Effect of diesel exhaust generated by a city bus engine on stress responses and innate immunity in primary bronchial epithelial cell cultures.

Harmful effects of diesel emissions can be investigated via exposures of human epithelial cells, but most of previous studies have largely focused on the use of diesel particles or emission sources that are poorly representative of engines used in current traffic. We studied the cellular response of primary bronchial epithelial cells (PBECs) at the air-liquid interface (ALI) to the exposure to whole diesel exhaust (DE) generated by a Euro V bus engine, followed by treatment with UV-inactivated non-typeable Haemophilus influenzae (NTHi) bacteria to mimic microbial exposure. The effect of prolonged exposures was investigated, as well as the difference in the responses of cells from COPD and control donors and the effect of emissions generated during a cold start. HMOX1 and NQO1 expression was transiently induced after DE exposure. DE inhibited the NTHi-induced expression of human beta-defensin-2 (DEFB4A) and of the chaperone HSPA5/BiP. In contrast, expression of the stress-induced PPP1R15A/GADD34 and the chemokine CXCL8 was increased in cells exposed to DE and NTHi. HMOX1 induction was significant in both COPD and controls, while inhibition of DEFB4A expression by DE was significant only in COPD cells. No significant differences were observed when comparing cellular responses to cold engine start and prewarmed engine emissions.

Regulation of YKL-40 expression by corticosteroids: effect on pro-inflammatory macrophages in vitro and its modulation in COPD in vivo.

BACKGROUND: Macrophages constitute a heterogeneous cell population with pro- (MΦ1) and anti-inflammatory (MΦ2) cells. The soluble chitinase-like-protein YKL-40 is expressed in macrophages and various other cell types, and has been linked to a variety of inflammatory diseases, including COPD. Dexamethasone strongly reduces YKL-40 expression in peripheral blood mononuclear cells (PBMC) in vitro. We hypothesized that: a) YKL-40 is differentially expressed by MΦ1 and MΦ2, b) is decreased by corticosteroids and c) that long-term treatment with inhaled corticosteroids (ICS) affects YKL-40 levels in serum and sputum of COPD patients. METHODS: Monocytes of healthy subjects were cultured in vitro for 7 days with either GM-CSF or M-CSF (for MΦ1 and MΦ2, respectively) and stimulated for 24 h with LPS, TNFα, or oncostatin M (OSM). MΦ1 and MΦ2 differentiation was assessed by measuring secretion of IL-12p40 and IL-10, respectively. YKL-40 expression in macrophages was measured by quantitative RT-PCR (qPCR) and ELISA; serum and sputum YKL-40 levels were analyzed by ELISA. RESULTS: Pro-inflammatory MΦ1 cells secreted significantly more YKL-40 than MΦ2, which was independent of stimulation with LPS, TNFα or OSM (p < 0.001) and confirmed by qPCR. Dexamethasone dose-dependently and significantly inhibited YKL-40 protein and mRNA levels in MΦ1. Serum YKL-40 levels of COPD patients were significantly higher than sputum YKL-40 levels but were not significantly changed by ICS treatment. CONCLUSIONS: YKL-40 secretion from MΦ1 cells is higher than from MΦ2 cells and is unaffected by further stimulation with pro-inflammatory agents. Furthermore, YKL-40 release from cultured monocyte-derived macrophages is inhibited by dexamethasone especially in MΦ1, but ICS treatment did not change YKL-40 serum and sputum levels in COPD. These results indicate that YKL-40 expression could be used as a marker for MΦ1 macrophages in vitro, but not for monitoring the effect of ICS in COPD. TRIAL REGISTRATION: ClinicalTrials.gov, registration number: NCT00158847.

Exhaled nitric oxide is related to bronchial eosinophilia and airway hyperresponsiveness to bradykinin in allergen-induced asthma exacerbation.

Exhaled nitric oxide (FeNO) has been associated with bronchial eosinophilia and with airway hyperresponsiveness (AHR) in mild stable asthma. We previously demonstrated in a large project that allergen exposure is able to raise FeNO and to worsen AHR to bradykinin. We postulated that allergen-induced increase in FeNO could be related to heightened mucosal eosinophils and AHR to bradykinin in atopic asthma. We performed a new immunohistochemical analysis on bronchial biopsy specimens, previously obtained from the same large project, in order to assess the number of mucosal eosinophils (EG-2+ cell) and other inflammatory cells at 48 hours after diluent and allergen exposures. Inflammatory cell counts were related to FeNO and AHR to BK (expressed as logPD20 bradykinin). In 10 atopic mild asthmatics, we found that the numbers of EG-2+ and CD4+ cells in bronchial submucosa were significantly increased after allergen compared to the respective counts after diluent (p < 0.01). EG-2+ cells in the bronchial submucosa were negatively correlated with logPD20 bradykinin only after allergen challenge (rho = -0.709, p = 0.027). We also found a positive strong correlation between EG-2+ cells and FeNO values in atopic asthmatics at 48 hours after both diluent (rho = 0.746, p = 0.017) and allergen (rho = 0.644, p = 0.049) challenge. FeNO values negatively correlated with responsiveness to bradykinin only after allergen challenge (rho = -0.675, p = 0.039). This study indicates that after allergen exposure heightened level of exhaled NO may reflect augmented airway eosinophilic inflammation and airway responsiveness to bradykinin indicating loss of asthma control.

Molecular characterization of allium virus X, a new potexvirus in the family Alphaflexiviridae, infecting ornamental allium.

ABTRACT: A new potexvirus affecting ornamental allium spp in the Netherlands was identified and characterized at the molecular level. The virus had a single-stranded RNA genome of 7100 bp (excluding the 18 bp poly A tail). The genome organization was found to be typical of members of the genus Potexvirus and consisted of five open reading frames (ORF). Nucleotide and amino acid sequence comparisons with those of known potexvirus members showed that this virus is related to Hosta virus X and Hydrangea ringspot virus. Sequence similarities and phylogenetic relationships suggested that the allium virus is a new and distinct species in the genus Potexvirus and the name, Allium virus X (AlVX) is proposed.

High-affinity immunoglobulin E receptor expression is increased in large and small airways in fatal asthma.

BACKGROUND: IgE and its high-affinity receptor FcɛRI play an important role in allergy and asthma. The distribution of FcɛRI expression in the airways and within the airway wall, however, is largely unknown. OBJECTIVE: In this study, we aimed to map the distribution of FcɛRI in different layers of large airways (LA) and small airways (SA) in lung tissue from non-smoking and smoking patients who died of asthma [fatal asthma (FA)] and non-smoking controls (CTR). METHODS: Postmortem lung tissue from 24 cases of non-smoking FA, 13 smoking FA patients and from 19 subjects who died of non-pulmonary causes (CTR) was immunohistochemically stained for FcɛRI and AA1 (mast cell tryptase marker). The expression of these markers was analysed in inner, muscle, and outer layers of both LA and SA by image analysis. RESULTS: FcɛRI expression was higher in non-smoking and smoking FA compared with CTR in the inner and outer layer of SA. In the outer layer of LA, FcɛRI expression was higher in non-smoking FA compared with CTR. AA1 was higher in non-smoking FA compared with smoking FA and CTR in the outer layer of the SA, which was correlated with FcɛRI in this layer. CONCLUSION: Our results show that the expression of FcɛRI is higher in both LA and SA in FA compared with CTR. These differences are predominantly found in the outer layer where they can be attributed in part to the increased mast cell numbers. These results indicate an increased capacity to mount IgE-mediated reactions in FA, both in LA and SA.

Nucleotide sequence and genome organization of a member of a new and distinct Caulimovirus species from dahlia.

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I-VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%.

Incidence and Relative Prevalence of Distinct Caulimoviruses (Genus Caulimovirus, Family Caulimoviridae) Associated with Dahlia Mosaic in Dahlia variabilis.

