bluuebunny/crossref_metadata_2025 huggingface dataset:
Human autoantibodies to lamin B receptor are also anti‐idiotypic to certain anti‐lamin B antibodies
Authors: Lassoued Kaiss , Danon Françoise , Brouet Jean‐Claude
Published: March 2007

Autoantibodies reactive with nuclear envelope proteins are mainly detected in human sera from patients with liver diseases. Some of these antibodies are directed to lamin B, lamins A and C, or to the lamin B receptor (LBR). We show here that the latter one are anti‐idiotypic to certain anti‐lamin B antibodies. Using an enzyme‐linked immunosorbent assay specific for lamins we found that serum M containing anti‐LBR antibodies inhibited the binding to lamins of anti‐lamin B autoantibodies from three of five sera tested. Similar results were obtained using patient's M purified IgG. The binding of monoclonal IgM, λ anti‐lamin B antibodies produced by a lymphoblastoid cell line derived from the patient's blood lymphocytes was also inhibited. Absorption of serum M with nuclei abolished the inhibitory activity. No inhibition was recorded with normal sera or sera containing other antinuclear specificities. Anti‐LBR antibodies did not alter the binding to lamins of sera containing anti‐lamins A and C antibodies. Altogether these findings demonstrate that anti‐LBR antibodies are also combining site related anti‐idiotypic antibodies (Ab2) to certain anti‐lamin B antibodies, provide further evidence for discrete specificities among anti‐lamin B antibodies and suggest that the occurrence of autoantibodies to nuclear envelope antigens may be under idiotypic regulation.
Glucocorticoid‐dependent and ‐independent mechanisms involved in lipopolysaccharide tolerance
Authors: Evans Glenn F. , Zuckerman Steven H.
Published: March 2007

Injection of bacterial lipopolysaccharide (LPS) into animals results in a transient increase in serum tumor necrosis factor (TNF). Maximal increases in TNF were detected by 1 h and by 3–4 h serum TNF was no longer apparent. These animals were LPS tolerant and a repetitive LPS stimulus did not result in an additional peak in TNF. Regulation of TNF expression in LPS‐tolerant animals was at the transcriptional level as TNF mRNA was not apparent in spleen or peritoneal macrophages following a second LPS stimulus. Adrenalectomized (adrex) mice, in contrast, did not become LPS tolerant and sera from these animals demonstrated an additional peak in TNF 1 h following a second LPS stimulus. Concomitant with the secondary rise in serum TNF in adrex mice was an increase in splenic TNF mRNA. The ability of adrex mice to become LPS tolerant was restored by exogenous glucocorticoids. LPS tolerance was also investigated in the galactosamine LPS model which like the adrex model is characterized by a thousandfold increase in the sensitivity of these animals to the lethal effects of LPS. Consistent with the absence of LPS tolerance in adrex mice, galactosamine‐sensitized mice were also responsive to a second LPS stimulus and did not become LPS tolerant. While LPS‐treated adrex mice had no significant increases in serum corticosterone, corticosterone levels in LPS‐treated galactosamine‐sensitized mice were comparable to LPS‐stimulated normals suggesting that LPS tolerance involves both glucocorticoid‐dependent and ‐independent components. Finally, prophylactic administration of a monoclonal antibody against murine TNF protected normal and galactosamine‐sensitized mice from a lethal dose of LPS and yet had no protective effect in adrex animals.
Antigen processing by endosomal proteases determines which sites of sperm‐whale myoglobin are eventually recognized by T cells
Authors: Van Noort Johannes M. , Boon Jacqueline , Van der Drift Alfons C. M. , Wagenaar Josée P. A. , Boots Annemieke M. H. , Boog Claire J. P.
Published: March 2007

This study reports an identification of the major processing products of an exogenous protein antigen, viz. sperm‐whale myoglobin, as obtained after cell‐free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages.

The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C‐terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin‐specific helper T cells.

Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC‐binding sequences as well as of T cell epitopes in their naturally occurring form.
Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line
Authors: Bikoff Elizabeth K. , Otten Gillis R. , Robertson Elizabeth J.
Published: March 2007

Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post‐implantation‐stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well‐defined class I product, we introduced the H‐2Dd gene under control of the human β‐actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H‐2Dd heavy chains and β2‐microglobulin protein, but only small amounts of H‐2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I‐negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H‐2Dd‐transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly‐defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post‐implantation‐stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post‐translationally at the level of MHC class I assembly.
Biased VH gene expression in murine CD5 B cells results from age‐dependent cellular selection
Authors: Andrade Luiz , Huetz François , Poncet Pascal , Thomas‐Vaslin Véronique , Goodhadt Michéle , Coutinho Antonio
Published: March 2007

Flow cytometry‐purified, peritoneal and splenic CD5+ and CD5− B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain‐treated mouse red blood cells (BrMRBC), single‐stranded DNA, trimethyl ammonium and bovine γ‐globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D‐proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti‐BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age‐dependent cellular selection mechanisms.
Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo
Authors: Imhof Beat A. , Schlienger Claude , Handloser Klaus , Hesse Barbara , Slanicka Michaela , Gisler Roland
Published: March 2007

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB‐1 and SAB‐2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic μ‐chain‐positive pre‐B cells or mature B cells to the BM stromal cell line. Injection of both SAB‐1 and SAB‐2 antibodies into pregnant mice reduced by 90% the number of B220+ B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU‐preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB‐1 is present on CS 1.3 stroma cells and on a pre‐B cell line while SAB‐2 is present on pro‐B and pre‐B cell lines, but not on CS 1.3 stromal cells. SAB‐1 and SAB‐2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB‐1 and SAB‐2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB‐1 and SAB‐2 probably recognize novel cell‐cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.
Tolerance to a self peptide from the third hypervariable region of the E chain. Implications for molecular mimicry models of autoimmune disease
Authors: Roudier Jean , Sette Alessandro , Lamont Alan , Albani Salvatore , Karras James G. , Carson Dennis A.
Published: March 2007

As a first step in the analysis of a molecular mimicry model of rheumatoid arthritis, we addressed the question of whether tolerance to self‐major histocompatibility complex (MHC) class II molecules includes tolerance to peptides from the third hypervariable region of their β chain. We studied T cell responses to a peptide from the third hypervariable region of the Esβ chain, Esβ peptide (PEFLEQRRAAVDTYC), in different mouse strains after footpad priming with peptide in complete‐Freund's adjuvant.

Strains of mice of the k or d haplotype (B10D2; H‐2d, B10BR; H‐2k) mounted a vigorous T cell response to the Esβ peptide. In mice expressing the Esβ chain either on the cell surface (B10S9R) or in the cytoplasm as free unassociated chain (B10S), no response could be detected. Binding studies using purified MHC class II molecules and competition for antigen presentation showed that the Esβ peptide binds Ak, Ad and As but not Ek. Thus, the nonresponder status of B10S and B10S9R mice appears to reflect self tolerance. Tolerance was also suggested by the observation that responder × nonresponder F1 crosses such as (B10D2 × B10S9R) and (B10BR × B10S9R) did not respond to Esβ peptide.

Interestingly, mice derived from the (B10BR × B10S) cross responded to the Esβ peptide, suggesting that the immune system may not always tolerate peptides from the third hypervariable region of self‐MHC class II molecules.
The early cellular and humoral immune response to primary and booster oral immunization with cholera toxin B subunit
Authors: Lewis David J. M. , Novotny Pavel , Dougan Gordon , Griffin George E.
Published: March 2007

The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers. A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization. Using an immunospot assay, cells spontaneously secreting anti‐toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization. There were 105 IgG‐ and 87 IgA‐secreting cells per 2 × 106 mononuclear cells 7 days after the first immunization, and 282 IgG‐ and 413 IgA‐secreting cells 5 days after the second immunization. A polyclonal increase in total IgM‐secreting cells was observed. Few anti‐toxinsecreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti‐toxin‐secreting cells after oral immunization.
Immunoregulation of mercuric chloride‐induced autoimmunity in Brown Norway rats: A role for CD8+ T cells revealed by in vivo depletion studies
Authors: Mathieson Peter W. , Stapleton Karen J. , Oliveira David B. G. , Lockwood C. Martin
Published: March 2007

