Time course of bleomycin-induced lung fibrosis.

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time-course of bleomycin-induced lung fibrosis in mice using computer-assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post-IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin-treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer-assisted morphometry demonstrated a 3-fold increase in fibrosis fraction and a 1.3-fold increase in wall area fraction in bleomycin-treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer-assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin-induced lung injury.

Human recombinant interferon-alpha2a and interferon-alphaA/D have different effects on bleomycin-induced lung injury.

BACKGROUND: Bleomycin (Bleo)-induced lung injury in mice serves as an animal model of pulmonary fibrosis. The pathogenesis of pulmonary fibrosis remains unclear, but it comprises both inflammatory and fibrotic components. The cytokine interferon (IFN)-alpha is produced by macrophages and may modulate both fibrogenesis and the determination of T lymphocyte phenotype in pulmonary fibrosis. OBJECTIVE: To investigate the effect of two preparations of recombinant IFN-alpha (IFN-alphaA/D and IFN-alpha2a) on Bleo-induced lung injury in C57BL/6 mice. METHODS: Mice were treated by a single intratracheal (IT) instillation of 0.06 mg of Bleo in 0.1 ml of saline or saline alone. One of two different IFN-alpha preparations, IFN-alphaA/D or IFN-alpha2a in saline, or saline alone were administered by daily intraperitoneal injections starting 1 day prior to IT instillation. The treatment groups were as follows: IT Bleo and intraperitoneal saline; IT Bleo and intraperitoneal IFN-alpha2a; IT Bleo and intraperitoneal IFN-alphaA/D; IT saline and intraperitoneal IFN-alphaA/D or IFN-alpha2a; IT saline and intraperitoneal saline. The animals were sacrificed 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage (BAL) fluid, by a semiquantitative morphological index of lung injury and a quantitative image analysis of cellularity and fibrosis fraction and by biochemical analysis of lung hydroxyproline content. RESULTS: In Bleo-treated mice, IFN-alpha2a treatment caused a significant rise in BAL lymphocytes and in cellularity and fibrosis fractions in lung tissue. In contrast, IFN-alphaA/D treatment had no effect on Bleo-induced lung injury. CONCLUSION: IFN-alpha may enhance Bleo-induced lung injury but this effect varies with different IFN preparations.

TGF-beta1 induces an accumulation of connexin43 in a lysosomal compartment in endothelial cells.

We have been studying the relationships between cell growth and the expression of the gap junction protein Connexin43 (Cx43) in cultured bovine aortic endothelial cells (BAEC). As part of these studies, we examined the effect of the growth inhibitory cytokine TGF-beta1 on Cx43 expression. We have shown recently that TGF-beta treatment increases Cx43 mRNA and synthesis, content, and half-life of the protein within 24 h, which leads, over the course of days, to an accumulation of Cx43 in large, intensely immunostaining vesicles, filling much of the perinuclear cytoplasmic space. In the current study, based on their distribution and markers, we identified these vesicles as lysosomes/autophagosomes. Cx43 immunostaining and staining with a fluorescent probe for acidic compartments are coincident, as retention of a fluorescent-labeled low-density lipoprotein occurs in a similar pattern and the same staining pattern can be detected in the treated cells using other markers for lysosomal compartments. TEM revealed prominent lysosomal figures with considerable heterogeneous material. After withdrawal of TGF-beta, the accumulated Cx43 was cleared only slowly, with some brightly immunoreactive cells remaining even after 72 h. The prolonged appearance (based on immunoreactivity in situ and in immunoblots) of intact vesicular Cx43 in the treated cells suggests decreased degradation, resulting from impaired lysosomal activity. These data not only emphasize the importance of the lysosome in connexin degradation, but also show that TGF-beta can cause an alteration in lysosomal functioning, with implications for cellular metabolism.

Hepatic alpha 2 mu-globulin localizes to the cytosol of rat proximal tubule cells.

