Socioeconomic geographical links to human immunodeficiency virus seroprevalence among childbearing women in Montreal, 1989-1993

Anti-idiotype (anti-Id) antibody can induce tumor dormancy in a murine B lymphoma, BCL1, by its ability to induce cell cycle arrest and apoptosis (negative signaling). In human B lymphoma, there is accumulating evidence that the antitumor effect of anti-Id or several other B cell-reactive antibodies relates to their ability to act as agonists rather than conventional effector antibodies. In this study, we sought to elucidate the role of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors in anti-IgM-induced cell cycle arrest to better understand the mechanisms underlying cancer dormancy. To accomplish this, we have performed in vitro studies with a human lymphoma cell line (Daudi) because its response to anti-Id (or anti-IgM) is similar to that of a BCL1 cell line, more reagents are available, and the results would be particularly pertinent to therapy of human B cell lymphomas. Our results show that cross-linking of membrane IgM on Daudi cells induces an arrest late in G1 and prevents pRb from becoming phosphorylated. The G1 arrest is correlated with an induction of the CDK inhibitor p21 and reduced CDK2 activity, although the level of CDK2 protein was not changed. Coprecipitation of CDK2 with p21 in anti-IgM-treated cells and the unchanged level of cyclin E suggest that p21 is responsible for the reduction of CDK2 activity and therefore blockade of the cell cycle. The induction of p21 was not accompanied by changes in p53 levels. As a result of the G1 block, cyclin A levels sharply declined by 24 h after anti-IgM treatment. There was no evidence for involvement of CDK4 or CDK6 in the blockade. These results provide evidence that membrane IgM cross-linking on Daudi cells induces expression of p21 and a subsequent inhibition of the cyclin E-CDK2 kinase complex resulting in a block to pRb phosphorylation and cell cycle arrest late in G1.     

In vivo retargeting of T cell effector function by recombinant bispecific single chain Fv (anti-CD3 x anti-idiotype) induces long-term survival in the murine BCL1 lymphoma model

As demonstrated in several preclinical models, bispecific Abs are attractive immunotherapeutic agents for tumor treatment. We have previously reported that a bacterially produced anti-CD3 x antitumor bispecific single chain variable fragment of Ab fragment (BsscFv), which is capable of retargeting CTLs toward BCL1 tumor cells, exhibits antitumor activity in vitro. To further facilitate BsscFv production, the coding sequence was subcloned in a eukaryotic expression vector and introduced into Chinese hamster ovary cells for large-scale production. In this report, we have determined the serum stability and the clearance rate from the circulation of BsscFv. Most important, we prove here the therapeutic value of BsscFv in the treatment of BCL1 lymphoma, a murine model for human non-Hodgkin's lymphoma. Tumor-bearing mice that were treated with rscFv in combination with staphylococcal enterotoxin B superantigen, human rIL-2, or murine rIL-12 showed long-term survival, whereas untreated mice all died. This is the first report of the successful in vivo use of BsscFv as an immunotherapeutic agent. Furthermore, long-term survival was the result of complete tumor removal and was not due to the induction of dormancy.     

Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy

Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo-passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These "reemerged," uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro-grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.     

Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)-transgenic mice were injected with plasmacytoma cells secreting a highly tumor-specific antigen, a monoclonal immunoglobulin (Ig), for which the transgene-encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen-presenting cells. The TCR-transgenic mice have previously been shown to be protected against an Id+ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id-specific CD4+ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4+ cells express endogenous, rather than transgene-encoded TCR alpha chains. Peripheral deletion, functional T cells unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, approximately 50 micrograms/ml (0.3 microM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.     

EblacZ tumor dormancy in bone marrow and lymph nodes: active control of proliferating tumor cells by CD8+ immune T cells

A well-defined lacZ gene tagged DBA/2 lymphoma (EblacZ) was used to examine the role of host immune responses in controlling tumor dissemination and persistence, as well as metastasis. In s.c. and intra-ear pinna-inoculated mice, low numbers of EblacZ cells homed to the bone marrow and lymph nodes. The frequency of bone marrow-residing tumor cells did not change with the growth of primary tumor or with multiple inoculations of tumor cells. The bone marrow-residing tumor cells expressed the proliferation-associated Ki67 antigen and expanded upon CD8+ depletion. In contrast, inoculation of nu/nu or severe combined immunodeficiency mice or of immune-suppressed DBA/2 mice led to the rapid outgrowth of EblacZ cells in the bone marrow and their metastasis to other organs. Transfer of bone marrow from EblacZ immunized MHC congenic or syngeneic DBA/2 donors, but not from naive donors, protected s.c.-inoculated DBA/2 mice. Protection was abrogated by in vitro depletion of CD8+ T cells prior to transfer of bone marrow. These experiments show that bone marrow and lymph nodes are privileged sites where potentially lethal tumor cells are controlled in a dormant state by the immune system. Metastasis may be a consequence of the breakdown of this immune control.     

