bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Natural Killer Cell Activity and Numbers of Leu‐7 and Leu‐11b (CD 16)‐Positive Cells in Bone Marrow of Children in Remission from Acute Lymphoblastic Leukaemia
Authors: SØRSKAAR D. , FØRRE Ø. , LIE S. O.
Published: June 2006

Natural killer (NK) cell activity and related markers were analysed in childhood acute lymphoblastic leukaemia (ALL). Children with untreated ALL, children with active disease, and children in remission for less than 1 month and undergoing induction therapy had significantly lower NK cell activity in peripheral blood than the control group (P<0.05, P=0.0005. and P<0.0025). Patients in remission for 1–3 months and undergoing consolidation chemotherapy had normal NK activity (P>0.05). Children in complete remission for more than 3 months and undergoing maintenance therapy also had a normal NK activity in their peripheral blood (P>0.05). However, their bone marrow cells showed an increased NK cell activity (P<0.0005). Cells positive for the Leu‐7 marker were reduced in the peripheral blood from untreated children (P<0.025) and children in remission for less than 1 month (P=0.025). The percentage of cells from peripheral blood expressing the marker Leu‐11b (CD 16) did not differ significantly from that of the controls (P>0.05). However, children in complete remission for more than 3 months had a higher number of bone marrow cells expressing the Leu‐7 (P=0.005) and the Leu‐11b (CD 16) markers (P=0.05) than controls. Stimulation of mononuclear cell suspensions with recombinant alpha interferon and recombinant interleukin 2 were shown to cause a normalization of the NK cell activity in peripheral blood and bone marrow.
High Rate of HLA Class II mRNA Synthesis in Rheumatoid Arthritis Joints and Its Persistence in Culture: Down‐Regulation by Recombinant Interleukin 2
Authors: KISSONERGHIS A. M. , MAINI R. N. , FELDMANN M.
Published: June 2006

The expression of HLA class II mRNA was investigated in the joints of patients with active rheumatoid arthritis (RA) in order to evaluate patterns of synthesis. Northern hybridization analysis showed that HLA class II gene transcripts in RA joints were of the correct sizes, and subsequent analyses were performed by slot blotting. All active RA samples expressed high levels of HLA‐DR, DP, and DQ mRNA with DP and DQ less than DR. Synovial fluid or membrane cells, chiefly B mixture of T cells and macrophages, were placed in culture, in the absence of any Stimulation. The levels of mRNA remained at a high level in vitro. The half life of HLA‐DR mRNA in joint cells was very brief(∼30 min), indicating that prolonged synthesis was due to re‐stimulation of the cells The effect of lymphokines on HLA class 11 regulation on joint cells was assessed. Gamma interferon was capable of augmenting HLA‐DR to some extent, but paradoxically interleukin 2 at concentrations optimal for stimulating T cells, diminished HLA‐DR expression.
Decreased Natural Killer (NK) Susceptibility of Human NK Target Cells after Phosphatidylinositol‐Specific Phospholipase C Treatment
Authors: UGGLA C. , UNE C. , AXBERG I. , JONDAL M. , KNOWLES R. W. , ÖRN A.
Published: June 2006

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) treatment of the human natural killer (NK) target cells Molt‐4, Jurkat, and U937 reduced their susceptibility to killing by human NK cells in a dose‐dependent fashion. This indicates that a cell surface Structure, anchored by a glycosyl‐Phosphatidylinositol (G‐P1) moiety, is important in NK cytotoxicity. In contrast, another common NK target cell line, K562, remained susceptible to NK killing after enzyme treatment, suggesting that distinct target structures are expressed by this cell line. PI‐PLC treatment of Molt‐4 cells also reduced their sensitivity to human lymphokine activated killer (LAK) cells, suggesting that NK and LAK cells share common specificity in the killing of Molt‐4. In contrast, PI‐PLC had no effect on the killing of the LAK target cell line. Daudi, which is only weakly sensitive to unactivated NK cells. Killing of a variety of murine target cells by murine NK cells was not affected by PI‐PLC treatment, but cross‐species killing of Molt‐4 by murine NK cells was inhibited by PI‐PLC, suggesting a common mechanism in the killing of this human target cell line. The PI‐PLC treatment of effector cells from either species did not reduce their NK activity. The reduction in sensitivity of the Molt‐4, Jurkat, and U937 target cells probably results from a loss of a target specific G‐PI linked membrane molecule, but other possible explanations for these results are also discussed.
Molecular Analysis of the Human Serum Amyloid A (SAA) Gene Family
Authors: SACK G. H. , TALBOT C. C. , SEUANEZ H. , O'BRIEN S. J.
Published: June 2006

We have assigned the human serum amyloid A (SAA) gene family to a 90 kb region on the short arm of human chromosome 11(11p) by hybridization of defined genomic fragments of human SAA genes to DNA from rodent‐human somatic cell hybrids and to large DNA fragments separated by transverse alternating field gel electrophoresis. We have also characterized SAA probe hybridization patterns in human DNA cleaved with restriction endonucleases Hind III, Pst I, Bg/II, Taq I, and Xba I and found invariant patterns except for a two‐allele restriction fragment length polymorphism (RFLP) with Hind III. These studies show that the SAA gene family comprises at least three members in the haploid human genome and will be useful in identifying variant patterns and establishing linkage between members of the SAA gene family and other markers on chromosome 11.
Inhibition of IgG‐Mediated Immunosuppression by a Monoclonal Anti‐Fc Receptor Antibody
Authors: HEYMAN B.
Published: June 2006

