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Introduction: Cardiovascular morbidity is a major burden in patients with rheumatoid arthritis (RA). In this study, we compare the effect of a targeted, intensified, multifactorial intervention with that of conventional treatment of modifiable risk factors for cardiovascular disease (CVD) in patients with early RA fulfilling the 2010 American College of Rheumatology European League Against Rheumatism (ACR/EULAR) criteria., Methods and Analysis: The study is a prospective, randomised, open label trial with blinded end point assessment and balanced randomisation (1:1) conducted in 10 outpatient clinics in Denmark. The primary end point after 5 years of follow-up is a composite of death from cardiovascular causes, non-fatal myocardial infarction, non-fatal stroke and cardiac revascularisation. Secondary outcomes are: the proportion of patients achieving low-density lipoprotein cholesterol <2.5 mmol/L, glycated haemoglobin <48 mmol/mol, blood pressure <140/90 mm  Hg for patients without diabetes and <130/80 mm Hg for patients with diabetes and normoalbuminuria (urinary albumin creatinine ratio <30 mg/g) after 1 year of follow-up and the proportion of patients in each treatment group achieving low RA disease activity after 1 year, defined as a disease activity score C-reactive protein (DAS28-CRP) <3.2 and a DAS28-CRP score <2.6 after 12, 24 and 60 months. Furthermore, all hospitalisations for acute and elective reasons will be adjudicated by the event committee after 12, 24 and 60 months. Three hundred treatment-naive patients with early RA will be randomly assigned (1:1) to receive either conventional treatment administered and monitored by their general practitioner according to national guidelines (control group) or a stepwise implementation administered and monitored in a quarterly rheumatological nurse-administered set-up of behaviour modification and pharmacological therapy targeting (1) hyperlipidaemia, (2) hypertension, (3) hyperglycaemia and (4) microalbuminuria (intervention group)., Ethics and Dissemination: This protocol is approved by the local ethics committee (DK-S-2014007) and The Danish Health and Medicines Authority. Dissemination will occur through presentations at National and International conferences and publications in international peer-reviewed journals., Trial Registration Number: NCT02246257., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The sst1, "supersusceptibility to tuberculosis," locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1(S) ) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1(S) mice. Infection of sst1(S) macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-β and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-β. Intriguingly macrophages from the sst1(S) mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.

Background: The radial artery's propensity for vasospasm and vulnerability to surgical trauma are well known. A less invasive endoscopic method to harvest the radial artery was recently introduced, but its effect on radial artery integrity is unknown., Methods: To compare the effects of harvest method on radial artery function, we prospectively randomized 54 patients undergoing coronary artery bypass grafting with the radial artery into 3 groups on the basis of harvest techniques: endoscopic, conventional with cautery, and conventional with harmonic scalpel. We assessed endothelium-dependent and endothelium-independent relaxation of radial artery segments to sequential doses of acetylcholine and nitroglycerin, respectively, using standard organ-chamber methodology. Vasospasm was assessed as the vasoconstrictor response to the thromboxane analog U46619. We assessed endothelial integrity using light and electron microscopy and by rating intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and P-selectin expression by means of immunohistochemistry on a semiquantitative 0- to 3-point scale. Harvest procedures were performed by a single surgeon, and data analyses were blinded to the harvesting method., Results: Maximal relaxation-contraction responses to acetylcholine, nitroglycerin, and U46619 and effective drug concentration yielding 50% response were similar in the 3 groups. Adhesion molecule expression and histologic changes, as assessed by means of light and electron microscopy, were similar in the 3 groups., Conclusions: Endoscopic harvest does not alter radial artery vasoreactivity or endothelial integrity compared with conventional harvest techniques. Because the endoscopic technique is less invasive, it might prove to be the technique of choice to harvest the radial artery.

