rats, gamma delta cell populations remain essentially unaltered in size and reactivity. Suppression of alpha beta T-cell development results in the loss of alloantigen reactivity and of B-cell help, suggesting that gamma delta and alpha beta populations differ in their functional capabilities. Indirect effects of the antibody treatment include quantitative changes in splenic B cells, as well as reduced sizes and weights of experimental animals. alpha beta-suppressed mice and rats may provide model systems for studies on gamma delta cell function in vivo.
We studied the production of immunoglobulin (Ig)E in cultures containing mononuclear cells obtain... more We studied the production of immunoglobulin (Ig)E in cultures containing mononuclear cells obtained from atopic and nonatopic subjects. The two groups did not differ in their baseline production of IgE. Pokeweed mitogen (PWM) and three different products of Staphylococcus aureus: purified protein A, heat-killed formalinized bacteria, and double-formalinized bacteria--induced a significant increase in the in vitro production of IgE. Mononuclear cells from both atopic and nonatopic individuals were stimulated to the same degree by all four mitogens. The increased production of IgE by PWM was totally abolished by cycloheximide; hence it was due to de novo protein synthesis.
ABSTRACT The immunologic response to immunotherapy with dog extract is not well characterized. Th... more ABSTRACT The immunologic response to immunotherapy with dog extract is not well characterized. The purpose of this study was to examine the immunologic response to 3 doses of dog extract expressed as their Can f 1 content. Cluster immunotherapy was administered to 28 patients with dog allergy who were randomly assigned to 1 of 4 treatment arms: placebo or acetone-precipitated extract containing 0.6 mug, 3.0 mug, or 15.0 mug Can f 1 per 0.5 mL maintenance dose. Studies included titrated skin prick tests, the late cutaneous response, titrated nasal challenge with dog extract, and serum allergen-specific IgE and IgG(4). Dog allergen-stimulated lymphocyte proliferation was performed with measurement of secreted cytokines by ELISA and of intracellular cytokines by flow cytometry. There was a significant dose-dependent response in suppression of titrated skin prick tests and suppression of the late cutaneous response. There was a significant increase from baseline in dog-specific IgG(4) in both the high-dose and low-dose groups and a dose-dependent suppression of secreted TNF-alpha and increase in secreted TGF-beta. There was a dose-dependent trend in suppression of secreted IL-4 with a significant decrease from baseline in the high-dose group. There were no significant changes in symptom scores; lymphocyte proliferation; secreted IFN-gamma, IL-10, or IL-5; or intracellular cytokine production. The dose-response in immunologic parameters after immunotherapy with dog extract is similar to that previously demonstrated with cat extract. The greatest and most consistent response is seen with a dose containing 15 mug Can f 1.
Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and ... more Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and are used in the screening and/or diagnosis of autoimmunity in patients as well as mouse models of systemic autoimmunity. Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for ANA screening. However, its usefulness is limited in diagnosis, prognosis and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. Various immunological techniques have been developed in an attempt to improve upon the method to quantify ANA, including enzyme-linked immunosorbent assays (ELISAs), line immunoassays (LIAs), multiplexed bead immunoassays and IIF on substrates other than HEp-2 cells. Yet IIF on HEp-2 cells remains the most common screening method for ANA. In this study, we describe a simple quantitative method to detect ANA which combines IIF on HEp-2 coated slides with analysis using a near-infrared imaging (NII) system. Using NII to determine ANA titer, 86.5% (32 of 37) of the titers for human patient samples were within 2 dilutions of those determined by IIF, which is the acceptable range for proficiency testing. Combining an initial screening for nuclear staining using microscopy with titration by NII resulted in 97.3% (36 of 37) of the titers detected to be within two dilutions of those determined by IIF. The NII method for quantitative ANA measurements using serum from both patients and mice with autoimmunity provides a fast, relatively simple, objective, sensitive and reproducible assay, which could easily be standardized for comparison between laboratories.
