bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of soil temperature on disease development in melon plants infected by Monosporascus cannonballus
Authors: Pivonia S. , Cohen R. , Kigel J. , Katan J.
Published: March 2003

The effect of soil temperature on melon collapse induced by Monosporascus cannonballus was studied in the laboratory and in the field. In the laboratory, ascospore germination and hyphal penetration into melon roots were enhanced by increasing the temperature from 20 to 32°C. The optimum temperature for mycelial growth of five isolates of M. cannonballus was 30°C. In the field, the effect of temperature was studied in experiments conducted during the winter and autumn cropping seasons from 1995 to 1998. Disease progress was much faster in the autumn than in the winter crop seasons. Disease incidence reached 100% in the three consecutive autumn seasons studied. In the winter seasons, however, planting date influenced disease incidence. Early planting, at the beginning of January, resulted in a low disease incidence (6–26%, 125 days after planting), whereas planting at the end of January resulted in higher disease incidence (72–88%, 95–119 days after planting). In plots in which the soil was artificially heated to 35°C during the winter season, disease incidence reached 85%, as in the autumn season. Plants grown during the winter in unheated soil, or in artificially heated soil disinfected with methyl bromide, did not collapse. Root colonization by the pathogen was higher in the autumn and in heated soil than in the winter season in nonheated soil. Fifty per cent of root segments were colonized 35, 42 and 67 days after planting in the winter‐heated, autumn and winter‐unheated plots, respectively. A high correlation was found between soil temperatures above 20°C during the first 30 days after planting and disease severity. It is suggested that soil temperature during the early stages of plant development is an important factor in disease development and the expression of melon collapse caused by M. cannonballus.
Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water
Authors: Pettitt T. R. , Wakeham A. J. , Wainwright M. F. , White J. G.
Published: March 2003

Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum, Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration‐dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis, tomato and Chrysanthemum. In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration‐dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.
The Human Mast Cell Line HMC‐1 Binds and Responds to C3a but not C3a(desArg)
Authors: Zwirner  , Götze  , Sieber  , Kapp  , Begemann  , Zuberbier  , Werfel
Published: March 2003

Controversial results have been published in the past regarding the functional reactivity of different cell types to the anaphylatoxin C3a and its degradation product C3a(desArg). To understand better the effects of C3a and C3a(desArg) on human mast cells, the authors performed binding experiments and calcium mobilization studies on the human mast cell line HMC‐1 which has been shown previously to express C3a binding sites. For this purpose, functionally active, recombinant C3a (rC3a) was constructed with an 11 amino acid peptide attached to the N‐terminus of the molecule. Using a monoclonal antibody (MoAb) against this tag, binding of rC3a to HMC‐1 cells could be demonstrated by flow cytometry. Its binding was specific as it could be blocked with serum‐derived C3a. In contrast, no binding of rC3a(desArg) to HMC‐1 cells was detectable. Recombinant C3a led to a transient mobilization of intracellular calcium [Ca2+]i in HMC‐1 which was inhibitable by the C3a‐specific MoAb K13/16. No increase of [Ca2+]i was detected when the cells were treated with C3a(desArg). The authors found C3a receptor (C3aR)‐specific mRNA in HMC‐1 cells indicating that this receptor represents the binding site for C3a on these cells. These results demonstrate a specific binding for C3a but not for C3a(desArg) on cells of the human mast cell line HMC‐1. As a consequence, functional activity was restricted to C3a with C3a(desArg) being completely inactive. Therefore, the data strongly suggest that the recently cloned high affinity C3aR which is assumed to represent the binding site for the anaphylatoxin on HMC‐1 cells is unresponsive to C3a(desArg).
Cell‐Mediated Immune Response Against Titin in Myasthenia Gravis: Evidence for the Involvement of Th1 and Th2 cells
Authors: Skeie  , Bentsen  , Freiburg  , Aarli  , Gilhus
Published: March 2003

Myasthenia gravis (MG) patients may have circulating autoantibodies against titin. In this study, we have stimulated T cells from MG patients with a recombinant polypeptide containing the main immunogenic region of titin, MGT‐30 (myasthenia gravis titin‐30 kDa). In an ELISpot assay, MGT‐30 reactive interferon (IFN)‐γ secreting cells (Th1 cells) were detected in six of 10 titin antibody positive MG patients. Such cells were not detected in any of the five titin antibody negative MG patients or in the seven blood donors. In three patients, the stimulated number of cells decreased when total remission of MG symptoms was achieved after thymectomy or following a period of intensive immunosuppressive medication. We detected MGT‐30 interleukin (IL)‐4 secreting cells (Th2 cells) in two of five titin antibody positive MG patients, but not in the two titin antibody negative patients or the five blood donors examined. We conclude that titin antibody positive MG patients have a combined Th1/Th2 cell mediated immunity against the muscle protein titin.
Priming of T‐Cell Responses in Mice by Porins of Salmonella typhimurium
Authors: Gupta
Published: March 2003

