Mitotic activity in non-neoplastic melanocytes in vivo as determined by histochemical, autoradiographic, and electron microscope studies.

Mitotic figures were demonstrated in the differentiated melanocytes of normal epidermal and nonepidermal tissues without the presence of external stimuli. These dividine melanocytes were present in human and mouse skin, mouse hair, chick feathers, and embryonic chick retinal pigment epithelium. In normal adult human epidermis, dividing melanocytes, though rare, were found in the nonstimulated areas. L-3,4-dihydroxyphenylalanine reaction on the melanocytes during mitosis demonstrated activity of the melanin-forming enzyme, tyrosinase, and ultrastructural studies demonstrated the characteristic melanosomes in variour stages of maturation. Other ultrastructural characteristics of the melanocytes during mitosis, except for the Golgi apparatus, which was smaller and less complex, were similar to those seen in well-differentiated nondividing melanocytes. Autoradiographic studies of thymidine incorporation into mouse skin indicated that 0.7% of epidermal melanocytes, when slightly stimulated, are in the S phase. Thus, in vivo differentiation of non-neoplastic melanocytes (to produce pyrosinase and melanosomes) does not preclude their replication by mitotic division.

Analysis of parathyroid hormone and its fragments in rat tissues: chemical identification and microscopical localization.

After intravenous injection of [(125)I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of (125)I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the (125)I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of (125)I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.

Interaction between the insulin-like growth factor family and the integrin receptor family in tissue repair processes. Evidence in a rabbit ear dermal ulcer model.

We have determined previously that IGF-I is dependent on the presence of IGF binding protein-1 (IGFBP-1) to act as a wound healing agent. We sought to determine the mechanism whereby IGFBP-1 is able to enhance IGF-I bioactivity. As IGFBP-1 binds both the alpha5beta1 integrin as well as IGF-I in vitro, we asked which of the following interactions were important: (a) the ability of IGFBP-1 to interact with an integrin receptor, and/or (b) the binding of IGF-I by IGFBP-1. We used an IGF-1 analogue (des(1-3)IGF-I) with a > 100-fold reduction in affinity for IGFBP-1 as well as an IGFBP-1 mutant (WGD-IGFBP-1) which does not associate with the alpha5beta1 integrin to selectively abrogate each of these interactions. We also tested the ability of IGFBP-2, a related binding protein which has an arginine-glycine-aspartate sequence but does not associate with integrin family members, to enhance IGF-I bioactivity. Full-thickness dermal wounds were created on rabbit ears; various combinations of native IGF-I, native IGFBP-1, native IGFBP-2, and their respective analogues/mutants were applied to each wound. Wounds were harvested 7 d later for analysis. Only native IGF-I in combination with native IGFBP-1 was effective as a wound healing agent, enhancing reepithelialization and granulation tissue deposition by 64+/-5 and 83+/-12% over controls (P = 0.008 and 0.016, respectively). The same doses of IGF-I/WGD-IGFBP-1, des(1-3)IGF-I/IGFBP-1, and IGF-I/IGFBP-2 were ineffective. We propose that IGF-I physically interacts with IGFBP-1 and that IGFBP-1 also binds to an integrin receptor, most likely the alpha5beta1 integrin. This interaction is unique to IGFBP-1 as the closely related IGFBP-2 had no effect, a finding consistent with its inability to bind to integrin receptors. Our results suggest that activation of both the IGF-I receptor and the alpha5beta1 integrin is required for IGF-I to stimulate wound healing.

Osseous changes and osteosacomas in mice continuously fed diets containing diethylstilbestrol or 17 beta-estradiol.

In a study on the long-term effects of dietary diethylstilbestrol or 17 beta-estradiol on C3H mice, estrogens induced a proliferation of osseous trabeculae and increased the incidence and hastened the development of osteofibrotic areas in the sterna. There were 6 osteosarcomas, 2 having metastases, in 1,242 mice fed dietary estrogens over 360 days, but none in 356 untreated controls. These tumors were reviewed along with 4 early sternal osteosarcomas selected from 17 osteosarcomas (only 1 in a control) found thus in two other ongoing comparable studies. In at least 1 case, and possibly in 2 other early cases, tumors were associated with areas of osteofibrosis, and 1 tumor was probably associated with proliferation of bony trabeculae in the medullary cavity.

