bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic Potency of CD22‐Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules
Authors: VAN HORSSEN P. J. , VAN OOSTERHOUT Y. V. J. M. , DE WITTE T. , PREIJERS F. W. M. B.
Published: July 2006

Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22‐ricin A IT to several B cell lines. The antigen density varied from 5.9 × 103 to 6.0 × 104 molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 × 10−12 to 3.8 × 10−1 M IT in absence and from 2.8 × 10−14 M to 5.2 × 10−12 M IT in presence of the cytotoxicity enhancer NH4C1 (6mM). In absence as well as in presence of NH4C1 no correlation could be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I‐labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos (154, 500), followed by Daudi (110, 300) and KM3 (69, 900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10−8 M IT was comparable with KM3. NH4C1 did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22‐ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22‐ricin A.
In Vitro Degradation of Serum Amyloid A by Cathepsin D and Other Acid Proteases: Possible Protection Against Amyloid Fibril Formation
Authors: YAMADA T. , KLUVE‐BECKERMAN B. , LIEPNIEKS J. J. , BENSON M. D.
Published: July 2006

The effects of acid proteases on degradation of serum amyloid A protein (SAA) were investigated in vitro. Human recombinant SAA1 (rSAA1), when incubated with human spleen extracts at pH 3.2, was degraded in the amino‐terminal portion of the molecule. This reaction was inhibited by an acid protease inhibitor, pepstatin. The degraded SAA molecules lacking nine or more amino‐terminal residues, when exposed to in vitro fibril‐forming conditions, failed to form Congo red positive precipitates and did not show amyloid fibril‐like structure by electron microscopy. This suggests that the amino‐terminal portion of SAA is essential for fibril formation. Cathepsin D, one of the lysosomal enzymes, also initiated degradation of rSAAl at the amino‐terminus. Cathepsin D immunoreactivity was detected in marginal areas of amyloid deposits in spleens from patients with reactive amyloidosis. These findings suggest that cathepsin D or similar acid proteases may be involved in SAA catabolism and may protect against amyloid formation.
Effects of Anti‐CD 18 and LPS on CD 14 Expression on Human Monocytes
Authors: OTTERLEI M. , SUNDAN A. , RYAN L. , ESPEVIK T.
Published: July 2006

In this study we show that the cytokine stimulatory effect of LPS on human monocytes is enhanced by addition of monoclonal antibodies against CD18 (aCD18 MoAbs). Incubation of monocytes with αCD18 MoAbs overnight increased the CD 14 expression as detected by Leu‐M3, but not with My‐4. These results indicate that CD18 participates in LPS‐induced TNF‐o production as well as in regulating CD14 expression on monocytes. Addition of LPS to monocytes resulted in a reduction in the CD14 expression after 1/2, 1, 2 and 4h, but increased CD 14 expression was seen after LPS stimulation overnight. By doing double labelling of the monocyte population for CD 14 and CD16 it was found that the reduction in CD 14 expression occurred in the CD14+/CD16+ sub‐population, while the increase in CD14 expression was seen in both the CD14+/CD16+ and the CD14+/CD16+ cells. αCDU MoAbs that were able to inhibit LPS‐induced cytokine production from monocytes (3C10 and My‐4) were considerably less able to detect the increase in CD14 expression after LPS stimulation than aCD14 MoAbs that did not inhibit LPS‐induced cytokine production (Leu‐M3 and αCD14serva)‐ Our data indicate that My‐4 and Leu‐M3 define two populations of CD14+ cells on LPS stimulated human monocytes.
Relationship Between the Reactivity to Hepatitis B Virus Vaccination and the Frequency of MHC Class I, II and III Alleles in Haemodialysis Patients
Authors: STACHOWSKI J. , KRAMER J. , FÜST G. , MACIEJEWSKI J. , BALDAMUS C. A. , PETRÁNYI G. G.
Published: July 2006

To study the immunoreactivity genes in a heterogeneous human population needs a large number of individuals. Associations between HLA antigens and immunoresponse to viral or bacterial antigens have been studied with controversial results. As a homogeneous population, the MHC class I, II and III allele distribution was studied in 153 end‐stage renal disease patients (ESRD, average duration of renal replacement: 8.2+5.1 years) immunized with a recombinant hepatitis B vaccine in accordance to the standard vaccination schedule. Thirty‐four patients with an antibody titre of less than 10 U/l following the last booster injection were considered as non‐responders while 119 patients with antibody titre equal to or more than 10 U/l were considered as responders. The responder group was divided into two subgroups: low responders (antibody titre: 1000 U/l) and high responders (antibody titre: > 1000 U/ 1). Marked differences were observed between responders and non‐responders in the occurrence of carriers of different MHC class I, II and III alleles. Homozygotes for HLA—A1, HLA—B8, HLA—DR3 and HLA—DQ2 were found almost exclusively in the non‐responder group and significantly more heterozygotes for these alleles were found in the non‐responder group compared to the responders. Similar albeit less marked differences were found in the frequency of some MHC class III alleles (C4A*6, C4A*QO, Bf*F, BPS0.7). Within the responder group, carriers of HLA—A2, HLA—B7 and HLA—DR4 were found to be clustered in the low responder sub‐group whereas carriers of HLA—A1, HLA—B27, HLA—Cw2, C4A*6 and Bf*F were observed more frequently in the group of high responders. Similar differences were found with extended haplotypes as well. For example, the extended haplotypes HLA—Al, B8, BfS, C4AQO, C4B1, DR3, DQ2 and HLA—A1, B8, BfF, C4A6, C4B2, DR3, DQ2 were present in nine of 34 cases of non‐responders but only in one of 119 case of responders (P <0.000001). These observations indicate that the presence or absence of certain MHC alleles even in heterozygous form determine the responsiveness to hepatitis B vaccination in end‐stage renal disease patients, and among responders, the intensity of antibody response is also markedly influence by immunogenetic factors.
Constitutive and Inducible Cytokine mRNA Expression in the Human Mast Cell Line HMC‐1
Authors: NILSSON G. , SVENSSON V. , NILSSON K.
Published: July 2006

