bluuebunny/crossref_metadata_2025 huggingface dataset:
A quantitative assay to measure the interaction between immunogenic peptides and purified class I major histocompatibility complex molecules
Authors: Olsen Anna Catharina , Pedersen Lars ØStergaard , Hansen Anette Stryhn , Buus Søren , Nissen Mogens Holst , Olsen Marianne , Hansen Paul Robert , Holm Arne
Published: March 2007

A direct and sensitive biochemical assay to measure the interaction in solution between peptides and affinity‐purified major histocompatibility complex (MHC) class I molecules has been generated. Specific binding reflecting the known class I restriction of cytotoxic T cell responses was obtained. Adding an excess of β2‐microglobulin (β2m) significantly increased the rate of peptide association, but it did not affect the rate of dissociation. Binding was complicated by a rapid and apparently irreversible loss of functional MHC class I at 37°C which might limit the life span of empty MHC class I thereby preventing the inadvertent exchange of peptides at the target cell surface. All class I molecules tested bound peptides of the canonical octa‐ to nona‐meric length. However, one class I molecule, Kk, also bound peptides, which were much longer suggesting that the preference of class I molecules for short epitopes is not absolute and may be caused by factors other than the peptide‐MHC class I binding event itself.
Transcriptional regulation of the gene for the second component of human complement: Promoter analysis
Authors: Sullivan Kathleen E. , Valle David , Winkelstein Jerry A. , Wu Lai‐Chu , Campbell R. Duncan
Published: March 2007

The 5′ flanking region of the human gene for the second component of complement was sequenced and analyzed functionally. RNase protection demonstrated a cluster of four initiation sites in the 5′ flanking region utilized in the hepatoma cell line, HepG2. Utilization of all four initiation sites increased in response to γ‐interferon (IFN‐γ). Transient transfection analysis was used to examine cis‐acting sequence motifs controlling transcription from the 5′‐flanking region. We identified a 228‐bp minimal promoter fragment which was able to direct basal and IFN‐γ inducible transcription from authentic initiation sites. Sequence motifs outside of this region may modulate the transcriptional regulation of the second component of complement. Although complement components are not coordinately regulated, we identified four regions of significant homology with the promoters of multiple other complement components. Three of these regions were within the minimal promoter fragment.
A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice
Authors: Miyawaki Shigeki , Nakamura Yutaka , Suzuka Hirotsugu , Koba Masahiro , Shibata Yoshihisa , Yasumizu Ryoji , Ikehara Susumu
Published: March 2007

We have found a new spontaneous autosomal recessive mutation in mice that causes a systemic absence of lymph nodes and Peyer's patches. The name “alymphoplasia”, with the gene symbol “uly”, is proposed for this mutant. The spleen of alylay mice is devoid of well‐defined lymphoid follicles, and the thymus does not show a clear cortical‐medullary distinction. The mutant homozygotes are deficient in both humoral and cell‐mediated immune functions, and are highly susceptible to infections. They have a reduced level of IgM and severely depressed levels of IgG and IgA in their sera, and do not reject allogeneic skin grafts. However, they have mature T and B cells as determined from their cell surface antigens. The results of bone marrow transplantation experiments suggest a mesenchymal disorder as a possible cause of the lack of lymph nodes and of immunodeficiency in the aly mouse. The aly mutant mouse may be a useful animal model of primary immunodeficiency, as are the nu (nude) and scid (severe combined immunodeficiency) mice.
Human monocytes are stimulated for nitric oxide release in vitro by some tumor cells but not by cytokines and lipopolysaccharide
Authors: Zembala Marek , Siedlar Maciej , Pryjma Juliusz , Marcinkiewicz Janusz
Published: March 2007

Nitric oxide (NO) has been recently identified as a potent mediator of tumoricidal activity of activated macrophages. Macrophages can be activated for tumor cell killing by microbial products, including lipopolysaccharide (LPS) and various cytokines. Here we report that in contrast to mouse macrophages, human peripheral blood monocytes stimulated with cytokines or LPS failed to release NO. Also priming of monocytes with interferon‐γ followed by activation with cytokines or LPS did not cause NO secretion. However, monocytes responded with NO production to stimulation with some human cancer cells but not with untransformed cells. NO production by monocytes was inhibited by NG‐monomethyl‐L‐arginine, specific inhibitor of NO synthase and emetine, an irreversible blocker of protein synthesis. This may imply that human monocytes are unique in their restricted capacity to produce NO following interaction with some tumor cells, but not with other stimulators, and in this respect they may be able to distinguish between malignant and normal cells.
B cell emigration directly from the cortex of lymphoid follicles in the bursa of Fabricius
Authors: Paramithiotis Eustache , Michael J. H. Ratcliffe
Published: March 2007

