Vol. 53, No. 4, 2005

Cynthia A. Pise-Masison, Soo-Jin Jeong and John N. Brady, (Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA)

Abstract. Human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus which is the causative agent of adult T cell leukemia (ATL). ATL occurs in about 4% of carriers and develops after a long latent period. Although the precise mechanism of HTLV-1 oncogenesis remains unclear, the pathogenesis has been linked to the pleiotropic activity of the viral transcriptional activator protein Tax. Tax has been shown to regulate viral and cellular gene expression and to functionally interfere with proteins involved in cell-cycle progression and DNA repair. This review will focus on the role of Tax in p53 inhibition.

Keywords: T cell; leukemia; Tax protein.

Bart L. Scott^{1, 2} and H. Joachim Deeg^{1, 2}, (¹Fred Hutchinson Cancer Research Center, Seattle, WA, USA, ²University of Washington, Seattle, WA, USA)

Abstract. Myelodysplastic syndromes (MDS) are hemopoietic stem cell disorders, and hemopoietic stem cell transplantation is currently the only therapeutic modality with curative potential. Among patients with less advanced/low-risk MDS (<5% marrow blasts), 3-year survivals of 65–70% are achievable with HLA-identical related and unrelated donors. The overall probability of disease recurrence in these patients is <5%. Among patients with more advanced disease (>=5% marrow blasts), the relapse probability is higher, ranging from 10–40%, and relapse-free survival is correspondingly lower. The criteria proposed by the International Prognostic Scoring System, derived from non-transplanted patients, also predict survival following transplantation. The development of reduced-intensity conditioning regimens and modification of conventional regimens, all aimed at optimizing the transplant approach, have permitted successful hemopoietic stem cell transplants even in patients 60–70 years of age. Improved survival with transplants from unrelated volunteer donors reflects to a large extent selection of donors on the basis of high resolution (allele-level) HLA typing. Graft-versus-host disease and associated problems remain major challenges after allogeneic transplantation. Autologous stem cell transplantation may be beneficial for selected patients who have obtained complete remissions with conventional chemotherapy.

Keywords: MDS; prognostic scoring systems; hemopoietic cell transplantation; conditioning regimens.

Kristin Hauff^{1, 2}, Christina Zamzow², Warren J. Law^{1, 3}, Jimmy De Melo^{1, 4}, Kieron Kennedy1 and Marek Los^{1, 3}, (¹Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada, ²Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada, ³Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada, ⁴Department of Anatomy, University of Manitoba, Winnipeg, Canada)

Abstract. In this review we focus on peptide- and peptidomimetic-based approaches that target autoimmune diseases and some pathologies of the central nervous system. Special attention is given to asthma, allergic rhinitis, osteoarthritis, and Alzheimer’s disease, but other related pathologies are also reviewed, although to a lesser degree. Among others, drugs like Diacerhein and its active form Rhein, Pralnacasan, Anakinra (Kineret), Omalizumab, an antibody “BION-1”, directed against the common b-chain of cytokine receptors, are described below as well as attempts to target b-amyloid peptide aggregation. Parts of the review are also dedicated to targeting of pathologic conditions in the brain and in other tissues with peptides as well as methods to deliver larger molecules through the “blood-brain barrier” by exploring receptor-mediated transport, or elsewhere in the body by using peptides as carriers through cellular membranes. In addition to highlighting current developments in the field, we also propose, for future drug targets, the components of the inflammasome protein complex, which is believed to initiate the activation of caspase-1 dependent signaling events, as well as other pathways that signal inflammation. Thus we discuss the possibility of targeting inflammasome components for negative or positive modulation of an inflammatory response.

Keywords: Anakinra; BION-1; b-amyloid; Diacerhein; Kineret; Omalizumab; osteoarthritis; Pralnacasan; Rhein; secretase; Zafirlukast.

