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An 8-year-old castrated male border terrier dog was diagnosed with acromegaly resulting from a growth hormone secreting pituitary tumor. Sixteen daily fractions of radiation therapy were delivered followed, approximately 1 year later, by administration of pasireotide. The aforementioned treatment was considered effective and should be further evaluated in similar cases., Competing Interests: Conflict of interest declaration: Dr. Herbert A. Schmid is employed by Novartis Pharma AG, Basel which manufactures and markets pasireotide for the treatment of hyperadrenocorticism and hypersomatotropism in humans.

Objectives: To investigate the association between domains of nutrition risk with hospitalisations and mortality for New Zealand Māori and non-Māori in advanced age., Methods: Within LiLACS NZ, 256 Māori and 399 non-Māori octogenarians were assessed for nutrition risk using the Seniors in the Community: Risk Evaluation for Eating and Nutrition (SCREEN II) questionnaire according to three domains of risk. Sociodemographic and health characteristics were established. Five years from inception, survival analyses examined associations between nutrition risk from the three domains of SCREEN II with all-cause hospital admissions and mortality., Results: For Māori but not non-Māori, lower nutrition risk in the Dietary Intake domain was associated with reduced hospitalisations and mortality (Hazard Ratios [HR] [95%CI] 0.97 [0.95-0.99], p=0.009 and 0.91 [0.86-0.98], p=0.005, respectively). The 'Factors Affecting Intake' domain was associated with mortality (HR, [95%CI] 0.94 [0.89-1.00], p=0.048), adjusted for age, gender, socioeconomic deprivation, education, previous hospital admissions, comorbidities and activities of daily living., Conclusion: Improved dietary adequacy may reduce poor outcomes for older Māori. Implications for public health: Nutrition risk among older Māori is identifiable and treatable. Effort is needed to engage relevant community and whānau (family) support to ensure older Māori have food security and cultural food practices are met., (© 2018 The Authors.)

The present study focuses on the use of copolymer nanoparticles as a dispersant for a model pigment (silica). Reversible addition-fragmentation chain transfer (RAFT) alcoholic dispersion polymerization was used to synthesize sterically stabilized diblock copolymer nanoparticles. The steric stabilizer block was poly(2-(dimethylamino)ethyl methacrylate) (PDMA) and the core-forming block was poly(benzyl methacrylate) (PBzMA). The mean degrees of polymerization for the PDMA and PBzMA blocks were 71 and 100, respectively. Transmission electron microscopy (TEM) studies confirmed a near-monodisperse spherical morphology, while dynamic light scattering (DLS) studies indicated an intensity-average diameter of 30 nm. Small-angle X-ray scattering (SAXS) reported a volume-average diameter of 29 ± 0.5 nm and a mean aggregation number of 154. Aqueous electrophoresis measurements confirmed that these PDMA 71 -PBzMA 100 nanoparticles acquired cationic character when transferred from ethanol to water as a result of protonation of the weakly basic PDMA chains. Electrostatic adsorption of these nanoparticles from aqueous solution onto 470 nm silica particles led to either flocculation at submonolayer coverage or steric stabilization at or above monolayer coverage, as judged by DLS. This technique indicated that saturation coverage was achieved on addition of approximately 465 copolymer nanoparticles per silica particle, which corresponds to a fractional surface coverage of around 0.42. These adsorption data were corroborated using thermogravimetry, UV spectroscopy and X-ray photoelectron spectroscopy. TEM studies indicated that the cationic nanoparticles remained intact on the silica surface after electrostatic adsorption, while aqueous electrophoresis confirmed that surface charge reversal occurred below pH 7. The relatively thick layer of adsorbed nanoparticles led to a significant reduction in the effective particle density of the silica particles from 1.99 g cm -3 to approximately 1.74 g cm -3 , as judged by disk centrifuge photosedimentometry (DCP). Combining the DCP and SAXS data suggests that essentially no deformation of the PBzMA cores occurs during nanoparticle adsorption onto the silica particles., Competing Interests: The authors declare no competing financial interest.