Early development and progression of lymphocyte-stimulatory cross-reactive idiotypes expressed on antibodies to soluble egg antigens during Schistosoma mansoni infection of mice

Idiotypes (Id) that stimulate immunoregulatory anti-Id T lymphocyte proliferation are expressed on murine and human antibodies (Ab) to soluble egg antigens (SEA) of Schistosoma mansoni. Kinetics of early expression of these stimulatory Id have now been studied using immunoaffinity-purified serum anti-SEA Ab from mice infected with S. mansoni for 6, 7, 8, 12, or 16 weeks. Rabbit anti-Id Ab specific for mouse anti-SEA Id expressed at 8 weeks post-infection (anti-8WkId) demonstrated the strongest interactions with Id present at 7 and 8 weeks post-infection by competitive enzyme-linked immunosorbent assay. Anti-8WkId Ab reacted progressively less well with 12 WkId, 6WkId, and 16WkId. Splenocytes from mice infected for 8 weeks demonstrated the highest blast transformation responses in vitro to anti-SEA Id from mice infected for 6 weeks, while 7, 8, 12, and 16 weeks post-infection Id preparations stimulated progressively less proliferation. These data indicate that although eventual Id-associated immunoregulatory events contribute to chronicity in this disease, production of anti-SEA Ab that express stimulatory cross-reactive immunoregulatory Id comprises a substantial portion of the initial, acute anti-SEA response in mice infected with Schistosoma mansoni. Furthermore, either this particular Id-expressing response is not maintained, or its proportional presence is greatly diminished by the cumulative production of other multiple anti-SEA Ab during the establishment of chronicity, perhaps in response to its immunoregulatory influence very early in infection.     

The morphologic spectrum of non-Hodgkin's lymphomas with BCL1/cyclin D1 gene rearrangements

BCL1/PRAD1 gene rearrangements involving the cyclin D1 gene are a feature of about 70% of centrocytic/mantle-cell lymphomas (CC/MCL) but are identified in only a small proportion of other B-cell non-Hodgkin's lymphomas. Of 37 lymphomas found to have BCL1/cyclin D1 (PRAD1, CCND1) gene rearrangements, 30 fit the morphologic and immunophenotypic criteria for typical CC/MCL. Seven cases with morphologic features atypical for CC/MCL were identified. CD5+ monoclonal B cells were documented in all these cases. Six cases were subsequently stained for cyclin D1 protein, and all showed nuclear positivity. Five cases had variably sized foci of cells with moderately abundant pale cytoplasm resembling parafollicular/monocytoid B cells, marginal zone cells, hairy cells, or even proliferation centers. Transformed-appearing cells were also present in some lymphomas. In one case, striking follicular colonization created a markedly nodular growth pattern mimicking a follicular lymphoma. A sixth case had a marked predominance of small, round lymphocytes at some sites, mimicking a small lymphocytic lymphoma. Five of these six cases also had areas more typical of CC/MCL. The seventh case was a CD5-positive splenic marginal zone-like lymphoma (SMZL) with plasmacytic differentiation and circulating villous lymphocytes consistent with a splenic lymphoma with villous lymphocytes (SLVL). These cases illustrate the morphologic spectrum of small B-cell lymphoid neoplasms that have BCL1/cyclin D1 gene rearrangements and overexpression of cyclin D1. Despite the BCL1 translocation and cyclin D1 overexpression, the splenic lymphoma with plasmacytic differentiation was definitely not a CC/MCL and fit the clinicopathologic entity of SMZL/SLVL. The other six cases are best considered CC/MCL variants based on a combined morphologic, immunophenotypic, and genotypic evaluation. Genotypic or immunophenotypic studies to identify cyclin D1 rearrangements and overexpression, although not pathognomonic, are useful in recognizing these variant CC/MCL cases, which can mimic almost any of the other well-described but more indolent low-grade B-cell lymphomas and leukemias. Some of the variant CC/MCL cases had features in common with the CD5+ cyclin D1+ SMZL/SLVL, suggesting a possible relationship between these two otherwise distinct entities.     

p53 induces angiogenesis-restricted dormancy in a mouse fibrosarcoma

The p53 tumor-suppressor gene is inactivated in over 50% of all human cancers. In normal cells, p53 induces growth arrest and apoptosis in response to DNA damage. We show that p53 acts as potent tumor-suppressor gene independent of its well-documented effects on tumor-cell proliferation and apoptosis. p53 activates target genes in a murine fibrosarcoma cell-line but does not affect tumor cell-cycle progression or survival. Exogenous expression of wt-p53 does, however, block the angiogenic potential of the tumor cells resulting in formation of dormant tumors in vivo. These data provide evidence that: (1) p53 acts as a tumor suppressor gene independent of its anti-proliferative effects; (2) By inhibiting angiogenesis p53 can indirectly induce apoptosis in vivo but not in vitro; (3) p53-gene therapy which alters a tumors angiogenic potential, can revert tumors to a dormant phenotype.     