IgG‐antibodies can efficiently suppress the antibody response against their specific antigen. The suppressive capacity is dependent on intact Fc regions. However, it is not clear which of the Fc‐mediated effector functions arc necessary for the induction of immunosuppression the monoclonal antibody 24G2, which binds to murine Fc receptors on macrophages and B cells, was used in the present study to address the question of whether IgG‐mediated suppression is in fact dependent on the binding of IgG antibodies to Fc receptors on splenocytes, In a murine in vitro immunization system, 24G2 is shown to reverse efficiently the suppression of the sheep erythrocyte‐specific antibody response. The role of B cells or macrophages as effector cells is discussed.
Dipeptidyl Peptidase IV in Human T Lymphocytes
Authors: SCHÖN E. , DEMUTH H.‐U. , EICHMANN E. , HORST H.‐J. , KÖRNER H.‐J. , KOPP J. , MATTERN T. , NEUBERT K. , NOLL F. , ULMER A. J. , BARTH A. , ANSORGE S.
Published: June 2006

Specific inhibitors of the membrane‐bound dipeptidyl peptidase IV (DP IV) and polyclonal antibodies against this enzyme were used to investigate the relationships between DP IV activity and the production and action of T cell‐derived lymphokines. Production of interleukin 2 (IL‐2) and gamma interferon by mitogen plus phorbol ester‐simulated mononuclear cells Bran human blood was found to be reduced in the presence of N‐Ala‐Pro‐O‐(nitrobenzoyl‐)‐hydroxylamine, epsilon‐(4′nitro) benzoxycarbonyl‐Lys‐Pro, and anti‐(DP IV) immunoglobulin in a dose‐dependent manner. Moreover, the proliferative response of mitogen‐stimulated mononuclear cells to IL‐2 is impaired in the presence of DP IV inhibitors. Therefore it is suggested that the membrane peptidase DP IV is involved in the induction and activation of cytokines controlling lymphocyte proliferation
Enhancement of Natural Killer Cell Activity by Nocardia opaca Fractions
Authors: BAROT‐CIORBARU R. , LINNA T. J. , PATEL M. R. , ALTMAN J. , CARNAUD C.
Published: June 2006

Three molecules derived from Nocardia opaca bacteria, NDCM, NWSMP, and PG, have been shown to express immunomodulating properties. The present study was aimed al assessing the effects of these derivatives on natural killer (NK) activity. Two experimental protocols were adopted, consisting of incubating whole or Percoll fractionated NK cells in vitro with those substances, and the other in which the derivatives were administered in vivo so mice and the activity assessed later. Incubation of spleen cells in vitro with NWSMP or its precursor NDCM promoted NK activity. This effect could be observed after only 2 h of incubation and continued until day 2. Percoll fractions 1–3, which contain most of the NK activity, were enhanced to a similar extent. Band 4, which is usually devoid of such activity, remained unresponsive even after contact with the N. opaca derivatives. PG was practically ineffective upon all the subsets. The results of experiments in vivo correlated with those obtained in vitro in that NWSMP and NDCM, but not PG, promoted NK activity. Bands 1–3 were similarly enhanced, the effect was observed after short treatment times, and could be partially cancelled by the concomitant administration of anti interferon antibodies (anti‐IFN Ab). All these findings suggest that the promoting effects of N. opaca derivatives are mediated through alpha/beta IFN. In contrast to the results observed on spleen NK cells. NK cells from the peritoneum displayed susceptibility mainly to PG, and much less to NWSMP or NDCM. The administration of PG to mice in vivo had a particularly marked promoting effect upon the cytotoxic activity of peritoneal cells. One logical explanation for the difference observed between PG and NWSMP or NDCM may be related to the specific IFN inducing properties of these compounds as well as to die different responsiveness of die NK cells present in the spleen and peritoneal cavity.
Trypsin Does Not Reconstitute Responsiveness to Lipopolysaccharide in the Strain C3H/HeJ, but Is a B‐Cell Mitogen‐Like Lipopolysaccharide, Stimulating a Different Subpopulation
Authors: CARDELL S. , MÖLLER G.
Published: June 2006

The effect of trypsin on mouse spleen cells and enriched B cells, added alone or together with lipopolysaccharide (LPS), was investigated. With trypsin, proliferation in serum free spleen cell cultures was 2–6 times greater than the background using cells from LPS responder strains, and 2–4 times the background with cells from the C3H/HeJ strain. Trypsin also induced the formation of a low number of IgM plaque forming cells (PFC). When added together with LPS, trypsin increased the proliferation caused by LPS alone by 10–50% with cells from LPS responder strains and by 50–100% with cells from the LPS non‐responder strain C3H/HeJ. Trypsin enhanced proliferation in cultures maximally stimulated by LPS. The increased proliferation obtained when trypsin was added to LPS‐stimulation of cells from the C3H/HeJ strain, was therefore not interpreted as a reconstitution of the LPS response. We conclude that trypsin has a moderate mitogenic effect on mouse B cells, stimulating the cells to proliferate and secrete IgM. The mechanism of action is unknown, but is different and independent from the action of LPS.
Recombinant Protein A of Non‐Staphylococcal Origin is Not Mitogenic for Human Peripheral Lymphocytes
Authors: PERSSON U. , INGANÄS M. , SMITH C. I. E. , HAMMARSTRÖM I , JOHANSSON S. G. O.
Published: June 2006

