Growth and spontaneous regression of swine melanoma: relationship of in vitro leukocyte reactivity.

A visual microcytotoxicity technique was used to evaluate the leukocyte reactivity of melanoma swine against allogeneic swine melanoma target cells. Peripheral blood leukocytes, which had been collected and cryopreserved in liquid nitrogen at various times during in vivo tumor growth and regression, were thawed and tested on the same day. Comparison of longitudinal leukocyte reactivity with in vivo tumor volume indicated that swine with regressing melanomas exhibited increased leukocyte reactivity during tumor regression. Swine with maximum tumor volumes less than 30,000 mm3 exhibited patterns of leukocyte reactivity that paralleled the patterns of in vivo tumor growth and regression. However, swine with maximum tumor growth and regression. However, swine with maximum tumor volumes greater than 30,000 mm3 demonstrated increased in vitro leukocyte reactivity at the time of maximum in vivo tumor volume. Histopathologic analyses revealed that increases in tumor volume were frequently a result of host inflammatory cells, particularly pigment-laden macrophages, infiltrating the tumors. Thus at the time of maximum tumor volume, malignant melanocytes were proportionately decreasing in number while pigment-laden macrophages were proportionately increasing. These studies provide additional evidence that the spontaneous tumor cell regression of swine melanoma is associated with immunologic events and that assays of leukocyte reactivity are useful in vitro correlates of host antitumor immunity.

Development of a new melanoma model in C57BL/6 mice.

In the present study, we induced melanomas in C57BL/6 mice by a single application of 7,12-dimethylbenz(a)anthracene to the scapular region of 4-day-old mice, followed by twice-weekly applications of croton oil. Of 20 mice treated, melanomas arose in two female littermates. The first melanoma (JB/MS) arose 16 weeks after initiation of treatment, and the second melanoma (JB/RH) arose 23 weeks later. The melanomas maintained their melanotic appearance after s.c. transplantation to normal C57BL/6 mice and metastasized spontaneously in the transplant recipients. To our knowledge, these are the first melanomas to have been induced in C57BL/6 mice since the B16 melanoma arose spontaneously in 1954. We feel that the JB/MS and JB/RH melanomas provide an excellent comparative system for studies done with the B16 melanoma. These melanomas of recent origin will also facilitate the investigation of biological, immunological, and biochemical parameters that influence the growth and metastasis of malignant melanomas.

Phenotypic instability of mouse melanomas after propagation in vivo and in vitro.

This study was designed to evaluate the phenotypic stability of the carcinogen-induced JB/MS and JB/RH melanomas. The JB/MS melanoma maintained its original slow-growing melanotic phenotype during in vivo passage over a 2-yr period. However, both melanin production and tumorigenicity decreased rapidly after propagation of JB/MS cells in vitro. The JB/RH melanoma became essentially amelanotic after the second transplantation in vivo. Cultured JB/RH cells produced tumors identical to those obtained by transplantation of JB/RH tumor fragments. However, after propagation in vitro for 2 yr, the JB/RH cell line decreased in tumorigenicity, requiring 10 times as many cells to produce tumors in C57BL/6 mice as did the original cell line. The JB/RH melanoma was highly immunogenic in syngeneic C57BL/6 mice, and passage of JB/RH cells through immunized mice resulted in tumors that were significantly more tumorigenic in normal mice than were JB/RH cells that had been passed through either nude or sublethally irradiated mice. These results indicate that, in studies of primary mouse melanomas, it is essential to: (a) limit the number of tumor passages; (b) choose methods of propagation that will preserve the original phenotype; and (c) distinguish those properties produced by technical manipulation from those produced by true tumor progression.

Adaptation of Sinclair swine melanoma cells to long-term growth in vitro.

Sinclair swine melanoma usually regresses in vivo. In the present study, swine melanoma cells were adapted to long-term growth in culture. The morphology of cultured melanoma cells ranged from dendritic to cuboidal, similar to that described for human melanoma cells. Doubling times of the swine melanoma cells were also similar to those of human melanoma cells in vitro. 3,4-Dihydroxy-L-phenylalanine oxidase-positive cells were detected by light microscopy, and melanin and premelanosomes were detected by electron microscopy. Cell cultures could be propagated from progressing, partially regressed, and primary cutaneous lesions, as well as from visceral metastases. Thus, it appears that, under these cell culture conditions, Sinclair swine melanoma cells can be adapted to prolonged growth in vitro.

Properties of an alkaline phosphatase from Sinclair swine melanoma.