An imbalance in signals delivered to T cells via T‐cell receptor and accessory molecules can lead to anergy, apoptosis, or both. In the present study we have demonstrated that Salmonella typhimurium infection in mice leads to a progressive loss of CD4+ T helper (Th) cell population, abnormal T‐cell death by apoptosis and loss of accessory molecules (B7 and intracellular adhesion molecule‐1) on macrophages. Quantification of interleukin‐2 (IL‐2), interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) secretion revealed a Th2‐type of response in lymphocytes isolated from spleen. However, preimmunization of mice with porins resulted in an increased CD4+ Th cell population and accessory molecules on the surface of macrophages. Quantification of cytokines revealed a Th1‐type of response. We conclude that preimmunization of mice with porins provides a microenvironment in which a well‐balanced accessory molecule and cytokine network is established, which results in the prevention of cell death by apoptosis.
Immunomodulation of Chiclero's Ulcer. Role of Eosinophils, T Cells, Tumour Necrosis Factor and Interleukin‐2
Authors: Lezama‐Dávila  , Isaac‐Márquez  , Padierna‐Olivos  , Aguilar‐Torrentera  , Chapa‐Ruiz
Published: March 2003

The progression of cutaneous leishmaniasis is controlled largely by cell‐mediated immunity. Two subpopulations of CD4+ T cells exist that control healing or immunopathology of murine and, perhaps, human leishmaniasis. To better understand the immunological pathways controlling outcome of the human disease, we analysed the pattern of tumour necrosis factor (TNF) and interleukin‐2 (IL‐2), both of which were present in the sera of humans with active or healed chiclero's ulcer, in relation to the development of delayed‐type hypersensitivity (DTH) responses and leucocyte counts in peripheral blood. Increased serum levels of IL‐2 and TNF‐α were apparent only in individuals with active lesions. All individuals with localized cutaneous leishmaniasis developed a strong DTH. The number of T cells was lower in the blood of diseased individuals and the CD4/CD8 ratio was reduced (from 1.5 to 1.0) when compared with the control group. However, diseased and recently cured individuals developed eosinophilia. We conclude that important alterations of the immune response exists in humans suffering from this normally self‐healing infection.
Receptor for α1‐Microglobulin on T Lymphocytes: Inhibition of Antigen‐Induced Interleukin‐2 Production
Authors: Wester  , Michaëlsson  , Holmdahl  , Olofsson  , Åkerström
Published: March 2003

The human plasma protein α1‐microglobulin (α1m) was found to inhibit the antigen‐induced interleukin‐2 (IL‐2) production of two different mouse T‐helper cell hybridomas. α1m isolated from human plasma and recombinant α1m isolated from baculovirus‐infected insect cell cultures had similar inhibitory effects. Flow cytometric analysis showed a binding of plasma and recombinant α1m to the T‐cell hybridomas as well as to a human T‐cell line. Radiolabelled plasma and recombinant α1m bound to the T‐cell hybridomas in a saturable manner and the binding could be eliminated by trypsination of the cells. The affinity constants for the cell binding were calculated to be 0.4–1 × 105 M−1 using Scatchard plotting, and the number of binding sites per cell was estimated to be 5 × 105–1 × 106. The cell‐surface proteins of one of the T‐cell hybridomas were radiolabelled, the cells lysed and α1m‐binding proteins isolated by affinity chromatography. SDS–PAGE and autoradiography analysis of the eluate revealed major bands with Mr‐values around 70, 35 and 15 kDa. The results thus suggest that α1m binds to a specific receptor on T cells and that the binding leads to inhibition of antigen‐stimulated IL‐2 production by T‐helper cells.
Differential Expression of T‐Cell Adhesion Molecules and LFA‐1‐Dependent Intercellular Adhesion in HgCl2‐Induced Autoimmunity and Immune Suppression
Authors: Roos  , Claessen  , Schilder‐Tol  , Weening  , Aten
Published: March 2003

Exposure of Brown Norway (BN) rats to HgCl2 induces Th2‐mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl2 induces immune suppression, mediated by CD8+ T cells. HgCl2 was previously found to enhance expression of LFA‐1, ICAM‐1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl2. CD8+ T lymphoblasts were significantly increased, which were predominantly CD45RChi, and which showed enhanced LFA‐1 expression. Furthermore, CD4+CD45RChi T cells showed increased numbers of ICAM‐1+ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4+ T lymphoblasts. Ex vivo experiments demonstrated that HgCl2‐ exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12‐myristate 13‐acetate]‐induced lymphocyte aggregation, mediated by LFA‐1 and ICAM‐1. In conclusion, HgCl2‐injected Lewis rats show early signs of T‐lymphocyte activation, predominantly on CD8+ cells. Strain‐dependent effects of HgCl2 on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.
Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa
Authors: Pilling  , Galvin  , Robins  , Sewell  , Mahida
Published: March 2003

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut‐associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α5 and α6 integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8‐positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α5 and α6‐positive cells than in the peripheral blood. In addition, there were significantly fewer α6 integrin molecules on the surface of CD4 and CD8‐positive lamina propria T‐cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down‐regulation of α5 and α6 integrin‐subunit expression on the surface of T cells. Molecules other than members of very late activation antigen‐5 (VLA‐5) (α5β1) and VLA‐6 (α6β1) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.
Independent Duplications of Bf and C3 Complement Genes in the Zebrafish
Authors: Gongora  , Figueroa  , Klein
Published: March 2003

