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A retrospective review of Mayo Clinic records through 1983 revealed 84 patients (24 male and 10 female; mean age, 41 years) with the diagnosis of pulmonary alveolar phospholipoproteinosis. The major clinical features were dyspnea, cough, fever, and chest pain. Chest roentgenograms usually showed bilateral symmetric alveolar infiltrates, but asymmetric, unilateral, and chronic patchy patterns were also noted. Diagnosis was established by thoracotomy-lung biopsy in 26 patients. Histologic analysis revealed uniform filling of the alveoli by periodic acid-Schiff-positive material and maintenance of normal alveolar architecture. Electron microscopy showed enlarged alveolar macrophages with lamellar osmiophilic inclusions, dense granules, and myeloid bodies. Of the 21 patients who underwent therapeutic bronchoalveolar lavage, 13 had no recurrence of the disease during a mean follow-up of 8.8 years. In patients who underwent pulmonary function testing both before and after lavage, significant restrictive dysfunctions present before the procedure were alleviated afterward. Three deaths occurred among the 34 patients. Pulmonary alveolar phospholipoproteinosis may result from defective clearance of phospholipids by the alveolar macrophages, excessive production of phospholipids by type II pneumocytes, or both. It is likely a nonspecific response to a variety of injuries to the alveolar macrophage or type II pneumocyte or both, including exposure to certain dusts and chemicals and occurrence of hematologic diseases or infections. The uncommon occurrence of this disorder suggests individual susceptibility.

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Subpopulations of human tumor-derived cell lines A101D, A204, and A549 were screened for Cd2+ cytotoxic response. Three of six A549, two of seven A101D, and four of seven A204 subpopulations were found to differ significantly from the parental line. A variant subpopulation of A101D (T3) was shown by flow cytometry to be comprised of cells having two distinct DNA histograms. One histogram, type 1, resembles that of normal human fibroblasts. The other, type 2, represents cells with one-third more DNA. Early passage T3 clonal populations were comprised primarily of type 1 cells. With passage, type 1 cells decreased relative to type 2 so that by passage 47 the culture was predominantly type 2. Correspondingly, the A101D T3 subpopulation became more Cd2+ sensitive with time in culture. Subclones having only type 1 DNA histograms were found to be Cd2+ resistant relative to subclones with type 2 histograms, and treatment of A101D T3 cultures having approximately equal amounts of type 1 and 2 cells with 2 microM Cd2+ resulted in the selection of type 1 cells. The enhanced Cd2+ resistance phenotype shown by A101D T3 type 1 cells correlated with reduced Cd2+ uptake and is not attributable to enhanced metallothionein synthesis.

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In these experiments, we assessed the role of hepatocyte lysosomes in biliary excretion of iron. We loaded rats with iron by feeding 2% carbonyl iron and collected bile for 24 h via bile fistulae from iron-loaded and control rats. In additional rats, bile was collected before and after the administration of colchicine. Rats were then killed and their livers were homogenized and fractionated for biochemical analyses or processed for electron microscopy and x-ray microanalysis. Inclusion of 2% carbonyl iron in the diet caused a 45-fold increase (P less than 0.001) in hepatic iron concentration compared with controls (1,826 +/- 159 vs. 38 +/- 6.7 micrograms/g liver, mean +/- SE). Electron microscopy with quantitative morphometry and x-ray microanalysis showed that the excess iron was sequestered in an increased number of lysosomes concentrated in the pericanalicular region of the hepatocyte. Iron loading was also associated with a twofold increase in biliary iron excretion (4.06 +/- 0.3 vs. 1.75 +/- 0.1 micrograms/g liver/24 h; P less than 0.001). In contrast, the biliary outputs of three lysosomal enzymes were significantly lower (P less than 0.0005) in iron-loaded rats compared with controls (mean +/- SE) expressed as mU/24 h/g liver: N-acetyl-beta-glucosaminidase, 26.7 +/- 4.6 vs. 66.2 +/- 13.4; beta-glucuronidase, 10.1 +/- 1.3 vs. 53.2 +/- 17.9; beta-galactosidase, 8.9 +/- 1.0 vs. 15.4 +/- 2.3. In iron-loaded rats but not in controls, biliary iron excretion was coupled to the release into bile of each of the three lysosomal hydrolases as assessed by linear regression analysis (P less than 0.001). In contrast, no relationships were found between biliary iron excretion and the biliary outputs of a plasma membrane marker enzyme (alkaline phosphodiesterase I) or total protein. After administration of colchicine, there was a parallel increase in biliary excretion of iron and lysosomal enzymes in iron-loaded rats, but not controls. We interpret these data to indicate that, in the rat, biliary iron excretion from hepatocyte lysosomes is an important excretory route for excess hepatic iron.

