Vol. 52, No. 1, 2004

Review

Eicosanoids: an emerging role in dendritic cell biology
Hedi Harizi and Norbert Gualde, (Laboratory of Immunology, Bordeaux University, Bordeaux, France)

Abstract. The arachidonic acid (AA)-derived metabolites, termed eicosanoids, are potent lipid mediators with a key role in immune and inflammatory responses. In the immune system, eicosanoids such as prostaglandins (PGs) and leukotrienes (LTs) are produced predominately by antigen-presenting cells (APC), including macrophages and dendritic cells (DC). DC constitue a family of bone marrow-derived professional APC that play a critical role in the induction and modulation of both innate and adaptive immunity. For many years, macrophages were considered as major producers of eicosanoids that are thought to drastically affect their function. Studies concerning the modulation of DC biology by eicosanoids show that PGs and LTs have the potential to affect the maturation, cytokine-producing capacity, Th cell-polarizing ability, and migration of DC. In addition, the development of DC from bone marrow progenitors appears to be under the control of some eicosanoids. Understanding the actions of eicosanoids and their receptors on APC functions is crucial for the generation of efficient DC for therapeutic purposes in patients. In this review, we summarize the current understanding of how DC functions are modulated by eicosanoids.

Keywords: DC; eicosanoids; immunomodulation.

5201
What came first: PKCq or the immune synapse?
Martin Villalba, (Molecular Genetic Institute, Montpellier, France)

Abstract. Stimulation of T cells by peptide-presenting antigen-presenting cells (APCs) induces formation of a highly organized complex of receptors, signaling molecules and cytoskeleton components at the immune synapse (IS), the contact site between T cells and APCs. Conjugate formation between T cells and APCs initiates the formation of the IS. After this event, micrometer-scale molecular movements occur in the T cell plasma membrane and the actin cytoskeleton undergoes reorganization. Our current knowledge suggests that formation of the IS is an essential step during T cell activation. This is probably related to the proper localization of certain proteins in specific compartments. One of these proteins is protein kinase Cq (PKCq), which is absolutely required for T cell activation. During the last years we have made great advances in understanding the function and targets of this kinase, and recent reviews have summarized these findings. In contrast, we do not know the exact mechanism that activates PKCq after TCR engagement and the role of PKCq activation in the formation of the IS. In this review I analyze the mechanism of the translocation of PKCq and discuss the function of PKCq in the formation of the IS and, vice versa, the role of the IS in the translocation of PKCq.

Keywords: T cell; PKCq; immune synapse; WASP; PLC.

5202
Recent advances in the regulation of CD44 expression and its role in inflammation and autoimmune diseases
Katrina Gee^1,3, Marko Kryworuchko^2,3 and Ashok Kumar^1,2,3, (¹Department of Biochemistry, Microbiology and Immunology, ²Department of Pathology and Laboratory Medicine, University of Ottawa, ³Division of Virology and Molecular Immunology, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada)

Abstract. Interaction of CD44, an adhesion molecule, with its extracellular matrix ligand, hyaluronan (HA), has been suggested to play a critical role in a number of biological manifestations, including cell migration, tumorigenesis, metastasis, and regulation of immune responses. CD44 comprises a large family of transmembrane glycoproteins that exhibit extensive molecular heterogeneity. This heterogeneity in size is generated by alternative RNA splicing of variable exons as well as by post-translational modifications. Most cell types express CD44 but do not bind HA. The biological functions of CD44, including the regulation of lymphocyte recruitment to the sites of inflammation, have been attributed to the generation of a functionally active, HA-adhesive phenotype. The molecular mechanisms underlying the regulation of CD44 expression and the generation of a functionally active HA-binding phenotype are not well understood. Recently, CD44-HA interactions have been reported to play a critical role in a number of autoimmune diseases in humans and experimental animal models. Initial studies have taken advantage of anti-CD44 antibodies which specifically block CD44-HA interactions. Administration of these antibodies in several experimental murine models of autoimmune diseases resulted in alleviation of inflammatory reactions. In addition, the generation of CD44-deficient animals has facilitated our understanding of the involvement of CD44 in inflammation and autoimmune diseases. This review will focus on the recent advances in the molecular mechanisms regulating CD44 expression, ligand binding, as well as the contribution of CD44 to the development of inflammation and autoimmune disorders.

Keywords: CD44; hyaluronan; inflammation; autoimmunity; cytokines.

5203
Role of RGS proteins in regulating the migration of B lymphocytes
Chantal Moratz, Kathleen Harrison and John H. Kehrl, (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20891, USA)

Abstract. The migration of B lymphocytes into distinct microenvironments in secondary lymphoid tissues and maintenance of cells in these micro-domains is strictly structured and likely supports the proper regulation of immune responses to both foreign and self-antigens. Chemokines’ and other chemoattactants’ signals serve as signposts to direct cell migration. They signal cells through heptahelical receptors, which couple to heterotrimeric G proteins (G protein-coupled receptors or GPCRs). The regulation of the signals transduced through these receptors ultimately determines the positioning of cells in lymphoid tissues. A variety of mechanisms regulate GPCR signaling including a family of approximately 25 proteins termed regulators of G protein signaling (RGS). These proteins act as GTPase activating proteins for Ga subunits and can also function as effector antagonists of specific Ga subunits, thereby attenuating signaling through GPCRs such as chemokine receptors. RGS proteins possess some degree of receptor and Ga subunit specificity. Thus, the particular spectrum of RGS proteins and their expression levels within a cell will determine the duration and magnitude of G protein signaling initiated by chemokines. In this review we illustrate the role RGS proteins have in regulating B cell signaling responses to chemoattractant stimuli during homeostasis as well as during an immune response.

Keywords: RGS proteins; chemokine; lymphoid tissue; germinal centers; G protein.

5204

Original Article

Reduction of soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin) and vascular endothelial growth factor levels in serum of rheumatoid arthritis patients following multiple intravenous infusions of infliximab
Piotr A. Klimiuk, Stanisław Sierakowski, Izabela Domysławska, Małgorzata Fiedorczyk and Justyna Chwiećko, (Department of Rheumatology and Internal Diseases, Medical University of Białystok, Białystok, Poland)

Abstract.

Introduction:
The purpose of this study was to determine the effect of repeated infusions of infliximab, a chimeric anti-tumor necrosis factor (anti-TNF)-a antibody, on the levels of soluble adhesion molecules and vascular endothelial growth factor (VEGF) in patients with active rheumatoid arthritis (RA).

Materials and Methods:
The treatment design consisted of 9 infusions of infliximab (3 mg/kg) at weeks 0, 2, 6, and every 8 weeks thereafter. All patients had been receiving methotrexate (MTX; 7.5–20 mg/week). Serum levels of soluble intercellular adhesion molecule (sICAM)-1, vascular cell adhesion molecule (sVCAM)-1, E-selectin (sE-selectin), and VEGF were measured by ELISA at weeks 0, 2, 6, 14, and 38 prior to infusion, and at week 62.

Results:
A remarkable decrease in serum sICAM-1 (p<0.001), sVCAM-1 (p<0.01), sE-selectin (p<0.01) and VEGF (p<0.001) levels was observed in RA patients after the initial dose of infliximab. The second administration of the drug was followed by an even more significant suppression of serum sICAM-1, sVCAM-1, sE-selectin, and VEGF (p<0.001 in all cases). Further infliximab infusions also significantly reduced serum soluble adhesion molecules and VEGF concentrations, although these were less effective. Infliximab treatment induced a significant decrease in the number of monocytes observed until the end of the study.

Conclusions:
Our study, besides a rapid suppression of disease activity, showed that serum soluble adhesion molecules and VEGF concentrations are down-regulated following anti-TNF-a antibody therapy combined with MTX. Repeated doses of infliximab sustained the reductions in the soluble adhesion molecules and VEGF concentrations, although they were less effective than the first and second infusions of infliximab.

Keywords: sICAM-1; sVCAM-1; VEGF; rheumatoid arthritis; infliximab.

5205
Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens O23
Agnieszka Torzewska¹, Nina A. Kocharova², Agnieszka Maszewska¹, Yuriy A. Knirel² and Antoni Różalski¹, (¹Institute of Microbiology and Immunology, University of Łódź, Łódź, Poland, ²N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia)

Abstract.

Introduction:
The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide – OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of Gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp.

Materials and Methods:
LPS and alkali-treated LPS of P. alcalifaciens O23 and serologically related P. rustigianii O14, P. mirabilis O13 and P. myxofaciens as well as O-antiserum against P. alcalifaciens O23 were used. Serological characterization of P. alcalifaciens O23 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot.

Results and Conclusions:
The OPS of P. alcalifaciens, O23, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-Llysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, O23, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifiaciens O23 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens O23 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of O23-specific antibodies.

Keywords: Providencia alcalifaciens; lipopolysaccharide; O-antigen; O-serogroups; N^e-[(R)-1-carboxyethyl]-L-lysine.

5206
Recognition of apoptotic cells by human peripheral blood monocytes does not alter their ability to phagocytize and kill Staphylococcus aureus
Ewa Zuba¹, Kazimierz Węglarczyk^1,2, Katarzyna Barczyk¹ and Juliusz Pryjma^1,2, (¹Department of Immunology, Faculty of Biotechnology, Jagiellonian University, Cracow, Poland, ²Chair of Clinical Immunology and Pathology, Institute of Pediatrics, Collegium Medicum, Jagiellonian University, Cracow, Poland)

Abstract.