BACKGROUND: Alpha 2 mu-Globulin (A2), an 18.6 kD protein of hepatic origin, accumulates in the proximal tubule as an abundant, 15.5 kD cleavage product termed "A2-fragment" (A2-f). A2-f facilitates proximal tubule fatty acid oxidation, presumably by binding hydrophobic ligands. This requires some A2-f to enter the cytosol of the renal epithelial cell (REC). The localization of A2/A2-f in the proximal tubule cell was evaluated in this study. METHODS: Immunoblot analysis of renal cortical homogenates separated by differential centrifugation and quantitative immunoelectron microscopy (IEM) was performed to localize A2/A2-f using an affinity-purified antibody that detects both proteins. To evaluate A2 as a physiologically relevant ligand, the accumulation of A2-f in the female rat kidney (normally devoid of A2-f) was examined after the induction of hepatic A2 synthesis. Ligand binding, uptake, and degradation assays were used to assess A2 processing by RECs in vitro. RESULTS: Although A2 and A2-f were detected in the "lysosomal" fraction, only A2-f was found in the soluble protein fraction. IEM confirmed the presence of significant signal in the vesicular and lysosomal as well as the cytosolic compartments. In contrast, both beta 2 mu globulin (B2) and cathepsin B were restricted to endosomes. In the female rat, induction of hepatic A2 production resulted in A2-f accumulation in the renal cortex. In RECs in culture, uptake of A2 and B2 demonstrated nonsaturable, nondisplacable surface binding and similar uptake rates. Compared with B2, A2 was markedly resistant to degradation. CONCLUSIONS: A fraction of A2 escapes lysosomal degradation, permitting A2-f to accumulate in the cytosol of the proximal tubule epithelial cell. A2 may represent an unusual example of a physiologic protein capable of accumulating in a distant cell type.

Effect of immunomodulators on bleomycin-induced lung injury.

BACKGROUND: The role of lymphocytes and their subpopulations in lung fibrosis is as yet unclear. OBJECTIVE: To define the role of immunomodulation in bleomycin-induced inflammatory fibrotic lung injury, by testing the effect of two known Th1 inhibitors: linomide and pentoxifylline. METHODS: C57BL/6 mice were treated by a single intratracheal instillation of 0.06 mg bleomycin in 0.01 ml saline or saline alone. Treatment groups included: (1) intratracheal bleomycin and daily treatment with linomide or pentoxifylline; (2) intratracheal bleomycin and daily water; (3) intratracheal saline and daily linomide or pentoxifylline; (4) intratracheal saline and daily water. Linomide and pentoxifylline were available per os in the drinking water from 1 day prior to intratracheal instillation. Animals were studied 14 days after intratracheal instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semiquantitative morphological index of lung injury and a quantitative image analysis of cellularity, fibrosis fraction and alveolar wall area fraction, and by biochemical analysis of lung hydroxyproline content. RESULTS: Linomide or pentoxifylline did not cause any lung injury in saline-treated control mice. Overt signs of lung injury were apparent in bleomycin-treated mice. These changes were not affected by daily treatment with linomide or pentoxifylline, which were given in the highest tolerable dose. CONCLUSION: This study does not support the use of linomide or pentoxifylline to prevent or ameliorate lung fibrosis and may suggest that drug-induced differentiation of T lymphocytes into Th1/th2 subpopulations does not affect the evolution of bleomycin-induced lung injury.

The effect of enoxaparin on bleomycin-induced lung injury in mice.

We have evaluated the effect of enoxaparin, a potent antithrombotic drug, on bleomycin (Bleo)-induced pulmonary inflammation in mice. Pulmonary injury was induced by a single intratracheal (i.t.) instillation of Bleo. Four groups of female C57BL/6 mice, each received one of four treatments: (1) i.t. Bleo and daily intraperitoneal (i.p.) injections of enoxaparin (EN) starting one day before i.t. instillation of Bleo (Bleo-EN); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. enoxaparin (Sal-EN); (4) i.t. saline and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated by analysis of bronchoalveolar lavage fluid and histologically by an overall semiquantitative index of lung injury and a quantitative image analysis assessing alveolar wall area fraction and fibrosis fraction. Treatment of mice with enoxaparin did not ameliorate Bleo-induced lung injury. Our study does not establish a critical role of procoagulant activity in the evolution of Bleo-induced lung injury and does not support the use of antithrombotic therapy for the prevention of pulmonary fibrosis.

Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells.