In the peripheral blood (PBL) of juvenile chickens three populations of B cells have previously been distinguished based on life‐span and origin of cells within each population. In this report we show that the largest PBL B cell subset, population 1 B cells, which are short‐lived cells corresponding to about 60% of PBL B cells and the vast majority of bursal emigrants, exit from the bursa directly from the follicular cortex. This conclusion is based on the specific labeling of rapidly dividing cortical lymphocytes with bromodeoxyuridine, followed by their detection in the periphery prior to the appearance of bromodeoxyuridine labeled cells in the bursa medulla. Furthermore, the rate of emigration of cortical lymphocytes, 1.00 ± 0.1% of PBL B cells per hour, is indistinguishable from the emigration rate of B cells from the bursa as a whole. The anatomical organization of the bursa has evolved to focus gut‐derived antigens from the bursal lumen into the lymphoid follicles. The emigration of cortical bursal cells is discussed in relation to the exposure of bursal lymphocytes to extrinsic antigen.
The functional state of follicular dendritic cells in severe combined immunodeficient (SCID) mice: Role of the lymphocytes
Authors: Yoshida Kikuyoshi , Berg Timo K. Van Den , Dijkstra Christine D.
Published: March 2007

In the present study, we have investigated the capacity of follicular dendritic cells (FDC) to trap immune complexes (IC) in the splenic white pulp of severe combined immunodeficient (SCID) mice and the influence of lymphocyte transfer on FDC function. FDC are absent in the splenic white pulp of naive SCID mice as revealed by in vitro IC trapping assay. One week after transfer of syngeneic lymphocytes, functional FDC with complement receptors appeared in the primary follicles coincident with B cell segregation, and IC were trapped on those FDC in a complement‐dependent manner. Next, we immunized the reconstituted SCID mouse to see whether another type of FDC could be induced in the secondary follicle. Antigenic stimulation induced FDC with an additional capacity to capture IC via FcR γ II. As seen in immunocompetent mice, this type of FDC was located only in the light zone of the secondary follicle. The newly generated FDC did not carry H‐2 antigen of transferred lymphocytes from F1 mice. In SCID mice, in which normally no functional FDC are detectable, the microenvironments of the splenic white pulp have a capacity to develop and differentiate normally after transfer of lymphocytes. Apparently, the generation of functional IC‐trapping FDC causes the induction of complement receptor(s) and Fc receptor on meshwork cells, which requires the presence of the lymphocytes.
The use of antibody‐secreting cell probes to reveal tissue‐restricted immune responses during infection
Authors: Meeusen Els , Brandon Malcolm
Published: March 2007

Antibody secreting cell probes (ASC‐probes) were generated from the hepatic lymph node (HLN), mesenteric lymph node (MLN) and spleen of rats after infection with the liver fluke, Fasciola hepatica, and used to probe Western blots of parasite antigens. In chronic primary infections, parasite‐specific antibodies were only detected in ASC‐probes derived from HLN. Seven days after a secondary infection, a restricted response was detected in ASC‐probes from the MLN, directed predominantly against an antigen specific to the newly excysted juvenile (NEJ) stage. This NEJ‐specific antigen was only recognized by HLN if the second infection was not rejected and the challenge flukes reached the liver. Measurement of the immunoglobulin levels present in the ASC‐probes showed significant elevation only in lymph nodes draining sites of recent infection. In addition, when the isotype profiles were determined in ASC‐probes derived from different lymph nodes, it was observed that they showed different isotype preferences, in particular IgA for the MLN, IgE for HLN and IgM for spleen. These results show that discrete and independent immune responses occur in different body compartments of a rat against different stages of a parasite.
Interleukin‐2 (IL‐2) receptor γ chain mutations in X‐linked severe combined immunodeficiency disease result in the loss of high‐affinity IL‐2 receptor binding
Authors: Disanto James P. , Certain Stéphanie , Fischer Alain , Basile Geneviève De Saint , Dautry‐Varsat Alice
Published: March 2007

Interactions of interleukin‐2 (IL‐2) with its high‐affinity, heterotrimeric receptor (IL‐2Rαβγ) play a pivotal role in the autocrine pathway of T lymphocyte expansion required in an immune response. Mutations in the IL‐2Rγ chain‐encoding gene have been found in SCIDX1, a primary immunodeficiency characterized by the absence of T cell and NK cell development. We have investigated six unrelated SCIDX1 patients for molecular abnormalities of the IL‐2Rγ gene. A variety of defects were identified, including the absence of transcripts, frame‐shift deletions and point mutations within canonical cytokine receptor motifs (conserved cysteines and the “WS” box). The ability of these mutated IL‐2Rγ chains to participate in the function of a high‐affinity IL‐2R complex was examined by radiolabeled IL‐2 binding studies using Epstein‐Barr virus‐transformed B lymphoblastoid cell lines (B‐LCL) derived from SCIDX1 patients. Although normal control B‐LCL express high‐affinity IL‐2 binding sites (Kd = 60 pM, 150 sites/cell), B‐LCL derived from SCIDX1 patients failed to bind IL‐2 under high‐affinity conditions. These SCIDX1 mutations confirm the critical role of the IL‐2Rγ chain in T cell and NK cell development. In addition, these data provide insight into the structure/function relationship of the IL‐2Rγ chain by identifying residues required for the formation of a high‐affinity IL‐2R complex.
Vβ gene repertoires in T cells expanded in local self‐healing and lethal systemic murine cutaneous leishmaniasis
Authors: Lohoff Michael , Steinert Meike , Weiss Angela , Röllinghoff Martin , Balderas Robert S. , Theofilopoulos Argyrios N.
Published: March 2007

Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self‐healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2‐type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor Vβ chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same Vβ gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar Vβ gene families, and argue against the engagement of a restricted set of Vβ by dominant determinants associated with L. major.
Selective expansion of activated Vδ4+ cells during experimental infection of mice with Leishmania major
Authors: Rosat Jean‐Pierre , Louis Jacques A. , Schreyer Magali , Ohteki Toshiaki , Waanders Gary A. , MacDonald H. Robson
Published: March 2007

Previous work from this laboratory has revealed that infection of mice with Leishmania major leads to an expansion of γδ+ T cells in the spleen. Further examination of the γδ+ T cells expanding in infected mice has shown that the majority of these cells in the spleen, lymph nodes, blood and liver expressed the Vβ4 gene segment.