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Eight of 92 consecutive silastic central venous catheters used for home parenteral nutrition occluded. Six of the eight had patency restored by the instillation of urokinase or streptokinase into the catheter. The thrombus in one of the two catheters that was not reopened with thrombolytic agents was studied in detail by electron microscopy, x-ray dispersive analysis, solubility in isopropyl alcohol-diethyl ether (1:1, v:v), and thin-layer chromatography of extracted lipids. Electron microscopy found the clot to be an amorphous mass without features to suggest crystalline properties. The x-ray dispersive analysis showed that the only elements which were significantly increased were chloride and silicon and the silicon detected was likely from the underlying catheter. Treatment with isopropyl alcohol-diethyl ether left an insoluble, flaky residue that resembled protein from a thrombus. Thin-layer chromatography detected a lipid profile suggestive of circulating endogenous fat instead of the fat that was infused through the catheter.

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Histologically seminal vesicle epithelium (SVE) of the intact adult guinea pig is a discrete and segregated monolayer of highly specialized tall columnar cells. The epithelial layer is so sharply demarcated from its attached stroma (primarily smooth muscle), that blunt dissection alone is sufficient to separate epithelium from muscle. After castration the epithelial cells decrease in both size and number so that by the fifth day, the surviving cells are greatly involuted structurally and comprise only about 12% of the original numerical population normally present in one seminal vesicle. Injected testosterone leads to restructuring of individual cells followed by cell replenishment. The major goal of this effort was to elaborate upon the processes of individual cell growth and cell replenishment during restoration of the tissue to normal cell size and number. The two separate processes were studied using light and electron microscopy, [3H]thymidine incorporation, and Northern blots with labeled histone gene probes. By approximately 48 hr of hormone repletion, parenchymal cell size had returned to normal as the result of a dramatic anabolic process of individual cell growth while cell number remained unchanged. During the subsequent 48-hr period of hormone repletion, the cell population was restored to normal as cell replenishment became the predominant process. Microscopic analysis at intervals throughout the 96-hr period failed to disclose any mitotic events to account for cell replenishment even when Colcemid had been administered. Nor could the increase in cell numbers be correlated with a great increase in [3H]thymidine incorporation or in histone mRNA synthesis. Thus, we could provide no evidence that mitotic division of the parenchymal cells themselves is responsible for cell replenishment. During the 24- to 48-hr interval of hormone repletion, electron microscopic examination disclosed the presence of small epithelial cells lying in a basal position. Some of these cells were seen to insert themselves between the basal regions of parenchymal cells and to expand from the basement membrane into the parenchyma. Possible origins of the cells which replenish the tissue are discussed.

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When rachitic chicks are given 1,25-dihydroxyvitamin D3 in amounts as low as 50 ng/bird, the appearance of the duodenal mucosa is altered within 2 h of the administration of the hormone. The changes are most readily apparent on scanning electron microscopy and include: a more plump appearance of villi with loss of furrows and pits on their surfaces, elongation of villi and a smoother, more uniform microvillus surface. These changes occur within 2 h of the administration of the hormone and persist for as long as 24 h. The morphological change precedes the increase in calcium absorption induced by 1,25-dihydroxyvitamin D3 in the intestine. These observations suggest that 1,25-dihydroxyvitamin D3 may play an important part in maintenance of the structure of the duodenal mucosa.

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Although patients with urinary incontinence have been treated successfully by periurethral injection of polytef paste, this study in continent animals demonstrates migration of polytef particles from the injection site. We injected polytef paste periurethrally into female dogs and male monkeys. Particles were found at 50 to 70 days in pelvic nodes in six of seven animals and lungs in four of seven (the kidneys and brain were not studied); and at 10 1/2 months in pelvic nodes, lungs, and brain in seven of seven; kidneys in four of seven; and spleen in two of seven. X-ray microanalysis confirmed that the particles were polytef. At 10 1/2 months, polytef granulomas were found at all injection sites and some sites of distant migration. Since these granulomas signify chronic foreign-body reaction, we believe that until the long-term effects in humans are known, polytef paste should not be used in children or young adults with normal life expectancy.

