The accelerated weight histogram method for alchemical free energy calculations.

The accelerated weight histogram method is an enhanced sampling technique used to explore free energy landscapes by applying an adaptive bias. The method is general and easy to extend. Herein, we show how it can be used to efficiently sample alchemical transformations, commonly used for, e.g., solvation and binding free energy calculations. We present calculations and convergence of the hydration free energy of testosterone, representing drug-like molecules. We also include methane and ethanol to validate the results. The protocol is easy to use, does not require a careful choice of parameters, and scales well to accessible resources, and the results converge at least as quickly as when using conventional methods. One benefit of the method is that it can easily be combined with other reaction coordinates, such as intermolecular distances.

Multidisciplinary educational programme for caregivers of children with atopic dermatitis- in South East Norway - an observational study.

BACKGROUND: Educational programmes for caregivers of children with atopic dermatitis (AD) are reported to reduce the severity of AD and improve quality of life (QOL). Oslo University Hospital (OUH) in Norway offers a multidisciplinary educational programme for caregivers of children with AD. We aimed to evaluate the AD educational programme by assessing QOL of the family, the severity of the disease and caregiver's fear of topical corticosteroid (TCS) before and after attending the programme. METHODS: This was a small observational prospective cohort study including 41 caregiver-child pairs. The children (mean age 3.4 years) had doctors' diagnosed AD with a difficult to treat eczema. The children's caregivers were referred from physicians to attend the AD educational programme at our hospital. At inclusion and at a 3 months follow-up QOL was assessed by Dermatitis Family Impact (DFI), the eczema severity by Patient-Orientated - SCORing Atopic Dermatitis (PO-SCORAD) and caregivers fear of TCS was recorded by asking a dichotomous "yes" or "no" question: "Are you worried about using TCS on your child?" RESULTS: Three months after caregivers attending the educational programme there was an improvement in QOL by reduced mean DFI from 9.6 (SD 6.3) to 6.8 (SD 5.4), the mean PO-SCORAD was reduced from 38.5 (SD 15.1) to 24.6 (SD13.6), the number of caregivers reporting fear of TCS use was reduced from 33/46 (72%) to 12/41 (29%). All results p < 0.001. CONCLUSION: Our study suggests beneficial effects by improving QOL of the family, the severity of the eczema and in reducing the fear of TCS when caregivers of children with difficult to treat AD attend an AD multidisciplinary educational programme. Lack of control group makes it difficult to draw definite conclusions.

Human skin barrier formation takes place via a cubic to lamellar lipid phase transition as analyzed by cryo-electron microscopy and EM-simulation.

The skin's permeability barrier consists of stacked lipid sheets of splayed ceramides, cholesterol and free fatty acids, positioned intercellularly in the stratum corneum. We report here on the early stage of skin barrier formation taking place inside the tubuloreticular system in the secretory cells of the topmost viable epidermis and in the intercellular space between viable epidermis and stratum corneum. The barrier formation process was analysed in situ in its near-native state, using cryo-EM combined with molecular dynamics modeling and EM simulation. Stacks of lamellae appear towards the periphery of the tubuloreticular system and they are closely associated with granular regions. Only models based on a bicontinuous cubic phase organization proved compatible with the granular cryo-EM patterns. Only models based on a dehydrated lamellar phase organization agreed with the lamellar cryo-EM patterns. The data support that human skin barrier formation takes place via a cubic to lamellar lipid phase transition.

Respiratory epithelial penetration and clearance of particle-borne benzo[a]pyrene.

