Murine lymphoma: augmented growth in mice with pertussis vaccine-induced lymphocytosis.

Injection of Bordetella pertussis vaccine caused an excessive lymphocytosis, associated with an augmented growth of a lymphoma cell homotransplant in mice. A markedly impaired reactive cell proliferation was revealed in the spleens of the vaccine-pretreated mice after stimulation with phytohemagglutinin or Freund's complete adjuvant.

Infiltration of interleukin-2-inducible killer cells in ascitic fluid and pleural effusions of advanced cancer patients.

Using ascitic fluid or pleural effusion obtained from 13 ovarian or metastatic breast cancer patients, we separated tumor cells from effusion-associated lymphocytes (EAL) with Percoll density centrifugation. Lymphocytes were incubated with recombinant interleukin 2 (IL-2) for 3-4 days and then assessed for tumoricidal activity in a 51chromium-release assay. The IL-2-activated EAL were found to lyse autologous fresh tumor cells, as well as allogeneic fresh tumor cells and FMEX tumor cells, a melanoma cell line which is resistant to natural killer cell activity but is sensitive to lysis by lymphokine-activated killer cells. There was little or no tumoricidal activity seen in freshly isolated EAL or in EAL which were cultured in medium without IL-2. Phenotypically, the IL-2-activated EAL were largely CD3-, although some cytolytic activity was found in CD3+ populations. Also, most activity was found in cells positive for CD2 (OKT11) and CD16 (Leu 11b), and negative for the monocyte marker Leu M3. These results indicate that the activated cell types found in EAL were predominantly natural killer/lymphokine-activated killer-like with a small contribution from T-cells. Finally, EAL were readily activated by IL-2 in medium containing autologous effusion fluid, indicating that in situ activation of tumoricidal activity by IL-2 can occur in the face of potentially inhibitory substances or cells that may exist in the effusions. Direct introduction of IL-2 may therefore be a potential therapeutic modality of effusion-forming cancers.

Oncogenes and growth factors.

Certain polypeptide gene products regulate mitosis, differentiation, and other basic biologic functions of cells. These genes and their products are well preserved throughout evolution. Other sets of genes encode receptors for these polypeptides. Polypeptide growth factors can stimulate the cells that express receptors for them. Autocrine growth occurs when a cell produces a growth factor and also expresses receptors for it. When certain genes remain in a permanently switched-on position or are amplified, excessive amounts of growth factors are produced and receptor-positive cells continuously respond, thus achieving illegitimate growth advantage over other cells. Permanently switched-on and/or point-mutated receptor-encoding genes direct the synthesis of truncated receptors that do not need to capture their ligand for signaling receptor activation, thus their cells remain in a permanently activated state. When immortalization and/or malignant transformation results from these activities, the gene is recognized as a protooncogene-oncogene. Acutely transforming retroviruses contain close derivatives of these cellular genes (c-onc----v-onc) obtained through transduction. Genes encoding the synthesis of nontransforming growth factors (angiogenesis factors, colony-stimulating factors, interleukins, etc.) imitate protooncogenes in stimulating the growth and differentiation-dedifferentiation on nonimmortalized cells. Immortalized and malignantly transformed cells may retain receptors to regulatory growth factors that may induced differentiation and/or cessation of mitosis. Growth factors or receptors produced in excess by transformed cells may be neutralized by monoclonal antibodies (McAb) breaking the chain of autocrine or paracrine growth. Protooncogenes-oncogenes may be deactivated by biological response modifiers (dexamethasone, interferons, bacterial toxins, etc.). These interventions may lead to a new treatment modality for the malignant process.

Hodgkin's disease revisited: Reed-Sternberg cells as natural hybridomas.