Mercuric chloride (HgCl2) induces the production of autoantibodies to glomerular basement membrane (GBM) in the Brown Norway (BN) rat. The autoimmune response is self‐limiting and thereafter the animals are resistant to rechallenge with HgCl2. Resistance can be transferred to naive animals by spleen cells from HgCl2‐treated rats. A similar state of resistance can be induced with a low dose of HgCl2, insufficient in itself to induce autoimmunity. We have examined the role of CD8+ T cells in the immunoregulation of this experimental model by depleting this subset in vivo. We have also used inhibition studies in a solid‐phase radioimmunoassay in an attempt to demonstrate any effect of anti‐idiotypic antibodies in the spontaneous resolution of the anti‐GBM antibody response. The initial induction and spontaneous resolution of anti‐GBM antibodies were unaffected by depletion of CD8+ T cells. However, CD8‐depleted animals were no longer resistant to rechallenge with HgCl2. Cell transfer studies showed that spleen cells from CD8‐depleted animals conferred less resistance to HgCl2 than those from animals which had received control antibody. CD8 depletion also reduced the resistance induced by pretreatment with low‐dose HgCl2. Studies in which peak sera were pre‐incubated with post‐recovery sera before testing in a solid‐phase anti‐GBM radioimmunoassay did not support an important role for anti‐idiotypic antibodies. We conclude that CD8+ T cells play an important role in the resistance to rechallenge with HgCl2 in the BN rat, although they are not required for the induction or spontaneous resolution of the initial autoimmune response. Demonstration of the reversal of a suppressive phenomenon in vivo using an anti‐CD8 monoclonal antibody is unusual.
Generation of monoclonal antibodies against soluble human T cell receptor polypeptides
Authors: Devaux Brigitte , Bjorkman Pamela J. , Stevenson Christine , Greif William , Ellio John F. , Sagerström Charles , Clayberger Carol , Krensky Alan M. , Davis Mark M.
Published: March 2007

One approach to the diagnosis and therapy of T cell‐mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen‐specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the “phosphatidylinositol linkage” strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA‐A2 and HLA‐Aw68/HLA‐Aw69, respectively. Monomeric TcR α and ß chains from the HLA‐A2‐specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti‐β chain mAb, denoted anti‐Vβ13, stain a significant (≈ 5%) fraction of human peripheral blood α/β T lymphocytes, immunoprecipitate native anti‐A2 TcR molecules, and activate T cells transfected with the relevant α and β chain cDNA. Anti‐α chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent‐solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.
Trypanosoma cruzi: Infection of T lymphocytes and their destruction by antibody‐dependent cell‐mediated cytotoxicity
Authors: Velge Philippe , Kusnierz Jean‐Pierre , Ouaissi Ali , Marty Béatrice , Pham Bach Nga , Capron André
Published: March 2007

We have demonstrated, with optical and transmission electron microscopy, that Trypanosoma cruzi trypomastigotes infect and multiply inside T lymphocytes. The infection rate we have observed in T cells was similar to that seen in the case of macrophage or polymorphonuclear cell infection. Flow cytofluorometric analysis of T lymphocytes purified from mice in the acute phase of the disease, revealed the presence of parasite‐derived antigens on their surface. These antigens appear to be specific to T. cruzi and they could be the result of intracellular parasite antigens as well as adsorption of T. cruzi antigens on the surface of noninfected T cells. Antibodies recognizing these surface antigens were present in both T. cruzi‐infected mouse and human sera. They were able to induce antibody‐dependent cell‐mediated cytotoxicity (ADCC) in the presence of nonimmune mononuclear cells both in autologous and in heterologous combinations. Consequently, we provided evidence suggesting that T lymphocytes could be destroyed during the acute phase of Chagas' disease either by cell infection or by an ADCC mechanism against cells bearing parasite antigens on their surface. Thus, the ability of trypomastigotes to invade T cells may play a crucial role in the immunopathogenesis characteristic of Chagas' disease.
Allospecific recognition of hemic cells in vitro by natural killer cells from athymic rats: Evidence that allodeterminants coded for by single major histocompatibility complex haplotypes are recognized
Authors: Vaage John Torgils , Dissen Erik , Ager Ann , Fossum Sigbjørn , Rolstad Bent
Published: March 2007