A dominant negative CREB (cAMP response element-binding protein) isoform inhibits thyrocyte growth, thyroid-specific gene expression, differentiation, and function.

cAMP mediates the effects of TSH by regulating thyroid follicular cell proliferation, differentiation, and function. To assess the functional importance of the cAMP response element binding protein (CREB) in thyroid follicular cell regulation in vivo, we targeted the expression of a dominant negative (DN) CREB isoform to the thyroid glands of transgenic mice using a tissue-specific promoter. Transgenic mice exhibited severe growth retardation and primary hypothyroidism. Serum levels of TSH were elevated 8-fold above normal levels, and T4 and T3 levels were low. Histologically, the mutant thyroid glands were characterized by poorly developed follicles that were heterogeneous in size with diminished colloid. Ciliated thyroid epithelial cells were observed in the transgenic thyroid glands, suggesting a failure of follicular cell differentiation. Consistent with this hypothesis, the DN CREB transgene inhibited the expression of an array of genes including thyroglobulin, thyroperoxidase, and the TSH receptor in semiquantitative RT-PCR experiments. Altered expression of the thyroid transcription factors Pax-8, TTF-1, and TTF-2 was also observed. These results demonstrate a critical role for CREB in thyroid growth, differentiation, and function in vivo.

Congenital muscle fiber-type disproportion in Krabbe's leukodystrophy.

Progressive spasticity, blindness, loss of skills, and neuropathy developed in a 4.5-month-old boy. When examined at 13 months, galactocerebrosidase and galactosylceramide-beta-galactosidase activities were deficient in leukocytes. Intramuscular nerves and a sural nerve biopsy specimen showed loss of nerve fibers, interstitial fibrosis, and axonal degeneration, rather than the segmental demyelination that predominates in most cases. A muscle biopsy specimen showed congenital muscle fiber-type disproportion (CMFTD). This case confirms a previous report of CMFTD in Krabbe's disease and supports a neurogenic mechanism as the basis for CMFTD.

Lipid storage myopathy in infantile Pompe's disease.

An infant died at 8 months of age with a history of developmental regression, hypotonia, severe weakness, cardiomegaly, congestive heart failure, and hepatomegaly. A diagnosis of Pompe's disease (glycogenosis type II) was established by muscle biopsy at 5 months of age. Vacuolar myopathy involved muscle fibers of histochemical type I more than type II. Many vacuoles were filled with glycogen. In addition, increased amounts of neutral lipid were demonstrated by oil red O stain, electron microscopy, and quantitative analysis. Acid alpha-1,4-glucosidase activity was demonstrated to be deficient. Biochemical studies failed to determine the cause of the lipid accumulation, but demonstrated a low total concentration of carnitine in the muscle (6.37 nmole/mg of protein), associated with elevated activities of carnitine palmityl-transferase and palmityl-coenzyme A dehydrogenase. Palmityl-coenzyme A synthetase activity was in the normal range.

Pneumocystis carinii choroiditis in patients with AIDS: clinical features, response to therapy, and outcome.

To further characterize the clinical features, response to therapy, and outcome of Pneumocystis carinii choroiditis in patients with AIDS, we retrospectively reviewed the course of choroiditis for eight patients identified from two institutions through April 1991. Seven patients had prior Pneumocystis carinii pneumonia and had received aerosolized pentamidine prophylaxis for a median of 10 months; one patient had no prior history of pneumonia or prophylaxis. The median CD4+ lymphocyte count for six patients was 11 cells/mm3. Choroiditis was a preterminal diagnosis for three patients--two with associated disseminated pneumocystosis. Ocular manifestations improved or resolved with therapy for five of the six treated patients. All five subsequently received prophylaxis with dapsone (n = 2), dapsone/trimethoprim (n = 2), or aerosolized pentamidine (n = 1). Choroiditis recurred at 15 months in the one patient receiving aerosolized pentamidine. The median survival from time of diagnosis was 44 weeks. A literature review including an additional 40 cases support the conclusions that (a) Pneumocystis choroiditis is a rare complication of advanced HIV disease, occurring often in the context of systemic pneumocystosis; (b) ocular signs and symptoms may improve or resolve with specific antipneumocystis therapy; and (c) relapse may occur, particularly in those not receiving systemic prophylaxis.

The ultracytochemical localization of cytidine-5'-monophosphatase in normal human thyroid follicle cells.