Dahlia mosaic, caused by Dahlia mosaic virus (DMV), is one of the most important viral diseases of dahlia. Molecular characterization of DMV showed the association of two distinct caulimoviruses (DMV-D10, DMV-Portland) and a D10-like sequence variant (DMV-Holland) with the disease. Using primers specific to these two viruses and the sequence variant, a polymerase chain reaction-based assay was used to determine their relative incidence in several dahlia samples from the United States and the Netherlands. Testing was done on samples collected in 2005 and 2006 in the United States and in 2006 in the Netherlands. Results indicated the predominance of DMV-D10 over DMV-Portland and DMV-Holland in both the United States and the Netherlands. Using conserved regions of the viral genome, primers were designed and used to detect all three sequences. Results suggested that DMV-D10 is predominantly associated with dahlia mosaic, but diagnostics should also include testing for DMV-Portland and DMV-Holland.

Increased number of B-cells in bronchial biopsies in COPD.

Recently, it has been shown that the accumulated volume of B-cells in small airways is increased in chronic obstructive pulmonary disease (COPD) Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 3 and 4. Little is known about the number of B-cells in central airways in COPD. The present authors hypothesised that the number of B-cells in bronchial biopsies of large airways is higher in patients with COPD than in controls without airflow limitation and higher in more severe COPD. Therefore, bronchial biopsies were collected from 114 COPD patients (postbronchodilator forced expiratory volume in one second (FEV1) 63+/-9 % predicted value, FEV1/inspiratory vital capacity (IVC) 48+/-9%) and 28 controls (postbronchodilator FEV1 108+/-12 % predicted value, FEV1/IVC 78+/-4%). Paraffin sections were stained for B-cells (CD20+) and their number was determined in the subepithelial area (excluding muscle, glands and vessels). B-cell numbers were higher in patients with COPD versus controls (8.5 versus 3.9 cells x mm(-2), respectively) and higher in patients with GOLD severity stage 3 (n = 11) than stage 2 (n = 103; 22.3 versus 7.8 cells x mm(-2)). No relationship was found between the number of B-cells and clinical characteristics within the chronic obstructive pulmonary disease group. The authors suggest that these increased B-cell numbers may have an important contribution to the pathogenesis of chronic obstructive pulmonary disease.

First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands.

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5'-AGC ATG GCG CCA TCT TGT GAC TG-3') was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5'-CTG AAG TCA CAA TGG GTG AAG AA-3') was located downstream at position 3969-3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus. References: (1) M. G. Bellardi and A. Pisi. Inf. Fitopatol. 37:33, 1987. (2) A. F. L. M. Derks. Crocus spp. Pages 260-264 in: Virus and Virus-like Diseases of Bulbs and Flower Crops. G. Loebenstein et al., eds. Wiley publishers, West Sussex, UK, 1995. (3) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991.

Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways.

OBJECTIVE: The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. MATERIALS AND METHODS: The presence of these molecules and the number of proliferating (Ki-67-positive) epithelial cells was analyzed by immunohistochemistry in bronchial epithelium from subjects with (n = 15) or without (n = 14) chronic obstructive pulmonary disease (COPD). RESULTS: Our data demonstrate higher numbers of defensin-positive (p = 0.0001), elastase-positive (p = 0.0001) and Ki-67-positive (p = 0.0001) cells in areas with squamous cell metaplasia as compared to areas with intact or damaged epithelium, while the reverse was observed for SLPI expression (p = 0.002). No differences were observed between subjects with or without COPD, nor between current smokers and those that had stopped smoking. CONCLUSIONS: These data are in line with a role of defensins in the hyperproliferative phenotype of squamous metaplastic lesions in the airways. This role does not seem to be restricted to patients with COPD.

Proliferation and inflammation in bronchial epithelium after allergen in atopic asthmatics.

