Ultrastructural changes in the pancreas of carbonyl iron-fed rats.

Diabetes mellitus is found with increased frequency in patients with both primary and secondary hemochromatosis. In these conditions, the pancreas shows fibrosis and iron overload of acini, interstitium, and islet B cells. Previous morphological studies have only described changes found in advanced stages of disease, while abnormalities of the initial stage of iron overload have, as yet, not been reported. Rats fed a carbonyl iron-supplemented diet for 4-15 months showed storage iron deposition (ferritin and hemosiderin) in many organs, in a pattern similar to primary human hemochromatosis. Electron microscopic examination of the pancreas showed ferritin particles segregated in lysosomes of acinar cells, as well as diffuse cytosiderosis of macrophages in the interstitial septa. In the islets, iron deposits were discrete and only in B cells. In the absence of electron-microscopic studies of incipient pancreatic cytosiderosis in human subjects, the present experimental animal study may contribute to a better understanding of the pathway leading to the extensive lesions found in the advanced stages of the human iron overloading diseases.

High-performance liquid chromatography separation of phospholipid classes and arachidonic acid on cyanopropyl columns.

An HPLC method for the separation and analysis of arachidonic acid and eight phospholipid classes is described: phosphatidylglycerol, phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and 2-lysophosphatidylcholine. The separation is carried out at 60 degrees C on 2 cyanopropyl columns using a gradient of acetonitrile and 5 mM sodium acetate (pH 5.0). Cyanopropyl columns require a lower proportion of water in the mobile phase to elute the more polar phospholipids than other types of columns and are thus less prone to equilibration problems. The method is highly reproducible (average coefficient of variation for each retention time less than or equal to 3.5%) and permits analysis of peaks by phosphorus content. Data obtained by analyzing lipid extracts from rat alveolar macrophages prelabeled with [G-3H]-arachidonic acid were analyzed by this HPLC method and compared to standard analysis by TLC. There was a significant correlation between the radioactivity profiles obtained with the two chromatographic methods (HPLC versus TLC) by linear regression analysis [HPLC = 0.83 (TLC) + 3.58, n = 25, r = 0.95, P less than 0.001].

Identification of mRNA coding for factor VII protein in human alveolar macrophages--coagulant expression may be limited due to deficient postribosomal processing.

Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.

Effect of irradiation on thermal sensitivity of bone marrow progenitors.

The heat sensitivity of murine CFU-GM and CFU-E following 2.5 Gy of total body irradiation (TBI) was studied. C3H f/Sed female mice were treated with 2.5 Gy TBI and femoral bone marrow was heated in vitro at 43 degrees C. CFU-GM show heat radiosensitization when bone marrow was heated immediately following irradiation. There was a brief decline in heat and radiation interaction when cells were heated 3 hours following 2.5 Gy of TBI, but heat radiosensitization returned to its maximum from 1 to 2 days following irradiation and remained significantly different from the control on days 5 and 7 following irradiation. The heat and radiation interaction disappeared by 30 days. CFU-E shows significant heat radiosensitization only on day 2 following 2.5 Gy of TBI. Total nucleated cells per femur showed a decrease by 70 per cent in days 1 to 2 following TBI, recovered to control values by day 5, and did not correlate with the changes in heat radiosensitization. Cell cycle analysis of CFU-GM using hydroxyurea showed no significant changes in cell cycle parameters on days 1 and 2 following 2.5 Gy, when maximum heat sensitization was observed. It is concluded that bone marrow progenitors may respond in a different way from other normal tissues to heat and irradiation sequencing, and that these differences must be considered when designing clinical trials.

A fish oil diet reduces the severity of collagen induced arthritis after onset of the disease.