Introduction:
During acute inflammation, leukocyte infiltration is mostly neutrophilic, but later monocytes prevail. The majority of inflammatory cells, particularly neutrophilic polymorphonuclear leukocytes (PMNs), become apoptotic at later stages of inflammation and are phagocytosed by neighboring cells, mostly by macrophages. Recently, it has been found that human peripheral blood monocytes also recognize apoptotic cells, which primes them to increased production of interleukin (IL)-10 – a cytokine known to reduce phagocytes’ ability to engulf and kill pathogens. Based on the above, we studied monocytes’ ability to phagocytose and kill Staphylococcus aureus while in contact with apoptotic cells.

Materials and Methods:
Monocytes isolated by elutriation were co-cultured with apoptotic PMNs or Jurkat cells and exposed to viable, human serum-opsonized S.aureus. To induce apoptosis PMNs were cultured overnight while Jurkat cells were UV-treated. Apoptosis, phagocytosis of bacteria and intracellular superoxide production were measured by flow cytometry. Production of reactive oxygen species was also followed by measurement of chemiluminescence. The bactericidal effect was determined by standard colony forming units method.

Results:
Data presented show that contact of monocytes with apoptotic neutrophils and Jurkat cells had no influence on monocyte phagocytosis of S. aureus, the generation of reactive oxygen species, or the killing of bacteria.

Conclusion:
The data obtained suggest that monocytes attracted to the inflammatory site are not deficient in their ability to cope with pathogens after contact with apoptotic cells despite increased production of IL-10.

Keywords: apoptotic cell recognition; monocytes; Staphylococcus aureus.

5207
Neuroleptics modulate cytokine and reactive oxygen species production in blood leukocytes of healthy volunteers
Agnieszka Szuster-Ciesielska¹, Maria Słotwińska², Anna Stachura², Halina Marmurowska-Michałowska³ and Martyna Kandefer-Szerszeń¹, (¹Department of Virology and Immunology, Maria Curie-Skłodowska University, Lublin, Poland, ²Department of Cell Biology, Maria Curie-Skłodowska University, Lublin, Poland, ³Department of Psychiatry, University Medical School, Lublin, Poland)

Abstract.

Introduction:
There have been several reports indicating that schizophrenia is related to the activation of the inflammatory response system (IRS), characterized by increased serum concentrations of several cytokines, and that antipsychotic drugs may have immunosuppressive or immunoregulatory effects. The aim of the present study was to examine the effects of neuroleptics on cytokine and reactive oxygen species production in vitro, in blood leukocytes.

Materials and Methods:
We studied the effect of haloperidol, chlorpromazine and clozapine on the unstimulated and stimulated (phytohemagglutinin+lipopolysaccharide – PHA+LPS) production of some cytokines which are known to be mainly products of T lymphocytes and monocytes (IL-2, lymphotoxin, IFN-g, IL-12, IL-4, IL-10 and TGF-ß) in peripheral blood mononuclear cells (PBMC) of healthy subjects. We also compared the effect of neuroleptics on superoxide anion and hydrogen peroxide production in blood neutrophils

Results:
All three antipsychotic drugs significantly increased PHA+LPS-stimulated production of anti-inflammatory cytokines such as IL-10 and TGF-ß as well as unstimulated production of IL- 10, but they did not influence IL-12 production. In the same in vitro conditions they inhibited PHA+LPS-stimulated production of IL-2 and lymphotoxin. IL-4 production was inhibited by haloperidol and chlorpromazine, but not by clozapine. IFN-g production was inhibited by haloperidol and chlorpromazine, but stimulated by clozapine. All neuroleptics examined at a high (100 µM) concentration, but not at a 1 µM concentration, significantly inhibited superoxide anion production by phorbol ester (PMA)-stimulated neutrophils in vitro.

Conclusion:
The results indicate that in vitro, typical antipsychotic drugs, such as haloperidol and chlorpromazine, and atypical ones, such as clozapine, modulate cytokines which are known to be produced by monocytes as well as by T helper (Th)1 and Th2 subpopulations.

Keywords: peripheral blood mononuclear cells; neutrophils; cytokines; reactive oxygen species; haloperidol; chlorpromazine; clozapine.

5208

Vol. 52, No. 2, 2004

Review

Targeting Janus kinase 3 in the treatment of leukemia and inflammatory diseases
Marina Cetkovic-Cvrlje¹, Fatih M. Uckun^{1, 2} (¹Department of Immunology, Parker Hughes Institute, St. Paul, MN 55113, USA, ²Drug Discovery Program, Parker Hughes Institute, St. Paul, MN 55113, USA)

Abstract. Janus tyrosine kinases (JAKs) are cytoplasmic protein tyrosine kinases that play a crucial role in the initial steps of cytokine signaling. JAK3, a member of JAK kinase family of four (JAK1, JAK2, JAK3 and TYK2), is abundantly expressed in lymphoid cells. JAK3 has been found to initiate signaling of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-13 and IL-15. Indispensable role of JAK3 in lymphocyte development and function has been revealed recently. Because of the involvement of JAK3 in T cell activation and proliferation, and the documented genetic evidence for the role of JAK3 in autoimmune or transplant-induced inflammatory disorders, the selective targeting of JAK3 in T cells may potentially be clinically beneficial in T cell-derived pathologic disorders. In this review we discuss inhibitors of JAK3 as a new class of immunomodulatory agents with immunosuppressive, anti-inflammatory, anti-allergic, and anti-leukemic properties. Preclinical data from multiple experimental model systems of autoimmune diabetes, allergy, solid organ transplantation, pancreatic islet transplantation and bone marrow transplantation are discussed in the context of the clinical need for new immunomodulatory agents with such properties.

Keywords: Janus kinase 3 (JAK3); JAK3 inhibitors; type 1 diabetes; allogeneic transplantation; allergy.

5236
Functions of human complement inhibitor C4b-binding protein in relation to its structure
Anna M. Blom¹, Bruno O. Villoutreix² and Björn Dahlbäck¹, (¹ Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden, ² INSERM U428, University of Paris V, Paris, France)

Abstract. Considering the destructive potential of the complement cascade, it is no surprise that there are several complement inhibitors present in blood and expressed on virtually all cells of the body to protect self tissue. C4b-binding protein (C4BP) is a potent soluble inhibitor of the classical and lectin pathways of complement. This large (500 kDa) plasma glycoprotein consists of seven identical 75 kDa ?-chains and a unique 40 kDa??-chain that are held together by disulphide bridges. Both types of subunit are almost exclusively composed of complement control protein (CCP) domains. In recent years, detailed studies of structure-function relationships have yielded new understanding of the interactions between C4BP and the activated complement factors C4b and C3b, heparin, and vitamin K-dependent anticoagulant protein S. This review describes the localization of binding sites for a number of C4BP ligands in relation to well-established and novel functions of C4BP such as complement inhibition, protection of apoptotic cells from complement, CD40-dependent stimulation of B cells, and the contribution of a number of human pathogens to pathogenesis.

Keywords: complement; C4b-binding protein; protein S; coagulation; heparin; B cells; CD40; apoptosis; C3b; C4b.

5237
Anti-GBM glomerulonephritis: a T cell-mediated autoimmune disease?
Ya-Huan Lou, (University of Texas Health Science Center at Houston, Houston, TX 77030, USA)

Abstract. Anti-glomerular basement membrane (GBM) glomerulonephritis, which was among the earliest recognized human autoimmune diseases, is characterized by the presence of anti-GBM antibody. It has been a prototypical example of autoantibody-mediated autoimmune disease. However, decades of research on this disease, based either on clinical observations or experimental models, have revealed that T cell-mediated cellular immunity may potentially be a more important mediator of glomerulonephritis. We have made several breakthroughs in understanding the T cell-mediated mechanism causing this disease in a rat model based on Goodpasture’s antigen, non-collagen domain 1 of ?3 chain of type IV collagen (Col4?3NC1). We demonstrated that anti-GBM glomerulonephritis was induced by either passive transfer of Col4?3NC1-specific T cells or active immunization with the nephritogenic T cell epitope of Col4?3NC1. Immunization with the T cell epitope also triggered production of anti-GBM antibodies to diversified GBM antigens. Thus, a single nephritogenic T cell epitope alone is sufficient to induce the clinical spectrum of anti-GBM glomerulonephritis, including proteinuria, glomerular injury, and anti-GBM antibody. A possible T cell-mediated mechanism for causing human anti-GBM disease is proposed.

Keywords: autoimmunity; T cells; autoantibody; glomerulonephritis.

5238
Leucine-rich repeats in host-pathogen interactions
Łukasz Kędzierski, Jacqui Montgomery, Joan Curtis and Emanuela Handman, (The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia)

Abstract. Leucine-rich repeats (LRRs) are versatile binding motifs found in a variety of proteins and are involved in protein-protein interactions. The LRR domain is composed of repeats forming a characteristic solenoid horse-shoe structure, which provides a scaffold for numerous insertions involved in binding to pathogen-associated molecular patterns and surface receptors. LRRs have been shown to be involved in the host defense systems of both plants (resistance genes) and mammals (Toll-like receptors and nucleotide-binding oligomerisation domain proteins), where they sense specific pathogen-associated molecules and activate the innate immune system. Paradoxically, LRRs have also been shown to be part of microbial virulence factors involved in the interaction with host cells and establishment of infection. The potential of LRRs to bind a vast array of structurally unrelated ligands and their well-documented involvement in microbial pathogenesis make them a potential target for vaccines and new drugs. The recent identification of LRRs in the obligate intracellular protozoan parasite Leishmania and their participation in the macrophage-parasite interaction have added new insight into the role of LRRs in the host cell invasion.

Keywords: leucine-rich repeat; host-pathogen interaction; Toll-like receptors; Leishmania.