We have previously shown that large granular lymphocyte (LGL)‐enriched cell populations have the capacity to spontaneously recognize and kill allogeneic small lymphocytes and bone marrow cells (BMC) in vitro in certain strain combinations of rats. Here, we have studied the alloreactivity of natural killer (NK) cells from PVG nude (RT1c) rats against a panel of major histocompatibility complex (MHC) incompatible hemic cells. Both lymphocytes and BMC from the AO (RT1u), DA (RT1a), BN (RT1n) as well as the MHC‐congenic PVG‐RT1u (RT1u) rat strains were efficiently killed in vitro, whereas cells from syngeneic PVG rats were spared. The structures recognized on lymphocytes and BMC were probably similar since the two cell populations inhibited each other in cross‐competition experiments. A number of features aligned the alloreactive effector cells with NK cells and not T cells. (a) Only about 5% of the effector cells from nude spleens expressed the T cell antigens CD3, CD5 or T cell receptor (TcR) α/β whereas > 50% of the cells expressed markers present on NK cells (CD2, CD8, OX52 and the rat NK cell‐specific marker NKR‐P1 recognized by the monoclonal antibody 3.2.3). (b) The alloreactive cells were granular since pretreatment of nude spleen cells with the lysosomotropic agent L‐leucine methyl ester which eliminated LGL, simultaneously abolished the cytolysis of both allogeneic lymphocytes and YAC‐1 tumor cells. (c) Nude spleen cells stimulated with human recombinant interleukin 2 for 1 week in vitro generated large granular proliferating cells which were CD3−. CD5−, TcR α/β−, but > 95% 3.2.3+. These cells efficiently killed allogeneic hemic cells from the same rat strains as did freshly isolated effector cells. (d) The cytolysis of allogeneic hemic cells could effectively be inhibited with unlabelled NK‐sensitive (YAC‐1 and K‐562), but not NK‐resistant (Roser leukemia) tumor cells.

Cross‐competition studies showed that PVG nude NK cells discriminated between AO, BN and DA BMC, suggesting that different alloantigens were positively recognized by subsets of NK cells. The mode of inheritance of the allodeterminant specifically recognized on AO BMC was investigated in crosses and backcrosses between AO and BN or DA rats. A gene dosage effect was observed in that this determinant was expressed at a slightly reduced level in F1 hybrids. Cells from the progeny of a BN × (BN × AO) or DA × (DA × AO) backcross used as inhibitor cells showed that the inhibition of killing of AO target cells co‐segregated completely with the expression of RT1u class I molecules on the inhibitor cells in 22 rats. Taken together, these results have shown that NK cells can recognize allogeneic cells in a specific manner and that one MHC haplotype is sufficient for the expression of at least one of the alloantigens recognized.
Late administration of monoclonal antibody to leukocyte function‐antigen 1 abrogates incipient murine cerebral malaria
Authors: Grau Georges E. , Pointaire Pascal , Piguet Pierre‐François , Vesin Christian , Rosen Hugh , Stamenkovic Ivan , Takei Fumio , Vassalli Pierre
Published: March 2007

We analyzed the role of adhesion molecules in the pathogenesis of experimental cerebral malaria (ECM), since tumor necrosis factor (TNF) plays a major role in this condition and has been shown to up‐regulate in vitro expression of cell adhesion molecules (CAM), particularly intercellular CAM‐1 (ICAM‐1). We found increased expression of ICAM‐1 on brain endothelial cells from mice with ECM. Treatment with monoclonal antibodies (mAb) directed against leukocyte function‐antigen 1 (LFA‐1, the ligand of ICAM‐1) on days 6, 8 and 10 almost totally prevented ECM, while decreasing blood TNF levels. To exclude the possibility that the effects of anti‐LFA‐1 mAb resulted from an even partial inhibition of TNF overproduction, mice with signs of imminent death (hypothermia and neurologic defects) were treated with the anti‐LFA‐1 mAb, with dramatically protective effect. In contrast, injection of anti‐ICAM‐1 mAb on day 6 caused rapid death, while it was innocuous in normal mice. An mAb directed against complement receptor type 3 (CR3) was ineffective, as were injections of soluble human ICAM‐1. These results suggest that adhesion of LFA‐1+ cells to endothelial cells, stimulated by TNF to express high levels of ICAM‐1, is critical in the pathogenesis of ECM. Emergency therapy at interfering with cytoadherence could be considered in the treatment of cerebral malaria in man, in which high blood TNF levels are also observed.
Negative signaling by surface IgM on B cells isolated from ileal Peyer's patch follicles of sheep
Authors: Griebel Philip J. , Davis William C. , Reynolds John D.
Published: March 2007