We have previously reported that compared to a corn oil diet a fish oil diet (5% by weight) fed to B10R.III mice before the induction of collagen induced arthritis markedly reduced disease severity. In this study we determine whether a fish oil diet could reduce the severity of collagen induced arthritis if begun after the arthritis was clinically apparent. Mice were initially fed either a fish oil or corn oil diet and immunized with bovine type II collagen 4 weeks later. At the onset of collagen-induced arthritis, half of the corn oil fed mice were switched to fish oil and arthritis assessed on a weekly basis. Four weeks after the diet change until killing 5 weeks later, the mice switched to fish oil developed much less severe arthritis than the corn oil fed controls. Thus the severity index of corn oil fed mice ranged between 9.4 and 7.1; the severity index of fish oil fed mice was between 6.8 and 4.3 while the mice switched to fish oil ranged between 7.2 and 5.6. Analysis of peritoneal macrophages 13 weeks after immunization showed that macrophages from fish oil fed mice incorporated eicosapentaenoic acid into phospholipids and produced less arachidonate products than corn oil fed mice. There was no difference between macrophages obtained from mice switched from corn oil to fish oil and those maintained on fish oil with respect to fatty acid composition of membrane phospholipids or prostaglandin profile. These results suggest that arthritis severity may be modulated after the onset of CIA by altering the PG profile of macrophages present at inflammatory sites.

Fatty acid composition of macrophage phospholipids in mice fed fish or borage oil.

The polyunsaturated fatty acid (PUFA) composition of murine peritoneal macrophage phospholipids was dramatically altered in vivo following the four-wk feeding of specific dietary oils. Fish oil (containing 20:5n-3 and 22:6n-3) feeding significantly increased macrophage 20:5n-3, 22:5n-3, and 22:6n-3 (P less than 0.05), while borage oil (containing 18:2n-6 and 18:3n-6) increased (P less than 0.05) the macrophage 20:3n-6/20:4n-6 ratio, relative to safflower oil (containing 18:2n-6) and hydrogenated coconut oil (containing 12:0)-fed animals. The macrophage phospholipid PUFA profiles were compared with those of the liver, lung and spleen. The significance of the PUFA alterations is discussed.

[Role of cyclic 3',5'-adenosine monophosphate in the development of Salmonella infection in an experiment]. / Issledovanie roli tsiklicheskogo 3',5'-adenozinmonofosfata v razvitii sal'monelleznoi infektsii v éksperimente.

The changes in cAMP levels in spleen macrophages of mice infected with low-virulent and virulent Salmonella strains and the effect of propranolol on Salmonella reproduction in the spleen, and the outcome of Salmonella-induced infection have been studied. A persistent increase in cAMP levels in spleen macrophages during Salmonella infection caused by virulent Salmonella strains has been demonstrated. Low-virulent Salmonella strains failed to cause the elevation of cAMP levels in spleen macrophages. Propranolol injection to mice prevented intensive Salmonella reproduction in the spleen and diminished the animal mortality rate.

Sequential histochemical staining for resident and recruited macrophages.

A new histochemical technique is described that permits differentiation of resident from recruited macrophages by staining of paraffin sections of tissues from rats and mice. Resident macrophages are identified by their ability to phagocytose and retain intravenously injected colloidal Prussian blue. New macrophages that emigrate into tissue are identified by phagocytosis of a second colloid, iron dextran. Paraffin sections of formalin-fixed tissues are sequentially stained for the presence of the two colloids with different chromogens, the endogenous pseudo-peroxidase activity of colloidal Prussian blue used to catalyze the polymerization of diaminobenzidine and after conversion of iron dextran to Prussian blue, the second colloid used to catalyze the polymerization of tetramethylbenzidine. The staining results in resident macrophages staining brown while newly recruited macrophages stain blue. The studies have shown that colloidal Prussian blue is stable in vivo and neither loses its catalytic activity nor undergoes extensive redistribution. They also show that the technique can be used to measure Kupffer cell recruitment stimulated by complete Freund's adjuvant in rats and tumor-associated macrophage recruitment in subcutaneous and spontaneous liver metastases in mice.

Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice.