Cell cycle analysis, using propidium iodide incorporation, demonstrated that while only 1% of αβ+ T cells in the spleen were in either S + G2/M phase, up to 10% of the γδ+ T cells were in cycling phase 8 weeks after infection. Comparison of the state of activation of the two populations in different organs after infection, confirmed that γδ+ T cells are actively dividing in lymph nodes, liver and blood, but not in the thymus or among intraepithelial lymphocytes. Examination of the expression of different activation markers on the surface of γδ+ T cells in the spleen of both normal and chronically infected BALB/c mice by FACS analysis, revealed increased expression of LFA‐1, CD25, CD44, 4F2, CD28 and the heat‐stable antigen, whereas Thy‐1 and CD5 decreased. Collectively, these results suggest an oligoclonal expansion and activation of γδ+ T cells in response to L. major infection.
Leishmania major parasites share an epitope with the murine CD3‐T cell receptor complex
Authors: Zimmermann Stefan , Becker‐Perez Inge , Beuscher Horst‐Ulrich , Kroczek Richard A. , Röllinghoff Martin , Solbach Werner
Published: March 2007

After immunization of BALB/c mice with a low molecular mass fraction (FrD; ≦ 31 kDa) isolated from a soluble extract of Leishmania major promastigotes, a panel of monoclonal antibodies (mAb) was obtained. One of these antibodies (mAb 9C) recognized a cytosol‐associated antigen from L. major of approximately 21 kDa as shown by Western blot and immunoprecipitation. In addition, mAb 9C reacted with surface structures of murine splenic T cells and T cell clones. Reactivity was confined to murine cells, but was not strain restricted. Immunoprecipitation studies and surface‐labeling experiments with CD4+ T cell clones and the T cell receptor (TCR)−CD3− T cell line TG40 transfected with V α/β chains from human TCR and concomitant co‐expression of murine CD3 suggested that mAb 9C binds to an epitope located within the murine CD3‐TCR complex. In addition, mAb 9C induced strong T cell proliferation. We conclude that L. major parasites share an epitope with the murine CD3‐TCR complex which is functionally important for T cell activation.
C3a activates reactive oxygen radical species production and intracellular calcium transients in human eosinophils
Authors: Elsner Jörn , Oppermann Martin , Czech Wolfgang , Dobos Gustav , Schöpf Erwin , Norgauer Johannes , Kapp Alexander
Published: March 2007

Whereas C5a is a well‐established potent activator of eosinophils, the functional role of C3a in the activation of eosinophils is, so far, poorly understood, Here, the activation of human eosinophils stimulated with C3a was analyzed and compared to C5a activation. Flow‐cytometrical measurements revealed that stimulation of eosinophils by C3a resulted in a transient elevation of the intracellular calcium concentration ([Ca2+]i) in a dose‐dependent manner. In addition, the production of reactive oxygen radical species (ROS) of eosinophils after C3a and C5a stimulation was measured by lucigenin‐dependent chemiluminescence and quantified by superoxide dimutase‐inhibitable reduction of ferricytochrom C. Half maximal and maximal ROS production in response to C3a was observed at 50 ng/ml and 1000 ng/ml, respectively, whereas C3a‐desArg was inactive. To ensure that C3a stimulation was not caused by contamination with C5a, monoclonal antibodies were used to demonstrate the specificity of C3a. The effect of C3a was completely abolished in the presence of monovalent antigen‐binding fragments of a functionally blocking anti‐C3a monoclonal antibody. In addition, blockade of the C5a receptor by the monoclonal anti‐C5a receptor antibody S5/1 totally inhibited the C5a‐evoked ROS production, whereas the C3a response in the presence of S5/1 was unaffected. Finally, desensitization experiments revealed a homologous desensitization of C3a after restimulation with C3a. In contrast, no cross‐desensitization was observed upon stimulation with C5a. Furthermore, the C3a‐ and C5a‐induced production of ROS of eosinophils was totally inhibited by pertussis toxin, indicating the involvement of guanine nucleotide‐binding proteins (Gi‐proteins). In summary, these results demonstrate that C3a is a potent activator for eosinophils initiating transient [Ca2+]i changes and production of reactive oxygen species. C3a therefore may play a part in the pathophysiology of diseases with eosinophil and complement activation.
Failure of correlation between B7 expression and activation of interleukin‐2‐secreting T cells
Authors: Lombardi Giovanna , Sidhu Sid , Dodi Tony , Batchelor Richard , Lechler Robert
Published: March 2007

It is well established that triggering interleukin‐2 (IL‐2) secretion by helper T cells requires the T cell to receive at least two discrete signals. One signal is transduced by the CD3 complex, usually as the result of T cell receptor (TcR) occupancy, the second, or co‐stimulatory, signal involves a non‐cognate interaction between cell surface accessory molecules on the antigen‐presenting cell (APC) and the T cell. A molecular interaction that has been implicated in the provision of co‐stimulatory signals is that between B7/BB1 on the APC and its ligands, CD28 and CTL‐A4 on the T cell.