The formation of natural hybridomas during the course of malignant lymphoproliferative diseases was first observed in 1969, and many times thereafter. This natural phenomenon remains experimentally unexplored even though it exerts fundamental influence on the natural history and outcome of neoplastic cell proliferations. Instead, hybridomas artificially constructed in the laboratory from 1975 on are being extensively studied and utilized for the production of highly specific monoclonal antibodies in the diagnosis and treatment of infectious and malignant diseases. This review lists examples of natural hybridomas formed in murine and human neoplasms. Indirect evidence supports the notion that hybridomas are generated in African Burkitt's lymphoma (BL) in the form of large, immunoresistant tetraploid BL cells emerging in immunoreactive relapsed patients; and in Hodgkin's disease (HD) in the form of Reed-Sternberg (RS) cells. RS cells may be formed when retroviral antigens expressed by the mononuclear HD cell attract reactive B and T cells, and instead of an immune attack by reactive cells, fusion with reactive cells takes place. The resulting RS cells may further fuse with other reactive cells (e.g., with two B cells, one expressing kappa, the other lambda light chains) or with each other, forming quadromas. RS cells appear multinucleated and hyperploid; they express the products of activated genes of the interdigitating dendritic cell (the retrovirally infected mononuclear HD cell) and those of reactive B and/or T cells. Thus, RS cells present themselves with a great variety of marker expression. Molecular mediators and immunoglobulins released from RS cells are responsible for the activities of proliferating polyclonal reactive cells and/or for the depletion of these cells in HD lesions. Multinucleated giant RS cells in anaplastic lymphomas and the syncytial subtype of RS cells of nodular sclerosing HD should be studied first for retro- or herpes viral antigen expressions and for fusion with reactive cells or with other RS cells.

Adoptive immunotherapy with activated peripheral blood lymphocytes.

Adoptive immunotherapy is used to treat malignant tumors resistant to conventional therapeutic modalities. Patients with metastatic melanoma, renal cell carcinoma or mesothelioma are most likely to benefit from this treatment. Tumor infiltrating lymphocytes (TIL) contain tumor specific killer cells and are found to be the most effective. When TIL is not available or until it can be produced in sufficient amount, autologous activated lymphocytes (AAL) are an alternative. AAL are leukapheresed lymphocytes, activated by conditioned medium from OKT3 stimulated autologous lymphocytes. Subcutaneous IL-2 and oral cimetidine are also administered to support the reinfused AAL and to inhibit activation of CD8+ suppressor cells, respectively. To improve the yield and activation of reinfused lymphocytes, addition of IL-2 to the culture medium was tested in different time intervals after the onset of the culture. Interleukin-2 added in the first or second day i) improved the yield of activated lymphocytes; ii) increased the expression of activation markers CD25 (IL-2 receptor) and HLA-DR and iii) augmented killing of tumor cells. Later addition of IL-2 had no or negative effects. In vitro priming of peripheral blood mononuclear cells with autologous or allogeneic but histologically identical tumors was used to increase tumor-specificity of AAL. Autologous serum, containing antibodies specific to tumor cells, facilitated antigen presentation and yielded cytotoxic lymphocytes capable of efficiently killing tumor cells.

Viral expressions in Reed-Sternberg cells.

In the murine system natural hybridoma formation was observed first in 1968-9. In the #620 to 818 system a mouse leukemia virus-(MLV-) producer diploid lymphoma cell fused with an immune plasma cell. The tetraploid fusion product cells grew in suspension cultures and as ascites tumors in mice and continued the production of MLV particles and MLV-neutralizing antibodies. Analogy between the #620 to 818 system and the origin of RS cells is proposed. Indirect evidence suggests retroviral infection of the mononuclear HD cell which presumably is an interdigitating reticulum (IR) cell. Reactive B and T cells interact in an abnormal manner and fuse with the retrovirally infected IR cell. The fusion product cells display hyperdiploidy and a disarray of markers as IR markers are lost due to dedifferentiation (and regained upon differentiation induction) and B and/or T cell markers are gained. Conventional theories for the origin of RS cells fail to explain the great heterogeneity of their markers. Derivation of RS cells from IR cells and B and/or T lymphocytes as natural hybridomas offers plausible explanation for all the features of RS cells.

The cell survival pathways of the primordial RNA-DNA complex remain conserved in the extant genomes and may function as proto-oncogenes.

