bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of Mannan‐Binding Protein from Mouse Serum
Authors: HOLT P. , HOLMSKOV U. , THIEL S. , TEISNER B. , HØJRUP P. , JENSENIUS J. C.
Published: July 2006

Mouse mannan‐binding protein (MBP) was identified in serum by its Ca2+ ‐dependent binding to mannan. On gel permeation chromatography, the protein eluted corresponding to a molecular weight of approximately 750 kDa. Analysed on SDS‐PAGE under reducing conditions, the polypeptide showed an apparent molecular weight of 28 kDa, while several high molecular weight bands were seen under non‐reducing conditions. The presence of collagen‐like domains within the molecule was indicated by a high glycine content (14.9%) and substantiated by sensitivity to collagenase. Rabbit anti‐mouse MBP antisera were raised. The concentration of MBP in serum from normal mice was measured by rocket immunoelectrophoresis and found to be from below 1 μg/ml to 100 μg/ml (average 50 μg/ml, n= 60). The binding of mouse MBP to mannan could be inhibited by mono‐ and disaccharides in the following order of potency: L‐fucose > D‐mannose > N‐acetyl‐D‐glucosamine > maltose > D‐mannoheptulose > D‐glucose > N‐acetyl‐D‐mannosamine ≫ lactose > D‐galactose ≫ N‐acetyl‐D‐galactosamine. Mouse MBP was shown to activate the classical complement cascade after binding to mannan. The sequence of 14 NH2‐ terminal amino acid residues of the molecule showed 93% identity to rat MBP‐A and complete identity to the translated cDNA sequences for mouse MBP‐A and mouse Ra‐reactive factor component P28b (RaRF P28b) published previously. The amino acid composition of mouse MBP showed a high degree of homology to MBPs from other species and mouse RaRF P28b.
Differential Effects of IL‐10 on Proliferation and Cytokine Production of Human γ/δ and α/β T cells
Authors: SCHLAAK J. F. , HERMANN E. , GALLATT H. , BOSCHENFELDE K‐H. MEYER ZUM , FLEISCHER B.
Published: July 2006

Gamma/delta TCR bearing T lymphocytes represent a T‐cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin‐10 on γ/δ and α/β TCR‐positive T‐cell clones and freshly isolated peripheral‐blood mononuclear cells (PBMC). IL‐10 has been shown previously to inhibit lectin and antigen‐induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL‐10 strongly inhibits lectin‐induced production of IFN‐γ, TNF‐α. IL‐2, and to a lesser degree proliferation and IL‐4 production of both T‐cell subsets. As IL‐10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T‐cell clones tested do not produce IL‐10 they can use it as a growth factor in combination with IL‐2, IL‐4 or alone. Furthermore, IL‐10 has the same properties on human α/β T‐cell clones and PBMC. In summary, it is shown that IL‐10 has pleiotropic effects on γ/δ and α/β TCR+ T cells by inhibiting lectin‐induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL‐2 or IL‐4.
Hamster Hepatic Amyloid A (AA) Protein is Derived from a Novel Member of the SAA Gene Family
Authors: NIEWOLD TH. A. , MURPHY C. , GRUYS E.
Published: July 2006

The nearly complete amino acid sequence of casein‐induced hepatic hamster AA is described. Hamster AA appeared to be C‐terminally ragged as found in other species. Furthermore, the N‐terminus was (partially) truncated. Hamster hepatic AA sequence showed high homology with, but was not identical to hamster SAA‐isotype sequences deduced earlier from mRNA in hepatic and extra‐hepatic tisstie. Therefore, hamster hepatic AA is derived from a novel member of the SAA gene family. However, the origin of hepatic AA‐amyloid remains unclear.
Intra‐Erythrocytic Plasmodium falciparum Induces Up‐Regulation of Inter‐Cellular Adhesion Molecule‐1 on Human Endothelial Cells In Vitro
Authors: ESSLINGER C. W. , PICOT S. , AMBROISE‐THOMAS P.
Published: July 2006

The stimulation of human vascular endothelial cells with P. falciparum‐infecterylhrocytes resulted in the non‐transient up‐regulation of ICAM expression on human endothelial cells. The induction was independent from TNF as assured in various controls including the application of a TNF‐neutralizing antibody. The possibility that TNF is produced by endothelial cells was investigated in a purified culture of human endothelial cells by TNF‐ELISA and TNF‐bio‐assay both with negative results. This result may be evidence of the existence of TNF‐independent mechanisms in the pathogenesis of human cerebral malaria.
Alkali‐Treated LPS of Yersinia enterocolitica does not Induce Expression of E‐Selectin, ICAM‐1 or VCAM‐1 on Endothelial Cells but may Mediate Antibody‐ and Complement‐Dependent Cell Injury
Authors: REPO H. , RENKONEN R. , HELANDER I. M. , LEIRISALO‐REPO M.
Published: July 2006

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enlerocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS‐OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS‐OH stimulated expression of endothelial cell adhesion molecules E‐selectin, VCAM‐1 and ICAM‐1, whereas both Intact J5 LPS and yersinia LPS were stimulatory. Impaired up‐regulation could not be explained by decreased binding of yersinia LPS‐OH to HUVECs. Furthermore, 51Cr‐labelled HUVECs treated with different concentrations of yersinia LPS‐OH released 51Cr in the presence of anti‐yersinia anti‐0 antibody and complement. J5 dLPS and yersinia LPS‐OH inhibited up‐regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha.

