Characterization of mannose-binding lectin (MBL) variants by allele-specific sequencing of MBL2 and determination of serum MBL protein levels.

Mannose-binding lectin (MBL) is a major component of the lectin pathway of complement activation. High and low MBL levels have been associated with susceptibility and severity of a variety of infectious and autoimmune diseases. Several single-nucleotide polymorphisms (SNPs) in the promoter region and exon 1 of the MBL2 gene are responsible for variations in serum MBL levels. We developed a sequence-based typing method for allele-specific MBL2 genotyping and measured serum MBL protein levels in 24 German blood donors. We identified the common MBL2 haplotypes including five promoter polymorphisms in linkage with the Q allele and correlated serum MBL levels with the respective genotypes. The genotyping method presented here could provide a basis for confirmatory studies in larger cohorts.

Characterization of four new HLA alleles: HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB 1*05:14.

We describe four novel HLA alleles, HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB1*05:14.

Characterization of three new alleles HLA-A*02:241, HLA-A*02:242 and HLA-A*30:04:02.

We describe three novel alleles HLA-A*02:241, HLA-A*02:242 and HLA-A*30:04:02.

Pitfalls of "hyper"-IgM syndrome: a new CD40 ligand mutation in the presence of low IgM levels. A case report and a critical review of the literature.

Here, we report on a male infant with low serum IgG, IgA and IgM levels who suffered from Pneumocystis jirovecii and cytomegalovirus (CMV) pneumonia. The patient was tested to be HIV-negative. Absolute and relative numbers of lymphocyte subsets were normal, excluding the diagnosis of an X-linked agammaglobulinaemia (Bruton's disease). Despite the decreased serum IgM level, an X-linked hyper-IgM syndrome (X-HIGM) was considered. X-HIGM is a rare immunodeficiency usually characterised by recurrent severe opportunistic infections, low serum IgG and IgA, but normal or increased serum IgM. The syndrome is caused by mutations of the CD40 ligand (CD40L) gene. In our patient, CD40L mutation analysis proved a novel mutation at codon 257 associated with non-detectable expression of CD40L on the surface of activated T cells. A literature search revealed that approximately 6.4% of X-HIGM patients had been found to have low serum IgM levels. Our statistical analysis of the IgM levels as reported by different studies arouses suspicion that many patients with low IgM levels may not have undergone diagnostic procedures for X-HIGM. In summary, in this report and critical review of the literature, we described a new mutation of CD40L and highlighted the pitfalls of the diagnosis of X-HIGM.

Long-term responses of canine lungs to acidic particles.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Particle clearance from the airways of subjects with bronchial hyperresponsiveness and with chronic obstructive pulmonary disease.

The aim of this study was to determine particle clearance and retention from non-alveolated airways of 14 healthy subjects (HS), 10 subjects with asymptomatic bronchial hyperresponsiveness (BHR), and 23 patients with chronic obstructive pulmonary disease (COPD). Monodisperse iron oxide particles of 1.6 micro m geometric and 3.5 micro m aerodynamic diameter labeled with (99m)Tc were delivered to the airways by inspiration of small aerosol boli into shallow volumetric lung depths. In each subject the penetration front depth of the aerosol boli was adjusted to 55% of the Fowler dead space of the airways. Particle deposition was enhanced by about 7 seconds of breath-holding after bolus inhalation. Retention of the particles in the airways during the 48 hours after their administration was assessed by measuring the decline in lung activity with a sensitive gamma counter. Particle deposition was not significantly different among study groups. Twenty-four hour particle retention in the airways was not different among study groups. Sixty-one percent of the particles were retained at 24 hours in HS, 58% in BHR, and 64% in COPD. However, subjects with BHR showed accelerated mucociliary clearance compared to healthy subjects, whereas clearance was retarded in COPD patients. This long-term particle retention in the airways has to be taken into account in aerosol toxicology risk assessment and aerosol therapy dose evaluation.

Aerosol-derived airway morphometry (ADAM) in patients with lung emphysema diagnosed by computed tomography--reproducibility, diagnostic information and modelling.

