Cell culture of rabbit meniscal fibrochondrocytes II. Sulfated proteoglycan synthesis.

Rabbit meniscal fibrochondrocytes were grown in vitro under culture conditions previously shown to foster growth of this cell type. Regardless of the culture regimen employed, the cells synthesized sulfated proteoglycans which could be differentiated by their solubility when dialyzed against water. The water soluble proteoglycans (WSPG) were monomeric in nature and could be separated into sub-types based on their hydrodynamic size when analyzed by gel-filtration chromatography. The water insoluble proteoglycans (WIPG) appeared to represent hyaluronic acid-dependent aggregates of the larger of the two WSPG. The proteoglycans contained approximately 87% chondroitin sulfate and 5% dermatan sulfate. Keratan sulfate could not be detected. Addition of ascorbate to the culture medium did not change the amount or the hydrodynamic size of the proteoglycan aggregates but did alter the quantity of the larger WSPG monomer synthesized depending upon the culture regimen used. Thus, these cells are capable of expressing their differentiated phenotype in short-term monolayer cell culture.

Cytokines in malignant lymphomas: review and prospective evaluation.

Cytokines play important roles in the pathogenesis of lymphomas. Cytokines either can be produced or exert effects on neoplastic or reactive cells. The secretion of cytokines can provide growth advantages for tumor cells in either an autocrine or a paracrine fashion. An elevated serum or tissue level of cytokines can contribute to the clinical and histopathologic alterations associated with malignant lymphomas. The effects of cytokines on the histopathologic changes are most noticeable in Hodgkin's disease (HD). The malignant (Hodgkin's-Reed-Sternberg) cells in HD have been shown to secrete interleukin-1 (IL-1), IL-5, IL-6, IL-9, tumor necrosis factor-alpha, macrophage colony-stimulating factor, transforming growth factor-beta, and, less frequently, IL-4 and granulocyte colony-stimulating factor. These cytokines may be responsible for the increased cellular reaction and fibrosis observed in tissues involved by HD and for the immunosuppression in patients with HD. In contrast to Hodgkin's-Reed-Sternberg cells, most non-HD lymphoma cells do not produce cytokines in excess amounts. Exceptions include T-cell-rich B-cell lymphoma (IL-4), angioimmunoblastic lymphadenopathy-like T-cell lymphoma with plasmacytosis and hypergammaglobulinemia (IL-6), anaplastic large-cell lymphoma (IL-9), polymorphic immunocytoma (IL-6), and immunoblastic lymphoma (IBL) (IL-6). Some cytokines are involved in the unique cellular reactions in each of these types of lymphoma. For example, IL-4 is responsible for the T-cell reaction in T-cell-rich B-cell lymphoma, while IL-6 is accountable for the plasma cell reaction in angioimmunoblastic lymphadenopathy-type T-cell lymphoma. Others may be directly involved in the tumor cell growth or differentiation. For instance, IL-9 may be important for the autocrine proliferation of anaplastic large cell lymphoma, whereas IL-6 is essential for plasmacytoid differentiation in polymorphic immunocytoma. Further studies of the roles of cytokines in lymphomas may lead to major advances in the understanding of the molecular processes involved in the histopathogenesis of malignant lymphomas. Elucidation of the autocrine or paracrine function of cytokines also may lead to new approaches to a rational intervention in these disease processes.

Rhinosporidiosis. A report of two cases from Arkansas.

Two human native cases of ocular-conjunctival rhinosporidiosis from Arkansas are believed to be the first documented reports in this part of the country. The common mode of infection was accidental injury to the eye by possible contaminated soil-dust. The appearance of the polypoid growth was relatively fast, 6-16 days, and unresponsive to topical antibiotic and steroid treatment. Surgical excision, with one recurrence in one case, was the elective treatment. Both patients are asymptomatic 10-12 months after treatment, respectively, with no evidence of other recurrence, dissemination, or major complications. From 1939 to September, 1983, only nine cases of conjunctival rhinosporidiosis were reported in the United States.