A xenogeneic DNA vaccination has been licensed for use in dogs with locally controlled stage II and III oral malignant melanoma (OMM). At present, there are limited outcome data for dogs with OMM treated with surgery and immunotherapy. The aim of this study is to retrospectively review the outcome and survival of 32 dogs affected by OMM that were treated with a combination of surgery and the xenogeneic DNA vaccination (with the addition of radiotherapy in some cases) and to determine the influence of surgical margins and delay in receiving vaccination. The overall median survival time (MST) was 335 days (95% CI: 301-540 days), and the overall median progression-free survival (PFS) was 160 days (mean 182 days, 95% CI: 132-232 days). Stage, completeness of surgical margins and delay in administration of the vaccine did not appear to statistically influence survival or PFS, although these results may reflect the low statistical power of the study due to small numbers. Further studies are required to assess whether the addition of any adjuvant treatment to surgery, including immunotherapy, is able to significantly prolong survival in cases of canine oral melanoma.

Objectives: To determine if proteinuria is more common in dogs with lymphoma when compared with healthy dogs and to assess the severity and frequency of proteinuria in dogs with lymphoma., Methods: Determination of urine protein:creatinine ratio in 32 dogs with lymphoma compared with 30 healthy dogs., Results: Canine patients with lymphoma are more likely to be proteinuric compared with healthy dogs. Proteinuria is common in dogs with lymphoma, although in most cases it is not severe. The presence of proteinuria is not linked with the stage or substage of lymphoma., Clinical Significance: Mild proteinuria is a common finding in dogs with lymphoma. The clinical impact of the proteinuria is probably low., (© 2012 British Small Animal Veterinary Association.)

Little information is available on the occurrence of neoplasms in dogs up to the age of 12 months. This is a retrospective review of histopathological diagnoses of neoplasia in dogs up to the age of 12 months based on biopsy specimens submitted to a commercial veterinary diagnostic laboratory in the United Kingdom between 1993 and 2008. In 20 280 histological submissions, 9522 neoplasms were identified. Canine cutaneous histiocytoma (n = 8465; 89%) was the most common histological type. Neoplasms other than histiocytoma (n = 1057; 11%) were grouped as benign epithelial (n = 375; 4%), haematopoietic (n = 229; 2%), benign mesenchymal (n = 145; 2%), miscellaneous (n = 118; 1%), non-hematopoietic malignant mesenchymal (n = 118; 1%) or malignant epithelial tumours (n = 72; <1%). Excluding canine cutaneous histiocytoma, 52% of tumours (n = 547) were benign, and 66% were from the skin or soft tissues. These data provide valuable epidemiological information on neoplasms occurring in juvenile dogs in the United Kingdom., (© 2010 Blackwell Publishing Ltd.)

Objectives: To determine which types of tumour occur in cats up to the age of 12 months based on biopsies submitted to Idexx Laboratories, Wetherby, UK., Methods: Retrospective review of histopathological diagnoses of tumours in cats up to the age of 12 months from biopsies received between September 1993 and March 2008., Results: A total of 4196 submissions from cats 12 months old or younger were identified; 233 biopsies (6%) were neoplastic and fulfilled the search criteria. Tumours were categorised as haematopoietic (n=73, 31%), malignant epithelial (n=44; 19%), malignant mesenchymal (n=38; 16%), benign epithelial (n=37; 16%), benign mesenchymal (n=30, 13%) and miscellaneous (n=11; 5%). The most frequent tumours were lymphoma (n=51; 22%), soft-tissue sarcoma (n=34; 15%), mast cell tumour (n=22; 9%) and squamous cell carcinoma (n=16; 7%). The most common tumour site was the skin and soft tissues (41% of tumours). In all, 164 neoplasms (70%) were malignant or had malignant potential., Clinical Significance: These data provide unique epidemiological information on a poorly characterised subgroup of feline cancer patients in the UK.

The primary goal of this study was to establish an objective baseline for subjects who participated in a study in an immersed environment created for the virtual reality therapy (VRT) situation. Since the effects of VRT on the subjects treated for neurosis have traditionally been measured by subjective measurements, there is a need to include objective measures. This will improve and validate the effectiveness of VRT. Fifteen college students participated in this study. Specifically, the researchers measured the activity of the subjects' brainwaves in response to the VRT using EEG technology. The preliminary data indicated that, in most cases, subjects had a decline in brain wave activity between what is deemed a normal / baseline brain activity and the brainwave activity recorded when they were when they were connected to the virtual reality equipment and under influence of an immersive scene. In rare instances, there were some subjects that showed extreme increases in brain activities. In addition, the data indicated that, in most cases, subjects are more relaxed while under the immersive influence with respect to brain activities than those that are not.