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

We describe a rare case of adult-onset neuronal ceroid lipofuscinosis (NCL) type B with probable autosomal dominant inheritance, exhibiting behavioral and cognitive abnormalities and extrapyramidal findings. Ultrastructural examination revealed abundant fingerprint profiles in several cell types. To our knowledge, this is the first reported case of an African-American with adult-onset NCL., ((c) 2005 Movement Disorder Society.)

IFN-gamma production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-gamma expression in bleomycin-induced lung injury. IFN-gamma mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-gamma protein levels were increased at 6 days, as was the percentage of LC expressing IFN-gamma. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-gamma production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-gamma production by these cells. To define the role of endogenous IFN-gamma in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-gamma gene (IFN-gamma knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-gamma knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-gamma production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-gamma in IFN-gamma knockout mice does not increase pulmonary fibrosis. Endogenous IFN-gamma may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-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.

Background: Intratracheal instillation of bleomycin (Bleo) into rodents serves as a model for human lung fibrosis. Various mouse strains respond differently to Bleo, and BALB/c mice are relatively resistant., Objective: Since T lymphocytes have been shown to play a major role in this model, the effect of the immunomodulator cyclosporin A (CyA) on lung fibrosis was studied in Bleo-'resistant' BALB/c mice., Methods: Pulmonary fibrosis was induced by a single intratracheal (IT) instillation of Bleo. One of the four following treatments was given to one of four groups of female BALB/c mice: (1) Bleo-CyA: IT Bleo and daily intraperitoneal (IP) injections of CyA 100 mg/day starting 1 day before IT instillation of Bleo; (2) Bleo-Sal: IT Bleo and IP injections of saline; (3) Sal-CyA: IT saline and IP CyA 100 mg/kg; (4) Sal-Sal: IT and IP saline. The animals were killed on day 14. Fibrosis was evaluated by analysis of hydroxyproline and by quantitative image analysis of the fibrosis fraction. Ex vivo IFN-gamma, IL-4, IL-13 and IL-5 secretion by peribronchial lymphatic tissue lymphocytes was measured., Results: Pretreatment with CyA upmodulated Bleo-induced lung fibrosis in the Bleo-'resistant' BALB/c mice; hydroxyproline was higher in Bleo-CyA compared to Bleo-Sal animals and both hydroxyproline and fibrosis fraction measurements were higher in Bleo-CyA compared to Sal-Sal. Cytokine secretion by lymphocytes demonstrated increased IFN-gamma/IL-4 and IFN-gamma/IL-13 mean secretion ratios in the Bleo-CyA animals compared to the Bleo-Sal animals., Conclusions: These results indicate that CyA upmodulates Bleo-induced lung fibrosis in Bleo-'resistant' BALB/c mice by allowing the emergence of a Th1 inflammatory response., (Copyright 2002 S. Karger AG, Basel)

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., (Copyright 2001 S. Karger AG, Basel)

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.

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.

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.

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.

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.

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.

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.

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)

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)

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.

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)

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.

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.

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.

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.

Studies were undertaken to evaluate the in vitro properties of recombinant human secretory leukocyte-protease inhibitor (rSLPI) that had been made in Escherichia coli in an inactive form and refolded, and to determine whether emphysema and bronchial secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be amelio-rated by prior intratracheal instillation of rSLPI. Chromatographic studies indicated that 3H-rSLPI formed a 1:1 complex with HNE. Blockage of the active site of HNE by a covalently bound tetrapeptide chloromethyl ketone reduced complex formation with 3H-rSLPI by more than 98%. Incubation of 3H-rSLPI-HNE complex with alpha 1-protease inhibitor for 3 hours at 37 degrees C decreased the amount of complex compared with incubation in the presence of bovine serum albumin (70% vs 27% dissociated). The calculated dissociation rate constant was 1.1 x 10(-4) sec-1, indicating a 1.8 hour dissociation half-life. Dissociated 3H-rSLPI retained its ability to recombine with HNE. rSLPI was as effective at inhibiting HNE released from stimulated neutrophils as 3H-rSLPI was at inhibiting purified HNE. Intratracheal pretreatment of hamsters with 3000 micrograms of rSLPI as long as 8 hours before the intratracheal instillation of 250 micrograms of HNE, resulted in significant protection against induction of emphysema and secretory cell metaplasia. One and 4 hours after instillation of rSLPI, 59% and 44%, respectively, of the initial functional activity was recovered in lung lavage supernatant, indicating a half-life of approximately 2 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