Exposure to diesel exhaust is a suspected risk factor for human lung cancer. The carbonaceous core of the soot particles found in diesel exhaust and the condensed organic compounds adsorbed (or bound) onto the surface of the particles are both possible contributors to this suspected risk. The extent and rate at which organic procarcinogens desorb from soot particles in the lungs after environmental and workplace exposures and the degree of metabolic activation in the lungs are also not known. We explored the relationship between a model polynuclear aromatic hydrocarbon (PAH)* and a typical carrier particle by measuring the rate of release, extent of release, and metabolic fate of benzo[a]pyrene (BaP) bound onto the carbonaceous core of diesel soot after bolus aerosol exposures of the dog's peripheral lung and trachea. Exogenous BaP was bound onto preextracted diesel soot at a surface concentration corresponding to 25% of a monomolecular layer. After deposition in the alveolar region, a fraction of BaP was rapidly desorbed from the soot and quickly absorbed into the circulating blood. Release rates then decreased drastically. When the BaP coating reached approximately 16% of a monolayer, it was not bioavailable and remained on the particles after 5.6 months in the lung. The bioavailability of BaP on particles retained in lymph nodes was markedly higher, however: after 5.6 months the surface coating of BaP was reduced to 10% of a monolayer. Fractions of BaP that remained bound to the soot surface during this 5.6 months had a low reactivity-nearly 30% of the radioactive compounds extracted from recovered soot particles were still BaP, the parent compound. In contrast, the rapidly released fraction of BaP, which was quickly absorbed through the alveolar epithelium after inhalation, appeared mostly unmetabolized in the circulation, along with low concentrations of phase I and phase II BaP metabolites. Within approximately 1 hour, however, this rapidly absorbed fraction of BaP was metabolized, most likely in the liver, with the metabolite spectrum being dominated by conjugated phase II metabolites. The fraction of BaP desorbed from particles deposited on the epithelium of the conducting airways was absorbed by the epithelium but slowly penetrated the capillary bed. The absorbed BaP was rapidly metabolized in the airway epithelium, as indicated by the influx of tritiated water (3H2O) from the lungs into the circulation. The results suggest that the dosimetry of inhaled, highly lipophilic BaP during typical exposures is bimodal. The larger fraction of bioavailable BaP deposited in the alveolar region was absorbed mostly unaltered into the blood through the alveolar type I cells and was metabolized systemically. A smaller fraction of bioavailable BaP was deposited on the airway mucosa and rapidly metabolized, most likely in the airway epithelium. The substrate levels of BaP in the epithelium of the conducting airways exceeded the systemic levels by up to two orders of magnitude. This dramatic site-of-entry to systemic duality in the dosimetry of inhaled BaP is likely to be similar in most mammalian species and should be considered in risk assessment models for PAHs in humans.

Human alveolar macrophage phagocytic function is impaired by aggregates of ultrafine carbon particles.

Alveolar macrophages (AM) were collected by bronchoalveolar lavage from healthy volunteers. The AM were loaded with small masses (0.03-3 microg/10(6) AM) of ultrafine carbon particle aggregates. The phagocytic activity of the cells was studied 20 h after the loading. Fluorescein-labeled silica particles (3 microm) were used as test particles and the attachment and ingestion processes were studied separately. In some experiments, AM were incubated with interferon-gamma (IFN-gamma) for 20 h before and during the test of phagocytic activity and during measurement of oxidative metabolism. The ingested carbon particles induced a dose-related impairment of both the attachment and the ingestion processes with a marked impairment down to a carbon particle dose around 0.2 microg/10(6) AM. Such levels should reasonably occur after inhalation of existing concentrations of urban air particles, which to a considerable extent consist of aggregates of ultrafine particles with a carbon skeleton. Incubation with IFN-gamma (12.5 U/ml) also induced significant impairments in both the attachment and the ingestion processes. Loading with carbon further aggravated the effect of IFN-gamma. In contrast to earlier studies in rat AM, IFN-gamma did not impair the oxidative metabolism at rest in these human AM; instead the oxidative metabolism was increased. This difference was due to a difference between rat and human AM and not between rat and human IFN-gamma. Our results suggest that ingested environmental particles in AM, e.g., after an episode of high particle concentration, may impair phagocytic capacity of the cells, especially after infections that induce an increased production of IFN-gamma. Consequently, there might be a risk for additional infections. Moreover, inhaled particles not phagocytized by AM might damage the lung tissue.

The rapid alveolar absorption of diesel soot-adsorbed benzo[a]pyrene: bioavailability, metabolism and dosimetry of an inhaled particle-borne carcinogen.