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Nuclear protein matrix was isolated from guinea pig seminal vesicle epithelium and liver. The two matrices were similar in fine structure as seen by transmission electron microscopy, in protein electropherograms, and in percent composition relative to protein, DNA, and RNA. Scanning electron microscopy was used to examine intact seminal vesicle nuclei, nuclei after treatment with Triton X-100 and DNAse I, and purified nuclear matrix. The matrix surface presented a 'porous' appearance by both scanning and transmission electron microscopy. The matrices of liver and seminal vesicle epithelium (SVE) and the intact nuclei of SVE were assayed for specific binding of free synthetic androgen, 17 alpha-methyltrienolone (R1881). Saturable specific binding was demonstrable for seminal vesicle matrix but not for liver matrix. Maximal binding of androgen occurred at a concentration of approximately 12 nM and was demonstrated to be 1.34 +/- 0.22 pmol of R1881 per mg of seminal vesicle matrix protein; the Kd was approximately 8 nM. The binding of labeled R1881 to matrix could be inhibited with low concentrations of unlabeled androgens, but not with estrogens or other steroids. Our data indicate that the binding of androgen to matrix could account for at least 21% of the binding to intact nuclei.

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We tested the hypothesis that hepatocyte microtubules modulate the biliary excretion of endogenous and exogenous constituents of hepatocyte lysosomes. We collected bile via bile fistulas from male rats before and after acute administration of colchicine and vinblastine, agents known to bind to hepatocyte microtubules; rats were then killed and livers were homogenized for biochemical analyses or processed for electron microscopy. Colchicine caused biphasic, parallel alterations in the biliary excretion of three lysosomal enzymes compared with control rats given saline or lumicolchicine; a peak rise in enzyme outputs of approximately 175% at 45-60 min after colchicine administration was followed by a sustained fall to approximately 25% of control values, which persisted for 2-4 h. When hepatocyte lysosomes were prelabeled in vivo by administration of [3H]Triton WR-1339, a nonionic detergent that is sequestered in hepatic lysosomes, the biliary excretion of radiolabel in response to colchicine paralleled the biliary excretion of the three lysosomal enzymes. Vinblastine also induced a biphasic response in biliary lysosomal enzyme output that was similar to that produced by colchicine administration. Morphometric analysis of electron micrographs of rat livers demonstrated changes in the number of lysosomelike vesicles in the vicinity of bile canaliculi after colchicine and vinblastine administration; the initial increase in lysosomal enzyme secretion was associated with a significant decrease in the number of pericanalicular lysosomes after both agents, while the subsequent decrease in enzyme secretion coincided with an increase in the number of pericanalicular lysosomes after vinblastine.(ABSTRACT TRUNCATED AT 250 WORDS)

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The proliferative potential of nonmalignant and malignant cells obtained from the human large bowel was assessed with a soft agar culture colony-forming technique and by flow cytometry to analyze cell cycle kinetics. No colony formation occurred with samples of normal human adult colon mucosa. The absence of colony formation in all but 1 of 24 evaluable colorectal adenomas suggested that malignancy of tissue in vivo is generally necessary for colony formation to occur in soft agar cultures. A high proliferative index (S + G2/M), as assessed by flow cytometry, was significantly associated with higher soft agar culture counts in a series of 25 colorectal carcinomas.