Recombinant protein A(SpA) produced in Escherichia coli or Bacillus subtilis bacteria did not induce activation of human peripheral mononuclear cells, whereas SpA preparations obtained from naturally occurring Staphylococcus aureus bacteria as well as recombinant SpA from Staphylococcus xylosus were potent mitogens. Further purification of SpA from S. aureus showed that the mitogenic material was concentrated in the side fractions containing more basic molecules. Some staphylococcal enterotoxins are mitogenic for human cells and in order to test whether contaminating enterotoxins would be responsible for the mitogenic effect of SpA preparations, rabbit antibodies were produced against enterotoxin A and B. These antibodies inhibited activation of human cells induced by the enterotoxins used for immunization but did not affect the activation induced by SpA preparation. The addition of selected human sera to in vitro cultures resulted in an inhibition of the response induced by low doses of SpA. There was no clear relationship between these effects and the content of IgG antibodies against staphylococcal enterotoxins A, B, and C1 in the sera. Thus, we conclude that the mitogenicity of SpA preparations is caused by contaminating molecules, probably not enterotoxins A, B, or C1.
Amyloidosis Related to a λ IV Immunoglobulin Light Chain Protein
Authors: BENSON M. D. , DWULET F. E. , MADURA D. , WHEELER G.
Published: June 2006

Amyloid subunit proteins related to the λ IV subgroup of immunoglobulin light chains have not been previously reported. We have determined the amino acid sequence of an AL amyloid protein BAK and shown that it has the structure typical of λ IV light chain proteins. This protein, which was isolated from the spleen of a patient with AL amyloidosis, has 111 residues in the variable domain and also includes the first tryptic peptide of the constant domain for a total of 130 residues. Comparison of the primary structure of this protein with the only other completely characterized λ IV protein (SH) reveals that they are highly homologous with only one amino acid change in FR1, two changes in FR2, and one change in FR3. The CDR regions also show few changes, with only three in CDR1, one in CDR2, and five in CDR3. To test the hypothesis that the formation of AL amyloid is related to changes in the FR regions which could affect molecular aggregation, the structure of BAK was compared with the myeloma protein SH with respect to the presumed tertiary structure. Only limited amino acid substitution was found in the surface positions that might affect intradimer and interdimer aggregation. These included an isoleucine for leucine change at position 43 and phenylalanine for valine at 45, which may affect intradimer interaction and a change of histidine to asparagine at position 67.
Tumour Regression after Treatment with Aminated β1‐3D Polyglucose Is Initiated by Circulatory Failure
Authors: SELJELID R.
Published: June 2006

Meth A sarcoma grew progressively when inoculated intradermally in CB6 mice. When the mice were treated on day 7 after inoculation with 10 mg aminated polyglucose (AG) [Bogwald, J., Hoffman, J. & Seljelid, R. Carbohydrate Res.148, 101, 1986], the tumours regressed completely in over 90% of the cases. During the first hours after AG treatment, tumour thymidine incorporation decreased, adenosine triphosphate (ATP) content decreased, and there were indication of circulatory disturbance as shown by decreased deposition of dye (trypan blue) in the tumour tissue after intravenous injection. Histological examination demonstrated a conspicuous thickening of the walls of small tumour vessels, statis of red blood cells, and perivascular collections of mononuclear cells only hours after AG treatment. In thymectomized animals, where regression does not occur after AG treatment [Seljelid. R. Bioscience Reports 6, 845. 1986], there was no evidence of circulatory failure, no tumour diameter reduction, and no decrease in colouring following intravenous injection of trypan blue. On the basis of these findings, we conclude that the early phase of events after AG treatment leading to tumour regression involves a vascular phenomenon that causes circulatory disturbance and necrosis. The data also indicate that this initial circulatory failure requires the involvement of functional T cells
Migration Pattern of Lymphocyte Subsets in the Normal Rat and the Influence of Splenic Tissue
Authors: WESTERMANN J. , WILLFÜHR K. U. , ROTHKÖTTER H. J. , FRITZ F. J. , PABST R.
Published: June 2006

Lymphocyte subsets leave the blood and appear in the thoracic duct of normal rats at different rates. The aim of the present study was to investigate their migration pattern through blood, spleen, bone marrow, mesenteric lymph nodes, and Peyer's patches in normal Lewis rats and to study the role of the spleen using splenectomized and spleen‐transplanted animals. Fluorescein isothiocyanate (FITC)‐labelled thoracic duct lymphocytes (TDL) were injected intravenously into rats and after 15 min, 1, 6, and 24 h the percentages of B, T, T helper (TH) and T‐cytotoxic/ suppressor (TC/S) lymphocytes in the FITC+ cells were determined in cell suspensions by means of monoclonal antibodies B and T lymphocytes are preferentially localized in different organs, e.g. B, cells in Peyer's patches and T cells in mesenteric lymph nodes The migration of TH lymphocytes differed from that of TC/S lymphocytes in all the organs investigated. In the late phase after injection the migration of B and TH lymphocytes was influenced by the spleen, since after splenectomy the number of injected B lymphocytes increased and that of TH lymphocytes decreased in all organs investigated except the bone marrow. Splenic autotransplantation could not normalize the disturbed migration.
Generation of Inositol Phosphates during Triggering of Cytotoxicity in Human Natural Killer and Lymphokine‐Activated Killer Cells
Authors: STÅHLS A. K. , CARPÉN O.
Published: June 2006