In Sinclair swine, there is an increase in alkaline phosphatase activity in spontaneously arising melanoma tumors when compared to normal skin. While alkaline phosphatase activity could be detected in melanomas from animals 1 day old, the maximum levels of alkaline phosphatase activity occurred in tumors from animals greater than 30 days old. The alkaline phosphatase was purified from cutaneous melanomas using chloroform precipitation, Phenyl-Sepharose chromatography, and concanavalin A Sepharose chromatography approximately 146-fold, with an overall recovery of 15%. The purified enzyme exhibited optimal activity over the pH range of 8.9-10.6. The apparent Km of the enzyme for p-nitrophenyl phosphate was 0.15 mM. The enzyme exhibited a relative mobility of 0.04 in nondenaturing polyacrylamide gels. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 122,000 and it was composed of two identical subunits each having a molecular weight of 67,000. The enzyme was thermolabile at 56 degrees C (T50, 18 min) and its activity was inhibited by L-homoarginine, levamisole, and vanadate, but not by L-phenylalanine or L-phenylalanylglycylglycine. These characteristics distinguished the enzyme from the intestinal isoenzyme that is found in normal swine skin but were similar to those exhibited by the porcine placental isoenzyme of alkaline phosphatase. These results suggest that the development of malignant melanoma in Sinclair swine is accompanied by the expression of a placental-like alkaline phosphatase activity.

Growth of Sinclair swine melanoma in the hamster cheek pouch.

Specimens from seven Sinclair swine melanomas were transplanted to the cheek pouches of Syrian Golden hamsters. The specimens were taken from young swine and were derived from raised tumors that either were present at birth or developed after birth from flat melanocytic lesions as well as from apparently normal skin. All seven specimens grew in the hamster cheek pouch. One lesion, derived from a 3-day-old piglet, exhibited the most aggressive growth in the hamster and was successfully transferred to other hamster cheek pouches. These results confirm the malignancy of Sinclair swine melanoma and indicate that tumors of neonatal swine contain more malignant cells than those of older animals.

Uveitis caused by cytotoxic immune response to cutaneous malignant melanoma in swine: destruction of uveal melanocytes during tumor regression.

In a substrain of Sinclair miniature black swine, bred for increasing incidence of cutaneous malignant melanomas, tumor regression occurs spontaneously and is accompanied by depigmentation of the skin, hair, and eyes. We conducted a 12-month longitudinal study of the ocular phenomena in 30 swine beginning at 3 weeks of age. The clinically observed sequence of depigmentation of the fundus and iris was correlated with histopathologic changes in selected enucleated eyes. Normal melanocytes of the uveal tract are destroyed between the 4th and 16th week of life. Melanocyte destruction is preceded by an invasion of the uveal tract by mononuclear cells having the ultrastructural features of lymphocytes and monocytes. Melanin and other cellular debris of ruptured melanocytes are ingested by macrophages which then migrate to the walls of blood vessels. Cataracts and band keratopathy develop secondary to the uveitis in some animals. Pilot electroretinograms show diminished electrical activity in photoreceptors of totally depigmented eyes possibly indicating ischemic or toxic damage to the retina. The retinal pigment epithelium remains essentially normal during the acute stages of uveal inflammation; later some damage and reparative hyperplasia may occur. The death of normal uveal melanocytes that occurs during the systemic attack on the cutaneous malignant melanomas appears to be an "innocent bystander" error in the immune recognition mechanism. The antigenic basis of this immunologic cross reaction is under investigation.

Antigenic diversity in flagellar epitopes among Bacillus piliformis isolates.

Monoclonal antibodies (MAbs) were developed to Bacillus piliformis isolate-specific flagellar epitopes and used to group B. piliformis isolates on the basis of epitope expression. BALB/c mice immunised with flagella purified from various B. piliformis isolates served as the source of immune spleen cells for fusion with SP2/0Ag14 myeloma cells. Evaluation of hybridoma culture medium by ELISA against various bacterial species and B. piliformis isolates indicated that 482 of 2127 hybridomas secreted antibodies specific for B. piliformis. Specificity of MAbs for flagellar epitopes was demonstrated by indirect fluorescent antibody assays and Western blot analyses. Probing of 10 B. piliformis isolates with MAbs indicated that four B. piliformis isolates each possessed a distinct and isolate-specific flagellar epitope; five other isolates shared a common flagellar epitope. One isolate did not react with any of the MAbs specific for flagellar epitopes. Thus, B. piliformis isolates could be grouped into six antigenically distinct groups based upon flagellar epitope expression. Additionally, a MAb reactive with a cell-associated component recognised all but one isolate. This serological grouping of B. piliformis isolates agrees with the grouping of isolates based upon genetic and physiological characteristics, and supports the assertion that there are different strains among B. piliformis isolates.