This pilot study is the first known in-depth case study of the effectiveness of virtual reality therapy (VRT) as a treatment for Test Anxiety (TA). The subject of the study was a 28-year-old male, whose anxiety and avoidance behavior was interfering with his normal academic activities. For treatment, he was placed in a virtual classroom and later in a virtual auditorium. The subject was exposed to six moderately increasing in difficulty level virtual situations. The subject rated each situation for discomfort. As a simple measure of anxiety, a modified version of the Subjective Units of Disturbance (SUD) scale was used every five minutes during exposure. This case study showed VRT to be an effective treatment method for reducing self-reported TA. Symptoms experienced by the subject during VRT sessions were just as real to the subject as actual test taking and general TA situations. They included increased heart rate, mild dizziness, and headaches. This case study of TA indicates that VRT may be used as an effective treatment method for reducing self-reported anxiety and improving the performance of subject(s) who suffer from TA.

Behavioral therapy techniques for treating phobias often includes graded exposure of the patient to anxiety-producing stimuli (Systematic Desensitization). However, in utilizing systematic desensitization, research reviews demonstrate that many patients appear to have difficulty in applying imaginative techniques. This chapter describes the Virtual Reality Therapy (VRT), a new therapeutical approach that can be used to overcome some of the difficulties inherent in the traditional treatment of phobias. VRT, like current imaginal and in vivo modalities, can generate stimuli that could be utilized in desensitization therapy. Like systematic desensitization therapy, VRT can provide stimuli for patients who have difficulty in imagining scenes and/or are too phobic to experience real situations. As far as we know, the idea of using virtual reality technology to combat psychological disorders was first conceived within the Human-Computer Interaction Group at Clark Atlanta University in November 1992. Since then, we have successfully conducted the first known pilot experiments in the use of virtual reality technologies in the treatment of specific phobias: fear of flying, fear of heights, fear of being in certain situations (such as a dark barn, an enclosed bridge over a river, and in the presence of an animal [a black cat] in a dark room), and fear of public speaking. The results of these experiments are described.

Behavioral therapy techniques for treating phobias often includes graded exposure of the patient to anxiety-producing stimuli (Systematic Desensitization). However, in utilizing systematic desensitization, research reviews demonstrate that many patients appear to have difficulty imaging the prescribed anxiety-evoking scene. They also express strong aversion to experiencing real situations. This chapter describes the Virtual Reality Therapy (VRT), a new therapeutical approach that can be used to overcome some of the difficulties inherent in the traditional treatment of phobias. VRT, like current imaginal and in vivo modalities, can generate stimuli that could be utilized in desensitization therapy. Like systematic desensitization therapy, VRT can provide stimuli for patients who have difficulty in imagining scenes and/or are too phobic to experience real situations. Unlike in vivo systematic desensitization, VRT can be performed within the privacy of a room, thus avoiding public embarrassment and violation of patient confidentiality. VRT can generate stimuli of much greater magnitude than standard in vivo techniques. Since VRT is under patient control, it appears safer than in vivo desensitization and at the same time more realistic than imaginal desensitization. Finally, VRT adds the advantage of greater efficiency and economy in delivering the equivalent of in vivo systematic desensitization within the therapist's office. The chapter also describes how to use virtual reality in the treatment of specific phobias: fear of flying, fear of heights, fear of being in certain situations (such as a dark barn, an enclosed bridge over a river, and in the presence of an animal [a black cat] in a dark room), and fear of public speaking.

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1 alpha and tumor necrosis factor-alpha on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.

