bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment and Characterization of a Human T‐Lymphocyte Cell Line Immortalized by SV40 and with Abnormal Expression of TCR/CD3
Authors: Chatelut  , Caspar‐Bauguil  , Tkaczuk  , Alibaud  , Pieraggi  , Roudani  , Vacaresse  , Feunteun  , Laharrague  , Duchayne  , Demur  , Vincent  , Thiers  , Salvayre  , Levade
Published: March 2003

Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein–Barr virus (EBV) and human T‐cell leukaemia virus (HTLV)‐1 genomes. The immortalized cell line was assigned to the T‐lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T‐cell receptor (TCR) α‐chains but not β‐chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin‐2 (IL‐2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL‐2 was secreted. This new human immortalized T‐lymphocytic cell line, which is cell‐surface TCR/CD3‐negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.
CD23 Antigen Expression on B Lymphocytes and Soluble CD23 Levels in Peripheral Blood of High‐Risk Type 1 Diabetes Subjects
Authors: KRĘTOWSKI  , SZELACHOWSKA  , PIETRUCZUK  , KINALSKA
Published: March 2003

Soluble CD23 (sCD23), a recently discovered multifunctional cytokine, is a 25‐kDa molecule released by autoproteolysis from the 45‐kDa CD23 molecule which is found mainly on the surface of B lymphocytes. In the present study we aimed to evaluate, in association with humoral immune and metabolic markers, the changes in CD23 antigen expression on B lymphocytes and levels of sCD23 in the peripheral blood of subjects at high risk of type 1 diabetes. The study was carried out in 28 first‐degree relatives of type 1 diabetes patients (versus a control group of 28 age‐ and sex‐matched healthy volunteers) using antibodies against different B‐cell antigens: ICA, GADA, IAA, IA‐2. Flow cytometry was used to measure the percentage of CD20+ (B lymphocytes) and CD20+CD23+ lymphocyte subsets, and sCD23 levels in serum were determined by enzyme immunoassay. Prediabetic subjects had a significantly (P < 0.01) lower percentage of CD20+CD23+ lymphocytes in comparison with healthy age‐ and sex‐matched controls. Expression of CD23+ on B lymphocytes was similar in subjects with ICA only and with two or more antibodies against pancreatic antigens. In the prediabetic group, the median concentration of sCD23 was lower than in the control group and was statistically significant (P < 0.02) in the subgroup of subjects with the most impaired function of pancreatic β‐cells (the lowest values of first phase of insulin release). In conclusion, our study suggests that CD23 molecule expression on B lymphocytes and sCD23 levels in peripheral blood could be additional markers for monitoring the development of type 1 diabetes and play a role in determining the efficacy of prevention trials. However, further prospective studies are needed.
The Role of Amoebocytes in Endotoxin‐Mediated Coagulation in the Innate Immunity of Achatina fulica Snails
Authors: BISWAS  , MANDAL
Published: March 2003

Achatina amoebocyte lysate (AAL) derived from amoebocytes of Achatina fulica was activated by Gram‐negative bacterial endotoxins in a time‐dependent manner resulting in gel formation/coagulation. The activation and maximum proliferation of amoebocytes was observed 40 min after intramuscular injection (20 μg/snail) of endotoxin. Endotoxin‐mediated proteolytic activity of AAL towards a serine‐protease‐specific chromogenic substrate was maximum at pH 8.0, 37°C and within 15 min in a divalent‐cation‐dependent manner. The AAL activity induced by the endotoxin was directly dependent on the endotoxin concentration, showed a high specificity and saturated at higher endotoxin concentrations. An endotoxin‐sensitive factor (ESF) was purified from AAL to apparent homogeneity by single‐step affinity chromatography on a heparin‐Sepharose 4B column. Native ESF of molecular weight 140 000 was composed of two identical subunits of molecular weight 70 000 attached through non‐covalent association. A strong binding to endotoxin (Escherichia coli 055:B5) was exhibited by ESF with a 40‐fold higher biological activity than AAL. The ESF was shown to have a unique Phe–Ile active site with regard to its alternate activation by α‐chymotrypsin instead of endotoxin. The ESF was characterized as a serine protease type as evidenced by potent inhibition with specific inhibitors.
The Effects of CD40 Ligation on Peripheral Blood Mononuclear Cell Interleukin‐12 and Interleukin‐15 Production and on Monocyte CD14 Surface Antigen Expression in Human Immunodeficiency Virus‐Positive Patients
Authors: ZANUSSI  , D'ANDREA  , SIMONELLI  , TRABATTONI  , BORTOLIN  , CAGGIARI  , TIRELLI  , CLERICI  , DE PAOLI
Published: March 2003

Human immunodeficiency virus (HIV) infection causes dysregulation of surface phenotype, of accessory function and of cytokine production from peripheral blood mononuclear cells (PBMCs). As CD40 ligation induces several functional activities in these cells, this stimulation may partially mimic the situation occurring in vivo during an antigen‐driven immune response. The aim of this study was to measure cytokine production and immunophenotypic changes induced by CD40 stimulation of PBMCs from HIV‐positive patients. Under these experimental conditions, total and heterodimeric interleukin (IL)‐12 production from PBMCs was similar, while IL‐10 production was increased in HIV‐positive patients compared with controls. On the contrary, CD40 ligation did not induce IL‐15 production by PBMCs. Surface CD14 was down‐modulated, as a consequence of CD40 stimulation, on monocytes from healthy controls but not on monocytes from HIV‐positive patients. These data demonstrate that some of the CD40‐mediated signals are disturbed in HIV‐positive patients. These disturbances may contribute to the immune dysfunction seen in HIV infection.
Repertoire Shift Occurs During the Memory Maintenance Phase of the Immune Responses and is Not Affinity‐Driven
Authors: ANDERSSON  , WRAMMERT  , LEANDERSON
Published: March 2003