Malignantly transformed (cancer) cells of multicellular hosts, including human cells, operate activated biochemical pathways that recognizably derived from unicellular ancestors. The descendant heat shock proteins of thermophile archaea now chaperon oncoproteins. The ABC cassettes of toxin-producer zooxantella Symbiodinia algae pump out the cytoplasmic toxin molecules; malignantly transformed cells utilize the derivatives of these cassettes to get rid of chemotherapeuticals. High mobility group helix-loop-helix proteins, protein arginine methyltransferases, proliferating cell nuclear antigens, and Ki-67 nuclear proteins, that protect and repair DNA in unicellular life forms, support oncogenes in transformed cells. The cell survival pathways of Wnt-ß-catenin, Hedgehog, PI3K, MAPK-ERK, STAT, Ets, JAK, Pak, Myb, achaete scute, circadian rhythms, Bruton kinase and others, which are physiological in uni- and early multicellular eukaryotic life forms, are constitutively encoded in complex oncogenic pathways in selected single cells of advanced multicellular eukaryotic hosts. Oncogenes and oncoproteins in advanced multicellular hosts recreate selected independently living and immortalized unicellular life forms, which are similar to extinct and extant protists. These unicellular life forms are recognized at the clinics as autologous "cancer cells".

Kaposi's sarcoma: its 'oncogenes' and growth factors.

Viral genes capable of inducing vascular tumors in the skin of transgenic mice are the tat gene of HIV-1 and polyoma virus' middle T antigen gene. Instead of vascular tumors, the tat gene of HTLV-I causes thymic atrophy and mesenchymal tumors in transgenic mice. No proof exists that any of these genes contribute to the induction of KS but HIV-1 tat is a strong suspect. The gene product K-FGF of the oncogene K-fgf/hst (int) uses bFGF receptors, is homologous with bFGF and acts as a mitogen for fibroblasts, endothelial cells and melanocytes. The overexpression of the K-fgf gene in KS is not proven unequivocally; some doubts exist suggesting the activation of this gene during the laboratory procedure of transfection with KS cell heavy DNA. Growth factor(s) not well identified (IL-6?) are released from HTLV-I- or II, or HIV-1- or 2-infected T4 lymphocytes and in particular from HIV-1-infected macrophages. This growth factor(s) promote(s) the continuous proliferation of endothelial cells and KS cells. AIDS-KS cells release other growth factors identical with or closely related to basic FGF, a major inducer of angioneogenesis. In addition, acidic FGF, IL-1 alpha and -beta, GM-CSF, PDGF-B and TGF-beta are released from AIDS-KS cells. The release of GM-CSF is induced by IL-1. GM-CSF promotes granulocytic, monocytic and endothelial cell proliferation. TGF-beta is known to suppress lymphocyte-mediated cytotoxicity and may act as a local immunosuppressive factor together with interferon inactivators. We theorize that when TGF-beta production ceases, TNF-beta (lymphotoxin) production switches on leading to programmed cell death (apoptosis) of KS cells resulting in regression of these lesions. The newly discovered angiogenesis factors VEGF/VPF may emerge as protooncogene-oncogene products analogous to PDGF and c-sis activation. AIDS-KS heavy DNA transfects NIH3T3 cells. NIH3T3 cells carrying this gene induced angiosarcomas when implanted in mice. An as yet unidentified large virus (mycoplasma?) was derived from these cells during passages in culture. No causative relationship between this agent and Kaposi sarcoma has as yet been established. Even though IFN-alpha exerts antiretroviral effects in AIDS, we propose that the therapeutic effect of IFN-alpha in AIDS-KS is based on antiangiogenesis activity by suppressing protooncogenes-oncogenes of the FGF family.(ABSTRACT TRUNCATED AT 400 WORDS)

Viral oncolysates as human tumor vaccines.

Postoncolytic immunity entails immune reactions acquired through an oncolytic virus infection or through repeated immunizations with viral oncolysates (or virally modified tumor cell membranes) that are valid and operational also against virally not modified tumor cells of the same type. NK cells react to budding virions, induce target cell lysis primarily but not exclusively by the production of granzymes and pore-forming proteins and operate without direction from memory cells. In contrast, immune T cells (including some TIL) are MHC-restricted, act under the direction of memory cells and lyse target cells primarily but not exclusively by the release of lymphotoxin (TNF beta) causing programmed cell death (apoptosis) through endonuclease activation and target cell DNA fragmentation. This author proposes that it is not NK, but the immune T cells that mediate postoncolytic immunity. Oncogene amplification may protect immortalized tumor cells even when expressing peptide antigens through MHC molecules against lymphotoxin-mediated apoptosis; but virally-infected tumor cells releasing budding virions remain susceptible to NK cells. Highly immunogenic viral oncolysates should present both budding virions for NK cells and processed viral and tumoral peptide antigens co-jointly for immune T cells.