The DiFi colorectal carcinoma cell line, derived from a patient with familial adenomatous polyposis, was examined for gene expression and production of the autocrine growth factor transforming growth factor alpha (TGF-alpha) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-alpha transcripts as identified on Northern (RNA) blots. Addition of TGF-alpha (10 ng/ml) or EGF (10 ng/ml) to DiFi cell cultures (lacking EGF or serum) up-regulated DiFi cell basal TGF-alpha mRNA levels, suggesting that autoinduction of TGF-alpha occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-alpha (347 pg/10(6) cells/24 h) into their culture medium, as determined by radioimmunoassay. DiFi cells showed strong overexpression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed enhanced EGFR expression in a cell subpopulation among the original (uncultured) ascitic fluid cells from which the DiFi cell line was established. This cell subpopulation was observed to expand dramatically between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphorylate. The EGFR gene in these cells was not found to be rearranged or genetically altered using Southern blot analysis. Dot blot analysis of DiFi cell DNA revealed EGFR gene amplification in the range of 60-80 copies/cell, which is approximately twice the copy number seen in A-431 epidermoid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because DiFi colorectal cancer cells uniquely show production and auto-induction of TGF-alpha in addition to amplification and overexpression of the EGFR gene, these cells represent a valuable tool for studying the role(s) of the EGFR in the regulation of tumor cell growth.

We used immunohistochemical procedures to study the cellular expression and distribution of cytokeratins (CKs) in rat 13762NF mammary adenocarcinoma cells growing at mammary fat pad sites and at spontaneous lymph node and lung sites. In order to establish CK distribution in normal rat mammary epithelia, immature, resting, and lactating rat mammary glands were probed with a panel of monospecific antibodies that recognize individual CKs. Basal/myepithelial cells were distinguished by expression of CKs 5 and 14 and coexpression of vimentin from luminal cells, which expressed CKs 8, 18, and 19. Antibody to CK 7 recognized luminal epithelium of immature and resting, but not lactating, mammary glands. Myoepithelial cells of lactating mammary gland were weakly recognized by antibodies to CKs 7 and 19. Tumors formed by cell lines and clones derived from parental 13762NF tumor (MTPa, MTC, MTA, and MTF7) were not recognized by any of the anti-CK antibodies. Only vimentin was expressed in these tumors and their metastases. In tumors and metastases generated from cell lines and clones derived from lymph node (MTLY) and lung metastases (MTLn2 and MTLn3) of the 13762NF tumor we observed heterogeneous CK phenotypes. Expression of CKs 5 and 18 was greatly reduced or lacking, while CK 14 was coexpressed with CKs 7, 8, and 19 with or without vimentin. Tumors from the highly metastatic clone MTLn3 had a dominant cellular phenotype, expressing CKs 7, 8, 14, and 19 and vimentin, a pattern that did not match normal mammary epithelia, whether luminal, basal/myoepithelial, or the dual-phenotype stem cell, in which CKs 5, 8, 14, and 18 were coexpressed. MTLn3 lymph node and lung metastases expressed the same cellular phenotype as the s.c. growing MTLn3 tumor. The results appear to contradict the belief that malignant mammary tumors may be distinguished from benign tumors or hyperplastic growths by the lack of basal/myoepithelial markers.

Monoclonal antibody (mAb) MT10:21, a rat IgG2a, reacts with antigens expressed on the metastatic subclone MTLn3 of the 13762NF rat mammary adrenocarcinoma (North, Steck & Nicolson, 1986). The clearance of mAb MT10:21 from circulation was monitored 15 days after s.c. injection of MTLn3 tumour cells into the mammary fat pad of syngeneic rats. At this point, when tumour-burden was small (< 1 cm average diameter), mAb MT10:21 was injected i.v. and serum samples were taken over the 7 days following injection. It was observed that when mAb MT10:21 was injected i.v. into syngeneic rats it induced a humoral immune response. Four days after injection of mAb, IgM serum antibodies which bound to the MTLn3 cell line were detected in both tumour and non-tumour bearing rats. Using a binding assay, tumour-bearer sera showed a steady increase in MTLn3-reactive antibodies over the 7-day assay period. These MTLn3-reactive antibodies were detectable in non-tumour-bearer sera, but reactivity was not as pronounced. Tumour-bearing rats also had serum IgM antibodies which bound to mAb MT10:21 in vitro, but not to a non-specific, isotype-identical control mAb, MC9:13. These serum antibodies were able to partially inhibit (up to 47%) the binding of 125I-labelled mAb MT10:21 to MTLn3 cells. This anti-idiotypic immune response was not observed in the non-tumour bearers. When the tumour was allowed to grow for a period of 30 days in vivo (average tumour diameter 2 cm), these serum antibodies were not readily detectable, suggesting that tumour burden had a significant effect on suppressing the humoral immune responsiveness of these tumour-bearing rats. In a standard delayed-type hypersensitivity (DTH) assay specific immunity to mAb MT10:21 was induced in vivo.

