Active immunoprophylaxis and immunotherapy in two mouse lymphoma models.

Injections of appropriate numbers of irradiated tumor cells produced antibodies against tumor cell-surface antigen(s) in both syngeneic tumor models studied: the early transplant generations of the spontaneous L2 lymphoma in AKR/J mice and the chemically induced EL 4 lymphoma in C57BL/6J mice. No antibody was detected in normal or nonimmunized tumor-bearing mice. Tumor inhibitory or enhancing activity was not demonstrated by these antibodies. Immunoprophylaxis or cell-mediated immunity against the L2 lymphoma was not observed after injections of irradiated L2 cells and/or BCG into AKR mice. However, injections of irradiated EL 4 cells alone were effective in immunoprophylaxis against as many as 10(6) EL 4 cells and in immunotherapy against 10(2) EL 4 cells per mouse. The addition of BCG injections made immunotherapy with irradiated EL 4 cells effective against a load of 10(4) EL 4 cells/mouse, though BCG alone was not effective for immunoprophylaxis against EL 4 cells. Resistance to EL 4 could be transferred with viable syngeneic peritoneal or nucleated spleen cells. In both tumor models, an ongoing delayed hypersensitivity reaction to BCG alone apparently did not inhibit bystander tumor cells even when tumor cells were mixed before inoculation with viable BCG. In neither tumor model were concanavalin A-coated tumor cells more potent for immunoprophylaxis than were irradiated tumor cells alone.

Immunochemotherapy of malignant melanoma with chlorambucil-bound antimelanoma globulins: preliminary results in patients with disseminated disease.

Thirteen consecutive patients with inoperable recurrent malignant melanoma were treated by immunochemotherapy with the use of chlorambucil noncovalently bound to goat or rabbit antihuman melanoma globulins. The next consecutive 11 patients fulfilling the criteria for admission into this study were treated with chemotherapy only, i.e., dimethyltriazenoimidazole carboxamide (DTIC). Follow-up was for a minimum of 29 months or until death. Two patients showing an objective response to immunochemotherapy had disease confined to lymph nodes and cutaneous sites; 5 others showed stabilization of cutaneous, nodal, and visceral disease, and 6 patients showed progression of their disease. The median survival of the responders and stabilizers was 20 months, but only 3.5 months for patients with disease progression. None of the 11 patients treated with DTIC had objective tumor regression, and all died within 11 months of the start of treatment with a median survival of 3 months. Immunochemotherapy significantly prolonged the survival compared to that in the DTIC-treated group (P less than 0.05). No hematologic or renal toxicity was detected after immunochemotherapy, but 2 patients in this group developed anaphylactic reactions. Skin reactivity tests to dinitrochlorobenzene and purified protein derivative were of no prognostic value

Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody.

External photoscanning with display of radioactivity data as a color-scaled image detected xenografts of human melanoma in male nude inbred mice of BALB/c background 48 hours after injection of 131I-labeled monoclonal IgG 225.28S that is specific for human melanoma. A 131I-labeled polyclonal goat IgG against human melanoma-associated antigens could also image the tumor, but with this preparation there was considerable localization of radioactivity in normal tissues, resulting in less satisfactory tumor definition. Labeled normal mouse IgG did not image the melanoma grafts. Assay of radioactivity in tissues of melanoma-grafted mice confirmed tumor-specific localization of the antimelanoma antibodies. The tumor:blood ratio of radioactivity was 6.55 with the monoclonal antimelanoma IgG and 0.45 with the polyclonal IgG.

Desmosomes: intercellular adhesive junctions specialized for attachment of intermediate filaments.

Cell-cell adhesion is thought to play important roles in development, in tissue morphogenesis, and in the regulation of cell migration and proliferation. Desmosomes are adhesive intercellular junctions that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role in maintaining tissue integrity. Recent observations indicate that tissue integrity is severely compromised in autoimmune and genetic diseases in which the function of desmosomal molecules is impaired. In addition, the structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have now been elucidated. Finally, the molecular constituents of desmosomes and other adhesive complexes are now known to function not only in cell adhesion, but also in the transduction of intracellular signals that regulate cell behavior.

Assembly of desmosomal cadherins into desmosomes is isoform dependent.