Taken together, the results suggest that although yersinia LPS‐OH can depress development of acute inflammation by inhibiting up‐regulation of etidothelial‐cell adhesion molecules, sufiicient LPS‐OH is bound to induce cell injury and thereby inflammation in the prescence of specific antibody and complement. The findings may have pathogenetic implications in yersinia‐triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.
Formation of the Terminal Complement Complex on Agarose Beads: Further Evidence that Vitronectin (Complement S‐Protein) Inhibits C9 Polymerization
Authors: JOHNSON E. , BERGE V. , HOGASEN K.
Published: July 2006

Vitronectin occupies the metastable binding site of C5b‐7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane‐associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9‐neoepitopes. Soluble SC5b‐7 and TCC (SC5b‐9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9‐neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b‐8 of the agarose‐bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane‐bound TCC by inhibiting C9 polymerization.
Somatic Hypermutations in the Immunoglobulin Genes of Two New Human Lymphoma Lines of Lymphatic Follicle Origin
Authors: WU H. Y. , TUOMIKOSKI T. , ERAY M. , MATTILA P. , KNUUTILA S. , KAARTINEN M.
Published: July 2006

Variable immunoglobulin heavy‐chain regions (VDJ) of two newly established human lymphoma cell lines (HF‐1 and HF‐4) were sequenced. The most homologous germline VH gene found for both the HF‐1 and HF‐4 sequences was VH26 of the VH3a (V gene) family (82% and 91 % homologies, respectively). The JH region of the HF‐4 heavy‐chain sequence contained two nucleotide differences compared to the published germline JH3 gene. The DHJH region of the HF‐1 gene had a record high number (20%) of somatic mutations.

The numerous hypermutations found in the HF‐1 cell line support the hypothesis that in some human follicular lymphomas, mutations continue to accumulate in immunoglobulin genes during the malignant growth. Follicular lymphoma cell lines, which have an active mutational machinery, in future may help to solve the molecular events behind the somatic hypermutations modifying immunoglobulin genes of B lymphocytes.
HLA DQ and DP Locus Mismatches and their Effect on MLC in HLA Class I‐ and DRB1‐Matched Unrelated Patient/Donor Pairs Waiting for Allogeneic Bone‐Marrow Transplantation
Authors: PARTANEN J. , KOSKIMIES S.
Published: July 2006

The frequency of HLA DQA1, DQB1, and DPB1 mismatches was evaluated in 110 HLA class I‐ and DRBl‐matched, unrelated pairs who were tested in mixed lymphocyte culture (MLC) in connection with a potential allogeneic bone‐marrow transplantation between unrelated persons. After typing of the DQA1, DQB1. and DPB1 genes retrospectively, it was found that only 22% of the pairs were matched for all the loci tested. However, the frequency of this fully matched group was higher (37%) in pairs where both individuals were of Finnish origin as compared to those where the donor originated from a foreign registry. This suggests that the variety of haplotypes may be limited in small populations and justifies local national registries. The overall correlation between class‐II match and stimulation in MLC was clear: better matches gave lower responses. However, the correlation was not definitive; for example, no response was observed in some cases with mismatches only in DPB1.
Characterization of a 58‐ and a 78‐kD Monocytic Membrane Protein with Affinity to the Acetylcholine Receptor in Myasthenia Gravis Patients
Authors: STUHLMÜLLER B. , KALIES I. , KACHELRIES H. , DRUSCHKY K.‐F. , KALDEN J. R.
Published: July 2006

The autoimmune disease myasthenia gravis (MG), caused by the effect of specific antibodies, directed towards the nicotinic acetylcholine receptor, is triggered by autoantigen‐specific T cells. In order to investigate cellular parts of the immune response in MG, the authors investigated the binding of the nicotinic acetylcholine receptor (AChR) to peripheral blood mononuclear cells (PBMC) from MG patients. AChR binding cells were identified by resetting experiments using AChR‐coated fluoresceine beads. Applying this technique, a significant percentage of PBMC (21.2 × 7.65%) from MG patients formed rosettes with AChR‐coated beads.

Membrane preparations of nycodenz‐ or percoll‐separated monocytes from MG patients or T‐cell depleted monocytic subpopulations were applied to SDS‐PAGE under reducing conditions. Ligand‐blotting studies with biotinylated AChRs revealed two cell‐membrane proteins with molecular weights of 58‐ and 78‐kD. In parallel the same results were obtained by affinity chromatography of monocytic membrane proteins using AChR‐sepharose. A possible interference of anti‐AChR IgG was excluded. The 58‐ and the 78‐kD proteins are detectable under reducing conditions by ligand blotting with AChR‐biotin, while under non‐reducing conditions only the 58‐kD protein can be detected. Furthermore, in experiments using Endoglycosidase‐H, the 58‐kD protein appears to be non‐glycosylated, while the 78‐kD protein bears carbohydrates.