Spleen cells from rats bearing syngeneic metastatic 13762NF mammary adenocarcinoma clone MTLn3 tumors were fused with the rat myeloma Y3 Ag1.2.3 to generate a panel of monoclonal antibodies (MAbs). The MAbs could be divided into three groups: those cross-reactive with all 13762NF cells; those reactive with cloned MTLn3 and MTC cells; and those predominantly reactive with the highly metastatic MTLn3 cells. One of these MAbs, MT10:21 (an immunoglobulin G2a), binds predominantly to highly metastatic MTLn3 cells and has a high tumor-cell affinity as determined by its saturation kinetics. MAb MT10:21 has a 6-h half-life on the MTLn3 cell surface and a 24-h half-life in the blood of syngeneic rats. Immunoblotting experiments using lysates from the cloned 13762NF sublines revealed that MAb MT10:21 binds to several proteins having relative molecular weights of 72,000, 73,000, and 120,000. Using an immunohistochemical procedure with frozen tissue sections, MAb MT10:21 shows little reactivity with normal rat mammary tissue, irrespective of the stage of the estrous cycle, and it failed to react with a number of other normal fetal and adult tissues. Furthermore, MAb MT10:21 is heterogeneous in its reactivity to cloned sublines of the 13762NF mammary adenocarcinoma, on both tissue cultured cells and tissue sections prepared from tumors growing in situ in the mammary fat pads of syngeneic rats. MAb MT10:21 reacted with certain human breast cancer cell lines and with a subpopulation of metastatic human breast cancer cells in frozen tissue sections from biopsies and autopsies. Metastases from breast cancers reacted more intensely than the primary tumors from which they were derived.

We analyzed the epidermal growth factor receptor gene using a complementary DNA probe of the epidermal growth factor receptor gene in 21 uncultured primary breast carcinomas and found that the gene was amplified in three of these tumors. We further demonstrated by immunohistochemistry using a monoclonal antibody to the epidermal growth factor receptor that the receptor protein product of this gene was overexpressed and displayed elevated kinase activity. Our data indicate that one of the molecular mechanisms for overexpression of epidermal growth factor receptor in human breast cancer is epidermal growth factor receptor gene amplification without rearrangement in a subset of tumors.

Hybridomas secreting monoclonal antibodies with specificity for Hooded rat fibrosarcomata have been obtained by fusion of the rat myeloma Y3 Ag 1.2.3. (Galfre, Milstein & Wright, 1979) with cells taken from spleens or lymph nodes of immunized syngeneic and allogeneic donors. Two of the monoclonal antibodies, both derived from he spleens of tumour bearers, showed specificity for individual tumours one for MC24 (M10/76) and the other for HSNTC (11/160). These two antibodies had a long half-life in the blood when injected intravenously showing that they had a low affinity for normal tissue antigens. Monoclonal antibodies exhibiting broad tumour specificity or extensive cross-reactivity with normal cells were secreted by many of the hybridomas derived from both syngeneic and allogeneic rats that had been hyperimmunized with tumour cells. These results are discussed in relation to the production of monoclonal anti-tumour antibodies for use in experimental therapy.

Rat 13762NF mammary adenocarcinoma cells (clone MTC) were heated to 42 degrees C either in vivo as a subcutaneous tumour in the rat mammary fat pad or in vitro as attached cells. Labelling in vivo or in vitro detected very similar heat-stress proteins (hsp) at 160, 112, 90, 70 and 56 kDa. Syngeneic rat endothelial and macrophage cells synthesized several cellular proteins in vitro differently than did the tumour cells in vitro, but both types of normal cells were similar to tumour cells in the hsp synthesized. Although the quantitative aspects of induction and repression of hsp may depend on cell type and microenvironment, the major tumour hsp being studied for function in vitro were qualitatively similar to those produced and labelled in vivo in response to a similar heat dose. Hsp were similar in both normal cells and tumour cells from the same host. These observations support the concept that hsp function in fundamental processes in the different microenvironmental and metabolic conditions found in vivo and in vitro. In addition, these observations suggest that prediction of tumour thermal response by measuring hsp levels may be influenced by host cell components.