The discovery that mast cells are a potential source of cytokines has suggested new ways in which mast cells can act in immunological and inflammatory responses. In this study we have used the HMC‐1 cell line as a model for human mast cells to study the constitutive and inducible mRNA expression of interleukins, colony‐stimulating factors, interferons, tumour necrosis factors α and β, tumour growth factor β and platelet‐derived growth factor A and B. We found that HMC‐1 cells constitutively expressed mRNA for TNF‐α and TGF‐β, and a low level of M‐CSF. After treatment with the phorbol ester TPA or the calcium ionophore ionomycin expression of several cytokines, i. e. IL‐1β, IL‐3, IL‐6, GM‐CSF, TNF‐β and PDGF‐A, could be detected. Both TPA and ionomycin induced the same set of cytokines, but the effect of TPA was more prominent. The relative induction was calculated to be 70X for IL‐1β and IL‐3, 30X for GM‐CSF and PDGF‐A and 3 ‐ 10X for IL‐6, M‐CSF and TNF‐β. This study shows that human mast cells have the capacity to express not only cytokines mediating an immune response but also cytokines affecting other cell types, e. g. fibroblasts and endothelial cells, involved in later steps of the inflammatory response.
Structural Analysis of VH4–21 Encoded Human IgM Allo‐ and Autoantibodies Against Red Blood Cells
Authors: BØRRETZEN M. , CHAPMAN C. , STEVENSON F. K. , NATVIG J. B. , THOMPSON K. M.
Published: July 2006

We have sequenced the variable heavy chain regions of a number of VH4–21 encoded monoclonal IgM anti‐Rh(D) antibodies produced in response to deliberate immunization. These were compared with the sequences of similarly encoded IgM anti‐I cold agglutinins (CA) derived from patients with lympho‐proliferative diseases. The anti‐Rh(D) antibodies show evidence of clonal expansion and somatic diversification. Even though they are produced in response to an antigenic stimulus, they demonstrate limited hypermutation in the variable heavy chain (VH) segments and there is no evidence of selective pressure acting on the complementarity determining regions (CDRs). The CA demonstrate a higher rate of mutation and yet this results in a lower ratio of replacement to silent mutations (R:S) in the CDRs than seen in the anti‐Rh(D) antibodies. It is not clear whether the different pattern of mutations seen in the CA is related to their auto‐reactivity or their tumour origin. In both groups of antibodies the region encoded by the VH4–21 segment can be found in germline configuration at the amino‐acid level indicating that other V‐gene structures, i. e. light chains or CDRH3s, are crucial to the generation of either specificity. A role of the CDRH3 is indicated by the identification of a motif shared by four CAs and one Rh(D) antibody which also demonstrates CA activity independent of its anti‐Rh(D) specificity. Amongst the anti‐Rh(D) antibodies there seems to be an obligatory combination with VL having closest homology to the DPL16 germline segment indicating this as particularly important in generation anti‐Rh(D) specificity.
T Cell‐Mediated Cytotoxicity Against p53‐Protein Derived Peptides in Bulk and Limiting Dilution Cultures of Healthy Donors
Authors: RÖPKE M. , REGNER M. , CLAESSON M. H.
Published: July 2006

The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over‐expression of wild type and mutant determinants of the p53 protein. This process might generate MHC‐I restricted epitopes for T cell recognition and p53‐derived peptides have been suggested as targets for tumour‐specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53‐peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA‐A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA‐A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53 peptides as stimulator cells. T cell reactivity was observed towards both wild type and mutated p53 peptide epitopes with CTL precursor frequencies varying from 1: 2 × 104 to 1: 1.5 × 105. These results might suggest the presence of an ongoing immune response in normal individuals against cells expressing increased levels of p53 protein.
Lymphocytic Choriomeningitis Virus Infection is Associated with Long‐Standing Perturbation of LFA‐1 Expression on CD8+ T Cells
Authors: ANDERSSON E. C. , CHRISTENSEN J. PRAVSGAARD , SCHEYNIUS A. , MARKER O. , THOMSEN A. RANDRUP
Published: July 2006

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long‐standing up‐regulation of LFA‐1 expression on CD8+, but not on CD4+ T cells. Appearance of CD8+ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4+ T cells or their products. Cell sorting experiments defined virus‐specific CTL to be included in this population (LFA‐1hiMEL‐14lo), but since about 80% of splenic CD8+ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8+LFA‐lhi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL‐2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA‐lhi subset and, correspondingly, the ligand ICAM‐1 was found to be up‐regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV‐primed donor splenocytes with anti‐LFA‐1 markedly inhibited the transfer of virus‐specific delayed‐type hypersensitivity to naive recipients. Together, these findings indicate that up‐regulation of LFA‐1 expression is a critical factor involved in directing activated CD8+ T cells to sites of viral infection.
Influence of CD 14, LBP and BPI in the Monocyte Response to LPS of Different Polysaccharide Chain Length
Authors: JAHR T. G. , SUNDAN A. , LICHENSTEIN H. S. , ESPEVIK T.
Published: July 2006