These findings suggest that monocytes which bind the AChR via specific membrane proteins on their surface might act as antigen‐presenting cells and may lead to an induction of the T‐cell response, in the early phase of the disease.
Human Complement Activation by Polygeline and Dextran 70
Authors: VIDEM V. , MOLLNES T. E.
Published: July 2006

The complement cascade is activated during cardiopulmonary bypass (CPB) and in trauma victims. In order to study whether plasma expanders may contribute to complement activation in these settings, fresh human serum was incubated with dextran 70 and polygetine. C3‐activation products and the terminal complement complex (TCC) were quantified in enzyme immunoassays. At 37 C, dextran caused immediate, dose‐dependent C3 activation and TCC formation with decline later in the rate of TCC formation relative to serum‐saline controls. High doses of polygeline caused slight activation after 15–30 min. The authors tested also the activation under conditions comparable to those found during clinical CPB. Activation induced by dextran and polygeline was increased by dilution of the serum. Furthermore, the plasma expanders counteracted the well‐known inhibitory effects of heparin (4 IU/ml) and moderate hypothermia (30°C) on complement activation. The authors conclude that dextran, and to a lesser extent polygeline. may contribute to complement activation during CPB and other clinical settings such as shock treatment.
Evidence for a Pathway Independent from 2′‐Deoxyguanosine and Reversible by IL‐2 by which Purine Nucleoside Phosphorylase Inhibitors Block T‐Cell Proliferation
Authors: BOEHNCKE W.‐H. , GILBERTSEN R. B. , HEMMER J. , STERRY W.
Published: July 2006

Patients with homozygous deficiency of purine nucleoside phosphorylase (PNP) present with a T‐cell selective immune deficiency. To elucidate the potential use of PNP inhibitors in the therapy of cutaneous T‐cell lymphomas (CTCLs) the authors studied the effects of CI‐1000 (formerly PD 141955‐2) and CI‐972 on a T‐cell line My La established from a patient with mycosis fungoides. Both PNP inhibitors had significant, dose‐dependent, inhibitory effects on the proliferation of the T‐cell line. CI‐1000 (ED50: 3.7 μM) was approximately six‐fold more potent in blocking 3H‐thymidine uptake than CI‐972 (ED50: 22.5 μM). The inhibitory effect of either substance could not be increased by addition of deoxyguanosine. Flow cytometric analysis revealed that both PNP inhibitors caused a block in the S‐phase of the cell cycle. The inhibitory effect on proliferation was reversible partially by addition of IL‐2. When testing proliferation inhibition of both substances on an IL‐2‐dependent T‐cell line (SeAx), their inhibitory effects were reduced significantly. These data document a mechanism of action of the PNP inhibitors independent of deoxyguanosine and partially reversible by IL‐2. The authors' observations suggest the potential use of PNP inhibitors in the therapy of cutaneous T‐cell lymphomas and provide evidence for a pathway independent from deoxyguanosine by which PNP inhibitors might function in T cells.
Thiol‐Proteindisulfide–Oxidoreductase (Proteindisulfide Isomerase): a New Plasma Membrane Constituent of Mature Human B Lymphocytes
Authors: KRÖNING H. , KÄHNE T. , ITTENSON A. , FRANKE A. , ANSORGE S.
Published: July 2006

The thiol‐proteindisulfide–oxidoreductase (TPO. EC 1.8, 4.2, proteindisulfide isomerase, EC 5.3.4.1.) is known as an cytoplasmatic enzyme, and is thought to be involved in the post–translational folding of disulfide containing proteins. Using monoclonal and polyclonal antibodies the authors were able to prove that this enzyme or an unknown homologous protein is localized also to the plasma membrane of B lymphocytes. In peripheral blood from healthy donors 11% of the mononuclear cells (PBMNC) expressed this surface antigen whereas in PBMNC of patients with B–cell chronic lymphocytic leukaemia 76% of the MNC were positive. This value correlates well with the known B–cell markers CD19 and CD20. However, this antigen is different from all known clustered B–cell markers. Immunoprecipitation analysis of PHA–stimulated PBMNC and of cells from patients suffering from chronic lymphocytic leukaemia revealed a membrane protein with the same molecular weight (61 kDa) as the TPO. These data suggest that this enzyme is present not only in the cytoplasm but also on the surface of B cells and that it is possibly involved in the regulation of the SH‐SS status of the cell membrane proteins of B lymphocytes.
Kinetics and Specificities of the T Helper–Cell Response to GP120 in the Asymptomatic Stage of HIV–1 Infection
Authors: GERETTI A. M. , VAN BAALEN C. A. , BORLEFFS J. C. C. , VAN ELS C. A. C. M. , OSTERHAUS A. D M. E.
Published: July 2006

