bluuebunny/crossref_metadata_2025 huggingface dataset:
The Combination of Major Histocompatibility Complex (MHC) and Non‐MHC Genes Influences Murine Lymphocytic Choriomeningitis Virus Pathogenesis
Authors: EYLER Y. L. , PFAU C. J. , BROOMHALL K. S. , THOMSEN A. R.
Published: June 2006

Resistance to the acute lethal disease caused by the docile strain of lymphocytic choriomeningitis (LCM) virus varies widely between different mouse strains. In order to study the inheritance of host influence on susceptibility to this strain of LCM virus, we crossed the F1 to the parent with the recessive disease phenotype. In all cases, susceptibility was dominant. In backcross progeny obtained from matings of parental strains differing in both major histocompatibility complex (MHC) and non‐MHC (SWR; C3H), 90% of the challenged mice died, indicating that at least three loci controlled susceptibility to the disease. When the parental strains carried similar MHC haplotypes but dissimilar background genes (B10.BR; CBA), 78% of the backcross mice succumbed, indicating that at least two non‐MHC loci influenced disease susceptibility. It is unlikely, however, that the same two non‐MHC loci are critical in all genetic combinations, since F1 produced from two H‐2 identical, resistant strains (B10.BR; C3H) were found to be fully susceptible. When congenic mice, differing only in the D‐end of the MHC region, were analysed, 50% of the backcross animals died, indicating that one gene in the MHC region was important; segregation analysis comparing MHC serotype and disease outcome indicated the H‐2D locus itself as the determining factor.
T‐Cell Recognition of Measles Virus Haemagglutinin Studied in a Mouse Model
Authors: MÄKELÄ M. J. , SMITH R. H. , LUND G. A. , SALMI A. A.
Published: June 2006

BALB/c mice were pretreated with cyclophosphamide and immunized 2 days later with inactivated, purified measles virus (MV) mixed with dimethyl dioctadecyl ammonium bromide (DDA). Seven days later, lymph nodes (LN) were removed and lymphocytes cultured in the presence of purified MV antigens. MV haemagglutinin (H) was found to be a major antigen responsible for proliferation of the lymphocytes Incorporation of purified H into liposomes significantly enhanced the proliferative response compared with purified H alone. Response to MV nucleocapsid protein was only moderate, and insertion of this protein into liposomes did not improve the response. As un attempt to analyse T‐cell epitopes of MVH, three synthetic peptides previously found to elicit a strong antibody response were used both as priming and stimulating antigens. None of the peptides was able to elicit a secondary response when MV‐primed LN cells were stimulated in vitro. However, each peptide primed T cells for a secondary challenge with purified, inactivated MV, which was demonstrated by proliferation and a delayed‐type hypersensitivity assay and also by transfer experiments with peptide‐primed cells.
A Functional Study of Purified CD4+ and CD8+ Cells Isolated from Synovial Fluid of Patients with Rheumatoid Arthritis and Other Arthritides
Authors: HOVDENES J. , GAUDERNACK G. , KVIEN T. K. , EGELAND T. , MELLBYE O. J.
Published: June 2006

The purpose of this investigation was to study purified synovial fluid (SF) CD4+ and CD8+ cells from patients with rheumatoid arthritis (RA) and other inflammatory joint diseases (non‐RA) with respect to the proliferative response to mitogens and recombinant interleukin 2 (rIL‐2). Highly purified cell subsets were isolated by an immunomagnetic technique, and spontaneous proliferation as well as proliferative responses to rIL‐2 and a combination of phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) (to substitute for accessory cells) were measured. Some patients had SF CD4+ and/or CD8+ cells with moderately increased spontaneous proliferation, but only the CD4+ cells of the two patient groups differed significantly from the peripheral blood (PB) T‐cell subsets of healthy individuals who served as controls. The response to rIL‐2 was variable but generally low, although about 50% of the CD4+ and 20% of the SF CD8+ cells of both patient groups expressed the Tac antigen. The response to PHA/PMA was significantly lower for RA SF CD4+ cells than for non‐RA SF CD4+ cells, which again was lower than for normal PB CD4+ cells. SF CD8+ response to PMA/PHA by both groups of patients was somewhat decreased, but not significantly lower than in the controls. Thus, the CD4+ cells seemed functionally more deviant than the CD8+ cells in both patient groups, but the abnormality was most pronounced in the RA group. The results demonstrate that the previously reported diminished response to mitogens by SF mononuclear cells is present even when SF CD4+ cells are cultured alone. This indicates that these T cells have a reduced response, probably because of prior activation.
Characterization of the Blood Mononuclear Leucocytes Producing Alpha Interferon after Stimulation with Herpes Simplex Virus in Vitro, by Means of Combined Immunohistochemical Staining and in Situ RNA‐RNA Hybridization
Authors: SANDBERG K. , GOBL A. E. , FUNA K. , ALM G. V.
Published: June 2006

A procedure is described for combined immunohistochemical staining and in situ RNA‐RNA hybridization of human peripheral blood mononuclear leucocytes (PBMC). These cells were first stimulated in vitro by herpes simplex virus (HSV)‐infected fibroblasts and after 6 h fixed and stained with a panel of antibodies against differentiation antigens. Alpha interferon (IFN‐α)‐producing cells (IPC) were identified by in situ hybridization by means of a 35S‐labelled IFN‐α2 cRNA probe. The IPC were infrequent, one in 200–5000 PBMC, but heavily labelled with the cRNA probe. They lacked antigens typical of T and B lymphocytes, and were also essentially negative for the Leu‐M5 antigen, present on a majority of monocytes. However, 50% of IPC expressed OKM5 antigens, corresponding to the thrombospondin receptor. The IPC lacked the antigens present on null lymphocytes detected by OKT16, but most of them expressed HLA‐DR, ‐DP and ‐DQ antigens. The IPC may represent a small subpopulation in the monocyte/macrophage lineage, resembling cells described as antigen presenting and stimulators of autologous mixed lymphocyte reactions. Alternatively, they constitute a subpopulation among the null lymphocytes.
Human CD4‐8‐ ‐Derived Clones Phenotypic and Functional Characteristics and Variation between Donors in Patterns of T‐Cell Receptor y Gene Rearrangements
Authors: CHRISTMAS S. E.
Published: June 2006

