Induction and prevention of chondrocyte hypertrophy in culture.

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.

Isolation and characterization of rough endoplasmic reticulum associated with mitochondria from normal rat liver.

A subfraction of rough endoplasmic reticulum (RER) characterized by its close association with mitochondria (MITO) was isolated from low speed pellets of normal rat liver homogenate under defined ionic conditions. This fraction enriched in MITO-RER complexes contained 20% of cellular RNA, 20% of glucose-6-phosphatase and 47% of cytochrome c oxidase activities. Morphologically, the isolated MITO-RER complexes closely resembled physiological associations between the two organelles commonly seen in intact liver. Partial dissociation of RER from mitochondria of the MITO-RER fraction was achieved by either EDTA (0.5 mM) or by hypotonic/hypertonic treatment of MITO-RER complexes. With the latter procedure approx. 70% of RER (RERmito) with 50% of ribosomes still attached could be separated from the inner compartments of mitochondria. This RERmoto exhibited a higher glucose-6-phosphatase activity than RER isolated as rough microsomes from the postmitochondrial supernatant. Isopycnic centrifugation on linear metrizamide gradients revealed that the mitochondria-associated part of RER corresponds to the high density, ribosome-rich subfraction of rough microsomes isolated in cation-free sucrose solution. The combined data demonstrate that a morphologically and biochemically distinct portion of RER is associated with mitochondria and support the concept of considerable intracellular heterogeneities in distribution of enzymes and enzyme systems along the lateral plane of the endoplasmic reticulum membrane system.

Enteric infections and diarrhea in human immunodeficiency virus-infected persons: prospective community-based cohort study. Swiss HIV Cohort Study.

BACKGROUND: Persons infected with human immunodeficiency virus (HIV) are at increased risk for diarrhea and enteric infections. We studied (1) the epidemiology of enteric pathogens associated with diarrhea, (2) the diagnostic yield of stool examination and endoscopic evaluation, (3) risks to develop diarrhea, and (4) the impact of diarrhea on patients' survival. METHODS: A total of 1933 participants in the Swiss HIV Cohort Study were prospectively followed up for a median of 25.5 months. A total of 560 diarrheal episodes were evaluated by standardized stool examination. Endoscopic evaluation was performed in 25% of patients with chronic diarrhea. RESULTS: The incidence of diarrhea was 14.2 per 100 person-years (95% confidence interval, 13.0-15.4). Among patients with CD4 cell counts below 0.05 x 10(9)/L, the probability to develop diarrhea within 1, 2, and 3 years was 48.5%, 74.3%, and 95.6%, respectively. The risk to develop diarrhea was increased among patients with severe immunodeficiency, homosexual men, and patients taking antiretroviral therapy. Pneumocystis carinii chemoprophylaxis did not reduce the risk of diarrhea. Diarrhea was an independent negative predictor of survival. Enteric pathogens were detected in 16.5% of 212 acute diarrheal episodes and in 46% of 348 chronic diarrheal episodes. The sensitivity of histological and stool examination was similar except for the diagnosis of intestinal cytomegalovirus infection and leishmaniasis, which required invasive evaluation. CONCLUSIONS: Intestinal infections were diagnosed in less than 50% of chronic diarrheal episodes. The prevalence of enteric pathogens tended to decrease during the observation period, possibly because of improved antiretroviral therapy. Endoscopic evaluation did not improve the diagnostic yield compared with stool examination except for the diagnosis of cytomegalovirus enteritis and leishmaniasis.

Infantile systemic hyalinosis in siblings: clinical report, biochemical and ultrastructural findings, and review of the literature.

A boy presented at age 3.5 months with joint contractures, restlessness, and pain on handling. His skin was thickened and there were livid-red macular lesions over bony prominences. Infantile systemic hyalinosis (ISH) was diagnosed, a presumably autosomal recessive, progressive, and painful disorder of as yet unknown pathogenesis. Observation over three years confirmed the diagnosis as typical changes, such as nodules on both ears, pearly papules in the perinasal folds and on the neck, fleshy nodules in the perianal region, and gingival hypertrophy, developed. Skin lesions and painful joint contractures progressed in spite of intense physiotherapy, and at age 3, the child had marked motor disability. The central nervous system (CNS) appeared to be intact and the infant showed normal mental development. Radiologic findings included marked generalized osteopenia, osteolytic erosions in the metaphyses of the long bones, and cortical thinning. Electron microscopy of two skin biopsies demonstrated deposition of floccular amorphous substance that was abundant around, and appeared to originate from, small blood vessels in the dermis, partially interfering with collagen fiber formation. Lysosomal inclusions were not seen. Serum acid hyaluronidase activity was within the normal range, and the synthesis of hyaluronic acid and proteoglycans in cultured skin fibroblasts was similar to that of control cells. A younger sister presented at age two months with painful joint contractures and discrete livid-red macules over both malleoli, and showed a similar progression of the disorder over the first year of life. The diagnosis of ISH should be considered in infants and children presenting with painful joint contractures and skin lesions. The pathogenesis of this disabling and disfiguring disorder remains unclear. Our data confirm probable autosomal recessive inheritance, and do not support lysosomal storage, hyaluronidase deficiency, or a primary collagen disorder, but indicate that the amorphous material accumulating in the skin and articular soft tissues may originate from the blood circulation.