Peripheral blood mononuclear cells from 36 asymptomatic HIV‐1 seropositive individuals were tested longitudinally for in vitro T–cell proliferation and IL–2 production in response to synthetic peptides spanning the entire gp120 of HIV–1. At baseline, significant T–cell proliferation to pooled and individual peptides was observed in 15 of the 36 donors. After 12 months, proliferate responses to peptide pools were lost or decreased significantly in most donors. Responses appeared to fluctuate over time: at 12 months new recognition sites were detected in four of 10 donors showing T–cell proliferation at baseline, as well as in five of 15 donors with no previous proliferative responses. IL–2 production appeared to be a more sensitive and longer preserved parameter of T–helper cell function: at baseline the majority of donors with no T–cell proliferation produced IL–2 in response to pooled peptides. This response was not decreased significantly after 12 months. The overall patterns of response to both pooled and individual peptides were heterogeneous among donors. Multiple recognition sites were detected in both variable and conserved regions of gp120, but no pool or individual peptide was recognized by all responders. Functional T–cell responses were not statistically correlated to CD4° cell percentile and absolute numbers.
Resistance and Susceptibility to Cyclosporin A as CD3− 4− 8− Human Thymocytes Differentiate In Vitro
Authors: PILARSKI L. M. , HOWLAND K. L. , DEANS J. P.
Published: July 2006

Human T‐cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA mw/eroaspart of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3‐4‐8‐) thymocytes as a model system for thymic progenitor development in vitro. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL‐2 and on thymic epithelial cells (TEC) with or without lL‐2, either in ihe presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3and ofthe low Mr isofomi CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected., but selective inhibition of TCR5 expression occurred. At all doses of CsA (0.1–100 μg/ml), MN thymocytes continued to cycle indicating a CsA‐resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo‐synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRδ and of TCRδ was inhibited, but on TEC, TCRδ was actually up‐regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the ceils were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may from an as yet uncharacterized mechanism
Characterization of a Cyclosporin A‐Sensitive Activation Pathway in Cultured T and Natural Killer Cells
Authors: CANTONI C. , CAMBIAGGI A. , SFORZINI S. , POGGI A. , VIALE M. , BIASSONI R. , FERRINI S.
Published: July 2006

Previously, the authors have described a molecule, identified by the LD6 monoclonal antibody (MoAb), present at the cell surface of long‐term cultured T and Natural Killer (NK) cells which is involved in cell triggering. In the study described here the authors used biotin surface labelling and immunoprecipitation to show that LD6 MoAb recognizes a surface protein of approximately 65 kDa. In combination with submitogenic concentrations ofphorbol esters (PMA), LD6 MoAb was able to induce accumulation of mRNA specific for GM‐CSF, γ‐IFN and TNF‐α and release of these cytokines by LD6+ T‐cell lines. Both lymphokine production and lymphokine‐specific mRNA accumulation induced by the LD6 MoAb were blocked totally by Cyclosporin A (CsA). To investigate the mechanism(s) of signal transduction through this activatory pathway, the authors performed Ca++ mobilization experiments. The results of these experiments suggested a role for Ca++ in signal transduction. The Ca++ mobilization induced by LD6 MoAb cross‐linking could be inhibited totally by the use of pertus.sis toxin, indicating a possible role for G proteins in signalling through the LD6 MoAb‐reactive molecule. Western blot analysis performed with an anti‐phosphotyrosine antibody did not suggest that tyrosine kinase activation has a role.
Interactions between Staphylococcal Superantigens and Human T‐Cell Clones are Predominantly but not Exclusively Governed by their T‐Cell Receptor Vß Usage
Authors: LUNDIN K. E. A. , BRINCHMANN J. E. , HANSEN T.
Published: July 2006

Stapbylococcal exotoxins (SE) are potent mitogens for human and murine T cells. Extensive studies in mice bave demonstrated strict correlations between T‐cell responses to individual SE and TCR Vβ expression. Studies examining the TCR Vβ expression of SE‐activated buman peripheral blood T cells also suggest close correlations, whereas the data reported using human T‐cell clones (TCC) are conflicting. We bave determined tbe cDNA TCRB sequences of 52 different human TCC, expressing 35 different T‐cell receptor Vfi (TCRBV)‐encodcd sequences. The TCC were tested in proliferative assays using nine different SE. Most of these TCC express Vs wbicb have not been tested previously in studies examining interaction between TCC and SE. The SE stimulated a variable fractioti (1/48‐31/52) ofthe TCC. Tbe ability of a given SE to stimulate TCC in many cases correlated with Vβ expression, but several exceptions were found. With one possible exception, comparisons between deduced amino‐acid sequences within the ‘fourth hypervariable region’ of the TCR p chain and SE responsiveness did not reveal potential SE binding motifs. We conclude that the reactivity of T cells towards SE is governed mainly by their TCR Vβ expression. However, the authors results also suggest that the interaction between SE and human T cells may involve elements unidentified as yet which are in addition to the chain of the TCR.
Activation Signal Induces the Expression of B Cell‐Specific CD45R Epitope (6B2) on Murine T Cells
Authors: WATANABE Y. , AKAIKE T.
Published: July 2006

Tlymphocytes express multiple forms of the leukocyte‐common antigen CD45, transcribed by alternative usage of leukocyte‐common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3‐6B2 is established as a B cell‐type isoform (B220)‐specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T‐cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti‐CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4+ and CD8+ T cells and also was induced by SEB in vivo predominantly on CD8+ T cells. The 6B2+ T cells are IL‐2R+ and blasted cells according to flow cytometry analysis. Therefore, the 6B2+ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo‐D or endo‐H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT‐PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T‐cell subpopuiation bearing a 6B2 epitope.
Decreased CD8‐p561ck Activity in Peripheral Blood T‐Lymphocytes from Patients with Hereditary Haemochromatosis
Authors: AROSA F. A. , DA SILVA A. J. , GODINHO I. M. , TER STEEGE J. C. A. , PORTO G. , RUDD C. E. , DE SOUSA M.
Published: July 2006

