Pulmonary alveolar phospholipoproteinosis: experience with 34 cases and a review.

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.

Clonal variation of cadmium response in human tumor cell lines.

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.

Biliary excretion of iron from hepatocyte lysosomes in the rat. A major excretory pathway in experimental iron overload.

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.

Analytical assessment of Broviac catheter occlusion.

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.

The growth of individual seminal vesicle epithelial cells and their proliferation.

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.

Migration and granulomatous reaction after periurethral injection of polytef (Teflon).

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.

Proliferative potential of cells from normal human colon epithelium, adenomas, and carcinomas of the large bowel.

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.

Biological availability of nickel arsenides: toxic effects of particulate Ni5As2.

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.

Morphologic features of chronic hepatitis associated with primary sclerosing cholangitis and chronic ulcerative colitis.

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.

Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H1(0); IP25) from Chinese hamster cells.

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.

In vitro propagation of seminal vesicle epithelial cells.

The unique anatomic features of the guinea pig seminal vesicle allow establishment of nearly pure cultures of epithelial cells in vitro. Primary monolayer cultures can be maintained for up to 5 weeks and suspension cultures for 5 to 7 days. The rapidly proliferating cells in monolayer culture do not seem to be markedly sensitive to the presence of androgens or estrogens in the medium. Scanning and transmission electron microscopy indicates that the proliferating cells are epithelial in nature and are quite different from control seminal vesicle fibroblast cultures; transmission electron microscopy shows that these cells are markedly dedifferentiated vis-à-vis the native tissue in vivo. This is the first report of the successful cultivation of seminal vesicle epithelial cells in vitro.

The fine structural localization of testicular phosphatases in man: the control testis.

Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes-glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase-in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.