CONTENTS
Review
- DNA Tumor Vaccines
ANTHONY P. WLAZLO and HILDEGUND C. J. ERTL (The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA)Abstract. A new generation of vaccines are being developed to induce immune responses that fight off infectious agents, or erradicate cancerous cells. The new vaccines are based on a plasmid vector, which in transfected mammalian cells cause constitutive high-level expression of the target antigen. Expression of the target antigen, in turn, can induce a full-range of immunologic responses, including cell-mediated killing, cell-mediated cytokine release and the production of antigen-specific antibodies. Through molecular techniques, these nucleic acid vaccines can be enhanced to increase target antigen expression and faciliatate antigen presentation. Additionally, genetic adjuvants expressed simultaneously with the target antigens can induce the immune responses to disease-associated antigens. The ease with which these genetic vaccines can be generated and the potency of their ability to generate immune-mediated responses make them highly effective, which creates hope for developing effective treatment and prevention of various diseases, most notably cancer.
Keywords: DNA vaccine; T cells; cancer; immunity; cytokines
- CD8+ T cell Suppressor Factors and the Control of Infection, Replication and Transcription of Human Immunodeficiency Virus
KAREN F. T. COPELAND (Ottawa Hospital Research Institute, Ottawa, Ontario, Canada)Abstract. CD8+ T cells have been shown to produce factors which modulate HIV-1 replication in both T cells and monocytic cells. Examination of the literature reveals that this modulation may occur by the production of -chemokines which block viral entry. However, another CD8+ T cell-derived activity targets the replication of HIV-1 at the level of transcription. CD8+ T cell factors strongly suppress replication and transcription in T cells and T cell lines, and in contrast, the factors enhance both replication and transcription in cells of the monocyte/macrophage lineage. The enhancement of transcription and replication by CD8+ T cell factors is induced by increased production of TNF by the macrophages. The enhancement is sensitive to pertussis toxin, indicating a G protein-coupled pathway. Thus, CD8+ T cells produce factors which mediate effects on transcription and replication of HIV-1 in a cell type-dependent manner. In this review a summary of the effects of chemokines and CD8-derived factors on HIV-1 transcription and replication is presented. The virus-host cell interactions that participate in the persistent replication of HIV in macrophages and the suppression of these functions in T cells require definition.The identification of CD8+ T cell factors which exert these controls on HIV-1 may lead to promising new therapies for HIV infection.
Keywords: CD8+ T cell; transcription; replication; HIV-1; macrophage; pertussis toxin.
- Phage Display as a Tool for Rapid Cloning of Allergenic Proteins
ULRICH APPENZELLER, KURT BLASER and RETO CRAMERI (Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland)Strasse 22, CH-7270 Davos, Switzerland)Abstract. Allergic diseases represent an immune disorder associated with the production of immunoglobulin E (IgE) against normally innocuous antigens (allergens). Almost 20% of the population in industrialised countries suffer from Type I allergic symptoms like allergic rhinitis, conjunctivitis, urticaria or asthma. Although the mechanisms responsible for these allergic reactions are quite well understood, knowledge about the repertoire of molecules able to elicit Type I symptoms is still limited. To clone and characterise entire allergen repertoires from complex allergenic sources in a fast and efficient way, new technologies are required. The phage surface display of cDNA libraries described here has proven to be a versatile cloning system to selectively isolate allergens physically linked to their genetic information. The screening of cDNA libraries displayed on phage surface with immobilised serum IgE from allergic patients reduces the time required for the selection of candidate clones to a few weeks. Robot assisted high throughput screening of enriched library provides a fast and cost-effective way to isolate complete allergen repertoires. The biotechnological production of recombinant allergens derived from these sequences bear a high potential for the improvement of the diagnosis of allergic diseases.
Keywords: cloning; cDNA; phage surface display; recombinant allergens; allergy.