We investigated early molecular mechanisms involved in the triggering of cytolytic responses in natural killer (NK) and lymphokine‐activated (LAK) cells. When NK or LAK cells were conjugated to the sensitive target cells K562, an increased formation of both inositol monophosphate (IP1) and inositol trisphosphate (IP3) was detected. Target cells like Raji or Jok‐1, which form conjugates with NK cells but are insensitive to NK lysis, did not elicit IP1 formation. Treatment of NK cells with interleukin 2 increased the basal turnover of inositol phosphates and enhanced the phosphatidyl inositol breakdown upon confirmation with sensitive targets. These finding indicate that hydrolysis of phosphatidyl inositols is associated with the signal which triggers the cytolytic response in NK and LAK cells. These events therefore constitute an early marker of the cytolytic activation.
Comparative Analysis of Immunodeficiency in Patients with Monoclonal Gammopathy of Undetermined Significance and Patients with Untreated Multiple Myeloma
Authors: PILARSKI L. M. , ANDREWS E. J. , SERRA H. M. , RUETHER B. A. , MANT M. J.
Published: June 2006

The objectives of this study were firstly, to compare the immunophenotype of patients with monoclonal gammopathy of undetermined significance (MGUS) with that of patients with newly diagnosed, untreated multiple myeloma (Unt. MM). Our second objective was to determine which variables might distinguish patients with MGUS and early MM. The CD4/CD8 ratio in both patient groups differed significantly from normal as a result of a decrease in the proportion of CD4+ cells. Similarly, surface immunoglobulin‐positive (Ig+) B cells were significantly reduced in both groups. Also, some impairment of Ig secretion was observed. An in vitro specificity study of B cells showed an enriched proportion of B cells specific for tetanus toxoid (which may be indicative of enrichment for memory B cells) in both MGUS and Unt. MM patients. Further to this, in MM patients but not in MGUS patients, there was an enriched proportion of B cells specific for determinants on the F(ab′)2 fragment of Ig. This suggests an anomalous auto‐immune reactivity to polyclonal Ig molecules. In one of the two patients studied, who progressed from MGUS to MM, disease progression was accompanied by an increase in this anti‐Ig reactivity. In both patients there was a decrease in CD4/CD8 ratio.
Reduced Erythrocyte CR1 (CD 35) Receptor Function and Complement Opsonization in Factor I‐Deficient Patients is Restored by Plasma Infusion
Authors: JEPSEN H. H. , TEISNER B. , RASMUSSEN J. M. , SVEHAG S.‐E.
Published: June 2006

Erythrocytes (E) from three factor I‐deficient patients were investigated for surface‐bound complement factors and CR1 (CD 35) expression and function. The E were coated with C4b,C3b, and factor H. Following plasma infusion of in vitro incubation of the patients’ E with normal human serum (NHS) or purified factor I, cell‐bound C4b and C3b could no longer be detected. The E now expressed C3d, and factor H was unaffected, indicating that factor H was hound to the C3d part of the C3b molecules, providing the co‐factor for effective cleavage of E‐bound C3b when purified factor 1 was added. The binding of monoclonal anti‐CR1 antibodies (M710) to the patients’ E was markedly reduced compared with control E, and was not normalized by treatment with NHS, probably because covalently bound C3d/factor H interfered with the binding of M710. By contrast, the reduced ability of the patients’ E‐CR1 to bind complement‐opsonized immune complexes (IC) was normalized after plasma infusion. This shows that the impaired CR1 function was acquired and emphasizes the importance of performing functional CR1 assays. Complement opsonization of IC for binding to normal E was severely compromised in the patients’ sera due to consumption of factor B and C3. After plasma infusion the opsonization capacity of the patients’ sera was restored. Thus, two mechanisms of importance for normal clearance of IC were compromised in factor I‐deficient patients: (1) the opsonization of IC for binding to E‐CRI, and (2) the capacity of E‐CRI to bind opsonized complexes. Both dysfunctions were temporarily corrected by plasma infusion.
Monoclonal Antibodies Specific for Variable and Constant Domains of Murine λ Chains
Authors: BOGEN B.
Published: June 2006

Rat monoclonal antibodies directed against the BALB/c myeloma protein M315 (α,λ2) are described. 9A8 (IgG1) binds the V domain of λ2 and cross‐reacts with λ1 and λ3 chains. 2B6 (IgG2a) is directed to the C domain of λ2 and cross‐reacts λ3. The antibodies bind isolated chains as well us complete immunoglobulins. The monoclonals detect soluble immunoglobulin (radioimmunoassay), immunoglobulin immobilized on polystyrene (enzyme‐linked immunosorbent assay), immunoglobulin bound to nitrocellulose (immunoblotting), and surface immunoglobulin intercalated in cell membranes (immunofluorescence). The antibodies are easily purified on protein G immunosorbents and may be biotinylated or conjugated with fluorescein isothiocyanate without loss of capacity to bind. In addition to the anti‐λ antibodies, a Cα2/Cα3‐specific monoclonal antibody, 8D2 (IgG2a) is described.
The Generation of Guinea Pig T‐Cell Lines Reactive to Antigens from Mycobacterium tuberculosis. Selected Lines Induce Erythematous Skin Reactions
Authors: HASLØV K. , HERON I.
Published: June 2006