BACKGROUND: This study tested the following hypotheses: (a) renal tubular epithelial cells subjected to transient adenosine triphosphate (ATP) depletion undergo apoptosis, and (b) induction of heat stress proteins (HSPs) inhibits cell death following ATP depletion, possibly by interacting with anti-apoptotic signal proteins. METHODS: To simulate ischemia in vivo, cells derived from opossum kidney proximal tubule (OK) were subjected to ATP depletion (5 mM cyanide, 5 mM 2-deoxy-D-glucose, and 0 mM glucose) for 1 to 1. 5 hours, followed by recovery (10 mM glucose without cyanide). The presence of apoptosis was assessed by morphological and biochemical criteria. The effect of prior heat stress or caspase inhibition on apoptosis and cell survival were assessed. RESULTS: In the ATP-depleted cell, both Hoechst dye and electron microscopy revealed morphological features that are typical of apoptosis. On an agarose gel, a "ladder pattern" typical of endonucleosomal DNA degradation was observed. Prior heat stress reduced the number of apoptotic-appearing cells, significantly decreased DNA fragmentation, and improved cell survival compared with controls (73.0 +/- 1% vs. 53.0 +/- 1.5%; P < 0.05). Two different caspase inhibitors also improved survival, suggesting that apoptosis is a cause of cell death in this model. Compared with ATP-depleted controls, prior heat stress inhibited the pro-apoptotic changes in the ratio of Bcl2 to BAX, proteins known to regulate the apoptotic set point in renal cells. HSP 72, a known cytoprotectant, co-immunoprecipitated with Bcl2, an anti-apoptotic protein. Prior heat stress markedly increased the interaction between HSP 72 and Bcl2. CONCLUSIONS: Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. Novel interactions between HSP 72 and Bcl2 may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depletion injury.

Abnormal mucous cell phenotype induced by neutrophil elastase in hamster bronchi.

Bronchial mucous cell metaplasia (MCM) is a histologic component of chronic mucus hypersecretion. The hamster model of elastase-induced MCM appears to involve an irreversible conversion of Clara cells to mucous cells. The present study questioned whether the mucous cells seen in hamster bronchi exposed to neutrophil elastase produce and maintain a form of glycoconjugate secretory product different from that normally found in mucous cells or Clara cells. Ultrastructural cytochemistry using the gold-labeled lectin HPA revealed a difference in the cell surface and stored secretory granules of elastase-derived mucous cells compared to normal mucous cells and Clara cells at 3 weeks and 4 months following exposure. The results suggest that elastase irreversibly alters the glycoconjugate character of the Clara cells normally present so that they produce an abnormal form of mucus. Because secreted glycoconjugates can affect the rate of mucociliary clearance and receptor-mediated binding of microorganisms, this change in phenotype may be involved in the pathogenesis of diseases associated with chronic mucus hypersecretion in humans.

Phenotypic variation in hamster bronchial mucous cells induced by different airway irritants.

Chronic mucus hypersecretion (CMH), a common feature of various obstructive pulmonary diseases, is caused by a variety of airway irritants. Bronchial mucous cell metaplasia (MCM), a histological correlate of CMH, can be induced in hamster airways by a number of different irritants. Previous studies with the hamster model suggest that the secretory cell response to different agents is not stereotyped but can vary in the type of mucus glycoconjugate produced. The present ultrastructural study was conducted therefore to provide quantitative evidence of phenotypic variation in mucous cells induced independently by exposure to the metaplastic agents elastase and acid. HPA-gold lectin cytochemistry revealed an increase in N-acetyl galactosamine at the cell surface and secretory granules of mucous cells in elastase-treated vs. acid-treated animals. Although there was no quantitative difference between the acid-treated and untreated groups, a difference in the pattern of binding within granules indicated variation in the secretory product. Because mucus glycoconjugates serve as attachment sites for specific pathogens, phenotypically distinct mucous cells may promote differential microbial colonization. In humans therefore, variation in the severity and progression of CMH may be due in part to secretory cell susceptibility and response to different pathogenic stimuli.

Immunocytochemical evidence for extra-cellular initiation of elastase-induced bronchial secretory cell metaplasia in hamsters.