Hereditary haemochromatosis (HH) is an autosomal recessive disease linked to certain MHC class‐I specificities. The disease is characterized by increased iron absorption and, in some patients, abnormally low numbers of CDS8+ T cells in the periphery. We were interested in whether CD4‐ and CD8‐associated p561ck kinase activities were altered in patients with HH. In a study of 18 patients with HH (with and without low numbers of CD8+ cells), the level of autophosphorylation of the CD8‐associated p561ck as well as its phosphotransferase activity, as determined by phosphorylation of an exogenous substrate, was significantly reduced by two‐ to three‐fold relative to a control population of 23 healthy blood donors (P < 6 × 10−7). CD8‐p56 lck activity was decreased in 16 out of 18 patients (ranging from 1.5‐ to 10‐fold decrease). By contrast, the level of CD4‐p561ck activity did not show an overall decrease relative to controls. In addition to an occasional decrease in the amount of CD8‐associated lck, HH patient‐derived T cells showed a consistent decrease in the relative CD8‐p561ck specific activity. Immunofiuorescence staining showed further that the difference could not be accounted by a discrepancy in the expression of CD8αα or CD8αβ complexes or MHC class I molecules. Decreased CD8‐p561ck activity was seen both in patients undergoing intensive phlebotomy treatment and in patients in maintenance therapy (i. e. patients who had reached normal levels of iron stores), indicating that this abnormality does not appear to be corrected by iron depletion. To our knowledge, this is the first demonstration of an abnormality in a src‐like receptor associated kinase in a human disease state linked to MHC class‐I antigens.
The B‐Cell Adhesion Molecule CD22 is Cross‐Species Reactive and Recognizes Distinct Sialoglycoproteins on Different Functional T‐Cell Sub‐Populations
Authors: SGROI D. , STAMENKOVIC I.
Published: July 2006

CD22 is a B cell lineage restricted cell‐surface adhesion glycoprotein which recognizes ligands on human T and B cells and cell lines. A soluble recombinant form of human CD22 (hCD22Rg) has been used to identify and characterize CD22‐specific ligands on human T cells, one of which has been shown to be the receptor‐linked phosphotyrosine phosphatase CD45. Because CD45 plays a pivotal role in lymphocyte activation, we assessed whether human CD22 might display cross‐species reactivity with CD45. In the study presented here we demonstrate that human CD22Rg recognizes several murine cell‐surface sialoglycoproteins. including CD45, containing sialic acid in a2, 6 linkage. Furthermore, hCD22Rg recognizes different ligands on functionally distinct T helper‐cell subpopulations and selectively binds medullary thymocytes in rivo. Our results confirm and extend previous observations that CD22 is a sialic acid‐binding lectin which interacts with CD45 and other glycoproteins capable of presenting α2, 6‐linked sialic acid in a manner that promotes high affinity binding. The cross‐species reactivity ofCD22 with its ligands underscores the potential physiologic importance ofCD22‐mediated lymphocyte interactions.
Mannan‐Binding Protein and Bovine Conglutinin Mediate Enhancement of Herpes Simplex Virus Type 2 Infection in Mice
Authors: FISCHER P. B. , ELLERMANN‐ERIKSEN S. , THIEL S. , JENSENTUS J. C. , MOGENSEN S. C.
Published: July 2006

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type I (HSV‐1) in viiro. We decided to investigate the role of mammalian Icctins in infection witb herpes simplex virus. Two lectins, conglutinin and mannan‐binding protein (also called mannose‐binding protein. MBP). belonging to the collectin family of lectins, were examined.

Four week‐old BALB/c mice were injected subcutaneously with 100 μg bovine conglutinin or 50 μg human MBP 1 day before intravenous infection with 5 × 104 PFU of herpes simplex virus type 2 (HSV‐2). A three‐fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP. whereas no effect was seen on days I and 5.

In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre‐incubated with differing concentrations ofthe collectins. Tbe concentrations used were similar to tbose used by us in livo, and by others in in vitro experiments showing inhibition of the infectivity of HSV‐1 with plant lectins.

In an ELISA with HSV‐2 antigens captured on anti‐HSV‐2 antibodies, calcium‐dependent and carbohydrate inhibitabte binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions witb an alternative port of entry into cells leading to infection enhancement.
The Treatment of Autoimmune Disease in (NZB/NZW)F1 Mice with Syngeneic Photomodulated Splenocytes
Authors: LIN R.‐H. , WANG L.‐F.
Published: July 2006

(NZB X NZW)FI (B/W) mice spontaneously develop a disease which is remarkably similar to systemic lupus erythematosus (SLE) in humans. This disease is characterized by the appearance of autoantibodies to double‐stranded (ds)DNA and the subsequent development of fatal glomerulonephritis. The prophylactic treatment of B/W mice with syngeneic photomodulated autoimmune spleen cells was found to significantly improve survival, and to inhibit the outgrowth of autoreactive B cells and the production of high‐titre IgG anti‐dsDN A antibodies. The function of the autoreactive T cells in vitro, however, did not change significantly. Our findings suggested a novel treatment for spontaneously occurring autoanti‐body‐rciated autoimmune diseases.
Interaction of In Vitro‐ and In Vivo‐Generated Cytotoxic T Cells with SV40 T Antigen: Analysis with Synthetic Peptides
Authors: ALSHEIKHLY A‐R.
Published: July 2006