- Inhibition of Angiogenesis in the Treatment of Tumors
KAZIMIERZ OSTROWSKI (Fellow, Polish Academy of Sciences, Department of Histology, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw, Poland) and AGNIESZKA KINSNER (Department of Transplantology and Central Tissue Bank, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw, Poland)Abstract. Angiogenesis is essential for tumor progression, growth and metastases. Many substances present in a normal organism can inhibit or stimulate the process of new vessel formation in tumors. The use of natural or synthetic angiogenesis inhibitors as anticancer drugs is currently under intense investigation. Such agents can have lower toxicity and are less likely to generate drug resistance than conventional cytotoxic drugs. Clinical trials are now underway to develop optimum treatment strategies for antiangiogenic drugs. This paper reviews the present achievements in preclinical and clinical studies with antitumor drugs based on inhibitors of angiogenesis.
Keywords: inhibitors; angiogenesis; cancer treatment.
- Immunomodulatory Role of the Corticotropin Releasing Factor
MARKO RADULOVIC and JOACHIM SPIESS (Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein Str. 3, D-37075 Goettingen, Germany)Abstract. Corticotropin-releasing factor (CRF) was originally identified as a hypothalamic peptide which stimulates secretion of the hypophyseal adrenocorticotropic hormone. CRF exhibits its actions through G protein-dependent seven-membrane-domain receptors. Two subtypes of CRF receptors (CRFR1 and CRFR2) have been characterized thus far. CRF and its receptors were found in a number of brain regions, where they function by neuromodulation, and also in several peripheral organs. Besides CRF, another naturally occurring CRF-like peptide, urocortin, has been characterized. In the immune system, CRF and CRFR1 have so far been detected at both mRNA and protein lelvels in several lymphoid organs and at sites of inflammation. Locally injected CRF was shown to modulate the severity of inflammation. This effect was not only a result of hemodynamic changes known to be induced by CRF or by activation of the hypothalamo-pituitary adrenal axis, as CRF-binding sites were also found on immune cells. CRF was shown to directly modulate secretion of cytokines and neuropeptides, proliferation, chemotaxis and degranulation of purified macrophage and lymphocyte populations in vitro . The presence of functional CRFR was more recently demonstrated also on polymorphonuclear cells and significant amounts of CRF were shown to be produced in lymphoid organs, or delivered to lymphoid organs by peripheral nerves. Taken together, the experimental results obtained so far strongly point to the importance of CRF as a signaling molecule in lymphoid tissues and at the sites of inflammation.
Keywords: corticotropin-releasing factor; inflammation; neutrophil; stress; immune system.
- Expression and Functional Significance of CTLA-4, a Negative Regulator of T Cell Activation
AGATA KOSMACZEWSKA (1), LIDIA CISZAK (1), DOROTA BOĆKO (1) and IRENA FRYDECKA (1, 2); (1) Laboratory of Immunopathology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland; (2) Department of Hematology, University Medical School, Pasteura 4, 50-367 Wrocław, PolandAbstract. The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the Ag-specific TCR and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response characterized by proliferation, differentiation, clonal expansion and effector function or, in absence of appropriate costimulation, to a state of long-lasting unresponsiveness termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between costimulatory molecule CD28 on T cells with members of the B7 family on APC results in upregulation of T cell proliferation, cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of those findings, the two-signal requirement model for T cell activation is today generally accepted. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue CTLA-4 (cytotoxic T lymphocyte antigen-4) in T cell activation has been both obscure and conteroversional. CTLA-4 was initially proposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is actually not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in dynamic interplay of activatory and inhibitory signals.
Keywords: CTLA-4; T cell activation; negative regulation.
- The Murine Ly49 Family: Form and Function
ANDREW P. MAKRIGIANNIS (Laboratory of Experimental Immunology, Division of Basic Sciences) and STEPHEN K. ANDERSON (Intramural Research Support Program, SAIC Frederick, National Cancer Institute-FCRDC, Frederick, MD 21702-201, USA)Abstract. The activity of natural killer (NK) cells is regulated by surface receptors that recognize class I MHC. Murine NK cells express a large family of lectin-related receptors (Ly49s) to perform this function, while human NK cells utilize a separate group of proteins containing Ig-related domains (KIRs). Although these receptor families not structurally related, the Ly49 family appears to be the functional equivalent of human KIRs, since it uses similar signal transduction pathways for either activation or inhibition of NK cell function. Therefore, lessons learned from the study of the murine MHC class I receptor system may be relevant to human NK function. This review summarizes the current state of knowledge of the Ly49 family.