We have examined the immunoglobulin variable gene usage and antibody affinities during the memory maintenance phase of the immune response to the hapten phenyl‐oxazolone. Hapten‐specific hybridomas representing the memory population were generated 4–6 months postimmunization. The V‐gene expression of these hybridomas was determined by reverse transcriptase–polymerase chain reaction screening and antibody affinities were estimated by biointeraction analysis (BIA) using the BIAcore biosensor. Our results show that the V‐gene repertoire has already been shifted during the memory maintenance phase of the immune response, i.e. prior to a second antigenic challenge, and did not entail any advantages in terms of antigen‐binding capacities. Our results concur with the view that antibody affinities are modulated mainly through differences in dissociation rates rather than in association rates, and the implications of this with respect to affinity maturation is discussed.
Selection of Distinct Vα/β T‐Cell Receptor Families During In Vivo and In Vitro T‐Cell Maturation
Authors: Caccamo  , Sireci  , Ivanyi  , Salerno  , Dieli
Published: March 2003

The experimental conditions influencing the use of Vαβ TCR families were examined in lymph node (LN) cells from peptide‐immunized C57BL/6 and Vβ8.2 transgenic mice. Expanded proportions of Vβ5, Vβ8.2, Vβ9, Vβ12 and Vβ14 positive cells and an association of Vβ8.2 with Vα11 was found in freshly harvested 8‐day or 34‐day immune LN cells. In contrast, peptide‐specific T‐cell lines generated in vitro from 8‐day immune lymph node cells were found to be almost exclusively of the Vα2/Vβ12 family. However, T‐cell lines originating from Vβ8.2 transgenic mice did not show preferential Vα usage. Anti‐Vβ8.2 antibody produced different effects: when added to cultures of LN cells from C57BL/6 or Vβ8.2 transgenic strains, the peptide‐induced proliferation was suppressed; however, following the injection of mice, subsequent in vitro proliferation and cytokine production induced by both peptide and Concanavalin A was suppressed in Vβ8.2 transgenic, but much less in C57BL/6 mice. Hence, compensatory expansion of different Vβ gene products occurred in vivo, but not under the employed in vitro conditions. In conclusion, these results suggest that the TCR family usage is influenced by the experimental conditions in which the T cells are selected and expanded and by the genetic potentials of the precursor pool.
Differential Expression of Major Histocompatibility Complex Class II Variants in Cells Infiltrating the Meth A Sarcoma
Authors: Busund  , Killie  , Leon  , Fandrem  , Bartnes
Published: March 2003

CD4+ T cells are involved in immune responses against the Meth A sarcoma and infiltrate tumours arising from Meth A cells inoculated intradermally in (BALB/c × C57BL/6)F1 (H‐2d/b) mice. In order to investigate whether the prerequisites for tumour antigen presentation to CD4+ T cells are fulfilled within the malignant lesion, expression of class II major histocompatibility complex (MHC) molecules and the costimulatory ligand B7 were examined. The I‐Ab and I‐Ed allomorphs were abundantly expressed by virtually all B cells and 50% of macrophages infiltrating the tumours. In striking contrast, the I‐Ad variant was hardly detectable. This pattern of class II expression appeared to be unique for the tumour microenvironment. Thus the proportion of I‐Ad+ spleen B cells and peritoneal macrophages did not significantly differ from the proportion expressing I‐Ab and I‐Ed, and these extratumoral I‐Ad+ cells stained as brightly as did cells from healthy mice. Expression of B7 was weak by tumour‐infiltrating cells. The profound capacity of the Meth A sarcoma to confer low local I‐Ad and B7 expression might preclude T‐cell‐dependent anti‐tumour immune responses and thus promote survival of malignant cells. Whereas MHC class II genes are generally found to be co‐ordinately transcribed, this study demonstrates that the expression of MHC class II allelic variants can be differentially regulated in vivo.
Bulk Culture Levels of Specific Cytotoxic T‐Cell Activity Against HIV‐1 Proteins are not Associated with Risk of Death
Authors: Aladdin  , Ullum  , Cozzi Lepri  , Leffers  , Katzenstein  , Gerstoft  , Gjedde  , Phillips  , Skinhøj  , Klarlund Pedersen
Published: March 2003

The ability of cytotoxic T lymphocytes (CTL) to control and influence the outcome of human immunodeficiency virus (HIV) infection is not fully understood. The association between HIV‐CTL activity and disease progression was evaluated prospectively in 36 HIV‐1‐infected individuals with a median follow‐up of 3.0 years. HIV‐CTL activity was measured in a 4 h Cr* release assay using autologous target cells expressing HIV‐1 BRU isolate gene products (gp‐120, gag, pol, nef) and a bulk culture of autologous effector cells. The CD4 count was measured at enrolment and plasma HIV RNA was measured retrospectively. The present study failed to support the hypothesis that HIV‐CTL activity, as measured using the present method, is important in reducing the risk of death in HIV‐infected individuals. However, using other approaches and methods could possibly yield other conclusions, and further prospective studies are needed to examine the relationship between CTL and disease progression.
Methods for Simultaneous Quantitative Analysis of Eosinophil and Neutrophil Adhesion and Transmigration
Authors: Moshfegh  , Halldén  , Lundahl
Published: March 2003