Vaccination against human cancers (review).

Classical and molecular immunological means of active tumor-specific immunization against human cancers yielded whole cell or tumor cell lysate vaccines of preventive value (reduced relapse rates) and dendritic cell-peptide or genetically engineered vaccines that may induce remissions even in metastatic disease. Active tumor-specific immunization was often successful in the past 50 years against experimental tumors maintained in the laboratory. During the epochs of classical and molecular immunology several vaccines were generated and used for the reduction of relapse rates of human cancer after surgical removal of the primary or metastatic tumors. Whole cell vaccines consist of X-irradiated autologous or allogeneic tumor cells coadministered with immunostimulants (BCG, Detox). Tumor cells haptenized biologically (as in viral oncolysates) or chemically were also used. Dendritic cell vaccines are prepared by transfection or transduction with tumor antigen-encoding DNA or by pulsing the cells with antigenic peptides in vitro; or collecting dendritic cells that engulfed apoptotic tumor cell DNA and/or peptide antigens in vivo for reinjection into the patient. Genetically engineered tumor cells are prepared in vitro to express MHC and peptides, costimulatory molecules (B7.1) and cyto- or lymphokines (interferons, interleukins, hematopoietic growth factors) for vaccination of patients. Antibody- and immune T cell-mediated immune reactions to autologous tumor cells are newly generated and/or quantitatively increased in immunized patients but do not always correlate with clinical response. Most vaccines are claimed to have reduced relapse rates presumably by inducing effective host immunity against micrometastases. Dendritic cell-peptide vaccines could induce partial or occasionally complete remissions in metastatic disease. The wrong antigenic presentation may result in tolerance induction toward the tumor; occasionally tumor enhancement may occur. Human tumor antigens when presented appropriately (with costimulatory molecules and with IL-2, IL-12) break the host's natural tolerance toward its tumor and induce rejection strength immune reactions even in patients with metastatic disease. Immune T cells thus generated could be collected for adoptive immunotherapy. For successful active specific immunization against human cancers the understanding of the immunoevasive maneuvers of the tumor cell (through FasL --> Fas; TRAIL; CD40L --> CD40; TGFbeta etc. systems) is essential.

Virological and immunological connotations of apoptotic and anti-apoptotic forces in neoplasia.

Against the background of its earliest recognition, programmed cell death (PCD) or apoptosis (A) is presented in its fundamental biological contexts. Techniques of its demonstration are listed. Former original works of the authors encompass designs for genetically engineered oncolytic viruses. Presented here are observations on mesenchymal stromal cells of the bone marrow serving as feeder layers to chronic lymphocytic leukemia (CLL) cells (recently rediscovered elsewhere as subverted "nurse cells" protecting CLL cells from A). A-resistant human melanoma cells are shown to expropriate the Fas ligand to Fas receptor (CD95; APO-1) (FasL-->FasR) system for their autocrine growth loop not only in melanoma cells coexpressing CD95 and its ligand but also in CD95-positive melanoma cells undergoing divisions when exposed to CD95 ligand. Bi-directional A-induction is demonstrated upon the encounter of cytotoxic lymphocytes and targeted tumor cells as exemplified with lymphomas; and chemotherapy-induced A of malignant cells as exemplified by paclitaxel-induced PCD of Reed-Sternberg (RS) cells in a case of chemotherapy-resistant Hodgkin's disease (HD). A list of interventions capable of inducing A in tumor cells is provided. These interventions are of potential therapeutic value. The balance of apoptotic and anti-apoptotic forces in virally infected normal and malignant cells is discussed.

Kaposi's sarcoma: breeding ground of herpesviridae - A tour de force over viral evolution (review).