We have studied the ability of HLA‐class II antigen‐positive human T cells and a population of DR1‐expressing transfected human fibroblasts to stimulate a proliferative response by human T cell clones, and by freshly isolated peripheral blood T cells. Despite their high levels of B7 expression, the T cell clones, were unable to induce proliferation or IL‐2 secretion by DR‐restricted, antigen‐specific T cells. In contrast, the DR1‐expressing transfectants, that were B7 negative, induced a strong proliferative response. When these two populations of DR‐expressing cells were used to stimulate a primary alloresponse the results were reversed, in that the T cell clones induced a strong alloresponse but the transfected fibroblasts induced no proliferation.

These results suggest that the expression of B7 may be necessary for co‐stimulation of unprimed T cells, but not of established T cell clones. Furthermore the data show that the expression of B7 by an APC does not necessarily lead to IL‐2 production or protection from the induction of tolerance. The mechanisms responsible for the inability of these T cells to provide full activation signals when used as APC is currently under investigation.
Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site‐directed mutagenesis
Authors: Kim Jin‐Kyoo , Tsen May‐Fang , Ghetie Victor , Sally Ward E.
Published: March 2007

Site‐directed mutagenesis has been used to change amino acid residues of a recombinant Fc‐hinge fragment derived from the murine immunoglobulin (Ig)G1 molecule, and the effects of these mutations on the pharmacokinetics of the Fc‐hinge fragment have been determined. Specifically, Ile‐253, His‐310 and Gln‐311 of the CH2 domain and His‐433 and Asn‐434 of the CH3 domain have been changed. In the three dimensional structure of an antibody, these amino acids are in close proximity to each other at the CH2‐CH3 domain interface. The mutated Fc‐hinge fragments have been purified from recombinant Escherichia coli cells and their pharmacokinetic parameters determined in mice and compared with those of the wild‐type Fc‐hinge fragment. The results show that the site of the IgG1 molecule that controls the catabolic rate (the ‘catabolic site’) is located at the CH2‐CH3 domain interface and overlaps with the Staphylococcal protein A binding site.
Two distinct non‐T helper type 2 interleukin‐4 cell subsets in mice as revealed by single‐cell cytokine analysis
Authors: Kariv Ilona , Hardy Richard R. , Hayakawa Kyoko
Published: March 2007

We have previously defined four murine CD4+ peripheral T cell subsets, fractions (Fr.) I – IV, based on expression of the 6C10 and 3G11 determinants (Hayakawa, K. and Hardy, R. R., J. Exp. Med. 1988. 168: 1825). These subsets also show distinctive levels of other cell surface markers: the two minor subsets, Fr. III and Fr. IV, are both CD45RBlow/‐, L‐selectin (Mel‐14)− and CD44hi, characteristic of secondary T cells. The patterns and levels of cytokine production by individual cells in each subset were determined by bioassay for interleukin (IL)‐2/IL‐4 or IL‐4/interferon (IFN)‐γ production after anti‐CD3 stimulation. Our data revealed that these four phenotypically defined subsets largely coincide with clusters of cells showing uniform distinctive cytokine profiles, i.e. IL‐2+/IFN‐γ−/IL‐4− (Fr. I and Fr. II, L‐selectin+), IL‐2+/IFN‐γ+/IL‐4+ (Fr. III, L‐selectin−), and IL‐2−/IFN‐γlow/‐/IL‐4+ (Fr. IV, L‐selectin−). Besides these subsets, an L‐selectin‐negative cell subfraction within Fr. II appears to represent a transitional population between the IL‐2+/IFN‐γ−/IL‐4− stage and the IL‐2+/IFN‐γ+/IL‐4+ stage. Taken together, these results demonstrate the presence of two IL‐4+ secondary T cell subsets with distinct cytokine production patterns, and show that the majority of IL‐4+ cells found in healthy adult laboratory mice co‐produce IFN‐γ, and thus are not typical T helper type 2 cells.
Reversal of hyporesponsiveness in lpr CD4−CD8− T cells is achieved by induction of cell cycling and normalization of CD2 and p59fyn expression
Authors: Clements James L. , Wolfe Julie , Cooper Sheldon M. , Budd Ralph C.
Published: March 2007

T cells freshly isolated from the peripheral lymph nodes of autoimmune MRL lpr/lpr (lpr) mice contain a large proportion of functionally non‐mature T cell receptor (TcR)‐αβ+CD3+CD2−CD4−CD8− T cells displaying the B cell isoform of CD45, B220. These cells are hyporesponsive as defined by minimal interleukin‐2 (IL‐2) production and proliferation in response to stimulation. However, increased levels of inositol phosphates and a rapid mobilization of Ca2+ do occur upon stimulation of the TcR/CD3 complex. Furthermore, lpr CD4−CD8− T cells contain high levels of transcripts for the src‐family tyrosine kinase p59fyn, and express a constitutively tyrosine‐phosphorylated CD3‐ζ chain. These features bear a certain resemblance to anergized T cells. These similarities are extended to show that culturing of lpr CD4−CD8− T cells in the presence of IL‐2 in combination with phorbol 12‐myristate 13‐acetate and ionomycin initiates cell cycling and results in the gain of function; re‐stimulation now yields IL‐2‐dependent proliferation in the absence of exogenous IL‐2. In parallel with this gain in function, the population of cells obtained after 1 week in culture retains the TcR‐αβ+CD4−CD8− phenotype, yet displays increased levels of CD2, decreased surface B220, and normal amounts of p59fyn‐specific transcripts. These findings show that cell cycling is associated with the recovery of functional capabilities by lpr CD4−CD8− T cells and is closely allied with surface CD2 expression. Thus, the hyporesponsiveness of lpr T cells is not a fixed state.
Binding of adenosine deaminase to the lymphocyte surface via CD26
Authors: De Meester Ingrid , Vanham Guido , Kestens Luc , Vanhoof Greet , Bosmans Eugène , Gigase Paul , Scharpé Simon
Published: March 2007