Desmosomes are intercellular adhesive junctions that exhibit cell- and differentiation-specific differences in their molecular composition. In complex epithelia, desmosomes contain multiple representatives of the desmosomal cadherin family, which includes three desmogleins and three desmocollins. Rules governing the assembly of desmosomal cadherin isoforms into desmosomes of different cell types are unknown. Here we compared the assembly properties of desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), which are widely expressed, with Dsg1 and Dsc1, which are expressed in the differentiated layers of complex epithelia, by introducing myc-tagged forms into simple and squamous epithelial cells that do not express Dsg1 or Dsc1. Dsc2.myc and Dsg2.myc assembled efficiently into desmosomes in every cell type in spite of significant shifts in the stoichiometric relationship between desmogleins and desmocollins. In contrast, Dsc1a.myc, Dsc1b.myc, and Dsg1.myc did not stably incorporate into desmosomes in any line. Coexpression of Dsc1a.myc or Dsc1b.myc and Dsg1.myc did not lead to their colocalization and failed to enhance incorporation of either cadherin into desmosomes. Dsg1.myc, but not Dsc1a, Dsc1b, disrupted desmosome assembly in a cell-type-specific manner, and disruption correlated with the recruitment of Dsg1.myc, but not Dsc1a or Dsc1b, into a Triton-insoluble pool. The plakoglobin:E-cadherin ratio decreased in Dsg1-expressing cells with disrupted desmosomes, but a decrease was also observed in a Dsc1a line. Thus, a modest reduction of plakoglobin associated with E-cadherin is apparently not sufficient to disrupt desmosome assembly. Our results demonstrate that desmosome assembly tolerates large shifts in cadherin stoichiometry, but is sensitive to isoform-specific differences exhibited by desmogleins and desmocollins.

Radionuclide imaging of primary renal-cell carcinoma by I-131-labeled antitumor antibody.

A goat antibody against human renal-cell carcinoma reacted on immunofluorescence with renal-cell carcinomas from 20 patients, but not with normal adult human tissues, including kidney. After i.v. administration the I-131-linked antibody showed preferential tumor localization in six of seven patients with primary renal carcinoma. Labeled antitumor antibodies may have the specificity for tumor imaging that current radiopharmaceuticals lack.

Generalized lentiginosis.

A patient with generalized lentiginosis and no other associated problems is described. The various syndromes and anomalies associated with generalized lentiginosis are reviewed. Inheritance patterns, long-term prognosis, and recommendations for evaluation and follow-up are discussed.

Cutaneous malignant melanoma.

In Canada melanoma causes 400 deaths a year, often in young adult patients. Excessive ultraviolet radiation to unprotected skin is an important cause. Certain moles should be removed as a precautionary measure; an experienced clinician can recognize many melanomas. Both microstaging by the pathologist and clinical staging must be carried out before definitive treatment is planned. The only 'curative' treatment is surgical. Any patient so treated requires ten years of careful follow up. The results of chemotherapy for disseminated disease are poor.

Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing beta-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG.

When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10(6)B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, or neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells.

Antibody as carrier of 131I in cancer diagnosis and treatment.

Cell-surface localizing heterologous antibodies against mouse EL4 lymphoma, Ehrlich ascites carcinoma, and several human malignant tumors could be bound to varying amounts of 131I without interfering with the reactivity of these antibodies with their respective tumor cells. Exposure of the mouse tumor cells to radio-iodinated antitumor antibodies in vitro, or the injection of radio-iodinated antitumor antibodies into mice preinoculated with tumor cells resulted in either partial or complete tumor inhibition depending upon the amount of 131I activity carried by the antibodies. Injection of comparable amounts of the immunoglobulin alone or of 131I bound to normal globulin did not cause any tumor inhibition. Intraperitoneally injected radio-iodinated anti-EL4 antibody was found to localize preferentially in the subcutaneous transplants of EL4 lymphoma. Similar localization of intravenously injected radio-iodinated antibodies was observed in the metastases of two cancer patients.

Tumor localization of 131-I-labeled antibodies by radionuclide inaging.

Intravenous injections of 131-I-labeled anti-EL4 lymphoma antibodies showed progressive localization of radioactivity in EL4 transplants but not in B16 melanoma in mice carrying both tumors. Normal rabbit globulin labeled with 131-I did not localize in either tumor and cleared more slowly from the internal organs. Metastatic localization of intravenous 131-I-labeled anti-tumor antibodies was also observed in 2 cancer patients.

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton.

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

Fluid shear stress induces beta-catenin signaling in osteoblasts.

beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.

Prophylactic node dissection for malignant melanoma.

The value of prophylactic node dissection was studied in 147 patients with nonsuperficial malignant melanoma of cutaneous origin; all had clinical stage I disease. Seventy-three patients had prophylactic node dissection and 74 did not. Survival rates were calculated by the actuarial method and were age and sex adjusted. Five-year crude survival rates for these two groups were 62 and 29%, respectively, and the adjusted rates were 70 and 33%, respectively. These significant differences (P less than 0.001) were maintained at 10 years. The difference in survival in the two groups cannot be explained on the basis of age, sex, year of operation, size or location of the primary tumour, or previous incisional biopsy. It is concluded that prophylactic node dissection contributed appreciably to increased survival in this study.