The bronchus is the only region of the hamster conducting airways to develop secretory cell metaplasia after an intratracheal instillation of human neutrophil elastase (HNE). We tested the hypothesis that this pathological change occurs because of cellular uptake of the enzyme that is specific to this region. HNE, dissolved in saline, was instilled into the trachea of hamsters, that were sacrificed 5, 15, 30 or 60 min later for immunocytochemical localization of the enzyme. Saline-treated animals served as controls. By light microscopy, HNE was evident only in the lumen and upon the epithelial surface in all airways, at all time points. Saline control tissues were negative. Electron microscopic immunogold staining revealed HNE within luminal macrophages and associated with mucus and, to a limited extent, upon the apical cell surface both in trachea and bronchus. A small amount of HNE staining occurred in the intercellular space and lamina propria of bronchi. Cytoplasmic gold particles were sparse both in treated and control animals. We conclude that instilled neutrophil elastase is excluded from the epithelial cytoplasm regardless of region. We thus reject the hypothesis of airway cellular uptake of HNE and suggest that stimulation of bronchial secretory cells to accumulate mucin granules is initiated at the cell surface, possibly by unmasking or altering region-specific receptors involved in signal transduction pathways governing mucin granule synthesis.

Quantitative ultrastructural analysis of the relationship between cell growth, shape change, and mucosecretory differentiation in cultured hamster tracheal epithelial cells exposed to retinoic acid.

Hamster tracheal epithelial cells in extended (32 degrees C) primary culture with and without supplemental retinoic acid (RA) were studied during the proliferative (5 days) and differentiation phases (11 days) by correlative transmission electron microscopy (EM) and light microscopic (LM) autoradiography to quantify the relationship between cell proliferation, shape change, and mucin granule expression. In retinyl acetate-containing control medium, cell numerical density was higher and [3H]thymidine labeling index (LI) lower at day 11 compared with day 5. The addition of 10(-7) M RA to the medium caused an increase in cell numerical density at both times. LI was increased by RA at 5 days and decreased at 11 days. Measurements of cell shape in ultrathin sections adjacent to LM autoradiographs made in the vertical plane demonstrated an RA-induced change from flat to cuboidal at 5 days and a more columnar phenotype at 11 days. Cells containing mucin granules were of two main types based on their ultrastructure. One type, seen at 5 and 11 days, contained diminutive mucin granules and had an LI of 50% at 11 days. Its LI and frequency (26%) were unaltered by RA. The other type, less frequent (15%) and present only at 11 days, was more columnar and contained mucous granules similar to those found in vivo. RA doubled the frequency of this cell type but did not affect its LI (11%). Cells of this type with more than five mucin granules in EM profile did not incorporate thymidine. The data indicate that RA accelerates and enhances cell shape change toward a more cuboidal phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of secretory granule content and proliferative intensity in the secretory compartment of the hamster bronchial epithelium.

We have previously shown that normal hamster airway epithelial secretory cells have a lower proliferative intensity than basal cells, but because of their high frequency are a major contributor to cell renewal (Am. J. Respir. Cell Mol. Biol. 1990; 2:51-58). In the present experiment, the relation between proliferative intensity and secretory granule content in bronchial epithelial cells is studied. [3H]thymidine (2 microCi/g wt) was given intraperitoneally, 1 h before killing, to 5 hamsters treated 21 days earlier with intratracheal saline and to six hamsters in which secretory cell metaplasia had been induced by intratracheal treatment with 300 micrograms of human neutrophil elastase given 21 days earlier. Light microscopic autoradiograms were prepared from 2-microns-thick Epon sections of left intrapulmonary hilar bronchi. Cells were categorized as basal, ciliated, secretory, or indeterminate. Secretory cells were classified as either: S1, with 0 to 4 granules; S2, with > or = 5 granules with intervening cytoplasm; or S3, with abundant granules and no apparent supranuclear cytoplasm. Proliferative intensity was defined by the categorical labeling index (LIc) at 1 h after [3H]thymidine injection. LIc was determined by the number of labeled cells in a category as the percent of labeled and unlabeled cells of that category. LIc of each of the cell categories were similar in the elastase and saline groups. LIc was highest for basal cells, reflecting their proliferative intensity, and lowest for ciliated cells. In the saline group, LIc of S1 (0.25%) was significantly higher compared with S2 (0.13%); S3 cells were rare (0.2%) and none were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)

Elastase causes secretory discharge in bronchi of hamsters with elastase-induced secretory cell metaplasia.