Virus‐specific cytotoxic T cells recognize antigens in the form of peptides (8 or 9 amino acids long) bound to MHC class‐I molecules. Exposure of unprimed murine splenocytes to synthetic peptides of viral antigens elicits primary CTL in vitro. The fme specificity of such CTL as well as the correlation between binding affinity of peptides to class‐I molecules and CTL induction was analysed using synthetic peptides corresponding to overlapping and distinct amino‐acid residues in SV40 T antigen (Tag) Db‐restricted T‐cell epitopes I, II‐III, and V. The peptides induced cross‐reactive CD8+ primary CTL in spienocytes of naive C57 BL/6 mice. This reactivity was seen regardless of the peptides allelic anchor motifs or their abilities to stabilize empty class‐I molecules. However, none of the primary CTL and CTL lines lysed Tag‐expressing cells. In contrast, CTL generated in vivo by immunizing mice with Tag‐expressing cells recognized endogenously processed Tag as well as synthetic peptides. The peptides recognized by these CTL depended on the intracellular concentration of Tag antigen in the immunizing cells. The reactivity of these CTL was peptide specific as shown by a functional peptide competition assay. Moreover, three peptides bound to and were recognized in the context of both Kb and Db molecules. These results have revealed a flexible disposition of MHC class‐I molecules with regard to peptide binding and also reflected lack of correlation between binding affinity to class‐1 molecules and the capacity of peptides to induce primary CTL or to serve as potential targets. The significance of these findings in relation to identifying major T‐cell epitopes using allele specific peptide motif and in vitro maintained CTL clones is discussed.
High Serum‐Soluble Interleukin‐2 Receptor is not Associated with the Immunosuppression in Diffuse Cutaneous Leishmaniasis
Authors: AKUFFO H. , MAASHO K.
Published: July 2006

Diffuse Cutaneous Leishmaniasis (DCL) is a rare complication of Leishmania aethiopica‐induced cutaneous leishmaniasis which is associated with non‐self healing and in vivo and in vitro antigen‐specific non‐responsiveness. Such antigen‐specific unresponsiveness is also observed in visceral leishmaniasis (VL). The non‐responsiveness seen in VL disease is believed to be due, in part, to serum‐derived factors, including raised serum soluble IL‐2 receptor (sIL‐2R). Raised sIL‐2R in serum was not a consistent feature of DCL in our study (range: 787–4546 U/ml) but was frequently observed in sera of patients with other dermatological disorders (range: 474–3313 U/ml) and some patients with the simple local cutaneous leishmaniasis (LCL; range: 556–4247 U/ml). The level of sIL‐2R in the sera of DCL patients was not indicative of the disease state. Sera from DCL patients did not reduce proliferation of the IL‐2‐dependent CTLL cell line nor reduce PHA‐driven mononuclear cell proliferation, although sera from VL patients could. Both DCL and VL sera could reduce the L. aethiopica‐driven proliferation. Furthermore addition of serial dilutions of recombinant IL‐2 to CTLL cultured in VL or DCL sera containing high sIL‐2R levels did not alter the effect of such sera on proliferation. We conclude therefore, that raised sIL‐2R in serum is not associated with the immunosuppression in DCL.
Analysis of Oct2‐Isoform Expression in Lipopolysaccharide‐Stimulated B Lymphocytes
Authors: JOHANSSON K. , SIGVARDSSON M. , LEANDERSON T.
Published: July 2006

Oct2‐isoform expression in splenic B cells stimulated with lipopolysaccharide or lipopolysaccharide plus phorbol‐di‐butyrate was analysed by cDNA cloning. The frequency of Oct2‐positivc clones was 1/15, 000 in both libraries. Two new isoforms were found that generate novel amino‐ or carboxy‐terminal sequences. An isoform lacking exon 11 destroyed the carboxy‐terminal leucin‐zipper region and introduced a frame shift creating a novel, proline‐rich carboxy terminus. A new exon containing a highly basic region (4c) was characterized, between exons 4 and 5. This exon was inserted between glutamine‐rich regions 2 and 3, carboxy terminal of a tentative leucine‐zipper structure. In addition, a new combination isoform containing Oct2a's amino terminal insert (exon 7a) and Oct2b's carboxy terminal insert (exon 13) was found that created a novel large isoform, Oct2ab. More frequent use of the classical Oct2a and Oct2b isoforms was observed in the lipopolysaccharide‐stimulated B cells, while a preference for the Oct2ab and Oct2ba isoforms was observed in lipopolysaccharide plus phorbol‐di‐butyrate‐lreated cells.
Indirect Effects of Oral Tolerance in Mice
Authors: CARVALHO C. R. , VERDOLIN B. A. , DE SOUZA A. V. , VAZ N. M.
Published: July 2006

Anti‐DNP antibody formation resulting from intraperitoneal (i. p.) immunization with DNP‐KLH may be blocked by simultaneous (i. p.) injection of DNP‐Ova or native Ova in mice orally tolerant to Ova, but not in normal mice. In Ova‐tolerant mice the inhibition of anti‐DNP antibody formation also occurred when DNP‐Ova and DNP‐KLH were given by separate routes of immunization: subcutaneous (s. c.) and i. p. A second exposure to Ova by gastric intubation (gavage) or intravenous administration simultaneously with i. p. immunization with DNP‐KLH failed to inhibit anti‐DNP antibody formation. There was inhibition of responses to DNP‐KLH i. p. by DNP‐Ova given 24 h before, but not 24 h after, and in the Ova‐tolerant mice, addition of DNP‐Ova only to the primary immunization with DNP‐KLH inhibited secondary and tertiary responses to DNP‐KLH in the absence of further exposures lo DNP‐Ova.