Clones were derived from highly purified human CD4‐8‐ lymphocytes from three different donors and maintained in the presence of interleukin 2 and phytohaemagglutinin. Considerable variation was noted between donors in the phenotype and T‐cell receptor (TCR) γ gene rearrangements of CD4‐8‐ ‐derived clones. In one donor, most clones remained CD4‐8‐ and all were CD3+WT31‐ and therefore expressed γ/δ heterodimers. TCR γ gene rearrangements almost all involved Cγ1. In contrast, most clones from a second donor were CD3+WT31‐, and therefore expressed α/β heterodimers, and many were positive for CD4 or CD8. Most clones from a third donor were CD3+ WT31‐ with a high proportion of TCRγ gene rearrangements involving Cγ2. The Vγ9JP rearrangement was exclusively confined to CD3+WT31‐ clones and was present in the majority of clones. Almost all CD3+WT31‐ clones showed TCR β as well as γ gene rearrangements. Most CD3+WT31‐ clones with at least one chromosome rearranged to Cγ1 exhibited high non‐major histocompatibility complex (MHC)‐restricted cytotoxic activity, while most of those with two Cγ2 rearrangements, and therefore expressing a non‐disulphide‐linked γ/δ heterodimer, had low activity. Preincubation of effector cells with anti‐CD3 strongly inhibited the cytotoxicity of CD3+WT31‐ clones while that of CD3+WT31‐ clones was enhanced. This implicates the CD3‐γ/δ complex in target cell recognition by cytotoxic γ/δ‐bearing T‐cell clones. The results show that there is heterogeneity between donors in the relative proportions of CD4‐8‐ ‐derived clones expressing α/β heterodimers and the different forms of the γ/δ heterodimer.
Specificity of γδ Receptor‐Bearing Cytotoxic T Lymphocytes Isolated from Human Peripheral Blood
Authors: BOSNES V. , HALVORSEN R. , SKAFTADOTTIR I. , GAUDERNACK G. , THORSBY E.
Published: June 2006

T‐cell receptor (TcR)‐γδ‐bearing lymphocytes were isolated from the peripheral blood of two healthy donors by immunomagnetic separation and subsequent cultured. The cell lines generated showed two distinct patterns of cytotoxicity. One TcR‐γδ+ cell line (HG.D) lysed K562 and U937 target cells, three TcR‐γδ+ cell lines lysed Daudi cells, and one TcR‐γδ+ cell line showed a shift from the former to the latter specificity during culture Cold target inhibition experiments showed that the HG.D effector cells which were cytotoxic against 119.17 cells also lysed K562 cells. The cytotoxicity against Daudi cells was strongly inhibited by monoclonal antibodies (MoAb) against the CD3 complex, whereas the cytotoxicity of the HG.D cell line against K562 and U937 was unaffected by such antibodies. The cytotoxicity against Daudi cells was also strongly inhibited in the presence of anti‐TcR‐γδ MoAb. However, in two of the Daudi‐specific cell lines, strong cytotoxicity against K562 cells was induced by Anti‐TcR‐γδ MoAb. Anti‐LFA‐1 MoAb caused only a partial inhibition of cytotoxicity, while anli‐CD2and anti‐TcR‐αβ MoAb were found to have no effect. The results indicate that human γδ receptor‐bearing T cells demonstrate a certain degree of target cell specificity, and that recognition of some target cells may be mediated through the TcR‐γδ.
Natural Killer Cells and Cytotoxic T Cells Induce DNA Fragmentation in Both Human and Murine Target Cells in Vitro
Authors: LIU Y. , MÜLLBACHER A. , WARING P.
Published: June 2006

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC‐1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC‐1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.
Increased Expression of Leucocyte Adherence‐Related Glycoproteins by Polymorphonuclear Leucocytes during Phagocytosis of Staphylococci on an Endothelial Surface
Authors: VANDENBROUCKE‐GRAULS C. M. J. E. , THIJSSEN H. M. W. M. , TETTEROO P. A. T. , ROZEMULLER E. , VERHOEF J.
Published: June 2006

Phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN) on the surface of endothelial cells is accompanied by adherence of the PMN to the endothelial surface and detachment of the endothelial cells from the culture monolayer. We studied the role of the leucocyte adherence‐related glycoproteins (Leu‐CAM: Mol/LFA‐1/150,95 or CDI 1a‐c CD18 complex) in these processes. Phagocytosis of S. aureus induced increased expression of the common β chain (CD18) of Leu‐CAM as demonstrated by flow cytometric analysis of PMN treated with a monoclonal antibody (MoAb) (CLB‐LFA‐1/1) directed against CD 18 and fluorescein isothiocyanate (FITC)‐conjugated anti‐MoAb. This same MoAb also inhibited the increased adherence of the PMN to the endothelial cells which occurs during phagocytosis. Blocking of adherence during phagocytosis with MoAb CLT‐LFA‐1/1 had no effect on the detaching activity of the PMN on the endothelial cells. We conclude that adherence of PMN to endothelial cells during phagocytosis of S. aureus is mediated by the Leu‐CAM complex. Adherence through the Leu‐CAM, however, is not necessary for endothelial damage by the phagocytosing PMN.
The Influence of C3‐Coated Homologous Erythrocytes on Pokeweed‐Mitogen‐Induced Polyclonal Differentiation of Human B Cells
Authors: RASMUSSEN J. MØLLER , SVEHAG S.‐E.
Published: June 2006

