Light and electron microscopic analysis of sequential liver biopsy samples from rheumatoid arthritis patients receiving long-term methotrexate therapy. Followup over long treatment intervals and correlation with clinical and laboratory variables.

OBJECTIVE: To describe liver histopathologic features and ultrastructural changes in a prospectively studied cohort of rheumatoid arthritis (RA) patients receiving long-term methotrexate (MTX) therapy, and to seek correlations between these changes and simultaneously measured laboratory indices of liver function. METHODS: This was a long-term, prospective, open observational study. Twenty-seven outpatients with RA who began therapy with MTX and continued treatment for extended periods underwent baseline and followup liver biopsies. One hundred seventy liver biopsy specimens were analyzed by light microscopy (LM) and assessed according to a modified Roenigk score and a newly devised numerical grading system. Ninety-three biopsy specimens were also analyzed by electron microscopy (EM). Blood samples were obtained at 4-6-week intervals for determination of bilirubin, alkaline phosphatase, aspartate aminotransferase (AST), and albumin levels, and the weekly dosage of MTX was adjusted if there were abnormalities in the AST or albumin level. A mean of 6.3 liver biopsies per patient were obtained over a mean followup period of 8.2 years (range 2-13 years). RESULTS: The modified Roenigk score was significantly different from baseline at year 3, when it increased from a mean of 1.8 to 2.3 (P = 0.05) and at year 6, when it increased to 2.4 (P = 0.04), but this was not considered clinically meaningful. No other significant changes from baseline were observed by either LM grading system. No significant progression was observed by EM over the course of the investigation. Increases in serial measurements of AST correlated with both the modified Roenigk score (r = 0.21, P = 0.016) and the numerical rating score (r = 0.19, P = 0.027). CONCLUSION: Patients with RA who are receiving weekly single-dose oral MTX therapy exhibit little deterioration in hepatic architecture by LM or EM when the dosage of the drug is adjusted for abnormalities in AST and serum albumin, monitored at frequent intervals.

Cadmium toxicity to the cornea of pregnant rats: electron microscopy and x-ray microanalysis.

Cadmium toxicity to the cornea of pregnant rats was studied using the electron microscope and x-ray microanalyzer. In in-vivo experiments, severe corneal edema occurred in pregnant dams that received intraperitoneal injections of cadmium sulphate for 4 days during gestation, but not in nonpregnant rats. Prominent swelling of mitochondria and the occurrence of intra- and intercellular vacuoles in the corneal endothelium were observed only in pregnant dams. In in-vitro experiments, electron-dense deposits consisting of cadmium-oxine complexes were preferentially found in swollen mitochondria of the endothelial cells. Cadmium peaks were obtained from these deposits with x-ray microanalysis. These data suggest that the corneal edema observed after administration of cadmium may imply the disturbance of pump function and barrier function of the corneal endothelium due to the primary toxic effects of this metal on mitochondria.

Comparison of osmium/sonication and EDTA/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon.

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique had a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.

Electron microscopic analysis of sequential liver biopsy samples from patients with rheumatoid arthritis. Correlation with light microscopic findings.

We used electron microscopy (EM) to analyze 52 biopsy samples from 22 patients who were receiving long-term weekly oral doses of methotrexate (MTX) for the treatment of rheumatoid arthritis. Forty-eight biopsy samples were obtained after 2-6 years of continuous treatment, and 4 samples were obtained before treatment was begun. Specimens were graded for neutral fat, secondary and tertiary lysosomes, and smooth endoplasmic reticulum (SER) in hepatocytes, and for collagen in the perisinusoidal space (Disse's space). We examined the correlations between the EM findings and the light microscopic (LM) findings in the same biopsy specimens, and between the EM findings and the results of simultaneous monthly measures of aspartate transaminase, alkaline phosphatase, bilirubin, and albumin levels, as well as history of alcohol consumption before MTX treatment and monthly assessments of clinical status during the course of treatment. The presence of collagen was minimally increased in these sequential biopsy samples, whereas fat, lysosomes, and SER were decreased. The SER decrease was statistically significant. EM findings of collagen in the space of Disse did not correlate with early fibrotic changes observed with LM. Thus, after as long as 6 years of weekly oral treatment with MTX, hepatic ultrastructural changes are minimal and are not clinically significant. The use of EM for sequential biopsy studies allows the quantitation of long-term hepatic changes that may be more limited than the impression gained after LM analysis.

Effects on mouse embryos of in utero exposure to saccharin: teratogenic and chromosome effects.