These results suggest that the indirect effects of parenteral exposure of tolerant mice to the tolerated immunogen may inhibit unrelated immune responses. This inhibition is not due to ‘innocent bystanding’ suppression, i, e., to inhibitory cytokines provided locally by specific suppressor lymphocytes; it may derive from more durable perturbations of immune system.
Amino Acids in the Peptide‐Binding Groove Influence an Antibody‐Defined, Disease‐Associated HLA‐DR Epitope
Authors: DROVER S. , MARSHALL W. H. , KWOK W. W. , NEPOM G. T. , KARR R. W.
Published: July 2006

A shared amino‐acid sequence on the a helix of certain DRβ1 chains is predicted to generate a ‘shared epitope’ that is implicated in susceptibility to the development of rheumatoid arthritis (RA). Different relative risks (RR) for disease susceptibility and severity conferred by these DRβ31 chains suggest that their ‘shared epitopes’ are not equivalent. A set of monoclonal antibodies (MoAb) that map to the critical region, and for which optimal binding depends on DR context and cell lineage, was used to test this idea. Mapping experiments using mutated DRβ1* molecules showed that the antibody‐binding epitopes are overlapping; residue 70Q is pivotal for each, but neighbouring residues on the a helix and on the floor of the groove are also involved. Importantly, these epitopes are profoundly modified by peptide loading of DRβ31*0401 molecules. These data suggest that ‘shared epitopes’ on DR molecules that are associated with RA are influenced by their context; such structural modifications may be the basis for the varying susceptibilities conferred by these DR molecules for the development of RA.
IL‐1β‐Stimulated Leucocyte–Endothelial Adhesion is Regulated, in Part, by the Cyclic‐GMP‐Dependent Signal Transduction Pathway
Authors: LESZCZYNSKI D. , JOSEPHS M. D. , FOEGH M. L.
Published: July 2006

It is well known that the exposure of endothelial cells to IL‐1β induces an increase in endothelial cell adhesiveness for leucocytes. Using rat heart endothelial cells we found that exposure of endothelial cells to IL‐1β (100U/ml) induces a 133‐fold increase in the intracellular concentration of cyclic‐GMP; from 11.5×0.2fm to 1530 × 117.8 fm (per 106 cells). Therefore, we examined whether cyclie‐GMP is involved in the regulation of endothelial adhesiveness for leucocytes. Cyclic‐GMP analogue, dibutyryl cyclic‐GMP (0.01–0.05mm), similarly to IL‐1β, increased endothelial cell adhesiveness for leucocytes. Methylene blue, an inhibitor of guanylate cyclase, and KT5823, a specific inhibitor of cyclic‐GMP‐dependent protein kinase, inhibited both basal as well as IL‐1β‐induced endothelial cell adhesiveness for leucocytes, and KT5823 abolished the dibutyryl‐cyclic‐GMP‐induced increase in endothelial adhesive ness. The effect of cyclic‐GMP, induced by IL‐1β treatment, on the endothelial adhesiveness may be either direct or indirect because of the time‐gap between the rise in cyclic‐GMP level and the increase of endothelial adhesiveness.

IL‐1β (100 U/ml) and dibuthyryl‐cyclic‐GMP (0.01 mm) both induced an increase in the expression of intercellular adhesion molecule‐1 by endothelial cells. However, the fact that KT5823 failed to prevent this increase, suggests that, although the IL‐lγb‐induced increase in adhesiveness is caused by the increase in intracellular levels of cyclic‐GMP, it may not be mediated through intercellular adhesion molecule‐1.

In conclusion, the results obtained indicate that endothelial cell adhesiveness for leucocytes is, in part, regulated by the cyclic‐GMP‐dependent signal transduction pathway.
Basic Amino Acids Predominate in the Sequential Autoantigenic Determinants of the Small Nuclear 70K Ribonucleoprotein
Authors: JAMES J. A. , SCOFIELD R. H. , HARLEY J. B.
Published: July 2006