Keywords: NK cells; MHC class I; receptors; signal transduction; Ly49; gene family.
Clinical Immunology
- A Method for Directly Determining the Number of Dendritic Cells and for Evaluation of Their Function in Small Amounts of Human Peripheral Blood
Sergiusz Markowicz, Henryk M. Skurzak and Jan Walewski (The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, 5 W.K. Roentgen Str., 02-781 Warszawa, Poland)Abstract. Bone marrow-derived dendritic cells (DC) are highly potent antigen-presenting cells capable of initiating primary responses of naive T lymphocytes to antigen. Studies on DC in disease have been impeded by the lack of a defined method for accurate DC counting and for evaluation of their function in a small amount of blood. In order to detect and enumerate DC in whole peripheral blood preparations, we applied a direct two-color immunofluorescence method. Blood from healthy donors was stained with a mixture of FITC-conjugated monoclonal antibodies (moAbs) recognizing lineage-associated molecules (CD3, CD14, CD16, CD20, CD57) and PE-conjugated anti-HLA-DR moAb. DC were identified as lineage marker negative (lin-), HLA-DR highly positive cells. The mean percentage of these cells present in peripheral blood leucocytes (PBL) was 0.54%, and the mean absolute DC count was 31.4 x 106/l of blood. DC stained directly in whole blood were heterogeneous with regard to their expression of CD2 and CD4 molecules, and did not express CD80 and CD83 molecules. Expression of CD80 and CD83 on DC was induced following a multistep isolation procedure, including overnight culture. We demonstrated a significant primary proliferative response to keyhole limpet hemocyanin (KLH) in cultures of peripheral blood mononuclear cells (PBMNC). Since primary proliferative response to neoantigens is entirely dependent on DC as antigen-presenting cells, the cultures of unseparated PBMNC stimulated with KLH can be used to evaluate DC function in a relatively simple test. This test does not require previous isolation of DC and T lymphocytes and, therefore, can be performed on a small amount of blood. The elaborated flow cytometric method of DC counting in blood and the proliferative test of DC-dependent primary response to neoantigen are currently being applied in an ongoing study on the effect of chemotherapy on DC number and function in cancer patients.
Keywords: dendritic cells; dendritic cell phenotyping; primary proliferative response.
- Malformations of Angiogenesis in the Low Differentiated Human Carcinomas. Immunohistochemical Study
ALBINA ŻÓŁTOWSKA, JAN STĘPIŃSKI, BARBARA LEWKO, BARBARA ZAMORSKA (Department of Immunopathology), ANDRZEJ ROSZKIEWICZ (Department of Pathomorphology), KRYSTYNA SERKIES (Department of Oncology and Radiotherapy) and WIESŁAW J. KRUSZEWSKI (Department of Surgical Oncology, Medical University of Gdańsk, Dębinki 7, 80-211 Gdańsk, Poland)Abstract. Our previous observations showed that the perivascular mesenchyma of the thin-walled vessels (capillaries) in cancers may be the source of organ-specific stem cells. We suggested that the cells forming vascular channels in altered stroma participate in the tumor development. This study was designed to examine the distribution of the vessels and their appearance in the breast, lung and colon cancers. Using immunohistochemical methods, we have shown that in the low differentiated tumors both CD31 and factor VIII antigens may be expressed in capillaries chiefly on the periphery of neoplastic foci. Many of these vessels were discontinuous, with interruptions or unformed tubules. Sporadically, CD31 protein and factor VIII antigens were not expressed in capillaries inside the very low differentiated cancer cases. It is difficult to assess by immunohistochemichal means whether the vascular malformations are the primary or secondary phenomena in the malignancy and why these abnormalities were especially visible in some low differentiated cancers.
Keywords: cancer; malformation of angiogenesis; immunohistochemistry.