5239
Genetic and biochemical background of chronic granulomatous disease
Monika Jurkowska^1,2, Ewa Bernatowska³ and Jerzy Bal¹, (¹National Research Institute of Mother and Child, Warsaw, Poland,²Postgraduate School of Molecular Medicine, Medical University of Warsaw, Poland,³ Children’s Memorial Health Institute, Warsaw, Poland)

Abstract. Chronic granulomatous disease (CGD) is a rare inherited immunodeficency syndrome caused by a profound defect in the oxygen metabolic burst machinery. Activity of NADPH oxidase is absent or profoundly diminished, as at least one of its components (gp91phox, p22phox, p47phox and p67phox) is lacking or non-functional. This review explains the molecular basis of NADPH oxidase dysfunction by the effects of mutations in genes coding for particular oxidase components. Among the four types of CGD, the most common is X-linked CGD (approximately 65%), with defects in the CYBB gene encoding gp91phox. A wide spectrum of mutations has been described in the CYBB gene with no predominant genotype. The second most common subtype of CGD caused by NCF1 mutation accounts for 30% of CGD patients and is inherited in an autosomal recessive manner, with predominance of a homozygotous ?GT deletion in the genotype. The other two CGD subtypes having an autosomal recessive pattern together account for no more than 10% of CGD cases. A strategy for the molecular diagnostics in CGD patients is proposed and principles of genetic counseling are discussed here.

Keywords: CGD; NADPH oxidase; genes; molecular diagnostics.

5240

Original Article

Characterization and serological classification of a collection of Proteus penneri clinical strains
Dominika Drzewiecka, Krystyna Zych and Zygmunt Sidorczyk, (Department of General Microbiology, Institute of Microbiology and Immunology, University of Łódź, Łódź, Poland)

Abstract.

Introduction:
Bacteria of the genus Proteus, which are a common cause of urinary tract infections, are divided into four species: P. mirabilis, P. vulgaris, P. penneri, and P. hauseri, and three unnamed genomospecies, Proteus 4, 5, and 6 (single-strain species P. myxofaciens was isolated from the gypsy moth). Establishing the serological classification of these species would aid in completing the classification scheme of the whole genus Proteus and in applying serological methods in diagnostic procedures and epidemiological investigations for these opportunistic pathogens. The aim of this research was a serological characterization and classification of 57 Proteus penneri clinical strains, isolated from patients from different countries all over the world, into Proteus O serogroups.

Materials and Methods:
Purified lipopolysaccharides (LPSs) extracted from 57 P. penneri strains were used as antigens in enzyme immunosorbent assay (EIA), SDS/PAGE, and Western blot techniques, and alkali-treated LPSs in passive immunohemolysis test (PIH), inhibition of PIH, and absorption of rabbit polyclonal O-antisera.

Results and Conclusions:As a result of serological studies of LPSs extracted from the P. penneri strains, one new Proteus serogroup, represented by the P. penneri 97 strain, was established. Three further strains were classified into the Proteus serogroup O8, which had not contained any P. penneri strains before. All the remaining strains were classified into 11 already existing Proteus O serogroups. It is important to emphasize that 72% of studied strains were classified into serogroups that contain P. penneri strains only. That result confirms the serological distinction of this species within the genus Proteus, and may have diagnostic significance.

Keywords: Proteus penneri; O antigen; lipopolysaccharide; O serogroups; O serotyping; serological classification.

5241
Effect on peripheral blood natural killer cytotoxic cell activity in rats after intraperitoneal implantation of double veloured polyester (Dacron) prosthesis
Marek K. Jurkowski¹, Barbara Bobek-Billewicz², Małgorzata Ćwiklińska-Jurkowska³ and Piotr Jurkowski⁴, (¹Department of Pediatrics, Hematology and Oncology, University of Medical Sciences, Bydgoszcz, Poland, ²Regional Cancer Center, Bydgoszcz, Poland, ³Department of Computer Science, University of Medical Sciences, Bydgoszcz, Poland, ⁴Department of Computer Science and the Methodology of Scientific Research, University of Medical Sciences, Bydgoszcz, Poland)

Abstract.

Introduction:
The aim of this study was to assess the changes affecting natural killer cytotoxic cell (NKCC) activity following intraperitoneal implantation of a double veloured polyester prosthesis in a rat model.

Materials and Methods:
Blood samples were taken by cardiac puncture 1 h before (base line) and 14, 28, 100 and 180 days post-implantation. Peripheral blood mononuclear cells were separated from heparinized blood by density centrifugation. A standard, 4 h 51Cr-release assay against YAC-1 target cells at effector to target ratios of 12:1; 25:1 and 50:1 was performed and the number of total leukocytes, lymphocytes, granulocytes, monocytes, and large granular lymphocytes (LGLs), as well as serum corticosterone levels (radioimmunoassay method) were determined.

Results:
Comparative analysis of the results obtained from animals with implants, baseline samples, and a control group (laparotomy only) revealed lower NKCC, LGL, leukocyte and lymphocyte counts and elevated plasma corticosterone levels in animals receiving the implant on the 14th day post-implantation.

Conclusions:
Our findings indicate that the polyester implant can transiently modulate immune system activities. Since NK cells are important in the control of viral infection and carcinogenesis in humans, it is possible that the stress generated by polyester prostheses can exacerbate the surgical stress and put patients at a higher risk for viral infection and/or metastases.

Keywords: Dacron • NKCC • LGL • rat • lymphocytes • leukocytes

5242

Vol. 52, No. 3, 2004

Reviews

Stroke, myocardial infarction, acute and chronic inflammatory diseases: caspases and other apoptotic molecules as targets for drug development
Michael Kreuter¹, Claus Langer², Claus Kerkhoff³, Pallu Reddanna⁴, Anna L. Kania⁵, Subbareddy Maddika^4,5, Katerina Chlichlia⁶, Truc Nguyen Bui⁷ and Marek Los^3,5 (¹Department of Medicine/Hematology and Oncology, Muenster, Germany ²Central Clinical Laboratory, Medical Clinic, Muenster, Germany ³Institute of Experimental Dermatology, University of Muenster, Germany ⁴School of Life Sciences, University of Hyderabad, India ⁵Manitoba Institute of Cell Biology, Winnipeg, Canada ⁶Department of Tropical Hygiene, University of Heidelberg, Heidelberg, Germany ⁷Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637, USA)

Abstract. Mapping of the human and other eukaryotic genomes has provided the pharmacological industry with excellent models for drug discovery. Control of cell proliferation, differentiation, activation and cell removal is crucial for the development and existence of multicellular organisms. Each cell cycle progression, with sequences of DNA replication, mitosis, and cell division, is a tightly controlled and complicated process that, when deregulated, may become dangerous not only to a single cell, but also to the whole organism. Regulation and the proper control of the cell cycle and of programmed cell death (apoptosis) is therefore essential for mammalian development and the homeostasis of the immune system. The molecular networks that regulate these processes are critical targets for drug development, gene therapy, and metabolic engineering. In addition to the primary, intracellular apoptotic suicide machinery, components of the immune system can detect and remove cells and tissue fragments that no longer serve their defined functions. In this review we will focus on apoptotic pathways converging on caspase family proteases, summarizing pharmacological attempts that target genes, proteins, and intermolecular interactions capable of modulating apoptosis and the inflammatory response. The upcoming pharmacological development for treatment of acute pathologies, such as sepsis, SIRS, stroke, traumatic brain injury, myocardial infarction, spinal cord injury, acute liver failure, as well as chronic disorders such as Huntington’s disease, Parkinson’s disease, ALS, and rheumatoid arthritis, will be discussed in details. We also suggest new potential molecular targets that may prove to be effective in controlling apoptosis and the immune response in vivo.

Keywords: apoptosis; Bcl-2, inflammation; myocardial infarct; stroke; sepsis

5533
Mechanisms of type I interferon signaling in normal and malignant cells
Yongzhong Li, Kishore K. Srivastava and Leonidas C. Platanias Robert H. Lurie (Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School and Lakeside Veterans, Administration Medical Center, Chicago, IL 60611, USA)

Abstract. Type I interferons (IFNs) are cytokines that induce multiple biological effects on target cells, including antiviral, antiproliferative, and immunomodulatory activities. Consistent with the pleiotropic nature of these cytokines, multiple signaling pathways are activated during binding of IFNs to the type I IFN receptor. An important signaling cascade activated by type I IFNs is the Jak-Stat pathway. Activation of the Tyk-2 and Jak-1 kinases, and downstream formation of various Stat complexes, mediates IFN-dependent gene transcription for IFN-stimulated genes. In addition to the classic Jak-Stat pathway, type I IFNs activate multiple other pathways, including the insulin receptor substrate-phosphatidylinositol 3’-kinase cascade, the CBL-CrkL pathway, and mitogen-activated protein kinase pathways. There is accumulating evidence that non-Stat IFN-regulated signaling pathways play important roles in the generation of the antiproliferative effects of type I IFNs. In this review, the regulation of various signaling cascades by the type I IFN receptor is summarized and an update on recent advances in the field is provided.

Keywords: Interferon; cytokines; signaling; malignat cells

5534
Targeting the chemokine network in renal inflammation
Vaclav Eis^1,2, Volker Vielhauer¹ and Hans-Joachim Anders¹ (¹Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Muenich, Germany ²Department of Pathology, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic)

Abstract. Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocyte recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocyte subpopulations contribute to renal damage by releasing inflammatory and profibrotic cytokines. All intrinsic renal cells are capable of chemokine secretion on stimulation in vitro. Expression of inflammatory chemokines correlates with renal damage and local accumulation of chemokine receptor-bearing leukocytes in a variety of animal models of renal diseases as well as in human biopsy studies. Chemokines and their respective receptors could represent new targets for therapeutic intervention in renal inflammatory disease states that often tend to progress to end-stage renal disease. This article summarizes the present data on the role of chemokines and their receptors in renal inflammation with special emphasis on our efforts to identify the chemokine receptors CCR1 and CCR2 as promising targets for therapeutic intervention.