To establish the progenitor role of bronchial epithelial cells in the steady state, we undertook a quantitative autoradiographic study in normal hamsters. Groups of 7 hamsters were killed 1 h and 1, 2, 3, 4, 7, and 14 d after an intraperitoneal injection of [3H]thymidine (2 microCi/g body wt). Autoradiograms were prepared from 861 Epon sections, 2 microns thick, of left intrapulmonary hilar bronchi. Epithelial cells were classified into 1 of 7 categories: basal-1 (B1) and basal-2 (B2), depending on nuclear height; secretory cells denoted as S1 with zero to 4 granules, S2 with 5 or more granules with intervening cytoplasm, and S3 with abundant granules completely filling the cytoplasm; ciliated (C); and indeterminate (IN). Mean silver grain counts decreased significantly over time only for B1 cells (P less than 0.05), with a cell cycle time of 20.6 d and a DNA synthetic time of 7.5 h. Labeled cells, 1 h after thymidine injection, comprised 30.5% S1, 27.8% B1, 22.8% B2, 6.8% IN, 6.4% S2, 5.7% C, and 0% S3 cells. Labeling indices of individual cell categories (LIc), at 1 h after labeling, were highest for B1 followed by B2 cells, reflecting their proliferative intensity. Labeling index of all epithelial cells combined did not change with time, indicating that there was no major cell death or label dilution. The LIc decreased significantly over time only for B1 and B2 cells (P less than 0.001 and P less than 0.002, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The authors wished to determine whether secretory cell metaplasia (SCM) induced in the bronchi of hamsters by human neutrophil elastase (HNE) was enzymatically mediated. We also wished to determine whether SCM could be induced by a proteolytic enzyme devoid of elastolytic activity. Accordingly, groups of weight-matched hamsters were given a single intratracheal instillation of 0.5 ml of saline solution containing one of the following: 300 micrograms of HNE purified from blood neutrophils, n = 14; 300 micrograms of HNE inactivated with Suc-Ala-Ala-Pro-Val chloromethyl ketone (CMK), n = 10; 500 micrograms of porcine pancreatic trypsin treated with CMK to eliminate residual active elastase, n = 10; 500 micrograms of trypsin inactivated by tosyl lysine chloromethyl ketone, n = 10; 2 micrograms CMK, n = 10; and saline alone, n = 10. Seven untreated animals served as additional controls. Twenty-one days post treatment, 5-6 micron paraffin-embedded sections, from the left lung hilar region, stained by Alcian blue and periodic acid-Schiff reaction were graded on a five-point scale for determination of the secretory cell index, which reflects SCM. Both elastase and trypsin produced severe SCM: mean +/- SEM secretory cell indices were 2.96 +/- 0.11 and 2.72 +/- 0.19, respectively, compared with values of 0.90 +/- 0.35 for the untreated group and 0.93 +/- 0.46 for the saline group (p less than .05). The secretory cell indices of the groups treated with inactivated elastase or trypsin were comparable to those of the saline-treated and untreated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous peroxidase activity was demonstrated by cytochemistry in mucous cells of the submucosal glands and tracheobronchial epithelium of guinea pigs. It is localized in the nuclear envelope, in cisternae of rough endoplasmic reticulum, and in secretory granules. It was not seen in Golgi saccules or in the airway lumen. By contrast, all epithelial cells within the lung including mucous (goblet) cells lack activity. Reaction product is also absent from alveolar macrophages and mast cells. The appearance of peroxidase in mucous cells is age-related. No activity was seen at 1.5 ms of age. A few mucous cells were positive at 2.5 and 3 ms while the proportion of positive cells increased substantially up to 7 ms. Thus, the age of guinea pigs in HRP transport studies must receive careful consideration in order to avoid misinterpretation of results. The function of mucous cell peroxidase is unknown. The results of this study suggest that it is secreted. Whether it plays a significant role in lung defense through its well documented anti-infectious properties remains to be determined.