Gravitational deposition of monodisperse particles can be used to determine effective airway diameter (EAD). The aim of our study was to assess intraindividual variability of EAD in healthy subjects and patients with emphysema, to compare EAD in patients with different degree and type of emphysema, and to evaluate whether parametric or model analysis would improve the results. EAD was measured vs volumetric lung depth (LD) in 11 healthy subjects (FEV subset1 107%pred) and 41 patients with emphysema (FEV subset1 60%pred; 8/9/24 mild/moderate/severe, 18/7/16 centriacinar/panacinar/bullous according to HRCT). Repeated measurements in LDs of 6-30% showed coefficients of variation of 7.0-10.4% in healthy subjects and 8.3-11.9% in emphysema. Average EAD in 10-16% LD was increased in emphysema, in particular moderate and severe (p<0.05, each). The slope of EAD in 10-16% LD differed between healthy subjects and emphysema, especially bullous and centriacinar. Patients with severe emphysema also showed a different slope compared to mild emphysema and controls. The parameters of the power function used for data fitting also showed differences between controls and emphysema, as well as between centriacinar vs panacinar and bullous emphysema. In a three-compartment lung model only the diameter of the intermediate compartment was enlarged in emphysema. We conclude that in using aerosol-derived airway morphometry, reproducibility of repeated measurements is acceptable. Average values and slopes of the EAD curve, as well as a power function for data fitting, were sensitive in the detection of type and severity of emphysema. In contrast, application of a lung model did not improve the results.

Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air-liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95+/-5 nm were produced by spark discharge at a stable concentration of about 2 x 10(6) cm(-3) and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6h yielded a total particle deposition of (2.6+/-0.4) x 10(8) cm(-2) corresponding to (87+/-23) ng cm(-2). The 2.7-fold (p < or = 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs.

Recently it was speculated that ultrafine particles (UFP) may translocate from deposition sites in the lungs to systemic circulation and whether long-term clearance differs between ultrafine and micrometer-sized particles. We have studied lung retention and clearance kinetics in 12 healthy male adult WKY rats up to 6 mo after an inhalation of (192)Ir-radiolabeled, insoluble, ultrafine 15- to 20-nm iridium particles. Whole-body retention was followed by external gamma counting, and particle clearance kinetics were determined by excretion radioanalysis. Four rats each were sacrificed after 3 wk and 2 and 6 mo; all organs as well as tissues and the carcass were radioanalyzed to balance the entire deposited radioactivity of the particles. The most prominent fraction was retained in the lungs at each time point of sacrifice (26%, 15%, 6%, respectively), and clearance out of the body was solely via excretion. Extrapulmonary particle uptake did not continue to increase but decreased with time in liver, spleen, heart, and brain when compared to previous data obtained during the first 7 days after inhalation (Kreyling et al., 2002). UFP long-term lung retention derived from whole-body measurements was comparable to previously reported data using insoluble micrometer-sized particles (Bellmann et al., 1994; Lehnert et al., 1989). In addition, differential analysis including daily excretion data revealed a pattern of fractional particle clearance rate of the ultrafine iridium particles similar to that of micrometer-sized particles reported by Snipes et al. (1983) and Bailey et al. (1985).

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 microg Cd/m(3) (1 x 10(6)/cm(3)) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)-mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53 +/- 0.12 microg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 microg Cd/m(3) for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 microg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Effects of inhaled CdO particles on the sphingolipid synthesis of rat lungs.

Surfactant lipids of the alveolar space protect the lung from various environmental stimuli. We investigated the influence of ultrafine (UF) CdO particles inhalation on two key enzymes involved in lung sphingolipid metabolism, serine palmitoyltransferase (SPT), and sphingomyelinase (SMase). Rats inhaled either 0.63 mg UF-CdO/m(3) for 6 h (group 1), or 1.08 mg UF-CdO/m(3) 12 h/day for 10 days (group 2). Two corresponding control groups inhaled filtered clean air. Additional rats intratracheally instilled with lipopolysaccharide (LPS) were used as positive controls. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) of lung tissue showed a significant increase in the level of SPT mRNA (LCB2 subunit) expression in group 2 compared to the corresponding controls (p <.01). Group 1 and LPS were not statistically different from control. No alteration in the mRNA level of SMase was detected in any exposure group. The immunohistochemical analysis showed that SPT (LCB2 subunit) localization was stronger in the alveolar type II cells of group 2 lungs compared to the corresponding controls. These results were correlated with alterations in BALF cellular and biochemical parameters and lung morphology. Since SPT is the key enzyme for de novo sphingolipid synthesis in lung surfactant and SMase is responsible for sphingomyelin catabolism, we can postulate that high-dose UF-CdO exposure for 10 days induces an increase in sphingolipid synthesis in the type II cells of rat lungs that would not be promptly followed by its degradation.

[Pneumological diagnostics by means of models aerosols. IV. Clearance of aerosol particles from the pulmonary alveoli. Foundations and first clinical results]. / Pneumologische Diagnostik mit Modellaerosolen - IV. Teil: Clearance von Aerosolpartikeln aus dem Alveolarbereich. Grundlagen und erste klinische Forschungsergebnisse.