Hooded Lister/Cbi rats bearing the HSN.TC fibrosarcoma produced a high-titre non-complement-binding IgG antibody, and tests in vitro indicated that the syngeneic antibody was specific for this tumour. About 1.4 X 10(5) antibody molecules were bound per cell, a figure one eighth that for cells treated with a high-titre allo-antiserum. When tumour-bearer serum was passively transferred into congenitally athymic rats bearing the HSN.TC tumour the antibody was absorbed out specifically, by comparison with control animals or athymic rats bearing an unrelated tumour that was also syngeneic in Hooded rats. The kinetics of loss of antibody from the surface of HSN.TC cells has been monitored in vitro and the antibody has been found to have an extended half-life at the cell surface (greater than 40 h).

Glycoprotein profiles of rat macrophages (M phi) at different stages of activation were studied by examining the reactivity of various lectins to the glycoproteins separated by polyacrylamide gel electrophoresis. Ricinus communis agglutinin 1 (RCA1) revealed several components including glycoproteins of Mr 160 kDa and 65 kDa prominent in resident M phi. A pokeweed mitogen (PWM) isolectin, Pa-4, recognizes branched poly(N-acetyllactosamine)-type carbohydrate chains, and revealed a significant increase in glycoproteins of Mr ranging from 70 kDa to 150 kDa on thioglycolate-elicited M phi. Increased reactivity of PWM to thioglycolate-elicited M phi was observed by direct binding of 125I-labeled Pa-4 to intact or glutaraldehyde-fixed M phi. Histochemical staining of formaldehyde-fixed M phi in vitro with biotinylated Pa-4 was consistent with the gel analysis, that is, resident M phi had no reactivity while thioglycolate-elicited M phi showed slight reactivity. Alveolar and intratumoral M phi bound more Pa-4 than resident or thioglycolate-elicited M phi. The PWM isolectin may therefore serve as a marker for an early stage of M phi activation.

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.

A rat hybridoma producing IgM monoclonal antibody (MAb) GP21:56 was generated with specificity for a high-molecular-weight, mucin-like glycoprotein (gp580) present on highly metastatic 13762NF rat mammary adenocarcinoma cells. The hybridoma was made by fusing rat Y3 Ag1.2.3 myeloma cells with spleen cells from a rat immunized i.d. with purified gp580. The gp580 appeared to be of low immunogenicity in syngeneic F344 rats because a total of 27 fusions were required to produce one hybridoma with specificity for this glycoprotein. Immunoblotting of purified gp580 after electrophoresis in 1% agarose and antibody-binding assays using purified gp580 linked to microtiter plates confirmed that MAb GP21:56 bound specifically to gp580. Other MAbs made against breast mucins were negative for gp580 reactivity. Enzyme-linked immunoabsorbent assays (ELISA) and radiolabelled antibody binding assays demonstrated that MAb GP21:56 bound to 13762NF adenocarcinoma cell lines and clones in relation to their spontaneous metastatic potentials; significantly more MAb GP21:56 bound to highly metastatic MTLn3 cells than to low metastatic MTC cells, and MAb GP21:56 showed little reactivity towards the majority of other cell lines tested, whether of rodent or of human origin. Kinetic binding studies indicated that MAb GP21:56 does not have a high affinity for gp580 but, once bound, it shows high avidity for this sialogalactoprotein. Localization studies using frozen tissue sections of 13762NF tumors indicated that MAb GP21:56 reacts with tumor cells grown in vivo in an analogous manner to in vitro cultured cells. Using immunoperoxidase techniques, less than 50% of the highly metastatic MTLn3 tumor cells were stained, whereas approximately 20% of the intermediate metastatic MTF7 and MTLn2 cells and less than 10% of low metastatic MTC and MTPa cells were stained with MAb GP21:56. The cell-to-cell reactivity was heterogeneous and mainly associated with the tumor-cell surface and extracellular matrix.