Granulocytes play important roles in various inflammatory responses. The aim of this study was to develop in vitro methods to enable simultaneous analysis of eosinophil and neutrophil adhesion and transmigration in mixed granulocyte preparations. We used fibronectin‐coated plates, with or without semipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)‐5 and eotaxin or N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), during incubation in the wells. Three different detergents: n‐octyl‐β‐
d‐glucopyranoside (OG), Triton‐X‐100 or N‐cetyl‐N,N,N‐trimethylammonium bromide (CTAB), were tested for their ability to cause lysis of granulocytes with minimal effect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigenicity. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the most efficient lysing reagent. Cell‐recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased in a time‐dependent manner upon stimulation with IL‐5 and eotaxin. Eosinophil adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigration upon activation with fMLP, reaching respective plateaux at 30 and 90 min. Our study shows that CTAB is an effective detergent for lysing granulocytes, yielding high and reproducible recovery rates of ECP and MPO. Measurement of ECP and MPO, as markers for cell counts, can therefore be used to quantify adhesive and transmigration properties of eosinophils and neutrophils in mixed granulocyte populations.
Cytokine Profiles in the Central Nervous System and the Spleen During the Early Course of Experimental African Trypanosomiasis
Authors: Sharafeldin  , Hamadien  , Diab  , Li  , Shi  , Bakhiet
Published: March 2003

Cytokines are important signalling proteins, which have been shown to contribute to immunopathogenesis of several inflammatory and infectious diseases such as African trypanosomiasis. The present study was conducted in order to evaluate the early induction of five potential cytokines in the central nervous system (CNS) and spleens from Trypanosoma brucei brucei (T. b. brucei)‐inoculated and uninfected control Sprague–Dawley rats. In brain, choroid plexus and spleen, cytokine levels were examined by in situ hybridization and immunohistochemistry, while ELISA was used to measure cytokine levels in cerebrospinal fluid (CSF). Our results showed that interferon (IFN)‐γ and transforming growth factor (TGF)‐β were highly expressed in all compartments, but low interleukin (IL)‐4, IL‐10 and tumour necrosis factor (TNF)‐α mRNA levels were registered. The pattern of these cytokines is in context with the severity of the disease because (i) IFN‐γ was previously demonstrated to promote parasite growth (ii) TNF‐α was previously demonstrated to kill the parasites and (iii) IL‐4 was previously demonstrated to promote antibody production necessary for elimination of the infection. These data support the hypothesis that cytokines may have a role in developing the disease either by enhancing the parasite growth or by suppressing the immune response.
LFA‐3 (CD58) Mediates T‐Lymphocyte Adhesion in Chronic Inflammatory Infiltrates
Authors: Kirby  , Cahen  , Porter  , Olsen
Published: March 2003

Previous studies have suggested that LFA‐3 has an important role in a number of chronic inflammatory pathologies, although an active role for LFA‐3 within in vivo inflammatory reactions has not previously been directly observed in humans. To assess the importance of LFA‐3 in this process, this study used an adaptation of the Stamper–Woodruff lymphocyte adhesion assay to measure the binding of exogenous activated lymphocytes to the T‐cell‐dominated chronic inflammatory infiltrate of oral lichen planus. Antibody blockade experiments showed that anti‐LFA‐3 monoclonal antibody reduced lymphocyte adhesion by ≈ 29%, while anti‐ICAM‐1 produced a reduction of 26%. These results thus suggest that both LFA‐3 and ICAM‐1 are likely to mediate cell–cell interactions within lesional tissues in vivo. Moreover, these findings are also the first to directly demonstrate that LFA‐3‐mediated adhesion, like that of ICAM‐1, is functionally important in the molecular pathology of inflammatory mucosal disease.
Low Levels of Interferon‐α Induce CD86 (B7.2) Expression and Accelerates Dendritic Cell Maturation from Human Peripheral Blood Mononuclear Cells
Authors: Radvanyi  , Banerjee  , Weir  , Messner
Published: March 2003

Interferon‐α (IFN‐α) (IFN‐α2b) is an immunoregulatory cytokine that is presently used in a recombinant form for the treatment of tumours and chronic viral infection. However, its mechanism of action remains largely undefined. In this paper, we studied the effects of low doses of IFN‐α (0–100 U/ml) on the generation of dendritic cells with granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐4 (IL‐4), and tumour necrosis factor (TNF)‐α in cultures of human peripheral blood mononuclear cells (PBMCs). An addition of IFN‐α to the PBMC cultures greatly increased the HLA class II and the CD86 expression on developing dendritic cells (DCs) during a 7‐day culture period. When added at the initiation of the PBMC culture, as little as 10 U/ml dramatically increased the HLA class II and CD86 expression, with maximal effects observed between 50 and 100 U/ml in all PBMC preparations tested. Almost all of the nonadherent cells induced with added IFN‐α possessed a phenotype of mature DCs, being CD1alow, CD83+, HLA class IIhigh, CD86high, CD40high, and CD80low, while being negative for the monocyte/macrophage and lymphocyte markers. In contrast, the floating cells isolated from cultures grown without IFN‐α were mostly immature DCs with a CD1ahigh, CD83−, HLA class IIint/high, CD86low/int, CD80low phenotype. An addition of 50 U/ml IFN‐α at the time of the culture initiation greatly increased both the number of mature DCs generated and their rate of appearance; by 3 days of culture, many large floating aggregates were present containing mature CD83+, CD1alow DCs, while much fewer aggregates of mature DCs were found without added IFN‐α. Histochemical staining confirmed that the floating cells induced with IFN‐α had typical DC features, including irregularly shaped nuclei, few cytoplasmic granules, and absent or diffuse perinuclear staining for esterase. Our results suggest that IFN‐α is a potent accelerator of DC maturation in vitro. These effects on DC maturation may explain its clinical success in the treatment of cancer and viral infection as well as its ability to promote autoimmunity.
Early Changes in Peripheral Blood T Cell Subsets Induced by Antiretroviral Treatment of Human Immunodeficiency Virus‐1 Positive Individuals
Authors: Dyrhol‐Riise  , Brantsæter  , Dunlop  , Baklien  , Skar  , Åsjö  , Bruun  , Røsok
Published: March 2003