In this study we examined the involvement of human serum, recombinant lipopolysaccharide binding protein (rLBP), recombinant (r)CD14, CD 14 antibodies and recombinant bactericidal permeability‐increasing factor (rBPI) in the induction of TNF by Salmonella minnesota LPS of different polysaccharide chain lengths. Soluble rCD14 and rLBP markedly enhanced LPS 6261 TNF production and to a lesser degree also enhanced TNF production from Re 595 LPS and lipid A DP. Addition of anti‐CD 14 antibodies resulted in nearly complete inhibition of LPS 6261‐induced TNF production and partial inhibition of Re 595 LPS and Hpid A DP‐induced TNF release. The ability of lipid A MP to induce TNF production increased with addition of rCD14. Addition of rLBP or anti‐CD 14 antibodies had no detectable effect on lipid A MP‐induced TNF production. The effect of rBPI was also tested and the results showed that only the TNF‐inducing ability from smooth LPS was completely inhibited by rBPI. Recombinant BPI was considerably less effective in inhibiting Re 595 LPS‐induced TNF production, and lipid A DP was not affected by rBPI. Our data suggest that the ability of rLBP, rCDI4, CD14 antibodies and rBPI to modulate LPS induced TNF production is strongly dependent on the LPS polysaccharide chain length.
TNF‐α Dominates Cytokine mRNA Expression in Lymphoid Tissues of Rats Developing Collagen‐ and Oil‐Induced Arthritis
Authors: MÜSSENER Å. , KLARESKOG L. , LORENTZEN J. C. , KLEINAU S.
Published: July 2006

Experimental arthritis can be induced in the DA rat strain with rat type II collagen (RCII) administered in Freund's incomplete adjuvant oil (FIA) or with only FLA. If ovalbumin (Ova), is added to these arthritogens the development of arthritis is blocked. To investigate the mechanisms responsible for induction of arthritis, as well as inhibition of arthritis, a kinetic study of the local cytokine expression in lymph nodes has been performed after immunization with the above mentioned agents. By using in situ hybridization techniques, mRNA expression of TNF‐α, IL‐2, IFN‐γ and IL‐4 was determined. The results show a rapid and pronounced accumulation of TNF‐α mRNA expression, in RCII/FIA and FIA immunized rats. This pronounced expression of TNF‐α mRNA was not recorded in the Ova/FIA immunized animals, which instead were the only animals in which the IL‐4 gene was expressed. The expression of IFN‐γ mRNA was limited in RCII/FIA‐ and FIA‐immunized rats, whereas IL‐2 mRNA expression was detected only after RCII/FIA injection. Lymph node cells from RCII‐immunized animals generated a high amount of TNF‐α mRNA after restimulation with RCII, whereas restimulation with the mitogen Con A generated a cytokine mRNA response dominated by IL‐2 and IFN‐γ.

These and other results indicate that a strong local expression of TNF‐α, induced by arthritogenic stimuli, may be important for the induction of arthritis. Moreover, the elicitation of an immune reaction against Ova, may inhibit arthritis development by contributing to a shift in the initial arthritogenic cytokine response.
ADCC‐Mediating Capacity in Children with Cow's Milk Protein Intolerance in Relation to IgG Subclass Profile of Serum Antibodies to β‐Lactoglobulin
Authors: SAALMAN R. , DAHLGREN U. I. , FÄLLSTRÖM S. P. , HANSON L. A. , WOLD A. E. , AHLSTEDT S.
Published: July 2006

In a previous study sera from children with cow's milk protein intolerance (CMPI) exhibiting gastrointestinal symptoms were found to efficiently induce antibody‐dependent cell‐mediated cyto‐toxicity (ADCC) to β‐lactoglobulin‐coated cells. In contrast, sera from children with coeliac disease showed a low ADCC‐mediating capacity, despite high levels of IgG anti‐β‐lactoglobulin antibodies. The study described here was undertaken to evaluate whether differences in IgG subclass profile of anti‐β‐lactoglobulin antibodies could explain the observed variations in the ADCC‐mediating capacity. Forty‐eight sera from the following groups of children were investigated: CMPI with predominantly gastrointestinal symptoms, CMPI with skin symptoms of immediate‐onset, children with untreated coeliac disease and healthy references. Absorption experiments indicated that primarily IgGl antibodies were responsible for the ADCC‐mediating capacity of the sera. Accordingly, the ADCC reactivity of individual sera correlated with their IgGl antibody levels. Sera from CMPI children with gastrointes tinal symptoms, most of which had a high ADCC reactivity, also demonstrated a distinctive subclass pattern of their anti‐β‐lactoglobulin antibodies with higher relative proportions of IgGl (ratios: IgGl/IgG, IgGl/IgG3 and IgGl/IgG4) than those from the other diagnostic groups. Using logistic regression analysis, the diagnostic potential of ADCC as well as of different IgG subclass variables for the recognition of gastrointestinal symptoms caused by CMPI was evaluated. The ADCC reactivity of sera was found to be the best predictor in this model.
Gliadin‐Specific T Cell Responses in Peripheral Blood of Healthy Individuals Involve T Cells Restricted by the Coeliac Disease Associated DQ2 Heterodimer
Authors: JENSEN K. , SOLLID L. M. , SCOTT H. , PAULSEN G. , KETT K. , THORSBY E. , LUNDIN K. E. A.
Published: July 2006