We describe methods for the development and partial characterization of antigen‐specific T‐cell lines from the guinea pig, which is the classical experimental animal in tuberculosis research. T cells were obtained from strain 2 guinea pigs immunized with BCG vaccine or with killed Mycobacterium tuberculosis in oil. T‐cell lines were obtained from limited dilution cloning of antigen‐stimulated, blast‐enriched lymphocyte cultures. The lines were grown with weekly reseedings in the alternating absence and presence of mycobacterial antigen. Antigen reactivity of the cell lines was studied with lymphocyte stimulation tests. With these methods we have consistently obtained antigen‐reactive cell lines. When injected in small numbers intradermally in the presence of antigen in syngeneic guinea pigs, some of these cell lines gave rise to antigen‐specific erythematous tuberculin‐like skin reactions. The skin reactions, which were usually without induration, were most pronounced after 24 h. Histological examinations of skin undergoing such reactions showed that the erythemas were not accompanied by mononuclear infiltrations. We expect that transfer experiments with guinea pig T‐cell lines will prove useful tools in the analysis of the contribution of defined mycobacterial antigen preparations to tuberculosis immunity.
Immunochemical and Biological Reactivity of Human Anti‐Lipopolysaccharide IgG Obtained by Screening of Blood Donors
Authors: FOMSGAARD A. , ZHANG G.‐H. , SHAND G. H. , BENDTZEN K , BAEK L.
Published: June 2006

An anti‐lipopolysaccharide (LPS) preparation for the intravenous treatment of septic endotoxic shock was prepared by purifying immunoglobulin G (IgG) from pooled serum from Danish blood donors. The sera were selected by the use of an enzyme‐linked immunosorbent assay (ELISA) to screen blood donors for high concentrations of antibodies to a mixture of LPS from 11 different Gram‐negative bacteria. ELISA was also used for indirect quantification of IgG antibodies to lipid A. and to rough LPS from Escherichia coli Ra and Salmonella minnesota R60 (Ra). The concentration of human antibodies to the LPS mixture correlated with the concentration of antibodies to the E. coli and S. minnesota rough LPS and to lipid A. The specificity of sera with high concentrations of anti‐LPS IgG was investigated by immunoblotting. Sites from individual donors reacted with LPS from different bacteria and recognized different sites on the LPS molecules. The range of specificities to different LPS was increased by the pooling of selected sera. The IgG fraction from the high titre donor pool neutralized biological activities of LPS such as activation of the Limulus amoebocyte lysate reaction and induction of tumour necrosis factor secretion from human monocytes.
B‐Cell Differentiation in the Chicken: Expression of Immunoglobulin Genes in the Bursal and Peripheral Lymphocytes
Authors: MANSIKKA A. , VEROMAA T. , VAINIO O. , TOIVANEN P.
Published: June 2006

We have studied the expression of immunoglobulin genes in the chicken B‐cell precursors, and of a B‐cell surface marker (Bu‐1) on the bursal and peripheral B‐cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post‐bursal, and post‐bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the γ and μ transcripts and expression of Bu‐I antigen in the chicken B‐cell precursors were found to be unchanged during the post‐hatching period. In the light of these experiments, the later events of B‐cell differentiation, i.e. the development from the bursal to post‐bursal B lymphocytes, occurs without the γ, μ, and Bu‐1 gene loci involved. On the other hand, the higher level of γ and μ expression in the splenic B lymphocytes indicates that the post‐bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.
Measurement of Complement Activation in Rabbit Plasma or Serum Using Monoclonal Antibodies against C5a
Authors: BERGH K. , IVERSEN O.‐J.
Published: June 2006

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan‐induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan‐induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat‐inactivated plasma. A cross‐reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme‐linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan‐induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan‐activated scrum. MoAb 2A5E3 was able to bind 125I‐labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen‐antibody complexes, or cobra venom factor.
Responsiveness of T cells to Mutant Major Histocompatibility Complex Class I Antigen
Authors: WATANABE H , SAITOH T. , FUJIWARA M.
Published: June 2006

Mechanisms of the activation of T cells responding to major histocompatibility complex (MHC) class I antigen were investigated with special reference lo interleukin 1 (IL‐1) production from stimulator‐type accessory cells. For this purpose, we used mainly fractionated Lyt‐2+ T cells of C57BL/6 (B6) mice as responder cells and irradiated spleen cells or those deprived of adherent cells of B6. C‐H‐2bm1 (bm 1) mice as stimulator ceils. Lyt‐2+ T cells of B6 mice proliferated in the presence of irradiated whole spleen cells of bm1 mice but did not to Sephadex G‐10 column‐passed bm1 spleen cells. The unresponsiveness in the latter case was overcome by the supplement of recombinant IL‐1 and/or IL‐2 in the culture medium. These interleukins were shown to promote the proliferative response of B6 Lyt‐2+ T cells in the presence of stimulator‐type for B cells. Both interleukins also facilitated the generation of cytotoxic T cells from BA Lyt‐2+ cells to H‐2Kbm1 antigen in the mixed lymphocyte culture deficient in stimulator‐type accessory cells. IL‐1 was shown to enhance the expression of IL‐2 receptor on the responding Lyt‐2+ T cells as assessed by flow cytometry. IL‐1 binding to responding T cells were also assayed by means of iodinated IL‐1 and was shown to increase significantly on responding Lyt‐2+ cells. Overall results indicate that accessory cells might play dual roles in the activation of Lyt‐2+ T cells responding to allogeneic MHC class 1 antigen: direct presentation of the antigen to responder T cells and production of IL‐1. Both signals are essentially required for Lyt‐2+ T cells responding to allogeneic MHC class I antigen to initiate proliferation and also to differentiate into cytotoxic T cells.
Studies on the Role of CD43 in Human B‐Cell Activation and Differentiation
Authors: WIKÉN M. , BJÖRCK P. , AXELSSON B. , PERLMANN P.
Published: June 2006