Keywords: chemokines; inflammation; kidney disease; fibrosis

5535
Tracking lymphocytes in vivo
Claire L. Adams, Natalie Kobets, Gordon R. Meiklejohn, Owain R. Millington, Angela M. Grierson, Catherine M. Rush, Karen M. Smith and Paul Garside (University of Glasgow, Western Infirmary, Glasgow, Scotland, UK)

Abstract. The ability to track antigen (Ag)-specific lymphocyte populations in vivo has greatly increased our understanding of the location and functional status of these cells throughout the course of an immune response. Recent technical advances have enhanced researchers’ capability to follow migration, activation and cellular interactions of Ag-specific lymphocytes in situ. It is now possible to monitor changes in T cell subsets, co-stimulatory molecules, and chemokine expression within the physiological context of secondary lymphoid organs. Furthermore, the Ag-presenting cell-T cell interaction can be studied, thus dissecting the role and timing of Ag presentation of particular dendritic cell subsets in the initiation of the immune response. The capacity to adoptively transfer small populations of Ag-specific T lymphocytes has also increased our knowledge of the physiologically important role of regulatory T cells in autoimmunity and immunosuppression. New fluorescence imaging techniques such as multicolor video microscopy, laser scanning cytometry, and multiphoton tissue imaging have provided new ways in which researchers can track cellular changes within Ag-specific lymphocytes in vivo. This review summarizes some of the ways in which these techniques have led to discoveries in the role of signaling cascades, cell cycle progression, and apoptosis in maintaining an Ag-specific immune response.

Keywords: adoptive transfer; Ag-specific; cellular interactions; fluorescence; lymphocytes; transgenic

5536

Original Articles

Structure of the O-polysaccharide of Proteus mirabilis O19 and reclassification of certain Proteus strains that were formerly classified in serogroup O19
Andrei V. Perepelov¹, Antoni Różalski², Beata Bartodziejska² , Sof’ya N. Senchenkova1 and Yuriy A. Knirel¹ (¹N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia. ² Institute of Microbiology and Immunology, University of Łódź, Łódź, Poland)

Abstract. Introduction: Bacteria of the genus Proteus are a common cause of urinary tract infections. The O-polysaccharide chain of their LPS (O-antigen) defines the serological specificity of these bacteria. Based on the immunospecificity of the O-antigens, two species, P. mirabilis and P. vulgaris, were classified into 49 O-serogroups, and more O-serogroups for strains of these species and P. penneri have been subsequently proposed.
Materials and Methods: The lipopolysaccharide of P. mirabilis CCUG 19011 from serogroup O19 was degraded under mildly acidic and mildly alkaline conditions. Polysaccharides thus obtained were studied by chemical methods, including O-deacetylation, sugar and methylation analyses, and ¹H- and ¹³C-NMR spectroscopy. Antisera were obtained by immunization of New Zealand white rabbits with heat-killed bacteria. In serological studies, enzyme immunosorbent assay, passive hemolysis test, and inhibition of passive hemolysis were used.
Results: The following structure of the O-polysaccharide repeating unit was established: →3)-b-D-GlcpNAc-(1→3)-a-D-GalpNAc4,6(R-Pyr)-(1→4)-a-D-GalpA-(1→3)-a-l-Rhap2Ac-(1→ where R-Pyr is (R)-1-carboxyethylidene (an acetal-linked pyruvic acid). This structure is significantly different from the O-polysaccharide structures of P. vulgaris, P. hauseri and P. penneri strains from the same Proteus serogroup O19.
Conclusions: Based on immunochemical studies of the lipopolysaccharides, it is suggested 1) to keep P. vulgaris CCUG 4654 and P. penneri 31 in serogroup O19 as two subgroups, 2) to reclassify P. mirabilis CCUG 19011 into a new Proteus serogroup, O51, and 3) to classify serologically related strains, including P. vulgaris ATCC 49990, P. hauseri 1732-80 and 1086-80, P. penneri 15, and some other P. penneri strains, in yet another Proteus serogroup, O52.

Keywords: Proteus mirabilis; lipopolysaccharide; O-polysaccharide; structure; serological classification; pyruvic acid

5537
Human sera with precipitating antibodies to human soluble immune complexes.A brief communication
Felix Milgrom and Donna Czechowski (Department of Microbiology and Immunology, State University of New York, Buffalo, NY, USA)

Abstract.
Introduction: Previous numerous papers by the senior author dealt with the human serum factor referred to as antiantibody which is specifically directed against IgG antibodies that underwent molecular transformation in the course of the reactions with their corresponding antigens. The reactions of this serum factor could be conveniently detected by means of agglutination of Rh-positive erythrocytes sensitized by anti Rh antibodies. No precipitation tests could be developed.
Materials: Most studies were conducted by means of double diffusion in gel precipitation.
Methods and Results: A rheumatoid arthritis serum, G, was noted that produced a strong reaction of double diffusion in gel precipitation with serum samples of a renal graft recipient, T. Further screening detected one more rheumatoid arthritis serum reacting with T; of 28 sera from renal graft recipients, 6 reacted in a similar way to T, but the reactions were considerably weaker and poorly reproducible. Evidence was presented that the precipitin in the two rheumatoid arthritis sera under study had properties of previously described anti-antibody.
Conclusions: Sera with precipitating anti-antibodies may serve as exquisite reagents for detection of soluble immune complexes in human sera.

Keywords: rheumatoid arthritis; renal graft; circulating immune complexes

5538
Serum concentrations of MCP-1 and RANTES in patients during aortic surgery: the relationship with ischemia-reperfusion
Monika Jedynak¹ , Andrzej Siemiątkowski.¹ , Marek Gacko², Barbara Mroczko³ and Jacek Borkowski¹ (¹Department of Anesthesiology and Intensive Therapy, Medical University, Białystok, Poland. ²Department of Vascular Surgery and Transplantology, Medical University, Białystok, Poland. ³Department of Biochemical Diagnostics, Medical University, Białystok, Poland)

Abstract.
Introduction: Surgical trauma is associated with depression of the immune system, which results in a high complication rate following abdominal aortic aneurysm (AAA) repair. Monocyte chemotactic protein-1 (MCP-1) and regulated-on-activation normal T cell expressed and secreted (RANTES) protein are important mediators of the immune and inflammatory response. The aim of this study was to determine whether there is any relationship between MCP-1 or RANTES and operative injury and ischemia-reperfusion during AAA surgery in human.
Materials and Methods: Peripheral blood samples were taken from 12 patients before surgery, after anesthesiainduction, before unclamping of aorta (PreX_off), 90 min after unclamping (90minX_off),and at 24 and 48 h after surgery.
Results: The MCP-1 and RANTES serum concentrations were measured with the ELISA technique. MCP-1 concentration significantly increased after reperfusion (90minX_off) in comparison with the PreX_off level (p=0.001). Twenty-four hours aft er AAA repair, MCP-1 significantly decreased 269–225 pg/ml (p=0.005) and reached preoperative value. RANTES level was higher in AAA patients before surgery than in controls (p=0.025) and decreased significantly after ischemia-reperfusion to 13 ng/ml (p<0.001) at 90minX_off. We showed increases in RANTES concentration to 26 ng/ml on the 1^st and to 31 ng/ml on the 2^nd day after surgery (p=0.020, p=0.012, respectively) compared with the 90minX_off level.
Conclusions: Ischemia-reperfusion during AAA repair results in an increase in MCP-1 and decrease in RANTES concentrations in serum. The changes in chemokine concentrations may influence the development of immunosuppression after AAA repair, contributing to the postoperative course.

Keywords: aorta; aneurysm; chemokines; ischemia-reperfusion

5539
Evaluation of selected peripheral blood leukocyte functions in patients with various forms of periodontal disease
Małgorzata Pietruska¹, Janusz Żak², Jolanta Wysocka², Barbara Jaworowska², Beata Żelazowska² , Alina Lipska² and Wanda Stokowska¹ (¹Department of Conservative Dentistry, Medical Academy, Białystok, Poland. ²Department of Pediatric Laboratory Diagnostics, Medical Academy, Białystok, Poland)

Abstract. introduction: Periodontitis (P) is an infectious disease that develops in the supporting tissues of the tooth. One of the risk factors leading to it may be dysfunction of some immune system cells. Therefore, the object of the study was to assess selected functions of peripheral blood leukocytes in patients with various forms of P. As leukocytes are able to secrete interleukin (IL)-4 and IL-6, concentrations of their soluble receptors and the expression of their membrane receptors were investigated.
Materials and Methods: Twenty generally healthy subjects with agressive (AP) and chronic periodontitis (CP) were enrolled in the study. The control group consisted of 8 healthy subjects, with no changes in periodontium. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured. Levels of IL-4, IL-6, and their soluble receptors sIL-4R and sIL-6R were determined in the supernatant by ELISA. The expressions of cell surface IL-4R and IL-6R were assayed on PBMC using flow cytometry.
Results: No statistically significant differences were found in the selected parameters between people with periodontal disease and healthy controls. However, in subjects with AP, there was an increasing tendency in IL-6 concentration and IL-4R expression on PBMCs.
Conclusions: Our results show that leukocytes play a significant part in P and their activity is probably lesion-dependent. Estimation of the cytokines secreted by leukocytes may facilitate differentiation and prognosis of the disease progression.