Autoantibodies binding the 70K nRNP polypeptide are commonly found in the serum of patients with systemic lupus erythematosus. IgG antibodies binding overlapping octapeptides of 70K nRNP have been evaluated in 10 patients with anti‐nRNP precipitins, seven patients with other autoimmune serology, and four normal human sera. Neither normal controls nor patients without an anti‐nRNP precipitin significantly bind any of the 70K nRNP octapeptides. Sera containing an anti‐nRNP precipitin strongly bind various combinations of eleven different regions of the 70K nRNP protein. One antigenic region is consistently the most reactive in nine often nRNP precipitin positive sera tested. This sequence, KDKDRDRKRRSSRSR, is highly charged and has a similar pattern of alternating basic amino acids also present in seven of the other purported humoral autoimmune epitopes of the 70K nRNP polypeptide. The closely related DRKR and ERKR are important components of two of these epitopes. All regions of the 70K peptide bound by human anti‐nRNP precipitin positive sera are very rich in the basic amino acids., especially lysine (chi‐square = 23.03, odds ratio = 13.3, P<0.000001).
T Cells from the Peripheral Blood of Coeliac Disease Patients Recognize Gluten Antigens when Presented by HLA‐DR, ‐DQ, or ‐DP Molecules
Authors: GJERTSEN H. A. , SOLLID L. M. , EK J. , THORSBY E. , LUNDIN K. E. A.
Published: July 2006

Coeliac disease (CD) is a T‐cell mediated immunological disease of the small intestine which is precipitated in susceptible individuals by ingestion of gluten. We recently reported that gliadin‐specific T cells can be found in the small intestinal mucosa of CD patients, and that a preponderance of these T cells was restricted by the CD‐associated DQ(%aL*0501, βl*0201)heterodimer. Here we report studies on whether the same is found for gliadin specific T cells in the peripheral blood of CD patients. T‐cell responses towards gluten antigens in vitro were found for both most CD patients and healthy controls. Gluten‐specific T‐cell clones (TCC) were established from four CD patients. Although a large proportion of these TCC were restricted by DQ molecules, including the CD‐associated DQ(α1*0501, β1*0201) heterodimer, several were restricted instead by DR or DP molecules. Thus, gluten‐derived peptides can be presented to T cells by several different HLA elass‐II molecules, and the preferential DQ(#aL1*0501, β1*0201) restriction of gluten‐specific T cells in the small intestinal mucosa of CD patients is less pronounced than for similar T eells in the peripheral blood.
Opsonophagocytic Activity Induced by Chimeric Antibodies of the Four Human IgG Subclasses With or Without Help from Complement
Authors: AASE A. , MICHAELSEN T. E.
Published: July 2006

The Opsonophagocytic activity of the four human IgG subclasses was studied using chimeric mouse‐human antibodies with specificity for the hapten NIP. As target cells we used haptenized sheep red blood cells and N. meningitidis, labelled with different amounts of hapten. We used polymorphonuclcar leucocytes (PMN) as effector cells to measure respiratory burst (RB), and U937 to measure phagocytosis/rosette formation. When the target cells were opsonized with antibody only, and PMN used as effector cells, lgG3 was highly efficient, while IgG1 revealed an intermediate activity and IgG2 and IgG4 were negative. The same pattern among the subclasses was obtained in the presence of complement source, when target cells with low hapten concentration were used. However, at high cpitope concentration on the target cells, in the presence of complement source, IgG2 was highly active. while IgG4 was still negative or only slightly positive. When U937 were used as effector cells and complement was omitted, IgG1, IgG3 and lgG4 all revealed high phagocylic/rosette‐forming activity, while IgG2 was negative. When the target cells were opsonized with antibody and complement, the phagocytic/rosette‐forming activity was often suppressed. Our results reveal that all four human IgG subclasses possess Opsonophagocytic capacity, but with different requirements concerning complement and Fc Rs. They also enlighten us as to how IgG2 might perform its protective effect against harmful bacteria displaying high density of carbohydrate epitopes on their outside surface.
Implanted Solid Human Tumours Grow Under the Renal Capsule of Cyclosporin‐Immunosuppressed Rats
Authors: KAARTINEN M. , ERAY M. , LEHTONEN E. , MATOSO‐FERREIRA A. , TIENARI J.
Published: July 2006

Cyclosporin (CsA) is a potent immunosuppressive drug widely used in organ transplantation. We transplanted fresh surgical samples from human solid malignant tumours into 45 CsA‐immunosuppressed rats. Eight out of nine tumour types grew and remained viable for 5 weeks or more in at least two of the transplanted rats. In 29 rats (64%) a distinct growth of primary human tumours was recorded.

Five malignancies (intestinal‐type gastric carcinoma, adenocarcinoma of the lung, lymph node metastasis of a testicular teratocarcinoma, soft tissue malignant fibrous histiocytoma (MFH), and small‐cell sarcoma) showed invasive and progressive growth. In all five cases the largest tumours were 0.9 cm or over in diameter when the rats were killed 5–9 weeks after transplantation. In three cases (adenocarcinoma of the colon, hypernephroma, and a second MFH) the growth of the implants under the kidney capsule was slow, but small living tumour transplants were still found 3–6 weeks later. In every case the microscopic morphology of the xenograft tumour was identical with the original tumour. In two cases the primary xenografts (teratocarcinoma and small‐cell sarcoma) were retransplanted into 11 CsA‐immunosuppressed rats. In both types the second passage tumours grew, and the take‐off and growth rates were comparable to the primary xenografts.

Cyclosporin‐treated laboratory rats are an alternative to immunodeficient nude and SCID mice for growing fresh human tumour transplants in vivo. Although a few infections were encountered, most of the rats survived the CsA treatment well for up to 2 months.