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Vascular endothelial growth factor (VEGF), an endothelial cell mitogen, plays a role in angiogenesis and progression in malignant melanoma. VEGF expression was examined in 62 biopsy specimens of melanocytic proliferations, including 45 malignant melanomas, 3 cellular blue nevi, 12 atypical compound nevi, and 2 Spitz nevi. The cases of malignant melanoma included 11 in situ melanomas, 18 Clark Level II, 9 Clark Level III, and 7 Clark Level IV tissue samples. All of the specimens were fixed in formalin and embedded in paraffin. Cytoplasmic immunoreactivity for VEGF was demonstrated in 19 (42%) of 45 melanoma samples, but there was no immunoreactivity for VEGF exhibited by any of the atypical compound melanocytic nevi, cellular blue nevi, or Spitz nevi (P < .009). Immunoreactivity for VEGF was found to be related to tumor thickness (as evidenced by Clark level [P < .03]) and to absence of regression (P < .04). Although VEGF is not a useful prognostic indicator for malignant melanoma, it may be useful as a discriminating factor between malignant melanoma and benign melanocytic lesions, and it may offer some insight into tumor growth.

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.

The pathologic description of the menisci is facilitated by grouping the various disorders into etiologic groups. These include congenital anomalies, traumatic conditions, inflammatory disorders, metabolic disorders, degenerative conditions, and neoplasms. In clinical practice, traumatic conditions, as exemplified by meniscal tears of the vertical-longitudinal or vertical-transverse type, are the most important pathologic disorders. Healing of meniscal tears occurs only when the tear involves the peripheral vascularized attachment of either the lateral or medial meniscus. Intensive clinical and animal experimentation suggests that serum-derived growth factors are necessary for meniscal healing. Chondrocalcinosis is extremely frequent in the menisci of older individuals and may or may not be associated with degenerative changes of the menisci, including fibrillation, loss of protein polysaccharide ground substance, and chondrocytic proliferation. The role of degenerative changes of the menisci, including tears of the horizontal type, in the development of osteoarthritis (OA) of the knee is a subject of intense interest, as is the relationship between chondrocalcinosis and OA. Neoplastic involvement of the menisci is essentially limited to invasion by tumors starting in or, more commonly, adjacent to the knee joints.

The effect of progesterone alone and in combination with estradiol was investigated in ovariectomized and gonadally intact female guinea pigs infected with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). The course of the infection, as determined by the percentage of cells with GPIC (chlamydia) inclusions in Giemsa-stained vaginal scrapings, was not affected in animals receiving 5.0 mg of progesterone daily. Progesterone had no influence on the enhancement of infection by estradiol. In comparison with sesame oil-treated controls, infection was prolonged by four to six days (P less than .05) in animals receiving a combination of 5.0 mg of progesterone plus 1.0 microgram of estradiol or 1.0 microgram of estradiol alone each day. In ovariectomized animals, estradiol delayed the appearance of IgA antibody in genital secretions, whereas progesterone alone had no effect. Guinea pigs treated with estradiol or progesterone plus estradiol manifested an acute endometritis not observed in animals treated with progesterone alone or in controls receiving sesame oil. Although cervical ectopy, analogous to that seen in women with high levels of progesterone, was identified by histopathology in animals treated with progesterone, no enhancement of the chlamydial infection was observed.

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.

Female guinea pigs were treated daily with 1 mg of beta-estradiol-3-benzoate intramuscularly beginning 14 days before intravaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis and continuing during the course of the infection. Treatment with estradiol was found to markedly influence the course of genital infection with the chlamydial agent of guinea pig inclusion conjunctivitis, producing infections of greater intensity and longer duration than those in control animals. Moreover, pathogenesis was altered in that ascending infection was observed, resulting in endometritis, cystic salpingitis, and cystitis. Infection in the controls was limited to the cervix and vagina. Estradiol treatment increased the apparent number of infected cells in the cervix and vagina as detected by histopathology and immunofluorescent staining. Humoral and cell-mediated immune responses to the chlamydial agent of guinea pig inclusion conjunctivitis were comparable in estradiol-treated and untreated animals. These data indicate that hormonal manipulation may have profound effects on the course of chlamydial genital infections.

Several preliminary reports have appeared in the medical literature in respect to eustachian tube substitution since Zollner first described his inability to open the obstructed eustachian tube. These procedures have the disadvantage that the distal end of the eustachian tube subsitute is inaccessible by other than another operation. This paper is follow-up report to our clinical experience. The purpose of the experiment was to determine whether the middle ear was ventilated by the tympano-oropharyngeal substitute eustachian tube and whether ascending infection occurred. Fourteen ears underwent eustachian tube substitution. The natrual eustachian tubes of these ears were obliterated three to five months after eustachian tube substitution had been performed. The animals were killed three to four months after obliteration of the natural eustachian tubes. Studies of the decalcified sections showed an absence of infection in six of the ears, with mild to severe inflammation in the rest of the specimens.