Incidence and disease associations of a proteinase 3-antineutrophil cytoplasmic antibody idiotype (5/7 Id) whose antiidiotype inhibits proteinase 3-antineutrophil cytoplasmic antibody antigen binding activity

OBJECTIVE: To evaluate the potential of a monoclonal proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA) antiidiotype autoantibody (5/7 anti-Id) as a candidate for specific immunotherapy in Wegener's granulomatosis (WG), and to estimate the immunodiagnostic value of the corresponding idiotype (5/7 Id). METHODS: We analyzed the incidence of 5/7 Id in patients with ANCA-associated vasculitides (WG, microscopic polyangiitis, Churg-Strauss syndrome), in disease controls (systemic lupus erythematosus patients), and in healthy donors. We then investigated the presence of 5/7 Id in relation to disease stage, clinical activity, and organ manifestations in 86 patients with WG. Finally, we investigated the ability of the 5/7 anti-Id reagent to inhibit the binding of PR3-ANCA to corresponding antigen in 19 WG patients. RESULTS: The incidence of 5/7 Id was significantly more frequent in WG patients (43 of 86; 50%). We did not find a significant correlation between the prevalence of idiotype expression and disease activity or organ manifestations. Further, we demonstrated in vitro suppression of PR3-ANCA antigen binding activity by 5/7 anti-Id in 11 of 19 WG patients who were positive for 5/7 Id. CONCLUSION: This study shows that 5/7 Id is a common idiotype with a significantly increased incidence in WG and that 5/7 anti-Id inhibits PR3-ANCA antigen binding activity. Based on these observations, we conclude that 5/7 anti-Id is a promising tool for the development of a specific immunotherapy for WG.     

Artificial neural networks in cancer research

Current therapy does not cure the majority of patients with B cell non-Hodgkin's lymphoma (NHL) and further intensification does not benefit the patient. Therefore, new approaches are necessary. Immunotherapy has become again a major interest as a new treatment modality for B cell lymphoma since the discovery that the lymphoma specific Id can be presented to antigen-specific T cells. Vaccination of the tumour-bearing host is one of the major strategies to induce a T cell mediated anti-tumour immunity in vivo. For B cell lymphomas the lymphoma specific Id can be used as a tumour-specific antigen to stimulate T cells. Alternatively, the malignant B cells can be modified to become efficient antigen presenting cells (APCs) and present peptides from their own tumour-specific antigens to the autologous T cells. Currently explored and future vaccination strategies for B cell lymphoma will be discussed here.     

Report of four cases of lymphocytic infundibuloneurohypophysitis

Multiple injections of intact irradiated BCL1 cells, a murine B-cell leukemia/lymphoma can trigger a dose-dependent anti-tumor immune response in naive syngeneic mice. The ability to induce anti-BCL1 immunity and the effect of various cell-modifications on BCL1 tumorigenicity and immunogenicity was evaluated. Newcastle disease virus (NDV) infection or transfer of cytokine genes by both retroviral and Adeno 5 vectors affect neither tumorigenicity nor immunogenicity of BCL1 cells given as a non-immunogenic cell-dose. New ways will have to be developed to elicit a reliable and reproducible anti-tumor effect in spontaneously arising and non-immunogenic hematological malignancies.     

Induction of autoantibody responses to GM-CSF by hyperimmunization with an Id-GM-CSF fusion protein

Fusion proteins consisting of an Ig containing xenogenic constant regions and granulocyte-macrophage colony stimulating factor (Id-GM-CSF) are potent immunogens capable of inducing anti-idiotypic Abs after two immunizations, without the usual need for adjuvants or carrier proteins. In this study, we investigated the effects of hyperimmunization with Id-GM-CSF and found that it induces anti-GM-CSF Abs that could bind to GM-CSF and neutralize its bioactivity in vitro. However, no detrimental effects of the anti-GM-CSF activity were apparent on the general health of the animals or on their base line white blood cell counts. Mice with the anti-GM-CSF activity reconstituted their peripheral white blood cells with identical kinetics as control mice after high dose cyclophosphamide treatment, sublethal irradiation, or lethal irradiation followed by syngeneic bone marrow transplantation. Primary and secondary Ab responses to a variety of protein Ags, including an unrelated Ig Id, were not affected. However, the anti-Id response induced by an unrelated GM-CSF fusion protein that is dependent upon the GM-CSF bioactivity was impaired. To avoid any potential problems associated with inducing anti-GM-CSF Abs, we show that priming with the Id-GM-CSF protein and boosting with the Id protein alone were sufficient to induce comparable anti-Id titers without inducing anti-GM-CSF Abs. We conclude that although hyperimmunization of mice with the GM-CSF fusion protein induced neutralizing anti-GM-CSF Abs, this was of little consequence to the animals. Nevertheless, we have devised a strategy to overcome this potential limitation on the use of GM-CSF fusion proteins for immunization.     