A single intratracheal instillation of pancreatic elastase in hamsters induces a lesion resembling human panacinar emphysema. This paper reports the occurrence of irreversible goblet cell metaplasia in the bronchial epithelium of hamsters similarly exposed to elastase. The goblet cell change was dose related; a dose of 0.1 mg/100 g body wt or less at 16 days, produced slight or moderate goblet cell metaplasia in fewer than half the animals, whereas 84% of animals treated with a dose between 0.2 and 0.5 mg/100 g body wt developed goblet cell metaplastic lesions, more than half of which were considered to be severe. The percentage of goblet cells in the epithelium of elastase-treated hamsters (32.5) was significantly higher (P less than 0.005) than that of unexposed (12.2) and saline-exposed controls (18.7). All hamsters examined 6 and 12 mo after elastase treatment showed the lesion. The pathogenesis of the changes is unclear but the possibility is raised that the bronchial changes may be due to disturbance of an endogenous protease-antiprotease system. The findings suggest the hypothesis that, under appropriate circumstances, a single pulmonary insult in man could lead to a permanent lung injury demonstrating the anatomic lesions of both chronic bronchitis and panacinar emphysema.

To determine whether purified human neutrophil cathepsin G (Cat-G) can act by itself or in concert with purified human neutrophil elastase (HNE) in the induction of emphysema and bronchial secretory cell metaplasia (SCM), we gave golden Syrian hamsters 100 micrograms of HNE alone or in combination with either 100 or 200 micrograms of Cat-G. Other groups of animals received intratracheal doses of up to 600 micrograms of Cat-G alone. The severity of emphysema was determined from measurements of lung volumes, compliance, forced expiratory flow, and the mean linear intercept. The severity of SCM in the main airways was graded on sections stained by the alcian blue and periodic acid-Schiff reaction. The Cat-G was a weak inducer of SCM; significant SCM was produced by 400 and 600 micrograms but not by 100 or 200 micrograms or 200 micrograms of Cat-G. The Cat-G (100 and 200 micrograms) did not potentiate the SCM induced by 100 micrograms of HNE. The Cat-G alone did not produce emphysema, and neither 100 nor 200 micrograms of Cat-G potentiated the mild emphysema induced by 100 micrograms of HNE. These results were not consonant with a report that Cat-G and HNE were synergistic in solubilizing human lung elastin. We therefore measured the ability of Cat-G and HNE to solubilize several radiolabeled elastins. The combination of Cat-G and HNE did not solubilize significantly more hamster lung elastin (23%) than the sum of their individual activities.(ABSTRACT TRUNCATED AT 250 WORDS)

In interstitial fibrosis of the lung, pulmonary vessels show priminent sclerotic changes. We have studied arteries in lung biopsies from patients with lung fibrosis of varied etiology using light and electron microscopy. It was found that the sclerotic change is essentially confined to arteries within fibrotic areas, areas of "nonfibrotic" lung having vessels with no intimal thickening. The changes are most striking in arteries with an external diameter of 500 micrometer. or greater (p less than 0.001), although smaller sized vessels also show significant changes. Ultrastructurally, the plaques are composed of typical smooth muscle cells with basement lamina, pinocytotic vesicles, dense bodies, and cytoplasmic microfilaments. These arterial changes could result from damage to the blood vessel which accompanies the mechanism producing lung fibrosis. Alternatively, the myointimal change could conceivably be part of an adaptive response following the establishment of fibrosis. In either instance, the narrowing would decrease blood flow to physiologically disadvantaged areas of the lung, and the muscle fibers might play an active role in reducing the blood flow.