Coeliac disease (CD) is probably caused by an abnormal immune response towards wheat gliadin in the small intestine. We found that gliadin‐specific T cells from the small intestinal mucosa of HLA‐DQ2 positive CD patients were almost exclusively restricted by the disease‐associated DQ2 molecule. In the peripheral blood of CD patients, a large proportion of gliadin‐specific T cells were found to be restricted by DQ molecules, including DQ2, but many were instead restricted by DR or DP molecules of the patient. We have now investigated gliadin‐specific T cell responses in peripheral blood from healthy individuals. Four of 20 persons tested had strong in vitro responses and were used as donors for gliadin‐specific T cell clones. We found gliadin‐specific T cells restricted by the CD‐associated DQ2 molecule in peripheral blood for two of these four individuals. It is the presence of such T cells also in the small intestinal mucosa which seems typical of CD.
Enhancement of Interleukin‐6 Production by Fibrinogen Degradation Product D in Human Peripheral Monocytes and Perfused Murine Liver
Authors: MANDL J. , CSALA M. , LÉRÁNT I. , BÁNHEGYI G. , BIRÓ J. , MACHOVICH R. , FALUS A.
Published: July 2006

The effect of fibrinogen degradation products D and E (FDP—D, FDP—E) on IL–6 production in perfused mouse livers and peripheral monocytes is studied. Similarly to bacterial endotoxin FDP—D is highly potent to augment the IL–6 production measured in perfused mouse livers, while FDP—E is not stimulatory. FDP—D but not FDP—E is able to stimulate the in vitro IL–6 production of human peripheral monocytes, as well. Plasmin alone is almost ineffective on IL–6 production both in perfused livers and monocytes. Our findings suggest a direct positive feedback circuit, among fibrinogen, FDP and IL–6.
The Aetiology of Mixed Cryoglobulinaemia Associated with Hepatitis C Virus Infection
Authors: AGNELLO V.
Published: July 2006

A strong association of hepatitis C infection (HCV) with ‘essential’ mixed cryoglobulinaemia has been established. The demonstration of HCV in Type II mixed cryoglobulins with monoclonal rheumatoid factors (mRF) that bear the WA crossidiotype has lead to the hypothesis that mixed cryoglobulins result from chronic stimulation by HCV‐lipoprotein of a population of Xld WA+B‐la cells. The reactivity of WA IgM intially produced is with the HCV‐self antigen complex with RF activity resulting secondarily from the pausi‐mutational process accompanying the T cell independent process. This benign proliferation progreses by multi step mutations to malignancy in a minority of patients. The implications of the hypothesis for understanding the physiology of certain natural auto antibodies and for therapeutic invervention in this disease are discussed.
The Role of Thymocytes in Regulating Thymic Epithelial Cell Growth and Function
Authors: MEILIN A. , SHOHAM J. , SCHREIBER L. , SHARABI Y.
Published: July 2006

The basic tenet underlying the present work and supported by recent studies is that there is a dialogue between developing thymocytes and thymic stromal cells. One direction in this dialogue, i. e. thymic stromal cell role in shaping thymocyte maturation, has been extensively studied. The other direction, thymocyte effect on stromal cell development and function, started to emerge only recently on the basis of in vivo observations in SCID and knockout mice. An in vitro approach to the analysis of this interaction may add substantial insight into the process, as demonstrated by the present work. We made use of a culture system of either murine thymic epithelial cells (TEC line) cultured alone or cocultured with thymocytes. Unstimulated thymocytes or their supernatant caused 40–80% inhibition of TEC cell proliferation, as measured by 3H‐thymidine incorporation. Cell cycle analysis by flow cytometry indicated that this inhibition can be attributed to reduction in G2/M phase cell number pari passu with an increase in Go/G1 cell number. This inhibitory effect was found to be partially mediated by TGF‐β produced by thymocytes. On the other hand, thymocytes augmented IL‐6 production by TEC cells in coculture, an effect which could not be reproduced by thymocyte culture supernatant and was not inhibited by thymocyte pretreatment with formaldehyde or emetine. Furthermore, antibodies against thymocyte adhesion molecules (CD2, LFA‐1) blocked the thymocyte‐induced IL‐6 secretion. IL‐6 was found to be an autocrine growth factor of TEC in culture, since a combination of anti IL‐6 and anti IL‐6 receptor antibodies caused 70% inhibition of TEC proliferation and addition of exogenous recombinant IL‐6 doubled the rate of proliferation. These results suggest that thymocytes regulate thymic epithelial cell growth by a complex set of inhibitory and enhancing signals mediated through either soluble factors or direct contact. The ultimate effect is dependent on the balance between different signals and may be different in different microenvironmental settings in vivo. In coculture in vitro the dominant effect was growth inhibition of the epithelial cells by thymocytes.
Platelets Contain Interleukin‐1 Alpha and Beta which are Detectable on the Cell Surface After Activation
Authors: SEDLMAYR P. , BLASCHITZ A. , WILDERS‐TRUSCHNIG M. , TIRAN A. , DOHR G.
Published: July 2006