Sebaceous carcinoma of meibomian gland origin. The diagnostic importance of pagetoid spread of neoplastic cells.

The clinical and pathologic features of four sebaceous carcinomas primary located in the eyelids were studied in detail. The neoplasms occurred in four patients of ages 62 to 76 years, were often misdiagnosed at initial excision, and recurred in all four cases. Three of the four cases required orbital exenteration, and preauricular lymph node metastases developed in two. The tumors were characterized by variable histologic growth patterns and evidence of sebaceous differentiation. Pagetoid spread of the neoplastic cells in the overlying epithelium was found in all four cases. Recognition of the pagetoid growth pattern in biopsy material was essential in correctly diagnosing two of the four cases. It is one of the important features enabling sebaceous carcinoma to be distinguished from basal cell carcinomas showing sebaceous differentiation.

Adrenal myelolipoma associated with Cushing's disease.

An 18-year-old man with a history of Cushing's disease was treated with a total right and a near total left adrenalectomy in 1956. Pathologic examination of the operative specimen revealed bilateral adrenal hyperplasia. After 13 years, recurrence of symptoms of cortisol excess necessitated cobalt irradiation to the pituitary, which was without clinical effect. After an initial response to the adrenolytic agent, o,p'-DDD, partial relapse occurred. At this time, the recognition of an abdominal mass prompted abdominal exploration revealing a huge adrenal myelolipoma containing adrenal cortical cells distributed diffusely throughout the tumor. Symptoms of adrenal insufficiency developed, and adrenal steroid secretion did not respond to exogenous adrenocorticotropic hormone postoperatively. The case illustrates that adrenal myelolipomas may become very large with continued stimulation by adrenocorticotropic hormone, may contain significant amounts of adrenal cortical tissue, and may be associated with clinical hypercortisolism.

Prognostic factors in adrenal cortical tumors. A mathematical analysis of clinical and morphologic data.

Forty-one patients with adrenal cortical tumors were evaluated retrospectively for the presence of histologic and non-histologic features associated with malignant behavior. The patients who survived operation were followed at least five years. The association of these features with subsequent metastasis was examined by both parametric and nonparametric statistical methods. Twelve criteria were statistically significant in predicting subsequent metastasis. The most significant of these were: clinical evidence of weight loss, broad fibrous bands traversing the tumor, a diffuse growth pattern, vascular invasion, tumor cell necrosis, and tumor mass. The tendency toward metastasis for each tumor was expressed in indices that correlated well with diagnoses made by conventional histologic means, but were more accurate in predicting subsequent clinical behavior than nonquantitative methods of evaluation.

Serum-free culture of rabbit meniscal fibrochondrocytes: proliferative response.

We have formulated a serum-free medium capable of supporting DNA synthesis in rabbit meniscal fibrochondrocytes at a level equivalent to 10% fetal bovine serum (FBS). The medium consists of a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 medium supplemented with transferrin (1 microgram/ml), selenium (1 pg/ml), trace metal mix (1:100), dexamethasone (100 ng/ml), insulin-like growth factors I and II (50 ng/ml each), pituitary fibroblast growth factor (100 ng/ml), and lactalbumin hydrolysate (2 micrograms/ml). Endothelial cell growth supplement could be substituted for lactalbumin hydrolysate to obtain similar results. Ventrex PC-1, a commercially available, low-protein, serum-free medium, was found to support proliferation of fibrochondrocytes but not as well as 10% FBS or our medium formulation. Lipid supplements, which are known to support the serum-free growth of hyaline chondrocytes, were found to be either of no value or antagonistic for the culture of fibrochondrocytes. Likewise, vitamin E alone, progesterone, putrescine, and hydrocortisone were also without benefit in our culture system. The cells had a more chondrocytic morphology when grown in defined medium as opposed to 10% FBS. The results of this study should now make it possible to identify and quantitate those factors necessary to affect meniscal repair by utilizing further techniques in vitro.