Exposure to diesel exhaust may contribute to lung cancer in humans. It remains unclear whether the carbonaceous core of the soot particle or its coat of adsorbed/condensed organics contributes most to cancer risk. Equally unclear are the extent and rate at which organic procarcinogens desorb from soot particles in the lungs following inhalation exposure and the extent of their metabolic activation in the lungs. To explore the basic relationship between a model polycyclic aromatic hydrocarbon (PAH) and a typical carrier particle, we investigated the rate and extent of release and metabolic fate of benzo[a]pyrene (BaP) adsorbed on the carbonaceous core of diesel soot. The native organic content of the soot had been denuded by toluene extraction. Exogenous BaP was adsorbed onto the denuded soot as a surface coating corresponding to 25% of a monomolecular layer. Dogs were exposed by inhalation to an aerosol bolus of the soot-adsorbed BAP: Following deposition in the alveolar region a fraction of BaP was rapidly desorbed from the soot and quickly absorbed into the circulation. Release rates then decreased drastically. When coatings reached approximately 16% of a monolayer the remaining BaP was not bioavailable and was retained on the particles after 5.6 months in the lung. However, the bioavailability of particles transported to the lymph nodes was markedly higher; after 5.6 months the surface coating of BaP was reduced to 10%. BaP that remained adsorbed on the soot surface after this period was approximately 30% parent compound. In contrast, the rapidly released pulse of BaP, which was quickly absorbed through the alveolar epithelium after inhalation, appeared mostly unmetabolized in the circulation, along with low concentrations of phase I and phase II BaP metabolites. However, within approximately 1 h this rapidly absorbed fraction of BaP was systemically metabolized into mostly conjugated phase II metabolites. The results indicate that absorption through the alveolar epithelium is an important route of entry to the circulation of unmetabolized PAHS:

Functional and morphological differences between human alveolar and interstitial macrophages.

Macrophages play an essential role in pulmonary host defense. They are, however, a heterogeneous cell population located in different lung compartments. This study was designed to elucidate differences between two macrophage populations obtained from the human lung, i.e., alveolar macrophages (AM) and interstitial macrophages (IM). Macroscopically tumor-free lung segments from nine patients undergoing lobectomy or pulmectomy were studied. All patients had a diagnosis of primary lung cancer. AM were recovered by bronchoalveolar lavage and IM were isolated by mechanical fragmentation of the lavaged lung segments followed by enzymatic treatment. The cell fractions were analyzed with respect to morphology (transmission electron microscopy) and function (phagocytosis). The cells in the IM fraction were smaller (7.6 +/- 1.8 microm (mean +/- SD) compared with 16.0 +/- 4.1 microm) and morphologically more heterogeneous than those in the AM fraction. Interestingly, a considerable portion of the cells in the IM fraction had a typical AM-like appearance. Despite this, the AM fraction had a higher phagocytic activity compared to IM, with faster attachment and ingestion processes (P <0.001 for both). We conclude that the heterogeneity of human lung macrophages must be taken into consideration when their role in the inflammatory response is studied.

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure.

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.

Ingested aggregates of ultrafine carbon particles and interferon-gamma impair rat alveolar macrophage function.

Alveolar macrophages (AM), obtained by lavage from the rat lung, were allowed to ingest aggregated ultrafine carbon particles, about 1 microgram/10(6) AM, which is a realistic result of long-term exposure to ambient air. The effects of the ingested carbon on the phagocytosis of test particles and oxidative metabolism of the AM were studied. In addition, the effects of short-term (40 min or 2 h) and long-term (28 or 44 h) incubation with interferon gamma (IFN-gamma) on AM loaded and unloaded with carbon were investigated. Phagocytic activity was studied using fluorescein-labeled 3.2-microgram silica particles. The attachment and ingestion processes were evaluated separately. The ingested carbon markedly impaired the phagocytosis of silica particles; the accumulated attachment (sum of attached and ingested particles per AM) decreased from 5.0 to 4.2 particles/AM and the ingested fraction (number of ingested particles per AM divided with accumulated attachment) from 0.42 to 0.27. The short-term incubation with IFN-gamma tended to increase the accumulated attachment (from 5.0 to 5.7 particles/AM) and decreased the ingested fraction (from 0.42 to 0.34) in unloaded AM. Long-term incubation with IFN-gamma markedly impaired both the accumulated attachment (to 3.8 particles/AM) and the ingested fraction (to 0.24) in unloaded AM and the carbon load further decreased the accumulated attachment to 2.8 particles/AM, and the ingested fraction to 0.21. The oxidative metabolism was not effected by the ingested carbon or the short-term incubation with IFN-gamma, but the long-term incubation with IFN-gamma increased it with a factor of almost 3. Our results suggest that ingested environmental particles in AM may markedly impair their phagocytic capacity, especially during long-term exposure to IFN-gamma as after infections, and there might be an increased risk for additional infections. Moreover, during an episode of high ambient particle concentration the inhaled particles will not be efficiently phagocytized and may thereby damage the Lung tissue.