The present study was undertaken in order to test the hypothesis that homologous erythrocytes (E) coated in vivo with C3d could modulate the immunoglobulin (Ig) synthesis of human peripheral blood mononuclear cells (PBMC), stimulated with pokeweed mitogen (PWM) in vitro. E from healthy individuals were found to enhance markedly the Ig synthesis of PBMC cultures stimulated with suboptimal doses (0.01 μg/ml) of PWM. E coated in vivo with increasing amounts of C3d (1.4–6.3 times the amounts on normal E), obtained from patients with systemic lupus erythematosus, failed to induce any significant increase in Ig synthesis of PBMC cultures stimulated with suboptimal PWM doses, compared with cultures co‐stimulated in parallel with normal E. In contrast, an increase in IgM and IgG synthesis was observed in about 50% of PBMC cultures from different donors when stimulated with PWM in the presence of L‐ touted with Ohm vivo (from a patient with congenital factor I deficiency), compared with the Ig synthesis in cultures co‐stimulated in parallel with normal E. In contrast to the inability of C3d‐coated E to modulate B‐cell proliferation, the monoclonal anti‐CR2 antibody OKB7 was found to be mitogenic for unstimulated peripheral B cells.
A Rapid Separation Procedure to Analyse Phosphorylation of Two Putative Protein Kinase C Substrates in Intact Lymphocytes
Authors: BRATTSAND G. , FRIEDRICH B. , GULLBERG M.
Published: June 2006

Activation of protein kinase C (PKC) has been implicated in the regulation of many biological processes including the regulation of T and B lymphocyte responses. A direct means of studying activation of this kinase is to analyse specific phosphorylation events of cellular substrates, in the present report we describe a fractionation method that allows quantitative analysis of phosphorylation of two distinct PKC substrates using normal human T cells and T‐cell tumour lines. This method, which selectively visualizes PKC‐dependent phosphorylation of both an 80‐and a 19‐kDa substrate, involves three simple fractionation steps and allows a large number of samples to be analysed simultaneously. Since specific antibodies to these cellular substrates are not commonly available, the present method provides an alternative approach which makes it feasible to UK phosphorylation of the 19‐ and 80‐kDa proteins as a sensitive marker for PKC activation. Finally, in a cellular system where PKC‐mediated phosphorylation of these substrates can be studied without prior purification, the present method results in greatly improved resolution of these phosphorylation events.
Induction of High‐Affinity Interleukin 2 Receptors on Human T Lymphocytes
Authors: LARSEN C. S. , CHRISTIANSEN N. O.
Published: June 2006

The relationship between free cytoplasmic calcium, activation of protein kinase C (PKC) and expression of high‐affinity interleukin 2 receptors (HA‐IL‐2R) on human T lymphocytes was studied. Induction of HA‐IL‐2R by phytohaemagglutinin (PHA) was associated with an increase in free cytoplasmic calcium and a transient increase in membrane‐associated PKC. However, whereas addition of EGTA inhibited induction of receptors by PHA, addition of the PKC‐inhibitor H7 did not. 12‐o‐tetradecanoyl‐phorbol‐13‐acetate (PMA) and 1‐oleoyl‐2‐acetyl‐racglycerol (OAG) were both found to activate and translocate PKC. However, only PMA induced expression of HA‐IL‐2R. Not surprisingly, the effect of PMA was independent of extracellular calcium, but was inhibited by H7. Furthermore, a correlation between the number of HA‐IL‐2R and free cytoplasmic calcium upon stimulation with ionomycin was observed. Associated with the rise in intracellular calcium, the ionophore caused a slight increase in membrane‐associat4ed PKC. Also, addition of 117 inhibited expression of HA‐IL‐2R. Finally, OAG and ionomycin acted synergistically on expression of HA‐IL‐2R. In conclusion, induction of HA‐IL‐2R requires at least two different signals and neither activation of PKC nor an increase in free cytoplasmic calcium is sufficient. However, these two signals may act synergistically. There is evidence for both a PKC‐ and calcium‐independent pathway.
Studies on Active Immunization with Self Antigens
Authors: MAHANA W. , GUILBERT B. , AVRAMEAS S.
Published: June 2006

Newborn BALB/c mice were repeatedly injected either with syngeneic (BALB/c) or xenogeneic (bovine) myosin, albumin, or actin in sterile physiological saline. The serum antibody response was evaluated by enzyme immunoassay 1 and 2 months alter birth and after two booster injections. At 1 month, higher antibody titres were found in the sera of mite injected with syngeneic than with xenogeneic antigens. At 2 months and after boosting, anti‐syngeneic actin antibodies were present in equal or higher amounts, anti‐syngeneic albumin antibodies were not detected, and anti‐syngeneic myosin antibodies were considerably decreased. Antibodies produced after booster injections of syngeneic actin were found to be highly specific and to belong mainly to the IgG isotype. These results suggest that newborn mice are better able than adult mice to respond to stimulation with self antigens, and that administration of self proteins during neonatal life may lead to the induction of immunological memory. They also indicate that one of the primary functions of the immune system in newborn mice is the recognition of self antigens.
Studies on Active Immunization with Self Antigens
Authors: MAHANA W. , GUILBERT B. , GONZALEZ R. , AVRAMEAS S.
Published: June 2006