The relationship between CD26/dipeptidyl peptidase IV, an ectopeptidase involved in T cell activation, and the binding protein for adenosine deaminase (ADAbp) was studied. Monoclonal antibodies (mAb) against CD26 and ADAbp, respectively, showed a similar binding profile on various lymphocyte subsets from the peripheral blood. The adenosine deaminase (ADA) itself blocked the binding of a specific set of anti‐CD26 mAb (among these the anti‐TA5.9 mAb) on lymphocytic CD26; ADA also hindered the binding of soluble CD26 to the same immobilized anti‐CD26 mAb. In addition, the interaction between immobilized ADA and purified CD26/DPP IV was inhibited by the anti‐TA5.9 mAb. ADA did not inhibit the specific peptidase activity of CD26. Neither soluble nor immobilized ADA was able to down‐modulate CD26 on the lymphocyte surface. Our data thus confirm the identity between ADAbp and CD26 and identify some epitopes, crucial in the binding of ADA to CD26.
T cell receptor γ cDNA in human fetal liver and thymus: Variable regions of γ chains are restricted to VγI or V9, due to the absence of splicing of the V10 and V11 leader intron
Authors: Zhang Xiao‐Min , Tonnelle Cécile , Lefranc Marie‐Paule , Huck Sylvie
Published: March 2007

Although complete in‐frame transcripts of the human T cell receptor γ V10 and V11 genes have been described, the corresponding γ chains have never been found in γδ T cell receptors. In this study, we show that the leader intron of all V10 and V11 cDNA isolated from fetal thymus, fetal liver and adult peripheral blood lymphocytes are unspliced. We demonstrate that, due to the absence of splicing, V10 and V11 are pseudogenes and cannot be expressed in γ chains. They are the first pseudogenes of this type described in a rearranging T cell receptor/immunoglobulin locus. Therefore the γ repertoire at the protein level is limited to subgroup VγI and to V9. By analysis of the γ polymerase chain reaction products from total cDNA, we find that the γ locus is active in early ontogeny (8 weeks), as shown by the presence of rearranged V9 and V10 gene transcripts in the liver. At 13 weeks, the VγI genes as well as V9 and V10 have undergone productive rearrangements in the liver, and in the thymus. Most rearrangements, if not all, involve the T cell receptor γ C1 region (JP1, JP, J1 segments) in both tissues, confirming the accessibility of the C1 region in early stages of development.
T cell clones from psoriasis skin lesions can promote keratinocyte proliferation in vitro via secreted products
Authors: Prinz Jörg Christoph , Groß Bernhard , Vollmer Sigrid , Trommler Paul , Strobel Isolde , Meurer Michael , Plewig Gerd
Published: March 2007

Psoriasis vulgaris has been recognized lately as an immunologically mediated inflammatory skin disease. To analyze the pathogenetic role of T lymphocytes in the generation of psoriatic skin lesions, 105 T cell clones (TCC) and 10 T cell lines (TCL) were differentially isolated from dermis and epidermis of psoriatic skin specimens. Supernatants prepared from these T cells were studied for their effects on keratinocyte proliferation in vitro. Conditioned media from 14 of 77 epidermal TCC, 7 of which were CD8+, and from 8 of 28 dermal TCC, 5 of which were CD8+, reproducibly enhanced keratinocyte proliferation, with more pronounced mitogenic activities found in dermal TCC. Another 9 epidermal and 3 dermal TCC did not affect keratinocyte growth and supernatants from the remaining clones, as well as from the 5 epidermal and 5 dermal TCL, inhibited keratinocyte replication to varying degrees. Both mitogenic and suppressive activities were largely abolished by addition of an antiserum to interferon‐gamma (IFN‐γ), while addition of epidermal growth factor or irradiated psoriatic TCL had little effect on the activities of the supernatants.