The properties and distribution of an enzyme specifically hydrolyzing cytidine-5'-monophosphate and the possible relationship of the enzyme to the synthesis and secretion of thyroid hormone in man were investigated using cytochemical methods. Activity due to cytidine-5'-monophosphatase was separated from that due to acid or alkaline phosphatase, both of which are also capable of hydrolyzing cytidine-5'-monophosphate. This distinction was established on the basis of manganese ion stimulation and differences in localization, levels of activity, and pH optimums. The localization of the enzyme along the face of Golgi apparatus involved in the formation of thyroglobulin suggests an association of the enzyme with the glycosylation of thyroglobulin.

Pathogenesis of experimental lipid keratopathy: corneal and plasma lipids.

Corneal and plasma lipids were studied in a rabbit model to gain insight into the pathogenesis of secondary lipid keratopathy. Rabbits were divided into four groups in which a high cholesterol diet and corneal suture placement were varied to produce lipid keratopathy. In rabbits with lipid keratopathy, quantitative thin layer chromatography revealed that cholesterol esters comprised most of the deposited lipid, with free cholesterol being deposited as well. The ratio of accumulated cholesterol ester to free cholesterol corresponded closely to the same ratio in hypercholesterolemic plasma total low and very low density lipoprotein (TLDL). Furthermore, gas chromatography showed that the cholesterol ester composition in the corneas with lipid keratopathy resembled that seen in hypercholesterolemic plasma TLDL but was different from the pattern observed in the normal cornea. These studies suggest that the direct source of the deposited cholesterol ester is primarily the plasma TLDL. Since phospholipids and triglycerides did not show a significant increase in the experimental corneas, they are presumably metabolized by the keratocytes after the uptake of TLDL. However, the amount of cholesterol ester carried by the lipoprotein exceeds the capacity of the cell for use and excretion and the lipid accumulates in the cornea.

The effect of platelet-activating factor (PAF), histamine, and ethanol on vascular permeability of the guinea pig conjunctiva.

Increased vascular permeability, one of the characteristic features of immediate hypersensitivity (Type I), is mediated through a variety of compounds, including histamine and platelet-activating factor (PAF), a phospholipid inflammatory mediator. The effects on vascular permeability of histamine, PAF, and ethanol, the solvent for PAF, were compared in the guinea pig conjunctiva. Permeability at 30 min was investigated by evaluation of conjunctival edema and Evans blue extravasation (clinically estimated and colorimetrically measured). Doses of PAF from 1 to 10 nmol produced an increase in vascular permeability, with a peak effect at 10 nmol. Ethanol had no effect on vascular permeability below 40 X 10(3) nmol; above this concentration, however, permeability increased, reaching a maximum at 175 X 10(3) nmol. At low doses of PAF and ethanol, the effects were additive, whereas at 20-80 nmol of PAF with high concentrations of ethanol there was no additive effect of PAF, producing a decrease in the net effect of PAF. Histamine increased vascular permeability, with a minimum effect at 10 nmol and a maximum effect at 450 nmol. The slopes of the dose-response curves for all three compounds were linear and parallel, with statistically different potencies. The potencies for each compound were identical by all three methods of evaluation. Therefore, we conclude that PAF is a potential mediator in hypersensitivity reaction in the guinea pig conjunctiva, and that its effect is similar to but much more potent than that of histamine or ethanol. Since ethanol alone has a significant effect on vascular permeability, studies on PAF effects using control solutions without ethanol may be difficult to interpret.

Pathogenesis of experimental lipid keratopathy. An ultrastructural study of an animal model system.

The histology and ultrastructure of experimental lipid keratopathy were studied in hypercholesterolemic rabbits in which the insertion of corneal sutures induced vascularization and subsequent lipid deposition in the anterior stroma. Lipid accumulated in the keratocytes, the pericytes and occasionally in the endothelial cells of the capillaries. The lipid-laden keratocytes were concentrated in the region of the capillaries. No lipid was seen in the control rabbits. In the hypercholesterolemic rabbit with sutures, intracellular lipid in the keratocytes was present largely in nonmembrane-limited droplets with smaller amounts of membrane-limited cholesterol crystals and rare numbers of myelin figures. In addition, large, lipid-engorged spherical cells were present. The numerous phagolysosomes seen ultrastructurally suggest that some of these cells probably represent macrophages. Keratocytes and the large, spherical lipid-engorged cells show focal degenerative changes, including pyknotic nuclei, cytoplasmic coagulation and membrane loss, leaving extracellular mixed accumulations of lipid and cytoplasmic organelles. Small numbers of lymphocytes and plasmacytoid cells were present. No corneal lipid was seen in animals with normocholesterolemia, with or without sutures. In hypercholesterolemic animals, a few lipid-laden keratocytes without macrophages were identified even in the absence of vessels. These morphologic studies support the hypothesis that the accumulation of the corneal lipid in this animal model of lipid keratopathy is the result of increased lysosomal uptake of lipid, probably as low density lipoprotein, from the extracellular space by the keratocytes. The rate of metabolism of this lipid is insufficient to clear the cells of the lipid and the subsequent lipid inspissation results in keratocyte death, leading to macrophage accumulation of lipid and free lipid in the stroma.