A single intratracheal instillation of human neutrophil elastase (HNE) into hamsters causes granule discharge from bronchial secretory cells followed by marked accumulation of granules, visible by light microscopy at 21 days and persisting through 18 months. To determine whether persistence of this secretory cell metaplasia (SCM) is due to inability of the metaplastic secretory cells to secrete their granules, hamsters having HNE-induced SCM were challenged with the potent secretagogue HNE. Four groups of 10 hamsters each received 300 micrograms HNE intratracheally. Twenty-one days later, hamsters were intratracheally treated with HNE or saline; the groups were designated HNE-HNE and HNE-SAL, respectively. Hamsters were killed 2 h or 21 days following the second treatment. Using light microscopy, nucleated epithelial cells were counted in plastic sections of the left main intrapulmonary bronchus. Cells were classified as ciliated (C), basal (B), indeterminate (IN), or secretory. Secretory cells were subcategorized as S0 (0 granules), S1 (1-4 granules), S2 (> or = 5 granules with intervening cytoplasm), and S3 (abundant granules completely filling the cytoplasm). At 2 h, S3 cell frequency in the HNE-HNE group was 13.0 +/- 2.2 (% mean +/- SE), significantly lower than in the 2 h HNE-SAL group (31.1 +/- 4.5). Concomitantly, higher cell frequencies were seen in the other secretory categories of the HNE-HNE group compared to the HNE-SAL group; S2 17.1 +/- 1.9 compared to 9.4 +/- 1.9, S1 2.4 +/- 0.4 compared to 1.1 +/- 0.5, and S0 2.4 +/- 0.5 compared to 1.1 +/- 0.5, respectively. The S3 cell frequency of the 21-day HNE-HNE group was 25.4 +/- 4.7, increased significantly compared to the 2 h HNE-HNE group; this change was concomitant with significant decrease in the frequency of the S0 secretory cells. Cell frequencies of C, B, and IN were not affected by treatment or time. It is concluded that metaplastic secretory cells discharge their granules in response to HNE; SCM returns to its original state after HNE rechallenge; persistent SCM is not due to the inability of metaplastic secretory cells to discharge their granules.

Oxidants from neutrophil myeloperoxidase do not enhance elastase-induced emphysema in the hamster.

Alpha-1-protease inhibitor is susceptible to oxidative impairment by the neutrophil myeloperoxidase (MPO) system. The purpose of this study was to assess the effect of the MPO oxidant system on elastase-induced emphysema in the hamster. Intratracheal instillation of 200 micrograms of human neutrophil elastase (HNE) induced a significant secretory cell metaplasia (SCM) and airspace enlargement [23% increase in mean linear intercept (MLI) as compared with control values]. Instillation of MPO system components [0.6 international units (U) of MPO, 5.5 U of glucose oxidase and glucose (0.02 M)] along with 200 micrograms HNE failed to enhance the severity of the SCM or emphysema induced by HNE alone. A second experiment was carried out using 50 micrograms of porcine pancreatic elastase (PPE) to induce emphysema. PPE produced a significant 45% increase in MLI, but the MPO system combined with PPE failed to enhance the emphysema induced by PPE alone. The MPO system alone had no measurable effect on airspace size or SCM. In vitro studies showed that PPE was partially inactivated by the MPO system; a 56% loss of elastolytic activity occurred during a 6-min incubation of PPE with the MPO system. This may explain why the MPO system did not exacerbate PPE-induced injury, but it does not explain the lack of enhancement for HNE. A 6-minute incubation of HNE with the MPO system resulted in a nonsignificant 10% decrease of elastolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the carbohydrate content of airway epithelium induced by human neutrophil elastase in the hamster.

Hamster airway epithelial secretory cells were investigated by light and electron microscopic cytochemistry to study possible changes in their carbohydrate content induced by human neutrophil elastase (HNE), an agent known to cause replacement of Clara cells by mucous cells in hamster bronchi. Characterization of secretory cell carbohydrates by the AB/PAS, PA-TCH-SP, HID-TCH-SP, and LID-TCH-SP sequences indicated the existence of periodate-reactive acidic glycoconjugates, but the absence of sulfated or carboxylated glycoconjugates in both treated and control animals. Differences were seen in the quality and quantity of historeactive carbohydrates throughout various regions in the lower respiratory tract. This was especially evident in the HNE-treated animals. It is concluded that the HNE-induced expression of the mucous cell phenotype is associated with an increase in the amount of neutral and acidic nonsulfated and noncarboxylated polysaccharides stored in the secretory granules of these cells.

Resistance of hamster bronchiolar epithelium to neutrophil elastase: investigation by cell surface lectin cytochemistry.