A Mr 74,000 phosphoglycoprotein (gp74) present on the surface of oncogene-transformed murine cells but not untransformed NIH 3T3 cells was previously identified with mouse monoclonal antibody 45-2D9. The original cell population used as the immunogen was found to consist of two cell populations. The purpose of this study was to characterize these cell populations; determine the distribution of gp74 on normal, transformed, and neoplastic cells; and to characterize the gp74 molecule. Southern hybridization studies of cloned cell populations demonstrated that the immunizing cell population consisted of c-Ha-ras-transfected NIH 3T3 cells and Kirsten sarcoma virus-transformed rat cells (TRF cells). TRF cells showed a high level of gp74 expression. We observed that the expression of gp74 was increased on chemically and spontaneously transformed rat cells compared to untransformed rat cells. No binding of monoclonal antibody 45-2D9 was detected to rat adult and fetal tissue. Immunoperoxidase staining, immunofluorescence flow cytometry, and immunoprecipitation analysis of dimethylbenz[a]anthracene-induced metastatic 13762NF rat mammary adenocarcinoma clonal sublines demonstrated an inverse relationship between gp74 expression and metastatic phenotype. gp74 was immunoprecipitated from two low and medium metastatic clonal sublines (MTC and MTF7), but not from highly metastatic clone MTLn3 cells. Biosynthetic labeling and immunoprecipitation studies demonstrated that gp74 was phosphorylated on serine residues and was not secreted from transformed cells. No detectable protein kinase activity in an immune complex assay was associated with this molecule. We conclude that increased gp74 expression by rat cells is associated with transformed and neoplastic cells.

The IgG2b monoclonal antibody M10/76, which is specific for the Hooded rat fibrosarcoma MC24, was obtained by fusion of the rat myeloma Y3 Ag 1.2.3 with spleen cells from an MC24 tumour-bearing rat. This antibody has been found to inhibit lung colonization when MC24 cells were injected i.v. into Hooded rats, and when passively transferred into athymic MC24 tumour bearers, it prevented or considerably reduced the incidence of spontaneous lung metastases in more than half the treated animals.

The importance of host immune status on the spontaneous metastasis of cloned cell lines of the 13762NF rat mammary adenocarcinoma was examined. Cell lines MTLn3 (high metastatic potential), MTF7 and MTLn2 (intermediate metastatic potential) and MTC (low metastatic potential) were subjected to a series of in vivo assays designed to assess how manipulation of the immune system in the syngeneic F344 host would affect the ability of these cells to metastasise. Treatment of tumour bearing rats with the immunosuppressive agents cyclosporin A or cyclophosphamide had little influence on metastasis in this system. Growth of tumours in congenitally athymic nude rats resulted in reduction of observed metastases. In addition, humoral immune response was not detectable during a 23-day period of tumour growth in F344 rats. Excision of the tumour growing in situ reduced the number of metastases when the tumours were resected early (less than 10 days), but at later times tumour resection did not influence the incidence of metastasis. The importance of initial lymphatic rather than haematogenous routes of dissemination was confirmed in experiments where the draining inguinal and axillary lymph nodes were removed at different times either before, or after, subcutaneous mammary fat pad injection of metastatic tumour cells.

The macrophage content of spontaneous metastases has been quantified morphometrically for a panel of rodent tumors at different stages of metastatic tumor growth. Using a histochemical technique to selectively stain macrophages, we have evaluated the relative content of macrophages in spontaneous pulmonary metastases from the 13762NF MTLn3 rat mammary adenocarcinoma and the B16-BL6 mouse melanoma, as well as in spontaneous hepatic metastases from the M5076 mouse reticulum cell sarcoma and from autochthonous reticulum cell sarcomas in SJL/J mice. Between 112 and 254 separate, individual metastases were evaluated for each of these tumors. The data show that the relative macrophage content of very small metastases is high. However, as metastases grow the relative macrophage content falls, reaching uniformly low levels by the time the metastases are 0.5 mm in diameter. These data are very similar to our previous observations on experimental metastases where the same pattern of high macrophage content in small metastases was seen. Finding the same pattern in more slowly growing, spontaneous metastases of tumors derived from several different tissues and in two species suggests that the fall in relative macrophage content is not a phenomenon isolated to experimental metastases, a particular site, or a tissue of origin for the tumor. The relative decrease in macrophage content may thus be a general phenomenon with important implications for immunotherapy directed to enhancing the tumoricidal activity of macrophages.