To explore the time course and the mechanism of development of bronchial secretory cell metaplasia (SCM) induced by human neutrophil elastase (HNE), anesthetized hamsters were injected intratracheally with 300 micrograms highly purified HNE in 0.5 ml saline solution; saline-injected and untreated animals served as controls. At 3, 8, 16, and 21 days after treatment, animals were killed and their lungs fixed by vascular perfusion. Samples from the hilar region of the left lung, containing the main axial airway and its proximal branches, were embedded in Epon-Araldite, and 1 micron sections were stained with methylene blue. Epithelial cells with a luminal border were categorized into three cell types and expressed as a percent of total cells counted (mean 1900 per animal); cells containing at least three mucin granules were classified as secretory, ciliated cells displayed cilia or basal bodies, and cells with none of these characteristics were classified as indeterminate. Percentages of the three cell types in saline-treated animals, at all time points, were comparable to those in the untreated controls. With HNE treatment the secretory cell percentages were higher at 16 days (mean +/- SEM, 36.4% +/- 3.2%) and at 21 days (35.7% +/- 2.9%) than in the untreated animals (18.2% +/- 1.8%, P less than 0.05). The percentage of the indeterminate cells in the HNE group was decreased at days 8, 16, and 21 (7.2% +/- 1.6%, 5.0% +/- 1.4%, and 8.2% +/- 2.5%, respectively) compared with that in the untreated group (21.7% +/- 2.5%, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Eglin-c is a naturally occurring polypeptide of 70 amino acids with a molecular mass of 8,100 daltons. It is a strong inhibitor of human neutrophil elastase (HNE) and cathepsin-G, and, when given intratracheally to hamsters 1 h before human neutrophil elastase, it can prevent or ameliorate the emphysema produced by HNE. The present experiments were designed to determine the duration of the effectiveness of eglin-c, prepared by DNA technology from Escherichia coli, in preventing the emphysema and secretory cell metaplasia induced by HNE. Eglin-c (2,000 micrograms in 0.5 ml saline) was effective in ameliorating emphysema, as determined histologically and physiologically, when it was given intratracheally to hamsters 1, 2, 4, and 8 h before the intratracheal instillation of 300 micrograms of HNE. Eglin-c ameliorated bronchial secretory cell metaplasia when given 1 h before HNE but not when the time intervals were 2 h or longer. The clearance of [3H]eglin-c from the lungs was assessed. Four h after intratracheal instillation of 446 micrograms of [3H]eglin-c, 33% of the tritium was found in the lung tissue and bronchoalveolar lavage fluid; 83% of the radioactivity in the lavage fluid supernatant was associated with functionally active eglin-c. No evidence of bronchopulmonary toxicity was seen in hamsters given 4 intratracheal instillations of 2,000 micrograms of eglin-c at 1-wk intervals.

Male golden hamsters were exposed to a solution of purified pancreatic elastase by intratracheal injection. They developed panlobular (panacinar) emphysema and, in addition, were found to show severe goblet cell metaplasia in the major bronchi. The metaplastic change in the respiratory epithelium was associated with persistence of a fenestrated sheet of mucus, widely present throughout the bronchial tree, which was greater in amount than that in either unexposed or saline exposed controls. Transmission electron micrography showed a striking increase in size of individual goblet cells, due to increased numbers of secretory droplets which were also much larger and paler than in control bronchi. Ciliated cells appeared smaller than normal due to compression by the swollen goblet cells. The presence of prominent dilatation of the endoplasmic reticulum and the increased frequency of secretory droplet release from the luminal surface stronly suggest that the goblet cell changes were due to increased formation and secretion of mucus into the damaged bronchi. These experiments show that a single exposure to elastase produces both severe panlobular emphysema and goblet cell metaplasia. The changes resemble several of the anatomic features of chronic obstructive lung disease in man for which this injury may be a suitable model.

