Comparison of hydrolases, peroxidase and protease inhibitors in bronchoalveolar fluid from Macacus cynomolgus and human controls

1. Serial bronchoalveolar lavages were performed on a subhuman primate (Macacus cynomolgus) in order to give an experimental model for silicosis. 2. We have measured glycosidases, proteases, peroxidase and antiproteases of the BAL fluids from seven normal monkeys. 3. The results obtained were similar to those found in human control BAL fluids. 4. For monkeys, the repetition of the bronchoalveolar procedure does not seem to have an important influence on the values obtained. 5. The present results will now permit sequential follow up studies during the course of experimental silicosis.     

Leukocyte immunophenotypes in bronchoalveolar lavage fluid and peripheral blood of paracoccidioidomycosis, sarcoidosis and silicosis.

Leukocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood of patients with paraccoccidioidomycosis, sarcoidosis and silicosis were characterized using monoclonal antibodies and an immunoperoxidase technique. In paraccocidioidomycosis, the number of T-helper/inducer CD4-positive lymphocytes was lower in peripheral blood than in BAL fluid. Additional analysis showed that the expression of HLA-DR was very similar in alveolar macrophages, lung and blood T-cells. In sarcoidosis and silicosis there were higher proportions of T-helper/inducer cells in peripheral blood than in BAL fluid. The alterations in the T-helper/inducer/T-suppressor/cytoxic CD4/CD8 ratio in sarcoidosis and silicosis were more appreciable in peripheral blood than in BAL fluid, contrasting with the results in paracoccidioidomycosis. The expression of HLA-DR by alveolar macrophages in sarcoidosis was the highest of all the disease studied. No statistically significant differences were observed between chronic multifocal and chronic unifocal paracoccidioidomycosis disease, stage II and stage III sarcoidosis, and chronic and accelerated silicosis. The three granulomatous diseases analyzed had a few alveolar macrophages expressing the CD4 molecule on their surface. These findings and the technique of analyzing both peripheral blood and BAL leukocyte subsets may help to understand the pathogenesis of interstitial lung diseases.     

Corticosteroids increase lipocortin I in BAL fluid from normal individuals and patients with lung disease

Lipocortin I is a corticosteroid-inducible protein that has potent anti-inflammatory activity. To determine whether lipocortin I is present on the epithelial surface of the human lung, we used a specific polyclonal antibody by the technique of Western blotting to evaluate bronchoalveolar lavage (BAL) fluid of normal individuals and patients with idiopathic pulmonary fibrosis. Lipocortin I was a normal constituent of the epithelial surface of the normal lung and comprised 0.23 +/- 0.03% of BAL fluid proteins. Four separate immunoreactive species were detected, at 37, 36, 34, and 33 kDa, consistent with previously published results. Corticosteroids increased the amounts of lipocortin present in normal volunteers and in patients with idiopathic pulmonary fibrosis. These results demonstrate that lipocortin I is normally present in the human lung and further suggest that lipocortin I may be an important modulator of the anti-inflammatory effects of corticosteroids in the lung.     

Profiling of bisenoic prostaglandins and thromboxane B2 in bronchoalveolar fluid from the lower respiratory tract of human subjects by combined capillary gas chromatography-mass spectrometry

Methods for the profiling of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 15(S),9 alpha,11 beta-trihydroxyprosta-5Z,13E-dien-1-oic acid (9 alpha,11 beta-PGF2), 6-keto-prostaglandin F1 alpha (6kPGF1 alpha), and thromboxane B2 (TxB2) in bronchoalveolar lavage (BAL) fluids from human subjects by combined capillary gas chromatography-mass spectrometry are described. Aliquots (5 ml) of BAL fluid obtained using a standardized lavage protocol were extracted on octadecylsilyl silica cartridges after addition of 0.8 to 2.0 nanograms of tetradeuterated analogs of PGE2, PGF2 alpha, and 6kPGF1 alpha as internal standards. Eluted analytes and internal standards were prepared for vapor phase analysis by sequential reactions resulting in the formation of methyloxime-pentafluorobenzyl ester-trimethylsilyl ether derivatives. The derivatized analytes were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The samples were of adequate purity for identification and quantitation of each of the prostanoids with detection limits of 0.1 to 0.2 picograms of each analyte per milliliter of BAL fluid. The time required for analysis of each sample was approximately 30 minutes. Standard curves of unlabeled species of the six prostanoids extracted after addition to BAL fluid were linear over a range from subpicogram to nanogram quantities. The differences between the amounts of prostanoid added and the amounts of prostanoid measured were typically less than 19%, and the intra-assay coefficients of variation for repeated measurements of a single sample were less than 20%. PGE2, PGD2, PGF2 alpha, and TxB2 were detectable in BAL fluids from normal subjects with levels of each of these compounds being less than 2.6 picograms/ml. BAL fluids from patients with lung disease presented qualitative and quantitative profiles of prostanoids markedly different than those from normal subjects. These analytical methods provide a basis for in vivo comparisons of prostanoid profiles in the lower respiratory tract of man and should be readily adaptable for use in a variety of clinical studies.     

Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa

In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model. The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge. Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge. Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge. Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection. Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid. Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected. When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge. No inhibition was observed by anti-CINC-1 antibody. These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P. aeruginosa.     