The collagenolytic responses of normal rat skin fibroblasts (NRS-F) and rat mammary MTLn3 tumor-derived fibroblasts (Ln3-F) were examined following exposure to rat macrophage (M phi-CM)- and lymphocyte (LYM-CM)-conditioned culture medium and/or tumor cell-conditioned medium. Alveolar, intratumoral, and peritoneal macrophages were prepared from mammary adenocarcinoma-bearing rats, as were the peritoneal lymphocytes. Incubation of the two fibroblast populations with LYM-CM produced a 10- and 7-fold stimulation of collagenolytic activity by NRS-F and Ln3-F cells, respectively. Similarly, exposure of NRS-F and Ln3-F fibroblasts to peritoneal M phi-CM produced a 7- and 4-fold increase in the expression of collagenolytic activity, respectively. Conditioned medium from MTLn2 tumor cells also stimulated the collagenolytic expression of both fibroblast populations. Incubation of tumor-associated Ln3-F or NRS-F fibroblasts with MTLn2 tumor cell-conditioned medium enhanced fibroblast collagenolytic activity approximately 20 and 17 times, respectively. When M phi-CM and LYM-CM were further "conditioned" by a subsequent incubation with MTLn2 tumor cells, each stimulated the expression of collagenolytic activity by both fibroblast populations and this was especially pronounced (120-fold increase) in the response of Ln3-F to LYM-CM further conditioned by MTLn2 tumor cells. The conditioned media derived from M phi, LYM, and MTLn2 tumor cells with or without trypsin activation contained low levels of interstitial-type collagenolytic activity which made no significant contribution to the collagenolytic activity of the stimulated fibroblasts. Some collagenase inhibitory activity, however, was detected in the M phi-CM, suggesting that the actual stimulation of collagenolysis by host fibroblasts is underestimated. We conclude that macrophages, lymphocytes, and tumor cells all have the potential to produce stimulatory factor(s) which enhance the collagenolytic activity of normal fibroblast populations. This study provides further evidence of the multifactorial control of collagenase production and supports the concept that host cell-tumor cell interactions can enhance the expression of collagenolytic enzymes.

We report a method for the production of rat hybridomas secreting IgA antibodies with biological activity. By fusing the rat myeloma Y3.AG.1.2.3 with mesenteric lymph node cells taken from animals that had been immunised via the Peyer's patches with allogeneic cells we have obtained a hybridoma secreting monoclonal antibodies of the IgA class with specificity for RT1c.

The susceptibility of cloned cell lines of the 13762NF rat mammary adenocarcinoma to macrophage-mediated cytolysis was investigated using both intra- and extratumoral macrophages. The percentage of Fc receptor-positive cells in tumors growing s.c. in syngeneic F344 rats ranged from 8 to 20%, but we could not demonstrate a significant correlation between the number of Fc receptor-positive cells within tumors and their spontaneous metastatic potentials. In macrophage-mediated cytolysis assays, cloned 13762NF cell lines of differing metastatic potential, established from tissue culture lines, fresh tumor explants, or short-term cultures (one passage in vitro), were used as targets. Effector cells were thioglycolate-elicited peritoneal macrophages (activated in vitro with bacterial lipopolysaccharide) or intratumoral macrophages (activated in vitro with lipopolysaccharide). When the effector cells were peritoneal macrophages, established cloned 13762NF cell lines showed little correlation in their susceptibility to macrophage-mediated cytolysis and metastatic potential, while this was not observed when fresh tumor explants were used. Highly metastatic MTLn3 cells were the least sensitive, less metastatic MTF7 and MTLn2 cells were more susceptible, and the low metastatic parental MTPa cells were the most sensitive in 72-h cytolysis assays. When the effector cells were intratumoral macrophages, all 13762NF cell lines showed less sensitivity in cytolysis assays than similar assays using thioglycolate-elicited peritoneal macrophages. With the exception of line MTLn2, short-term cultures (one passage in vitro) did not differ substantially in susceptibility to intratumoral macrophages compared to fresh explants. In this system, the sensitivity of 13762NF cells to macrophage-mediated cytolysis is a function of effector as well as target cell source.