The elimination of deposited particles (inorganic and organic particles, bacteria, viruses) from the periphery of the human lung (alveolar clearance) implies phagocytosis by alveolar macrophages, intracellular digestion, migration and translocation. Alveolar clearance of poorly soluble particles happens very slowly and primarily depends on the function of alveolar macrophages. In humans, the transport of particles to the bronchial tree is of secondary relevance, suggesting that the elimination of particles primarily depends on digestion (dissolution) processes within macrophages. The dissolved material is excreted via urine, if there is no further metabolization within the body. The pathophysiology of the alveolar clearance mechanisms in the human lung can be studied by a magnetic tracer technique (magnetopneumography). Ferromagnetic magnetite test-particles are deposited in the periphery of the lung by controlled inhalation. After magnetization and particle alignment in a strong external magnetic field pulse, the amount of retained particles can be detected by a sensitive magnetic field sensor (SQUID, superconducting quantum interference device). Long lasting cigarette smoking and chronic lung inflammations (sarcoidosis, interstitial lung fibrosis) induce a significant impairment of alveolar clearance capacity, while patients with chronic bronchitis show only a moderate impairment of alveolar clearance.

Respiratory mechanics in mice: strain and sex specific differences.

To assess the contribution of genetic background to respiratory mechanics, we developed a ventilator unit to measure lung function parameters in the mouse. We studied two commonly used inbred mice strains originating from Mus musculus domesticus (C57BL/6 and C3HeB/FeJ) and a third strain derived from Mus musculus molossinus [Japanese fancy mouse 1 (JF1)]. The ventilator allows for accurate performance of the different breathing manoeuvres required for measuring in- and expiratory reserve capacity, quasi-static and dynamic compliance, and airway resistance. In combination with a mass spectrometer for monitoring gas concentrations, single-breath manoeuvres were performed and He-expirograms obtained, from which dead space volume and slope of phase III were determined. From each strain and each sex, 10, 2-month old animals were studied immediately after being killed by an intraperitoneal overdose of xylazine and ketamine. C3HeB/FeJ and C57BL/6 exhibited comparable lung volumes. In male C3HeB/FeJ mice, e.g. vital capacity (VC) was 1072 +/- 79 microL, inspiratory reserve capacity 782 +/- 88 microL, and dead space volume at total lung inflation 216 +/- 18 microL. Lung volumes of JF1 were significantly lower (e.g. VC 611 +/- 53 microL, P < 0.01) even when normalized to body weight. In all three strains, specific lung volumes were significantly higher in females than in males, possibly explained by a higher oxygen demand during pregnancy and lactation, both of which fill most of their life times. Static compliance in C3HeB/FeJ was 64.3 +/- 5.4 microL cmH2O-1. It was smaller in C57BL/6 and JF1 mice, even when related to the lung volume. Analysis of the degree of genetic vs. non-genetic components of the phenotypic variation revealed that at least 80% of the total variation of lung volumes and static compliance in the mixed population is attributable to genetic differences between individuals. These differences will be verified in further studies by segregation and genetic linkage analysis.

Increased sputum IL-8 and IL-5 in asymptomatic nonspecific airway hyperresponsiveness.

Since asymptomatic, nonspecific airway hyperresponsiveness (BHR) may be due to an enhanced local inflammatory response, we studied molecular markers of inflammation in induced sputum from subjects with asymptomatic BHR (n = 14) compared with control subjects (n = 13) and patients with chronic obstructive pulmonary disease (COPD) (n = 10). Pulmonary lung function parameters were measured by spirometry and body plethysmography. Hyperresponsiveness was defined based on histamine challenge. Induced sputum samples were collected and the solid phase was isolated and analyzed for leukocyte numbers and differential and for cytokines (ELISA). IL-8 was 2.4-fold increased (p = 0.036) in the sputum of subjects with asymptomatic BHR (24.8 +/- 22.0 ng/mL; +/- SD) and 11.2-fold enhanced in patients with COPD (117.8 +/- 106.3 ng/mL) as compared with control subjects (10.5 +/- 7.7 ng/mL). In control subjects, no IL-5 was measured, however, sputum of those with asymptomatic BHR contained IL-5 at 0.044 +/- 0.090 ng/mL fluid and COPD patients at 1.00 +/- 2.01 ng/mL. GM-CSF could not be detected in sputum samples of any subjects investigated. Number of total leukocytes was higher in those with asymptomatic BHR and COPD (with BHR: 9.4 +/- 10.8 x 10(5); COPD: 83.5 +/- 182.5 x 10(5)) compared with persons without BHR (2.9 +/- 3.4 x 10(5)). PMN were increased in patients with asymptomatic BHR (4.1 +/- 5.3 x 10(5)) (38.8 +/- 24.7%) and COPD (32.9 +/- 71.0 x 10(5)) (75.4 +/- 18.6%) compared with controls (0.7 +/- 0.9 x 10(5)) (25.8 +/- 25.7%). In contrast to PMN counts in those with asymptomatic BHR (0.06 +/- 0.11 x 10(5)) (1.5 +/- 3.7%), eosinophil counts were only slightly increased compared with control subjects (0.01 +/- 0.02 x 10(5)) (0.6 +/- 0.9%). This study supports the hypothesis that BHR in asymptomatic people is associated with airway inflammation that may predispose to development of chronic diseases such as COPD.