Human immunodeficiency virus (HIV)‐1 infection causes a gradual decline in peripheral blood CD4+ T cells. Shortly after the primary infection, an expansion of the activated memory CD8+ T‐cell pool is also observed paralleling increased levels of plasma viraemia. In the present study we investigated the immediate effects of zidovudine therapy on peripheral blood T‐cell subsets during the first 3 weeks of therapy in a group of HIV‐1 positive individuals receiving influenza vaccine. HIV‐1 positive individuals who received vaccine, but no treatment, were included as controls. Both the number of CD4+ and CD8+ T cells increased during the first week of therapy in parallel with a decline in plasma viraemia. The majority of CD4+ T cells contributing to this expansion expressed CD28, CD45RO and Fas, whereas the expanded CD8+ T cells were predominantly CD28−, CD45RO+, CD38+, Fas+ and Fas+ (CD95). We propose that the increase in the number of activated memory T cells observed in peripheral blood immediately after the onset of antiretroviral treatment is most likely caused by the redistribution of cells from various lymphoid organs in response to decreased levels of viral load in these compartments. The degree of T‐cell redistribution is probably dependent on the magnitude of virus suppression.
Diversity of Lung and Spleen Immune Responses in Mice with Slowly Progressive Primary Tuberculosis
Authors: Phyu  , Tadesse  , Mustafa  , Tadesse  , Jonsson  , Bjune
Published: March 2003

The aim of the present study was to assess the compartmentalized immune response, in terms of cytokine secretion and cell activation, in lungs and spleens of mice with slowly progressive primary tuberculosis. Immunocyte populations from both organs were isolated and stimulated with concanavalin A, purified protein derivatives and MPT 59. Production of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) was measured using an enzyme‐linked immunosorbent assay, and cell activation was measured using a tetrazolium colorimetric assay. The IFN‐γ and IL‐4 levels in the supernatants of Mycobacterium tuberculosis antigen (Ag)‐stimulated lung immunocytes from infected mice were higher than the levels from uninfected mice. However, only IL‐4 levels were raised in the supernatants of Ag‐stimulated spleen immunocytes from infected mice. Spontaneous and Ag‐stimulated immunocyte activation was lower only in the lungs of infected mice compared with uninfected mice. The level of lung immunocyte activation was inversely associated with the extent of gross pulmonary pathology. In conclusion, cytokine secretion and cell activation were different between lungs and spleens in slowly progressive primary murine tuberculosis. Cytokine diversity may explain the confinement of tuberculous lesions in the lungs and the absence of lesions in the spleens of mice with slowly progressive tuberculosis.
Schistosoma mansoni in Mice: the Pattern of Primary Cercarial Exposure Determines Whether a Secondary Infection Post‐chemotherapy Elicits a T Helper 1‐ or a T Helper 2‐Associated Immune Response
Authors: Farah  , Johansson  , Lövgren‐Bengtson  , Hau
Published: March 2003

Reinfection with Schistosoma mansoni following chemotherapy often results in an ameliorated granulomatous reaction and hence a mild disease. This study examined some of the immunological mechanisms that could be associated with this residual protection. BALB/c mice were infected with either a single dose (group A) of 100 S. mansoni cercariae or with 10 doses of 10 cercariae each (group B) given at 3‐day intervals. The mice were treated with praziquantel 8 weeks postinfection and, 2 weeks later, together with another group of naive mice (group C), they were infected with a single dose of 100 cercariae each. All the animals were killed 8 weeks later and schistosome egg antigen (SEA)‐ and soluble adult worm antigen preparation (SWAP)‐induced cytokine recall responses in splenocytes, as well as serum immunoglobulin levels, were quantified and hepatic granuloma sizes measured. Group A animals had higher levels of SEA‐induced interferon‐γ (IFN‐γ) but lower levels of interleukin (IL)‐5 than groups B and C (P < 0.01). Group B animals had low SEA‐induced IFN‐γ levels and elevated IL‐5 levels, although these were lower than group C. SEA‐induced IL‐10 was low in both groups A and B as compared to group C (P < 0.01). SWAP was less effective as an inducer of splenocyte cytokine production than SEA but both SWAP‐induced IFN‐γ and IL‐5 were detected in groups A and C. SEA‐ and SWAP‐specific immunoglobulin E (IgE) and immunoglobulin G (IgG) titres were not significantly different between the three groups. Granuloma diameters were larger in group C (mean 297 ± 51.3 μm) as compared to groups A (174 ± 49 μm, P < 0.01) and B (247.5 ± 44 μm, P < 0.05). Taken together, these results demonstrate that granuloma size is reduced during a reinfection exposure compared with a primary infection. This reduction is associated with a T helper 1 response in mice exposed to a single large dose of cercariae in the primary infection and with a predominantly T helper 2 response in those infected with multiple small doses.
Immunological and Virological Changes in Antiretroviral Naïve Human Immunodeficiency Virus Infected Patients Randomized to G‐CSF or Placebo Simultaneously with Initiation of HAART
Authors: Aladdin  , Ullum  , Katzenstein  , Gerstoft  , Skinhoslash;j  , Klarlund Pedersen
Published: March 2003