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

Genetic polymorphism of alpha 1-antitrypsin in green monkeys studied by isoelectric focusing and family analysis

24 variants of alpha 1-antitrypsin (alpha 1-AT) were recognized in sera of 120 wild and capture-born African green monkeys by isoelectrofocusing in Ampholine PAG-plates (pH 4-6.5) and western blotting with antihuman alpha 1-AT serum. All variants had much more cathodal position than human alpha 1-AT and revealed very high microheterogeneity which was slightly different from the observed in human alpha 1-AT. The alpha 1-AT banding pattern allowed to postulate existence of 10 codominant alleles in the Pi locus of African green monkeys. The reality of 8 alleles was proved by family analysis which included 45 monkey birth cases. Two other alleles were absent in the parents available. Thus, alpha 1-AT is the most polymorphic among the known serum proteins of African green monkeys. The latter can be useful for molecular systematics of these primates.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Biochemical factors in pulmonary inflammatory disease

Various biochemical events taking place during pulmonary inflammation were examined in the bronchoalveolar lavage (BAL) fluids from patients with acute respiratory distress syndrome (ARDS) and in experimental animal models. In patients with ARDS, active neutrophil elastase was found in the BAL fluids. In these fluids, inactivation of the major elastase inhibitor alpha 1-protease inhibitor (alpha 1-PI) occurred. This was caused by oxidation of a methionine residue at the active site of the alpha 1-PI, and offered indirect evidence of oxidation occurring in the inflamed pulmonary tissues. Studies with experimental animals have been initiated to gain understanding of the relative roles of proteases, oxidants, arachidonate metabolites, complement and contact system components, and other mediators in the pathogenesis of pulmonary inflammation. Intrabronchial instillation of glucose oxidase/glucose to produce oxidants or formylated norleucylleucylphenylalanine or phorbol myristate acetate as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits and rhesus monkeys. The injury was accompanied by leukocytic protease (acid cathepsins) release in rabbit lungs and oxidant formation, and could be inhibited by neutrophil depletion. Oxidant formation was demonstrated by the inactivation of catalase by 3-amino-1,2,4-triazole in the presence of H2O2, a drop in intracellular glutathione levels, and in the rhesus monkey by inactivation of alpha 1-PI.     

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.     

Prostanoids in bronchoalveolar lavage fluid do not predict outcome in congenital diaphragmatic hernia patients

Vasoactive prostanoids may be involved in persistent pulmonary hypertension (PPH) in infants with a congenital diaphragmatic hernia (CDH). We hypothesized that increased levels of prostanoids in bronchoalveolar lavage (BAL) fluid would predict clinical outcome. We measured the concentrations of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), thromboxane B(2) (TxB(2)), protein, albumin, total cell count, and elastase-alpha1-proteinase-inhibitor complex in BAL fluid of 18 CDH patients and of 13 control subjects without PPH. We found different concentrations of prostanoids in BAL fluid of CDH patients with PPH: infants with a poor prognosis had either high levels of both 6-keto-PGF(1alpha) and TxB(2) compared to controls, or high levels of 6-keto-PGF(1alpha) only. TxB(2) levels showed a large variability in all CDH patients irrespective of outcome. We conclude that prostanoid levels in BAL fluid do not predict clinical outcome in CDH patients.     

Comparison of casein of cynomolgus monkey (Macaca fascicularis) with human casein

Casein of cynomolgus monkey was compared with those from human and bovine milk. Cynomolgus monkey casein showed similar electrophoretical patterns to those of human casein on Disc- and SDS-electrophoresis. It consisted of beta- and kappa-casein-like components. The component corresponding to bovine alpha s1-casein was not detected. The beta-casein-like fraction of cynomolgus monkey showed 9 bands on Disc-PAGE. These were suggested to be the same protein binding different levels of phosphorus by dephosphorylation experiment using an acid phosphatase. The kappa-casein-like component of cynomolgus monkey was highly glycosylated (about 50% carbohydrate) similarly as human kappa-casein and the constituent carbohydrates were same as those detected in human kappa-casein (galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid). Amino acid composition of cynomolgus monkey kappa-casein bore a resemblance to those of both human and bovine kappa-caseins. Amino acid composition of cynomolgus monkey beta-casein was also similar to those of human and bovine beta-caseins.     

Effects of fluticasone propionate inhalation on levels of arachidonic acid metabolites in patients with chronic obstructive pulmonary disease

BACKGROUND: In smoking COPD patients the bronchoalveolar lavage (BAL) fluid contains high numbers of inflammatory cells. These cells might produce arachidonic acid (AA) metabolites, which contribute to inflammation and an increased bronchomotor tone. AIMS: To investigate levels of AA metabolites in BAL fluid, before and after inhaled glucocorticoid therapy: fluticasone propionate (FP) 1 mg per day, or placebo. METHODS: A double-blind placebo controlled trial lasting six months. COPD patients were selected by clinical criteria and the presence of bronchial hyper-responsiveness (BHR). Lung function was recorded and in BAL fluid we counted cell numbers and measured LTB4, LTC4/D4/E4, PGE2, 6kPGF1alpha, PGF2alpha and TxB2. A control group consisted of asymptomatic smokers (n=6). RESULTS: Paired data were obtained from 9 FP treated and 11 placebo patients. BAL cells were almost exclusively alveolar macrophages. In patients and controls both cellularity and levels of AA metabolites were equal Cell numbers did not change after treatment. Statistically significant decreases after FP therapy were noticed for PGE2 (30%), 6kPGF1alpha (41%) and PGF2alpha (54%). CONCLUSIONS: In COPD, the capability of inflammatory cells to produce certain AA metabolites was decreased after inhaled FP treatment. This result is discussed in its relation to clinical effects, the influence of smoking, and the results of an earlier, similar study in asthma patients.     

The differentiation of hepatocyte-like cells from monkey embryonic stem cells

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.