As part of an ongoing project aimed at the characterization of the MHC system in bony fishes, we have attempted to identify the class III region in the zebrafish (a teleost), a region that in higher vertebrates contains genes coding for complement proteins C2, Bf and C4. We obtained several genomic PAC clones by hybridization with a zebrafish Bf probe, previously identified in our laboratory, and searched these for the presence of other class III genes. We were able to obtain a second Bf‐like gene, however, we were unable to detect any C2‐ or C4‐like genes. By using highly degenerated primers, we extended our search to a hepatopancreas cDNA library and amplified from it clones corresponding to three different C3‐like genes, and also the Bf genes, but not any C2‐ or C4‐like genes. The zebrafish therefore contains two Bf and three C3 loci but apparently no C2 and C4 loci. Independent duplications of the Bf and C3 genes in bony fishes suggest that complement plays a prominent part in the immune response of this class of vertebrates.
Normal T‐Helper 1/T‐Helper 2 Balance in Peripheral Blood of Coeliac Disease Patients
Authors: KERTTULA  , . COLLIN  , MÄKI  , HURME
Published: March 2003

Activated T cells, with their secretion of cytokines, probably play an important role in the pathogenesis of mucosal lesions in coeliac disease (CoD) and the prominence of a T‐helper (Th)1‐type cytokine pattern has been reported. As the process of immunological activation in the jejunal mucosa in active CoD has been shown to also cause some differences in peripheral blood lymphocyte populations, we sought to establish any changes in the Th1/Th2 balance in peripheral blood of patients, at different stages of CoD, relative to healthy individuals. Twenty‐two CoD patients and 10 healthy controls were included in the study. The Th1/Th2 balance was examined both in resting cells and after polyclonal stimulation using two different methods: intracytoplasmic cytokine contents were measured using an intracellular staining method and three‐colour flow cytometry and cytokine contents of cell culture supernatants were measured using traditional enzyme‐linked immunosorbent assays (ELISAs). Interferon‐γ (IFN‐γ)‐producing cells (Th1) were as prominent in untreated CoD patients and treated CoD patients as in healthy controls, while cells fitting a Th2 or Th0‐type cytokine pattern were few in all groups. In ELISA assays, Th1 type (IFN‐γ or interleukin (IL)‐2) cytokines were again prominent in all study groups but no statistically significant differences were found in IFN‐γ, IL‐4 or IL‐2 levels among the three groups. These results suggest that the increased shift towards a Th1 response is mainly restricted to the actual site of inflammation and that circulating T cells do not show a similar response, presumably because activated cells in peripheral blood are too few. Further research on cytokine profiles measuring T‐cell activation in CoD should be focused on the actual tissue of inflammation.
Characterization of Subpopulations of T‐Cell Receptor Intermediate (TCRint) T Cells
Authors: SKÖLD  , RYTTER  , IVARS  , CARDELL
Published: March 2003

CD1‐autoreactive T cells of two types have been demonstrated among T cells expressing the T‐cell receptor (TCR) αβ at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an α‐chain with an invariant Vα14‐J281 rearrangement, and a β‐chain using Vβ8.2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1‐reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1.1− TCRint subsets from MHC class II‐deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)‐4 and interferon (IFN)‐γ after activation. Importantly, no IL‐10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T‐helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi‐invariant Vα14/Vβ8.2‐type TCR was lower in NK1.1+ cells from MHC class II‐negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Vα14‐J281 rearrangement was also demonstrated among NK1.1− TCRint cells.
Interaction with Complement Receptor 1 (CD35) Leads to Amelioration of Sepsis‐Triggered Mortality but Aggravation of Arthritis During Staphylococcus aureus Infection
Authors: Sakiniene  , Heyman  , Tarkowski
Published: March 2003

The aim of this study was to assess the importance of complement receptor 1 (CR1, CD35) in Staphylococcus aureus arthritis and sepsis. The murine model of haematogenously acquired septic arthritis was used, injecting toxic shock syndrome toxin 1 (TSST‐1)‐producing S. aureus LS‐1 intravenously. CR1 was blocked using immunoglobulin G (IgG) rat antimouse CR1 monoclonal antibody (MoAb) (8C12). Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of blocking CR1 was assessed on the phagocytic activity of leucocytes and on T‐cell dependent and independent inflammation. Seven days after inoculation with bacteria, 96% of CR1 MoAb‐treated mice had clinical symptoms of arthritis compared with 58% of the control animals (P < 0.01). The severity of arthritis, expressed as mean arthritic index, was 2.9 ± 0.5 and 1.4 ± 0.5, respectively (P = 0.004). Fifteen days after bacterial inoculation, all CR1 MoAb‐treated mice had severe arthritis (mean arthritic index 6.3 ± 0.6), while only 77% of controls were affected (mean arthritic index 2.9 ± 0.6; P = 0.002). The potential explanation of these findings is that treatment with CR1 MoAb significantly increases the polymorphonuclear cell‐dependent inflammatory response as a result of enhanced vasodilatation in treated animals. We conclude that treatment with CR1 MoAb leads to amelioration of sepsis‐induced mortality during S. aureus infection, possibly as a result of the increased phagocytic activity of peripheral phagocytes.
In Vitro Interleukin‐13 Production by Peripheral Blood in Patients with Newly Diagnosed Insulin‐Dependent Diabetes Mellitus and Their First Degree Relatives
Authors: Kre¸towski  , Myśliwiec  , Kinalska
Published: March 2003