American Journal of Respiratory and Critical Care Medicine, May 1, 2008
Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether ... more Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether the established allergic airway inflammation compromises lung innate immunity and subsequently predisposes patients with asthma to Mp infection remains unknown. To test whether the established allergic airway inflammation compromises host innate immunity (e.g., Toll-like receptor 2 [TLR2]) to hinder the elimination of Mp from the lungs. We used mouse models of ovalbumin (OVA)-induced allergic airway inflammation with an ensuing Mp infection, and cultures of mouse primary lung dendritic cells (DCs) and bone marrow-derived DCs. Lung Mp clearance in allergic mice and TLR2 and IL-6 levels in lung cells, including DCs as well as cultured primary lung DCs and bone marrow-derived DCs, were assessed. The established OVA-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, inhibited TLR2 expression and IL-6 production in lung cells, including lung DCs, and eventually led to impaired host defense against Mp. Studies in IL-6 knockout mice indicated that IL-6 directly promoted Mp clearance from the lungs. IL-4- and IL-13-induced suppression of TLR2 was mediated by inhibiting nuclear factor-kappaB activation through signal transducer and activator of transcription 6 (STAT6) signaling pathway. The established OVA-induced allergic airway inflammation impairs TLR2 expression and host defense cytokine (e.g., IL-6) production, and subsequently delays lung bacterial clearance. This could offer novel therapeutic strategies to reinstate TLR2 activation by using TLR2 ligands and/or blocking IL-4 and IL-13 to ameliorate persisting respiratory bacterial infections in allergic lungs.
The Journal of Allergy and Clinical Immunology, Jul 1, 2009
Background: To date, there have been no randomized, doubleblind studies showing the effectiveness... more Background: To date, there have been no randomized, doubleblind studies showing the effectiveness of sublingual immunotherapy with multiple allergens. Objective: The purpose of this study was to examine whether the efficacy of sublingual immunotherapy (SLIT) with standardized timothy extract was reduced by combination with other allergen extracts. Methods: A single-center, randomized, double-blind, placebocontrolled trial with SLIT was conducted. After an observational grass season, SLIT was administered for 10 months to 54 patients randomized to 1 of 3 treatment arms: placebo, timothy extract (19 mg Phl p 5 daily) as monotherapy, or the same dose of timothy extract plus 9 additional pollen extracts. Symptom and medication scores were collected and titrated nasal challenges, titrated skin prick tests, specific IgE, IgG 4 and cytokines release by timothy-stimulated lymphocyte proliferation were performed. Results: Perhaps because of a very low grass pollen season in 2008, there were no significant differences in medication or symptom scores in either treatment group compared with placebo. Compared with placebo, in the timothy monotherapy group, thresholds for titrated nasal challenge and skin prick tests (P 5 .03 and P 5 .001, respectively), and serum-specific IgG 4 levels (P 5 .005) significantly increased, and IFN-g levels decreased (P 5 .02), whereas in the multiallergen group, there was significant improvement only in the titrated skin prick tests (P 5 .04) which was less than in the monotherapy group. There were no significant differences between the 2 active groups in any outcome measure, and both active groups experienced more adverse events than placebo. There were no systemic reactions. Conclusion: Improvement in multiple relevant outcomes strongly suggests that SLIT with timothy extract alone was effective; however, the results for symptom and medication scores were not significant. The differences between multiple allergen SLIT and placebo only in skin sensitivity to timothy suggest a reduction in SLIT efficacy in this group. However, further studies are required to confirm these observations. (J Allergy Clin Immunol 2009;124:150-6.)
Page 1. Anti-native DNA Antibodies Mickey J. Mandel, MD; Ronald I. Carr, MD, PhD; William L. West... more Page 1. Anti-native DNA Antibodies Mickey J. Mandel, MD; Ronald I. Carr, MD, PhD; William L. Weston, MD; W. Mitchell Sams, Jr., MD; Ronald J. Harbeck, PhD; and Gerald G. Krueger, MD, Denver ... 12. Burch PRJ, Rowell NR: Lupus erythema-tosus. ...
Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitry... more Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare-induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs Bai et al. AAT and NTM in Macrophages macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.