Purified human neutrophil elastase (HNE) and a crude extract of human neutrophils (EXT) were administered intratracheally to hamsters, and their effects were determined 21 and 56 days later by measurement of lung statics and dynamics and by light microscopy and morphometry of the lungs. A dose of 450 micrograms of HNE (HNE 450) produced focal emphysema of moderate severity. The combination of HNE 450 and EXT (HNE 450 + EXT) produced no more severe emphysema than HNE 450 alone; EXT alone did not produce emphysema. The HNE 450, HNE 450 + EXT, and EXT all produced persistent secretory cell metaplasia in large intrapulmonary airways. In a second experiment, 40 micrograms of HNE (elastolytic activity equivalent to that in EXT), designated HNE 40, did not produce secretory cell metaplasia. Neither EXT nor EXT treated with the elastase inhibitor suc-ala-ala-pro-val chloromethyl ketone (EXT + CMK), which had a residual elastolytic activity equivalent to 15 micrograms of HNE, produced emphysema; both preparations caused bronchial secretory cell metaplasia 21 days after treatment. Values of maximal expiratory flow, measured 21 days after treatment, were reduced at points between 20 and 50% of vital capacity for HNE 450 and between 30 and 80% of vital capacity for HNE 450 + EXT; maximal expiratory flows were not significantly different from saline controls for HNE 40, EXT, or EXT + CMK.(ABSTRACT TRUNCATED AT 250 WORDS)

The lower respiratory tract of the hamster was examined for evidence of endogenous peroxidase activity. Using the standard diaminobenzidine cytochemical technique with controls to distinguish peroxidase from other hemoproteins, brown peroxidase reaction product was observed in the tracheal lumen and within epithelial secretory cells. The lumen and secretory cells of submucosal glands also contained peroxidase activity. Peroxidase-positive cells were most numerous in the upper trachea. Activity gradually decreased distally so that the least number of positive cells occurred in the extrapulmonary bronchus. Older animals contained many more positive cells than did younger animals. Within the lung, all epithelial cell types in both conducting and respiratory zones lacked activity. Peroxidase-positive cells in the tracheo-bronchial epithelium were identified as mucous cells by electron microscopy. Within these cells, peroxidase activity was found in the nuclear envelope, cisternae of rough endoplasmic reticulum, Golgi saccules, condensing vacuoles, and secretory granules. Discharge of the granules into the lumen appeared to result from a merocrine type of secretion. These ultrastructural findings are similar to those described for the secretory peroxidase in mammary and salivary glands. The peroxidase in these glands plays a key role in a nonspecific antibacterial system. Although the function of airway peroxidase is presently unknown, it is quite possible that it too possesses anti-infectious properties, thus forming an important adjunct to the well-known physical, cellular, and immunologic processes that protect the respiratory tract from microbial and toxic injury.

N,N-dimethylformamide (DMF) induces differentiation of human colon carcinoma (DLD-1) cells in culture and reduces their tumorigenicity in nude mice. The current investigation analyzed DLD-1 (clone D) cells for ultrastructural evidence of differentiation. Examination of treated and untreated confluent monolayers by transmission electron microscopy revealed an occasional intracytoplasmic lumen indicative of adenocarcinoma. DMF-treated cells showed no signs of a toxic reaction. Cytoplasmic organelles were essentially unchanged except for an increase in tonofilaments and associated desmosomes. The number of desmosomes per unit length of contiguous cell border increased almost sixfold in treated monolayers. No other type of cell junction was seen. The increased frequency of desmosomes in DMF-treated cultures is significant because of the direct correlation known to exist between the number of desmosomes and degree of differentiation of some human carcinomas. Desmosomes serve as foci of cell adhesion and are reduced in number in some invasive tumors. Whether the supernumerary desmosomes in DMF-treated cells contribute to the reduction in malignant behavior of these cells in vivo remains to be determined.