Krzysztof Laudanski and Dorota Wyczechowska, (University of Rochester Medical School, Department of Surgery, Rochester, NY 14642, USA)

Abstract. Mechanical trauma is one of the most important causes of morbidity in the developed world. The response of the immune system to mechanical insult is of paramount importance for the patient’s recovery. Shortly after trauma, the indiscriminate saystemic inflammatory response syndrome (SIRS) is mediated by circulating monocytes (MΦs) and other innate immunity components. Then acquired immunity, limited to the offending pathogen and the site of injury, gradually preponderates. SIRS is followed by the compensatory anti-inflammatory response syndrome (CARS), where the initial inflammatory response is quenched by anti-inflammatory mediators. This precisely regulated process of immune system activation in response to trauma can be easily deviated, resulting in multiorgan failure (MOF) and increased mortality. Excessive activation of inflammatory MΦs in the SIRS phase, premature or exorbitant CARS, a predominance of macrophages (Macs) in the blood stream and peripheral tissues, as well as a depletion of dendritic cells are often seen in trauma patients and contribute to the development of MOF. Here we explore several mechanisms of pathological MΦ activation in patients with severe mechanical traumatic injury without accompanying sepsis.

Keywords: mechanical trauma; monocyte; dendritic cell; macrophage; inflammatory monocyte.

Xiaobo Li, Zhiren Zhang, Thomas Beiter and Hermann J. Schluesener, (Institute of Brain Research, D-72076 Tuebingen, Germany)

Abstract. Exosomes are small membrane vesicles derived from late endosome. They are about 30–100 nm in diameter. The secretion of exosomes is a process in which multivesicular bodies fuse with the cell membrane, and all cells that contain multivesicular endocytic compartments could theoretically secrete exosomes. The surprising biological functions of exosomes are only slowly being unveiled, but it is already clear that they serve to remove obsolete membrane proteins and act as messages of inter-cellular communication. Exosomes derived from tumor or antigen-presenting cells have been extensively investigated. They are released into the extracellular environment and fuse with the membranes of neighboring cells, delivering membrane and cytoplasmic proteins from one cell to another. Exosomes carry immunorelevant structures which play important roles in immune response, such as MHC molecules, costimulatory molecules, heat shock proteins, and naive tumor antigens. Therefore they have been suggested as potential vaccines. Consequently, exosomes have shown considerable anti-tumor effect in several studies and are in phase I clinical trials.

Keywords: exosomes; immunotherapy; biogenesis; tumor; vaccine.

Edyta Pawlak¹, Iwona Ewa Kochanowska¹, Irena Frydecka^{1, 2}, Marek Kiełbiński², Stanisław Potoczek² and Małgorzata Bilińska³, (¹ Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland, ² Department of Hematology, Medical University, Wrocław, Poland, ³ Department of Neurology, Medical University, Wrocław, Poland)

Abstract. A soluble form of cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4) was recently found and shown to possess B7 binding activity. sCTLA-4 is generated by alternatively spliced mRNA. The mRNA encoding sCTLA-4 consists of 3 exons: exon 1 encodes a leader peptide, exon 2 the ligand binding domain, and exon 4 the cytoplasmic tail, but it lacks the transmembrane domain encoded by exon 3. The altered transcript is detected in resting CD4 and CD8 T cells and its expression is inhibited after 24–48 h of activation and returns to the prestimulation level after 72–120 h of activation. Low levels of sCTLA-4 have been detected in normal human serum and increased serum levels have been observed in several autoimmune diseases (e.g. Graves’ disease, myasthenia gravis, systemic lupus erythematosus, and systemic sclerosis). The biological significance of increased sCTLA-4 serum level has not been clarified. On one hand, sCTLA-4 may bind B7 expressed on antigen-presenting cells and is thus able to interfere with the B7:CD28-mediated costimulation of T cell responses. On the other hand, sCTLA-4 may also be capable of interfering with B7:CTLA-4 interactions, thereby blocking the negative signal imparted via the full-length form of CTLA-4. This double-edged nature of B7 blocking by sCTLA-4 may result in different outcomes of the clinical course of disease.

Keywords: sCTLA-4; alternative splicing; autoimmune diseases.