Keywords: periodontitis; IL-4; IL-6; receptors

5540

Vol. 52, No. 4, 2004

Reviews

Role of lipid rafts in T cells
Sunil Thomas, Rajeev Kumar S. and Teodor-D. Brumeanu (Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA)

Abstract. The plasma membrane of T cells is made up of a combination of phospholipids and proteins organized as glycolipoprotein microdomains termed lipid rafts. The structural assembly of lipid rafts was investigated by various physical and biochemical assays. Depending on the differentiation status of T cells, the lipid rafts seclude various protein receptors instrumental for the early T cell signaling, cytoskeleton reorganization, protein and membrane trafficking, and the entry of infectious organisms into the cells. This review article summarizes recent information on the assembly of lipid rafts in plasma membrane of T cells and their signaling output in mature and thymic precursors towards cell growth and differentiation, and possible modalities by which the function of lipid rafts can be altered by drugs and T cell ligands. The concept of using lipid rafts as a target for pharmaceutical compounds and biological T cell ligands to ultimately alter the T cell function is discussed.

Keywords: lipid rafts; T cells; thymic selection; immune modulation

5885
Rethinking oxidized low-density lipoprotein, its role in atherogenesis and the immune responses associated with it
Peter X. Shaw University of California, San Diego, La Jolla, CA 92093, USA

Abstract. Atherosclerosis is a chronic inflammatory disease, resulting from hyperlipidemia and a complex interplay of many environmental, metabolic, and genetic risk factors. The unregulated macrophage uptake of cholesterol and lipids through modified forms of lowdensity lipoprotein (LDL), such as “OxLDL”, transforms macrophages into “foam cells” to form the initial morphological lesion (the fatty streak). The modification of LDL not only enhances its uptake by macrophages, but also changes the natural structures of these otherwise ubiquitous molecules to generate a variety of modified lipids and proteins that represent highly immunogenic neo-determinants. For example, in ApoE–/– mice, autoantibody titers to epitopes on OxLDL are correlated with the extent of atherosclerosis. Similarly, oxidative stress on cellular membranes could also give rise to “oxidation-specific” epitopes and common autoantibodies. However, OxLDL is not uniform, but rather contains complex structures, ranging from a small conformational change in surface lipids to the breakdown of the peptide chain. Therefore, the immune responses to the variety of OxLDL and their association to atherosclerosis progression are very different. For example, phosphorylcholine (PC) is a natural component of phospholipids and exists in LDL and plasma membranes. “Natural” antibodies against PC can distinctively react to PC on bacteria, OxLDL and apoptotic cells, but not to those on unoxidized phospholipids, native LDL and viable cells, which suggests the broader role of such autoantibodies in maintaining the homeostasis of the host. While malondialdehyde-modified structures resemble more the exogenous changes and associate with advanced stage of lesion, they are more likely to associate with adaptive immunity.

Keywords: oxidized LDL; MDA-LDL; atherosclerosis; immune response; anti-phosphorylcholine; inflammation

5886
Polymorphisms within the genes encoding TNF-a and TNF-b associate with the incidence of post-transplant complications in recipients of allogeneic hematopoietic stem cell transplants
Katarzyna Bogunia-Kubik (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland)

Abstract. Hematopoietic stem cell transplantation (HSCT) is a curative treatment of many hematological disorders. Recent studies have shown the associations between polymorphic features of cytokine-encoding genes and the incidence of post-transplant complications in the recipients of allogeneic HSCT. This review focuses on the relationship between the polymorphic patterns of patient genes encoding tumor necrosis factor (TNF)-a and TNF-b and the manifestation of post-transplant complications, acute graft-versus-host disease (aGvHD), generation of toxic lesions, and mortality. Discussed in more detail are the relationships of TNFd microsatellites and polymorphisms within the promoter region of the TNF-a-encoding gene (TNFA) in the position (–308) and within the first intron of the TNF-ß-encoding gene (TNFB). It appeared that heterozygosity within the TNFA promoter and the first intron of the TNFB gene increased the susceptibility to severe grades III–IV of toxic complications, while the presence of the TNFd3 homozygous genotype was associated with a higher risk of severe aGvHD and early mortality in patients after allogeneic HSCT. These results imply that donor-recipient genotyping, extended to cytokine loci, may be of prognostic value for transplantation outcome.

Keywords: allogeneic HSCT; TNF polymorphism; GvHD; toxicity

5887
The role of PTEN in allergic inflammation
Yong C. Lee (Department of Internal Medicine, Research Center for Allergic Immune Diseases, Chonbuk National University Medical School, Jeonju, South Korea)

Abstract. Bronchial asthma is a chronic inflammatory disease of the airways, characterized by airway eosinophilia, goblet cell hyperplasia with mucus hyper-secretion, and hyper-responsiveness to inhaled allergens and to non-specific stimuli. Eosinophil accumulation and subsequent activation in bronchial tissues play critical roles in the pathophysiology of bronchial asthma. Many inflammatory mediators attract and activate eosinophils via signal transduction pathways involving an enzyme phosphatidylinositol 3-kinase (PI3-kinase). Studies using wortmannin, a specific inhibitor of PI3-kinase, have revealed the involvement of PI3-kinase in the biochemical transduction of activation signals generated by many inflammatory mediators in eosinophils. Wortmannin prevents the development of airway inflammation, either by inhibiting the eosinophil infiltration of bronchial tissues or their activation on arrival. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN opposes the action of PI3-kinase by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. Recently we have demonstrated that PTEN expression is diminished in airway epithelial cells of antigen-sensitized and -challenged mice. Administration of PI3-kinase inhibitors or adenoviruses carrying PTEN complementary DNA remarkably reduces eosinophil levels and inflammation. One likely mechanism for this reduction is PTEN-mediated eosinophil degranulation and suppression of interleukin (IL)-4 and IL-5. These findings indicate that use of PTEN may be a good therapeutic strategy for the management of allergic inflammation.

Keywords: PTEN; bronchial asthma; eosinophilia; phosphatidylinositol 3-kinase; signal transduction; inflammation

5888
Mouse Ly-6 proteins and their extended family: markers of cell differentiation and regulators of cell signaling
Anil Bamezai (Biology Department, Villanova University, Villanova, PA 19085, USA)

Abstract. The Ly-6 locus on mouse chromosome 15 encodes a family of 10–12 kDa proteins that are linked to the cell surface by a glycosylphosphatidyl-inositol anchor and have cell signaling and cell adhesion properties. Expression of Ly-6 proteins is tightly regulated during development; these proteins continue to serve as excellent differentiation surface markers on normal and abnormal cells, but their role in driving cellular differentiation is still emerging. Recent studies suggest that Ly-6 gene products participate in regulating signaling through other cell type-specific receptors, perhaps by virtue of these proteins being localized in lipid rafts that play a key role in relaying signals from the membrane to the nucleus. Ligands for some Ly-6 proteins have been reported; the consequence of their interactions with the Ly-6 receptor remains to be fully uncovered. Mouse Ly-6-like proteins have also been reported from a variety of life forms ranging from Caenorhabditis elegans to humans that show a limited amino acid dentity and share structural features with members of mouse Ly-6. Despite these similarities, the non-murine Ly-6 proteins bind distinct ligands and appear to have different cellular functions. All members of the Ly-6 super gene family perhaps evolved from an ancestral gene by a gene duplication mechanism.

Keywords: Ly-6; cell adhesion; cell signaling; differentiation marker; disease; lipid raft

5889
The common gc-cytokines and transplantation tolerance
Minh Diem Vu and Xian Chang Li (Department of Medicine, Harvard Medical School, Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA)

Abstract. Transplant rejection, like tolerance, is a T cell-dependent event. There is compelling evidence to suggest that induction of transplant tolerance is an actively learned process in which T cells need to engage the alloantigens in order to learn to tolerate the allograft. A family of cytokines whose receptors use the same IL-2 receptor g chain (also called the common gc) plays an important role in regulating multiple aspects of the allograft response (i.e. rejection vs. tolerance). It is undeniable that gc-cytokines can drive clonal expansion and effector maturation of alloreactive T cells, and therefore, targeting such cytokines or their receptor components remains an attractive way of blocking transplant rejection. However, we just started to appreciate that gc-cytokines also regulate the acquisition of transplant tolerance via programming activated T cells for apoptotic cell death and via guiding the evolution of regulatory T cells. Thus, understanding precisely the role of gc-cytokines in regulating T cell homeostasis and T cell regulation is critically important in the induction of transplant tolerance.

Keywords: common gc; cytokines; T cell regulation; tolerance; transplantation

5890

Original Articles

Antichlamydial antibodies in the serum and expressed prostatic secretion in prostatitis
Iwona Ostaszewska-Puchalska¹, Bożena Zdrodowska-Stefanow², Jerzy Badyda³, Violetta Bułhak-Kozioł¹ , Katarzyna Puciło¹ and Barbara Darewicz⁴. (¹ Center for Sexually Transmitted Disease Research and Diagnostics, Białystok, Poland, ² Department of Dermatology and Venerology, Medical School, Białystok, Poland, ³ Outpatients Urology Clinic, Białystok, Poland, ⁴ Department of Urology, Medical School, Białystok, Poland)

Abstract. Introduction:The aim of the study was to evaluate the prevalence of anti-Chlamydia trachomatis (C. trachomatis) antibodies in serum and expressed prostatic secretion (EPS) in chronic prostatitis.
Materials and Methods:Thirty-six patients with chronic prostatitis were examined. The presence of C. trachomatis was determined in urethral smears and EPS. Specific antibodies were determined in the serum (IgM, IgA, IgG) and in the EPS (IgA, IgG). In the direct diagnosis of chlamydial infection, the direct immunofluorescence method and the ligase chain reaction were employed, and for the serological diagnosis, the immunoenzymatic method.
Results: C. trachomatis infection was detected in the urethra of 3 (8.3%) patients and in the prostatic gland of 3 (8.3%) patients; only one of these patients was found to have C. trachomatis in both the urethra and the EPS. In the control group, C. trachomatis was detected in the urethra of 1/50 (2%) of the men, but the EPS of all of them was free of C. trachomatis. Specific IgM antibodies were found in 7 (19.4%), IgA in 9 (25%), and IgG in 18 (50%) of the patients’ serum, whereas IgAs were detected in 12 (33.3%) and IgGs in 13 (36.1%) of the patients’ EPS. In the control group, anti-C. trachomatis antibodies of the IgG were detected in the serum of 2/35 (5.7%) of the men, whereas in the EPS neither IgA nor IgG antibodies were detected in any of these patients.
Conclusions: Serological tests of the serum and EPS are useful as a complementary method in the diagnosis of chronic prostatitis.

