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Idiopathic pulmonary hemosiderosis is primarily a disorder of childhood, which is characterized by hemoptysis, iron deficiency anemia, and diffuse parenchymal infiltrates on chest x-ray secondary to recurrent attacks of alveolar hemorrhage. It can be diagnosed by showing hemosiderin laden macrophages in bronchoalveolar lavage fluid after other specific causes of diffuse alveolar hemorrhage are definitely excluded. A 5-year-old male patient was admitted to our clinic with sudden-onset pallor during iron therapy given for anemia. While he was being investigated for clinical and laboratory signs mimicking hemolytic anemia, he developed cough and dyspnea. He had infiltrates on chest x-ray and scattered patchy infiltrates in both lungs on high-resolution computed tomography. Hemosiderin laden macrophages were identified in fasting gastric juice and bronchoalveolar lavage fluid. The patient was diagnosed with idiopathic pulmonary hemosiderosis and started corticosteroid therapy.

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv(+)) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4(+) cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Gastroesophageal reflux (GER) is frequently associated with pulmonary diseases. Esophageal acid-induced bronchoconstriction and recurrent microaspirations of gastric content are the proposed mechanisms. At present there is not a sensitive test available to prove a causal relationship between pulmonary symptoms and GER. In this study we aimed to investigate the value of a marker of aspiration: lipid-laden alveolar macrophage index (LLAM) in induced sputum, in the diagnosis of pulmonary symptoms highly suspected to be due to GER., Methods: Twenty-two patients with the endoscopic diagnosis of erosive esophagitis who had various pulmonary symptoms that could not be attributed to any apparent etiology constituted the study group. Fifteen healthy volunteers with no prior diagnosis of gastroesophageal and lung disease constituted the control group. Subjects were questioned for pulmonary and abdominal symptoms and underwent physical examination, chest radiography, pulmonary function tests, and sputum induction. Prepared cytospins were stained with oil red-O to detect cytoplasmic lipid droplets and LLAM index was calculated., Results: There was no statistically significant difference between the LLAM indexes of the study (1.9 +/- 3.3) and control group (4.2 +/- 4.5). LLAM index had a significant positive correlation with the duration of reflux symptoms (p=0.01, r=0.5)., Conclusions: LLAM index is not found to be a valuable method in the differential diagnosis of pulmonary symptoms suspected to be due to GER.

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise-induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2-5 years was used. Horses were grouped as either control (EIPH-negative) or EIPH-positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocentrifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P < .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa = 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.

Pulmonary dysfunction represents one of the least studied complications in thalassemic patients. Probably, it is due to the absence of pulmonary symptoms. There are few works in the literature, and contradictory results have been published. The aim of this study was to define the spirometric pattern and the possible causes of lung impairment by testing bronchoalveolar lavage (BAL) with pathological pulmonary function tests (PFTs). Furthermore, diffusion capacity tests for carbon monoxide corrected for hemoglobin value (Dco*) were performed. We studied 48 thalassemic patients (27 F and 21 M), with an age range from 8 to 23 years, divided into two groups on the basis of PFTs results. Thus, group A was formed by 16 patients with restrictive spirometric patterns of whom 14 had also reduced Dco* values and group B consisted of 32 patients with normal PFTs and Dco* values. Patients of group A underwent chest high-resolution computing tomography (CHRCT) and BAL whose fluid was analyzed by microbiologic and cytological assays. A pathological CHRCT picture was present in 8 patients. Nine out of 16 patients who accepted to undergo BAL had a chronological age greater than 17 years with a mean bone age of 13.9 years. BAL results showed lymphocyte alveolitis in 6 patients and a normal cytogram in 3, while alveolar iron-laden macrophages were present in 4 out of 6 patients with alveolitis and 2 out of 3 patients with normal cytogram. Moreover, all examined BAL fluids showed a normal CD4/CD8 ratio, while only 2 patients showed an altered serum CD4/CD8 ratio. We demonstrated the presence of (1) lung-restrictive syndrome in 16 of the oldest thalassemic patients; (2) lymphocyte alveolitis in 6 patients, and (3) a picture of interstitial fibrosis by CHRCT in 8 of them. All these data are suggestive of a diagnosis of interstitial lung disease secondary to thalassemia. BAL helped to identify the presence of alveolar iron-laden macrophages that represented a local defense mechanism against free iron. This latter finding therefore might be the primary cause of the lung impairment promoting an oxidative damage. Further studies are needed to investigate this hypothesis and therapeutical potentials., (Copyright 2000 S. Karger AG, Basel)

A 34-year-old man with an 18-year history of hypertrophic cardiomyopathy died of worsening right-sided heart failure. Central venous pressure was greatly increased to 25 cm H2O before death. Postmortem examination revealed features of severe congestive vasculopathy, including those of pulmonary capillary hemangiomatosis in the lungs. Marked proliferation of capillaries was seen chiefly in alveolar septa and extending into pulmonary veins and arteries, causing severe luminal occlusion with recanalization. Diffusely distributed intra-alveolar edema and hemorrhage with collections of hemosiderin-laden macrophages were also seen, which suggested that the pulmonary capillary hemangiomatosis was associated with longstanding chronic passive congestion of the lung. It is possible that severe pulmonary passive congestion may be one of the causes of development of idiopathic pulmonary capillary hemangiomatosis.