Differences between rat alveolar and interstitial macrophages 5 wk after quartz exposure.

Macrophages play an essential role in pulmonary host defense. We investigated differences between rat alveolar (AM) and interstitial (IM) macrophages after exposure in vivo to quartz, an inducer of intensive alveolitis. Rats were exposed to 0.5 ml of saline without (n = 8) or with (n = 8) 10 mg of quartz by intratracheal instillation. In a third group (n = 8), 10 mg of surfactant were added to the quartz mixture. Five weeks later, AM were recovered by bronchoalveolar lavage and IM by mechanical fragmentation of the lung, followed by enzymatic treatment. Contamination of AM to the IM fraction was calculated to be 12-15%. After quartz exposure, the expression of major histocompatibility complex class Ia was increased in both AM and IM fractions. The receptor corresponding to human complement receptor 3 increased in AM after quartz exposure, and AM from quartz-exposed animals had a lower metabolic activation. Our findings indicate that IM are immunocompetent cells and that differences between AM and IM fractions occur upon quartz-induced inflammation. This response is not affected by addition of surfactant.

Effects of low concentrations of 3-carene on alveolar macrophages in vitro.

Rat alveolar macrophages (AM) were incubated with the monoterpene 3-carene in vitro at the concentrations 0, 0.005, 0.05, 0.5 and 5.0 microM in culture medium for 75 min. A dose-dependent relationship was found between the cell viability and the 3-carene concentration. At 5.0 microM 3-carene, 98% of the AM were dead. The phagocytosis of heat-killed yeast particles was significantly decreased and the attachment of particles to the cell surface significantly increased at 0.5 microM 3-carene. Electron microscopy showed that about 50% of the AM lacked or had very few surface protrusions after incubation in 0.5 microM 3-carene. Thus, 3-carene seemed to affect mainly the engulfment of particles. Surfactant, 0.5 mg/ml as Curosurf, added to the incubation medium, did not affect the AM reaction to 3-carene exposure.

Rapid diagnosis of respiratory Chlamydia pneumoniae infection by nested touchdown polymerase chain reaction compared with culture and antigen detection by EIA.

Chlamydia pneumoniae is a common cause of respiratory tract infection and community-acquired pneumonia. During an extensive outbreak of C. pneumoniae in northern Sweden, 319 respiratory samples from 129 persons were collected. Sputum, throat, and nasopharyngeal samples were obtained and analyzed by nested touchdown polymerase chain reaction (PCR), EIA, and culture in Hep-2 and McCoy cells. Serology was performed by complement fixation and microimmunofluorescence tests. By PCR, 30 patients were diagnosed with C. pneumoniae compared with 26 positive by EIA and 23 by culture. The finding of C. pneumoniae in the respiratory samples was accompanied by serology indicating acute infection in 26 (96%) of 27 patients for whom adequate sera were available. Nested PCR was sensitive and reliable for diagnosing acute respiratory C. pneumoniae infection. Sputum samples had the highest diagnostic efficacy, and the nested type of PCR was superior to one-step PCR. EIA and culture were less sensitive than nested PCR.

Functional, morphological, and phenotypical differences between rat alveolar and interstitial macrophages.