Lymphoid follicles from the sheep ileal Peyer's patch (PP) were used to prepare a cell suspension consisting of 98% surface IgM‐positive (sIgM+) B cells and 1% T cells. Co‐stimulation of follicular cells with pokeweed mitogen and either recombinant bovine interleukin 1 (IL 1) or IL 2 resulted in a marked proliferative response. In contrast, the addition of soluble F(ab')2 rabbit anti‐sheep Ig completely inhibited the proliferative response induced by pokeweed mitogen and IL 1 or IL 2 co‐stimulation. Anti‐Ig inhibition of B cell proliferation was specific for ileal PP follicular cells and was not observed with mesenteric lymph node cells or splenocytes. Furthermore, suppression of ileal PP follicular B cell proliferation required at most divalent cross‐linking of sIg was independent of Fc receptors, but was dependent on the concentration of anti‐Ig and required 48 h for maximal effect. Negative signaling by sIgM indicates that ileal PP follicular B cells are functionally distinct from B cells in other secondary lymphoid tissues. Also, the present observations are consistent with previous reports indicating that B cell proliferation in ileal PP follicles is antigen independent.
Germ‐line origin of functional idiotypic interactions: Identification of two idiotypically connected, natural antibodies that are encoded by germ‐line gene elements
Authors: Carlsson Leif , Andersson Åsa , Holmberg Dan
Published: March 2007

Two monoclonal, natural IgM antibodies derived from normal BALB/c mice were selected on the basis of being idiotypically complementary and functionally connected. Nucleotide sequence analysis of their respective heavy and light chain V regions showed that both of the clones expressed VH, D, JH, VL and JL gene segments of germ‐line origin. Furthermore, none of the clones displayed N‐sequence additions. These data suggest a germ‐line origin of a functional idiotypic network and confirm a minimized contribution of somatic diversification through template‐independent addition of N‐nucleotides in neonatal B cell repertoires.
Evidence for an association between human resistance to Schistosoma mansoni and high anti‐larval IgE levels
Authors: Rihet Pascal , Demeure Christian E. , Bourgois Alain , Prata Aluizio , Dessein Alain J.
Published: March 2007

The anti‐larval IgE antibody response of adolescents with high or low resistance to infection by Schistosoma mansoni was evaluated before parasitological cure with oxamniquine and over an extended post‐treatment period during which the least resistant subjects regained high infections. IgE from most sera, taken at several bleeding times before and after treatment, reacted, on immunoblots, with a large number of antigens (Ag) in schistosomular tegument extract. A family of 120‐165‐kDa cross‐reacting molecules and a 85‐kDa Ag were the most prominent Ag. Some of these determinants were shown to be located on the outer tegumental membrane and to be accessible to IgE on living larvae. The comparison of IgE between the two study groups showed that IgE levels were on average six‐ to eightfold higher (p<0.01) in the sera of the most resistant adolescents whereas there was no difference in patterns of Ag recognition between study groups. In contrast to IgE, anti‐larval IgG and IgM levels were either similar in both groups or higher in the least resistant subjects when these exhibited high reinfection levels. IgG that competed for the binding of IgE to larval Ag were detected in most sera and their levels were higher in the least resistant group after reinfection. Finally, the treatment had no observable long‐lasting effects on the levels and on the specificity of the anti‐larval IgE. Altogether, these observations can be taken as evidence supporting a role of IgE in human resistance to infection by S. mansoni.
Chemotaxis of germinal centers B cells in response to C5a
Authors: Kupp Leo I. , Kosco Marie H. , Schenkein Harvey A. , Tew John G.
Published: March 2007