An immortalized hamster corneal epithelial cell line for studies of the pathogenesis of Acanthamoeba keratitis.

PURPOSE: The protozoan Acanthamoeba produces a severe keratitis in a small percentage of people, especially contact lens-wearers. The purpose of this work was to develop and characterize an immortalized line of hamster corneal epithelial cells to be used in studies of the pathogenesis of Acanthamoeba keratitis. METHODS: Hamster corneal epithelial cells were maintained in primary culture and immortalized using simian virus 40 (SV40). Foci of transformed cells were cloned and subsequently characterized by phase-contrast microscopy and immunocytochemistry. Growth characteristics of the clone that were analyzed included loss of dependence on conditioned medium and ability to grow in soft agar. Cytotoxicity experiments were performed, to determine whether the selected clone was susceptible to Acanthamoeba infection in vitro. RESULTS: A cell line which exhibited epithelial morphology, as determined by phase contrast microscopy, was selected and cloned. Immunocytochemistry demonstrated the presence of keratin in the cloned cells, confirming the epithelial nature of the cell line. Immortalization was shown by loss of dependence on fibroblast-conditioned medium, ability to form colonies in soft agar and no apparent senescence following numerous passages in culture. This cell line was found to be sensitive to the cytotoxic effects of a pathogenic strain of Acanthamoeba. CONCLUSIONS: An immortalized line of hamster corneal epithelial cells was developed. This clone is susceptible to infection with Acanthamoeba and will be a useful tool with which to investigate the pathogenesis of Acanthamoeba keratitis.

Seroanalysis of Tyzzer's disease in horses: implications that multiple strains can infect Equidae.

A monoclonal antibody based competitive inhibition assay was used to detect antibodies in horse sera to purified flagellar antigens from distinct Clostridium piliforme isolates. Sequential absorption of hyperimmune rat serum to C. piliforme isolate E (horse-origin isolate), a positive C. piliforme-immune horse serum, and other suspected immune horse sera with unrelated bacteria or C. piliforme isolates E or isolate R1 (rat-origin isolate) alone demonstrated the specificity of this assay for C. piliforme. This specificity was associated with the inhibition of monoclonal antibody binding to C. piliforme flagella, rather than to C. piliforme somatic antigens, by horse immunoglobulins partially purified from serum. Thirty seven of 162 horse sera possessed large amounts of antibody to the flagella of C. piliforme isolate E and 23 of the 162 had large amounts of antibody to the flagella of C. piliforme isolate R1; 9 of the sera possessed large amounts of antibody to both flagellar antigens. Absorption of these sera with isolate E or R1 demonstrated that antibody reactivity to the 2 C. piliforme isolates was isolate-specific and not due to antibody cross-reactive with both isolates. These results suggest that infection of horses with C. piliforme may be relatively common; and that they are susceptible to at least 2 distinct strains.

Experimental production of proliferative ileitis in Syrian hamsters (Mesocricetus auratus) by using an ileal homogenate free of Campylobacter jejuni.

The role of Campylobacter jejuni in the pathogenesis of proliferative ileitis of Syrian hamsters (Mesocricetus auratus) has been uncertain. C. jejuni has been implicated as the etiologic agent on the basis of the campylobacter-type morphology of the intracellular organism and the repeated microbiologic isolation of C. jejuni from hamsters with proliferative ileitis. The inability to reproduce the disease with pure culture inocula, coupled with immunohistochemical studies, however, has suggested that although C. jejuni may be present in the ilea of infected hamsters, its involvement in the pathogenesis of proliferative ileitis is questionable. In this study hamsters were inoculated with infective ileal homogenates prepared from ilea which were extensively washed to remove the ileal contents before grinding. The ilea from hamsters inoculated with this homogenate were also washed before being ground and used to experimentally inoculate a second group of hamsters. Of the 20 hamsters from this second group, 12 developed lesions typical of proliferative ileitis. Extensive microbiologic cultures from these hamsters were negative for C. jejuni. Immunofluorescence studies with a C. jejuni-specific monoclonal antibody were also negative. The use of a Campylobacter genus-specific monoclonal antibody, however, revealed numerous campylobacter-type organisms within the ileal epithelial cells of the crypts and villi. The presence of C. jejuni is therefore apparently not necessary for the production of proliferative ileitis in hamsters, and the intracellular campylobacter-type organism present in the ileal epithelial cells of infected hamsters is probably not C. jejuni.