It is generally accepted that proinflammatory cytokines secreted by macrophages/monocytes as well as cytotoxic T cells are responsible for pancreatic B‐cell destruction in animal models of autoimmune diabetes and presumably in insulin‐dependent diabetes mellitus (IDDM) in humans. The aim of the present study was to evaluate the production of interleukin (IL)‐13–a Th2 cells derived anti‐inflammatory cytokine, by peripheral blood of newly diagnosed IDDM patients and their first degree relatives with a low or high risk of IDDM development. The study was carried out in 20 patients with a recent onset of type 1 diabetes, their first degree relatives with high (with DRB1*03 and/or DRB1*04 HLA class II alleles and two or more autoantibodies directed against pancreatic B‐cell antigens) (n = 20) or a low (with DQB1*0602 allele) risk of type 1 diabetes development (n = 10) and a control age matched group of healthy volunteers (n = 18). IL‐13 concentrations in supernatant of 72 h cultures of peripheral blood after incubation with phytohemagglutinin (PHA) or PHA+ insulin were quantified by enzyme‐linked immunosorbent assay (ELISA). The levels of IL‐13 in the supernatants were significantly lower in at high risk of IDDM first degree relatives of diabetic patients (P < 0.02), higher in subjects with low genetic risk of diabetes type 1 (P < 0.02), and normal in IDDM patients in comparison to the control group. We have also observed that the adding of human insulin to the cultures resulted in a significant increase of in vitro IL‐13 production in prediabetics, but not in the other studied groups. In conclusion our findings suggest that the IL‐13 alterations could play an important role in the pathogenesis of type 1 diabetes. We would speculate that IL‐13 as an anti‐inflammatory cytokine and a mediator of the Th2 pathway could be the potential therapeutic approach in the prevention of type 1 diabetes.
Immune Abnormalities in Aneurysmal Subarachnoid Haemorrhage Patients: Relation to Delayed Cerebral Vasospasm
Authors: Chrapusta  , Górski  , Mrowiec  , Grieb  , Andrychowski  , Ryba
Published: March 2003

Peripheral blood CD3+, CD19+, CD4+, CD8+ and CD45RO+ mononuclear cell subsets, T‐cell proliferative responses to combinations of coimmobilized OKT3 antibody and an ECM protein (collagen I, collagen IV, fibronectin or elastin), and T‐cell adhesion to collagen IV, fibronectin and elastin were studied in patients with aneurysmal subarachnoid haemorrhage. No significant difference was found in the major lymphocyte subsets between subarachnoid haemorrhage patients receiving no dexamethasone for brain oedema treatment and healthy blood donors. Compared with the latter, both the dexamethasone‐untreated and ‐treated subarachnoid haemorrhage patients showed decreased relative proliferative responses of circulating T cells to OKT3 combinations with collagen IV and fibronectin, and an increased PHA‐activated T‐cell adhesion to elastin. CD45RO+, CD4+ and CD19+ peripheral blood cell subsets, CD4+/CD8+ cell ratio, PHA‐activated T‐cell adhesion to fibronectin and collagen IV, and OKT3‐triggered T‐cell costimulatory responses to elastin, collagen IV and fibronectin were significantly higher in subarachnoid haemorrhage patients presenting with delayed cerebral vasospasm (DCV) than in their DCV‐free counterparts. The DCV‐related differences in circulating lymphocyte subsets showed no apparent relationship to the glucocorticoid treatment, whereas the differences in the other indices were confined to the dexamethasone‐untreated subarachnoid haemorrhage patients. The above results suggest that the CD4+/CD8+ ratio and T cell–ECM interactions play a role in the emergence of subarachnoid haemorrhage/DCV and may represent potential targets for subarachnoid haemorrhage therapy.
Neutralizing Human Anti Crotoxin scFv Isolated from a Nonimmunized Phage Library
Authors: Cardoso  , Nato  , England  , Ferreira  , Vaughan  , Mota  , Mazie  , Choumet  , Lafaye
Published: March 2003

Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South‐American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus.
Anti‐CD3 Induced Thymocyte Apoptosis In Vivo Require the Antibody Fc Domain
Authors: Xue  , Castanos‐Velez  , Biberfeld  , Jondal
Published: March 2003

We have earlier found that explanted thymic epithelial cells (TEC) can produce glucocorticoid (GC) activity in vitro and that the GC receptor (GR) antagonist RU486 partially inhibit thymic apoptosis induced by the anti‐CD3 monoclonal antibodies (MoAb) 2C11, both in vivo and in new‐born thymic organ cultures. To explain the inhibitory effect of RU486 in this system we have now investigated the importance of the 2C11 Fc as this MoAb bind with high affinity to cellular FcR. We have found both that whole 2C11 MoAb can bind to explanted TEC in vitro and that F(ab)′2 fragments from this MoAb loose this ability, in addition with the capacity to induce thymic apoptosis in vivo. We interpret our results to indicate that the injected 2C11 MoAb may establish a close contact between GC producing, FcR positive TEC cells and CD3 positive thymocytes and thereby subject the later to high paracrine GC concentrations and subsequent induction of apoptosis.
Nitric Oxide Promotes Growth and Major Histocompatibility Complex‐Unrestricted Cytotoxicity of Interleukin‐2‐Activated Rat Lymphocytes
Authors: Badovinac  , Trajkovic  , Mostarica‐Stojkovic
Published: March 2003