After reviewing the molecular biological basis of prominent theories for the integration of viruses into the earliest forms of living matter, an account is given on the immunoevasive strategies viruses have had to acquire in order to secure their existence against the most sophisticated anti-viral defensive mechanisms evolving in their hosts. Herpes-viridae and Kaposi's sarcoma illustrate the complexity of host-virus relationship. In following the evolutionary steps of simians and hominoids to Homo, it becomes evident that: a) Epstein-Barr virus evolved in Africa and its ancestral viruses are present in cercopithecines and hominoids; b) human herpes-virus-8-related viruses are present in macaques, in S. American primates and in Homo but such isolates from the great apes are missing. Thus interspecies transfer occurred from lower monkeys to Homo but when and at what geographical location? The human retrolentiviruses also jumped species barriers: this occurred recently in Africa, from great apes (chimpanzee and bonobo) to Homo sapiens (except when HIV-2 was transferred to mankind from sooty mangabeys). The matter is further complicated by the long coevolutionary cooperative interactions between herpes- and retrolentiviruses. Of pathological entities suspected to be etiologically affected by such complex viral cooperation, the origin of Reed-Sternberg cells of Hodgkin's disease is singled out for critical analysis. In this article the senior author summarizes his own 52 years of studentship in virology.

Newcastle disease virus (NDV): brief history of its oncolytic strains.

BACKGROUND: While genetically engineered viruses are now being tested for the virus therapy of human cancers, some naturally occurring viruses display unmatched oncolytic activity. Newcastle disease virus (NDV) excels as an oncolytic agent. OBJECTIVES: As its virulence versus attenuation can be explained on molecular biological bases, it may be possible to develop or select highly oncolytic strains of NDV without adverse toxicity. STUDY DESIGN: Questions are posed as to the mechanisms of viral oncolysis, the appropriateness of tests to predict oncolytic activity of a given NDV strain and the best modes of administration for oncolytic effects. Answers are provided based on specific data or on considerations drawn from experience (the authors use NDV oncolysates to immunize against melanoma and kidney carcinoma) or from analogous clinical situations (therapeutic use of mumps or measles viruses). RESULTS AND CONCLUSIONS: NDV oncolysates probably suit better for immunotherapy (providing also active tumor-specific immunization) than massive repeated inoculations of NDV strains, especially when the NDV strain used is not proven to be oncolytic by appropriate pre-clinical tests.

Chemotherapy for advanced Kaposi sarcoma.

Kaposi sarcoma is a rare disorder, usually controlled with conservative localized treatment. However, prognosis is serious for patients with aggressive cutaneous disease, and aminous for those with visceral involvement. Two cases of advanced Kaposi sarcoma are reviewed to illustrate a chemotherapeutic approach to patients with this disease. The first case represents a verified response of visceral Kaposi sarcoma to chemotherapy. The second provides an example of the control of aggressive dermatologic disease.

Oncogenes-antioncogenes and virus therapy of cancer.

Viruses can render services to mankind. 1. Retroviruses pinpoint and transduce cellular oncogenes. 2. Retroviral vectors can introduce antioncogenes (the RB gene) into malignant cells thus rendering the recipient cells nonmalignant. 3. Oncolytic viruses lyse tumor cells. 4. Parvoviruses replicate only in dividing cells and exert lysis and antioncogene effect in tumor cells without affecting resting normal cells. 5. Myxo- and paramyxoviruses (and other viruses) upgrade the immunogenicity of cell surface antigens thus eliciting rejection type host immunity against these cells which is operational against not virus-infected cells of the same type (post-oncolytic antitumor immunity). 6. Viruses or virally infected cells (including tumor cells) induce the production of lymphokines and cytokines (interferons, interleukins and tumor necrosis factor) and activate NK cells and specific immune T cells cytotoxic to virus-infected cells (including tumor cells). 7. Measles virus may activate suppressor cells and both directly (by infecting lymphoma cells) and indirectly (by inducing molecular mediators of suppressor mononuclear cells inhibitory to the growth of neoplastic lymphoid and hematopoietic cells) induce remissions of lympho- and hematopoietic malignancies. 8. Retroviral vectors deliver genes into tumor cells for encoding new surface antigens that render the tumor cells highly antigenic and more vulnerable to rejection type immune reactions of the host. Examples illustrate each statement. Immunotherapy of tumors with active tumor-specific immunization after the induction of suppressor cells by fetal antigens and the elimination of the proliferating suppressor clones by cyclophosphamide will again be proposed.

Retroviral and human cellular oncogenes.