Activated platelets have been shown previously to exhibit membrane‐bound IL‐1 bioactivity, which leads to the question of localization of the cytokine in platelets. Using immunocytological and flow cytometric techniques, we found IL‐la and IL‐Iβ in the cytoplasma of both resting and thrombin‐activated platelets. Immunogold‐silver staining of the cell surface of activated platelets as well as pre‐embedding antibody treatment of platelets revealed the presence of IL‐1 (α and β) in low density on the surface of intact cells in contrast to distinct enrichment in the cytoplasma of damaged platelets. Fibrin fibres present between cells indicated adsorbance of IL‐1. There was also weak binding of anti‐IL‐1α to the surface of thrombin‐activated platelets as shown by flow cytometry. Following activation there appears to be some transfer of IL‐1 onto the cell surface of activated cells, the bulk of the cytokine, however, is probably not released prior to platelet disintegration. In summary, we present evidence for the presence of both IL‐1α and IL‐1β in resting and activated platelets without being able to demonstrate localization of the cytokines to specific subcellular structures.
Construction and Adhesive Properties of a Soluble MAdCAM‐1‐Fc Chimera Expressed in a Baculovirus System: Phylogenetic Conservation of Receptor‐Ligand Interaction
Authors: YANG Y. , SAMMAR M. , HARRISON J. E. B. , LEHNERT K. , PRINT C. G. , LEUNG E. , PRESTTDGE R. , KRISSANSEN G. W.
Published: July 2006

MAdCAM‐1 is a high endothelial venule adhesion molecule composed of immunoglobulin and mucin‐like domains which binds the leucocyte integrin LPAM‐1 (α4β7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mouse MAdCAM‐1 which is normally membrane bound has been produced by joining the extracellular region of the receptor to the Fc domain of human IgGl. The MAdCAM‐1‐Fc cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda insect cells infected with the recombinant virus produced MAdCAM‐1‐Fc as a disulfide‐linked homodimer of 82kDa polypeptides, which was secreted into the culture medium at > 1 μg/ml. The product purified by Protein G‐Sepharose was identified as authentic MAdCAM‐1‐Fc by the anti‐MAdCAM‐l monoclonal antibody (MoAb) MECA‐367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in supporting the binding of mouse α4β1+α4β7+ mesenteric lymph node lymphocytes, and the α4β1−αβ7+ TK1 T cell lymphoma. Binding was enhanced by Mn++ ‐induced integrin activation, and specifically blocked by anti‐integrin α4 subunit and anti‐MAdCAM‐1 MoAbs. Binding was blocked by pretreatment of cells with sodium azide, and EDTA, indicating that binding is an energy‐dependent process which requires divalent cations. Thus the mouse MAdCAM‐1‐Fc chimera produced in insect cells retains certain functional properties that typify the native receptor, and should be valuable in analysing the role of MAdCAM‐1 in lymphocyte recirculation and emigration. However it was not sialylated despite being post‐translational modified with N‐ and O‐linked carbohydrate moieties, suggesting that the ability of MAdCAM‐1 to support cell adhesion under static conditions is sialylation‐independent. A rabbit polyclonal antibody raised against the entire cytopiasmic domain of the human integrin β7 subunit recognized LPAM‐1‐like molecules in human, rat, and mouse cells, suggesting a high degree of conservation of the MAdCAM‐1 receptor across species. In agreement with this notion MAdCAM‐1‐Fc immobilized on glass was fully functional in supporting the cation‐dependent binding of peripheral blood or spleen cells from a range of other species including human, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by o4 integrins.
The Influence of Non‐HLA Genes on the Human T‐Cell Receptor Repertoire
Authors: AKOLKAR P. N. , GULWANI‐AKOLKAR B. , ROBINSON M.‐A. , SILVER J.
Published: July 2006

We previously demonstrated a central role for HLA genes in determining the T‐cell receptor (TCR) repertoire. However, these studies also suggested that other genetic factors might also play a role in the development of this repertoire. In order to assess the role of non‐HLA genes in the development of the TCR repertoire, we have analysed and compared the TCR repertoires of individuals in three families consisting of both monozygotic twins as well as an HLA‐identical sib. TCR repertoire analysis was performed with both V‐segment‐specific MoAb and the polymerase chain reaction using TCRBV segment‐specific oligonucleotide primers. We observed that in every case the TCR repertoires of identical twins were more similar to each other than to their HLA‐identical sib. Furthermore, in one family we were able to show by genotype analysis that most of the differences in repertoire between the identical twins and their HLA‐identical sib were caused by polymorphisms in the TCR genes that influence expression levels. These studies document an important role for non‐HLA genes in determining the TCR repertoire in man and raise the possibility that such TCR polymorphisms may play a signiflcant role in determining disease susceptibility.
Alterations of the Post Transplant Blood Lymphocyte Phenotype Subsets as a Marker of Rejection in Renal Allograft Recipients
Authors: WRAMNER L. , MJÖRNSTEDT L. , BLOHME I. , TUFVESON G. , SöDERSTRöM T. , OLAUSSON M.
Published: July 2006

The postoperative alterations of absolute levels of lymphocyte phenotype subsets in peripheral blood were studied in recipients of living donor renal allografts and in kidney donors. The results were expressed as per cent changes of the preoperative values. The lymphocyte subsets, CD3, CD4 and CD8 cells, decreased to approximately 50% following the surgical trauma, with rapid recovery to preoperative levels within 1 week in kidney donors and in recipients without rejection episodes. In contrast, the T‐cell levels in recipients with rejection episodes remained low after 1 week, before clinical signs of rejection, and was predictive for the later occurrence of acute rejection episodes. The T‐cell levels in the recipients with rejection episodes remained low during the first 6 weeks, maybe due to the rejection treatments given during this period. The B‐lymphocytes were not affected in any of the recipient groups. The alterations observed were not explained by CMV infections, which occurred mainly after the observation period of 6 weeks. In conclusion, the operation per se induced alteration in circulating T‐lymphocyte subsets and low T‐cell levels after 1 week were predictive of rejection episodes.
High Endothelial Cells of Postcapillary Venules Express the Scavenger Receptor in Human Peripheral Lymph Nodes
Authors: GENG Y.‐J. , HANSSON G. K.
Published: July 2006