Rat lungs were histologically examined at 1, 7, 14 and 28 days following a single intratracheal instillation of zinc hydroxide (1 mM). After one day of treatment, no confirmatory findings were noted. The zinc hydroxide injections were followed by an increase in proliferating cell nuclear antigen labeling indices in both alveolar macrophages and terminal bronchioles. After 7 days, the zinc hydroxide-treated lungs showed thickening of the interstitium with infiltration by alveolar macrophages, and an increase in the grade of Masson's trichrome staining (collagen fiber) in the alveolar interstitium. Thereafter, these morphological changes disappeared. The vehicle- and zinc sulfate (1 mM)-exposed lungs had no abnormalities at any time point. Formazan deposits in alveolar macrophages, formed as a result of nitro blue tetrazolium reduction, were increased in zinc hydroxide-treated lung slices, suggesting that zinc hydroxide stimulated super oxide anion generation from alveolar macrophages. These results show that zinc hydroxide can induce morphological alterations of rat lungs.

Corynebacterium pseudotuberculosis infection is a common cause of pyogranulomas in ovine lungs and often occurs as a dual infection with lentiviruses. This coinfection usually leads to the development of chronic pneumonia and cachexia that is similar to the clinical syndrome seen in human beings with AIDS-related pneumonias. Recent in vitro studies indicate that monokines such as tumor necrosis factor-alpha (TNF alpha) are induced by C. pseudotuberculosis, suggesting that TNF alpha is involved in the pathogenesis of corynebacterial lesions in vivo. To substantiate in vitro observations concerning bacterial induction of TNF alpha in ovine pulmonary macrophages, immunohistochemical labeling techniques were used in combination with in situ hybridization to identify TNF-producing cells in corynebacterial lesion sites in vivo. TNF alpha message and translation product were found in macrophages comprising pyogranulomas that were induced by naturally acquired and experimental pulmonary C. pseudotuberculosis infections.

Human migration inhibitory factor (MIF) is suggested to play a notable role in regulation of macrophage functions in host defense. A major binding component for the lymphokine in human tissue is the interferon antagonist sarcolectin. This high-affinity interaction gives access to MIF by affinity chromatography on immobilized sarcolectin and may be of significance for in situ activity of MIF. Localization of MIF is one step towards answering this question. Labelled sarcolectin and MIF-specific antibodies can be employed to analyze the expression of the factor. Surgical specimens of 74 patients, who underwent lobe/lung resection or diagnostic biopsy, were fixed with buffered formalin and embedded in paraffin. The material consisted of 36 cases of morphologically normal lung parenchyma of patients, suffering from bronchial carcinoma, of 16 cases with sarcoidosis, of 15 cases with tuberculosis and of 7 cases with idiopathic interstitial pneumonitis. The two types of probe to visualize presence of MIF invariably showed the same level of reactivity, underscoring the potential physiological significance of sarcolectin-MIF interaction. In detail, all cases with pneumonitis, most tuberculosis-affected as well as normal cases and 44% of the cases with sarcoidosis were positive. All positive cases with sarcoidosis and some cases from the other groups revealed accessible binding sites for biotinylated MIF.

This study was designed to investigate the expression and functional properties of leukocyte adhesion molecules (LeuCAM; CD11/CD18 family) on human alveolar macrophages (AM) from patients with sarcoidosis and idiopathic pulmonary fibrosis. Cells were obtained by bronchoalveolar lavage (BAL) from 17 patients with sarcoidosis (SA), 15 with idiopathic pulmonary fibrosis (IPF), and 14 nonsmokers (NS). Expression of LeuCAM on freshly isolated cells was studied using the peroxidase-antiperoxidase method with monoclonal antibodies (MoAb) detecting CD11a, CD11b, CD11c, and CD18. The functional properties of the adhesion molecules were studied by measuring superoxide anion production (O2-) of SA and IPF AM after blocking the CD18 molecule by an MoAb. Compared with nonsmokers, the samples from SA and IPF patients contained an increased number of AM expressing CD11a, CD11b, CD11c, and CD18 (all p < 0.008), which was correlated to the number of AM/ml BAL (p < 0.008). Spontaneous O2- secretion of AM was higher in SA (6.4 +/- 1.2 nMO2-/10(6) AM/120 min) and IPF (12.0 +/- 1.1 nMO2-/10(6) AM/120 min) compared with NS (2.5 +/- 0.2 nMO2-/10(6) AM/120 min) (both p < 0.008). Incubation of the AM with the MoAb anti-CD18 reduced the spontaneous O2- release from SA AM by 52 +/- 8% and from IPF AM by 49 +/- 3% but did not influence O2- release from NS AM (92 +/- 4%). Our data indicate that the increased expression of LeuCAM on AM in subjects with SA and IPF seems to be involved in the increased O2- production of these cells in both diseases.