Ultrastructure of the guinea pig pancreas in acute hypercalcemia.

The effect of acute hypercalcemia on pancreatic ultrastructure and the ultrastructural localization of calcium during hypercalcemia were studied in the guinea pig pancreas. After 3 h of i.v. calcium infusion (0.6 mmol/kg/h), hypertrophy and distention of the Golgi apparatus and an increased number of condensing vacuoles were seen. At 6 h, vacuolar fusion and displacement of zymogen granules occurred. At 9 h, irregular distribution of zymogen granules, indentation of the nucleus with chromatin clumping, and inclusion of intact cell organelles were present. Disruption of the plasma membrane and release of cell organelles into the interstitial space were seen. Control animals receiving saline solution (0.9% NaCl) revealed normal pancreatic ultrastructure. The serum ionized calcium values were 0.65 +/- 0.36 mM in controls and 0.71 +/- 0.14, 0.79 +/- 0.21, and 1.22 +/- 0.50 mM at 3, 6, and 9 h of calcium infusion, respectively. The ultrastructural localization of calcium was performed with the pyroantimonate staining technique after 3 h of calcium and saline infusion. Large calcium deposits were found in calcium-treated animals along the plasma membrane and in the Golgi region. The findings indicate that calcium exerts a strong stimulatory effect that eventually leads to the degeneration of the pancreatic acinar cell.

Infantile phytanic acid storage disease, a disorder of peroxisome biogenesis: a case report.

The infantile and classic forms of phytanic acid storage disease belong to the newly recognized group of peroxisomal disorders. In this paper we report the full clinical, morphological and biochemical results in a patient with infantile phytanic acid storage disease. The results indicate a generalized loss of peroxisomal functions due to a deficiency of peroxisomes as demonstrated in hepatocytes and cultured skin fibroblasts.

Hypercalcemic-type of small cell carcinoma of the ovary: characterization of a new tumor line.

BACKGROUND: The aim of this study was to develop and characterize a mouse xenograft model for the hypercalcemic-type of small cell carcinoma of the ovary (HTSCCO). PATIENTS AND METHODS: Tumor fragments were removed from a patient and cultured in six subsequent generations of nude mice. Histology, comparative genomic hybridization (CGH), electron microscopy and serum calcium levels were investigated. RESULTS: Morphology remained the same from the primary tumor of the patient through the 6th passage in the mouse. Serum calcium levels were significantly higher in the tumor-bearing mice compared to controls. CGH of the HTSCCO did not show evidence of a close relationship to either a germ cell tumor or an epithelial ovarian cancer. CONCLUSION: Some evidence was provided that the HTSCCO is an inhomogeneous tumor that is neither related to a germ cell tumor nor to an epithelial ovarian cancer, but is a distinct tumor entity.

Electron microscopy: a method for the diagnosis of inherited metabolic storage diseases. Electron microscopy in diagnosis.

In the diagnosis of inherited metabolic diseases, electron microscopy has become an important method complementary to clinical, histological and biochemical assays. The characteristic ultrastructure of stored material as well as the site of accumulation in the cell are shown in a number of metabolic disorders. The most prominent advantages of electron microscopical techniques as compared to alternative techniques are discussed. One of the advantages is the fact that ultrastructural investigation requires only tissue samples of very small size, and another that its results may be obtained within two days. Moreover, transmission electron microscopy permits new and promising analytical methods such as quantitative estimation of organellar changes (morphometry) and energy dispersive X-ray elemental analysis (EDX).

Variation of quantitative and qualitative changes of enchondral ossification in heterozygous achondroplasia.

Whereas the radiologic features of achondroplasia--the most common type of skeletal dysplasia in adults--are clearly defined there is still some debate about the severity and the type of histologic changes. Earlier descriptions reported severe disturbance of enchondral ossification to be typical of achondroplasia: They are, however, misleading by dealing mostly with cases of lethal neonatal dwarfism (e.g. thanatophoric dysplasia). Newer findings confirmed that only minor, quantitative lesions are typical of heterozygous achondroplasia. But even in recent years some observers noted more severe changes of enchondral ossification. An extensive histologic skeletal survey in a newborn achondroplastic male revealed remarkable findings: It clearly showed that in addition to a generalized, but moderate narrowing of the zones of enchondral ossification focal severe changes were present in various epiphyseal plates including clusterlike arrangement of enlarged chondrocytes, vacuolization, premature calcification and important fibrosis of cartilagineous matrix with membranous ossification. Our findings thus enable us to reconcile the seemingly divergent statements made before.