Hanna Stankowiak-Kulpa^{1, 2, 3}, Jane Sanders¹, Hilde Depraetere¹, Jennifer Jeffreys¹, Michele Evans¹, Tonya Richards¹, Jadwiga Furmaniak^{1, 2} and Bernard Rees Smith^{1, 2}, (¹FIRS Laboratories, RSR Ltd., Llanishen, Cardiff, UK, ²Department of Medicine University of Wales College of Medicine, Cardiff, UK, ³Department of Medicine, Poznań University of Medical Sciences, Poznań, Poland)

Abstract. Introduction:
Mouse monoclonal antibodies (mAbs) with the ability to inhibit thyrotropin (TSH) binding to the TSH receptor (TSHR) are useful tools to study TSH-TSHR interaction. The 3C3 mAb we produced was found to inhibit binding of TSH to human (h)TSHR but not to porcine (p)TSHR.

Materials and Methods:
Purified 3C3 immunoglobulin G (IgG) and its antibody-binding fragment were prepared using standard methods and their ability to inhibit TSH binding to hTSHR or pTSHR was analyzed using a coated tube assay. The TSHR epitope reactive with 3C3 IgG was determined using Western blotting, ELISA based on peptides corresponding to the TSHR sequence, and the SPOT synthesis technique. RNA was isolated from 3C3 hybridoma cells and the mAb variable (V) region genes were sequenced and analyzed.

Results:
3C3 mAb had a 1×10⁸l/mol binding affinity to the hTSHR as assessed by Scatchard analysis. 3C3 reacted with the hTSHR region between amino acids (aa) 212-230, and two aa differences were found between the corresponding regions in the hTSHR and pTSHR. The light chain (LC) genes of 3C3 were derived from the Vk21 germ-line (97.6% homology) and Jk2 genes. The heavy chain (HC) genes were from the V130 germ-line (94.6% homology) combined with a D gene (not identified) and JH3 gene. The replacement/silent mutation ratios of 6.0 and 6.5 for the LC and the HC V regions, respectively, indicated that 3C3 underwent antigen-driven maturation.

Conclusions:
Mouse mAbs of this type should be useful in studying the interactions between the TSHR, TSH, and mAbs in more detail.

Keywords: thyrotropin receptor; monoclonal antibodies; thyroid; Graves’s disease.

Jadwiga Sawecka¹, Joanna Skulimowska¹, Jerzy Windyga², Stanisław Łopaciuk² and Jerzy Kościelak¹,(¹Department of Biochemistry, Institute of Hematology and Blood Transfusion, Warsaw, Poland, ²Department of Blood Coagulation and Hemostasis, Institute of Hematology and Blood Transfusion, Warsaw, Poland)

Abstract. Introduction:
Patients with severe hemophilia A often develop inhibitors (antibodies) against transfused factor VIII.

Materials and Methods:
One hundred thirteen Polish patients with severe hemophilia A, who had been treated on and Methods: demand with cryoprecipitate until 1992 and exclusively with factor VIII concentrates after 1995, were examined for intron 22 inversion by Southern blotting and the presence and magnitude of inhibitor activity in blood as determined by the Bethesda assay. The patients’ ages ranged 4–67 years (mean: 33.7±12.4 years, median: 32 years).

Results:
The number of patients with the inversion amounted to 57, while in 56 patients the mutation types were unknown; 47 patients had a distal and 10 patients a proximal type of inversion. Thirteen patients with inversions (22.8%) were found to have inhibitor in their blood. Most patients (14 out of 15) who developed inhibitors in the course of cryoprecipitate therapy were high responders. Conversely, 4 of 5 patients treated between 1992 and 1995 with both cryoprecipitate and intermediate-purity factor VIII concentrates were low responders. One multitransfused patient who had remained inhibitor-free on cryoprecipitate therapy developed inhibitor after receiving a large dose of factor VIII concentrate during surgery. None of these 5 patients developed inhibitors during their 12–40 years of treatment with cryoprecipitate, suggesting that it was less immunogenic than factor VIII concentrates.

Conclusions:
The prevalence of the intron 22 inversion mutation of the factor VIII gene in Polish hemophiliacs is similar to that in other European countries. Treatment regimens with either cryoprecipitate or virus-inactivated plasma-derived factor VIII concentrates may affect inhibitor formation in hemophilia A patients.