Endothelial viral inclusions in Fuchs' corneal dystrophy.

A case of Fuchs' corneal dystrophy is presented. The patient, a 70-year-old white woman, had bilateral decreasing vision, especially while reading and driving at night. Clinical features were characteristic of Fuchs' corneal dystrophy. Ultrastructural studies demonstrated findings in Descemet's membrane typical of those previously reported with numerous tactoids of fibrous long-spacing collagen in the posterior collagenous layer and in the guttata. In the endothelial cells were encapsulated ellipsoid viral particles, 400 nm long and 225 nm in diameter. They contained an outer and inner membrane with an electron-dense intervening region and a central dense core. Nucleocapsids were present in the endothelial cells and stromal keratocytes. The authors suggest that the pathogenesis of Fuchs' corneal dystrophy may be endothelial damage, and that in this case, the etiology is a viral infection.

Parathyromatosis in hyperparathyroidism.

Recurrent hyperparathyroidism after parathyroidectomy may present a difficult diagnostic problem. A rare etiology is parathyromatosis (multiple nodules of hyperfunctioning parathyroid tissue scattered through the neck and mediastinum) due to spillage of otherwise benign parathyroid tissue during surgery. We present a case of recurrent hyperparathyroidism and parathyromatosis due to tissue spillage during surgical removal of probable double adenomas, a rare cause of primary hyperparathyroidism. Thus, parathyromatosis must be included in the differential diagnosis of recurrent or persistent hyperparathyroidism, distinguished from parathyroid carcinoma by histologic criteria. The surgeon must be careful of parathyroid spillage during surgery, even of benign tumors of the parathyroids.

Typhlitis complicating autologous blood stem cell transplantation for breast cancer.

Three cases of typhlitis occurring during autologous blood stem cell transplantation (ABSCT) for metastatic breast cancer are described. Typhlitis is a rare complication of neutropenia and has uncommonly been reported in the autologous transplant setting. Although it has been most commonly described in children with leukemia, typhlitis has increasingly been reported in adult leukemias and in association with neutropenia secondary to chemotherapy for a number of solid tumors. Only five previous cases of typhlitis in the setting of ABSCT have been described. Whereas diarrhea and fever are common toxicities associated with high-dose chemotherapy, it is likely that many cases of typhlitis go unrecognized. Bone Marrow Transplantation (2000) 25, 321-326.

Desquamating endotheliopathy. An incipient iridocorneal endothelial syndrome?

Unilateral, noninfectious, nontraumatic corneal endotheliopathy was noted in a 34-year-old man who had had blurred vision for five years without evidence of iridic disease or glaucoma. Ultrastructural studies demonstrated focal necrosis of the corneal endothelial cells, with desquamation of the cells into the anterior chamber. The corneal endothelium appeared to expand beneath the dying endothelial cells, indicating reendothelialization of the cornea. There was no epithelialization of the endothelium, as evidenced by the lack of keratin production or desmosome formation. Descemet's membrane was thickened with edema, a posterior collagenous layer, and fibrous, long-spacing collagen. These alterations in Descemet's membrane were similar to those described for other corneal dystrophies. It is proposed that this unilateral desquamating endotheliopathy represents an incipient form or a forme fruste of the iridocorneal endothelial syndrome.

Lattice corneal dystrophy type IIIA. Clinical and histopathologic correlations.