The monoclonal antibody (MoAb) B1B6 to human leucocyte sialoglycoprotein, CD43, induces aggregation of T cells and drivers progression signals early during activation of both T and B cells in the presence of primary activators of proton kinase C. In this report we further studied the role of CD43 in human B‐cell activation and differentiation. About 5–10% of resting tonsillar B cells are CD43+. In the presence of TPA or antibodies to CDw40, the proportions of CD43+ cells drastically increased. The expression was optimal on day 3 of culture, when up to 80% and 50%, respectively, were CD43+. Whereas MoAb B1B6 together with TPA induced a three‐ to fivefold higher proliferative response as compared to TPA alone, antibodies to CDw40 did not synergize with MoAb B1B6 in B‐cell proliferation Tonsillar populations depleted of CD43+ B cells responded with lower proliferation to TPA alone or to TPA and B1B6 or anti‐CDw40antibodies. MoAb BlB6 did not affect the production of IgM or IgG as induced by pokeweed mitogen in the presence of autologous T cells, from either peripheral blood or tonsillar B cells. Neither did it affect the IgG production from the CD43+ BSF‐2 sensitive Epstein Barr virus‐transformed lymphoblastoid cell line CESS. The results show that CD43 is upregulated on B cells during activation. Furthermore, CD43+ B cells are included in the population which responds to signals delivered by TPA, anti‐CD43 or anti‐CDw40 antibodies, and the proliferation of this population is not merely due to an expansion of the small population of CD43+ cells present among these cells. Moreover, the epitopes recognized by MoAb B1B6 are not involved in the differentiation of and ultimate Ig‐secretion from activated B cells.
Macrophages and Dendritic Cells during the Early Stages of Antigen‐Induced Arthritis in Rats: Immunohistochemical Analysis of Cryostat Sections of the Whole Knee Joint
Authors: VERSCHURE P. J. , VAN NOORDEN C. J. F. , DIJKSTRA C. D.
Published: June 2006

The appearance of different macrophage subpopulations, Ia‐positive antigen‐presenting dendritic cells and of T and B lymphocytes was studied in early phases of antigen‐induced arthritis in rat knee joints. Cryostat sections of whole knee joints were analysed with immunohistochemical techniques using monoclonal antibodies against rat macrophages, Ia‐antigen, and lymphocyte subpopulations. The results showed that in the early phases of the development of arthritis, the synovium was already infiltrated by many monocytes, young macrophages, granulocytes, perivascular Ia‐positive non‐lymphoid cells, some mature tissue macrophages, and only few T lymphocytes. In later phases not only monocytes, young macrophages and Ia‐positive cells became more prominent but also the more mature ED2 positive macrophages and the ED3 positive macrophages that are normally confined to lymphoid organs became increasingly important. The T‐cell population increased to some extent in later phases of arthritis induction, possibly induced by clustering with the Ia‐positive cells.
Effect of Physical Exercise on Blood Mononuclear Cell Subpopulations and in Vitro Proliferative Responses
Authors: TVEDE N. , PEDERSEN B. K. , HANSEN F. R. , BENDIX T. , CHRISTENSEN L. D. , GALBO H. , HALKJÆR‐KRISTENSEN J.
Published: June 2006

The present study was designed to examine the effect of physical exercise on subsets and proliferative responses of blood mononuclear cells. Sixteen young, healthy volunteers underwent 60min of bicycle exercise at 75% of maximal oxygen uptake (VO2max). After an interval of at least 1 week, six of the subjects underwent a 60‐min back muscle training period at up to 30% of VO2max. Blood samples were collected before and during the last minutes of exercise, as well as 2 and 24 h later. Blood mononuclear cell (BMNC) subpopulations were determined and the proliferate responses after incubation with phytohaemagglutinin (PHA) or purified derivative of tuberculin (PPD), were quantified by [3H]thymidine incorporation. During bicycle exercise the relative blood concentration of T cells (CD3+ cells) declined, mainly due to a fall in T helper cells (CD4+ cells). The natural killer (NK) cell subset (CD16+ cells) increased during work, but reverted after; the monocytes (CD14+ cells) increased 2 h after work, whereas the B‐cell subset (CD20+ cells) did not change. BMNC subsets were not significantly changed by back muscle exercise. The PHA‐induced proliferative response decreased during bicycle exercise, whereas the PPD‐induced response did not change. No significant changes occurred during back muscle exercise. Investigation of subgroups after incubation with [3H]thymidine showed that the proliferative response per CD4+ cell did not change in relation to exercise, but the contribution of the CD4+ subgroup to proliferation declined during bicycle exercise due to the decreased proportion of CD4+ cells. The suppression of the PHA response during bicycle exercise can be explained in part by a relative fall in CD4+ cells. The pool sizes of BMNC subfraction may be elicited by increased catecholamine and cortisol levels.
Opioid Peptides Modulate the Organization of Vimentin Filaments, Phagocytic Activity, and Expression of Surface Molecules in Monocytes
Authors: PRIETO J. , SUBIRÁ M. L. , CASTILLA A. , ARROYO J. L. , SERRANO M.
Published: June 2006