The macrophage scavenger receptor (MSR) is a multipotent receptor that mediates uptake of macromolecules with clustered negative charges, such as endotoxins and oxidized proteins and lipoproteins. It may also serve as an adhesion molecule for monocytes and macrophages. We have analysed MSR expression in human peripheral lymph nodes and show, by reverse transcription‐PCR, that the type I isoform of MSR is abundantly expressed in this tissue. With the use of a new set of isoform‐specific peptide antibodies, both isoforms of MSR were detected in the cuboidal endothelium of high‐endothelial venules (HEV). MSR antibodies also stained macrophages and follicular dendritic cells of germinal centres. These data indicate that the MSR gene is expressed in HEV endothelium in vivo. It is suggested that MSR expression may be important for antigen uptake and leucocyte adhesion to HEV in the lymph node.
T Cell‐Dependent Antibody Response to Staphylococcal Enterotoxin B
Authors: WILLIAMS O. , AROEIRA L. S. , MENGEL J.
Published: July 2006

Treatment of mice with staphylococcal enterotoxin B (SEB) induces specific T‐cell tolerance to this superantigen, characterized by partial deletion of Vβ8+ T cells in vivo and T cell anergy in vitro. In this study we examined the humoral response to SEB in BALB/c mice. Immunization of mice with SEB results in a detectable anti‐SEB antibody response. Upon further treatment of mice with SEB, specific antibody levels increase significantly and the response is accelerated—characteristics of a secondary humoral response. The secondary antibody response is T cell dependent, can be transferred to T cell deficient mice with splenocytes and is composed mainly of IgM, IgG1 and IgG2b isotypes, suggesting that Th2 cells provide B cell help in this response. These data demonstrate that at the same time as inducing in vitro unresponsiveness, SEB primes SEB‐specific T helper cells to provide help for B cells in a secondary antibody response.
β‐Endorphin Enhances Phagocytosis of Latex Particles in Mouse Peritoneal Macrophages
Authors: ICHINOSE M. , ASAI M. , SAWADA M.
Published: July 2006

The effects of β‐endorphin (βEnd) on phagocytosis in peritoneal macrophages were examined by using flow cytometry (FCM). βEnd enhanced phagocytosis in a dose‐dependent manner. Leucine—enkephalin (Leu‐Enk), methionine—enkephalin (Met—Enk), α‐endorphin (αEnd), γ‐endorphin (γEnd), αEnd (18–31) and βEnd (28–31) had no such activity. βEnd (1–27) and βEnd (6–31) enhanced phagocytosis less effectively than βEnd did. Naloxone did not inhibit the enhancement of phagocytosis induced by βEnd. Unstimulated control phagocytosis was partially suppressed in Ca2+‐free EGTA‐containing solution and even in this solution βEnd enhanced phagocytosis. However, the enhancement was suppressed in the solution containing BAPTA‐AM. The present study showed that βEnd enhanced extracellular Ca2+ ([Ca2+]o)‐dependent and ‐independent phagocytosis and that the enhancement is largely dependent on intracellular Ca2+ ([Ca2+]i). These results support the contention that βEnd is one of the mediators that modulates the immune system.
Identification of H‐2Kb Binding and Immunogenic Peptides from Human Papilloma Virus Tumour Antigens E6 and E7
Authors: BAUER S. , HEEG K. , WAGNER H. , LIPFORD G. B.
Published: July 2006

Peptides can be used to induce MHC class I restricted cytotoxic T cells (CTL) tbrough in vivo immunization. This approach may enable the development of peptide vaccination schemes for immunization against viral infection in humans. Human papillomavirus (HPV) is one of a few viruses associated with human cancer and the development of an anti‐cancer vaccine seems possible. As a model approach, we searched the E6 and E7 proteins of the human papillomavirus type 16 for possible murine MHC class I restricted peptide epitopes. We utilized the mouse H2‐Kb peptide binding motif which consists of phenylalanine or tyrosine at position five and leucine at the carboxy‐terminus with the modification that leucine could be replaced by other aliphatic but non‐aromatic amino acids. Four peptide sequences from E6 and two from E7 were selected. These peptides were tested for their ability to bind and stabilize Kb and for their immunogenicity in vivo. It was shown tbat one peptide from E6, E6.1 (50–57), bound Kb, but was not able to prime mice in vivo. In contrast, the two selected E7 peptides E7.1 (21–28) and E7.2 (48–55) bound Kb and were immunogenic in vivo. The peptide induced CTL lysed syngeneic EL‐4 cells transfected with the open reading frame of E7 but not vector only transfectants. This implies tbat both peptides were naturally processed and presented by Kb on the surface of target cells. MHC class I peptide binding motifs therefore appear to be an effective and useful tool to predict peptide epitopes of proteins associated with cancer.
Administration of Antibodies to Hyaluronanreceptor (CD44) Delays the Start and Ameliorates the Severity of Collagen II Arthritis
Authors: VERDRENGH M. , HOLMDAHL R. , TARKOWSKI A.
Published: July 2006