Keywords: prostatitis; Chlamydia trachomatis; IgG; IgM; IgA antichlamydial antibodies; expressed prostatic secretion

5891
Oxidative modification of type II collagen differentially affects its arthritogenic and tolerogenic capacity in experimental arthritis
Janusz Marcinkiewicz¹, Rafał Biedroń¹, Katarzyna Maresz¹, Beata Kwaśny-Krochin², Małgorzata Bobek¹, Ewa Kontny³, Włodzimierz Maśliński³ and Benjamin Chain⁴ (¹ Department of Immunology, Jagiellonian University Medical College, Kraków, Poland, ² Department of Rheumatology, Jagiellonian University Medical College, Kraków, Poland, ³ Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland, ⁴Department of Immunology and Molecular Pathology, The Windeyer Institute for Infectious Diseases, University College London, UK)

Abstract. Introduction: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly use in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen.
Materials and Methods: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined.
Results: Mice immunized with C_NAT in adjuvant developed arthritis (CIA) with an incidence of 69%. C_NAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-C_NAT IgG antibodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-C_NAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with C_NAT.
Conclusions: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil-derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.

Keywords: collagen-induced arthritis; DBA/1 mice; protein oxidation; neutrophils; myeloperoxidase; hypochlorous acid

5917

Vol. 52, No. 5, 2004

Reviews

Ludwik Hirszfeld Memorial Lecture: HIV-1 reservoirs: major molecular obstacles to viral eradication
Roger J. Pomerantz (Center for Human Virology and Biodefense, Thomas Jefferson University, Philadelphia, PA 19107, USA)

Abstract. Over the last 18 years of study in one of our laboratories, we have observed the development of residual disease and latent reservoirs as major problems in the long-term therapy of HIV-1-infected individuals on highly active antiretroviral therapy (HAART). It was shown in the early 1990’s that HAART, as it is presently configured, is unlikely to lead to viral eradication due to several mechanisms of viral persistence. The two general mechanisms involved with persistence during HAART include low-level residual, cryptic replication and proviral latently-infected cells. As such, these are key areas of potential study for depletion and, hopefully in the future, eradication of residual disease in patients on suppressive HAART. To deplete these residual disease mechanisms will require multipronged approaches. These will include induction of HIV-1 latent proviruses, suppression of residual viral replication and destruction of long-lived cellular sanctuaries, such as tissue-bound macrophages.

Keywords: HIV-1; eradication; reservoirs; latency

6334
Nucleic acid aptamers in human viral disease
Zhiren Zhang¹, Michael Blank² and Hermann J. Schluesener¹, (¹ Institute of Brain Research, University of Tuebingen, Tuebingen, Germany, ² NascaCell IP GmbH, München, Germany)

Abstract. Nucleic acid aptamers are short, single-stranded oligonucleotides or their modified analogues which avidly and specifically interact with targeted ligands through their 3-dimensional structure. Aptamers can be selected out of a large combinatorial oligonucleotide library through an in vitro evolution process termed SELEX. Since 1990, a wide variety of aptamers targeted to ligands ranging from small molecules to complex mixtures have been isolated. Most selected aptamers have shown high specificity to and affinity for their ligands and are potential detection and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, resulting in potent therapeutic candidates in disease models. Some recent advances to increase the stability of aptamers, extend their in vivo circulation time and their in vivo expression have pushed aptamers closer to therapeutic applications. This review presents recent developments in the field of aptamer research and focuses on their applications to human viral diseases, particularly HIV induced diseases.

Keywords: aptamer; SELEX; therapeutic applications; analytic applications; human viral diseases; HIV; HCV

6336
Genetics of experimental autoimmune encephalomyelitis in the mouse
Asa Andersson and Jenny Karlsson (Medical Inflammation Research, I11, BMC, Lund University, Lund, Sweden)

Abstract. Multiple sclerosis (MS) is an inflammatory, demyelinating disease in the central nervous system (CNS) affecting approximately 0.1% of the population in the northern part of the world. The factors behind the initiation of the inflammatory response are not known at present, but MS is considered as a complex disease depending on genetic as well as environmental factors. Experimental autoimmune encephalomyelitis (EAE) is the prevailing experimental rodent model for multiple sclerosis (MS). Disease is induced in genetically susceptible mice or rats by immunization with myelin proteins or peptides, which leads to an infiltration of leukocytes into the CNS. EAE has been subjected to investigations of genetic susceptibility to disease development. By the identification of genes predisposing to EAE, the hope is to get clues as to what genetic elements are also important in MS. To date, more than 25 Eae loci have been described in the mouse. The quantitative trait loci are linked to different disease traits and several show sex specificity. Here we discuss the current state of the genetics controlling susceptibility to EAE.

Keywords: autoimmunity;experimental models; genetics; EAE; multiple sclerosis

6337
Protein catabolism in chronic uremia: is it due to malnutrition?
Tejinder S. Ahuja and William E. Mitch 9University of Texas Medical Branch, Galveston, TX 77555-0562, USA)

Abstract. The high prevalence of anthropometric measurements and laboratory values that are similar to those in patients with protein-energy malnutrition has lead to the classification of a large number of dialysis patients as being malnourished. However, malnutrition in the strict sense implies that abnormalities will be reversed if more food is eaten. There is virtually no evidence, however, that simply providing more nutrients in the diet of dialysispatients will reverse the abnormalities attributed to malnutrition. This suggests that the diagnosis of malnutrition is a misdiagnosis. In this review, we discuss mechanisms that will cause the loss of protein stores, including albumin, other plasma proteins, and muscle mass in dialysis patients. We will also review the shortcomings of techniques that are used to measure the nutritional status of these patients.

Keywords: malnutrition; muscle atrophy; dialysis

6338
New target against inflammatory diseases: transglutaminase 2
Soo-Youl Kim (Department of Neurology and Neuroscience, Weill Medical College of Cornell University and Burke Medical Research Institute, White Plains, NY 10605, USA)

Abstract. Transglutaminase (TGase) 2 is an enzyme that is widely used in many biological systems for generic tissue stabilization or immediate defense for wounds. Many reports showed that TGase 2 is aberrantly activated in tissues and cells and contributes to a variety of diseases, including neurodegenerative diseases and autoimmune diseases. In most cases, TGase 2 appears to be a factor in the formation of inappropriate proteinaceous aggregates that may be cytotoxic. However, in other cases, such as celiac disease, arthritis, lupus, and amyotrophic lateral sclerosis, TGase 2 is involved in the generation of autoantibodies. This suggests the possibility that inappropriate expression and/or presentation of TGase 2 to T cells might contribute to these diseases in genetically predisposed individuals. We and others have found that TGase 2 expression is also increased in the inflammation process. Furthermore, we also demonstrated a reversal of inflammation by TGase inhibition. This review will examine a possibility of TGase inhibitors as therapeutic agents in a variety of inflammatory diseases.

Keywords: transglutaminase; transglutaminase inhibitor; inflammation; autoimmune disease

6339
Neurokinin receptors: relevance to the emerging immune system
Helen S. Kang^{1, 2}, Katarzyna A. Trzaska^{1, 2}, Kelly Corcoran^{1, 2}, Victor T. Chang^{1, 3} and Pranela Rameshwar¹ (¹ Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ, USA, ² Graduate School of Biomedical Sciences, UMDNJ, Newark, NJ, USA, ³VA New Jersey Health Care System, East Orange, NJ, USA)

Abstract. The adult bone marrow (BM) is the major site of the emerging immune system. Hematopoiesis is the process whereby immune cells are generated from a finite number of hematopoietic stem cells. Hematopoiesis is regulated by soluble mediators and intercellular interactions. A major regulatory mechanism of hematopoiesis involves bidirectional crosstalk with the neural system. This communication mainly occurs by the release of neurotransmitters from innervated fibers. The neurotransmitters interact with specific receptors on BM resident cells and release other hematopoietic regulators such as cytokines. Together, the neurotransmitters and cytokines form a complex network to regulate hematopoiesis. Among BM resident cells, the stromal cells are particularly relevant for two reasons: 1) they represent non-neural sources of neurotransmitters, and 2) stromal cells express specific receptors for neurotransmitters. This review focuses on the hematopoietic effects of neurotransmitters belonging to the tachykinins. The two major tachykinins focused in this review are substance P and neurokinin (NK)-A, 11 and 10 amino acid peptides. In BM, the tachykinins interact with two major NK receptors: NK-1 and NK-2. These two receptors appear to limit tachykinin-mediated effects on hematopoiesis. The central roles of NK receptors within a network comprising of cytokines and tachykinins are reviewed.