B10.RIII and B10.G mice were transferred from a diet of laboratory rodent chow to a standard diet in which all the fat (5% by weight) was supplied as either fish oil (17% eicosapentaenoic acid [EPA], 12% docosahexaenoic acid [DHA], 0% arachidonic acid [AA], and 2% linoleic acid) or corn oil (0% EPA, 0% DHA, 0% AA, and 65% linoleic acid). The fatty acid composition of the macrophage phospholipids from mice on the chow diet was similar to that of mice on a corn oil diet. Mice fed the fish oil diet for only 1 wk showed substantial increases in macrophage phospholipid levels of the omega-3 fatty acids (of total fatty acid 4% was EPA, 10% docosapentaenoic acid [DPA], and 10% DHA), and decreases in omega-6 fatty acids (12% was AA, 2% docosatetraenoic acid [DTA], and 4% linoleic acid) compared to corn oil-fed mice (0% EPA, 0% DPA, 6% DHA, 20% AA, 9% DTA, and 8% linoleic acid). After 5 wk this difference between the fish oil-fed and corn oil-fed mice was even more pronounced. Further small changes occurred at 5-9 wk. We studied the prostaglandin (PG) and thromboxane (TX) profile of macrophages prepared from mice fed the two diets just before being immunized with collagen. Irrespective of diet, macrophages prepared from female mice and incubated for 24 h had significantly more PG and TX in the medium than similarly prepared macrophages from male mice. The increased percentage of EPA and decreased percentage of AA in the phospholipids of the macrophages prepared from the fish oil-fed mice was reflected in a reduction in the amount of PGE2 and PGI2 in the medium relative to identically incubated macrophages prepared from corn oil-fed mice. When this same fish oil diet was fed to B10.RIII mice for 26 d before immunization with type II collagen, the time of onset of arthritis was increased, and the incidence and severity of arthritis was reduced compared to arthritis induced in corn oil-fed mice. The females, especially those on the fish oil diet, tended to have less arthritis than the males. These alterations in the fatty acid pool available for PG and leukotriene synthesis suggest a pivotal role for the macrophage and PG in the immune and/or inflammatory response to type II collagen.

Quantitative x-ray microanalysis of spleen lysosomes after acid phosphatase reaction.

A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H2PO4)2 is the main reaction product of the histochemical acid phosphatase reaction.

Intramural extravasation of barium simulating carcinoma of the rectum.

This report describes two patients found to have barium granuloma of the rectum. The lesions appeared as indurated, ulcerated rectal masses that resembled carcinoma on endoscopic examination. Deep mucosal biopsy results demonstrated no malignancy and barium sulfate crystals in tissue macrophages. Radiographs showed persistent soft-tissue barium in the rectum. Past reports of barium granuloma have described ulcerated or polypoid masses in the rectum and anus. Rectal intramural extravasation of barium occurs as a result of asymmetric enema balloon inflation and impaction of the enema tip against the rectal mucosa.

Complement synthesis by guinea pig peritoneal macrophages: failure to detect chemical carcinogens.

In vitro production of the second and fourth components of complement (C2 and C4, respectively) by peritoneal macrophages from noninbred Hartley guinea pigs was tested after the animals had been inoculated with known carcinogens. The system demonstrated the capacity of N-nitrosodimethylamine to decrease C2 and C4 production. However, a similar decrease in C2 and C4 production was seen with only CCl4, 1 of the 10 chemical carcinogens studied. This system had little usefulness as a short-term screening procedure for the detection of carcinogenicity. The effect of the carcinogens on several other functions of peritoneal macrophages was also determined. The number of peritoneal exudate cells (PEC) was significantly lower in carcinogen-inoculated animals than in solvent-inoculated controls for three carcinogens: BeSO4, P < 0.005; CHCl3, P < 0.025; and CCl4, P < 0.01. However, the capacity of the PEC to adhere to plastic was decreased by only CHCl3 (P < 0.05), and adherent cells from all guinea pigs produced normal amounts of total secreted protein.

Ion microanalysis of cells.