These studies reveal that a subpopulation of lesional psoriatic T lymphocytes is capable of enhancing keratinocyte proliferation in vitro via secreted products. Their mitogenic capacity most likely requires IFN‐γ, but the ultimate effect is apparently determined by the presence of additional cytokines. Activation of T cells secreting such combinations of factors in vivo may contribute to the keratinocyte alterations characteristic of psoriatic skin lesions.
C3 binds with similar efficiency to Fab and Fc regions of IgG immune aggregates
Authors: Antón Luis C. , Ruiz Sol , Barrio Elena , Marqués Guillermo , Sánchez Angel , Vivanco Fernando
Published: March 2007

The covalent binding reaction of the third component of complement (C3) with rabbit IgG immune aggregates has been studied by enzymic digestion of C3b‐IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal thioester of C3. Trypsin digestion of 14C‐labeled C3b‐IgG adducts degrades C3b to a small antibody‐bound 14C‐labeled C3 fragment (14C‐C3frg), whereas the antibody remains unaltered. Papain digestion of trypsin‐treated 14C‐C3frg‐IgG complexes generated Fc and Fab fragments bearing equivalent amounts of covalently bound 14C‐C3frg (43% and 40%, of the total C3 present in the aggregates, respectively). Hydroxylamine treatment of the 14C‐C3frg‐Fab and 14C‐C3frg‐Fc complexes released a 14C‐C3frg of similar size (about 3–4 kDa) in which the N‐terminal residue was the radiolabeled Cys1010. A fragment with the same radioactive N terminus and characteristics was obtained by sequential trypsin and papain digestion of purified C3 labeled with iodo–[14C] acetamide.

Affinity‐purified 14C‐C3frg‐Fc complexes digested with pepsin generated a mixture of radioactive peptides, most probably complexes formed by 14C‐C3frg and Cγ2 or the hinge digestion products, and 14C‐C3frg‐pFc' complexes. The latter was also immunoprecipitated with anti‐Fc‐Sepharose from the pepsin digestion supernatants of 14C‐labeled‐C3b‐IgG complexes.

Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune aggregates, C3 is not bound to a single site on the antibody molecule. Both Fab and Fc regions of IgG are equally efficient targets for C3 anchorage. In addition, the data confirm the pFc' as a region of C3 attachment within the Fc portion, and strongly suggest that C3b is bound either to the Cγ2 domain or the hinge or both.
Characterization of a novel human T cell receptor β chain variable region family by transspecies DNA hybridization
Authors: Hall Margaret A. , Jaeger Emma E. M. , Bontrop Ronald E. , Lanchbury Jerry S.
Published: March 2007

One essential component in the generation of diversity of T cell receptor (TcR) α and β chains is the existence of multiple tandemly arranged variable regions in the germ line which are subsequently rearranged to form functional units. In attempting to establish the extent of the human TcR α and β chain repertoire an approach of randomly sequencing cDNA for rearranged receptors has been generally pursued. So far, 24 human Vβ families have been characterized and they encompass some 51 transcriptionally active gene segments. Recently, surveys of several hundred TcR β cDNA clones have failed to establish further Vβ families which suggests that if they exist, they are likely to be poorly represented in the peripheral blood repertoire. It is likely, therefore, that the majority of human Vβ regions which are rearranged conventionally and are transcriptionally active have already been described. To investigate whether additional Vβ families characterize the human TcR repertoire we have examined Vβ sequences from an inverse polymerase chain reaction (PCR) TcR β cDNA library of the chimpanzee, the most closely related creature to man. One clone which could not be assigned to any human Vβ family cross‐hybridized with human TcR β chain cDNA. This new human family has been called Vβ25. Comparison of chimpanzee and human Vβ25 sequences using the Lipman‐Pearson method gives a similarity index of 94.6% over a consensus length of 112 amino acids. On the evidence of Southern blotting with cDNA probes, Vβ25 segments in human and chimpanzee appear to represent single member gene families and no restriction fragment length polymorphism was evident with the restriction enzymes used. Equalization of TcR β cDNA followed by family‐specific quantitative PCR showed that the gene is expressed at similar levels in the chimpanzee and the human. The actual level of expression in the human is less that 0.5% of the total TcR β repertoire. Transspecies DNA hybridization using novel sequences from closely related species may be a powerful tool for establishing the full extent of TcR α and β and immunoglobulin repertoires.
Expression of the transforming growth factor‐α gene by human eosinophils is regulated by interleukin‐3, interleukin‐5, and granulocyte‐macrophage colony‐stimulating factor
Authors: Brach Marion A. , Sott Claudia , Kiehntopf Michael , Herrmann Friedhelm
Published: March 2007

Transforming growth factor (TGF)‐α is a pleiotropic polypeptide which mediates a variety of tissue‐specific cellular responses such as induction of proliferation, cell migration, vascularization, and formation of extracellular matrix. TGF‐α is produced by certain tumor cells and embryogenic tissues, as well as by normal cells of different origin. Within the granulocytic lineage. TGF‐α production has been shown in promyelocytic leukemia cells induced to differentiate, as well as in blood eosinophils of patients with the idiopathic hypereosinophilic syndrome. The present study was carried out in order to examine expression of the TGF‐α gene in polymorphonuclear (PMN) and mononuclear (MN) blood cells of normal healthy donors. While MN and neutrophilic PMN failed to synthesize TGF‐α transcripts and protein, eosinophils constitutively exhibited TGF‐α transcripts accompanied by the release of immunoreactive TGF‐α protein. Exposure of PMN and MN cells to the leukocyte‐activating cytokines interleukin (IL)‐3, IL‐5, and granulocyte‐macrophage colony‐stimulating factor resulted in a several‐fold increase of TGF‐α mRNA expression and protein release by eosinophils, but not by neutrophils and MN cells. PMN and MN were insensitive to induction of TGF‐α release by IL‐8 and granulocyte colony‐stimulating factor. These results point to a functional role of eosinophils in disorders characterized by unbalanced TGF‐α production such as disease states associated with abnormal matrix formation and neovascularization which may be explained by the present demonstration of TGF‐α production in these cells.
Protein‐G binding material from synovial fluid of rhematoid arthritis patients induces unorthodox autoantibodies (IgG1 rheumatoid factor) in NZB, NZW and (NZB x NZW)F1 mice
Authors: Abedi‐Valugerdi Manuchehr , Ridderstad Anna , Lettesjö Helene , Ström HÅKan , Möller Erna
Published: March 2007