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.

Meniscal fibrochondrocytes from male and female New Zealand white rabbits, ages 6, 12, and 24 months, were grown in primary and secondary monolayer cell culture. Neither age nor sex affected the majority of their cell culture characteristics. Cells from young males (6 months old) synthesized greater amounts of sulfated proteoglycans than did those from young females, but by 2 years of age, this result was reversed. All age groups synthesized 2 classes of proteoglycans, based on hydrodynamic size, but the ratio of the 2 classes changed as a function of age. Overall, the meniscal fibrochondrocytes from both skeletally immature and skeletally mature rabbits of both sexes were capable of proliferation and matrix synthesis in vitro.

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.

The metabolism of arachidonic and linoleic acids by VX2 carcinoma tissue in vitro was determined. Prostaglandin E2 was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3- 8% of arachidonic acid metabolism were II-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (II-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40-80% 9-HODD and 20-60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX2 carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E2 biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E2 biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX2 carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E2 via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.

The purpose of this investigation was to determine the natural history and pathogenesis of the acute arthritis induced by inoculation of a viable Chlamydia trachomatis biovar (mouse pneumonitis agent or MoPn) in C57Bl/6 mice. Immunologically naive (previously unsensitized) mice as well as mice immunized against Chlamydia (MoPn) by vaginal infection were employed. Both intravenous and intraarticular inoculations were employed. No arthritis developed after intravenous injections of MoPn although statistically significant antibody titers and splenic enlargement ensued. Intra-articular inoculation into knee joints produced a definite arthritis of 7 to 10 days duration marked by granulocyte and mononuclear cell infiltration of the joint and vacuolated synovial macrophages that stained heavily for chlamydial antigen by immunoperoxidase technique. Statistically significant increases in articular acute and chronic inflammation (P less than 0.02 were observed in previously sensitized, but not unsensitized, female mice at 2 but not 7 days after intra-articular chlamydial challenge. Chlamydiae were isolated from injected joints up to day 5, but not at day 10, after challenge. Chlamydial antigen disappeared rapidly from knee joints between day 10 and 15 after challenge. Electron micrographs demonstrated vacuolated synovial cells of the macrophage type, many of which contained degenerating chlamydial elementary bodies. Reticulate and intermediate bodies also were seen but were far less frequent than degenerating elementary bodies. Unaltered elementary bodies were difficult to identify beyond day 2 after articular inoculation. Thus, it appears likely that intra-articular chlamydial survival is shorter than the duration of the arthropathy. This may have important implications in attempts to identify chlamydiae in human joints in Reiter's Disease.

An angiographically demonstrated case of total occlusion of the abdominal aorta by a malignant retroperitoneal tumor is presented. Surgical exploration revealed a diffuse tumor of the retroperitoneum involving the wall and lumen of the abdominal aorta and vena cava, respectively. Histologic and ultrastructural evaluation of the tumor established a diagnosis of malignant fibrous histiocytoma. Total occlusion of the abdominal aorta thus represents another angiographic finding in retroperitoneal tumors.

The purpose of this investigation was to determine the role of the humoral immune response in the production of arthritis in mice immunized with the chlamydial agent of mouse pneumonitis (MoPn) (Chlamydia trachomatis biovar). Mice were made B cell deficient (BCD) by treatment with rabbit antiserum to murine IgM. Control mice included animals treated similarly with normal rabbit serum or phosphate-buffered saline. Male mice were immunized with MoPn inactivated with ultraviolet irradiation while female mice were immunized by genital tract infection with viable chlamydiae. Arthritis was elicited in all mice by intra-articular inoculation of inactivated MoPn. When knee joints were examined for pathologic changes at varying times after challenge, a marked enhancement of the arthritis was observed in both male and female BCD mice when compared with controls at all time points. These data indicated that the humoral immune response is not essential for the production of arthritic disease in this model but may have some role in the modulation of the process in immunologically intact animals. Persistence of chlamydial antigen in joint tissue of BCD mice suggested that antibody may play a role in the elimination of antigen, thus decreasing the stimulus for the development of cell-mediated immunologic injury. Regulatory role for T suppressor cells cannot be ruled out however, because B cell deficient mice have been shown to lack certain T suppressor cell subsets.