To determine the efficacy of combined G‐CSF and highly active antiretroviral treatment (HAART), a randomized, double blind, placebo controlled study was conducted. Treatment naive human immunodeficiency virus (HIV) infected patients were randomized to receive either placebo or G‐CSF (0.3 mg/ml, 3 times a week) for 12 weeks and HAART simultaneously. The trial was terminated prematurely after interim analysis performed because of a case of severe encephalopathia in the G‐CSF group. At that point 11 HIV infected patients with a CD4+ T cell count < 350/mm3 had been randomized to the G‐CSF group (n = 6) or placebo group (n = 5). In both groups plasma HIV RNA decreased significantly in response to HAART. However, plasma HIV RNA changed significantly different between the two groups with the decrease being less pronounced in the G‐CSF group (P = 0.02). The concentrations of CD4+ memory T cells and CD8+ naive and memory T cells increased in response to HAART, and there was a trend towards more pronounced increases in several T‐cell subpopulations in the G‐CSF group. The CD56+ NK cells increased significantly more in the G‐CSF group compared with placebo (P = 0.000). All patients in the G‐CSF group reported bone pain. The present data do not support simultaneous administration of G‐CSF with initiation of HAART in treatment naive HIV infected patients.
Serum Amyloid A Secretion from Monocytic Leukaemia Cell Line THP‐1 and Cultured Human Peripheral Monocytes
Authors: Yamada  , Wada  , Itoh  , Igari
Published: March 2003

Serum amyloid A (SAA), an acute‐phase protein and a precursor of fibrous components in reactive amyloid deposits, is synthesized mainly in the liver under the stimulation of inflammation‐related cytokines. In addition, the SAA gene is expressed in monocytes/macrophages, which are believed to play a central role in amyloid fibrillogenesis. Consequently, the pathogenic implication of SAA produced from these cells has been of major concern. Because SAA synthesis at the protein level in such cells has never been analyzed quantitatively, in this study an enzyme‐linked immunosorbent assay was generated with a detection level sufficiently high to measure SAA concentrations in the culture supernatants of the human monocytic leukaemia cell line THP‐1. SAA secretion by THP‐1 with interleukin (IL)‐1β required the presence of dexamethasone as proposed previously. We also found that unidentified components in fetal calf serum (FCS) could induce SAA production by THP‐1 in the presence of dexamethasone. These findings are in contrast to the results obtained from hepatoma cell line HepG2, in which IL‐1β alone could induce SAA secretion, while dexamethasone‐supplemented FCS could not. The method was able to quantify SAA secreted from cultured human peripheral monocytes. The findings suggest that monocytes produce SAA in almost the same manner as THP‐1. Thus, THP‐1 cells can be utilized to investigate a distinctive manner of SAA production from monocytes.
Molecular Analysis of Human Immunoglobulin Heavy Chain Variable Region Associated Determinants Recognized by Anti‐VH3 Antibodies 7B4, B6 and D12
Authors: Suleyman S. , Thompson Keith M. , Mageed R. A. , Natvig J. B.
Published: March 2003

7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3–30, V3–30.3 and V3–33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3–11 and V3–7 genes; D12 also reacts with some V3–43 gene Ig. We show here, by site‐directed mutagensis, that the lysine at position 57 in the complementarity‐determining region 2 (CDR‐2) of the V3–30 gene product is crucial for epitope recognition by all three anti‐VH3 MoAbs. Further analysis of the amino‐acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR‐2. Comparing the efficiency of 7B4 reactivity with VH3 gene‐encoded human Ig indicates that amino‐acid position 4 in the frame region 1 (FR‐1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3–30, V3–30.3 and V3–33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)‐2, SIV and HTLV‐4. We also report that other tetrapeptide sequences found on the 56–59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV‐1 isolates. The possible significance of these observations is discussed.
The Effect of Long‐Term Treatment with Granulocyte Colony‐Stimulating Factor on Hematopoiesis in HIV‐Infected Individuals
Authors: Nielsen  , SøRensen  , Aladdin  , ErsbøLl  , Mathiesen  , Ullum  , Gerstoft  , Nielsen  , Pedersen
Published: March 2003

This randomized, placebo‐controlled trial examine the long‐term effect of granulocyte colony‐stimulating factor (G‐CSF) on absolute numbers of CD34+ progenitor cells and progenitor cell function in human immunodeficiency virus (HIV)‐infected patients. G‐CSF (300 μg filgrastim) or placebo was given three times weekly for 12 weeks to 30 HIV‐infected patients that had been treated with HAART for at least 24 weeks and not yet achieved CD4 counts above 350 CD4+ cells/μl. Blood samples were collected at weeks 0, 2, 4, 8, and 12, and again 12 weeks after termination of the G‐CSF treatment. Significant increase in absolute numbers of circulating CD34+ cells was detected in the treatment group (P = 0.006). The function of progenitor cells was examined in vitro using a colony‐forming unit (CFU) assay, and increase in the number of CFU/ml was detected (P = 0.005). In order to estimate the effect of G‐CSF on in vivo function of progenitors the white‐blood count was determined. Significant increase in white‐blood count was found (P < 0.001), while hemoglobin and platelet count decreased (P = 0.001 and P = 0.013, respectively). Significant increase in the CD4 count occurred, but correlation between the numbers of progenitors and the CD4 count was not found. These data suggest that G‐CSF mainly increases the number and differentiation of myeloid progenitors.
Antigenic Diversity of Plasmodium falciparum and Antibody‐Mediated Parasite Neutralization
Authors: Bolad  , Berzins
Published: March 2003