An infiltrate of B cells and plasma cells is characteristic of certain chronic inflammatory lesions. However, mechanisms involved in the local accumulation of these cells have not been established. Efforts to demonstrate that B cells from normal animals can migrate in response to inflammation‐induced chemoattractants have been inconclusive. The objective of this study was to determine if murine germinal center (GC) B cells could respond chemotactically to a C5a gradient. On successive days after secondary immunization, draining lymph nodes were harvested and the activated GC B cells isolated. These GC B cells were placed in modified Boyden chambers, incubated for 3 h and the distance the leading front of cells migrated through the filters was determined. The results show that GC B cells migrated to factors in zymosan‐ and lipopolysaccharide‐activated serum. The migratory response demonstrated distinct kinetics. Cells isolated between 2 to 4 days after secondary immunization migrated, whereas cells isolated at day 0 and beyond day 6 did not. Checkerboard analysis revealed that the migratory response was attributable to both chemokinesis and chemotaxis. Anti‐C5 inhibited the migration of day‐3 GC B cells implicating C5 in the migration mechanism. Studies using recombinant C5a established that this C5 fragment was chemotactically active. In conclusion, GC B cells generally were not chemotactically active. However, at a particular stage of maturation B cells in the GC become responsive to C5a as a chemotactic agent. Thus, B cells from normal animals may respond chemotactically, and C5a may play a role in recruitment of recently activated B cells into inflammatory sites.
Allelic exclusion in transgenic mice expressing a heavy chain disease‐like human μ protein
Authors: Corcos Daniel , Iglesias Antonio , Dunda Olga , Jami Jacques
Published: March 2007

Heavy chain diseases (HCD) are neoplastic proliferations of B cells which secrete truncated immunoglobulin heavy chains without associated light chains. These proteins are encoded by mutated genes which may also give rise to truncated membrane immunoglobulins. The neoplastic cells proliferate in vivo although they cannot bind any antigen, due to deletions in the variable domain of their antigen receptors. The reason for the clonal proliferation of HCD cells and the biological effects of the truncated membrane‐bound chains are presently unknown. We wanted to determine whether the expression of HCD proteins would interfere with B cell development. To this end we made transgenic mice with a human μ gene, lacking the VDJ exon, that encodes a protein similar to that produced in two cases of HCD. Transgenic mice were also produced with a similar construct but encoding only the membrane‐bound form of the truncated μ chain. Transgene encoded Cμ proteins are expressed on the cell surface without associated light chains and are responsible for allelic exclusion of murine heavy chains.
Induction of antigen‐specific CD8+ cytolytic T cells by the exogenous bacterial antigen streptolysin O in rhesus monkeys
Authors: Chizzolini Carlo , Millet Pascal G. , Olsen‐Rasmussen Melissa A. , Collins William E.
Published: March 2007

We have characterized the T cell responses induced by streptolysin O (SLO), a sulfydryl‐activated hemolysin secreted by streptococci, by applying long‐term in vitro culture and cloning rhesus monkey (Macaca mulatta) T cells. T cell lines specific for SLO were obtained from three rhesus monkeys. These T cell lines required autologous antigen‐presenting cells (APC) to proliferate in response to SLO and did not respond to purified protein derivative. Phenotypic analysis showed that the cells from two of three SLO‐specific T cell lines were more than 85% CD3+CD4−CD8+ after prolonged in vitro culture. The rh 1842 CD8+ T cell line proliferative response to SLO was inhibited by the addition of anti‐major histocompatibility complex (MHC) class I and anti‐CD8 but not of anti‐MHC class II and anti‐CD4 monoclonal antibody (mAb). This cell line was able to lyse P815 target cells in the presence of anti‐CD3 mAb and did not show natural killer activity. Moreover, specific lysis of autologous but not allogeneic non‐rosetting E− cell targets pulsed with SLO was observed. Such lysis was inhibited by the addition of anti‐MHC class I mAb.