- TNF-a, IL-6 and Their Soluble Receptor Serum Levels and Secretion by Neutrophils from Cancer Patients
EWA JABŁOŃSKA (Department of Immunopathology), MAREK KILUK, WŁODZIMIERZ MARKIEWICZ (Department of Surgery, Regional Center of Oncology), LESZEK PIOTROWSKI, ZYTA GRABOWSKA (Department of Oral and Maxillofacial Surgery) and JAKUB JABŁOŃSKI (Department of Toxicology, Medical Academy of Bialystok, Kilińskiego 1, 15-230 Białystok, Poland)EWA JABŁOŃSKA (Department of Immunopathology), MAREK KILUK, WŁODZIMIERZ MARKIEWICZ (Department of Surgery, Regional Center of Oncology), LESZEK PIOTROWSKI, ZYTA GRABOWSKA (Department of Oral and Maxillofacial Surgery) and JAKUB JABŁOŃSKI (Department of Toxicology, Medical Academy of Bialystok, Kilińskiego 1, 15-230 Białystok, Poland)Abstract. Simultaneous evaluation of cytokines and their soluble receptor production and serum levels can be helpful in understanding the local and systemic immune response of a tumor-bearing host. In the present study we examined the serum levels of TNF-(, IL-6 and their soluble receptors sTNFRp55, sTNFRp75 and sIL-6R compareded with their production by the polymorphonuclear neutrophils (PMN) from cancer patients. Examinations were carried out in patients with adenocarcinoma breast cancer and squamous cell carcinoma of the oral cavity and related to the clinical course and to different phases of therapy. Secretion of IL-6, sTNFRp55 and sTNFRp75 by PMN appeared to be dependent on tumor type, clinical progression of disease as well as on therapy, suggesting a significant role of these cells at different phases of the immune response to cancer associated with these mediators. Changes in values of TNF-(, IL-6 and their soluble receptors in sera of both cancer groups, dependent on tumor type, clinical progression and cancer therapy, could have a diagnostic and prognostic role in cancer disease.
Keywords: tumor necrosis factor-a (TNF-a); interleukin 6 (IL-6); soluble IL-6 receptor; soluble TNF receptor; breast cancer; oral cavity cancer.
- Cytokine Production in Whole Blood Cell Cultures of Patients with B-Lineage Acute Lymphoblastic Leukemia. The Influence of Granulocyte-Macrophage Colony Stimulating Factor
TERESA KAMIŃSKA, MARTYNA KANDEFER-SZERSZEŃ (Department of Virology and Immunology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland), IWONA HUS, ANNA DMOSZYŃSKA (Department of Hematology, University School of Medicine, Jaczewskiego 8, 20-950 Lublin, Poland)
Abstract. We investigated the levels of 6 different cytokines in the sera of 10 newly diagnosed patients with B-cell lineage acute lymphoblastic leukemia (ALL) and detected a significant increase in IL-6 and IFN-( serum levels in comparison to that of healthy controls. Whole blood cell cultures of 10 ALL patients and 20 control individuals were induced with classical cytokine inducers, such as virus, PHA and LPS, and their ability to produce 9 different cytokines was compared. Blood cells of ALL patients produced significantly less IL-1(, IL-1(, IL-10 and TNF-( than control cells and not significanly lower levels of IL-6, but comparable with control levels of IL-2, IL-4. rHuGM-CSF added to cell cultures 24 hr before induction significantly enhanced the production of IL-1(, IL-1( and TNF-( in controls, but only IL-1( and IL-1( in the blood cell cultures of patients with ALL. GM-CSF did not significantly influence the production of IFN-(, IFN-(, IL-2, IL-4 and IL-10 in the control cells and the cells of ALL patients. The patients examined differed not only in the expression of CD10 and CD34 antigens on blast cells, but also in the reaction to GM-CSF treatment, which was found as very high standard deviation values. We suppose that these differences can partially explain the different effects of GM-CSF when used to ameliorate neutropenia of ALL patients after chemotherapy and to reduce the incidence of microbial infections.
Keywords: GM-CSF; acute lymphoblastic leukemia; IL-1; IL-2; IL-4; IL-6; IL-10; TNF-a; IFN-a; IFN-a.