The effect of nitric oxide (NO) on growth and major histocompatibility complex(MHC)‐unrestricted cytotoxicity of interleukin(IL)‐2‐cultivated rat spleen nonadherent mononuclear cells was examined. NO donor sodium nitroprusside (SNP) at relatively low concentrations increased magnitude, as well as duration of IL‐2‐induced proliferative response of nonadherent splenocytes. SNP effect depended completely on released NO, because it was prevented by NO scavenger haemoglobin, but not mimicked by expired SNP solution, unable to generate NO, or ferricyanide, a second breakdown product of SNP. Other NO donors — SIN‐1, SNAP and GSNO failed to exert SNP‐like growth‐enhancing action, probably as a consequence of rapid NO generation, compared to sustained NO release by SNP. All NO‐releasing chemicals at high concentration blocked IL‐2‐induced proliferation. Growth‐promoting effect of SNP‐derived NO was independent of guanilat cyclase activation, because dibutyryl cGMP did not affect IL‐2‐triggered splenocyte proliferation. Macrophage NO acted in a manner similar to SNP; at low concentrations it promoted IL‐2‐induced splenocyte growth, however higher amounts were suppressive. Cytotoxicity of IL‐2‐activated splenocytes against NK‐sensitive K562 cell line was significantly increased when SNP was present during cultivation with IL‐2. Proportion of NKR‐P1+ and CD25+ cells, as well as per cell expression of these important activation molecules were increased upon SNP treatment, suggesting possible mechanism for the observed NO action.
Effects of G‐CSF on Telomere Lengths in PBMCs from Human Immunodeficiency Virus‐Infected Patients: Results From a Randomized, Placebo‐Controlled Trial
Authors: Aladdin  , Ullum  , Schjerling  , Skov Jensen  , Dam Nielsen  , Mathiesen  , Gerstoft  , SkinhøJ  , Klarlund Pedersen
Published: March 2003

Telomeres are unique terminal chromosomal structures, the length of which has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. In human immunodeficiency virus (HIV)‐1 infection, decrease of telomere length is primarily found in CD8+ T cells, and not in CD4+ T cells. In this double‐blind placebo‐controlled study, we investigated the effect of granulocyte colony stimulating factor (G‐CSF) treatment combined with highly active antiretroviral therapy (HAART) on mean telomere length in peripheral blood mononuclear cells (PBMC). The terminal restriction fragment (TRF) length showed no changes during G‐CSF treatment although the number of lymphocytes increased significantly. The mean TRF length correlated positively (R = 0.552, P = 0.009) and negatively (R = −0.503, P = 0.02) to the proportion of CD4+ memory and naïve cells, respectively. Our data suggest that during G‐CSF treatment lymphocytes are recruited by a combination of central and peripheral proliferation.
Localization of Human Serum Amyloid P Component and Heparan Sulfate Proteoglycan in In Vitro‐Formed Aβ Fibrils
Authors: Holm Nielsen  , Nybo  , Junker  , Toftedal Hansen  , Rasmussen  , Svehag
Published: March 2003

Ultrastructural studies of the localization of serum amyloid P component (SAP) in amyloid fibrils have given divergent results. We here report for the first time that electron microscopy of SAP coincubated with Aβ1–42 peptides or with mature Aβ1–42 fibrils, revealed SAP molecules coating the surface of the mature fibrils and that protofibrils of Aβ1–42 did not bind SAP. Also when incubated with extracted amyloid light chain (AL)‐fibrils the SAP molecules aligned on the fibril surface. Heparan sulfate proteoglycan bound to the surface of the Aβ fibrils with a spacing of about 50 nm. We conclude that SAP does not bind to protofibrils but to the surface of mature Aβ fibrils and that it may stabilize and protect the fibrils.
Interaction Between Human Complement and a Pectin Type Polysaccharide Fraction, PMII, from the Leaves of Plantago major L.
Authors: Michaelsen T. E. , Gilje A. , Samuelsen A. B. , Høgåsen K. , Paulsen B. S.
Published: March 2003