Hyaluronanreceptor (CD44) has been shown to be involved in lymphocyte homing during normal leucocyte circulation and during leucocyte extravasation into sites of tissue inflammation. In addition, interaction with CD44 molecule induces T‐cell activation and production of cytokines, such as interferon‐γ. In this study we have examined what influence interaction with the CD44 receptor would have on collagen II‐induced arthritis in mice. Mice were immunized with rat collagen II and administered with injections of a monoclonal anti‐CD44 antibody. Seventeen days after the outbreak of the disease, all of the anti‐CD44 treated animals remained clinically healthy, whereas 37% of the controls displayed arthritis (P<0.001). Ten days later the prevalence of arthritis was 26% and 65% (P<0.05), respectively. Furthermore, the severity of the arthritis was significantly ameliorated by the anti‐CD44 treatment. Serum levels of interferon‐γ were significantly higher in collagen II immunized animals having been treated with anti‐CD44, compared to the controls. Delayed‐type hypersensitivity (DTH) response was significantly decreased in the anti‐CD44 treated animals, indicating a functional suppression of T cells. In contrast, T cell independent experimental inflammation was not affected by the administration of CD44 antibodies. Our results suggest that interaction with CD44 down‐regulates T lymphocyte/monocyte mediated inflammatory reaction, possibly by triggering of interferon‐γ release.
Proliferation of IL‐2 Activated Lymphocytes Preferably Occurs in Aggregates by Cells Expressing the CD57 Antigen
Authors: VOLLENWEIDER I. , LAZZARATO M. , GROSCURTH P.
Published: July 2006

Aggregates of lymphocytes were investigated in long‐term cultures of IL‐2 stimulated peripheral blood mononuciear cells. Cells in clusters formed broad contact areas where coated pits and vesicles were often detectable. Proliferating cells were preferably found in aggregates indicating that the cell clusters represent proliferation centres in which cell division may be promoted by accessory signal transduction mediated by the close cell‐to‐cell contact. In bulk cultures CD57+ cells yielded increased proliferation capacity compared to CD57‐ cells. In addition, CD57+ cells were preferably localized in aggregates where they occupied the same position at the periphery of the clusters than the proliferating cells, suggesting that both cell types may be identical. It is discussed that the CD57 antigen represents a differentiation marker which is upregulated when CD57‐ cells start to proliferate assisted in cell aggregates.
Variable Region Usage in B Cell Deficient Mice in a Model of Experimental Herpes Simplex Virus Retinitis
Authors: ARRUNATEGUI‐CORREA V. R. , DUTT J. E. , FOSTER C. S.
Published: July 2006

By using PCR, we have found previously that differences in T cell receptor alpha/beta chain variable region (TCR α/β) gene expression in lymph nodes (LN) existed between susceptible and resistant mice following anterior chamber (AC) inoculation with herpes simplex virus (HSV). We now report and compare the immunoglobulin gene variable region (Ig VH) and the TCR Vβ mRNA expression in normal congenic mice (BALB/c and C. B‐17) and also in B cell modulated C. B‐17 mice (B‐). RNA prepared from spleen and LN of these mice before and after HSV‐AC inoculation was analysed by PCR using oligoprimers specific for 11 VH gene families and 19 Vβ gene families. Densitometry analysis revealed that VH family mRNA expression levels in spleen and lymph nodes did not correlate with HSV susceptibility or resistance patterns in B‐, BALB/c and C. B‐17 mice. Analysis of TCR Vβ chain genes showed that spleen of resistant C. B‐17 and B‐ Ab transferred mice showed a preference for Vβl 1 gene expression not seen in susceptible mice. In contrast, LN of BALB/c and B‐, both susceptible mice, made TCR Vβ transcripts that were indistinguishable from those generated by resistant C. B‐17 mice, following HSV‐AC inoculation. Finally, TCR Vβ gene family repertoire appears to be severely diminished in uninfected B‐ mice (50% in LN and 45% in spleen) by anti‐μ treatment.
Metalloproteases and Serineproteases are Involved in the Cleavage of the Two Tumour Necrosis Factor (TNF) Receptors to Soluble Forms in the Myeloid Cell Lines U‐937 and THP‐1
Authors: BJÖRNBERG F. , LANTZ M. , GULLBERG U.
Published: July 2006

The proteolytic processing of the two TNF receptors (TNF‐R55 and TNF‐R75) into soluble forms was investigated in the myeloid cell lines U‐937 and THP‐1. Phorbol myristate acetate (PMA) rapidly stimulated release of soluble forms of both TNF‐receptors. Incubations were made with PMA and protease inhibitors directed against different target protease groups. The serineprotease inhibitors TPCK and dichloroisocoumarin and the metalloprotease inhibitor 1, 10‐phenanthroline reduced PMA‐induced release of both soluble receptor forms with about 60–70%. Furthermore, 1, 10‐phenanthroline also reduced PMA‐induced down‐regulation of TNF‐receptors in both cell lines as judged by TNF‐binding to cells. Reduced down‐regulation and TNF‐receptor shedding by 1, 10‐phenanthroline was reversed by Zn2+, indicating involvement of a Zn2+‐dependent metalloprotease. Thus, both serine proteases and metalloproteases are involved in the processing of TNF‐receptors.
Allosteric and Temperature Effects on the Plasma Protein Binding by Streptococcal M Protein Family Members
Authors: CEDERVALL T. , ÅKESSON P. , STENBERG L. , HERRMANN A. , ÅKERSTRÖM B.
Published: July 2006