American Journal of Respiratory and Critical Care Medicine, 1995
Recent studies indicate that chronic asthma is associated with a spectrum of glucocorticoid recep... more Recent studies indicate that chronic asthma is associated with a spectrum of glucocorticoid receptor (GCR) binding abnormalities that are cytokine-inducible. These GCR abnormalities may contribute to poor asthma control and failure to respond to glucocorticoid (GC) therapy. The purpose of this study was to determine whether GCR defects are associated with poorly controlled asthma, and whether diminished GCR binding is reversible following a course of GC therapy. We enrolled 12 patients with poorly controlled asthma characterized by nocturnal awakening with cough or wheezing, AM FEV1 < 70%, or FEV1 variability of > 25% requiring a short course of high dose GC therapy. GCR binding affinity was measured in peripheral blood mononuclear cells using a radioligand binding assay before and after the GC course. Spirometry, serum cortisol, eosinophil cationic protein (ECP), and soluble IL-2 receptor (sIL-2R) levels were also performed before and after the GC course. At baseline, all subjects had airflow obstruction that significantly improved (median FEV1 increased from 65.0% to 89.5% of predicted, median FEV1/FVC ratio increased from 0.60 to 0.72) with therapy. A diminished GCR binding affinity at baseline was noted with an elevated median dissociation constant (Kd) of 29.0 nM (interquartile range at the 25th and 75th percentile [IQ] of 22.3 and 44.5 nM) compared with normal controls (Kd 8.0 nM [IQ 7.0, 9.2]). Following the GC course, a significant decrease in the Kd was seen. Serum ECP and sIL-2R levels at baseline were elevated, with serum ECP demonstrating a significant reduction following the GC course.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, Sep 1, 2009
A new analytical method suitable for high throughput measurements of LTE(4) in human urine is des... more A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.
We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidi... more We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidia. Rat SP-D but not rat SP-A bound the conidia, and the binding was inhibited by EDTA, mannose, glucose, maltose, and inositol. Binding studies using a mutant recombinant rat SP-D with altered carbohydrate recognition but normal structural organization clearly established a role for the carbohydrate recognition domain in binding to conidia. However, neither rat SP-A nor SP-D increased the association of fluorescein isothiocyanate-labeled conidia with rat alveolar macrophages as determined by flow cytometry. Both human SP-A (isolated from normal and alveolar proteinosis lungs) and SP-D (recombinant protein and protein isolated from alveolar proteinosis lungs) bound the conidia. These data indicate that important differences exist between rat and human SP-A in binding to certain fungi. Human SP-A and SP-D binding to conidia was also examined in the presence of hydrophobic surfactant components (HSC), containing both the phospholipid and hydrophobic proteins of surfactant. We found that HSC inhibited but did not eliminate human SP-A binding to Aspergillus conidia. In contrast, the SP-D binding to conidia was unaffected by HSC. These findings indicate that SP-D plays a major role in the recognition of Aspergillus conidia in alveolar fluid.
Employing a hydroxyapatite double-stranded (ds) DNA column for affinity chromatography, anti-ds-D... more Employing a hydroxyapatite double-stranded (ds) DNA column for affinity chromatography, anti-ds-DNA antibodies were isolated from the sera of aging NZB/W mice. These antibodies were radiolabeled and injected intravenously into NZB/W mice at various ages. Significant deposition of radiolabeled antibodies were shown to occur in NZBlW mice 4 months of age and older. While the injected antibodies displayed heterogeneity in their isoelectric points (PI'S), those antibodies which bound to the kidney were restricted to a more alkaline pI range. Glomerular cultures were prepared from NZB/W mice at various ages and incubated with radiolabeled anti-ds-DNA antibodies with restricted pl's. Antibodies having pi's between 7 and 9 bound in vitro to glomeruli obtained from 9-month-old NZBlW mice but not to glomeruli obtained from 2-monthold mice. In contrast, antibodies with pi's between 5 and 7 were unable to bind to glomeruli obtained from either age group. An exchange between free antibodies and antibodies bound to glomeruli was also demonstrated to occur in vitro. These findings suggest the presence of subpopulations of anti-ds-DNA antibodies which preferentially bind to glomeruli containing immune complexes.
Adult mice can be depleted of essentially all mature alpha beta T lymphocytes by chronic treatmen... more Adult mice can be depleted of essentially all mature alpha beta T lymphocytes by chronic treatment with the framework-recognizing, pan-specific anti-TCR alpha beta mAb, H57-597. Similar findings have been reported in rats, gamma delta cell populations remain essentially unaltered in size and reactivity. Suppression of alpha beta T-cell development results in the loss of alloantigen reactivity and of B-cell help, suggesting that gamma delta and alpha beta populations differ in their functional capabilities. Indirect effects of the antibody treatment include quantitative changes in splenic B cells, as well as reduced sizes and weights of experimental animals. alpha beta-suppressed mice and rats may provide model systems for studies on gamma delta cell function in vivo.
We studied the production of immunoglobulin (Ig)E in cultures containing mononuclear cells obtain... more We studied the production of immunoglobulin (Ig)E in cultures containing mononuclear cells obtained from atopic and nonatopic subjects. The two groups did not differ in their baseline production of IgE. Pokeweed mitogen (PWM) and three different products of Staphylococcus aureus: purified protein A, heat-killed formalinized bacteria, and double-formalinized bacteria--induced a significant increase in the in vitro production of IgE. Mononuclear cells from both atopic and nonatopic individuals were stimulated to the same degree by all four mitogens. The increased production of IgE by PWM was totally abolished by cycloheximide; hence it was due to de novo protein synthesis.
ABSTRACT The immunologic response to immunotherapy with dog extract is not well characterized. Th... more ABSTRACT The immunologic response to immunotherapy with dog extract is not well characterized. The purpose of this study was to examine the immunologic response to 3 doses of dog extract expressed as their Can f 1 content. Cluster immunotherapy was administered to 28 patients with dog allergy who were randomly assigned to 1 of 4 treatment arms: placebo or acetone-precipitated extract containing 0.6 mug, 3.0 mug, or 15.0 mug Can f 1 per 0.5 mL maintenance dose. Studies included titrated skin prick tests, the late cutaneous response, titrated nasal challenge with dog extract, and serum allergen-specific IgE and IgG(4). Dog allergen-stimulated lymphocyte proliferation was performed with measurement of secreted cytokines by ELISA and of intracellular cytokines by flow cytometry. There was a significant dose-dependent response in suppression of titrated skin prick tests and suppression of the late cutaneous response. There was a significant increase from baseline in dog-specific IgG(4) in both the high-dose and low-dose groups and a dose-dependent suppression of secreted TNF-alpha and increase in secreted TGF-beta. There was a dose-dependent trend in suppression of secreted IL-4 with a significant decrease from baseline in the high-dose group. There were no significant changes in symptom scores; lymphocyte proliferation; secreted IFN-gamma, IL-10, or IL-5; or intracellular cytokine production. The dose-response in immunologic parameters after immunotherapy with dog extract is similar to that previously demonstrated with cat extract. The greatest and most consistent response is seen with a dose containing 15 mug Can f 1.
Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and ... more Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and are used in the screening and/or diagnosis of autoimmunity in patients as well as mouse models of systemic autoimmunity. Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for ANA screening. However, its usefulness is limited in diagnosis, prognosis and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. Various immunological techniques have been developed in an attempt to improve upon the method to quantify ANA, including enzyme-linked immunosorbent assays (ELISAs), line immunoassays (LIAs), multiplexed bead immunoassays and IIF on substrates other than HEp-2 cells. Yet IIF on HEp-2 cells remains the most common screening method for ANA. In this study, we describe a simple quantitative method to detect ANA which combines IIF on HEp-2 coated slides with analysis using a near-infrared imaging (NII) system. Using NII to determine ANA titer, 86.5% (32 of 37) of the titers for human patient samples were within 2 dilutions of those determined by IIF, which is the acceptable range for proficiency testing. Combining an initial screening for nuclear staining using microscopy with titration by NII resulted in 97.3% (36 of 37) of the titers detected to be within two dilutions of those determined by IIF. The NII method for quantitative ANA measurements using serum from both patients and mice with autoimmunity provides a fast, relatively simple, objective, sensitive and reproducible assay, which could easily be standardized for comparison between laboratories.
American Journal of Respiratory and Critical Care Medicine, May 1, 2008
Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether ... more Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether the established allergic airway inflammation compromises lung innate immunity and subsequently predisposes patients with asthma to Mp infection remains unknown. To test whether the established allergic airway inflammation compromises host innate immunity (e.g., Toll-like receptor 2 [TLR2]) to hinder the elimination of Mp from the lungs. We used mouse models of ovalbumin (OVA)-induced allergic airway inflammation with an ensuing Mp infection, and cultures of mouse primary lung dendritic cells (DCs) and bone marrow-derived DCs. Lung Mp clearance in allergic mice and TLR2 and IL-6 levels in lung cells, including DCs as well as cultured primary lung DCs and bone marrow-derived DCs, were assessed. The established OVA-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, inhibited TLR2 expression and IL-6 production in lung cells, including lung DCs, and eventually led to impaired host defense against Mp. Studies in IL-6 knockout mice indicated that IL-6 directly promoted Mp clearance from the lungs. IL-4- and IL-13-induced suppression of TLR2 was mediated by inhibiting nuclear factor-kappaB activation through signal transducer and activator of transcription 6 (STAT6) signaling pathway. The established OVA-induced allergic airway inflammation impairs TLR2 expression and host defense cytokine (e.g., IL-6) production, and subsequently delays lung bacterial clearance. This could offer novel therapeutic strategies to reinstate TLR2 activation by using TLR2 ligands and/or blocking IL-4 and IL-13 to ameliorate persisting respiratory bacterial infections in allergic lungs.
The Journal of Allergy and Clinical Immunology, Jul 1, 2009
Background: To date, there have been no randomized, doubleblind studies showing the effectiveness... more Background: To date, there have been no randomized, doubleblind studies showing the effectiveness of sublingual immunotherapy with multiple allergens. Objective: The purpose of this study was to examine whether the efficacy of sublingual immunotherapy (SLIT) with standardized timothy extract was reduced by combination with other allergen extracts. Methods: A single-center, randomized, double-blind, placebocontrolled trial with SLIT was conducted. After an observational grass season, SLIT was administered for 10 months to 54 patients randomized to 1 of 3 treatment arms: placebo, timothy extract (19 mg Phl p 5 daily) as monotherapy, or the same dose of timothy extract plus 9 additional pollen extracts. Symptom and medication scores were collected and titrated nasal challenges, titrated skin prick tests, specific IgE, IgG 4 and cytokines release by timothy-stimulated lymphocyte proliferation were performed. Results: Perhaps because of a very low grass pollen season in 2008, there were no significant differences in medication or symptom scores in either treatment group compared with placebo. Compared with placebo, in the timothy monotherapy group, thresholds for titrated nasal challenge and skin prick tests (P 5 .03 and P 5 .001, respectively), and serum-specific IgG 4 levels (P 5 .005) significantly increased, and IFN-g levels decreased (P 5 .02), whereas in the multiallergen group, there was significant improvement only in the titrated skin prick tests (P 5 .04) which was less than in the monotherapy group. There were no significant differences between the 2 active groups in any outcome measure, and both active groups experienced more adverse events than placebo. There were no systemic reactions. Conclusion: Improvement in multiple relevant outcomes strongly suggests that SLIT with timothy extract alone was effective; however, the results for symptom and medication scores were not significant. The differences between multiple allergen SLIT and placebo only in skin sensitivity to timothy suggest a reduction in SLIT efficacy in this group. However, further studies are required to confirm these observations. (J Allergy Clin Immunol 2009;124:150-6.)
Page 1. Anti-native DNA Antibodies Mickey J. Mandel, MD; Ronald I. Carr, MD, PhD; William L. West... more Page 1. Anti-native DNA Antibodies Mickey J. Mandel, MD; Ronald I. Carr, MD, PhD; William L. Weston, MD; W. Mitchell Sams, Jr., MD; Ronald J. Harbeck, PhD; and Gerald G. Krueger, MD, Denver ... 12. Burch PRJ, Rowell NR: Lupus erythema-tosus. ...
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