The malaria parasite Plasmodium falciparum, causing the most severe form of the disease in humans, is characterized by a broad antigenic diversity between different strains and isolates of the parasite. The antigenic diversity reflects on the one hand polymorphisms in allelic gene products and, on the other hand, antigenic variation as a result of expression of alternative genes in multigene families. Using selected polymorphic regions in two merozoite surface antigens, a method for genotyping P. falciparum parasites has been developed. This has resulted in new information on the clonal multiplicity and dynamics of parasite populations. Observations from in vivo and in vitro studies have identified many potential parasite‐neutralizing immune responses and several of the target antigens are being explored as vaccine candidates. Studies of antibody‐mediated neutralization of parasites in P. falciparum in vitro cultures, with or without leukocytes as effector cells, have been instrumental in identifying potential target antigens for protective immunity and for elucidation of the effects of immune pressure on the dynamics of parasite populations and their antigenic plasticity.
Neutrophil Response of Transgenic Mice Expressing Human Group IIA Phospholipase A2 in Bacterial Infections
Authors: Laine V. J. O. , Rajamäki A. , Grass D. S. , Nevalainen T. J.
Published: March 2003

Group IIA phospholipase A2 (PLA2) is a newly recognized acute phase protein with marked antibacterial properties. We have shown previously that transgenic C57BL/6 J mice expressing human group IIA PLA2 (PLA2+ mice) are more resistant to bacterial infections than nontransgenic C57BL/6 J mice that, among mice, are unusual in that they lack the mouse analogue of group IIA PLA2 (PLA2− mice). To elucidate the possible mechanisms involved in the host response of these mice in bacterial infection, peripheral inflammatory cell responses of PLA2+ and PLA2− mice were studied after i.p. administration of Escherichia coli, E. coli lipopolysaccharide or Staphylococcus aureus. Uninfected PLA2+ mice had higher numbers of lymphocytes and polymorphonuclear neutrophil leukocytes (PMNs) in their blood than PLA2− mice. In PLA2+ mice, the number of PMNs increased in peripheral blood in parallel with the concentration of group IIA PLA2 after the administration of bacteria, whereas these responses were not seen in PLA2− mice. High concentrations of group IIA PLA2 in PLA2+ mice may increase the synthesis of bioactive molecules, such as prostaglandins, which in turn may mobilize PMNs into circulation. Our results support the hypothesis that group IIA PLA2 is an important inflammatory mediator in bacterial infections.
Longevity of Immune Complexes and Abnormal Germinal Centre Formation in NZB Mice
Authors: Nordström E. , Abedi‐Valugerdi M. , Möller E.
Published: March 2003

Rheumatoid factor (RF)‐like (antibody‐antibody) immune complexes induce a selective and intensive immunoglobulin (Ig)G1‐RF response after a single injection in mice. However, the longevity of the response differs between mouse strains: serum IgG1‐RF antibody titres decline 40 days after injection in C57Bl/6 mice whereas levels are maintained for more than 100 days in NZB mice. In order to elucidate whether this difference was owing to a lower ability of NZB mice to clear the injected immune complexes, sections of kidney, spleen, liver and mesenteric lymph nodes were harvested at different time points after injection with RF‐like immune complexes. Immunohistochemical staining revealed that NZB mice have a delayed clearance of the injected immune complexes, because the immune complexes are retained for more than 40 days in their spleens and 100 days in their kidneys, compared to only 14 days in C57Bl/6 mice. Germinal centres were also present for a longer period in the spleens of the NZB mice, accompanying the presence of the immune complexes, and were abnormally large compared to C57Bl/6 mice. The clearance of immune complexes from the spleen coincided with the decline in serum levels of IgG1‐RF, indicating that prolonged retention of immune complexes is responsible for the sustained IgG1‐RF response.
Garlic Induces a Shift in Cytokine Pattern in Leishmania major‐Infected BALB/c Mice
Authors: Ghazanfari T. , Hassan Z. M. , Ebtekar M. , Ahmadiani A. , Naderi G. , Azar A.
Published: March 2003

The regulation of T helper (Th)1‐ and Th2‐type cytokine patterns is important in the final outcome of leishmaniasis in human and murine models. We examined the efficacy of garlic therapy or a combination of garlic and an antimonial drug (glucantime) in promoting healing and regulation of Th1/Th2 cytokine patterns in highly susceptible BALB/c mice infected with Leishmania major. Separate groups of infected mice received 20 mg/kg/day garlic, 60 mg/kg/day glucantime or a combination of the two, from day 30 after infection for 2 weeks. An enzyme‐linked immunosorbant assay (
ELISA) was performed on spleen cell culture supernatants for interferon(IFN)‐γ interleukin(IL)‐2, IL‐4 and IL‐10. The results indicate that garlic therapy is more effective than the usual antileishmanial drug in curing the infection. Garlic‐treated mice developed Th1‐type cytokine responses. In contrast, glucantime therapy led to a Th2‐type response in the control group with a lower level of IL‐2. However, a combination of garlic and glucantime treatment was more effective than either treatment alone, and resulted in a Th1‐type response similar to that which developed with garlic treatment. These results suggest that garlic extract in combination with an antimonial drug, may provide effective therapy against L. major. The immunomodulatory properties of garlic were elucidated in terms of shifting the cytokine response to a Th1‐type pattern and therefore causing the protective response.
Interleukin‐10 is an Unequivocal Th2 Parameter in the Rat, whereas Interleukin‐4 is Not *
Authors: Vandebriel R. J. , Meredith C. , Scott M. P. , Van Dijk M. , Van Loveren H.
Published: March 2003