The interaction between a pectin type polysaccharide fraction, PMII, isolated from the leaves of Plantago major, and human complement was tested in two different hemolytic complement‐fixation tests and in addition by two
elisa methods detecting complement‐activation products. Sera were used as a complement source of 10 arbitrary human volunteers, individually and as a pool. The complement‐fixation tests were designed to measure the concentration of the pectin necessary to inhibit 50% of the hemolysis (ICH50). The
elisa tests for complement‐activation products were measured in AU/mg using a fully activated serum as a standard. We observed a more than 200‐fold difference in ICH50 activity of the PMII pectin in one of the hemolytic tests by varying the individual sera used as complement‐source. On the other hand, the
elisa complement‐activation tests showed no significant variation in activity of the PMII depending on the complement‐serum used. The level of antibodies against PMII detected in the complement‐sera did not correlate with the ICH50 activity of PMII. The results show that PMII is a potent complement activator with an activity of the same order of magnitude on a weight basis as that of aggregated human immunoglobulin (Ig)G. This activation leads to a complement consumption probably explaining the PMII's effect in the complement‐fixation tests. PMII seems to be an activator both on the classical and the alternative pathway of activation. The results might be related to the reported wound‐healing effect of the leaves of Plantago major.
Human Autologous Monocytes and Monocyte‐Derived Macrophages in Co‐Culture with Carcinoma F‐Spheroids Secrete IL‐6 by aNon‐CD14‐Dependent Pathway
Authors: Heimdal J.‐H. , Aarstad H. J. , Olsnes C. , Olofsson J.
Published: March 2003

The secretion of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α were compared when freshly isolated autologous monocytes or monocytederived macrophages (MDMs) were co‐cultured in vitro with autologous fragment (F)‐spheroids established from a series of head and neck squamous cell carcinoma (HNSCC) patients. F‐spheroids were generated from the malignant tumour (M‐spheroids) or from benign mucosa (B‐spheroids) from which the tumour originated control. If monocytes maturated towards MDMs before co‐culture, the IL‐6 secretion declined dependent on the extent of the MDM maturation by both M‐ and B‐spheroid stimulation. When MDMs maturated in continuous co‐culture, a steady‐state secretion of IL‐6 continued for several days but diminished when the culture medium was changed every 24 h. No co‐culture‐induced IL‐1β or TNF‐α was determined. Both the cytokine secretion and the mRNA gene expression revealed a different monocyte/MDM activation when co‐culture and lipopolysaccharide (LPS)‐stimulation were compared. Addition of anti‐CD14 (10 µg/ml) decreased monocyte LPS‐stimulated, but increased monocyte co‐culture stimulated IL‐6 secretion. In conclusion, M‐ and B‐spheroids similarly stimulated monocytes and to a lesser extent MDMs. MDMs that maturated with F‐spheroids present, retained responsiveness at the monocyte level. Co‐culture‐induced monocyte stimulation, as measured by IL‐6 secretion, was not dependent on activation via the CD14 molecule.
Activation of Cell‐Mediated Immunity Following Immunization with Pneumococcal Conjugate or Polysaccharide Vaccine*
Authors: Wuorimaa T. , Käyhty H. , Eskola J. , Bloigu A. , Leroy O. , Surcel H. M.
Published: March 2003

The immunogenicity of pneumococcal polysaccharide (PS) vaccines can be improved by conjugating PS to a polypeptide carrier that alters the immune response from T‐cell independent to T‐cell dependent. In order to study the influence of PS or protein antigens as inducers of cell‐mediated responses, 30 adults were immunized with a 23‐valent pneumococcal PS vaccine (PS‐group) or an 11‐valent, tetanus and diphtheria mixed carrier conjugate vaccine with (adjuvant group) or without aluminium adjuvant (nonadjuvant group). Cell‐mediated responses were analyzed on days 0, 14 and 28 after vaccination by measuring lymphocyte proliferation and production of interferon (IFN)‐γ (Th1 marker) or interleukin (IL)‐4 and IL‐5 (Th2 markers) cytokines after in vitro stimulation with the PS and protein components of the vaccines. Tetanus and diphtheria proteins were the main inducers of lymphocyte proliferative and cytokine responses. Conjugate vaccines induced increased proliferative responses to the tetanus or diphtheria protein, but not to the PS components. In the PS‐group, a lymphocyte proliferative response to protein antigens was not observed. The number of antigen‐specific and nonspecific IFN‐γ‐secreting cells detected by ELISPOT tended to increase in all three groups in response to protein or to PS antigen. No major differences were detected in the number of IL‐4‐secreting cells measured 14 and 28 days after vaccination. The conjugate vaccine with adjuvant was associated with Th2 type of activation indicated by an enhanced IL‐5 secretion in response to the tetanus and diphtheria protein antigens.
Fc‐Signalling in the Modulation of Immune Responses by Passive Antibody
Authors: Sinclair N. R. StC.
Published: March 2003

Passive antibody can both suppress and augment immune responses. Until recently, there was virtual unanimity on the importance of the interaction of the Fc portion of modulating antibody with Fc‐receptors (Fc‐signalling), especially in experiments involving the suppression by antibody. Experiments reported in the last few years, that do not demonstrate the range of Fc‐portion/Fc‐receptor influences on the suppression of immune responses by passive antibody, have introduced new uncertainty into this field. The purpose of this paper is to review how the initial controversy on the influence of Fc‐signalling in inhibition by passive antibody was resolved. Old and new approaches are suggested that may help in resolving the current uncertainty engendered by recent experimental results that were interpreted to mean that passive suppressive antibody does not utilize the inhibitory FcγRIIB receptor. An understanding of the factors that influence negative Fc‐signalling is needed in order to optimize clinical therapies whose action depends on the suppressive property of antibody.
C1qRp Elicits a Ca++ Response in Rat NK Cells but Does not Influence NK‐Mediated Cytotoxicity
Authors: Løvik G. , Larsen Sand K. , Iversen J. G. , Rolstad B.
Published: March 2003