Keywords: neurokinin;substance P; cytokines; neuropeptides; hematopoiesis

6340
b₃-Integrin cytoplasmic binding proteins
Renyi Zhao, Alokkumar S. Pathak and George A. Stouffer (Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC, USA)

Abstract. Integrins are cell-surface adhesion receptors that play an important role in mediating numerous physiological processes, including inflammation, migration, adhesion, and proliferation. Integrin regulation by events within the cell has been termed “inside-out” signaling; this is a capacity that is unique to integrin receptors. As is typical of other cell-surface receptors, integrins can also transduce signals from outside the cell into the cytoplasm on binding extracellular ligands (“outside-in signaling”). Integrins are composed of an a and a b subunit, which form a heterodimer. The b₃-integrin family consists of a_IIbb₃ found on platelets and megakaryocytes, and the more widely distributed a_vb₃. ß Subunits consist of a large extracellular domain, a single transmembrane segment, and a relatively short cytoplasmic tail. The cytoplasmic domains do not contain intrinsic tyrosine kinase activity, and therefore signaling occurs primarily via recruitment of intracellular signaling molecules. Integrins form transmembrane connections, and the interactions between integrin cytoplasmic domains, intracellular factors (cytoplasmic proteins and intracellular signaling pathways), and membrane-anchored proteins play an important role in integrin- mediated events. There are at least 21 proteins that associate with integrin b tails to regulate cell motility, proliferation, differentiation, and apoptosis. In this review, we will focus on 10 of these proteins and their function in integrin-mediated events.

Keywords: integrinscytoplasmic domains; intracellular factors

6341
Treatment options for severe lupus nephritis
Oliver Lenz and Gabriel Contreras (Division of Nephrology and Hypertension, University of Miami, Miami, FL 33136, USA)

Abstract. Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Landmark trials conducted by the National Institutes of Health established cyclophosphamide as the mainstay of therapy. Since then, the prognosis of patients with lupus nephritis has markedly improved, and 10-year survival rates now surpass 75%. These superior outcomes have come at the expense of adverse events such as serious infections and gonadal failure in a significant number of patients, and the relapsing nature of the disease continues to pose a problem. For these reasons, new treatment protocols, such as mycophenolate mofetil induction or sequential therapies using azathioprine or mycophenolate mofetil in the maintenance phase, have been developed in recent years with the goal to maintain remission and reduce adverse events. In addition, ongoing research into the pathogenesis of lupus nephritis has confirmed the importance of B and T cell activation, leading to the identification of potential new therapeutic targets. This article discusses established and novel treatment options for patients with severe lupus nephritis corresponding to WHO c asses III, IV, and V with III or V with IV.

Keywords: lupus erythematosus; systemic; lupus nephritis; drug therapy

6342

Original Articles

CTLA-4 (CD152) gene polymorphism at position 49 in exon 1 in Graves’ disease in a Polish population of the Lower Silesia region
Irena Frydecka^{1, 2} , Jacek Daroszewski³ , Katarzyna Suwalska¹ , Magdalena Żołędziewska⁴ , Anna Tutak¹ , Mirosław Słowik⁵ , Stanisław Potoczek² and Tadeusz Dobosz⁴ (¹ Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland, ²Department of Hematology, Blood Neoplastic Diseases and Bone Marrow Transplantation, Medical University, Wrocław, Poland, ²Department of Endocrinology and Diabetics, Medical University, Wrocław, Poland, ⁴Department of Forensic Medicine, Medical University, Wrocław, Poland ⁵ Department of Ophthalmology, Medical University, Wrocław, Poland)

Abstract. Introduction:Graves’ disease (GD) is an autoimmune disease believed to be caused by a combination of environmental and genetic factors. The gene encoding cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of the candidate genes for conferring susceptibility to thyroid autoimmunity. The aim of the study was to investigate the association between the exon 1 CTLA-4 gene polymorphism A(49)G and susceptibility to GD and Graves’ ophthalmopathy (GO) as well as its severity in a Polish population of the Lower Silesia region.
Materials and Methods: We analyzed the A(49)G exon 1 CTLA-4 gene polymorphism in 99 unrelated Polish patients with GD, of whom 50 had clinically evident GO (NOSPECS class III and higher), and 154 matched healthy subjects from the Lower Silesia region. Genomic DNA was isolated from whole frozen blood using the NucleoSpinR Blood kit. A/G transition was genotyped by polymerase chain reaction followed by labeling with the SnaPshot kit of PE Applied Biosystems and detected usng an ABI PRISM 310 capillary genetic analyzer.
Results: The distribution of CTLA-4 exon 1 A(49)G genotype, allele, and phenotypic frequencies did not differ between patients with GD and healthy subjects. There was a significantly lower frequency of the AA genotype in the group of patients with clinically evident GO than in patients without severe GO (22% vs. 43%; p=0.02, OR=2.6).
Conclusions: Our results showed that the AA genotype in patients with GD is associated with a lower risk of GO severity.

Keywords: CTLA-4;A(49)G; Graves’ disease; Lower Silesia

6343

Vol. 52, No. 6, 2004

Reviews

Factors underlying chronic inflammation in rheumatoid arthritis
See Heng Wong and Janet M. Lord, (MRC Centre for Immune Regulation, Department of Immunology, Birmingham University Medical School, Birmingham B15 2TT, UK)

Abstract. Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease whose characteristic pathology includes swollen, painful, and deformed joints. In recent decades, both clinical and basic scientific research have tried to determine the factors involved in the pathogenesis of this common disease. Although the cause of RA is still unknown, several factors that contribute to RA have been identified. Among these are the discoveries of: susceptibility genes, disease-causing immune cells, and cytokine and signal transduction networks involved in promoting persistence of inflammation. Various therapeutic strategies, including anti-tumor necrosis factor ? therapy, have been developed to target one or more of these factors. Although none of these therapeutic strategies can actually cure the disease, some of these novel agents have proven to be more effective than others. This implies that the success of a therapy is very much dependent on the therapeutic targets chosen. Therefore, improved understanding of the cellular and molecular events occurring in the rheumatoid joint during the pathogenesis of the disease is particularly important if we are to better combined therapeutic strategies. In this article we summarize current understanding of the factors that contribute to disease pathogenesis in RA and identify cellular and molecular events that could drive the development of the disease and represent potential new therapeutic targets.

Keywords: inflammation; fibroblasts; cytokines; apoptosis; chemokines.

6557
Macrophage migration inhibitory factor and its role in autoimmune diseases
Claudia M. Denkinger^1,2, Christine Metz³, Günter Fingerle-Rowson^3,4, Michael D. Denkinger¹ and Thomas G. Forsthuber¹, (¹Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA, ²Institute of Immunology, University of Würzburg, D-97080 Würzburg, Germany, ³North Shore Long Island Jewish Institute, Manhasset, NY 11030, USA, ⁴Klinikum der Universität Köln, Medizinische Klinik I, Hämatologie and Onkologie, D-50924 Köln, Germany)

Abstract. After several decades of research into the macrophage migration inhibitory factor (MIF), its diverse actions in the immune system are yet to be fully revealed. What has become clear is that MIF plays an important role in both innate and adaptive immunity. However, while several pathways mediating the function of MIF in the immune system have been established, its role in pathogenic states such as autoimmune diseases has remained unresolved. MIF has been implicated in different autoimmune diseases, including rheumatoid arthritis, glomerulonephritis, and multiple sclerosis, but knowledge about the underlying cellular and molecular mechanisms is just emerging. However, overall it appears that the inhibition of its proinflammatory action is likely to be a successful new therapeutic strategy for some autoimmune diseases, possibly by reducing the need for steroids. As more aspects of the role of this cytokine in the pathogenesis of autoimmune diseases are elucidated, better strategies to target it therapeutically can be expected.

Keywords: MIF; multiple sclerosis; EAE; glomerulonephritis; rheumatoid arthritis; autoimmune disease.

6558
RNA interference: a potential novel therapeutic combating HIV-1 in the central nervous system
Roger J. Pomerantz, (Center for Human Virology and Biodefense, Thomas Jefferson University, Philadelphia, PA 19107, USA)

Abstract. RNA interference (RNAi) is a conserved process by which eukaryotic cells protect their genomes utilizing small, double-stranded RNAs to degrade target RNAs. This occurs in a sequence-specific manner and is different from the interferon effect of larger doublestranded RNAs. Post-transcriptional gene silencing by these nucleic acids can lead to degradation of either cellular or viral RNAs. It has been recently shown that doublestranded, small interfering RNAs (siRNAs) of 21 to 25 nucleotides can be transfected into relevant cells to target specific RNAs. In addition, utilizing hairpin motifs, siRNAs can be expressed intracellularly using molecular therapeutic vectors. This potent approach has been utilized to both inhibit pathogens, including viruses, as well as to dissect cellular molecular mechanisms via a potent knockout effect. At this time in the HIV-1-pandemic, one of the remaining, most enigmatic, and still vitally important areas of HIV-1 pathogenesis occurs in the central nervous system (CNS). HIV-1-induced encephalopathy remains difficult to treat in the developing world and in parts of the developed world, even in the era of highly active anti-retroviral therapy. As such, novel approaches which could lead to intracellular immunization, and life-long resistance against HIV-1 encephalopathy would be of important impact worldwide. Thus, we now seek to combine our background in molecular therapeutics and RNAi with our long-standing interest in HIV-1 neuropathogenesis to target the CNS using siRNAs.

Keywords: RNA interference; siRNA; therapeutics; HIV-1.

6560
The influence of aging in one tauopathy: Alzheimer’s disease
Jesús Avila, (Centro de Biología Molecular “Severo Ochoa”, Facultad de Ciencias, Campus de Cantoblanco, Universidad Autónoma de Madrid, 28049 Madrid, Spain)

Abstract. In this short review, the link between aging and the onset of Alzheimer’s disease is discussed. It has been widely suggested that aging is the greatest risk factor for Alzheimer’s disease, in which a failure in the insulin signal-transduction pathway could occur with age and, thereby, the assembly of senile plaques and neurofibrillary tangles (two aberrant structures present in Alzheimer’s disease) could be promoted. The main component of neurofibrillary tangles is the microtubule-associated protein tau, and the assembly of tau protein appears to occur after its modification by phosphorylation. In this phosphorylation, some protein kinases related to the insulin-transduction pathway could play a role.