BALB/c mice were injected during neonatal life with conjugates in buffered physiological saline, prepared by coupling trinitrophenyl groups (TNP) at various densities to either syngeneic mouse serum albumin (TNP MSA) or xenogeneic bovine serum albumin (TNP USA). Serum samples were obtained on days 30 and 60 after birth, mid on days 75 and 88 after two booster injections, and monoclonal antibodies were prepared from spleens of neonatally treated mice. The antibody titres, isotypes, and specificities were evaluated by enzyme‐immunoassay. It was found that the extent of the anti‐TNP immune response to TNP MSA conjugates depends on the degree of hapten substitution, which is not the case for the anti‐TNP BSA. All the TNP‐MSA conjugates induced mainly IgG and only a few IgM antibodies. These antibodies reacted essentially with the TNP group but seemed to have a higher avidity for the TNP protein conjugate used in their induction. During the course of the immunization, decreasing quantities of TNP‐MSA conjugates were needed to inhibit antibody binding. A large amount of monoclonal anti‐TNP antibodies was found in hybridomas obtained after neonatal treatment either with TNP‐MSA or TNP‐BSA. Therefore, it appears that the anti‐TNP‐immune response obtained after antigenic stimulation with sufficiently substituted TNP‐MSA conjugates possesses all the characteristics of a normally occurring humoral immune response.
Capping and Co‐Capping of Membrane Immunoglobulin and Lipid‐Conjugated Immunoglobulin Inserted in the Cell Membrane of B Lymphocytes
Authors: FARO‐RIVAS J. , CLINCHY B. , HÖIDÉN I. , MÖLLER G.
Published: June 2006

Human and mouse immunoglobulins (Ig) or F(ab′)2 fragments of rabbit Ig were conjugated to lipids and the conjugates inserted into the membrane of mouse spleen cells. It was found that nearly all B cells, but not T cells, became decorated with lipid Immunoglobulin. Both endogenous Ig receptors and inserted Ig capped after the addition of cross‐linking F(ab′)2 antibodies and in both cases capping required energy. Capping of endogenous mouse Ig led to co‐capping of inserted human Ig, but the reverse was not true.
Candida albicans: Mannan and Protein Activation of Cells from Various Human Lymphoid Organs
Authors: TOLLEMAR J. , RINGDEN O. , HOLMBERG K.
Published: June 2006

Challenging human lymphocytes with soluble cytoplasmic Candida albicans protein antigen (CP) and cell‐wall‐derived C. albicans mannan (CM) induced DNA synthesis and antibody secretion measured as plaque‐forming cells (PFC) in an indirect haemolytic assay. Enriched T and B lymphocytes responded poorly to CP and CM However, when adherent cells were added. T and B cells responded well to CM, but not to CP. Peripheral blood lymphocytes responded more strongly to CP than to CM, with peak stimulation on day 7. Bone marrow cells displayed similar DNA synthetic responses as blood lymphocytes, but peak Stimulation occurred on days 4 and 5 for CP and CM, respectively. Cells from the various parenchymal organs, including adenoid, tonsil, and spleen, showed peak DNA synthesis ranging from days 3 to 5 with a tendency for CM to produce an earlier response than CP. In contrast to that observed in blood and bone marrow cells. CM induced a higher DNA synthesis in cells from parenchymal organs than CP, which gave low stimulations. PFC, like DNA synthesis, in blood and bone marrow cells responded best to CP, whereas spleen cells showed the highest response to CM. In blood and bone marrow cells, only IgG PFC were induced, but in spleen cells IgA and IgM PFC were also stimulated by CM. Fetal liver cells responded poorly to these two Candida preparations. CM but not CP stimulated DNA synthesis in umbilical cord blood lymphocytes and IgG, IgA, and IgM in spleen cells, suggesting that CM is a polyclonal activator and that CP acts as an antigen.
Characterization of PPD‐Specific T‐Cell Lines Generated in Type I (Insulin‐Dependent) Diabetic and Healthy Individuals
Authors: Mølvig J. , MCDEVITT G. SØNDERSTRUP , THOMSEN M. , BOSMANN B. , BLANGERO C. , DE PREVAL C. , BAEK L. , NERUP J.
Published: June 2006

The particular susceptibility to insulin‐dependent diabetes mellitus (IDDM) conferred by HLA‐DR3,4 heterozygosity has been suggested to be an effect of transcomplementation of HLA class II molecules. To test this hypothesis of special IDDM‐specific hybrid determinants and to evaluate the T‐cell repertoire towards a specific antigen in IDDM patients we generated a total of 352 PPD‐specific T‐cell lines by the soft‐agar cloning technique and studied their restriction by HLA class II molecules. Of these lines, 227 were from nine IDDM patients, of whom six were DR3,4 heterozygotes, and 125 from 10 healthy controls Forty‐six T‐cell lines elicited specific responses in at least two experiments and in addition to T‐cell lines demonstration class‐II‐restricted PPD specificity, lines with an alloreactivity occurred. HLA‐DQ‐restricted PPD‐specific T‐cell line were not identified and a possible DP restriction (DPw2) was only observed with one line. These data indicate that PPD is preferentially presented to T cells in the context of HLA‐DR/Dw, Presentation of PPD by hybrid molecules in IDDM patients or by IDDM‐specific class II epitopes recognized by the T‐cell lines was not demonstrated. By restriction fragment length polymorphism analysis using a probe for the joining region of the T‐cell receptor gamma gene, T‐cell lines generated by the soft‐agar cloning technique were found to be oligoclonal. It is concluded that soft‐agar cloning should be followed by subsequent limiting dilution in order to assure monoclonality. Different preparations of antigen‐presenting cells (APC) were tested. In several cases the T‐cell lines were not able to respond to PPD presenting by Epstein ‐Barr‐virus transformed lymphoblastoid cell lines (LCL). It was demonstrated that lipopolysaccharides (LPS) of E. coli potently reduce the proliferative response of antigen‐specific and alloreactive T cells when T‐cell‐depleted peripheral blood mononuclear cells (E− cells) were used as APC, whereas only limited inhibition was observed when LCL were used as APC in the presence of LPS. This effect of LPS is suggested to be mediated by increased prostaglandin secretion by monocytes among the E− cells since indomethacin abolished the effect of LPS. This observation may have implications for T cell cloning procedures since we have found that most commercially available culture media are heavily contaminated with endotoxin.
Cyclosporine A Prevents the Generation of Single Positive (Lyt2+ L3T4−, Lyt2− L3T4+) Mature T Cells, but not Single Positive (Lyt2+ T3−) Immature Thymocytes, in Newborn Mice
Authors: HEEG K. , BENDIGS S. , WAGNER H
Published: June 2006

The influence of cyclosporine A (CsA) on T‐cell maturation was investigated in newborn mice. CsA treatment during the pre‐and postnatal periods resulted in a hypoplasia of peripheral lymphatic organs, absence of mature T3+ T cells in lymph nodes and spleens; no functional T‐cell reactivity was observed. In thymuses of CsA‐treated mice, no T3+ single positive Lyt2+ or T3+ L3T4+ thymocytes could be found, but double positive (DP) cells were readily detected. A thymocyte subset with the phenotype Lyt2+ L3T4− T3− was still discernible; this population was non‐functional in vitro. The data show that the maturation of single positive (SP) T cells is critically influenced by CsA; under the conditions used here we found no evidence that ‘leaky’ autoreactive SP T cells develop in CsA‐treated newborn mice.
Hereditary, Complete Deficiency of Complement Factor H Associated with Recurrent Meningococcal Disease
Authors: NIELSEN H. E. , CHRISTENSEN K. C. , KOCH C. , THOMSEN B. S. , HEEGAARD N. H. H. , TRANUM‐JENSEN J.
Published: June 2006

Complement factor H (β‐1H globulin) is an important regulatory protein which inhibits the spontaneous complement activation via the alternative pathway. We describe a 15‐year‐old girl without any detectable factor H in plasma. She has had two episodes of meningococcal disease, but is otherwise completely healthy. Secondary to the factor‐H deficiency, the levels of factor B, properdin, C3, and C5‐C9 were strongly reduced due to spontaneous in vivo activation of the alternative complement pathway. Plasma C3dg was strongly elevated in spite of the Factor‐H deficiency; apparently erythrocyte CR1 substitutes for factor H in C3 degradation. Neither C3 nor complement lesions were demonstrable on her erythrocytes which did, however, show increased, spontaneous haemolysis in vitro in citrate plasma, but not in serum. The patient is a single child and her parents, who are unrelated and healthy, had half‐normal levels of factor H. This reduction of factor H is sufficient to cause increased, spontaneous activation of the alternative pathway.
The T‐Lymphocyte Proliferative Response to Synthetic Peptide Antigens of Defined Secondary Structure
Authors: MOURITSEN S. , MELDAL M. , RUBIN B. , HOLM A. , WERDELIN O.
Published: June 2006

Immunodominant sites in proteins recognized by T lymphocytes are segments consisting of at least 7–8 amino acids. It has previously been proposed that these sties in proteins are α‐helical and amphipatic structures. We synthesized and investigated the immunogenicity of three synthetic peptides (MP7, MP8, and MP9), each consisting of the same 15 amino acids, but differing with respect to sequence. Based on information analysis and circular dichroism measurements, MP7 was shown to have an α‐helical secondary structure and, based on previously assigned hydrophilicity indices, was also strongly longitudinally amphipatic. MP8 also was conformed as an α‐helix, but was amphipatic in the sense that the N‐terminal half of the molecule was hydrophilic and the C‐terminal half hydrophobic. MP9 had neither an amphipatic nor α‐helical structure. All three peptides were immunogenic in some strains of mice but none was immunogenic in all strains. This supports other studies concluding that amphipaticity per se is neither a necessary nor sufficient requirement for immunogenicity of a peptide. On the other hand, the present experimental data suggest that longitudinally amphipatic α‐helical peptides may function better as T‐cell determinants than the other peptides investigated.
The Stimulation of T Cells with Anti‐CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B‐Cell Responses but does not Enhance the Activation of Antigen‐Specific B Cells
Authors: TRIFILETTI R. , HYMAN B. , LaVIA M. , KNAPP W. , VIRELLA G.
Published: June 2006

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function‐associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)‐stimulated PBMC results in ihe enhancement of polyclonal immunoglobulin synthesis and of antigen‐specific B‐cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti‐CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented wilh interleukin 2 (IL‐2). Specific responses to telanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM‐stimulated PBMC. Significant anti‐TT responses were observed in cultures stimulated with E+TT+IL‐2. In contrast, stimulation of PBMC wilh VIT13+Leu5b+IL‐2+antigen was not effective in inducing anli‐TT antibody and only weakly effective in inducing anti‐KLH antibodies. Replacing Leu5b by anti‐CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti‐TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B‐cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.
Accumulation of CD8+ Cells after Immunization with Soluble Antigen
Authors: NILSSON G. , MATSSON P. , AHLSTEDT S.
Published: June 2006

Rats were immunized with ovalbumin, either subculaneously or by aerosol inhalation. The lymphocyte distribution in lymph nodes, peripheral blood, and spleen was investigated by flow cytometry after labelling with T pan (0X19 and W3/13), T helper lymphocytes (W3/25), T cytotoxic/suppressor lymphocytes (OX8), k light chain (MAR 18‐5), or MHC class II (0X6) monoclonal antibodies. The influence of the neurotoxic agent capsaicin on the lymphocyte distribution was also analysed. Subcutaneous immunization resulted in an increased number of OX8+ cells in mesenteric lymph nodes, spleen, and peripheral blood but not in the draining lymph nodes, axillary, brachial, and mediastinal lymph nodes. The number of positive cells for the other cell markers used were not affected by immunization. The neuromodulatory effect of capsaicin had no effect on the lymphocyte distribution. The results showed that the type of immunization used, low amounts of antigen without adjuvant given during a prolonged period, selectively induced OX8+ cells. The patterns were unaffected by neuromodulation using capsaicin.
Characterization of Murine Liver‐Derived Inhibitory Protein
Authors: WANG S. R. , HUANG M. H. , CHANG K. L. , YU C. L. , CHIANG B. N. , HAN S. H.
Published: June 2006

Murine liver‐derived inhibitory protein (LIP) capable of inhibiting human lymphocyte proliferation was highly purified from liver extract. Its molecular weight determined by gel filtration and SDS‐PAGE was 105,000 and 38,400 respectively. LIP moved electrophoretically at the gammaglobulin region. Its activity in inhibiting lymphocyte proliferation was temperature‐stable up to 60° C, and pH‐stable between 4 and 11. It was not cytotoxic to lymphocytes as shown in 51 Crrelease experiments. The purified LIP possessed arginase activity.
Tumour Necrosis Factor in Hepatosplenic Schistosomiasis
Authors: ZWINGENBERGER K. , IRSCHICK E. , SIQUEIRA J. G. VERGETTI , DACAL A. R. CORREIA , FELDMEIER H.
Published: June 2006

Periportal fibroplasia is the dominating feature of hepatosplenic schistosomiasis. Since monokines play an Important role in the regulation of fibroplasia, tumour necrosis factor (TNF) and interleukin 1β (IL‐1β) were assessed in sera and cell culture supernatants from patients with intestinal and hepatosplenic schistosomiasis before and 3‐6 months after treatment with praziquantel. Uninfected controls were from the study area in Alagoas, Brazil. TNF was measured using an L‐M mouse fibroblast bioassay and radioimmunoassays specific for TNF‐α. Whereas TNF‐α was elevated three fold in the patients' sera, three‐to five‐fold reductions of TNF were observed by radioimmunoassay and bioassay, respectively, in cell culture supernatants of hepatosplenic schistosomiasis patients. Significant deviations, in opposite directions, from TNF levels in control sera and supernatants are most plausible in the event of a sequestration of TNFα‐producing cells from the circulation. This process may be disease stage‐specific since a dichotomy between incipient and advanced cases of hepatosplenic schistosomiasis became apparent in the amplitude and kinetics of changes during the follow‐up after treatment.
Structural Analyses of Human Developmentally Regulated Vh3 Genes
Authors: CHEN P. P.
Published: June 2006

In mice, a restricted set of the Jh‐proximal Vh genes are preferentially expressed during early ontogeny. Recently, analyses of human Ig cDNA from a fetal liver revealed a restricted set of Vh genes which belong to the VhI, 3, 4, and 6 families. Although the Vh6 and some Vh5 genes are proximal to the Jh region, no Vh5 gene was found in the fetal liver, suggesting that the distance between the Jh genes and some early‐expressed Vh genes may not be the only factor responsible for Vh gene expression during early development. As an initial step in searching for other underlying mechanisms, we characterized two human germline Vh3 genes which belong to the developmentally restricted Vh repertoire, and found that they contain many enhancer‐like sequences which are identical, or highly homologous to, various transcriptional enhancer motifs. Hence, it is conceivable that, in addition to the established positional effects, cis regulatory elements may be important in the programmed expression of some Vh genes during early B‐lymphocyte development.
A New Technique Using an Aggregating Antibody Against Glycophorin‐A for Purging Ficoll‐Paque‐Separated Leucocytes of Contaminating Erythroid Lineage Cells
Authors: HELDRUP J.
Published: June 2006

Ficoll‐separated leucocytes used for phenotyping may be contaminated with erythroblasts or erythrocytes, which can cause problems in cytofluorography, especially if erythroid lineage cells outnumber leucocytes in the preparation. Different means have been described for removing erythroid cells from leucocyte preparations, such as BD‐FACS lysing solution, hypotonic shock and ammonium chloride, but none of these is entirely satisfactory. In my hands all these techniques damaged the leucocytes to a greater or lesser extent and did not adequately eliminate the erythroblasts, sometimes not even the reticulocytes and erythrocytes. To obtain pure leucocyte suspensions, I developed a new technique based on an aggregating antibody (α‐Gly‐A, 733/8F7, from BioCarb, Sweden) directed against glycophorin‐A which is found on the cell surface of erythroid lineage cells. Agglutinated erythroid cells could be removed by filtration or low‐speed centrifugation. With the new technique there was no loss or damage of the leucocytes.
Cross‐linking of Both Types of IgG Fc Receptors, FcγyRI and FcγyRII, Enhances Intracellular Free Ca2+ in the Monocytic Cell Line U937
Authors: VAN DE WINKEL J. G. J. , TAX W. J. M , JACOBS C. W. M. , HUIZINGA T. W. J. , WILLEMS P. H. G. M.
Published: June 2006

We have studied the effects of Fc receptor triggering on the free cytosolic Ca2+ concentration, [Ca2+]i, in U937. These cells express two types of IgG Fc receptors, FcγRI and FcγRII. Binding of several anti‐FcγRI and anti‐FcγRII mouse monoclonal antibodies (MoAb) to Quin2‐ or Indo‐I‐loaded U937 cells had no direct effect on [Ca2+]i. After addition of a bridging anti‐mouse Ig antibody however, transient increases in [Ca2+]i were observed for both types of FcγR. One of the anti‐FcγRII MoAb, CIKM5, was exceptional in that it could induce Ca2+ increases in U937 cells by itself. Studies with F(ab′)2 fragments of CIK M5 revealed that this MoAb simultaneously binds to FcγRII, via both its Fab and Fc fragments, which might induce cross‐linking of two FcγRII molecules. One anti‐FcγRI MoAb, 197, a mouse (m)IgG2a antibody directed to an epitope outside the IgG‐binding region, remarkably also caused an immediate increase in [Ca2+]i, but only when added to U937 precultured with gamma interferon (IFN‐γ). FcγRI can bind monomeric human IgG as well as mIgG2a, and cross‐linking of cytophilic Ig induced an increase in [Ca2+]i. Our results show that [Ca2+]i increases can be induced only after cross‐linking of FcγR, either via anti‐FcγR MoAb or via Fc‐FcR interactions. Furthermore, we show that FcγR cross‐linking results in activation of the Ca2+/phosphatidylinositol (PI) signal transduction pathway.
Activation of Human T Lymphocytes by Phorbol‐12,13‐Dibutyrate and Ionomycin
Authors: LARSEN C. S.
Published: June 2006

The calcium ionophore ionomycin and the phorbol ester phorbol‐12,13‐dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL‐2) production, interleukin 2 receptor expression, and T‐lymphocyte proliferation.

The proliferative response was inhibited by addition of a monoclonal antibody directed against the IL‐2R (Tac antigen) demonstrating that PDBu and ionomycin induce T‐cell growth through an IL‐2‐dependent autocrine pathway.

Sequential stimulation with PDBu and ionomycin failed to induce IL‐2 production, IL‐2R expression, and consequently proliferation of the T cells, indicating that T‐cell activation requires simultaneous activation of protein kinase C (PKC) and elevation of cytosolic calcium.

Exposure of T cells to both agents for different times resulted in IL‐2 production, IL‐2R expression, and proliferation in proportion to the duration of incubation with at least 4 h required for maximal T‐cell activation. Further, in the presence of PDBu maximal T‐cell activation was found to require stimulation with ionomycin for 4 h, indicating that a sustained increase in free cytoplasmic calcium of several hours' duration is essential for T‐cell activation. In contrast T cells incubated with ionomycin were induced to produce IL‐2 and express IL‐2Rs upon brief exposure to PDBu with a 2‐h incubation period being sufficient for maximal T‐cell activation. Thus transient activation of PKC seems to be sufficient for activation of the IL‐2 gene and IL‐2R gene. However, maximal T‐cell activation requires activation of PKC for at least 2 h.
Whole Cholera Toxin and B Subunit Act Synergistically as an Adjuvant for the Mucosal Immune Response of Mice to Keyhole Limpet Haemocyanin
Authors: WILSON A. D. , CLARKE C. J. , STOKES C. R.
Published: June 2006

Cholera toxin (CT) is a potent stimulator of IgA responses when adtninistered orally and has been shown to promote IgA responses to a second protein such as keyhole limpet haemocyanin (KLH) if this is fed simultaneously. ln this paper we show that whilst feeding 5 mg KLH with either 0.5 μg CT or 10 μg B subunit fails to stimulate a mucosal IgA response to KLH, feeding 0.5μg CT and 10 μg B subunit together with 5 mg KLH produces a local IgA anti‐KLH response as greal as that produced by 10 μg of whole CT. ln addition to stimulating IgA responses in the lamina propria, preliminary results indicate that cellular responses are also stimulated, as we have demonstrated KLH antigen‐driven proliferation of cells isolated from groups of mice fed either 10 μg CT + 5 mg KLH or O.5 μg CT + 10 μg CTB + 5 mg KLH but not mice fed KLH alone or with either 10 μg CTB or O.5 μg CT.

These results indicate that the mucosal adjuvant action of CT is due to a synergistic effect involving both the GMl binding of the B subunit and adenylate cyelase activation by the A subunit.
A New Surface Antigen on Human Lymphocytes Defined by the Murine Monoclonal Antibody IVF7
Authors: GRUNEWALD J. , JANSON C. H. , TEHRANI M. J. , MATSUO Y. , MELLSTEDT H. , WIGZELL H.
Published: June 2006

The murine monoclonal antibody (MoAb) IVF7 was produced against tumour cells from a patient with a CD 3+, CD4+, CD8− T‐cell chronic lymphatic leukaemia (T‐CLL). The MoAb IVF7 showed reactivity with subpopulations of normal peripheral blood lymphocytes (PBL), as well as with a few cell lines of haematopoietic origin. Thirty‐six percent of PBL were stained with IVF7. Analysing subpopulations, we found that 80% of NK cells, 25% of T cells, and 10–20% of B cells were positive. The myelomonocytic cell line KG‐1 was also stained. The molecular weight of the molecule was 40 kDa under reducing conditions. The antigen was found to be trypsinsensitive.

MoAb IVF7 could modulate the antigen from the cell surface. The antibody did not stimulate PBL to DNA synthesis, nor did it significantly lnfluence NK cell‐mediaied kilting.
Comparison of the Immunological Activity of Five Defined Antigens from Mycobacterium tuberculosis in Seven Inbred Guinea Pig Strains. The 38‐kDa Antigen is Immunodominant
Authors: HASLØV K. , ANDERSEN Å. B. , LJUNGQVIST L. , BENTZON M. WEIS
Published: June 2006

We have examined the immunological activity of five affinity‐purified protein antigens from Mycobacterium tuberculosis in seven inbred and one outbred guinea pig strains. The test systems were measurements of delayed‐type hypersensitivity (Dth) responses, lymphocyte stimulation assays (LS), and antibody response measurements. The results showed significant differences in the immunogenicity of the single‐protein antigens and, when the antigens were considered separately, highly significant guinea pig strain differences The outbred guinea pig strain behaved as a Dth high responder to all antigens studied. The order of magnitude of the Dth responses was not usually correlated with that of the corresponding antibody responses for the individual guinea pig strain‐antigen combinations. In particular, when compared with the other strains, strain 2 guinea pigs generally gave the lowest Dth, but the highest antibody responses.

A 38,000 molecular weight protein, possessing M. tuberculosis complex‐specific B‐cell determinants, appeared immunodominant in 5 out of 7 strains. Our Dlh data in the inbred strains further suggest the presence of an M. turberculosis‐specific‐ T‐cell epitope. A T‐cell line, 11D9, derived from the high‐responder guinea pig strain 13 reactive to this prolein, was shown to be able to confer a tuberculin‐like skin reaction in vivo. LS assays with recombinant 38‐kDa protein and truncated versions of the protein mapped the 1lD9‐defined T‐cell epitope to the middle parl of the molecule.
Signal Transduction during Platelet‐Activating Factor‐Induced Lymphocyte Binding to Endothelial Cells
Authors: RENKONEN R. , MATTILA P. , USTINOV J.
Published: June 2006

Platelet‐activating factor (PAF) is a lipid mediator of inflammation. PAF pretreatment of cultured endothelial cells leads to an increase in lymphocyte binding. We have analysed the intracellular signal transduction during this PAF‐induced effect. The protein kinase C activator, phorbol 12‐myristate, 13‐acetate, mimicked PAF in the binding assay. Concomitantly, the protein kinase C (PKC) inhibitor H7 down‐regulated the PAF‐induced binding to nearly control level. Also dibutyryl‐cAMP treatment of endothelial cells increased lymphocyte binding, but the protein kinase A inhibitor HA1004 did not alter the PAF‐induced binding. Furthermore, PAF did not increase the level of cytosolic cAMP in the endothelial cells. Other second messengers. cGMPandCa2+, had no effect on lymphocyte binding. These fmdings suggest that protein kinase C, but no other signal transduction pathway, is essential in the PAF‐induced lymphocyte binding to endothelial cells.
A Comparative Study on the Effects of rIL‐4, rIL‐2, rIFN‐γ, and rTNF‐α on Specific T‐Cell Non‐Responsiveness to Mycobacterial Antigens in Lepromatous Leprosy Patients in Vitro
Authors: OTTENHOFF T. H. M. , WONDIMU A. , REDDY N. N. B.
Published: June 2006

We have studied lepromatous leprosy (LL) as a human model disease for T‐cell non‐responsiveness to specific mycobacterial antigens and studied the effect of rlL‐4, rIL‐2, rlFN‐y and rTNF‐x thereon. T‐cell non‐responsiveness to Mycobacierium bovis bacillus Calmette‐Guerin (BCG) or purified protein derivative of M. tuberculosis (PPD) antigens could be overcome in 5 out of 8 non‐responder patients by rlL‐2 and in 2 out of 8 by rlL‐4. The ability of rIL‐4 to overcome BCG/PPD non‐responsiveness was strongly dose‐dependent. When rIL‐2 and rIL‐4 were added simultaneously, they seemed to synergize in their effect. T‐cell non‐responsiveness to M. leprae could be overcome only in 2 out of 18 non‐responders by rIL‐2 but not by rIL‐4 alone. The ability of rlL‐2 to overcome T‐cell non‐responsiveness to M. leprae antigens became particularly marked when the recombinant 65‐kDa heat shock antigen of M. leprae was used instead of whole bacilli. Exogenously added rIL‐4, and to a lesser extent rlL‐2, strongly enhanced existing T‐cell responses to BCG or M. leprae in the majority (8 out of 11) of responders. These findings may have implications for the in vivo manipulation of the immune response by recombinant lymphokines and vaccines.
Inhibition of Polyphosphoinositide Breakdown and c‐myc Induction Accompanying Inhibition of Human B‐Cell Activation by Two Monoclonal Antibodies Against the Leucocyte Common Antigen (CD45)
Authors: SMELAND E. B. , HOLTE H. , BLOMHOFF H. KIIL , ÅSHEIM H. C. , STOKKE T. , TORJESEN P. , FUNDERUD S.
Published: June 2006

In this report we show that the two monoclonal anti‐CD45 antibodies, EO‐1 and FN‐I26, potently inhibit G0 to G1 transition and S phase entry in human B cells stimulated with anti‐μ and low molecular weight B‐cell growth factor. Both antibodies were found to inhibit anti‐μ‐induced inositol phospholipid breakdown and c‐myc mRNA induction. In contrast, EO‐I and FN‐I26 only partially inhibited the early anti‐μ‐induced increase in cytoplasmic Ca2+ levels, both in normal and in Ca2+‐depleted medium. B‐cell activation provoked by 12‐O‐tetradecanoylphorbol 13‐acetate(TPA) was not inhibited by these antibodies, except when using high concentrations of EO‐1. In addition, both antibodies were found to inhibit G1 entry induced by the anti‐CD20 antibody IF5, which confers an activation of B cells without any detectable increase in [Ca2+]1 or in phospholipid metabolism. This indicates that alternative mechanisms in addition to the inhibition of polyphosphoinositide (PI) breakdown are involved in the inhibitory action of these antibodies.
Characterization by Monoclonal Antibody of a Highly Conserved Antigenic Determinant Expressed on Human Platelet Membranes and Intermediate Filament Type III
Authors: ALESSIO M. , BELLONE G. , FUNARO A. , DE MONTE L. B. , ROGGERO S. , PERUZZI L. , ZACCOLO M. , MOMO M. , CAPPA A. P. M. , MALAVASI F.
Published: June 2006

The murine monoclonal antibody (MoAb) CB21, raised after immunization with sonicated extracts of human platelets, has been shown to react with a line‐restricted surface molecule and also a cytoplasmic structure displaying no restriction in terms of lineage and species. The surface structure recognized by the CB2I MoAb is exclusively expressed on the surface membrane of human platelets, being undetectabie on other cells or lines so far tested. After permeabilizalion. The majority of the cells and lines tested with the CB21 MoAb displayed strong cytoplasmic reactivity with a constant typical filamentous distribution. Biochemical and morphological analyses showed that the cytoplasmic counterpart recognized by the CB2I MoAb is the intermediate filament type III.