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In these experiments, we tested the hypothesis that chloroquine, a lysosomotropic agent which modifies protein and lipid metabolism by hepatocyte lysosomes, would alter the biliary excretion of lipids and lysosomal enzymes. We treated male rats for 5 days with intraperitoneal chloroquine (50 mg/kg body wt, n = 9) or saline (n = 8) and collected bile for 6 h via bile fistulas; rats were then killed and livers homogenized for biochemical analyses or processed for electron microscopy. Chloroquine markedly increased the biliary excretion of three lysosomal enzymes (mean +/- SEM) expressed as milliunits of activity per gram liver: N-acetyl-beta-glucosaminidase (24.4 +/- 2.7 vs. 12.5 +/- 1.4, p less than 0.01), beta-glucuronidase (26.4 +/- 4.7 vs. 10.9 +/- 1.4, p less than 0.01), and beta-galactosidase (9.8 +/- 1.7 vs. 5.5 +/- 0.8, p less than 0.05). In contrast, biliary outputs of enzymes associated with other organelles (e.g., alkaline phosphodiesterase I and lactic dehydrogenase) were unaffected by chloroquine treatment. Biliary cholesterol secretion was decreased after chloroquine administration (0.28 +/- 0.02 mumol/g liver vs. 0.39 +/- 0.03 mumol/g liver, p less than 0.01), but bile acid and phospholipid secretion were not altered; as a result, cholesterol saturation of bile decreased by 22% (p less than 0.05). Hepatic activities of all three lysosomal enzymes were increased after chloroquine administration (p less than 0.04); activities of enzymes associated with mitochondria, plasma membrane, endoplasmic reticulum, and cell sap were not altered. Morphometric analysis of electron micrographs of rat livers demonstrated a marked increase (p less than 0.001) in the number of lysosomelike vesicles and autophagic vacuoles in the vicinity of bile canaliculi after chloroquine administration; also, the number of canalicular microvilli decreased (p less than 0.003) after chloroquine treatment. We conclude that altered hepatic lysosomal morphology and function after chloroquine is accompanied by marked changes in outputs of lipids and lysosomal enzymes into bile. These findings call attention to a possible role for hepatic lysosomes in modulating biliary protein and lipid secretion.

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To determine if fugitive nickel arsenides from an oil shale retort could pose a threat to living organisms, we studied the effects of particulate Ni5As2 on cultured mammalian cells. Culture growth rate was greatly reduced at the lowest suspension concentration tested (10 microM), even though much of the Ni5As2 powder remained insoluble. FCM analysis indicated Ni5As2 arrested cell-cycle traverse in G1 and G2. Cell survival studies indicated cells could overcome this toxicity if exposure levels were low (less than or equal to 25 microM in suspension) and restricted to less than or equal to 24 h. At higher powder levels, survival was greatly reduced. Transmission electron microscopy (TEM) demonstrated that cells exposed to less than or equal to 100 microM powder did not phagocytize the Ni5As2 particles. At higher concentrations, TEM X-ray microanalysis demonstrated that As was preferentially extracted from the Ni5As2 particle surface and free Ni was deposited inside the cell. These observations suggest that the toxicity of Ni5As2 particles may be caused by some soluble product of Ni5As2.

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Pulmonary alveolar microlithiasis is a rare disease of unknown cause in which calcium phosphate microliths are deposited throughout the lungs. These deposits are of sufficient density to be almost diagnostic on chest roentgenograms. The Mayo Clinic experience with 8 patients is added to the approximately 120 cases reported in the world literature. The age range of all patients is from newborn to 80 years, with a mean age at diagnosis of about 35 years. No sexual predominance has been noted, but in about half of the reported cases a familial pattern has been found. The progression of the disease is generally very slow, some patients having been followed up for more than 30 years without evidence of change. No specific treatment is available. Pulmonary function studies demonstrate a tendency toward a restrictive pattern. Technetium-99m scanning and scanning and transmission electron microscopy are useful procedures for analysis of pulmonary alveolar microliths.

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After intraperitoneal injection of paraquat, rats showed evidence of neurologic and respiratory damage and had a mortality rate of 41% in 3 days. The lungs quadrupled in weight between the third and the fifth day. Pulmonary edema and extravascular fibrin and platelets were identified by light and transmission electron microscopy. As early as 4 h after injection of the paraquat, 51Cr from labeled platelets began accumulating in the lungs. The peak was reached by 48 h; 125I from labeled fibrinogen also concentrated in the lungs of treated rats. Total complement was unchanged. The paraquat-treated rat is a suitable model for study of the behavior of fibrin and platelets in permeability pulmonary edema. Disturbances of copper metabolism deserve further investigation.