It is theorized that intermediate filaments are important in the modulation of membrane activity and cell motility; however, their functions are unknown. The assembly and organization of these filaments are under hormonal regulation. We investigated in human monocytes the in vitro effects of Met‐enkephalin, Leu‐enkephalin, and β‐endorphin on the expression of immunoreactive cytoskeletal vimentin filaments. We simultaneously examined their effect on the phagocytosis of Candida albicans and on the membrane display of surface molecules. The three opioid peptides markedly reduced the expression of vimentin filaments, the phagocytic activity, and the display of HLA‐DR molecules at concentrations of 10‐6, 10‐8, and 10‐10m. On the other hand, the intravenous administration of fentanyl, a synthetic opiate agonist, to patients undergoing surgery induced similar changes in monocytes. In other experiments. 10‐8mβ‐endorphin alto decreased the expression of CR3 but did not influence the display of CD13, a surface protein of unknown function. Expression of vimentin filaments correlated directly with the display of HLA‐DR antigens and CR3 and with the phagocytic activity. The results of this, paper indicate that opiates and opioids, neuropeptides known to be released during stress, can directly depress several monocyte functions. Furthermore, from these data it may be speculated that intermediate filaments may regulate the membrane expression of some surface molecules and the phagocytic process.
Dynamics of Complex Formation between Thymocytes and Thymic Medullary Epithelial Cells
Authors: HUGO P. , POTWOROWSKI E. F.
Published: June 2006

Direct cell contact is an intrinsic part of several differentiation processes. A case in point is the formation of complexes between thymic lymphocytes and stromal cells, which are essential for T‐cell maturation. The objective of the present work was to gain an insight into the mechanisms underlying the formation and dissociation of such lymphostromal complexes. Using an in vitro system, we show that the number of thymocytes adhering to a thymic medullary epithelial cell line (E‐5) increases with time and reaches a plateau, after which some thymocytes spontaneously detach, while the rest remain attached to epithelium. The detached and forcibly removed thymocytes were analysed for their expression of L3T4 and Ly‐2 antigens. The detached thymocytes showed a markedly lowered expression of both antigens. Both of these subtypes were shown to be totally refractory to form further complexes after a first encounter with L‐5 cells. We also show that, once a mean adherence level of one thymocyte per E‐5 cell was reached, (a) the level of further adherence increased exponentially, and (b) during this phase, binding of thymocytes to E‐5 cells occurred in clusters, indicating pre‐existing polarity or contact‐induced polarization of the receptors at the E‐5 cell surface.
Kinetic Analysis of Lymphokine‐Activated Killer (LAK) Cells: Lytic Parameters and Determination of LAK Cell Frequency
Authors: LEFEVER A. , MICHA S.
Published: June 2006

Kinetic analysis was used to define lytic events in murine lymphokine‐activated killer (LAK) cell‐mediated tumour cell lysis. The maximum rate of target cell lysis (Vmax) and Km (target cell number resulting in 1/2 Vmax) were determined. Single cell lytic assays demonstrated that only LAK effector cells bound to target cells (i.e. non‐lytic, bystander lymphocytes did not influence the determination of kinetic parameters) in contrast to natural killer (NK) cell lysis. This finding allowed for LAK cell frequency determinations where Km approximates the concentration of lytic LAK effector cells within a given number of lymphocytes. Frequencies determined in this manner were not significantly different from those obtained using the more cumbersome single cell lytic assay. Furthermore, frequencies determined for the same lymphocyte population against four different NK‐resistant tumour targets, that varied in their sensitivity to LAK cell lysis, were not significantly different. In addition, LAK cell lytic programming of target cells was found to be the rate limiting lytic event. This study provides a means of determining reliable estimates of LAK cell frequencies within a lymphocyte population, which will be useful in studies evaluating LAK cytolytic mechanisms and the effects of drugs, biological response modifiers, or disease states on LAK cell lytic activity.
The Immune Response to Bacterial Dextrans
Authors: LUNDKVIST I. , PORTNOÏ D. , COUTINHO A.
Published: June 2006

C57BL/6 mice respond to Dextran B512 (Den) with a predominant idiotype (Id) (17‐9) while no such Id‐positive antibodies are identified in the specific antibody response of BALB/c mice. We used limiting dilution systems to determine the absolute frequencies of clonal B‐cell precursors producing the 17–9 Id in these two mouse strains and analysed the correlation between Id‐expression and antibody activity at the clonal level The results show very similar frequencies of anti‐Dex und Id‐positive B cells in both strains, but C57BL/6 mice contained fourfold higher frequencies of Dex‐specific clonal precursors which are Id‐positive. This kind of clone, although not used in the specific response of BALB/c mice, constitute roughly 20% of their anti‐Dex repertoire and they are readily induced in this strain. Thus, immunization of both strains with anti‐idiotypic antibodies results in the production of Id‐positive anti‐Dex antibodies with serum titres that directly correlate with precursor frequencies. The results show, therefore, that the section of clonal repertoires utilized in a primary immune response vanes with the immunogen, even if thymus‐independent. These observations are discussed in the context of the genetic controls of anti‐Dex antibody responses and on the general question of the utilization of available antibody repertoires in immune responses.
Dual Immunoregulatory Effects of Monoclonal IgG‐Antibodies: Suppression and Enhancement of the Antibody Response
Authors: WIERSMA E. J. , COULIE P. G. , HEYMAN B.
Published: June 2006

Nine monoclonal IgG‐anti‐TNP antibodies were investigated for their ability to modulate anti‐carrier responses in mice immunized with sheep red blood cells–2,4,6‐trinilrophenyl (SRBC–TNP) or keyhole limpet haemocyanin–TNP (KLH–TNP). The antibodies enhanced the anti‐carrier response when KLH–TNP was used as antigen but suppressed it when SRBC–TNP was used. The enhancing and suppressive effects were not exerted by entirely the same sets of antibodies. The suppression was correlated to efficient antigen binding, but not complement activation, haemagglutination, or isotype of the monoclonal antibodies. In contrast, enhancement was correlated to isotype and complement activation but not to antigen binding capacity. Both the enhancing and the suppressive effects seem to require Fc‐mediated functions of the IgG molecules since they modulate the anti‐carrier response although they recognize hapten determinants. Thus, one and the same monoclonal hapten‐specific IgG‐antibody can enhance the anti‐KLH response up to 38‐fold whereas it suppresses the anti‐SRBC response by more that 10‐fold.
N‐α‐Benzyloxycarbonyl‐l‐Lysine Thiobenzyl Ester (BLT) Serine Esterase in Human Cytolytic Effector Cells and Cell Line Targets
Authors: SOMERSALO K. , SAKSELA E.
Published: June 2006