Alveolar macrophages (AM) and interstitial macrophages (IM) from rat lungs were characterized with respect to morphology, phagocytosis, adhesion properties, and phenotype. AM were recovered by lung lavage and IM by treatment of the lung tissue with DNAse and collagenase. The AM were enzyme treated in the same way as the IM. The IM were smaller than AM and had a higher nuclear to cytoplasm ratio. They had markedly lower phagocytic capacity. The attachment of particles to the cell surface was significantly lower in IM than in AM, but the capacity to ingest the particles was the same. Adherence to vitronectin- as well as fibronectin-coated surfaces was significantly higher in AM. The phagolysosomal pH was similar in IM and AM, around pH 5, indicating that dissolution of inorganic particles can take place effectively also in IM. Five surface receptors were studied, and the expression differed significantly in all five between AM and IM. The expression of OX-1 (CD 45), a common leukocyte antigen, was significantly higher on AM as was the expression of CD 71 (transferrin receptor). The receptor density for OX-42 was higher on a fraction of IM. This might be compatible with a stronger interaction between these cells and, for example, matrix components. IM had more surface antigen expressing MHC class Ia (OX-6) and CD 54. Both receptors are important for the antigen presentation capacity of macrophages. These findings show profound differences in phenotype between AM and IM and indicate that IM is a highly immunocompetent cell and should not be regarded only as a precursor to AM.

Pulmonary macrophage function in systemic gadolinium chloride-pretreated rats.

The potential use of systemic gadolinium chloride pretreatment of rats as a tool to inhibit pulmonary macrophage function was studied by measurement of its effect on phagocytosis and nitroblue tetrazolium reduction by alveolar and interstitial macrophages. Gadolinium chloride solution was administered through the tail vein 48 h before the animals were killed. At 10 mg/kg, a dose which is generally used to inhibit Kupffer cell function, no effect on pulmonary macrophages was observed. Neither did a higher dose, 20 mg/kg, affect pulmonary macrophage function. It is concluded that gadolinium chloride pretreatment does not affect alveolar and interstitial macrophages upon systemic administration at doses and time after administration that generally depress Kupffer cell functioning.

DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products.

A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO2) was tested for its potency to induce DNA damage measured as single-strand breaks (SSB) in lungs of mice. Both gases were also tested separately. After 16 h exposure a UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO2, but not 20 ppm butadiene + 10 ppm NO2 + UV, induced a significant increase in SSB as measured by the alkaline unwinding technique. There was no increase in the level of SSB using the alkaline elution technique during the same testing conditions. However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO2 + UV both methods demonstrated a significant increase in SSB. Mice were also exposed to butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of exposure using the unwinding technique. Using the alkaline elution assay, a significant increase in the level of SSB in lung and liver was found only after 5 h of exposure. When mice were exposed to 30 ppm NO2 for 16 h or 50 ppm for 5 h, a significant increase in SSB was found with the unwinding technique. Alveolar macrophages from mice were also exposed in vitro to the gas mixture and to butadiene and NO2 separately. In these experiments, the DNA damage was studied with the unwinding technique. A significant effect was demonstrated with 40 ppm butadiene + 20 ppm NO2 + UV. NO2 itself contributed to some extent to the increase. Reasons for the discrepancies between the unwinding and the alkaline elution techniques are discussed.

Phagolysosomal morphology and dissolution of cobalt oxide particles by human and rabbit alveolar macrophages.

The effect of phagolysosomal size on dissolution of cobalt oxide particles was evaluated in two different macrophage systems: alveolar macrophages (AM) of human smokers with phagolysosomes enlarged by ingested cigarette smoke products, and rabbit AM incubated in vitro with sucrose, which causes swelling of the phagolysosomes by osmosis. Human AM from smokers and nonsmokers were studied in vitro. There was no significant difference in particle dissolution between AM obtained from smokers and nonsmokers, although there was a clear difference in the morphological appearance of AM, including significantly larger phagolysosomes in smokers. Rabbit AM were incubated for 24 or 72 h with or without 80 mM sucrose in the medium. The sucrose-treated cells had 3-4 times larger phagolysosomes than untreated cells, with no major change in phagolysosomal pH. The increased size of the phagolysosomes did not affect the ability of the AM to dissolve cobalt oxide particles. Furthermore, rabbit AM showed the same ability as human AM to dissolve the cobalt oxide particles, in spite of the fact that they had markedly smaller phagolysosomes. Another difference between human and rabbit AM was that phagolysosomes in human AMs increased in size with time in culture, while rabbit AM phagolysosomes decreased in size.

Role of EDRF in the regulation of regional blood flow and vascular resistance at rest and during exercise in conscious dogs.