Our previous studies have demonstrated that injection of rheumatoid arthritis (RA) synovial fluid (SF) induces a marked increase mainly of IgG1 antibody‐producing cells in autoimmune disease prone (NZB X NZW)F1 mice but not in CBA mice. In the present study, the in vivo effect of RA‐SF on autoantibody production was tested in different strains of mice. Injection of RA‐SF induced the production of unorthodox autoantibodies (IgG1 rheumatoid factor, RF) in young (NZB X NZW)F1 mice as well as in their parental strains NZB and NZW, but not in normal mice (CBA) or in mice with severe combined immunodeficiency, indicating that the response is not caused by a conventional immune response against RA‐SF material. IgG1 RF production was rapidly induced and reached high levels already on day 7 and lasted for more than 90 days. The induction of IgG1 RF was not the result of polyclonal activation, since RA‐SF did not stimulate the production of other antibodies, such as autoantibodies against double‐stranded DNA, bromelain‐treated mouse red blood cells, myosin, transferrin, cytochrome c, thyroglobulin or myoglobin or antibodies reactive with the hapten TNP. To elucidate the identity of the active substance in RA‐SF, responsible for IgG1 RF production, bound and unbound material of RA‐SF, eluted from a protein‐G column was injected into (NZB X NZW)F1 mice. Only the protein‐G binding material was active, indicating that the effect is mediated by autoantibodies or immune complexes in the synovial fluid. Further studies demonstrated that identical concentrations of protein obtained from a pool of normal human IgG or SF from seronegative RA and non‐RA arthritides patients did not contain the same activity.
Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS
Authors: Pascual Manuel , Danielsson Carina , Steiger Gertraud , Schifferli Jürg Alfred
Published: March 2007

The number of complement receptor type 1 (CR1; CD35) on human erythrocytes (E) decreases during normal in vivo aging. Patients with acquired immunodefiziency syndrome (AIDS) have an acquired deficiency of CR1 on E. The possible mechanisms responsible for the loss of CR1 from E include the release of small vesicles from the E membrane and proteolytic cleavage of CR1. When compared to E of normal donors and of asymptomatic human immunodeficiency virus HIV+ subjects, E of patients with AIDS had fewer CR1/E (p < 0.001), but had the same number of two glycosylphosphatidyl‐inositol‐anchored proteins, decay‐accelerating‐factor (DAF) and CD59. When compared to young E, old E separated by density gradients on Percoll had fewer CR1 [six normal subjects, mean loss: 50.4 ± 4.9 (SEM) %], DAF (34.4 ± 1.2%) and CD59 (34.5 ± 2.7%). The loss of CR1 was significantly higher than the loss of DAF and CD59 (p < 0.02). In vitro, ATP depletion of E is responsible for the release of vesicles from the E surface, a reaction that has been called in vitro aging. CR1, DAF and CD59 were lost on ATP‐depleted E; however, the loss of CR1 and DAF were identical (six experiments, mean loss for CR1: 28.7 ± 2.7%, DAF: 26.3 ± 4.6% and CD59: 20.5 ± 4%). Thus, the release of vesicles from E cannot explain the specific loss of CR1 in patients with AIDS and would explain only incompletely the loss of CR1 during in vivo aging. In vitro experiments indicated that CR1 was more sensitive to trypsin and papain cleavage than DAF and CD59. Enhanced chemiluminescence Western blotting, using a monoclonal antibody (E11) recognizing fragments of CR1 down to 43 kDa on E exposed to trypsin or papain, indicated that normal E bear fragments of CR1, which are not found on polymorphonulcear leukocytes or on CR1‐bearing vesicles in urine. The relative amount of these fragments was increased in patients with AIDS. Taken together these data suggest that the specific loss of CR1 on E in AIDS is due to proteolytic cleavage. The loss of CR1 during in vivo aging also involves proteolytic cleavage, although part of the loss might be explained by other mechanisms including the release of vesicles by E.
Interleukin‐7 regulates c‐myc expression in murine T cells and thymocytes: a role for tyrosine kinase(s) and calcium mobilization
Authors: Seckinger Philippe , Milili Michèle , Schiff Claudine , Fougereau Michel
Published: March 2007

Interleukin‐7 (IL‐7) was originally identified as a pre‐B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c‐myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL‐7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL‐7‐dependent D10 G4.1 cell proliferation and c‐myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short‐lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c‐myc mRNA expression in the murine T cell line. IL‐7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL‐7 regulates c‐myc mRNA expression in a protein kinase C‐independent manner and these data are strengthened by protein kinase C depletion which does not modify IL‐7 c‐myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL‐7 induction of c‐myc mRNA expression and may represent a physiological mechanism whereby IL‐7 bioactivity is regulated. The activity of IL‐7 on c‐myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL‐7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL‐7 c‐myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL‐7 induced gene induction and cell proliferation.
In vitro development of B cells and macrophages from early mouse fetal thymocytes
Authors: Péault Bruno , Khazaal Ibrahim , Weissman Irving L.
Published: March 2007