Most group A Streptococcal strains bind immunoglobulins (Ig) and fibrinogen to their cell walls. It is shown in this paper that the Ig‐binding of three different strains was much weaker at 37°C than at room temperature (20°C), whereas the fibrinogen binding was unaffected by temperature. The binding properties and molecular sizes of two purified group A Streptococcal cell surface proteins from the M protein family were studied at various temperatures, M1 protein with affinity for IgG, fibrinogen and albumin, and protein Sir22 with affinity for IgA and IgG. Both proteins appeared as monomers which bound all their ligands, including fibrinogen, very weakly at 37°C, and as strongly binding dimers at 10 and 20°C. Furthermore, the results demonstrated that the plasma protein binding of the bacterial proteins was allosterically regulated, i. e. the binding of a ligand to one site modulated the binding of a ligand to a second site. For example, the binding of albumin or IgG to purified M1 protein at 10 and 20°C strongly enhanced the binding of fibrinogen at 37°C. This indicates that the high affinity dimer form of the bacterial proteins can be stabilized at 37°C, a possible explanation for the strong fibrinogen binding of whole bacteria. Finally, the sizes and binding properties of three M1 protein fragments were studied and the results indicated that the centrally located C‐repeats, which are conserved among the members of the M protein family, are important for the formation of the high‐affinity dimers of the bacterial proteins.
T‐Cell Receptor Jβ Gene Segment Usage in Immature and Mature Human Thymocytes
Authors: JEDDI‐TEHRANI M. , HODARA V. , ESIN S. , GRUNEWALD J. , WIGZELL H. , ANDERSSON R.
Published: July 2006

Immature double positive (DP, CD4+CD8+) and mature single positive (SP, CD4+CD8− and CD4−CD8+) human thymocytes from nine thymi were analysed for their complete patterns of relative TCR Jβ multigene member usage in relation to six rearranged Vβ family exons (Vβ 5.1, 6.1–3, 8, 9, 12 and 18). Each sample tested contained mRNA transcripts corresponding to all potential Vβ(Dβ)Jβ combinations. Individual Jβ gene segments were expressed in a similar, highly non‐random manner both in SP and DP thymocytes, irrespective of original genomic position of the individual associated Vβ exon. In addition, ranges of family usage and frequency of individual over‐representations of Jβ gene segments, as determined in DP and SP thymocyte populations, displayed no significant differences. Upon comparison of DP and SP thymocytes, however, a discrepancy in one aspect of Jβ gene utilization was established: decreasing Jβ family 1/ Jβ family 2 ratios were determined to be positively correlated with increasing maturity of thymocytes, a condition further supported by data previously obtained from studies of PBL T cells. At the individual Jβ gene level, the observed gradual modification of the relative family usage can largely be explained by a significant shift from a higher Jβ 1.1 /Jβ 2.7 ratio in DP to a higher Jβ 2.7/Jβ 1.1 ratio in SP thymocytes. Altogether, the present results imply that selectional processes in the thymus appear to have only minor consequences on the distribution pattern of expressed Jβ exons. Hence, the disproportionate pattern of TCR Jβ gene usage seems to be established mainly at the recombinatorial level followed by minor adjustments during thymic and post‐thymic events.
Dysregulation of T Helper Cell Cytokines in Autoimmune Prone NZB x NZW Fl Mice
Authors: LIN LONG‐CHYI , CHEN YEN‐CHEN , CHOU CHEN‐CHENG , HSIEH KUE‐HSIUNG , CHIANG BOR‐LUEN
Published: July 2006

Multifactorial involvement in the pathogenesis of autoimmune NZB/W F1 mice has been well documented. To further elucidate the role of cytokines in the disease development of murine lupus, single spleen cells isolated from NZB/W Fl and non‐autoimmune C57BL/6 mice were stimulated with T cell mitogens or anti‐CD3 antibody at pre‐determined optimal concentration. Supernatants were collected and assayed for production of cytokines including IL‐2, γ‐IFN, IL‐3, IL‐4, IL‐5 and IL‐10. In both young and old mice, cytokine profiles by mitogen‐stimulated T cells showed higher TH2 (type 2 T helper) cell‐related cytokine production in NZB/W Fl mice compared to those in non‐autoimmune C57BL/6 mice. In contrast, cytokines produced by TH1 (type 1 T helper) cells, such as γ‐IFN and IL‐2, were lower in NZB/W F1 mice by stimulation with either mitogen or anti‐CD3 antibody. In addition, cytokine production at different time points also demonstrated decreased γ‐IFN and increased IL‐4 levels by anti‐CD3 stimulated splenic cells in autoimmune NZB/W F1 mice. Furthermore, the IL‐10 levels produced by lipopolysaccharide (LPS)‐stimulated splenic and peritoneal exudate cells were higher in young NZB/W Fl mice compared to those in C57BL/6 mice. Our data suggest that dysregulation between TH1 and TH2 cells may play an important role in the pathogenesis of autoimmunity in NZB/W F1 mice.
Immunophenotypic and Functional Characteristics of Haemopoietic Cells from Human Cord Blood
Authors: MILOSEVITS J. , PÓCSIK É. , SCHMIDT B. , REMÉNYI P. , INTÖDI Z. S. , RÉTI M. , BÁTAI Á. , ILLÉS P. , MIHALIK R. , PETRÁNYI G. G. , PÁLÓCZI K.
Published: July 2006

Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL‐2, IFN‐γ and IL‐6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3+ T cells, CD4+ T subpopulation, activated T cells and CD3+ CD16/CD56+ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA‐DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27‐ and 5‐fold reduction was observed in the production of IFN‐γ by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL‐2 in PHA‐stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3+ T cells. However, an increase was observed in the production of IL‐6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.