The application of ion microanalysis (IMA) to the chemical characterization of freeze-fixed, freeze-dried cells is reviewed. Particular emphasis is given to pathological studies involving the determination of the chemical composition of isolated cells (e.g. rabbit alveolar macrophages--RAMs) exposed in vitro to toxic species (e.g. Pb3O4 particles). Ion microscopic results indicated that lead from Pb3O4 migrated into the RAMs and subsequently formed phosphorous-containing compounds. Quantitative comparisons of the relative concentrations of physiologic elements in Pb3O4-treated versus control RAMs also were made using ion microanalytical techniques. The Pb3O4 results illustrate that the three-dimensional analysis capabilities of the IMA may be exploited for the in situ observation of the penetration of xenobiotic agents into cell interiors and their subsequent intracellular chemistry. The potential advantages of ion microanalysis for the characterization of cells include high elemental sensitivity (including low atomic number elements and diffusible ions), broad elemental coverage, three dimensional analysis, and isotopic information. The major limitations include non-idealities of the ion sputtering process, the constraints on the lateral resolution available to identify subcellular features, and the difficulties inherent in the determination of absolute elemental concentrations.

Human colostral cells. I. Separation and characterization.

Analyses of the cells present in human colostrum obtained from fifty-four healthy donors during the first four days of lactation revealed that there were 3.3 x 10(6) (range 1.1 x 10(5)--1.2 x 10(7)) cells per ml of colostrum. Based on histochemical examinations, it was found that this population consisted of 30--47% macrophages, 40--60% polymorphonuclear leucocytes, 5.2--8.9% lymphocytes, and 1.3--2.8% colostral corpuscles; epithelial cells were rarely encountered. The identity of various cell types was confirmed by Wright's stain and by a series of histochemical techniques which disclosed the presence of non-specific esterase, peroxidase, and lipids. For further characterization, the different types of cells were separated by various methods, such as Ficoll-Hypaque density centrifugation, isokinetic centrifugation on a linear Ficoll gradient, adherence to glass or plastic, and phagocytosis of carbonyl iron. Immunohistochemical staining with FITC- and/or TRITC-labelled reagents to IgA, IgM, IgG, K- and lambda-chains, secretory component, lactoferrin, and alpha-lactalbumin were applied to unseparated as well as separated colostral cells. Polymorphonuclear leucocytes (staining for peroxidase) as well as macrophages and colostral corpuscles (staining for non-specific esterase) exhibited numerous intracellular vesicles that contained lipids as well as various combinations of milk proteins. Lymphoid cells did not stain with any of these reagents and plasma cells were not detected among the colostral cells. Individual phagocytic cells contained immunoglobulins of the IgA and IgM classes, both K and lambda light chains, secretory component, lactoferrin, and alpha-lactalbumin. The coincidental appearance of these proteins in single, phagocytic cells but not in lymphoid cells indicate that the cells acquired these proteins by ingestion from the environment. Markers commonly used for the identification of B lymphocytes (surface immunoglobulins) and T lymphocytes (receptors for sheep red blood cells) were unreliable for the analysis of colostral cells (unless accompanied by subsequent morphological characterization) because strong fluorescence was observed on the surface of many non-lymphoid cells and because numerous macrophages and colostral corpuscles formed rosettes with sheep red blood cells (SRBC). Lymphocytes, often found in association with colostral macrophages or corpuscles, were classified as T cells.

Energy dispersive x-ray microanalysis of mitochondrial deposits in sideroblastic anemia.

Energy dispersive x-ray microanalysis was used to analyze mitochondrial and lysosomal iron-containing deposits in sideroblastic anemia. Although it has been previously known that these deposits contain iron by inference from Prussian blue staining, the possible presence of other cations as well as the nature of the anions present has not been identified. The results show that the mitochondrial deposits in erythroid cells have peaks for iron and phosphorus indicating that they do not represent calcifications which commonly occur following injury and that the principal anion may be phosphorus. Studies of hemosiderin and ferritin aggregates in lysosomes of macrophages in the same bone marrow samples again reveal similar peaks for iron and phosphorus. The results also indicate the probable similarity of mitochondrial and macrophage deposists although ferritin itself was never identified in the mitochondrial deposits. The results illustrate the potential of this method for diagnostic and investigative pathology.

X-ray microanalytical studies of lead-implanted rat brains.

Various cytoplasmic and intranuclear inclusions found in macrophages and astrocytes of lead-implanted rat brains were studied with an energy dispersive x-ray microanalytical technique. Cytoplasmic inclusions contained large quantities of lead, calcium and phosphorus. The proportions of these elements were different within each inclusion. Intranuclear inclusions also contained small amounts of lead and, occasionally, calcium.