Exposure of Wistar rats to the immunotoxic compounds hexachlorobenzene (HCB), bis(tri‐n‐butyltin)oxide, and benzo(a)pyrene was previously found to affect mRNA expression of interleukin (IL)‐2, IL‐2R α‐chain, and interferon (IFN)‐γ, the prototypic Th1 cytokine. In contrast, the mRNA expression of IL‐4, the prototypic Th2 cytokine, was unaffected. This latter finding suggested that the IL‐4 mRNA expression may not be an unequivocal parameter for Th2 responses in the rat. In order to obtain such a parameter the present study was performed, consisting of two types of experiments. Expression and production of IL‐4 as well as IL‐10, a second Th2 cytokine, were measured. First, Lewis (Th1 prone) and Brown Norway (BN; Th2 prone) rats were exposed to HCB. Exposure was previously found to increase the serum immunoglobulin (Ig)E levels, an IL‐4‐dependent response, in BN but not Lewis rats, and in Lewis rats to aggravate experimental allergic encephalomyelitis (EAE), severity being inversely related to IL‐10 levels. Secondly, BN rats were infected with Trichinella spiralis, an infection previously found to induce IL‐4 production. HCB exposure did not affect IL‐4 mRNA expression in either strain, while IL‐4 production was decreased in Lewis and unaffected in BN rats. In Lewis rats both the mRNA expression and the production of IL‐10 were decreased. The T. spiralis infection induced IL‐4 and IL‐10 mRNA expression, as well as IL‐10 production. In contrast, the IL‐4 production was strongly reduced. Thus, both the IL‐10 mRNA expression and production correlated with the EAE development and T. spiralis infection. In HCB exposed Lewis rats and T. spiralis infected BN rats the IL‐4 mRNA expression correlated with IgE levels and T. spiralis infection, respectively, whereas the IL‐4 production lacked correlation in all cases. Collectively, these results suggest that IL‐10 is an unequivocal Th2 parameter in the rat, whereas IL‐4 is not.
No Evidence for Generation of Th‐2‐like MBP‐Specific T‐Cell Lines by Blockade of the Costimulatory Molecule B7‐1
Authors: Zipp F.
Published: March 2003

The T helper‐1 (Th‐1)/T helper‐2 (Th‐2) paradigm is relevant for the pathogenesis and therapy of multiple sclerosis. In experimental autoimmune encephalomyelitis, a shift towards a Th‐2 immune response serves as treatment of the disease. In the human immune system, the factors which determine and modulate the differentiation of CD4+ T cells into the Th‐1 or Th‐2 phenotype have yet to be elucidated completely. Here, the split‐well approach was used to analyse costimulatory requirements for the generation of myelin basic protein‐specific T‐cell subsets considered to play a major role in the pathogenesis of multiple sclerosis. Myelin basic protein‐specific T‐cell lines were isolated from peripheral blood cells of healthy individuals in the presence or absence of a blockade of the costimulatory molecule B7‐1, previously reported to be involved in the development of Th‐1 cells. T‐helper type was determined by the interferon/interleukin ratio. Blockade of B7‐1 did not increase the number of Th‐2‐like myelin basic protein‐specific T‐cell lines. Thus, these data show no evidence for an influence of B7‐1 blockade on the development of human myelin basic protein‐specific T‐cell subsets. These results have to be taken into account when discussing whether antibody‐mediated B7‐1 blockade might be a suitable therapy in multiple sclerosis, as demonstrated in experimental autoimmune encephalomyelitis.
Decreased T Cell Stimulatory Capacity of Monocyte‐Derived Human Macrophages following Herpes simplex Virus Type 1 Infection
Authors: Hoves S. , Niller H.‐H. , Krause S. W. , Straub R. , Glück T. , Mountz J. D. , Schölmerich J. , Fleck M.
Published: March 2003

Macrophages play a central role in establishing a specific immune response by acting as professional antigen presenting cells (APC) for T cells leading to a vigorous immune response. In order to analyze if Herpes simplex Virus (HSV) type 1 infection might affect the macrophage APC‐function, monocyte‐derived human macrophages were infected with HSV‐1 strain F in vitro. Cocultures with allogeneic T cells revealed a strongly impaired stimulatory capacity of HSV‐infected macrophages compared to uninfected controls which was not owing to a productive viral infection in macrophages. An increased expression of Fas ligand (FasL/CD95L) was detected in HSV‐infected macrophages by FACS analysis. Although the majority of the macrophages expressed high levels of Fas (CD95/Apo‐1), the HSV‐induced upregulation of FasL did not result in an increased autocrine apoptosis of macrophages which might be related to endogenous expression of the apoptosis inhibitor FLICE inhibitory protein (FLIP). However, substantial apoptosis occurred in peripheral T cells as well as Fas‐sensitive Jurkat T cells when cocultured with HSV‐infected macrophages. These findings suggest that the paracrine killing of activated T cells by FasL expressing APC might be a novel strategy of immune evasion by HSV.
Signalling via MHC Class II Molecules Modifies the Composition of GEMs in APC
Authors: Setterblad N. , Becart S. , Charron D. , Mooney N.
Published: March 2003