Interleukin-2 receptor (IL-2R) expression on bronchoalveolar lavage (BAL) cells was studied in patients with sarcoidosis using immune cytochemistry and cytometric analysis. A low percentage of alveolar lymphocytes (AL) was found positive for IL-2R, with 7% in patients with impaired lung function and 6% in patients with normal lung function (0.4% in control subjects). Expression of IL-2R on alveolar macrophages (AM) was considerably higher, with 25% in patients with lung function impairment compared with 14% in patients without lung function impairment (1.5% in control subjects). Serum-soluble IL-2R (ssIL-2R) was significantly elevated only in patients with impaired lung function (140.0 pM), but not in patients with normal lung function (52.2 pM; control subjects, 40.0 pM). These elevated levels of ssIL-2R positively correlated with the percentage of IL-2R positive AM (p < 0.001). Immunosuppressive treatment in three patients resulted in a decrease of IL-2R+ AM and in a decrease of ssIL-2R, whereas IL-2R+ AL were unaffected. The positive correlation and the concomitant decrease of IL-2R+ AM and ssIL-2R are consistent with the idea that in sarcoidosis with clinically apparent lung involvement, elevated levels of ssIL-2R may be derived from AM and may thus be a useful indicator of the degree of activation of these cells.

Diffuse pulmonary hemorrhage syndromes occasionally present diagnostic problems if the clinical picture is not very typical. The presence of hemosiderin-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid is a useful diagnostic criterion for this entity. However, in many diffuse interstitial pulmonary diseases (DIPD) there is a lesion at the alveolocapillary barrier, and an exit of red blood cells could occur from the blood vessels, leading to the appearance of siderophages. The aim of this work was dual: to evaluate the presence and number of siderophages in different types of interstitial pulmonary disease and to compare the diagnostic yield of two ways of quantifying hemosiderin-laden macrophages. Three groups of patients--controls (n = 5), DIPD (n = 32) and diffuse pulmonary hemorrhage (n = 3)--were subjected to BAL, and a differential count was made on cytocentrifuged Diff-Quik- and Perl-stained preparations. On the latter, two different measurements were made: the number of macrophages laden with hemosiderin and a quantitative "score." The results of a conventional count (percent of Perl-positive macrophages) showed significant differences between the three groups considered overall. Applying a cutoff value of 20%, the sensitivity of this method was 100% and the specificity, 91.6%. The results of the hemosiderin score showed significant differences between the three groups. Applying a value of 50 as a cutoff, the sensitivity and specificity of the method were 100%. In control patients and carriers of DIPD, the percentage of alveolar macrophages was higher than in healthy subjects. Quantification of the hemosiderin content of alveolar macrophages improved the specificity of the diagnosis of diffuse pulmonary hemorrhage by BAL.

Leukocyte function associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) are cell adhesion molecules that play an important role in the capacity of monoculear phagocytes (MPs) to present antigens to T lymphocytes. Since in pulmonary sarcoidosis (PS) this capacity is increased at sites of disease activity, we studied the expression of LFA-1 and ICAM-1 on peripheral blood monocytes (BMs) and alveolar macrophages (AM) obtained by bronchoalveolar lavage (BAL) from normal subjects (n = 7) and patients with PS (n = 14). To accomplish this, immunocytochemical stainings were made on cytocentrifuge preparations using anti-LFA-1 (anti-CD 11a) and anti-ICAM-1 (anti-CD 54) monoclonal antibodies (MoAbs). Normal and sarcoid BMs displayed a high percentage of positivity with both MoAbs with no difference between study groups (LFA-1: control BM 87.8 +/- 8.8 percent; sarcoid BM 84.7 +/- 9.5 percent; ICAM-1: control BM 80.8 +/- 10 percent; sarcoid BM 88.0 +/- 4.2 percent; p = NS for all comparisons). In both groups the percentage of cells expressing LFA-1 and ICAM-1 molecules among AMs was lower than among autologous BMs (LFA-1: control AM 46.5 +/- 13.2 percent, p less than 0.001 vs control BM; sarcoid AM 64.2 +/- 15.9; p less than 0.001 vs sarcoid BM) (ICAM-1: control AM 42.7 +/- 8.5 percent, p less than 0.001 vs control BM; sarcoid AM 72.1 +/- 10.6, p less than 0.001 vs sarcoid BM). AMs from patients with PS showed a higher degree of positivity for LFA-1 and ICAM-1 than normal AMs (p less than 0.02 and p less than 0.001, respectively). The positivity for LFA-1 and ICAM-1 molecules on sarcoid AMs was not correlated with the positivity for two different BM-associated markers (ie, the CD 11b and the CD 14 molecules) and was not correlated with the percentage of T lymphocytes in BAL, selected as a marker of the intensity of the alveolitis. These results suggest that the increased ability of sarcoid AMs to induce the proliferation of T lymphocytes may be related, at least in part, to the increased expression of LFA-1 and ICAM-1 molecules on their surfaces.