Effect of recombinant human manganese superoxide dismutase on radiosensitivity of murine B cell leukemia (BCL1) cells

Recombinant human manganese superoxide dismutase (SOD) protects cells from oxidative damage and is known to ameliorate post-irradiation damage in mice exposed to whole body or localized chest irradiation. The concept behind the present experiments was to investigate whether it is possible to improve the outcome in leukemia following total body irradiation used as part of the conditioning prior to allogeneic bone marrow transplantation. We determined whether SOD protects leukemic cells from the effects of ionizing irradiation both in vitro and in vivo. Murine B cell leukemia (BCL1) cells, derived from tumor-bearing mice, were irradiated in vitro with or without SOD and injected into BALB/c mice. All mice receiving 10(4) unirradiated BLC1 cells developed leukemia and died within 19-39 days. In vitro exposure of BCL1 cells to 800 cGy or 1600 cGy abolished the potential to induce leukemia by inoculation with 10(4) or 10(6) BCL1 cells, respectively. Addition of SOD in vitro during irradiation increased the resistance of BCL1 cells to ionizing irradiation; all mice receiving 10(6) BCL1 cells previously exposed in vitro to 1200 cGy in the presence of SOD died of leukemia, whereas only 40% of mice receiving a similar inoculum of irradiated BCL1 cells died of leukemia. In contrast, when BCL1-bearing mice were irradiated with 600-800 cGy with or without intravenous injection of SOD (100 mg/kg) 30 minutes prior to irradiation, development of leukemia was unaffected. Residual leukemia cells following therapy were assessed by adoptive transfer of 10(5) spleen cells to secondary BALB/c recipients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stage-specific expression of c-kit protein by murine hematopoietic progenitors

We have analyzed c-kit expression by hematopoietic progenitors from normal and 5-fluorouracil (5-FU)-treated mice by staining with monoclonal anti-c-kit antibody ACK-4. Marrow cells that were enriched for progenitors by a combination of metrizamide density separation and negative immunomagnetic selection with lineage-specific monoclonal antibodies (MoAbs) were separated into three populations based on the level of c-kit expression, c-kit(high), c-kit(low), and c-kit-. The majority of colony-forming cells from normal mice were in c-kit(high) population, whereas most of the progenitors from 5-FU-treated mice were in the c-kit(low) population. Optimal colony formation from c-kit(low) cells from 5-FU-treated mice required the interactions of at least two factors among interleukin-3 (IL-3), IL-11 and steel factor (SF) whereas colony formation from c-kit(high) cells of normal mice was supported well by IL-3 alone. Blast cells that were derived from 5-day culture of c-kit(low) post 5-FU cells were c-kit(high). These observations suggest that the primitive hematopoietic progenitors in cell cycle dormancy are c-kit(low) whereas actively cell cycling maturer progenitors are c-kit(high). Mature cells, with the exception of mast cells, derived from secondary culture of the c-kit(high) blast cells expressed little, if any, c-kit. These results are consistent with a model in which c-kit expression progresses from low levels on primitive, dormant multipotent progenitors to high levels on later, actively cycling progenitors, and finally, decreases to very low or undetectable levels on most mature blood cells, with the exception of mast cells.     

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.     

Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases

In cancer metastasis, only a small percentage of cells released from a primary tumor successfully form distant lesions, but it is uncertain at which steps in the process cells are lost. Our goal was to determine what proportions of B16F1 melanoma cells injected intraportally to target mouse liver 1) survive and extravasate, 2) form micrometastases (4 to 16 cells) by day 3, 3) develop into macroscopic tumors by day 13, and 4) remain as solitary dormant cells. Using in vivo videomicroscopy, a novel cell accounting assay, and immunohistochemical markers for proliferation (Ki-67) and apoptosis (TUNEL), we found that 1) 80% of injected cells survived in the liver microcirculation and extravasated by day 3, 2) only a small subset of extravasated cells began to grow, with 1 in 40 forming micrometastases by day 3, 3) only a small subset of micrometastases continued to grow, with 1 in 100 progressing to form macroscopic tumors by day 13 (in fact, most micrometastases disappeared), and 4) 36% of injected cells remained by day 13 as solitary cancer cells, most of which were dormant (proliferation, 2%; apoptosis, 3%; in contrast to cells within macroscopic tumors: proliferation, 91%; apoptosis/necrosis, 6%). Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: failure of solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors.