bluuebunny/crossref_metadata_2025 huggingface dataset:
T‐Cell Responsiveness to LCMV Segregates as a Single Locus in Crosses between BALB/cA and C.B‐17 Mice. Evidence for Regulation by a Gene Outside the Igh Region
Authors: CHRISTENSEN J. P. , MARKER O. , THOMSEN A. R.
Published: July 2006

The course of systemic infection with lymphocytic choriomeningitis virus (LCMV) was studied in BALB/ cA and C.B‐17 mouse strains differing in the immunoglobulin heavy chain region (Igh). Susceptibility to intracerebral infection and the ability to clear the virus differed significantly between these presumably congenic strains, suggesting that a gene in the Igh region might influence the course of this infection. A difference in virus spread prior to appearance of the immune response could not explain the observed differences. On the other hand, the differences in course of infection correlated with a difference in virus‐specific T‐cell responsiveness measured in terms of virus‐specific cytotoxicity in vitro and delayed‐lype hypersensitivity invivo. Analysis of F1, BC1 and F2 progeny showed that differential T‐cell responsiveness was influenced hy a single gene or gene complex; however, no linkage was found between this locus and the Igh‐C region. Taken together, these results indicate that an additional, and previously unknown, genetic difference exists between these two mouse strains, and that the involved locus carries a gene which significantly affects T‐cell responsiveness to LCMV.
Preferential Damage to IgM Production by Ultraviolet B in the Cells of Patients with Bloom's Syndrome
Authors: OZAWA T. , KONDO N. , MOTOYOSHI F. , KATO Y. , ORII T.
Published: July 2006

In most patients with Bloom's syndrome (BS), selective IgM deficiency is commonly found. We examined proliferative responses by incorporation of [3H]‐thymidine and the production of immunoglobulin after ultraviolet B (UVB) irradiation in the cells of two patients with BS. With regard to the proliferative responses in peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM), the patients' PBMC were more sensitive to UVB irradiation than controls. Although the effect of UVB irradiation in lymphoblastoid cell lines (LCL) after 0 days of culture showed no difference between one patient and controls, the patient's LCL were more sensitive to UVB than the controls after 3 and 7 days of culture. These results suggest that the proliferative responses of the patient's LCL recovered later than those of controls. IgM production was the most sensitive to UVB in the patients' PBMC and LCL. IgG and IgA production in the patients' PBMC and LCL showed the same sensitivity as controls. From our results, it is suspected that the preferential damage to IgM production by UVB is connected with the selective IgM deficiency of BS.
Specific Activation of Human Peripheral Blood γ/δ+ T Lymphocytes by Sonicated Antigens of Mycobacterium tuberculosis: Role In Vitro in Killing Human Bladder Carcinoma Cell Lines
Authors: WANG M.‐H. , CHEN Y.‐Q. , GERCKEN J. , ERNST M. , BÖHLE A. , FLAD H.‐D. , ULMER A. J.
Published: July 2006

Tumour regression induced in cancer patients hy local instillation of Bacillus Calmette‐Guérin (BCG) into the hiadder has been considered to be mainly mediated by activated cellular immunity and inflammatory reactions. In the present study we investigated the cytotoxicity of T cells bearing γ/δ T‐cell receptors (γ/δ+ T cells) against bladder carcinoma cells in vitro. Long‐term cultured γ/δ+ T‐cell lines from peripheral blood lymphocytes of healthy donors were established by stimulation with sonicated cell wall‐associated antigens of Mycobacterium tuberculosis (SMA). These γ/δ+ T cells lack the natural killer (NK) markers CD16 and CD56, as determined by flow cytometry. The SMA‐specific γ/δ+ T cells exhibited profound cytotoxicity against two NK‐resistant bladder tumour cell lines as well as against NK‐sensitive tumour eells in a non‐major histocompatibility complex‐restricted manner. The pattern of tumour cells killed by γ/δ+ T cells differed significantly from those of NK cells and lymphokine‐activated killer LAK cells. Furthermore, we tested the effects of recombinant human cytokines. including interleukin (IL)‐l, IL‐2, IL‐4, IL‐6, interferon (IFN)‐γ and tumour necrosis factor (TNE), on γ/δ+ T‐cell‐medlated cytotoxicity. It was shown that the addition of recombinant TNF in co‐incubation could augment γ/δ+ T‐cell‐mediated killing of two bladder tumour cell lines, but not of cells of the erythroleukaemia eell line K562. Based on these results it was concluded that mycobacterial antigens could specifically activate resting γ/δ+ T cells. The cytotoxicity of γ/δ+ T cells against bladder tumour cells and its selective enhancement by TNF may bean important mechanism involved in bladder tumour regression induced by intravesical instillation of BCG.
Phenotypic and Molecular Characterization of Human Monoclonal TCRγ/δT‐Cell Lines from Jejunum and Colon of Healthy Individuals
Authors: ROBIJN R. J. , BLOEMENDAL H. , JAINANDUNSING S. , WIEGMAN L. J. J. M. , VANBERGE‐HENEGOUWEN G. P. , LOGTENBERG T. , KONINGSBERGER J. C.
Published: July 2006

We have perfortned a phenotypic and molecular analysis of monoclonal TCRγ/δ T‐cell lines derived from jejunal and colonic biopsies of healthy individuals. Flow cytometric analysis employing a panel of 24 monoclonal antibodies (MoAbs) demonstrated that intestinal TCRγ/δ intraepithelial lymphocytes (IEL) constitute a phenotypically heterogeneous population. Nucleotide sequence analysis of expressed TCRδ variable (V) regions revealed the dominant utilization of the Vδ2 and Dδ3 gene segments and frequent rearrangement of Jδ3. IEL Vδ regions displayed extensive junctional diversity as a result of N and P insertion and the utilization of Dδ3 in all three reading frames. The results demonstrate that intestinal TCRγ/δ T cells from healthy individuals constitute a phenotypically heterogeneous population expressing Vδ regions that differ from their systemic counterparts.
Staphylococcal Enterotoxin B Promotes Deletion and Functional Inactivation of CD4Vβ8‐Positive Cells in the Absence of CD8 T Cells
Authors: WILLIAMS O. , GONZALO J. A. , MARTÍNEZA C.
Published: July 2006

The staphylococcal enterotoxins stimulate discrete subsets of T cells depending on their expression of particular V genes. Among these, staphylococcal enterotoxin B (SEB) vigorously stimulates Vβ8+ cells. This stimulation results in proliferation of both CD4 + Vβ8 + and CDS + T cells and eventually to anergy and clonal deletion in the former subset. We have examined the possible role of CD8+ T cells in the response of CD4+ cells to SEB, by in vivo CD8+ T‐cell‐depletion. We found no qualitative difference in the responses of untreated and CD8+ T‐cell depleted mice to SEB; however, a small quantitative difference in deletion was observed. Thus it appears that on the whole the response of CD4 +Vβ8 + T cells to SEB is independent of CD8+ T‐cell effector function, although the latter may play a partial role.
Proteolysis Increases the Fc‐Mediated Binding of Murine IgG2b to Human EBV‐Transformed B Cells, but Decreases the Expression of FcγRII and FcγRII
Authors: HOLTROP S. , RIJKE‐SCHILDER G. P. M. , VAN DOORN N. E. M. , JACOBS C. W. M. , KOENE R. A. P. , TAX W. J. M.
Published: July 2006

We have previously described a polymorphic human Fc receptor for murine IgG2b (mIgG2b). This receptor was defined by the binding of (complexed) mIgG2b to monocyles and Epstein‐Barr virus (EBV)‐transformed B cells. Three per cent of normal individuals were high responders with respect to mIgG2b (mIgG2b‐HR), whereas the other individuals were low responders for mIgG2b (mlgG2b‐LR). In the present study we investigated the effect of proteolytic enzymes on the Fc‐mediated binding of mIgG2b to EBV‐B cells. Pronase, human leucocyte elastase and cathepsin G caused an inereased binding (in an EA‐rosetting assay) of mIgG2b to EBV‐B cells from mIgG2b‐HR, but not from mIgG2b‐LR. Simultaneous immunofluorescence studies demonstrated that these protcolytic enzymes strongly reduced the expression of FcγRII and FcγRII on these cells, whereas HLA class I or HLA class II molecules were not affected. These findings strongly suggest that binding of mIgG2b is not mediated by FcγRII or FcγRII We also studied the effect of proteolysis on mIgG2b‐HR EBV‐B cells from an HLA class II‐negative individual. In this case EA‐mIgG2b rosetting was decreased after proteolysis, suggesting that HLA class II molecules may have a role in protecting the binding site for mIgG2b against proteolytic destruction.
Expression of Human IL‐2 Receptor α‐ and β‐Chains using the Baculovirus Expression System
Authors: LINDQVIST C. , ENGBERG C. , OJALA P. , KARP M. , ÅKERMAN K. , OKER‐BLOM C.
Published: July 2006

The genes encoding the α‐ and β‐chains of the human interleukin‐2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding genes were inserted under the polyhedrin promoter of the Autographa california nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line during viral infection. The recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and ELISA techniques. At 36 h post infection the corresponding proteins were clearly detected using anti‐IL‐2 α‐ and β‐receptor‐specific antibodies. A large amount of the α‐chain was also found in the supernatant culture media at 72 h post infection and metabolic labelling with [35S]‐methionine indicated that it was proteolytieally cleaved into a 32 kDa soluble form. A similar soluble or secreted form of the β‐chain was. however, not observed. Both receptor proteins were expressed on the surface of the insect cells as determined by flow cytometry analysis. Studies performed with the different IL‐2 receptor forms (α‐ and β‐chains alone or in combination) in the presence or absence of rIL‐2 suggest that the receptor proteins when expressed in infected insect cells are non‐functional with respect to tyrosine phosphorylation.
E‐Selectin Involvement in In Vitro Adhesion of Blood Dendritic Cells to Human Umbilical Cord Endothelial Cells
Authors: SRINIVAS U. , LARSSON M. , LUNDBLAD A. , FORSUM U.
Published: July 2006

Peripheral blood dendritic cells (BDC) are potent antigen‐presenting lymphoid cells. In the present study, we have examined the in vitro adhesion of BDC to human umbilical cord venous endothelial cells (HUVEC) and studied the expression of CD molecules and oligosaccharide haptens on BDC and endothelial cells. Immunohistochemistry showed that BDC were strongly positive for antibodies against HLA‐DR, CD 11c, CD 18, CD44 and CD54, and moderately positive for anti‐CD 11a, CD31,CD43 and CD58. In addition, BDC were moderately positive for anti‐Sialyl Lewis a and strongly positive for anti‐Sialyl Lewis x and CD77 (Galα l 4Galβl‐ 4Glc) Non‐stimulated HUVEC were positive for anti‐CD29, CD31 and CD77. An in vitro adhesion assay showed that only a small percentage of radiolabelled BDC bound to non‐stimulated HUVEC (16.9 ± 5.9%, mean±SD). Stimulation of the HUVEC with IL‐1 for 4 h produced a significant increase (P<0.002) in the percentage of radiolabelled BDC that bound to HUVEC (42.3 ± 7.1%). Preincubation of HUVEC with antibodies against E‐selectin (10μg/ml) significantly inhibited (P<0.02) the binding of radiolabelled BDC to activated HUVEC (32.2±1.3%) whereas preincubution of BDC with antibodies against CD54, CD18, CD11b, CD11c and Sialyl Lewis x did not produce any significant inhibition. Preincubation of BDC with Sialyl Lewis a antibody and with isotype‐matched control antibodies did not affect the increased binding of BDC to IL‐l‐activated HUVEC. Thus, E‐selectin seems to be involved in adhesion of BDC to IL‐1‐stimulated HUVEC.
Expression of Secreted Immunoglobulin Heavy Chain Genes and Immunoglobulin‐Secreting Cells in Human Lymphocytes
Authors: KONDO N. , INOUE R. , AGATA H. , SUZUKI Y. , SHIMOZAWA N. , TOMATSU S. , NAKASHIMA Y. , YAMAGISHI A. , OGAWA T. , ORII T.
Published: July 2006

The numbers of immunoglobulin‐secreting cells in peripheral blood mononuclear cells and the expression of mRNA for secreted type of immunoglobulin heavy chains were investigated in healthy children, compared with the percentages of surface immunoglobulin‐bearing cells and the expression of mRNA for membrane‐bound type of immunoglobulin heavy chains, respectively. Although a difference between expression of μs mRNA and μm mRNA was unclear, μmRNA was well transcribed. The expression of γs mRNA or αs mRNA was markedly higher than that of γm mRNA or αm mRNA. However, although the detection methods could be of different sensitivities, the percentage of IgM‐, IgG‐, or IgA‐secreting cells was markedly low, compared with the percentage of surface IgM‐, IgG‐, or IgA‐bearing cells, respectively. Therefore, additional regulation of the pattern of the immunoglobulin gene expression may be exerted at the translational and post‐translational stages.
Imbalance of the CD4+ Subpopulations Expressing CD45RA and CD29 Antigens in the Peripheral Blood of Adults and Children with Down Syndrome
Authors: BARRENA M. J. , ECHANIZ P. , GARCIA‐SERRANO C. , CUADRADO E.
Published: July 2006

Peripheral blood lymphoid subsets expressing either CD45RA or CD29 antigens, were quantified in 30 children and 59 adults with Down Syndrome and appropriate age‐matched controls, by dual immunofluorescence and flow cytometry, Down's patients, both adults and children, displayed a significant decrease of CD4+CD45RA+ cells in comparison with the observed in their age‐matched controls, but no difference was found in the CD4+CD29+ subset. These results show clearly the imbalance of these subpopulations in the peripheral blood of individuals with Down syndrome that result in the inversion of the CD45RA/CD29 ratio, due to a major reduction of the CD45RA subset. No obvious diflerence was found in the CD45RA/CD29 ratio within the CD4 negative cells.

Abnormalities of these subpopulations could be indicative of early senescence of the immune system, since age‐related changes in Down's persons were in parallel with those observed in normal individuals and the proportion of both subpopulations were roughly similar in Down's children and normal adults.
Histocompatibility Complex Gene Products and Exposure to Oestrogen: Two Independent Disease Accelerating Factors in Murine Lupus
Authors: CARLSTEN H. , TARKOWSKI A.
Published: July 2006

A number of studies have demonstrated that oestrogen exerts a significant impact on the course of experimental autoimmune diseases. Exposure to oestrogen aggravates SLE glomerulonephritis whereas the opposite outcome has been demonstrated in experimental arthritis, vasculitis, thyroiditis, and sialadenitis. In this report we have analysed the respective impact of H‐2z linked gene products and long‐term treatment with physiological doses of oestradiol on clinical and immunological variables in castrated backcrosses of lupus prone NZB/W and NZB mice.

Our results demonstrate that H‐2z linked gene products accelerate B‐cell activation and stimulate autoantibody production resulting in aggravation of glomerulonephritis and precocious death in renal failure. These H‐2z linked gene products do not influence T‐cell mediated sialadenitis. Irrespectively of the H‐2 haplotype of the mice, administration of oestrogen resulted in intense polyclonal B cell activation and aggravation of glomerulonephritis. However, exposure to oestrogen resulted in amelioration of sialadenitis. Notably, our result indicates that B‐cell activation achieved by oestrogen and H‐2z gene linked products, respectively is mediated by independent mechanisms.

In addition, we have developed a predictive in vivo test that permits forecasts regarding efficiency of oestrogen treatment for suppression of T‐cell mediated lesions. Using this test procedure in young, clinically healthy SLE mice we have been able to prove that animals displaying suppressed delayed type hypersensitivity (DTH) after short‐term oestrogen exposure showed significantly lower long‐term morbidity regarding development of sialadenitis upon continuous treatment with physiological doses of oestradiol.
Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A
Authors: IBRAHIM S.
Published: July 2006

Protein A binds immunoglobulins and it has two target structures, one in Feγ (CH) and the other in selected VH regions. The protein has five homology regions (domains). A, B, C, D, and E. Fc‐binding and VH‐binding have been reported to be non‐competitive, suggesting that different domains are responsible for the binding of the two ligands. On the other hand, all five domains have been reported to bind Fc. I studied binding of different immunoglobulins by protein A or its domain B (rBB). The results show that separate domains bind VH and Fc. If all five domains are capable of binding Fc, the ones that bind Vh have low affinity for Fc. Furthermore, the number of Fc‐binding domains varies depending on the type of the IgG being bound. Human IgG1 or lgG2 or rabbit IgG (Fc) seem to be bound by several domains (possibly four), and domain B is one of them. Mouse IgG1 or lgG2b are bound by fewer domains not including B. Murine lgG2a is also bound by fewer domains but B is one of them.
The Influence of LFA 1 on Human T Cells Stimulated by Solid‐Phase Immobilized HLA Class II‐Peptide Complexes
Authors: VAN SCHOOTEN W. C. A. , QUAN J.
Published: July 2006

Purified HLA class II‐peptide complexes immobilized to a solid support induce proliferation of human T‐cell clones, indicating that human T‐cell clones can proliferate in the absence of secondary signals from accessory cells. We hypothesized that T cells can provide co‐stimulatory signals to T cells. LFA‐1 molecules play an important role in homotypic interactions of T cells and murine monoclonal antibodies reactive with LFA 1 can inhibit T cell T cell interactions. LFA‐1 reactive monoclonal antibodies inhibited cytolysis of peptide‐pulsed T cells by T cells and partially inhibited T‐cell proliferation. To study the direct effect of the LFA 1 molecule on T‐cell activation, we co‐immobilized HLA‐class II‐peptide complexes with LFA‐1 reactive MoAbs. Co‐immobilization resulted in an enhanced proliferative response of the T‐cell clones. This could indicate that the LFA 1 molecule on T cells is not a passive adhesion molecule, but is capable of transducing a signal that synergizes with the stimulatory signal via the T‐cell receptor.
Characterization of a Multimeric Polypeptide Complex on the Surface of Thymus‐Derived Cells in the Mexican Axolotl
Authors: KERFOURN F. , GUILLET F. , CHARLEMAGNE J. , TOURNEFIER A.
Published: July 2006

We previously raised a rabbit antiserum (L12) against a 38 kD polypeptide which is expressed on the surface of thymocytes and peripheral T cells of an Urodele Amphibian, the Mexican axolotl (Ambystoma mexicanum). Here we show that L12 antibodies immunoprecipitate several labelled molecules from surface iodinated axolotl spleen cells, including the 38 kD molecule, but also two polypeptides of 43 and 22 kD which are covalently linked to other elements. Another rabbit antiserum (L10) was raised against detergent‐solubilized axolotl thymocyte membranes and shown to recognize the majority of thymocytes and about half of the splenocytes in immunofluorescence. In Western blotting, L10 antibodies recognized a limited number of surface polypeptides in thymocyte and splenocyte lysates, including 43,38, and 22 kD elements. Immune complexes formed between L10 antibodies and solubilized splenocyte membranes were used to immunize BALB/c mice intrasplenically in the aim of raising MoAbs specific for axolotl Tcells. Monoclonal antibody 87.16 was shown to stain in immunofluorescence 26.7% of thymocytes and 26.8% of spleen cells. This MoAb recognized a 43 kD polypeptide that can covalently associate on the T‐cell surface with several other molecules to form a multimeric complex.
Regulation of MHC Class I Membrane Expression by β2‐Microglobulin
Authors: MOTAL U. M. ABDEL , ZHOU X. , SIDDIQI A. R. , JONDAL M.
Published: July 2006

MHC‐I binding peptides and β2 microglobulin (β2‐m) can upregulate the MHC‐I heavy chain expression on certain peptide transporter mutant cells. We have further studied this with normal cells and non‐mutant cell lines. No MHC‐I upregulation was seen with normal, resting or activated T cells. On mouse cell lines P815 and B16, both peptides and human β2‐m gave an additive upregulation response. With the human small cell lung carcinoma H82, an optimal HLA.A2 binding peptide (GILGFVFTL) gave an upregulation response, whereas β2‐m alone or in combination with this peptide had no effect. However, β2‐m potentiated the response of H82 cells to a slightly longer peptide. Using mutant RMA‐S cells, it was found that both Brefeldin A (BFA) and chloroquine, but not leupeptin, inhibited MHC‐I upregulation response to both peptide and β2‐m. In contrast to chloroquine, BFA also gave a reduction of background membrane MHC‐I expression, presumably due to a block in Golgi transport. Human β2‐m, which binds to RMA‐S cells, and which is known to internalize into endosomes, did not reappear on the cell surface. When Db on RMA‐S cells was upregulated by human ft‐m, the sensitivity of these cells to Db restricted CTL cells increased. Even if β2‐m did not upregulate the overall MHC‐I expression on normal cells, it may still quantitatively increase the expression of optimally presented peptides and endosomal recycling may be important in this process.
Antibodies to dsDNA are Produced During Primary BK Virus Infection in Man, Indicating that Anti‐dsDNA Antibodies may be Related to Virus Replication In Vivo
Authors: FREDRIKSEN K. , SKOGSHOLM A. , FLÆGSTAD T. , TRAAVIK T. , REKVIG O. P.
Published: July 2006

Experimental immunizations with both the Polyomavirus BK and with the isolated viral genomic dsDNA regularly induce antibodies with a relative affinity for BK virus dsDNA. In the present study we demonstrate that the anti‐dsDNA responses to BK virus in experimental animals also appear during natural BK virus infection in man. Fifty‐nine children were examined over time for serological signs of primary BK virus infection. Of eight children found to undergo primary infection with BK virus, anti‐BK dsDNA antibodies appeared in all. In 4 of the 8 patients the antibodies cross‐reacted significantly with mammalian dsDNA, and weak cross‐reactions were also noted in at least three other patients. The antibodies resembled those induced in the experimental model with regard to their relative affinity for BK dsDNA. In contrast, most, but not all, anti‐dsDNA antibodies from 10 SLE patients cross‐reacted extensively with dsDNA from viral and mammalian origin. Thus, a dsDNA virus like BK virus may provoke immunological intolerance to dsDNA, but, with qualities different from those produced during SLE. The present observations demonstrate that induction of anti‐dsDNA antibodies is not restricted to experimental immunization of animals, but does also take place in humans during naturally acquired BK virus infection. The relevance of this model for the spontaneous production of anti‐dsDNA antibodies is discussed.
Identification of a Vimentin‐Like Function Associated Molecule (FAM) on Rat NK Cells: Evidence for Receptor Function
Authors: EVANS D. L. , HARRIS D. T. , LEARY J. H. , JOHN A. L. ST , JASO‐FRTEDMANN L.
Published: July 2006

Monoclonal antibody (MoAb) 5C6 specifically binds to fish, rat and human NK cells and inhibits cytotoxicity. The molecule recognized by this MoAb is a 50–53–kDa membrane protein on rat leukaemic NK (CRC) cells. In the present study, we have obtained a partial internal amino acid sequence from a purified 42‐kDa fragment of the CRC‐function associated molecule (FAM). Three tryptic peptide fragments were sequenccd and each showed homology to intermediate filament vimentin sequences as deduced from (GenBank) mouse cDNA sequences. Amino acid composition analysis indicated that similar to cytoskeletal vimentin, the FAM contained a high percentage of non‐polar amino acids. To further assess the similarities between this protein and vimentin., two commercially available anti‐vimentin MoAbsand one anti‐vimentin polyclonal antibody were tested for binding and inhibition of NK cytotoxicity. All anti‐vimentin MoAbs inhibited killing by rat NWNA cells of appropriate targets. Anti‐vimentin MoAb 13.2 bound to 41% of NWNA cells compared with approximately 58% binding for MoAb 5C6. Capping and sequential binding experiments showed that MoAb 5C6 effectively removed, from CRC‐cell membranes, the protein recognized by MoAb V9. Sequential addition of these two MoAbs (MoAb 13.2 followed by MoAb V9) to CRC cells did not produce competitive binding.

Biochemical and Western blot analysis of the vimentin‐like protein obtained from CRC cells indicated that this protein has a molecular weight of 48–50 kDa, with an isoelectric point of pH 6.1–6.3. This protein is cross‐reactive by Western blot analysis with anti‐vimentin and anti‐intermediate filament (IFA) antigen MoAbs but not with anti‐desmin or anti‐actin M oAbs. The molecular weight heterogeneity (43 versus 48 50 k Da) of the CRC protein was also examined. Western blot analysis of the CRC extract after different in vitro incubation times at 37°C and 4°C demonstrated that the 50 53–kDa ‘native’ protein degraded to a 42‐kDa protein by 24 and 48 h respectively. This degradation was inhibitable by 10 mm EGTA.

Evidence is presented which indicates that a vimentin‐like protein on transformed rat NK cells may be an antigen binding receptor which initiates target cell lysis.
Binding of Leukaemic Blasts to Various Subpopulations of Lymphokine‐Activated Killer Cells
Authors: PALUCKA A. K. , PORWIT A. , REIZENSTEIN P.
Published: July 2006

Conjugate formation by natural killer (NK)‐resistant and N K‐sensitive leukaemic cell lines with fresh and IL‐2‐stimulated (lymphokine‐activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining, A significant difference in binding of NK‐resistant T‐cell lymphoma (HuT 78) and NK‐sensitive myeloid (K562) blasts to fresh PBL was observed (P<0.01). Activation of lymphocytes with IL‐2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK‐resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulalions of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK‐cell‐derived. but also with T‐cell‐derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56+ subpopulation.

Our results indicate that the resistance of leukaemic blasts to cell‐mediated cytotoxicity may depend on different (and probably several) steps of this process.
The Human Fc Receptor for Mouse IgG2b on Monocytes and EBV‐B Cells is Functionally Inhibited by Anti‐HLA Class II Antibodies
Authors: HOLTROP S. , RIJKE‐SCHILDER G. P. M. , KOENE R. A. P. , TAX W. J. M.
Published: July 2006

We have recently described a polymorphic Fc receptor for murine IgG2b (mIgG2b), present on human monocytes and EBV‐transformed B lymphocytes. The present study shows that anti‐HLA class II monoclonal antibody (MoAb) completely inhibits both the (Fc receptor‐dependent) T‐cell proliferation, induced by mIgG2b anti‐CD3 MoAb, and rosetting with mIgG2b‐sensitized erythrocytes. This inhibition is also observed with F(ab′)2 fragments of anti‐HLA class II MoAb, and is therefore not Fc mediated. The Fc receptor for mIgG2b is also present on EBV‐transformed B cells obtained from a patient with‘bare lymphocyte syndrome′, that completely lack HLA class II antigens. Therefore, the Fc receptor for mIgG2b and HLA class II antigens are not identical. Since the low affinity receptor for IgE (FcɛII; CD23) was reported to be associated at the cell surface with HLA class II antigens, we have compared both types of Fc receptor, and observed that human IgE strongly inhibits the mitogenic effect of murine IgE anti‐CD3 but not of mIgG2b anti‐CD3 MoAb. We conclude that the human Fc receptor for mIgG2b is strongly inhibited by anti‐HLA class II MoAb, but is not identical to HLA class II or FcɛRII.
Characterization of a Tryptase mRNA Expressed in the Human Basophil Cell Line KU812
Authors: BLOM T. , HELLMAN L.
Published: July 2006

The expression of a tryptic serine protease was detected in the cell line KU812 by Northern blot analysis with an oligonucleotide probe directed against a conserved region present in all of the five presently cloned human mast cell tryptases. PCR primers designed for the amplification of a nearly full‐length copy of tryptase mRNAs were used to study the identity of the KU812 tryptase. Ten clones were characterized and all were found to be identical to one of the tryptases previously cloned from a human skin cDNA library. This tryptase has been thought to originate from mast cells of the skin.

Two possible explanations may account for the observed identity between the presumed mast cell tryptase and the KU812 tryptase. Firstly, it is possible that the KU812 tryptase is a basophil‐specific tryptase which has previously been cloned from a human skin cDNA library containing low levels of cDNA copies derived from basophils in the starting material. Secondly, the KU812 cell line, and possibly normal basophils, express a tryptase which is identical to one of the tryptases expressed in normal skin mast cells. We cannot at present rule out any of the two possibilities, but we favour the second explanation as being the most likely.
Seasonal Variation and Sex Differences of Circulating Macrophages, Immunoglobulins and Lymphocytes in Healthy School Children
Authors: AFOKE A. O. , EEG‐OLOFSSON O. , HED J. , KJELLMAN N‐I. M. , LINDBLOM B. , LUDVIGSSON J.
Published: July 2006

Subpopulations of T and B lymphocytes and levels of serum immunoglobulins G, A, M, E and subclasses G1, G2 and G3 were studied in 45 healthy school children aged 8–16 years during four seasons of the year. There were significant increases in CD4+ T helper cells, total T lymphocytes and CD4+/CD8+ (helper/cytotoxic) T‐cell ratio during the spring season. While the levels of CD8+ T cells and total B lymphocytes remained statistically unchanged during all four seasons, the levels of natural (HNK‐1) killer cells and macrophages increased significantly during the autumn and summer seasons respectively. The levels of immunoglobulins G, A, M and E remained statistically unchanged during all four seasons. Girls had higher levels of CD4+ T cells and a higher CD4+/CD8+ T‐cell ratio than boys. Girls also had slightly higher levels of immunoglobulin G and M. These observations suggest that seasonal variations of some immunological parameters occur in healthy children. This may be an adaptive response to variable climatic and other environmental factors. These natural variations due to seasonal changes should be taken into account when immunological tests are used in clinical investigations.
Interleukin‐4 Bypass of the Immunosuppressive Effect Mediated by Interleukin‐2 Receptor Antibodies
Authors: STEINMANN J. , HERWARTZ C. , MÜLLER‐RUCHHOLTZ W.
Published: July 2006

Numerous studies have demonstrated that the generation of alloreactive effector cells depends on cytokines. Conversely, there is evidence that cytokine metabolism is altered at the clonal level in tolerant chimaeras. This has led to preclinical and clinical studies using antibodies that antagonize interleukin‐2 (IL‐2), with the hope of achieving immunosuppression and inducing tolerance. Monoclonal antibodies against the α‐chain (p55) of the human IL‐2 receptor are being applied to prevent transplant rejection and graft‐versus‐host disease in several clinical trials. The antibodies that have been applied clinically so far antagonize the binding of IL‐2 to the IL‐2 receptor α‐chain which is part of the high affinity IL‐2 receptor, but they do not deplete the receptor‐bearing cells. Our study investigates the immunosuppressive effect of monoclonal antibodies against the α‐chain (p55) and β‐chain (p75). In mixed lymphocyte cultures the p55 antibody causes a reduction in T‐cell proliferation to about 50%. The generation of cytotoxic T cells is reduced more effectively (up to 80%). By additional blocking of the IL‐2 receptor β‐chain we achieved an additional but still incomplete immunosuppressive effect. Moreover we show that IL‐2 receptor‐blocked alloreactive T cells escape suppression by using IL‐4 as an alternative stimulating signal. To prevent T lymphocytes benefiting from this alternative and thwarting the immunosuppressive effect, cytotoxic IL‐2 receptor antibodies that deplete the high affinity receptor‐bearing cells are needed.
Interferon‐γ Stimulation of Messenger RNA for Human Secretory Component (poly‐Ig Receptor) Depends on Continuous Intermediate Protein Synthesis
Authors: KRAJČI P. , TASKÉN K. , KVALE D. , BRANDTZAEG P.
Published: July 2006

Secretory component (SC or poly‐Ig receptor) plays a key role in mucosal external body fluids. The aim of this study was to elucidate the molecular events underlying IFN‐γ‐dependent up‐regulation of SC. Using a human SC cDNA clone isolated by our laboratory, we found that IFN‐γ up‐regulated SC mRNA levels in a time‐ and concentration‐dependent manner. Moreover, in situ hybridization showed a striking increase of SC mRNA‐positive HT‐29 cells after IFN‐γ treatment. Inhibition with 5,6‐dichloro‐l‐β‐ribofuranosyl‐benzimidazole (DRB) indicated a half‐life for IFN‐γ‐induced SC mRNA of approximately 1 h. Cycloheximide (CHX) abolished the IFN‐γ‐induced accumulation of SC mRNA in a reversible manner; the time‐course suggested that de novo synthesis of protein factor(s) with a turnover time shorter than 6 h was required for accumulation of SC message. IFN‐γ‐stimulated up‐regulation of SC expression therefore appears to depend on molecular events similar to those taking place for the activation of several other genes in the Ig supergene family.
Preferential Vδ1 Expression among TcR γ/δ ‐Bearing T Cells in Human Oral Epithelium
Authors: PEPIN L. F. , ROGER T. , MORISSET J. , SEMAN M.
Published: July 2006

The oral cavity is a septic area colonized by various bacterial species, and the oral mucosa is frequently submitted to microtraumas. Several mechanisms are implicated in the defence of the oral tissue, but little is known concerning the eventual presence and role of γ/δ T cells at this site.

Samples of healthy keratinized oral mucosa were examined with immunochemica! techniques using anti‐CD3, CD4, CD8, CD22, TcRδ1, Vδl and Vδ2 monoclonal antibodies. Whatever the site examined. γ/δ T cells represent at most 2% of the T‐cell population, a value similar to that found in other tissues. In the connective tissue, under the basement membrane, Vδ2+γ/δ T cells are predominant whereas the epithelium mostly contains Vδ+γ/δ T cells. The significance of this preferential Vδ1 intraepithelial presence is discussed.
Pancreatic Gangliosides Delay the Onset of Insulitis and Hyperglycaemia in the Low‐Dose Streptozotocin Mouse Model
Authors: ANASTASI B. , TIBERTI C. , SENSI M. , PONTE E. , FILIPPETTI R. , DOTTA F. , VECCI E. , FIORI M. G. , MARIO U. DI
Published: July 2006

Gangliosides have been shown to modulate autoimmune phenomena in experimental diabetes. The effects of a pancreatic ganglioside preparation or of a commercial brain ganglioside mixture on the insulitis and blood glucose levels in the low‐dose streptozotocin mouse model of diabetes have been investigated. Fifty‐five C57BL/6J male mice were grouped as follows: Group 1 (n= 20) was injected intraperitoneally with repeated low doses of streptozotocin; Group 2 (n= 10) received streptozotocin as above but was also injected with a pancreatic ganglioside preparation equivalent to 2 μg sialic acid 2 h before each streptozotocin dose; Group 3 (n= 15) received streptozotocin and brain‐derived gangliosides in the same dose as that of pancreatic gangliosides; Group 4 (n= 10) consisted of normal animals. Half of the mice were killed on day 12 and the others on day 24 from the beginning of treatment. On day 12, among the streptozotocin‐injected animals only those treated with pancreatic gangliosides remained normoglycaemic, whereas on day 24 all streptozotocin mice were hyperglycaemic. Such a result paralleled the data pertaining to insulitis scores. In conclusion, pancreatic gangliosides have a short‐term protective role on the development of diabetes in the low‐dose streptozotocin model, an effect therefore linked to tissue‐related differences in the glycosphingolipid composition.
Effect of Heat Shock on Susceptibility of Normal Lymphoblasts and of a Heat Shock Protein 70‐Defective Tumour Cell Line to Cytotoxic T Lymphocytes In Vitro
Authors: GEGINAT G. , HEINE L. , GÜNTHER E.
Published: July 2006

The effect of heat shock pretreatment of target cells on their lysability by cytotoxic T lymphocytes was analysed. Killing of Concanavalin A‐stimulated normal lymphocytes by minor or major histocompatibi‐lity antigen‐specific cytotoxic T lymphocytes is unchanged or even slightly enhanced after heat shock, whereas cells of the myeloma line Y3, which is derived from one of the lymphocyte donor strains, become nearly resistant to killing after the same pretreatment. Cold target inhibition experiments show that heat‐shocked cells are recognized specifically and that untreated and heat‐shocked target cells possess similar inhibitory potential. Y3 cells are unable to express the strongly heat‐inducible heat shock protein of 70 kDa (hsp70) after heat shock; the acquired resistance is thus independent of hsp70 induction. Possible mechanisms of the different lysability seen in lymphoblasts and tumour cells after heat shock are discussed.
Recombinant Human Interleukin‐8 Restores Function in Neutrophils from Patients with Myelodysplastic Syndromes Without Stimulating Myeloid Progenitor Cells
Authors: ZWIERZINA H. , HOLZINGER I. , GAGGL S. , WOLF H. , SCHÖLLENBERGER S. , LAM C. , BAMMER T. , GEISSLER D. , LINDLEY I.
Published: July 2006

Prognosis in myelodysplastic syndrome (MDS) is not only correlated closely with blast cell count in bone marrow and chromosomal abnormalities but also correlated with decreased leucocyte count and function leading to acquisition of lethal infections. Recently, clinical trials in MDS have focused on the application of haemopoietic growth factors such as G‐CSF or GM‐CSF, which have proven to increase neutrophil count and function. However, these cytokines carry the risk of stimulating the malignant clone, particularly in patients with increased blast cell count. Therefore, investigation of cytokines which are able to stimulate neutrophil function without the potential risk of stimulating haemopoietic progenitor cells may be relevant for MDS.

As the stimulatory effect of interleukin‐8 on neutrophil function is well known, we investigated whether recombinant human IL‐8 is also able to improve the function of neutrophils gained from patients with MDS. Using three different techniques—the E. coli killing assay (8 patients), the production of reactive oxygen as determined by cytochrome c reduction (7 patients) and chemiluminescence (8 patients)—a significant stimulation of neutrophil function at a concentration of 10 nM IL‐8 was found in all test systems. No correlation with FAB classification was evident. On the other hand, IL‐8 only mildly stimulated growth of myeloid progenitor cells in bone marrow culture of healthy individuals and MDS patients. This minimal stimulation was blocked by a neutralizing antibody directed against GM‐CSF, suggesting an indirect effect of IL‐8 via secondary GM‐CSF release.

Thus, IL‐8 is able in vitro to repair the functional abnormalities of neutrophils from patients with MDS but has only a marginal influence on myeloid progenitor cells.
Responsiveness for Mouse IgG2b Antibodies to the Human α/β T‐Cell Receptor/CD3 Complex: Evidence for Genetic Determination and Low Grade Antibody Cross‐Linking Not Mediated by Known Feγ Receptors
Authors: SCHLITT H. J. , SCHWINZER R. , WONIGEIT K.
Published: July 2006

Mouse IgG2a and IgG3 antibodies directed to the human T‐cell receptor/CD3 complex stimulate peripheral blood mononuclear cells in almost all individuals. By contrast, responder and non‐responder individuals exist for the stimulatory effect of mouse IgG1 and IgG2b antibodies. Whereas responsiveness to IgG1 antibodies is rather frequent (60–70%) and is known to be determined by an FcγRII polymorphism on the accessory cells, little is known about the underlying factors of the rare (6%) IgG2b responsiveness. In this study it is shown that (1) IgG2b responsiveness is genetically determined with a dominant pattern of inheritance; (2) IgG2b responsiveness is determined by a radioresistant feature of responder accessory cells which can be substituted by artificial cross‐linking, but not by IL‐1β or IL‐2; (3) stimulation of peripheral blood mononuclear cells of an IgG2b responder by IgG2b antibodies requires rather high antibody concentration compared with stimulation by IgG2a antibodies; (4) the stimulation leads to proliferation and IL‐2 receptor expression, but no measurable IL‐2 production; (5) antibody binding to known Fey receptors (FcγRI, FcγRII, FcγRIIT) is not involved in the stimulatory effect of the IgG2b antibodies in responders. These results demonstrate that responsiveness to IgG2b antibodies against the TcR/CD3 complex depends on a genetically determined feature of accessory cells that is not identical with any of the known Fcγ receptors. Most likely, the stimulatory effect observed in IgG2b responders is explained by a low grade cross‐Unking of the antibody by a not yet identified polymorphic structure on the surface of accessory cells.
Association of Pepsin with Type II Collagen (CII) Breaks Control of CII Autoimmunity and Triggers Development of Arthritis in Rats
Authors: VINGSBO C. , LARSSON P. , ANDERSSON M. , HOLMDAHL R.
Published: July 2006

Lewis rats develop arthritis after immunization with heterologous but not homologous rat type II collagen (CII). We have observed that if the rat CII is prepared by pepsin digestion without subsequent extensive purification, it is arthritogenic in Lewis rats. To address whether pepsin in the CII preparations contributed to the development of arthritis and whether this was associated with the induction of an immune response to CII, Lewis rats were immunized with rat CII of various degrees of purity and with various pepsin contents. After immunization with a crude preparation of CII, containing relatively large amounts of pepsin, Lewis rats developed arthritis with an incidence of 80% together with a strong anti‐CII autoantibody production. Further purification of the CII on DEAE‐Sepharose, which removes pepsin, eliminated the arthritogenic properties and the capacity to activate CH‐specific B cells. Likewise, lathyritic CII, prepared without pepsin, induced neither a CII‐specific immune response nor arthritis. If, however, pepsin was added to non‐arthritogenic batches of rat CII, arthritis appeared at an incidence of 40%. By using an ELISPOT technique to detect antigen‐specific interferon‐γ‐producing T cells and antibody‐producing B cells, the immune response to CII and pepsin can be evaluated. Eleven days after immunization with lathyritic CII and pepsin, a B‐eell response towards both CII and pepsin was seen. Pepsin‐specific T cells were also seen at day 11, but CII‐specific T cells did not appear until day 14 after immunization. In addition, a weak CII ‐specific proliferative response of the T cells could be demonstrated at day 14 but not at day 11 or 12. These data show that pepsin plays an important role in the triggering of a CII‐specific immune response. We suggest a carrier‐hapten mechanism where pepsin acts as a carrier and CII as a ‘hapten’ which will activate CII‐specific B cells. Subsequently these CII‐specific B cells will break the T‐cell tolerance and evoke a T‐cell‐mediated immune response towards CII.
Modification of the Complement Binding Properties of Polystyrene: Effects of End‐Point Heparin Attachment
Authors: NILSSON U. R. , LARM O. , NILSSON B. , STORM K.‐E. , ELWING H. , EKDAHL K. NILSSON
Published: July 2006

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin‐treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways After end‐point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW‐mediated mechanisms. while adsorption and APW ‐mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for Clq which leads to binding of Cl and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I‐dependent down‐regulation of C3b and to the lowered protein‐adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt‐dependent activation of the complement system.
Intrathecal Synthesis of Anti‐Borrelia burgdorferi Antibodies in Neuroborreliosis: a Study with Special Emphasis on Oligoclonal IgM Antibody Bands
Authors: WANG Z.‐Y. , HANSEN K. , SIDÉN Å. , CRUZ M.
Published: July 2006

Pre‐ and post‐treatment (up to 3–26 months after antibiotic therapy) humoral immune responses were investigated in five neuroborreliosis patients. Anti‐Borrelia (B.) burgdorferi IgG and IgM antibodies in CSF and serum were quantitated by capture ELISA. Agarose gel isoelectric focusing (AIF) and protein blotting were used to detect oligoclonal IgG and IgM bands as well as oligoclonal anti‐B. burgdorferi IgG and IgM antibodies. These latter components were visualized by transfer to antigen‐coated membranes (immunoblot) and immunoenzymatic staining. By ELISA, intrathecal anti‐B. burgdorferi IgG and IgM antibody synthesis was detected in all initial specimens and continued 3–26 months after antibiotic therapy in four and three cases, respectively. AIF with protein blotting showed oligoclonal bands of total IgG as well as total IgM in the initial CSF specimens of all patients and persistence of such components occurred in four and five cases, respectively. By AIF and immunoblot, oligoclonal anti‐B. burgdorferi IgG and IgM antibody bands could be detected in the CSF of every patient. IgG antibody bands were present in all initial CSF samples. The first specimen of one patient was negative for IgM antibody bands but such components appeared 3 weeks later. Oligoclonal CSF anti‐B. burgdorferi IgG antibody components persisted over the entire follow‐up periods in all but one case where they disappeared 6 weeks after treatment. The oligoclonal IgM antibodies in CSF vanished in two cases (after being present up to 4 and 11 months after antibiotic therapy) while they persisted over the entire (3–6 months after antibiotic therapy) follow‐up periods in three cases. The specificity of the IgM antibody immunoblot technique was corroborated by control experiments, including antibody absorption studies and use of 41 kDa flagellar antigen.