Development of a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay for detection of Bacillus piliformis isolate-specific antibodies in laboratory animals.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect Bacillus piliformis isolate-specific antibodies in serum specimens from rats and gerbils experimentally infected with B. piliformis isolates R1, R2, or M. Detection was based on the ability of serum antibodies to block binding of B. piliformis isolate-specific monoclonal antibodies to purified B. piliformis flagella. Application of this assay to serum specimens collected from sham-infected or experimentally infected rats and gerbils demonstrated that the serum specimens were capable of specifically inhibiting the binding of B. piliformis isolate-specific monoclonal antibodies to homologous flagella preparations (> 70% inhibition) only when the serum specimens were from animals infected with the homologous B. piliformis isolate. Only one false-negative and false-positive result were obtained when 80 serum specimens were tested by this competitive inhibition ELISA. In addition, we demonstrated that little nonspecific inhibition of monoclonal antibody binding occurred (< 30% inhibition) in this immunoassay specific inhibition of monoclonal antibody binding by serum was due to serum antibody and a serum's ability to inhibit binding of monoclonal antibodies to purified B. piliformis flagella was correlated with antibody reactivity with B. piliformis flagella but not with serum antibody reactivity to whole B. piliformis organisms. These results suggest that this monoclonal antibody-based competitive inhibition assay could be successfully applied to the serologic identification of isolates involved in naturally occurring B. piliformis infections in laboratory animals.

Isolation of a Campylobacter-like organism from healthy Syrian hamsters (Mesocricetus auratus).

A Campylobacter-like organism was isolated from the ilea of normal hamsters. The organism was isolated from an ileal homogenate which was passed through a filter (0.65-micron pore size) and cultured on blood-agar plates in a microaerophilic atmosphere at 37 degrees C. Pinpoint translucent colonies were first observed after 120 h of incubation. The isolated organism measured 2.0 to 3.5 microns in length (excluding flagella) by 0.17 to 0.25 micron in width and typically had a single terminal sheathed flagellum. The organism was oxidase, catalase, and urease positive, reduced nitrates, and was susceptible to nalidixic acid (30-micrograms disk) and resistant to cephalothin (30-micrograms disk). Unlike Campylobacter pylori subsp. mustelae, this organism did not hydrolyze indoxylacetate. Immunofluorescence studies with a Campylobacter species-specific monoclonal antibody (8322-2E6) revealed the presence of numerous positively stained organisms within the crypt epithelial cells of the hamsters from which this organism was isolated. The role of this organism in the pathogenesis of proliferative ileitis in hamsters is uncertain, as is the taxonomic relationship of this organism to other members of the genus Campylobacter.

Utilization of monoclonal antibodies to evaluate the involvement of Campylobacter jejuni in proliferative ileitis in Syrian hamsters (Mesocricetis auratus).

The role of Campylobacter jejuni in the pathogenesis of proliferative ileitis in Syrian hamsters was evaluated with monoclonal antibodies of different specificities. Monoclonal antibodies were produced with two different specificities: one for all members of the genus Campylobacter tested (antibody 8322-2E6) and one for C. jejuni and Campylobacter coli (antibodies 841-2A11, 841-4C6, and 841-5B1). Heal sections from healthy hamsters, from hamsters with naturally occurring proliferative ileitis, and from hamsters with experimentally induced proliferative ileitis were examined by using fluorescein isothiocyanate-labeled, Campylobacter sp.-specific 8322-2E6 and tetramethylrhodamine isothiocyanate-labeled C. jejuni-C. coli-specific 841-2A11 for direct dual-labeling immunofluorescence. Organisms which stained with the C. jejuni-C. coli-specific monoclonal antibody were observed in the ileal lumens and along the distal tips of the villi of hamsters with either experimentally induced or naturally occurring proliferative ileitis. In contrast, organisms identified by the Campylobacter sp.-specific monoclonal antibody were present deep within the villus lumens and crypts and intracellularly within the apical portions of the epithelial cells. No organisms stained with the C. jejuni-C. coli-specific monoclonal antibody were observed in ileal sections from control hamsters; an occasional intracellular organism stained with the Campylobacter sp.-specific monoclonal antibody was observed in 2 of 10 control hamsters. Thus, at least two immunologically distinct patterns were identified in ileal sections from hamsters with proliferative ileitis. On the basis of these results, we conclude that the organism seen intracellularly in ileal sections from hamsters with proliferative ileitis is a member of the genus Campylobacter but that it probably is not C. jejuni or C. coli.