The cell surface receptor C1qRp (receptor for C1q, regulating phagocytosis) present on macrophages and neutrophils, is presumed to stimulate phagocytosis in these cells. However, C1qRp is also present on natural killer (NK) cells, and in these cells its physiological function is not currently known. We have investigated putative functions of this cell surface molecule in rat NK cells with the aid of two novel monoclonal antibodies (MoAb) LOV3 and LOV8 against rat C1qRp. NK cells are known to be potent cytotoxic effector cells, both through specific recognition of ligands on a target cell and killing of antibody‐coated target cells (antibody‐dependent cellular cytotoxicity, ADCC). NK cells prestimulated with MoAbs LOV3 or LOV8 did not exhibit altered ADCC. Furthermore, the addition of MoAb LOV3 or LOV8 to cytotoxic cultures of NK cells and Fc‐receptor positive tumour cells did not affect killing in a redirected killing assay, indicating that the receptor did not influence NK cytotoxicity. However, this is the first paper to show that an intracellular Ca++‐response is induced in rat NK cells upon stimulation of C1qRp with LOV3 and LOV8. The response induced by the antibodies was only minimally reduced in the presence of EGTA, indicating that most of the response is owing to the Ca++ mobilization from intracellular calcium stores.
Long‐Term Expression of a Transferred Gene in Epstein–Barr Virus Transformed Human B Cells
Authors: Shi G. X. , Wang Y. , Liu Y. , Cui W. , Zhao F. T. , Zhu L. P.
Published: March 2003

Delivering a gene into the Epstein–Barr virus (EBV)‐transformed B cells is useful in studying effects of the gene on B‐cell functions. However, although people have been able to efficiently transfer genes into and get them expressed in B‐lympho blastoidcells for a time probably long enough to kill the cells using vectors harbouring oriP, the expression time of the delivered gene is not long enough in order to study the gene function in B cells. To solve this problem, we constructed an adeno‐associated virus (AAV) plasmid pAGX(+) based on plasmids pSub201 and pRc/CMV. We developed and packaged recombinant AAV (rAAV) expression vectors containing an antisense or a sense DNA fragment of 6A8 cDNA encoding a human α‐mannosidase, or an antisense fragment of 5D4 cDNA encoding a human cell membrane protein, or EYFP DNA. EBV‐transformed B cell SKW6 and 3D5 were transduced with those rAAV or the mock. Transduction with the rAAV‐EYFP showed an infection frequency of 64 ± 3.5% and 58 ± 6.2% for SKW6 and 3D5 cell, respectively. Genomic polymerase chain reaction (PCR) for neoR gene indicated an integration of the transferred gene into the host DNA. After being cultured and propagated for over 12 months, the cells were detected for the expression of the transferred gene. The RT–PCR, enzymatic assay and Con A binding test demonstrated an inhibition of 6A8 α‐mannosidase in both SKW6 and 3D5 cells transduced with the antisense 6A8 DNA. Immunofluorescence staining with monoclonal antibodies (MoAb) 5D4 showed a reduction of the 5D4 protein expression on both the cells transduced with the antisense 5D4 DNA. The DNA fragmentation assay showed a resistance of the cells with 6A8 α‐mannosidase inhibition to apoptosis induction by anti‐Fas antibody. The data indicate that the AAV vector pAGX(+) can efficiently introduce genes into EBV‐transformed B cells and the delivered gene can be expressed in the cells for more than 12 months which may be long enough for the study of gene functions in B cells.
Quantification of Murine IFN‐γ mRNA and Protein Expression: Impact of Real‐Time Kinetic RT–PCR Using SYBR Green I Dye
Authors: Hein J. , Schellenberg U. , Bein G. , Hackstein H.
Published: March 2003

Reliable quantification of cytokine mRNA expression is an important technique for analyzing immune responses. Up until now, little to no information has been available as to whether different mRNA quantification techniques lead to similar results. Recently, real time quantitative reverse transcriptase (RT)‐PCR using SYBR®Green I as a double stranded DNA specific dye has been introduced. This novel method enables simple and rapid measurement of PCR product accumulation during the log‐linear reaction phase and obviates the need for expensive hybridization probes. Here, we analyzed murine gamma interferon (IFN)‐γ mRNA expression in splenocytes by this technique in comparison to semiquantitative noncompetitive RT–PCR, Northern blot analysis, and ELISA after stimulation of the cells with interleukin (IL)‐12, IL‐18 and a combination of both cytokines. The results clearly show that all of the techniques detect differences in the IFN‐γ gene expression induced by these distinctive stimuli qualitatively exactly in the same order. However, real‐time kinetic RT–PCR offers several advantages, notably its high sensitivity that allows the detection of basal IFN‐γ mRNA expression in unstimulated samples. In addition it provides the lowest interassay variability of all techniques investigated. Finally, the gene expression measured by this method eliminates any post‐PCR manipulations because the PCR product identification can be easily performed by melting curve analysis.
Regulation of IL‐18 Expression in Virus Infection
Authors: Pirhonen J.
Published: March 2003