Keywords: Alzheimer’s disease; tau phosphorylation; aging; tauopathies.

6562
Fatty acid synthase-catalyzed de novo fatty acid biosynthesis: from anabolic-energy-storage pathway in normal tissues to jack-of-all-trades in cancer cells
Javier A. Menendez^1,2 and Ruth Lupu^1,2, (¹Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL, USA, ²Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA)

Abstract. In 1994, Kuhajda and colleagues unambiguously identified the oncogenic antigen-519, a prognostic molecule found in breast cancer patients with markedly worsened prognosis, as fatty acid synthase (FAS), the key enzyme for the de novo fatty acid biosynthesis. It now appears that human carcinomas and their pre-neoplastic lesions constitutively overexpress FAS and undergo significant endogenous fatty acid biosynthesis. Moreover, FAS blockade specifically induces apoptotic cancer cell death and prolongs survival of cancer xenograft hosts. Therefore, FAS signaling seems to play a central role in the maintenance of the malignant phenotype by enhancing cancer cell survival and proliferation. This review documents the rapidly changing perspectives on the function of FAS in cancer biology. First, we describe molecular mechanism by which aberrant transduction cascades driven by oncogenic changes subvert the down-regulatory effects of dietary fatty acids, resulting in tumor-associated FAS insensitivity to nutritional signals. Second, we speculate on the putative function that hypoxia can play as the epigenetic factor that triggers and maintains FAS overexpression in cancer cells by inducing changes in gene expression and in metabolism for survival. Third, we explore the role that FAS exhibits in cancer evolution by specifically regulating cancer-related proteins such as Her-2/neu oncogene and estrogen receptor. Finally, we reveal previously unrecognized functions of FAS on the response of cancer cells to chemo-, endocrine-, and immuno-therapies. These findings, all together, should ultimately enhance our understanding of how FAS-dependent endogenous fatty acid metabolism, once considered a minor anabolic-energy-storage pathway in normal cells, has become a jack-of-all-trades in cancer cells.

Keywords: fatty acid synthase; fatty acids; oncogene; cancer; metabolism.

6563
Recent controversy surrounding lipid rafts
Martyna Skwarek, (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland)

Abstract. Lipid rafts (LRs) are highly enriched in glycosphingolipids, sphingomyelins, and cholesterol membrane microdomains, existing in a liquid-ordered phase of the plasma membrane. In the literature, LRs are also known as detergent-insoluble membranes, detergent-resistant membranes, glycosphingolipids-enriched membranes, detergent-insoluble glycolipid-rich membranes, and Triton-insoluble floating fractions. These properties enabled their separation from the rest of the phospholipid bilayer, providing new insight into the structure of the plasma membrane, which until then was believed to represent a two-dimensional liquid structure with proteins uniformly solubilized in the lipid solvent. Although there have been many articles concerning LRs, there is still controversy about their existence in the natural state, their size, definition, and function. Different techniques have been developed to visualize LRs in living cells, but the results are contradictory. In this minireview, some recent papers concerning LRs, their existence in vivo, their dynamics, size, methods of isolation, and their association with different proteins are discussed.

Keywords: lipid rafts

6564

Original Articles

Concentration of TBA-reactive substances in type II pneumocytes exposed to oxidative stress
Wojciech J. Piotrowski , Jerzy Marczak , Zofia Kurmanowska and Paweł Górski, (Department of Pneumology and Allergology, Medical University of Łódź, Poland)

Abstract. Introduction:
Oxidative lung damage may be associated with the destruction of alveolar cells. Type II alveolar epithelial cells (AECs), as progenitors of type I cells, are indispensable for the renovation of alveolar structure after lung injury. Extensive damage to type II cells could be responsible for unfavorable outcome. However, the susceptibility of type II AECs to oxidative stress is unclear.

Materials and Methods:
We investigated the susceptibility of freshly isolated and cultured rat type II AECs to oxidative stress (H2O2 and Fe2+). Thiobarbituric acid reactive substances (TBARS) were measured as indices of lipid peroxidation and cytotoxicity was estimated by the MTT test. Aminotriazol (ATZ), an inhibitor of intracellular catalase, was used to estimate the protective role of catalase.

Results:
TBARS concentration increased significantly in freshly isolated, oxidant-exposed cells (4.0±1.3 vs. 8.3±2.2 nmol/g protein, p=0.0313) and insignificantly in cultured cells (1.7±0.4 vs. 4.4±1.7 nmol/g protein). ATZ was toxic even to cells not exposed to oxidants. Inhibition of catalase in cells exposed to oxidants resulted in an insignificant increase in TBARs: 4.5±1.5 vs. 16.2±3.9 nmol/g protein, p=0.0625, and 4.0±0.8 vs.7.6±4.0 for freshly isolated and cultured cells, respectively. Oxidative stress itself did not increase cytotoxicity.

Conclusions:
Type II AECs are not resistant to oxidative stress. We cannot, however, explain why cells with evidence of lipid peroxidation do not show increased cytotoxicity. The toxicity of ATZ is not related to oxidative cell damage. In cells exposed to oxidants, TBARS may further increase when catalase is inhibited, which suggests an important protective role for catalase.

Keywords: lung diseases; TBA-reactive substances; lipid peroxidation; oxidative stress; antioxidants; alveolar epithelium; type II pneumocytes.

6566
The search for a genetic defect in Polish patients with chronic granulomatous disease
Monika Jurkowska¹, Magdalena Kurenko-Deptuch², Jerzy Bal¹ and Dirk Roos³, (¹Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland, ²Department of Immunology, Children’s Memorial Health Institute, Warsaw, Poland, ³Department of Experimental Immunohematology, CLB, Amsterdam, Holland)

Abstract. Introduction:
Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytic cells are unable to generate superoxide anions. Patients with CGD are predisposed to recurrent bacterial and fungal infections because the superoxide-generating NADPH oxidase activity is needed for efficient killing of microbes. Among the at least 5 subunits creating a functional NADPH oxidase, a molecular defect located in any of the gp91phox, p22phox, p47phox, or p67phox subunits may cause CGD.

Materials and Methods:
In this study, 8 patients were diagnosed with CGD on the basis of clinical findings and absence of nitroblue tetrazolium reduction in phagocytes. Southern blot analysis, GeneScan, and direct sequencing were performed to define particular DNA mutations.

Results:
Among 6 X-linked CGD (X-CGD) patients, 4 different mutations were identified in the X-linked CYBB gene (encoding gp91phox) by direct sequencing. A novel missense mutation, located in the NADPH-binding region of gp91phox, was found in 2 brothers. One frameshift 1578delA, one splicing 252G->A mutation, and one partial gene deletion were also identified. The molecular defect in the NCF1 gene (encoding p47phox) was established in 2 patients. One was a ?GT/?GT homozygote, the other carried, besides this GT deletion on one allele, a unique Phe118stop mutation on the other.

Conclusions:
In general, the X-CGD patients within the group followed a more severe clinical course than the patients with an NCF1 defect. However, the lack of a straightforward genotype-phenotype correlation indicates that the clinical severity of CGD depends also on other antimicrobial host-defense systems.

Keywords: CGD; NADPH oxidase; CYBB; NCF1; molecular diagnostics.

6568

Temperature can prime human peripheral blood neutrophils in a p38MAPKa-dependent manner
Grażyna Józefowicz-Okonkwo and Dariusz Nowak, (Institute of Physiology and Biochemistry, Medical University of Łódź, Poland)

Abstract. Introduction:
Previous ex vivo experiments by others suggest that elevated body temperature can prime the respiratory burst of human neutrophils. The mechanism of the priming phenomenon induced by temperature has not been addressed so far. Furthermore, the priming temperature range was not defined.

Materials and Methods:
In the present study we explored, under in vitro conditions, the influence of febrile-range temperatures on reactive and Methods: oxygen species (ROS) generation by human peripheral blood neutrophils. ROS production was measured using whole-blood luminol-dependent chemiluminescence. Two elements of signal transduction pathways, calcium and p38 mitogen-activated protein kinase a (p38MAPKa), frequently underlying neutrophil priming were also examined. Calcium levels in the cytosol of resting and fMLP-stimulated isolated neutrophils were measured with the Fura-2AM spectrofluorimetric method. The activity of p38MAPKa was assessed indirectly with a specific inhibitor of the kinase, SB 203580.

Results:
The study revealed a priming effect at 38°C toward human peripheral blood neutrophil ROS production. Any concomitant effect on calcium response was not observed. Instead, experiments with SB 203580, a specific inhibitor of p38MAPK?, pointed to an increased activity of the kinase as a molecular background of temperature-induced priming. However, the priming effect of temperature was confined to 38°C, while higher temperatures proved toexert no effect (39 and 40°C) or even inhibited ROS generation by neutrophils (43°C).

Conclusions:
Our study suggests a heterogeneous influence of temperature on human neutrophil functioning, including the priming of the cells by a low-febrile-range temperature. It also suggests a p38MAPKa-dependent molecular background of the priming phenomenon.

Keywords: chemiluminescence; reactive oxygen species; SB 203580; intracellular calcium.

6569

Vol. 52, 2004

Vol. 52, No. 1, 2004

CONTENTS

Review

Original Article

Vol. 52, No. 2, 2004

CONTENTS

Review

Original Article

Vol. 52, No. 3, 2004

CONTENTS

Reviews

Original Articles

Vol. 52, No. 4, 2004

CONTENTS

Reviews

Original Articles

Vol. 52, No. 5, 2004

CONTENTS

Reviews

Original Articles

Vol. 52, No. 6, 2004

CONTENTS

Reviews

Original Articles