Glycogenosis type I (glucose 6-phosphatase deficiency): I. Ultrastructural morphometric analysis of juvenile liver cells.

The essential biochemical characteristic of von Gierke's disease is an inborn glucose-6-phosphatase deficiency and glycogen storage in the liver and kidney. This expresses itself morphometrically as an increased volume of glycogen per unit volume of the hepatocellular cytoplasm. Since glucose-6-phosphatase activity in patients studied is practically at the zero level, and the endoplasmic reticulum loses a large part of its membrane values, we conclude that the remaining endoplasmic reticulum represents glucose-6-phosphatase free membranes. A typical structural feature of the endoplasmic reticulum in von Gieke's disease is the appearance of "double contoured vesicles" (= pockets). These vesicles comprise approximately 3,5% of the total membrane system. The mitochondria play an important role in glycolysis and glycogen synthesis. It is thus to be expected that these organelles change in terms of their morphometric parameters in the course of glycogenosis type I. An important point in this direction is numerical mitochondrion reduction in combination with an unchanged mitochondrial volume.

A case of congenital non-progressive sensory neuropathy with tonic pupils.

One case of a non-progressive congenital neuropathy is reported. Clinical findings included subtotal analgesia, and diminished temperature, vibration and proprioceptive sense in arms and legs. The sensory nerve action potentials were absent. Autonomic dysfunctions were restricted to tonic pupils. Sural nerve biopsy taken at the age of 8 1/2 showed fascicular hypoplasia, subtotal loss of myelinated nerve fibers and severe loss of unmyelinated nerve fibers with a pathological size distribution. In the skin biopsy, including several cutaneous nerves, no myelinated nerve fibers were present, the unmyelinated nerve fibers were severely reduced and the collagen density was increased. Perivascular and periglandular innervation was significantly reduced. The findings in this case are suggestive of a malformative or fetally acquired lesion and further illustrate the difficulties in classification of the hereditary sensory neuropathies.

Focal lacunar resorption in the articular cartilage of femoral heads.

Human articular cartilage taken from 92 femoral heads at autopsy was examined macroscopically and microscopically. Fifty-two showed no changes except for occasional slight degeneration in the non-pressure areas; these changes were visible only microscopically. In the remaining 40 heads, different degrees of osteoarthrosis were seen; half the heads also showed focal lacunar resorptive lesions in the cartilage. The origin of this focal cartilage resorption is discussed and its possible association with necrosis, pannus formation and enzymatic synovial activities. We conclude that there is no evidence of a direct relationship between focal cartilage resorption and osteoarthrosis.

Perhexiline maleate-induced hepatitis.

We report on a case of perhexiline maleate-induced hepatitis secondary to a long-term administration of recommended daily dosages of 300 mg. The patient had a spectacular weight loss of 29 kg. He developed hepatitis, which subsided after drug withdrawal. Our electron-microscopic findings with the typical inclusion bodies and impaired hydroxylation capacity point to an underlying metabolic disorder as the pathogenetic mechanism.

[Morphology of articular cartilage in fab2-induced arthritis of the knee-joint in the rabbit (author's transl)]. / Morphologie des Gelenkknorpels bei der Fab2-induzierten Kniegelenksarthritis des Kaninchens.

An arthritis closely resembling rheumatoid arthritis in man can be produced by the intra-articular injection of Fab2 into the knee joint of rabbits. This experimental model was used for the examination of ultrastructural alterations in articular cartilage. The lesion starts at the surface and advances gradually to the deeper zones of the cartilage. Morphologically, the lesion is characterized by progressive necrobiosis of the chondrocytes, as well as by a continually increasing thickening of the collagen fibres. It is suggested that the initial damage to the cartilage is not brought about by the pannus tissue, but is caused by a direct reaction to the pathologically-altered synovial fluid in response to inflammation.

Mucolipidosis type II in a domestic shorthair cat.

A seven-month-old, female domestic shorthair cat was presented to the Veterinary Teaching Hospital, University of Zurich, with abnormal facial features, retarded growth and progressive hindlimb paresis. On physical examination the cat had a flat, broad face with hypertelorism, frontal bossing, small ears and thickened upper and lower eyelids. The corneas of both eyes were clear and the pupils were dilated. The skin was generally thickened, most prominently on the dorsal aspect of the neck. Radiography of the entire skeleton revealed a severely deformed spinal column, bilateral hip luxation with hip dysplasia, an abnormally shaped skull and generalised decreased bone opacity. The clinical features and radiographic changes were suggestive of mucopolysaccharidosis. The toluidine blue spot test on a urine sample, however, was negative for glycosaminoglycans. Further biochemical investigations revealed a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) in peripheral leukocytes and an elevation of many lysosomal enzymes in the serum of the cat which is diagnostic for mucolipidosis type II. Histology and electron microscopy of different tissues are briefly summarised. The findings of this cat, the first reported case of mucolipidosis type II are compared with other similar storage diseases described in the cat.