Human alveolar long-term clearance of ferromagnetic iron oxide microparticles in healthy and diseased subjects.

Monodisperse ferrimagnetic microparticles (Fe3O4) with 1.3 microm geometric diameter were inhaled to study alveolar long-term clearance in healthy and diseased human subjects. Nineteen younger (age 20 to 39 years) and 20 older (age 40 to 65 years) healthy volunteers participated in the study as well as 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 15 patients with chronic obstructive bronchitis (COB). In each group the subjects were divided into never smokers (NS) and active smokers (S). Clearance was measured by magnetopneumography (MPG) for 300 days after inhalation. In COB, 50% of the deposited particles were removed from the lungs after 2 days, indicating high bronchial deposits due to bronchial obstructions. In healthy NS, only 10% of the particles were removed after 2 days and cigarette smoking enhanced the fraction of fast-cleared particles. In subjects who smoked, slow clearance was significantly impaired (P < . 02). Clearance half-lives (in days) for younger, healthy, NS were 124 +/- 66 (mean +/- SD) compared to 220 +/- 74 for S. Similarly for older subjects, the timeswere 162 +/- 120 for NS and 459 +/- 334 for S. The impairment of alveolar clearance due to cigarette smoking increases by 5.7 +/- 1.3 days/pack-year (P < .01). Alveolar clearance was impaired in SAR and in IPF; half-lives were 275 +/- 109 days (P < .05) and 756 +/- 345 days (P < .02), respectively, compared to healthy NS. Most COB patients were ex-smokers, their long-term clearance was 240 +/- 74 days, which is more than healthy NS (P < .01), but less than healthy S and might indicate a recovery of alveolar clearance. In view of studies using totally inert particles like Teflon, we conclude that the lung clearance measured with iron oxide tracer particles primarily reflects clearance by intraphagosomal particle dissolution within alveolar macrophages, which is impaired by cigarette smoke consumption and in patients.

Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats.

The cardiovascular system is currently considered a target for particulate matter, especially for ultrafine particles. In addition to autonomic or cytokine mediated effects, the direct interaction of inhaled materials with the target tissue must be examined to understand the underlying mechanisms. In the first approach, pulmonary and systemic distribution of inhaled ultrafine elemental silver (EAg) particles was investigated on the basis of morphology and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Rats were exposed for 6 hr at a concentration of 133 microg EAg m(3) (3 x 10(6) cm(3), 15 nm modal diameter) and were sacrificed on days 0, 1, 4, and 7. ICP-MS analysis showed that 1.7 microg Ag was found in the lungs immediately after the end of exposure. Amounts of Ag in the lungs decreased rapidly with time, and by day 7 only 4% of the initial burden remained. In the blood, significant amounts of Ag were detected on day 0 and thereafter decreased rapidly. In the liver, kidney, spleen, brain, and heart, low concentrations of Ag were observed. Nasal cavities, especially the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of Ag. For comparison, rats received by intratracheal instillation either 150 microL aqueous solution of 7 microg silver nitrate (AgNO(3) (4.4 microg Ag) or 150 microL aqueous suspension of 50 microg agglomerated ultrafine EAg particles. A portion of the agglomerates remained undissolved in the alveolar macrophages and in the septum for at least 7 days. In contrast, rapid clearance of instilled water-soluble AgNO(3) from the lung was observed. These findings show that although instilled agglomerates of ultrafine EAg particles were retained in the lung, Ag was rapidly cleared from the lung after inhalation of ultrafine EAg particles, as well as after instillation of AgNO(3), and entered systemic pathways.