Vascular endothelial growth factor expression in malignant melanoma: prognostic versus diagnostic usefulness.

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.

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.

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.

Pathology of the meniscus.

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.

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.

Pathogenesis of acute arthritis due to viable Chlamydia trachomatis (mouse pneumonitis agent) in C57Bl/6 mice.

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.

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.

Antibody-mediated modulation of arthritis induced by Chlamydia.

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.

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.

Induction of arthritis in C57B1/6 mice by chlamydial antigen. Effect of prior immunization or infection.

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.

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.

Renal pelvic carcinoma: morphological correlates of metastatic behavior.

We report a clinicopathological analysis of morphological parameters in relation to subsequent biological behavior in 48 patients with renal pelvic carcinoma. The relationships of subsequent metastasis to tumor stage and grade, as well as the presence of vascular, renal parenchymal or renal hilar invasion were evaluated by parametric and nonparametric statistical tests. Corrected 5-year survival rates for grades 1 to 3 tumors were 100, 67 and 5 per cent, respectively. Grade 3 tumors demonstrated more invasion of the blood vessels, hilus and renal parenchyma compared to grades 1 and 2 tumors (p less than 0.005, 46 patients). Moreover, invasion of renal hilar tissues had greater predictive value for subsequent distant metastatic spread (95 per cent, nonparametric) than either vascular (83 per cent) or renal parenchymal (77 per cent) invasion. Metastases developed in 27 of 48 patients during followup periods of 2 to 39 years. The pattern of metastatic lesions revealed that local spread to hilar soft tissues occurred in 92 per cent of the patients who subsequently had distant metastases. Spread to retroperitoneal lymph nodes and ipsilateral ureteral mucosa occurred in 84 and 44 per cent of the patients, respectively. Our study illustrates the potential value of analysis of individual histological parameters to evaluate the likelihood of subsequent metastasis.

The effect of rapid resuscitation upon cardiac index following thermal trauma in a porcine model.

Minipigs were divided into four experimental groups: nonburned control (C); burn, minimal resuscitation (MR); burn, standard resuscitation (SR); and burn, rapid resuscitation (RR). Resuscitation was begun 30 minutes postburn and hemodynamics were measured through 24 hours postburn. At 30 minutes postburn, the three burn groups demonstrated a significant decrease in cardiac index % (CI%). At 1 1/2 hours postburn, the RR group CI% was significantly greater than for the SR group and not significantly different from that of controls. At 2 hours postburn, fluid was abruptly decreased in the RR group and the salutary effect on CI promptly dissipated. In this model, the early decrement in CI following thermal injury is responsive to rapid resuscitation, concluding that the etiology of decreased CI following thermal trauma is at least partially due to inadequate volumes of fluid administration in the early postburn period.

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.

In vitro cell proliferation and proteoglycan synthesis of rabbit meniscal fibrochondrocytes as a function of age and sex.

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.