For teratogenesis studies, pregnant ICR albino mice were administered saccharin by three routes and at three different doses by each route as follows: Intraperitoneal injection of 500, 1,000, or 2,000 mg/kg saccharin on day 10 of gestation; intragastric tube delivery of 5, 10, or 25 mg/kg/day of saccharin on days 5-15 of gestation; and as drinking water containing a 5, 10, or 20% solution of saccharin from day 0 through day 17. Appropriate controls were used for each set. No increase in either resorptions or malformations was found in mouse embryos whose dams had received saccharin by any of the three routes. For chromosome studies, saccharin was administered IP to pregnant ICR albino mice on day 10 of gestation as either a 1,000 mg/kg or 2,000 mg/kg dose. To demonstrate sister chromatid exchanges (SCE), bromodeoxyuridine was administered as 16 consecutive half-hourly doses of 25 mg/kg during the 8 h prior to saccharin injection. In addition, one group received two doses of BrdU and 2,000 mg/kg saccharin 8 h apart to demonstrate SCE frequency following exposure to saccharin for two cell cycles. The mice were given colchicine (4 mg/kg) 6 h after the final injection and killed 2 h later. Embryonic cell suspensions and metaphase spreads were prepared by routine methods. Metaphase spreads were examined for breaks or gaps (after Giemsa staining), for G-banding (using the ASG technique), for C-banding (using Giemsa staining after exposure to 0.07 N NaOH and incubation in 2 X SSC at 60 degrees C), and for SCE by the Hoechst-Giemsa method. Fifty metaphase spreads were counted for each experimental condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences between two fractions of glycosaminoglycans of the corneal stroma in their structural relation to collagen.

Glycosaminoglycans (GAG) of bovine cornea were sequentially extracted by 0.15 M NaCl and by 1 M CaCl2, pH 8. The amounts of hexosamine (HexN) and hexuronic acid (HexUA), specific hexoses (Hex), and protein were determined in the extracts. The ultrastructure of the corneal stroma after NaCl and NaCl/CaCl2 extraction was also studied. Approximately 70% of the Hex-HexN extractable by 0.15 M NaCl is removed in the first NaCl extract, with the amount decreasing rapidly to the fifth NaCl extract. Only 20-30% of the extractable HexUA-HexN is removed in the first NaCl extract; subsequent extraction removes successively less HexUA-HexN. There is a sharp increase, however, in both Hex-HexN and HexUA-HexN removed in the first 1 M CaCl2 extract, ranging from 25 to 40% of the total extractable with NaCl. The stroma retained lamellar organization and normal spacing, diameter, and length of collagen fibers after NaCl extraction. Visibility of the 64-nm periodicity was enhanced because of loss of density in the ground substance. CaCl2 extraction led to loss of lamellar organization and apparent disruption of the collagen fibers. Only short thin fibrils remained, embedded in a material having the density and very fine fibrillar organization of basal lamina. Disruption of the collagen fibers is probably due to removal of a specific GAG component necessary to maintain the collagen as an extended morphologic structure but may also be due to a specific degradative effect of CaCl2 on collagen no longer covered by a protective layer of GAG and glycoproteins.

Studies on the cornea. X. Incorporation of 14C-glucosamine into glycosaminoglycans and glycoproteins of elasmobranch corneas, extractability of these components and morphological effects of such extractions.

Incorporation of 14C-glucosamine into dogfish (Squalus acanthias) corneas occurs primarily in the glycosaminoglycans (GAG) and glycoproteins (GP) extractable with elasmobranch Ringer's solution but the rate at which this component can be extracted varies inversely with the age (size) of the fish. Whereas this fraction represents only one-fourth of the total hexosamine (Hex-N) it contains nearly three-fourths of the newly incorporated 14C-glucosamine. The CaCl2-extractable HexN contains only 13% of the incorporated radioactivity. Collagen-associated GAG in both soluble and insoluble collagen fractions contain keratan sulfate, dermatan sulfate and chondroitin-4-sulfate, with the GAG associated with the salt-insoluble collagen having only half the radioactivity of those associated with the soluble fraction. However, the specific activities of both the salt-insoluble and the salt-soluble fractions were very low, suggesting that the GAG-GP which are tightly bound to collagen may have a high degree of stability. Partially de-epithelialized corneas incorporated 14C-glucosamine at rates ranging from 22% to 58% of the efficiency of intact corneas, suggesting a possibly significant role of the epithelium in the biosynthesis of GAG-GP in the adult dogfish. Morphologic results exactly paralleled those previously reported by us for the bovine cornea with the exception that the effects of CaCl2 extraction of GAG-GP were not evident on the collagen fibers until the second to fifth sequential CaCl2 extraction. The morphologic findings, therefore, exactly parallel the analytic findings which show that a significant portion of the collagen and collagen-associated GAG-GP of the dogfish cornea remains after the double extraction. How much of this remaining material represents sutural fibers and their associated GAG-GP is yet to be determined.

Sister chromatid exchange frequency in mouse embryo chromosomes after in utero ethanol exposure.