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In these experiments, we tested two hypothesis: first, that Triton WR-1339, a nonionic detergent which is sequestered in hepatocyte lysosomes, undergoes biliary excretion; and second, that Triton WR-1339, which also alters serum lipid levels and modifies hepatic catabolism of lipoproteins, affects the biliary output of proteins and lipids. When 3H-Triton WR-1339 was administered to rats, biochemical and morphologic studies showed that hepatocyte lysosomes sequestered Triton WR-1339: (i) the subcellular distribution of 3H was identical to that of lysosomal enzymes after liver fractionation by differential or isopycnic centrifugation, and (ii) lysosomes appeared engorged with Triton WR-1339 on electron microscopy. 3H was also excreted into bile in parallel to three lysosomal enzymes. Triton WR-1339 administration caused a coordinate increase in the biliary excretion of three lysosomal enzymes and also increased the biliary output of total protein, bile acids, and phospholipid. Triton WR-1339 administration did not affect bile flow or the biliary outputs of cholesterol, plasma membrane, and cytosolic enzymes, but did decrease biliary cholesterol saturation by 50%. These results demonstrate that an exogenous compound which is sequestered in hepatocyte lysosomes may be excreted directly into bile in parallel with endogenous lysosomal constituents. The data also show that such a lysosomotropic agent may also selectively modify the biliary excretion of proteins and lipids. The findings are consistent with the existence of a lysosome-to-bile hepatic excretory pathway and suggest that hepatocyte lysosomes may be important in modulating biliary protein and lipid secretion.

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Histologic, ultrastructural, chemical, and statistical methods were used to study liver biopsy and autopsy specimens from 43 patients who had primary sclerosing cholangitis (PSC), with or without chronic ulcerative colitis (CUC), and from 19 patients who had CUC without PSC. In all study groups, essentially the same abnormalities were found in the hepatic parenchyma outside the major bile ducts, although nondiagnostic tissue samples were observed also. Specimens from patients with extrahepatic PSC were indistinguishable from those patients with combined extra- and intrahepatic PSC. Common findings included periductal fibrosis and inflammation, portal edema and fibrosis, focal proliferation of bile ducts and ductules, focal bile duct obliteration and loss of bile ducts, copper deposition, and cholestasis. Proliferation of bile ducts in some portal tracts and obliteration or absence of bile duct in others were the most characteristic changes. In most specimens, inflammatory changes appeared mild, yet biliary cirrhosis had developed in 34% of the patients. Specimens from patients with PSC, with or without CUC, more often contained bile and strikingly increased stainable copper (Grades 2 and 3) than did specimens from patients with CUC without PSC. Hepatic copper contents, measured by atomic absorption spectrophotometry, also were higher in specimens from patients with PSC. Study of PCS specimens by transmission electron microscopy and by energy-dispersive X-ray microanalysis revealed that most copper was sequestered in lipolysosomes. The recognition of strikingly similar morphologic features in many liver specimens from patients with either PSC or CUC or both suggests that the causes of these conditions are closely related.

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A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approximately 20 amino acids so that the large fragment seen in sodium dodecyl sulfate--acrylamide gels has a molecular weight of approximately 20 000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. In G1-arrested cells, little or no phosphate is incorporated into BEP. As cells progress through interphase, BEP becomes phosphorylated so that 12--35% of the BEP molecules are phosphorylated at one to two sites by late interphase. During mitosis, all BEP molecules become phosphorylated at approximately four sites per molecule (BEPM). Electrophoresis and the analysis of cell populations by electron microscopy indicate that the appearance of BEPM is temporally correlated with the mitotic phosphorylation of histone H1 (H1M) and with chromosomal condensation during prophase, metaphase, and anaphase. During exit from mitosis, BEPM undergoes dephosphorylation. The dephosphorylation of BEPM is temporally correlated with dephosphorylation of H1M and with the unraveling of fully condensed chromosomes near the anaphase--telophase transition. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1(0) [Panyim, S., & Chalkley, R. (1969) Biochem. Biophys. Res. Commun. 37, 1042], rat H1(0) [Medvedev, Zh. A., Medvedeva, M. N., & Huschtscha, L. I. (1977) Gerontology (Basel) 23, 334], and IP25, a protein enhanced in differentiated Friend erythroleukemia cells [Keppel, F., Allet, B., & Eisen, H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 653]. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP.

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