The granules of in vitro primed cytotoxic mouse T cells and cytotoxic cell lines have been shown to contain high levels of N‐α‐benzyloxycarbonyl‐l‐lysine thiobenzyl ester (BLT) esterase. The enzyme activity has been suggested lo be associated with the cytotoxic capacity of killer cells. We investigated human leucocytes and found that neutrophils, monocytes, cytotoxic T lymphocytes (CTL), natural killer (NK) cells [large granular lymphocytes (LGL)], and interleukin 2 activated killer (LAK) cells, which all display efficient cytotoxic capacity, show only marginal BLT esterase activity. The low BLT esterase activity in human lymphocytes increases about twofold when cells are stimulated in vitro with interleukin 2 (IL‐2), phytohaemagglutinin (PHA), or cultured in mixed lymphocyte culture (MLC). Mouse T lymphocytes have about 20 times more BLT esterase activity than human T lymphocytes. The BLT activity in mouse T cells also increases about twofold in MLC. The human leukaemia cell lines (K562, U937, MOLT‐4, Jurkat) and the mouse mastocytoma line (P815), which are frequently used as target cells, contain more BLT esterase activity than human resting or activated lymphocytes. We did not find a direct correlation between the cytotoxic capacity and the BLT esterase activity of killer cells.
Antibody Responses to Hapten in Thymectomized Mice: Extraordinarily Pronounced Deficiency in IgG1 Production
Authors: SEPPÄLÄ I. J. T. , HURME M. , MÄKELÄ O.
Published: June 2006

The effect of thymectomy on the production of antibodies was studied by immunizing mice with hapten‐carrier conjugates. Antibody responses were analysed with monoclonal antibody‐based quantitative isotype‐resolving assays. In spite of bone marrow reconstitution, irradiation without thymectomy caused a long‐lasting relative deficiency in responsiveness to T‐independent antigens. Even when no visible remnants of the thymus could be observed at the autopsy of thymectomized mice, there appeared to be a gradual recovery of antibody‐forming capacity within 4 months, as assessed by the response to a T‐dependent antigen. Therefore, some of the thymectomized mice had to be regarded as having recovered with respect to the helper T‐cell effect. The antibody responses to T‐dependent antigens were improved in all isotypes by a functional T‐cell system, but the IgG isotypes seemed to benefit more than IgM. The most conspicuous deficit in antibody production in non‐recovered thymectomized mice was observed in the T‐dependent responses of the IgG1 isotype (2000‐fold reduction in contrast to about 50‐ to 100‐fold in IgG2a, IgG2b, and IgG3).
IgG Autoantibodies against Interleukin 1α in Sera of Normal Individuals
Authors: SVENSON M. , POULSEN L. K. , FOMSGAARD A. , BENDTZEN K.
Published: June 2006

A pool of human sera from healthy blood donors was found to interfere competitively with the binding of 125I‐labelled human recombinant interleukin 1α (rIL‐1α) to the murine T‐cell line EL4. The interference was reversible at the cellular level, and direct binding of the ligand to serum factors was therefore investigated. After preincubation of [125I]rIL‐1α with pooled serum, the 125I activity eluted in two peaks from a Sephadex G‐75 column. The first was located in the void volume. The second eluted together with monomer rIL‐1α. An almost complete displacement of the high molecular weight 125I fraction was achieved with an excess of unlabelled rIL‐1α but not with rIL‐1β. The serum factors binding to [125I]rIL‐1α were located in the molecular weight range 100,000–200,000, judged by fractionation on a Sephacryl S‐400 column, and the factors were bound to immobilized protein A. Furthermore, [125I]rIL‐1α preincubated with serum co‐precipitated with a specific rabbit anti‐human IgG antibody. Screening of 29 sera from normal individuals showed similar effects in three cases. We conclude that approximately 10% of normal human sera contains detectable IgG autoantibodies to IL‐1α.
Functional Heterogeneity in Allospecific Cytotoxic T Lymphocyte Clones
Authors: CLAESSON M. H. , MILLER R. G.
Published: June 2006

Two veto active allospecific cytotoxic T‐cell clones (CTL), 4A2 and 4B3, were studied for the development of autoreactivity. The 4A2 CTL became autoreactive within 6 days when cultured without appropriate allospecific stimulator cells, whereas the 4B3 CTL retained its alloreactivity without becoming autoreactive when cultured under these conditions. Simultaneously with the development of autoreactivity, the 4A2 CTL lost its veto activity. Limiting dilution cultures of normal allospecific 4A2 CTL cells and autoreactive 4A2 CTL cells and mixtures of both showed that the former CTL cells were capable of inhibiting clonal growth of the autoreactive CTL cells in a dose‐dependent manner. We suggest that this growth inhibition reflects veto activity, i.e. cells capable of growing are inhibited by autorecognition of veto active allospecific 4A2 CTL cells. Taken together, the present results indicate that the existence of intraclonal down‐regulation of potentially autoreactive cells is a normal feature of allospecific cytotoxic T‐cell clones.