Unexpected meeting of two separate lines of research resulted in the discovery of oncogenes. Oncogenes are deoxyribonucleic acid (DNA) sequences coding for polypeptide gene products which cause, or contribute to, neoplastic growth of cells. Oncogenes remain almost unchanged through evolution: oncogenes and their gene products of avian, murine, feline, simian and human species show close homology. Retroviruses possess three genes encoding virion structural proteins and envelope. The DNA copy of the viral genome (the provirus) recombines with DNA sequences of the host cell genome and thus acquires an additional DNA sequence of host origin (transduction). The newly acquired DNA sequences render the retrovirus oncogenic. Certain genomic DNA sequences extracted from human tumor cells induce malignant transformation in selected assay systems (transfection). The transforming genes of retroviruses show close homology to these cellular oncogenes. Retroviruses appear to have acquired cellular proto-oncogenes during past interactions with their host cells. In the cell, proto-oncogenes are presumed to fulfill fundamental functions of cell differentiation and mitosis. This is deduced from their preservation during evolution, i.e., proto-oncogenes of avian, lower and higher mammalian and human species display close DNA sequence homology and thus their gene products are also similar in distant species. When expressed in excess or in altered form or at a wrong chromosomal location or at an inappropriate time of the cell cycle, proto-oncogenes function as oncogenes by inducing mitoses and inhibiting differentiation of their host cells.

Cytotoxic lymphocytes.

Interactions between human lymphocytes and target cells are highly variable. Lymphocytes may enter fibroblasts, establish cytoplasmic bridges, and draw material through these bridges from the fibroblasts; these lymphocytes may undergo divisions within the fibroblasts, while the fibroblasts vacuolize and die. In this interaction, the fibroblasts may serve as feeder cells to the lymphocytes without any immunological principle involved. Small (presumably immune T) and large (presumably NK) lymphocytes of tumor-bearing patients lyse either autologous, or allogenic, or both autologous and allogeneic tumor cells. Serum factors can block or intensify these reactions. Healthy donors frequently yield lymphocytes cytotoxic to tumor cells. Serum factors blocking the cytotoxicity of patients' lymphocytes frequently fail to block the cytotoxicity of healthy donors' lymphocytes.

Can virus therapy of human cancer be improved by apoptosis induction?

Direct virus inoculations and viral oncolysates may induce temporary remissions and prolong life with reduced tumor burden, or decrease relapse rates but fall short of curing human cancers. We propose: (i) investigations of Cassel's 73-T, an Ehrlich's mouse ascites carcinoma-adapted Newcastle disease virus (NDV) strain that so effectively reduced relapse rates in malignant melanoma if it is an admixture or a recombinant with a murine parvovirus; (ii) transfection of prostatic carcinoma cells with the TRMP gene; (iii) transfection of sarcoma cells with the fas gene followed by treatment with anti-fas monoclonal antibodies, and (iv) treatment of metastatic tumors with a parvovirus incorporating the apoptosis-inducer Ad5 E1A gene.Thus, replicating virions and haphazard generation of cytokines in the inoculated host could be replaced with transfection of single genes of well-defined, limited but selected efficacy.

Multiple pathogens may induce growth factor cascade resulting in KS.

While lipopolysaccharide endotoxin is the most prominent inducer of the kinecascade (TNF alpha, IL-1, 4, 6, 8) that leads to shock and multiple organ failure, bacterial exotoxins and products of certain gram positive bacteria can induce the same end results. We theorize that more than one pathogen can induce the sequence of protooncogene activation and growth factor release that results in the formation of KS. If KS has its own unique viral etiology, this virus has not as yet been isolated or identified but we continue to search for it. However, it is entirely possible that these lesions do not have a single well-defined etiologic agent but are the result of multiple agents cooperating in a set sequence. An endogenous, or apathogenic exogenous, retrovirus may replace HIV for initiator growth factor induction in CD4 cells in the classical (Mediterranean) or iatrogenic disease; and other pathogens co-exist or sequentially replace each other in the African endemic disease; whereas an array of viral pathogens (prominent among them CMV) take over growth factor induction in endothelial cells proliferating in response to the initiator growth factor (oncostatin M) released from HIV-infected CD4 lymphocytes in AIDS-KS.

Progress in clinical immunotherapy for tumors.

Immunotherapy of human tumors is a promising new modality that remains highly investigational and poses unforeseen risks. It should preferably be conducted in patients brought into remission by conventional antitumor therapy and against the background of a complex, reliable monitoring system capable of quantitatively measuring the patient's antitumor immune reactions.