BACKGROUND: The mechanisms that regulate epithelial integrity and repair in asthma are poorly understood. We hypothesized that allergen exposure could alter epithelial inflammation, damage and proliferation in atopic asthma. OBJECTIVE: We studied epithelial cell infiltration, shedding, expression of the proliferation marker Ki-67 and the epithelial cell-cell adhesion molecules Ep-CAM and E-cadherin in bronchial biopsies of 10 atopic mild asthmatics 48 h after experimental diluent (D) and allergen (A) challenge in a cross-over design. METHODS: Epithelial shedding, expressed as percentage of not intact epithelium, Ki-67+, eosinophil/EG-2+, CD4+ and CD8+ cells were quantified by image analysis in bronchial epithelium, and adhesion molecules were analysed semi-quantitatively. RESULTS: Epithelial shedding was not altered by A (D: 88.1+/-3.1% vs. A: 89.2+/-3.7%; P=0.63). The numbers of Ki-67+ epithelial (D: 10.2+/-0.2 vs. A: 19.9+/-0.3 cells/mm; P=0.03), EG-2+ (D: 4.3+/-0.5 vs. A: 27+/-0.3 cells/mm; P=0.04) and CD4+ cells (D: 1.7+/-1.2 vs. A: 12.3+/-0.6 cells/mm; P=0.04) were significantly increased after A, whilst CD8+ numbers were not significantly changed (P>0.05). E-cadherin and Ep-CAM epithelial staining showed a similar intensity after D and A (P>0.05). We found a positive correlation between EG-2+ and Ki-67+ cells in the epithelium (Rs: 0.63; P=0.02). CONCLUSION: Our study indicates that allergen challenge increases epithelial proliferation in conjunction with inflammation at 2 days after exposure. This favours the hypothesis that long-lasting epithelial restitution is involved in the pathogenesis of asthma.

Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma.

BACKGROUND: Endogenous nitric oxide protects against airway hyperresponsiveness (AHR) to bradykinin in mild asthma, whereas AHR to bradykinin is enhanced by inhaled allergens. OBJECTIVE: Hypothesizing that allergen exposure impairs bronchoprotective nitric oxide within the airways, we studied the effect of the inhaled nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on AHR to bradykinin before and after allergen challenge in 10 subjects with atopic asthma. METHODS: The study consisted of 3 periods (1 diluent and 2 allergen challenges). AHR to bradykinin (PD(20)BK) was examined before and 48 hours after allergen challenge, both after double-blinded pretreatment with L-NMMA or placebo. The accompanying expression of the various NOS isoforms (ecNOS, nNOS, and iNOS) was examined by means of immunohistochemistry in bronchial biopsies obtained after diluent and allergen challenge. RESULTS: After placebo, AHR to BK worsened after allergen challenge in comparison with before allergen challenge (PD(20)BK, 70.8 nmol [range, 6.3-331] and 257 nmol [35.5-2041], respectively; P =.0004). After L-NMMA, preallergen and postallergen PD(20)BK values (50.1 nmol [1.8-200] vs 52.5 nmol [6.9-204]; P =.88) were similarly reduced (P <.01) and not different from the postplacebo/postallergen value (P >.05). After allergen challenge, the intensity of staining in bronchial epithelium decreased for ecNOS (P =.03) and increased for iNOS (P =.009). These changes in immunostaining were correlated with the accompanying worsening in AHR to BK (R(s) = -0.66 and 0.71; P <.04). CONCLUSIONS: These data indicate that allergen exposure in asthma induces increased airway hyperresponsiveness to bradykinin through impaired release of bronchoprotective nitric oxide associated with downregulation of ecNOS. This suggests that new therapeutic strategies towards restoring the balance among the NOS isoforms during asthma exacerbations are warranted.

Localization of gamma-glutamylcysteine synthetase messenger rna expression in lungs of smokers and patients with chronic obstructive pulmonary disease.

Cigarette smoking results in an oxidant/antioxidant imbalance in the lungs and inflammation, which are considered to be key factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). Glutathione (GSH) is an important protective antioxidant in lung epithelial cells and epithelial lining fluid. De novo GSH synthesis in cells occurs by a two-enzyme process. The rate-limiting enzyme is gamma-glutamylcysteine synthetase (gamma-GCS), in which the heavy subunit (HS) constitutes most of its catalytic activity. The localization and expression of gamma-GCS-HS in specific lung cells as well as possible differences in its expression between smokers with and without COPD have not yet been studied. The purpose of this study was to investigate gamma-GCS-HS expression using messenger RNA in situ hybridization in peripheral lung tissue. We studied 23 current or ex-smokers with similar smoking histories with (n = 11; forced expiratory volume in 1 s [FEV(1)] < 75% predicted) or without COPD (n = 12; FEV(1) < 84% predicted). We assessed the relations between pulmonary gamma-GCS-HS expression, FEV(1) and transforming growth factor-beta1 (TGFbeta(1)), because TGFbeta(1) can modulate gamma-GCS-HS expression in lung epithelial cells. Gamma-GCS-HS is predominantly expressed by airway and alveolar epithelial cells, alveolar CD68+ cells (macrophages), and endothelial cells of both arteries and veins. In subjects with COPD, semiquantitative analysis revealed higher levels of gamma-GCS-HS messenger RNA in alveolar epithelium (1.5 times, p <.04) and a trend for a higher expression in bronchiolar epithelium (1.3 times, p =.075) compared with subjects without COPD. We did not observe a significant correlation between airway and alveolar epithelial gamma-GCS-HS expression and TGFbeta(1) expression (r =.20), FEV(1) percentage predicted (r =.18), or FEV(1)/forced vital capacity ratio (r =.14; p.05). Our results show that gamma-GCS-HS is localized, particularly in lung epithelium, and shows higher expression in smokers with COPD. This suggests a specific role for enhanced GSH synthesis as a mechanism to provide an adaptive response against oxidative stress in patients with COPD.

Monocyte chemoattractant protein 1, interleukin 8, and chronic airways inflammation in COPD.

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death, with cigarette smoking among the main risk factors. Hallmarks of COPD include chronic airflow obstruction and chronic inflammation in the airway walls or alveolar septa. An earlier study reported elevated numbers of macrophages and mast cells within the bronchiolar epithelium in smokers with COPD, compared with smokers without. Since specific chemokines may be involved in this influx, the in situ protein and mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and of interleukin 8 (IL-8) were studied in tumour-free peripheral lung tissue resected for lung cancer of current or ex-smokers with COPD (FEV(1)<75%; n=14) and without COPD (FEV(1)>84; n=14). MCP-1 was expressed by macrophages, T cells, and endothelial and epithelial cells. Its receptor, CCR2, is expressed by macrophages, mast cells, and epithelial cells. IL-8 was found in neutrophils, epithelial cells, and macrophages. In subjects with COPD, semi-quantitative analysis revealed 1.5-fold higher levels of MCP-1 mRNA and IL-8 mRNA and protein in bronchiolar epithelium (p<0.01) and 1.4-fold higher levels of CCR2 in macrophages (p=0.014) than in subjects without COPD. The bronchiolar epithelial MCP-1 mRNA expression correlated with both CCR2 expression on macrophages and mast cells (p<0.05) and the numbers of intra-epithelial macrophages and mast cells (p<0.04). The epithelial IL-8 expression did not correlate with the numbers of neutrophils, macrophages, CD45RO+, CD8+, or mast cells. These data suggest that MCP-1 and CCR2 are involved in the recruitment of macrophages and mast cells into the airway epithelium in COPD.

Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease.

Chronic airways inflammation is one of the features of chronic obstructive pulmonary disease (COPD). We demonstrated previously that bronchiolar epithelium in COPD contains increased numbers of macrophages and mast cells. Transforming growth factor beta1 (TGF-beta1) may be involved in this influx because it has chemotactic activity for macrophages and mast cells. In this study, we examined expression patterns of TGF-beta1, TGF-beta receptors type I and II (TGF-betaRI and TGF-betaRII) by immunohistochemistry and mRNA in situ hybridization in peripheral lung tissue of 14 current or ex-smokers with COPD (FEV1 < 75%) and 14 without COPD (FEV1 > 84%). In both groups, TGF-beta1 and its receptors are present in airway and alveolar epithelial cells, airway and vascular smooth muscle cells, and tissue and alveolar CD68(+) cells (considered herein to be macrophages). In subjects with COPD, a semiquantitative analysis revealed approximately twofold higher levels of TGF-beta1 mRNA and protein in bronchiolar and alveolar epithelium (p < 0.02) as compared with subjects without COPD. With regard to bronchiolar epithelial cells, we found a significant correlation between TGF-beta1 mRNA and protein expression (r = 0.62; p < 0.002), and between the FEV1 of all subjects together and TGF-beta1 protein (r = -0.60; p < 0.0002) and mRNA (r = -0.67; p < 0. 002) levels. The epithelial expression of TGF-beta1 mRNA and TGF-beta1 protein correlates with the number of intraepithelial macrophages (both: r = 0.44; p < 0.03) whereas intraepithelial mast cell numbers correlate with epithelial TGF-beta1 mRNA expression. These data suggest a role for TGF-beta1 in recruiting macrophages into the airway epithelium in COPD.

A comparison of the detection sensitivity of lymphocyte membrane antigens using fluorescein and phosphor immunoconjugates.

In this study we compared the sensitivity of immunocytochemical procedures, using conventional and time-resolved fluorescent dyes, in a model system consisting of paraformaldehyde-fixed human lymphocytes. The lymphocytes were stained for the presence of the CD4 epitope by indirect immunofluorescence using FITC as label or by using time-resolved luminescent immunophosphors. These immunophosphors were primarily developed for use under time-resolved fluorescence conditions, but they are also very well suited for use in conventional fluorescence microscopes. The differently labeled cells were first examined visually with a conventional fluorescence microscope in a double-blind study. The fluorescence was also measured with a CCD camera mounted on a specially constructed time-resolved fluorescence microscope which allows the suppression of the fast decaying fluorescence, thereby permitting visualization of the specific, slowing decaying luminescence of the phosphor label. With this microscope FITC and immunophosphor labeled lymphocytes were compared under normal conditions (i.e., continuous excitation) and under conditions of time-resolved registration. Conventional fluorescence microscopy revealed a better sensitivity in favor of the phosphor conjugates. This difference became more prominent when the preparations were quantitatively assessed with the CCD-time-resolved microscope. Time-resolved microscopy permitted a suppression of fast decaying fluorescence better than 1 to 10(6).

Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers.

A new luminescent marker for the immunochemical detection of proteins and nucleic acids on filters is reported. The label consists of inorganic crystals, generally called phosphors, with a particle size of 0.1-0.3 microns, stabilized in suspension with polycarboxylic acids and subsequently conjugated to immunoreactive macromolecules. Immunophosphor conjugates exhibit slowly decaying fluorescence that is strong and practically nonfading and not sensitive to quenching by water molecules. They are therefore suited for conventional fluorescence detection as well as for time-resolved detection. The lifetime of the phosphors was in the micro/milliseconds range upon excitation with ultraviolet light. Proteins or nucleic acids immobilized on nitrocellulose filters were detected immunochemically or by hybridization, using haptenized nucleic acid probes followed by immunochemical detection, respectively. The ultimate detection limit of proteins, using phosphor-labeled macromolecules including an immunochemical amplification step, was found to be 10 fg. The detection limit of nucleic acids was 300 fg for demonstration of hapten-labeled probes and 10 pg in hybridization formats with hapten-labeled probes. The sensitivity of methods using phosphor-labeled macromolecules was in all cases as good as or better than that of methods using alkaline phosphatase developed to NBT/BCIP. The use of immunophosphors for detection of proteins and nucleic acids on Western and Southern blots is demonstrated. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.

Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy.

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.