The frequency of sister chromatid exchange (SCE) increased in mouse embryo chromosomes following a single maternal i.p. injection of ethanol on the tenth day of gestation, indicating a possible morphogenetic action of ethanol in the developing mouse embryo.

Detection of lateral asymmetry in the C-bands of mouse-embryo chromosomes exposed in utero to mitomycin C.

Chromosomes from 10-day mouse embryos were C-banded and examined for aymmetric bands between sister chromatids. No occurrences of lateral asymmetry (LA) were observed in this series. However, when 10-day embryos were exposed in utero to a single maternal injection of 10 mg/kg mitomycin C 4--5 h prior to metaphase arrest with colcemid, a significant level of asymmetric C-banding was demonstrated in their chromosomes. As many as 4 LAs per cell were seen in 2 of the 50 metaphase spreads examined.

Amyloid formation in insulinoma.

Local amyloid depositions were investigated with electron microscopy in a functioning beta cell adenoma of the pancreas. Beta granule-containing neoplastic cells adjoining small amyloid depositions were shown to have cellular invaginations containing radiating amyloid bundles, indicating the neoplastic cells were involved in the formation of amyloid. Seen in the larger mayloid depositions were attenuated, thin, cellular processes of the neoplastic cells, separating the amyloid stroma into globules. The globular separations of the amyloid correlated well with the light-microscopic globular appearance of amyloid stroma. Possible mechanisms are discussed for the amyloid deposition of insulinoma with relation to amyloidogenesis of other types of amyloidosis.

Ultrastructural evidence of myofibroblasts in circumscribed fibromatosis.

The myofibroblast, which exhibits ultrastructural features of both smooth muscle cells and fibroblasts, is the predominant cell type in the granulation tissue of healing wounds. Similar cells were identified in an ultrastructural study of a case of circumscribed fibromatosis. Myofibroblasts have been described in several apparently unrelated conditions, suggesting that the acquisition of smooth muscle-like features is one of a limited number of ways in which fibroblasts may react to abnormal stimuli.

Primary malignant melanocytic tumors of the sympathetic ganglia, with an ultrastructural study of one.

Three cases of melanocytic tumors arising in the sympathetic chain were studied. A metastasis from one of them was studied with electron microscopy. Both light and electron microscopy demonstrated the presence in these tumors of true melanogenesis and autophagic activity in melanocytes as well as phagocytosis of mature melanin by associated histiocytes. Review of the embryology, biochemistry and in vitro culture of sympathetic nervous tissue showed a close relationship between it and pigmented cells in other parts of the body which are also derived from neural crest. These findings support the clinical and morphological conclusion that these rare pigmented tumors are true melanocytic tumors originating from the sympathetic chain. The malignant potential of these tumors is emphasized by the fact that the two patients who survived initial surgery later died of metastatic disease.

Nodular regenerative hyperplasia of the liver.

Nodular regenerative hyperplasia of the liver, an uncommonly reported and poorly defined clinicopathological entity, obscured clinical diagnosis and was misdiagnosed on hepatic biopsy in a recent case. Approximately 19 cases are recorded in the English literature. Six patients had Felty's syndrome, about 12 patients had congestive heart failure, and the patient under discussion had subacute bacterial endocarditis. Light- and electron-microscopic examination was utilized to define nodular regenerative hyperplasia pathologically. Features common to all reported cases are discussed but elucidation of the pathogenesis of nodular regenerative hyperplasia must await further investigation.

Comparative electron-microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium. II. Variations in surface architecture found by scanning electron microscopy.

We have used the high resolution and the great depth of focus possible with the scanning electron microscope to define the surface features of normal human colon and human colonic polyps. This technique demonstrates that the surface of the normal colon has a territorialization which encompasses multiple crypts. The surface of the normal colon is covered with both absorptive cells and goblet cells. Hyperplastic polyps are covered with overdeveloped absorptive cells and the normal teritorialization is present but is distorted. Adenomatous polyps are covered with immature cells and, except for the villous adenomas, goblet cells are strikingly absent from the surface. The territorial patterns are totally obliterated in the adenomas.

Malignant soft tissue tumors of probable histiocytic origin (malignant fibrous histiocytomas): general considerations and electron microscopic and tissue culture studies.

Correlated light and electron microscopic study of four fibrous histiocytomas of proven malignancy has confirmed the presence of histiocyte-like and fibroblast-like cells in this tumor. In addition, an undifferentiated cell type, giant cells, xanthomatous cells, and rare cells with morphological characteristics intermediate between those of histiocytes and fibroblasts were seen. "Nuclear body" type inclusions were commonly present in both principal cell types in all four cases, as were the somewhat less common cytoplasmic inclusion bodies. Electron microscopic study of a tissue culture of one of these tumors demonstrated only fibroblast-like, histiocyte-like, and xanthomatous cells. The possibility is suggested that both principal cell types in this tumor may derive from the same undifferentiated stem cell.