Antigen from a Chlamydia trachomatis biovar, mouse pneumonitis agent (MoPn), was used to produce an inflammatory arthritis by inoculation in the knee joints of C57B1/6 mice. The production of arthritis was strongly dependent on prior sensitization to chlamydiae by subcutaneous immunization in either sex or genital infection in females. In unimmunized animals quantitatively less inflammation, fibrin exudation, and pannus formation developed than in immunized counterparts. In neither uninjected nor McCoy or HeLa Cell antigen-treated joints did arthritis develop. The arthritis produced was more pronounced at 7 than at 2 days after intraarticular challenge and showed minimal residual changes at 21 days. Residual changes included subsynovial hyperplasia. In genitally infected female mice less severe arthropathy developed than in subcutaneously immunized female mice after intraarticular challenge with formalin-inactivated chlamydial antigen. However, in infected mice challenged with chlamydiae inactivated by ultraviolet irradiation a severe arthritis with destructive pannus developed, suggesting alteration of antigenicity by formalin inactivation. The strong association of the arthritis with the presence of chlamydial antibody suggests its use as a model of human Reiter's disease in which prior sensitization to Chlamydia is an important factor.

One- to three-day-old guinea pigs were inoculated intranasally with the chlamydial agent of guinea pig inclusion conjunctivitis. Physical signs of infection included a marked increase in respiration rate on days 5 to 10 of infection and radiographic evidence of pneumonia on day 6. When animals were killed at various times after infection and lung tissue was examined by histopathology, evidence of pneumonia was found beginning on day 4 and lasting as long as day 12, with maximal pathological changes on days 6 to 8. The pneumonia was generally unilateral and consisted of an acute inflammatory component in the bronchioles with granulocytes in both the lumen and the wall of the bronchioles and an interstitial and intra-alveolar mononuclear infiltrate in the parenchyma of the lung. Chlamydial antigen was detected in the bronchial epithelial cells by immunoperoxidase staining, and the guinea pig inclusion conjunctivitis organism was isolated from lung tissue on days 6 to 9. No other significant bacteria were isolated from lung tissue or seen on gram stains of lung sections. Both immunoglobulin M and immunoglobulin G serum antibodies to the guinea pig inclusion conjunctivitis agent were detected as early as day 8 and reached peak levels on day 12. The infection was apparently self-limiting. This model presents the opportunity to investigate pathophysiological and immunological aspects of chlamydial respiratory infections in a neonatal animal.

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.

The effect of four different alkyltins (trimethyltin, triethyltin, dibutyltin, and dioctyltin) on the metabolism of rabbit articular and growth-plate chondrocytes was investigated using a monolayer cell-culture system. In most instances the compounds tested exhibited a general cytotoxic effect on these cells, inhibiting the synthesis of both DNA and sulfated proteoglycans. The effect of these compounds on proteoglycan synthesis was both quantitative and qualitative, as demonstrated by CsCl isopycnic density gradient centrifugation and gel exclusion chromatographic techniques. However, certain tin compounds tested, at specific concentrations, exerted a stimulatory effect on chondrocyte proliferation. Regarding DNA synthesis, growth-plate chondrocytes were more sensitive to the effect of the triethyltin, dibutyltin, and dioctyltin than were articular chondrocytes. The data are discussed in relation to the possible effects of the alkyltins on skeletal growth and development as well as the mechanism of action of the alkyltins at the molecular level.

In New Zealand white rabbits bearing the transplantable VX2 carcinoma intramuscularly in the hind limbs uniformly a granulocytosis syndrome developed, characterized by peripheral blood neutrophilia and marked granulocytic bone marrow hyperplasia, whereas the syndrome did not always develop in animals bearing the tumor intraabdominally. The relationship between the extent of pulmonary metastases and granulocytosis/bone marrow hyperplasia was significant (P less than 0.005). This positive correlation (correlation coefficient = 0.85) existed for both animals bearing intraabdominal tumors and animals bearing intramuscular tumors. The granulocytic syndrome occasionally occurred in rabbits with mild degrees of pulmonary metastatic involvement, but never with total absence of pulmonary metastases. Hypercalcemia occurred prior to the appearance of pulmonary metastases and was not essential to the development of granulocytosis. These data suggest that granulocytosis in VX2 carcinoma-bearing rabbits is dependent on the presence of tumor in the lungs and is not directly related to implantation site. This tumor-bearing animal model may be suitable for studies of tumor-associated granulocytosis in vivo.

The effect of various doses of estradiol on genital tract infection by the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC) was investigated in ovariectomized guinea pigs. Prolongation of infection, as determined by chlamydial inclusion counts of cells in Giemsa-stained smears of vaginal scrapings, was observed in animals receiving daily doses of 1.0, 10.0, 100.0, or 1000 micrograms of estradiol. In contrast to controls, ascending infection resulting in endometritis was found in animals receiving doses of greater than or equal to 1.0 microgram of estradiol per day. Response to estradiol treatment was reflected in an increase in cervical-uterine wet weight and uterine wall thickness. No differences were observed in time of appearance of antibody titers to GPIC in serum, but a delay in appearance of IgA antibody to GPIC in genital secretions was found in estradiol-treated animals receiving doses of greater than or equal to 1.0 microgram per day.

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.

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.

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.