The developmental potentialities of early mouse fetal thymocytes were analyzed by in vitro culturing cell suspensions obtained from 12–15‐gestational day thymus in contact with a bone marrow stroma clonal cell line that supports pre‐B and myeloid cell differentiation. B cell and macrophage development from fetal thymocytes was observed at all fetal stages tested, but limiting dilution analysis revealed that the frequency of cells forming colonies on bone marrow stroma is the highest in the fetal thymus at day 12, then dramatically decreases until day 15. These observations suggest that the thymus rudiment is seeded by multipotential precursor cells which are not immediately committed to T cell development in the thymic cellular environment.
Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes
Authors: Bellone Matteo , Karachunski Peter I. , Ostlie Norma , Lei Sijin , Conti‐Tronconi Bianca M.
Published: March 2007

T cell from H‐2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) α, γ and δ subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti‐peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes.

Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a “pure” B epitope (Tα63–80), induced in most cases anti‐peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide Tα360–378 was co‐injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides Tα360–378 and Tα63–80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide Tα360–378.

Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.
Preferential expression of interleukin‐12 or interleukin‐4 by murine bone marrow mast cells derived in mast cell growth factor or interleukin‐3
Authors: Smith Tracey J. , Ducharme Lori A. , Weis John H.
Published: March 2007

In this report we demonstrate that murine bone marrow cells cultured in either interleukin (IL)‐3 or mast cell growth factor (MGF, also known as c‐kit ligand and stem cell factor) differentially express cytokine genes. Bone marrow cells cultured in IL‐3 differentiate and proliferate, taking on a mucosal mast cell‐like phenotype. These cells express the IL‐4 gene. Bone marrow cells cultured in MGF take on a connective tissue mast cell‐like phenotype and possess transcripts for both of the subunits of the IL‐12 cytokine. Bone marrow cells cultured in both IL‐3 and MGF express the IL‐4 gene at lower levels than that seen for the IL‐3 culture alone, but do not possess IL‐12 gene transcripts. The level of IL‐12 subunit transcripts derived from the MGF‐derived bone marrow cells was compared to that found in splenocytes and activated macrophages, the only cells in which IL‐12 production has been previously documented. Both of the IL‐12 subunit transcripts were found, compared to a β‐actin control, to be present within MGF‐derived cells in the same if not higher quantities than the splenocyte or macrophage cultures. Mucosal mast cells have been previously implicated in the development of the T helper type 2 (TH2) T cell phenotype via their expression of IL‐4. The finding that the MGF‐derived connective tissue‐like mast cells possess IL‐12 transcripts suggests that the development of the TH1 T cell pathway may be positively influenced by this type of mast cell.
Bimodal distribution of Vβ2+CD4+ T cells in human peripheral blood
Authors: Clarke Gillian R. , Reyburn Hugh , Lancaster Fiona C. , Boylston Arthur W.
Published: March 2007

The distribution of T cells using the Vβ2 gene was studied in a group of 99 humans. The distribution of Vβ2+CD4+ levels was bimodal. Twelve individuals had levels of Vβ2+CD4+ less than 2% and 86 others had values greater than 5%. Only one individual had a value between 2% and 5%. The Vβ2 low (mean 1.3 ± 0.49) and Vβ high (mean 8.2 ± 1.65) phenotypes were stable. The Vβ low phenotype is inherited and not limited to HLA or T cell receptor Vβ gene complexes. The CD8Vβ2 levels of CD4Vβ2 low individuals are also low. The residual Vβ2+ T cells in Vβ2 low individuals were not anergic to Vβ2‐specific stimulation. These data are compatible with the effects of an endogenous superantigen.
A novel, complement factor H‐related regulatory protein expressed on the surface of human B cell lines
Authors: Erdei Anna , Julen Nathalie , Marschang Peter , Feifel Elisabeth , Kerekes Krisztina , Dierich Manfred P.
Published: March 2007

Complement regulatory proteins present on the surface of various mammalian cells play an important role in controlling homologous lysis, by interacting with C3 (and usually C4). These proteins have a similar structural motif (“short consensus repeat”) (Reid, K.B.M., Bentley, R.D., Campbell, R.D., Chung, L.P., Sim, R.B., Kristensen, T. and Tack, B.F., Immunol. Today 1986. 7: 230), and the genes encoding them are members of the family of regulators of complement activation. Here we describe a hitherto unknown member of this family, a molecule expressed by B lymphoblastoid cells. This protein is recognized by polyclonal antibodies fo factor H and by MAH4, a monoclonal antibody reacting with the N‐terminal portion of factor H. The cell surface protein is built up of two disulfide‐linked chains of approximately 68 and 75 kDa. Biosynthetic labeling studies confirmed that it is synthesized by B cells only, but not by the investigated lines of other origin. When tested for its functional activity, this molecule was shown to act as cofactor for factor I‐mediated cleavage of fluid‐phase C3b to C3bi. The protein appears to be encoded by a 3.5‐kb mRNA, hybridizing with a cDNA probe coding for the N‐terminal portion of factor H. Due to its cross‐reactivity with anti‐H antibodies, cofactor activity for factor I and hybridization with factor H cDNA, despite its two‐chain composition, it is considered a factor H‐like protein.