Since its discovery as an interferon (IFN)‐γ‐inducing factor, it has become evident that interleukin (IL)‐18 plays a crucial role in the generation of protective immunity against microbial infections. Macrophages are the major source of biologically active IL‐18, and they express constitutively IL‐18 mRNA and proIL‐18 protein. Microbial infections enhance the IL‐18 gene expression in macrophages but post‐translational processing of proIL‐18, rather than transcriptional activation, is predominant in the regulation of IL‐18 secretion. This review summarizes the current knowledge of proinflammatory and immunomodulatory properties of IL‐18, and focuses on the role of caspases in the proteolytic activation of IL‐18 in response to virus infection.
IgG2a‐Mediated Enhancement of Antibody Responses is dependent on FcRγ+ Bone Marrow‐Derived Cells
Authors: De Ståhl T. DÍaz , Heyman B.
Published: March 2003

Antibodies (Ab) administered in complex with antigens (Ag) have the capacity to regulate the out‐coming specific immune response. Primary immunization with complexes of bovine serum albumin‐2,4,6‐trinitrophenyl (BSA–TNP) and immunoglobulin (Ig)G2a anti‐TNP induced a significant enhancement of IgG1 and IgG2a BSA‐specific Ab response compared to immunization with the Ag alone. Enhancement was absent in nude mice, demonstrating the requirement of T cells for this regulation. Secondary immunization with BSA alone in mice previously primed with BSA‐TNP/IgG2a led to a dramatic increase of Ab production, showing that immune complexes are efficient inducers of immunological memory. IgG‐mediated enhancement of Ab responses has previously been shown to be impaired in mice lacking FcγRI, FcγRIII and FcεRI owing to gene targeting of the common FcRγ subunit (FcRγ−/–). Here we show that enhancement after immunization with BSA–TNP/IgG2a complexes is restored in irradiated FcRγ−/– recipients transferred with wild‐type (FcRγ+/+) bone marrow (BM) cells. In contrast, no enhancement is seen in FcRγ+/+ irradiated animals reconstituted with FcRγ−/– BM cells. We conclude that IgG2a‐mediated enhancement of Ab responses is dependent on the presence of FcγRI and/or FcγRIII on BM‐derived cells and that the presence of these receptors on the radioresistant follicular dendritic cell is not essential.
Primed B Cells Present Type‐II Collagen to T Cells
Authors: Holmdahl M. , Vestberg M. , Holmdahl R.
Published: March 2003

Development of type‐II collagen (CII)‐induced arthritis (CIA) is dependent on a T‐cell mediated activation of autoreactive B cells. However, it is still unclear if B cells can present CII to T cells. To investigate the role of B cells as antigen‐presenting cells (APCs) for CII, we purified B cells from lymph nodes of immunized and nonimmunized mice. These B cells were used as APC for antigen‐specific T‐cell hybridomas. B cells from naïve mice did present native, triple‐helical, CII (nCII) but also ovalbumin (OVA) and denatured CII (dCII) to antigen‐specific T‐cell hybridomas. In addition, B cells primed with nCII or OVA, but not dCII, activated the antigen‐specific T‐cell hybridomas two to three times better than naïve B cells. We conclude that antigen‐primed B cells have the capacity to process and present CII to primed T cells, and antigen‐primed antigen‐specific B cells are more efficient as APC than naïve B cells. We further conclude that B cells have the potential to play an important role as APC in the development of CIA.
Production of Antibodies against Glycolipids from the Mycobacterium tuberculosis Cell Wall in Aerosol Murine Models of Tuberculosis
Authors: Cardona P. J. , Julián E. , Vallès X. , Gordillo S. , Muñoz M. , Luquin M. , Ausina V.
Published: March 2003

Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL‐I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL‐I. Their evolution has a positive correlation with bacillary concentration in tissues.
Long‐Term Expression of an HLA‐DQ Molecule in the EBV‐Transformed Bare Lymphocyte Cell Line, BLS‐1, using a Plasmid Vector
Authors: Kelly M. A. , Rayner M. L. , Mijovic C. H. , Barnett A. H.
Published: March 2003

The HLA class II molecule, DQ6, confers strong natural protection against the development of type 1 diabetes. The mechanism of disease protection is unknown, but is likely to be related to the function of the molecule in antigen presentation. In order to investigate this function, we have created an in vitro model which expresses DQ6 in isolation by introducing the relevant DQ alleles into an Epstein–Barr virus (EBV)‐transformed, human leucocyte antigen (HLA) class II‐deficient B cell line, bare lymphocyte syndrome (BLS)‐1. A recent report suggested that the expression of transferred genes in human EBV‐transformed B cells might be limited in duration. We present a plasmid‐based transfection method that allows long‐term stable expression of the DQ molecule. The DQA1*0102 and DQB1*0602 alleles were cloned into the pCIneo expression vector and the constructs were introduced into BLS‐1 by electroporation. Stable transfectants were selected using magnetic sorting and cloned by limiting dilution. Two clones were shown to express functionally active DQ6 molecules even after 14 months of continuous culture. These clones will be used in functional studies to investigate the antigen binding and T‐cell activation properties of the DQ6 molecule.