All three types of lattice corneal dystrophy are inherited and localized, and they largely involve linear corneal amyloid deposits. We encountered two white families with lattice corneal dystrophy which closely resembled type III. Four generations of one family and three of another family exhibited lattice corneal dystrophy. Because both families are from Caccamo, Sicily, Italy, we believe it is likely that both are from a single mutation. Thick, ropy lattice lines were seen to traverse the corneas almost from limbus to limbus and were easily detected with direct illumination. Histopathologic examination revealed accumulations of varying sized amyloid deposits in the stroma and ribbons of amyloid between the stroma and Bowman's layer typical of lattice corneal dystrophy type III. We have named the disease in this family lattice corneal dystrophy type IIIA, because of three differences from lattice corneal dystrophy type III: the presence of corneal erosions, the occurrence in whites, and the autosomal dominant inheritance pattern.

Subepithelial mucinous corneal dystrophy. Clinical and pathological correlations.

We describe a family with an unusual autosomal dominant anterior corneal dystrophy. The onset was characterized by frequent, recurrent corneal erosions in the first decade. This subsided during adolescence and was followed by progressive decreased vision. Slit-lamp examination revealed bilateral subepithelial opacities and haze, involving the entire cornea, but most dense centrally. Histopathological study revealed a subepithelial band of eosinophilic, periodic acid-Schiff-positive, alcian blue-positive, hyaluronidase-sensitive material anterior to Bowman's layer. Electron microscopy demonstrated subepithelial deposition of fine fibrillar material consistent with glycosaminoglycan. Immunohistochemical analysis indicated that the accumulated material contained a combination of chondroitin 4-sulfate and dermatan sulfate. This unique condition clinically resembled Grayson-Wilbrandt dystrophy, but differed histochemically. To our knowledge, this anterior corneal dystrophy has not previously been reported, and it is best described by the name "subepithelial mucinous corneal dystrophy."

Structural evaluation of porcine heart valve prostheses with radiofrequency ultrasound.

BACKGROUND: Bioprosthetic heart valve use is limited by progressive degeneration. Early degenerative changes are often occult, making assessment of tissue integrity difficult. Ultrasound tissue characterization may detect alterations in tissue structure and allow early detection of leaflet degeneration. METHODS: Using a modified echocardiographic unit (Acuson), radiofrequency (RF) integrated backscatter amplitude (IBA) (integral/RF/dt) was measured in 38 leaflets from nine explanted and six control porcine valves. Regions of interest in each leaflet were studied using four ultrasound frequencies. Radiographic gray scale mean and leaflet thickness were measured at each region of interest. Percent collagen and mineral were calculated for each region of interest using color-image processing of histologic sections and compared to IBA. RESULTS: IBA values for control vs. explanted leaflets were (mean value+/-standard deviation): 8.2+/-4.69 dB vs. -4.7+/-4.64 dB at 7.0 MHz; -5.8+/-4.34 dB vs. -3.1+/-5.34 dB at 5.0 MHz; -3.8+/-3.38 dB vs. -2.1+/-3.18 dB at 3.5 MHz; and -9.0+/-4.58 dB vs. -7.1+/-4.25 dB at 2.5 MHz. Collagen content was 27.7+/-8.50% vs. 33.2+/-10.90%, mineral content was 0.1+/-0.10% vs. 2.1+/-4.30%, and radiographic gray scale mean was 150.6+/-1.96 vs. 145.3+/-5.14 for control vs. explanted leaflets, respectively. For control and explanted leaflets IBA, collagen content, mineral content, and radiographic gray scale mean were different (control vs. explanted P<0.05). Leaflet thickness was also noted to be different between the two groups. IBA was different among explanted leaflets with low, medium, and high mineral content. CONCLUSION: IBA was found to be a useful technique to differentiate normal from explanted porcine prosthetic valves in vitro. This method may be useful in the serial assessment of bioprosthetic leaflet degenerative properties in vivo.

Intracellular lipid droplets in functioning transitional parathyroid oxyphil adenomas. A caveat.

Histochemical demonstration of intracellular lipid droplets on frozen section has been used to distinguish normal parathyroid tissue from that of adenoma and chief cell hyperplasia. Differentiation is based on the observation that the cells of adenoma and chief-cell hyperplasia largely lack intracellular lipid, which is present in the suppressed chief cells of normal glands in patients with adenoma. We present two functional transitional oxyphil adenomas that contained abundant intracellular lipid. Failure to recognize that transitional oxyphilic adenomas may contain focal accumulation of intracellular lipid droplets could lead to confusion in histologic interpretation.