Keywords: hemophilia A; cryoprecipitate; factor VIII concentrates; intron 22 inversions; inhibitor in hemophilia.

Włodzimierz Łuczyński¹, Anna Stasiak-Barmuta², Maryna Krawczuk-Rybak¹ and Iwona Malinowska³, (¹Department of Pediatric Oncology, Medical University of Białystok, Poland, ²Flow Cytometry Unit, Medical University of Białystok, Poland, ³Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw, Poland)

Abstract. Introduction:
Recent years have seen a rise in the importance of cytokine production and co-stimulatory/activatory molecule expression in the immune response in leukemia. The aim of our study was to assess the function of T lymphocytes in children with acute lymphoblastic leukemia (ALL) during remission induction based on selected cytokine and co-stimulatory/activatory molecule expression.

Materials and Methods:
The study group consisted of 50 children with ALL (B cell precursor). Peripheral blood samples were taken before treatment (day 0), after the prednisone prophase (day 8), and during (day 15) and after (day 33) remission induction. The percentages of T cells with interferon (IFN)-γ (Th₁), interleukin (IL)-4 (Th₂) and IL-2 receptor (IL-2R), CD28, CTLA-4, CD38, ICAM-1, and HLA-DR expression were assessed by tricolor flow cytometry.

Results:
At the time of diagnosis we noted higher percentages of T cells with adhesion molecule ICAM-1, activation molecule CD38 expression, and an increased population of Th₂ cells (IL-4) compared with the control group. During and after remission induction we observed a decreased population of CD38⁺ T cells, elevated percentages of helper T lymphocytes with IL-2R expression, and a rise in helper T lymphocytes producing IFN-γ (Th₁). During fever/infection, higher levels of activated T lymphocytes (CD4⁺HLA-DR⁺, CD8⁺HLA-DR⁺), a rise in Th₁, and no change in Th₂ populations were observed.

Conclusions:
The results suggest T cell activation and Th₂ predominance at the time of diagnosis and during remission induction in ALL in children. These results confirm the involvement of cellular immunity in the leukemic process and can be used in immune therapy in leukemia.

Keywords: immunosuppression; cancer; IFN-γ IL-4; co-stimulatory molecules.

Wiesław Wiktor-Jędrzejczak¹, Małgorzata Rokicka¹, Elżbieta Urbanowska¹, Tigran Torosian¹, Elżbieta Graczyk-Pol¹, Agnieszka Tomaszewska¹, Małgorzata Król¹, Monika Paluszewska¹, Anna Gronkowska¹, Bogna Ziarkiewicz-Wróblewska¹, Justyna Jółkowska² and Michał Witt², (¹Department of Hematology, Oncology, and Internal Diseases and Department of Pathology, Medical University of Warsaw, Warsaw, Poland, ²Division of Molecular and Clinical Genetics, Institute of Human Genetics, Poznań, Poland)

Abstract. Introduction:
The major obstacle to the therapeutic use of hematopoietic transplantation is the unavailability of matched, unrelated marrow donors for the large number of potential patients, although all of them have the chance to find sufficiently matched, unrelated cord blood units. However, the use of cord blood as a source of cells for transplantation is limited by its cell number, usually below 1 billion, which allows for routine transplantation only in children weighting less than 30 kg, while most potential recipients possess a higher body mass. This led to the idea of the simultaneous use of several units of cord blood which, combined, would fulfill the requirements for the necessary cell number for an adult recipient.

Materials and Methods:
We attempted to simultaneously transplant an adult patient with refractory acute myeloblastic leukemia utilizing two different cord blood units, one fully matched and one mismatched at one locus.

Results:
The patient became reconstituted with only one unit, the mismatched, as determined using microsatellite markers, and had no signs of relapse of leukemia. Unfortunately, he died of persistent fungal (brain aspergilloma) infection on day +103.

Conclusions:
The successful engraftment may suggest that a method based on the principle of using more than one cord blood unit for transplantation is feasible in large adult patients and may reach routine application.

Keywords: placental blood; bone marrow transplantation; hematopoiesis.