The contribution of endothelium-derived relaxing factor (EDRF) to the regulation of regional vascular resistance and tissue blood flow at rest and during acute moderate exercise was studied in chronically instrumented conscious dogs. Radioactive microspheres were injected before and during exercise to measure regional blood flow. An infusion of nitro-L-arginine (L-NA), an analogue of L-arginine, was used to inhibit the synthesis of EDRF and resulted in a significant increase in mean arterial pressure, associated with significantly elevated vascular resistance in heart, skeletal muscle, renal and splanchnic circulations and with decreases in tissue blood flow in those regions at rest. Acute exercise caused a typical redistribution of blood flow, in which there was vasodilation in heart and working skeletal muscles, accompanied by vasoconstriction in kidney and splanchnic circulations. L-NA resulted in significantly elevated vascular resistance during vasodilation in heart and working skeletal muscles and also significantly increased vasoconstriction in renal cortex, stomach, pancreas, liver, and colon during exercise. Blood flows during exercise were largely unaffected by L-NA treatment. Our results suggest that whereas EDRF functions to regulate basal vascular tone and vascular resistance during exercise, EDRF has a minor role in determining the pattern of the redistribution of tissue blood flow during exercise.

Effects of spermidine on function and ultrastructure of alveolar macrophages.

It is known that spermidine and other naturally occurring polyamines accumulate in rat lung slices by an energy-dependent uptake process and that alveolar macrophages (AM) have a greater rate of uptake than has the total lung cell population. In the present study rat AMs were incubated with spermidine, which resulted in a marked and significant (P < 0.002) decrease in phagocytosis of heat-killed yeast cells at concentrations 0.2 and 0.5 mM and a tendency to decrease at 0.05 mM. The number of microtubules surrounding the centrioles was measured using electron microscopy and appeared to be decreased at concentrations 0.2 and 0.5 mM. There was no affect on phagolysosomal pH. The results suggest that spermidine might affect the defense against inhaled pollutants and microbes, especially when spermidine levels are increased, as they are under conditions with high mitotic activity, e.g., in tumors.

Inhalation of cobalt by sensitised guinea pigs: effects on the lungs.

Two groups, each of six guinea pigs, were sensitised by the application of cobalt chloride (CoCl2) on the skin on day 0, 2, 7, and 9 and the establishment of contact allergy was confirmed by patch testing on day 21. A further six animals were not sensitised. Starting on day 42 one sensitised group and the non-sensitised group were exposed by inhalation to 2.4 (0.8) mg (mean (SD)) Co in the form of CoCl2 for six hours a day for two weeks. After exposure the lungs were lavaged and the cells obtained were studied by light and electron microscopy. In the sensitised exposed group much more lavage liquid was retained in the lungs than in the other two groups; although more liquid was instilled in the lungs of this group, on average only 5 (range 2.5-10) ml were recovered compared with 10 ml in all animals in the other two groups. In the sensitised exposed group, the percentage of neutrophils and eosinophils tended to be higher than in the non-sensitised exposed group. The results indicate that the lungs of guinea pigs allergic to contact with Co react differently to inhaled Co compared with those of non-sensitised ones.

Phagolysosomal pH and dissolution of cobalt oxide particles by alveolar macrophages.

We studied phagolysosomal pH in rabbit alveolar macrophages (AM) incubated with 0-15 microM chloroquine. There was a dose-related increase in pH with chloroquine concentration. Electron microscopy showed that chloroquine increased lysosomal size. In a second experiment we studied dissolution of radiolabeled cobalt oxide particles by rabbit AM, phagolysosomal pH, and lysosomal size. The cells were incubated for 2 days with 0, 2.5, and 10 microM chloroquine. Size and pH increased with chloroquine concentration. Dissolution of cobalt particles by the AM did not clearly change with pH. In a third experiment, dissolution of cobalt oxide particles in 0.1 M acetate buffer in saline with pH 4.0, 5.0, and 6.0 was studied. At the same pH, dissolution in acetate buffer was faster than in the AM, and the dissolution appeared to decrease faster with increasing pH than in the AM. A simple model for dissolution of a particle in a phagolysosome was proposed. This model predicts the types of differences in dissolution between AM and buffered saline.