Major histocompatibility complex (MHC) class II molecules are responsible for peptide presentation to helper T lymphocytes and as such play an essential role in the immune response. These molecules transmit intracellular signals leading to diverse consequences in B lymphocytes including proliferation and apoptosis. Recent studies have revealed that glycolipid enriched membrane microdomains (GEMs) behave as signalling platforms for a variety of lymphocyte receptors. We have quantified human leucocyte antigen (HLA)‐DR molecules localized in GEMs in human B lymphocytes. Use of a model imitating the interaction of HLA‐DR with a T‐cell receptor (TCR) modified the constituents of the HLA–DR‐enriched GEMs. Confocal microscopy demonstrated a recruitment of HLA–DR and the ganglioside GM1 at the site of HLA–DR interaction with the stimulating ligand. Moreover, cholesterol depletion efficiently impaired this recruitment. Co‐localizing proteins detected in HLA–DR‐enriched GEMs include protein kinase C (PKC)‐δ and actin. These data reveal that MHC class II antigens are localized in GEMs in mature human B lymphocytes and indicates that the formation of the immunological synapse regulates the composition of HLA–DR enriched GEMs in the antigen presenting cell (APC).
Subcellular Redistribution and Surface Exposure of the Ro52, Ro60 and La48 Autoantigens During Apoptosis in Human Ductal Epithelial Cells: a Possible Mechanism in the Pathogenesis of Sjögren's Syndrome
Authors: Ohlsson M. , Jonsson R. , Brokstad K. A.
Published: March 2003

The Ro52, Ro60 and La48 autoantigens are associated with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The mechanisms behind tolerance breakdown of these self‐peptides remain unclear; however, apoptosis has been proposed to cause their presentation to the immune system. We have examined the localization of transiently expressed enhanced green fluorescent protein (EGFP)‐tagged Ro52, Ro60 and La48 autoantigens in a human salivary gland (HSG) cell line by laser confocal microscopy under normal growth conditions and during apoptosis. Surface exposure of Ro52, Ro60 and La48 was demonstrated on nonfixed apoptotic cells with monoclonal antibodies (MoAbs) or with primary SS patient antisera. Laser scanning cytometry determined the apoptotic frequency. EGFP alone was studied as control. We found that Ro52 mainly is cytoplasmic, Ro60 both nuclear and cytoplasmic, while La48 only resides in the nucleus under normal conditions. During early apoptosis, La48 is dramatically redistributed to the cytoplasm, while the localization of Ro52 and Ro60 is maintained. All three autoantigens filled apoptotic blebs and covered TUNEL (terminal‐deoxynucleotidyl‐transferase‐mediated dUTP–digoxigenin nick end labelling)‐positive apoptotic bodies. Identical results were obtained in COS‐7 cells. We have developed a transfection system to study the intracellular localization of the three autoantigens Ro52, Ro60 and La48, without antibody detection. During apoptosis, there is an intracellular redistribution of endogenous and EGFP‐tagged Ro52, Ro60 and La48, leading to surface exposure. These findings may indicate a role for apoptosis in the induction and facilitation of humoral responses to Ro52, Ro60 and La48 in the autoimmune exocrinopathy of SS.
Expression of Human Protein Tyrosine Phosphatase Epsilon in Leucocytes: a Potential ERK Pathway‐Regulating Phosphatase
Authors: WABAKKEN T. , HAUGE H. , FINNE E. F. , WIEDLOCHA A. , AASHEIM H.‐C.
Published: March 2003

The expression of protein tyrosine phosphatase epsilon (PTPε) was studied in human tissues and blood cells. High mRNA expression was observed in peripheral blood leucocytes, particularly in monocytes and granulocytes which revealed at least four distinct transcripts. In lymphocytes, PTPε expression was induced after 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or antigen‐receptor stimulation, indicating that PTPε plays a role in the events taking place after antigen engagement. Previously, PTPε has been shown to be involved in regulating voltage‐gated potassium channel activity, insulin‐receptor signalling and Janus kinase‐signal transducers and activators of transcription (STAT) signalling. Transfection of cells with different PTPε constructs and activator protein‐1 reporter gene indicates that the catalytic activity of PTPε is involved in the regulation of the mitogen‐activated protein kinase cascade. In particular, the extracellular signal‐regulated kinases (ERK1/2) were shown to be inhibited in both phosphorylation status and enzymatic activity after overexpression of PTPε. Thus, PTPε emerges as a phosphatase with a potential to regulate the ERK1/2 pathway either directly or indirectly through its catalytic activity.
The coastal transition at the mouth of a small mountainous river in Taiwan
Authors: LIU  , YUAN  , HUNG
Published: March 2003

The Tseng‐wen River is a small mountainous river in southern Taiwan that has distinct dry and flood seasons. Several lines of evidence have revealed that the depositional system at the mouth of the river has transformed from a deltaic system to an estuarine system. The evidence includes long to short‐term shoreline changes, geomorphology of the river mouth, mixed sediment sources inside the river mouth, mixing and suspended sediment characteristics inside the river mouth, and the grain‐size distribution patterns on the river bed. Unlike the long‐term evolution of many other coastal systems, the transformation at the Tseng‐wen River mouth is largely caused by the building of a reservoir in the middle reaches of the river. The present study provides an example that coastal environments can be influenced by human activities farther inland on a time scale much faster than what would occur naturally.