An intratracheal instillation of human neutrophil elastase (HNE) causes accumulation of an excess number of secretory granules in the epithelial secretory cells lining the hamster bronchus. This chronic lesion, which we refer to as secretory cell metaplasia (SCM), is not seen in the trachea or bronchioles. Because luminal cell surface lectin binding is much higher in the trachea than in the bronchus, we concluded that tracheal resistance may be due to a protective glycoconjugate coat. In the present ultrastructural study, we analyzed the lectin-binding capability of bronchiolar epithelial cells to determine whether their luminal cell surface glycoconjugate layer is similar to tracheal epithelial cells. None of the six ferritin-conjugated lectins showed higher binding in bronchioles compared to the bronchus, suggesting that a high level of surface oligosaccharides is not necessary for resistance to the metaplastic effects of HNE. HNE caused a significant reduction in bronchiolar surface binding of the gold-labeled, secretory cell-specific lectin, Helix pomatia agglutinin. The principal granulated secretory cell type in bronchioles was ultrastructurally similar to a form of bronchial Clara cell that converts to a mucous cell phenotype in response to HNE. The results suggest that absence of bronchiolar SCM is not attributable to a protective layer of cell surface oligosaccharides, a lack of cellular contact by HNE, or the presence of a morphologically distinct population of epithelial cells in bronchioles.

Flurbiprofen does not affect elastase-induced bronchial secretory cell metaplasia in hamsters.

The anti-inflammatory drug flurbiprofen speeds the repair of cigarette smoke-induced bronchial secretory cell metaplasia (SCM) in rats. We tested whether flurbiprofen would prevent or mitigate the development of elastase-induced bronchial SCM in hamsters. Twice daily injections of flurbiprofen (4 or 6 mg.kg-1.day-1) were begun immediately after intertracheal instillation of porcine pancreatic elastase (PPE) or its vehicle, saline. At 3 wks, the bronchi of these animals were compared to those of animals receiving PPE or saline. PPE-treated hamsters, with or without flurbiprofen, developed moderate to severe SCM. Control hamsters had normal airways. Flurbiprofen had no effect on the neutrophilic pulmonary infiltrate seen 2 h after PPE instillation. We conclude that the development of elastase-induced SCM in hamsters is not affected by a flurbiprofen regimen begun directly after elastase instillation. The pathogenesis of this lesion may involve factors which are insensitive to flurbiprofen or which trigger the lesion immediately upon exposure to the enzyme.

CHOP Versus Chlorambucil + Prednisolone in Chronic Lymphocytic Leukemia.

One hundred and fifty-seven previously untreated stage B or C B-CLL patients were randomized to treatment with either chlorambucil + prednisolone (CLBP) 5 days every 4 weeks or CHOP every 4 weeks. Significantly more patients achieved complete remission on CHOP, but duration of response and survival were equal in the two regimens. Non-responders on CLBP were switched to CHOP, so that finally most patients received nearly the same amount and quality of treatment, which possibly explains the lack of difference in survival. However, compared to previous studies, the study-designed intensive chemotherapy seems to prolong survival for patients with advanced disease, especially those in stage C.

Lectin cytochemistry reveals differences between hamster trachea and bronchus in the composition of epithelial surface glycoconjugates and in the response of secretory cells to neutrophil elastase.

Hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate an abnormally high number of secretory granules in bronchial but not tracheal epithelial cells. We employed lectin cytochemistry to investigate possible differences in the epithelial cell surface glycoconjugate layer in trachea compared to bronchus which might explain the regional dissimilarity in response to HNE. Portions of glutaraldehyde-fixed trachea and bronchi were incubated in one of several ferritin-labeled lectins prior to embedding for transmission electron microscopy. Lectins from Ricinus communis, Helix pomatia, and Triticum vulgaris bound to the surface of tracheal secretory cells in moderate to profuse amounts, while most bronchial secretory cells showed little or no label with these lectins. Gold-labeled Helix pomatia agglutinin (HPA), a lectin specific for secretory cells, showed a decrease in surface binding to all tracheal secretory cell types within 2 h of HNE instillation, compared to saline controls. In contrast, the majority of bronchial secretory cells showed an HNE-induced increase in surface label from extremely low levels in saline controls. The low levels of lectin binding to bronchial cells, in contrast to the trachea, may indicate the lack of a protective surface glycoconjugate coat, thus explaining the vulnerability of these cells to HNE. The rise in number of accessible HPA binding sites on the surface of bronchial secretory cells exposed to HNE may represent an important event in the pathologic accumulation of secretory granules by these cells.