Cell culture of rabbit meniscal fibrochondrocytes: proliferative and synthetic response to growth factors and ascorbate.

This study was undertaken to determine whether the cells of the fibrocartilaginous meniscal substance are capable of proliferation and matrix synthesis. Cells were isolated from the fibrocartilaginous menisci of young New Zealand white rabbits, and grown in two alternative culture regimens differing only in the basal nutrient medium used to initiate primary monolayer growth. Under each culture regimen, the cells attached and proliferated both initially and after passage into secondary (2 degrees) culture. Differences were noted in cell morphology and time to reach confluence in primary (1 degrees) culture. Upon passage into 2 degrees culture, the fibrochondrocytes assumed two distinct morphologies depending upon the type of medium used for 1 degree culture. These morphological changes were accompanied by differences in the population doubling time and incorporation of 35SO4 into sulfated proteoglycans. The proliferation of both fibrochondrocyte subtypes was stimulated by the addition of either pituitary fibroblast growth factor (FGF) or human platelet lysate in a dose-dependent manner. Both FGF (10 ng/ml) and ascorbate (40 micrograms/ml) decreased 35-sulfate incorporation, whereas only ascorbate was found to alter the amount of sulfated glycosaminoglycan in the pericellular coat. We conclude that the fibrochondrocytes of the meniscal substance are capable of replication and synthesis of matrix macromolecules if given the proper stimuli. Additionally, there may be two subpopulations of fibrochondrocytes that can be distinguished by their in vitro behavior.

Allograft bone in spinal fusion for paralytic scoliosis.

To determine the efficacy of allograft bone in spinal surgery, a retrospective study was carried out on thirty-two patients who had had spinal fusion with banked bone from the femoral head between 1977 and 1983. The diagnoses were paralytic and neuromuscular scoliosis. At final follow-up (average, 3.7 years), the infection rate was 9.3 per cent. There were no pseudarthroses, and all patients had well marginated trabecular markings by eighteen months. The rates of complications and of fusion were comparable with or better than those obtained using autogenous bone graft.

An organ culture model for assaying wound repair of the fibrocartilaginous knee joint meniscus.

We describe an in vitro organ culture system that can be used to test the effect of various substances and compounds on the wound healing process in the fibrocartilaginous substance of the knee joint meniscus. Using culture medium containing either 10% fetal bovine serum (FBS) or our recently developed serum-free, defined culture medium (DM), we have demonstrated the ability of meniscal fibrochondrocytes from intact rabbit menisci to extricate themselves from their surrounding matrix and migrate into an exogenous, purified fibrin clot in vitro. After 4 weeks of culture in FBS-containing medium, the cells which had invaded the clot had initiated the early aspects of a typical reparative response; the same response did not occur in DM alone. Morphologically, the cells on the surface of the clot resembled the original superficial fibrochondrocytes, whereas those cells within the substance of the clot more closely resembled the original deep fibrochondrocytes. After 10 weeks, the reparative response had progressed only to a certain point and then failed to progress further under these culture conditions. However, use of this culture system should now make it possible to rapidly identify and quantitate those factors which would most likely be useful in continuing the reparative response and in affecting meniscal wound repair. Elucidation of the mechanisms and requirements for meniscal healing will eventually allow the practicing orthopaedic surgeon to effect in situ meniscal repair and obviate the need for meniscectomy and its morbid sequelae.

Aging phenomena and osteoarthritis: cause or coincidence? Claude P. Brown memorial lecture.

Osteoarthritis is an ubiquitous disease, primarily occurring in older individuals. Any attempt to explain the disease in terms of biologic aging must first define the basic pathology of the disease, accurately classify the clinicopathologic variants of the disease, and develop a scientific approach to the recognition of the pathologic physiology of any known precursor states. This epidemiologic information must insure comparability between populations of patients or animals under study, since significant differences in susceptibility among groups do exist. Cumulative environmental influences on joints do have pathophysiologic consequences but require careful study to isolate cause and effect from coincidental occurrences. Musculoskeletal aging itself is a complex process including both post-synthetic changes in the extracellular matrix macromolecules and alteration of phenotypic expression by the connective tissue cells themselves. In contrast to older assertions that chondrocytes are terminally differentiated cells incapable of replication, evidence has accumulated that chondrocytes proliferate in vivo and in vitro under appropriate conditions. Studies in several laboratories have confirmed that articular chondrocytes from aged animals both proliferate and synthesize macromolecules in a fashion similar to comparable cells extracted from young animals. Further studies are necessary before osteoarthritis can be definitively separated into aging-dependent and aging-independent categories.

Cartilage in hemophilic arthropathy. Ultrastructural and microanalytical studies.

The presence of iron in articular cartilage was investigated in five human and two canine cases of factor VIII-deficiency hemophilic arthropathy. The lesions were mild in three cases. Advanced destruction of the cartilage was present in four cases, in one of which sufficient cartilage was preserved to permit recognition of hemosiderin in chondrocytes. The Fe was present in the form of siderosomes and ferritin granules free in the cytoplasm. Iron and phosphorus were demonstrated in the siderosomes by energy dispersion analysis of x-rays; but no Fe was identified by this means or by light or transmission electron microscopy in chondrocytes from the three subjects in an early stage of the disease or in the matrix from any of the seven subjects. The findings lend no support to the hypothesis that salt formation or chelation of Fe ions by matrix proteoglycans is responsible for destruction of cartilage in hemophilic or other chronic hemarthroses. A second, nonferruginous, bilirubin-like pigment (hematoidin) was present in the matrix at the surface of the cartilage in two specimens.

An introductory orientation to clinical pathology core and on-call responsibilities.

An introductory 4-week orientation for clinical pathology is described. There were 76 hours of lectures, 74 hours of conferences, and 68 hours of laboratories for a total of 221 hours. During the orientation, all calls handled by the residents were evaluated as to resolution, patient outcome, and interaction required. Eighty calls were received during the orientation from 57 technologists (71%), 16 physicians (20%), and seven nurses (9%). The calls originated concerning the following: blood banking, 37 (46%); hematology, 21 (27%); chemistry, 14 (18%); microbiology, five (6%); and administration, three (4%). Sixty percent of the calls were consultative and 40% were supervisory. Ninety-nine percent were handled appropriately by the residents. Patient outcome was moderately or significantly affected in 44% of all calls, divided between 67% of all consultative calls and 9% of all supervisory calls. Significant pathologist interaction was required in 49% of all calls, divided between 71% of the consultative calls and 16% of the supervisory calls. Using this integrated, dynamic system of resident instruction, on-call experience, and evaluation, residents quickly gain confidence in handling call, didactic clinical consultation, and patient management. The orientation and on-call system described provides for a relevant and dynamic system for resident education.

Effects of adrenalectomy and corticosterone on hippocampal lesions induced by trimethyltin.

The effects of adrenalectomy and corticosterone supplementation on the neurotoxicity of trimethyltin (TMT) were tested. CD-1 mice with or without adrenalectomy were injected with TMT at a dose of 3.0 mg/kg body wt. At 48 h post-TMT administration, the animals were killed for pathological examination. It was found that the adrenalectomized animals developed even more severe lesions in the hippocampal formation (fascia dentata granule cells) than the intact animals. When animals were given a supplement of corticosterone pellets at doses of 0.15, 1.5, and 7.5 mg, there was a reduction of lesion development. Total alleviation of pathology was seen at the two higher doses of supplementation. Our present investigation strongly indicates that there may be a close and important interrelationship between TMT-induced neurotoxicity and adrenal function.