In the attempt to identify the restriction elements involved in SLO presentation APC from six unrelated rhesus monkeys and three humans were used. A CD4+ rh 1842 T cell clone responded when SLO was presented by one of six, and a CD8+ rh 1842 T cell clone by four of six rhesus monkeys APC. Both CD4+ and CD8+ T cell clones did not respond when SLO was presented by human APC. However, both clones responded when APC from all donors were used in conjunction with anti‐CD3 mb. Furthermore, SLO required active processing to be presented to CD4+ and CD8+ T cell clones as glutaraldehyde fixation of APC before but not after antigen pulsing inhibited T cell proliferation.

The SLO‐specific CD8+ cytolytic T cells described here could play a role in the regulation of the immune response occurring during streptococcal infections and/or could participate in the pathogenesis of poststreptococcal nonsuppurative sequelae.
Comparison of class I‐ and II‐restricted T cell recognition of the identical peptide
Authors: Perkins David L. , Berriz Gabriel , Wang Yunsheng , Smith John A. , Gefter Malcolm L.
Published: March 2007

There is structural and functional evidence that both class I‐ and II‐restricted T cells recognize short processed peptides bound to MHC molecules. Although the structural conformation of bound peptides remains unknown, no evidence of distinct structural motifs of class I‐ or class II‐restricted peptides has been described. Conversely, two algorithims proprosed to predict T cell epitopes, and based on primary amino acid sequence or tertiary structure, are both compatible with many observed class I‐ and class II‐restricted peptides. We previously identified eight class I‐restricted peptides which were also recognized by class II‐restricted T cells. Based on functional and direct binding studies, additional examples of peptides with both class I and II restrictions have been identified. In this study, we have directly compared the fine specificity of T cell recognition of a single epitope in a single mouse strain in the context of both class I‐ and class II‐restricted responses. Based on a panel of analogue peptides with amino acid substitutions and peptides of various lengths, we observed several striking similarities in the recognition patterns of both class I‐ and class II‐ restricted T cells. In addition, some characteristics of recognition were different in the two systems indicating that the recognition processes were similar but not identical.
Signaling requirements for the expression of the transactivating factor NF‐AT in human T lymphocytes
Authors: Hivroz‐Burgaud Claire , Clipstone Neil A. , Cantrell Doreen A.
Published: March 2007

A protein called nuclear factor of activated T cells (NF‐AT) binds to DNA sequences within the enhancer region of the interleukin 2 (IL 2) gene and appears necessary for both the inducibility and T cell specificity of IL 2 expression. IL 2 production is regulated by multiple signals including those generated via activation of the T cell antigen receptor complex (TcR/CD3), CD2 antigen, protein kinase C (PKC) or elevation of intracellular free calcium concentration ([Ca2+]i). We have, therefore, explored the role of these different stimuli in regulating the nuclear expression of NF‐AT in human peripheral blood‐derived lymphocytes. Results presented herein indicate that maximal expression of NF‐AT in T cells requires at least two signals: PKC activation and TcR/CD3 or CD2 triggering, [Ca2+]j increases and TcR/CD3 or CD2 triggering. Data are presented that indicate that either the [Ca2+]i or PKC signal generated via the TcR/CD3 complex would not alone induce NF‐AT expression, and that the TcR/CD3 complex probably regulates NF‐AT expression because of its ability to regulate multiple intracellular signals in T cells, and not via any single biochemical event. The combination of CD2 mAb GT2/OKT11 used in the present study to trigger the CD2 antigen is able to act in synergy with phorbol 12,13‐dibutyrate or ionomycin to induce NF‐AT expression. However, these CD2 mAb do not elevate [Ca2+]i or activate PKC, suggesting that signals other than [Ca2+]i or PKC can regulate NF‐AT expression in peripheral blood‐derived T cells.
Phosphorylation of the β‐subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion
Authors: Valmu Leena , Autero Matti , Siljander Fia , Patarroyo Manuel , Gahmberg Carl G.
Published: March 2007

Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu‐CAM. We have now studied the phosphorylation of Leu‐CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the β subunit of Leu‐CAM (CD18), and the extent of cell‐to‐cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the β subunit of isolated Leu‐CAM in vitro. The phosphorylation occurred mainly on serine residues.
V genes of oxazolone antibodies in 10 strains of mice
Authors: Kaartinen Matti , Solin Marja‐Liisa , Mäkelä Olli
Published: March 2007

One pair of V genes (Vx45.1 and V11) code for a great portion of phenyloxazo‐lone (anti‐phOx) antibodies in 10 strains of mice. This combination replaces the first‐known major combination VHOx1‐VxOx1 in some strains, and is important in most strains. C57BL/10 and SJL mice have an additional subset of antibodies encoded by genes Vx45.1 and V13 (a relative of V11).

All three genes involved (Vx45.1, V11 and V13) have „allelic”︁ variation. Four alleles of V11 were found, one in Igh haplotypes a, c and g, the second in haplotypes d, j and n, the third in b, and the fourth in f. The most distant alleles d,j,n and f had 10 nucleotide differences out of 429 determined (97.7% homology). Only one allele of the V13 gene was found from anti‐phOx hybridomas but two others have been published. Three alleles of the Vx45.1 gene were found; one in NZB mice (Igx haplotype b) another in CE (haplotype f), and the third in eight strains including representatives of three Igx haplotypes (a, c and e). The three alleles had >99.0% homology.

The V11 and V13 genes that code for anti‐phOx antibodies in C57BL/10 and SJL mice were different from the related genes found from the C57BL/10 germ line. C57BL/10 mice must have a chromosome bearing two V11 and two V13 genes. RF mice were found to have two V11 genes, and both code for anti‐phOx antibodies.

Our data show that the majority of antibodies in the anti‐phOx response are encoded by the same restricted collection of V genes in most mouse strains. Antibody responses appear to be no less heritable than other functions of the body.
The role of C5a and antibody in the release of heparan sulfate from endothelial cells
Authors: Platt Jeffrey L. , Dalmasso Agustin P. , Lindman Bonnie J. , Ihrcke Nathan S. , Bach Fritz H.
Published: March 2007

The activation of endothelial cells is thought to contribute to the host response to infection and to the pathogenesis of autoimmune disease. It was recently shown that antibody and complement can activate endothelial cells leading to cleavage and release of heparan sulfate from the cells. We show here that release of heparan sulfate from endothelial cells is mediated by antibody and the complement fragment C5a and that assembly of the membrane attack complex and lysis of endothelial cells is not necessarily involved. These data suggest that the generation of C5a in conditions such as autoimmunity and infection in which anti‐endothelial cell antibodies may also be present, might amplify tissue injury by a novel mechanism involving endothelial cell activation and loss of heparan sulfate mediated by antibody and C5a.
Exact prediction of a natural T cell epitope
Authors: Rötzschke Olaf , Falk Kirsten , Stevanovic Stefan , Jung Günther , Walden Peter , Rammensee Hans‐Georg
Published: March 2007

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351: 290). These allele‐specific motifs should allow exact prediction of natural T cell epitopes. H‐2Kb‐restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb‐restricted epitope of ovalbumin, thought to be contained in the 19‐mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.
The λ/λ ratio in surface immunoglobulin molecules on B lymphocytes differentiating from DHJH‐rearranged murine pre‐B cell clones in vitro
Authors: Rolink Antonius , Streb Martin , Melchers Fritz
Published: March 2007

The expression of x and λ light chains in surface immunoglobulin (sIg) molecules on B lymphocytes differentiating from murine pre‐B cell clones in vitro was analyzed. The four pre‐B cell clones used represent a very early pre‐B cell stage. They have their heavy chain loci DJ rearranged and their light chain loci in germ‐line configuration. In order to grow in vitro, these clones require contact with stromal cells and the stimulatory activity of interleukin (IL) 7. Upon removal of IL 7 from the cultures, these clones differentiate within 3 days into sIg+ B cells. Between 7% and 12% of IgM+ B cells could be detected in these cultures. The majority (78%‐92%) of the IgM+ B cells co‐expressed x light chains. The percentage of λ light chain expressing B cells was below detectable level. Upon lipopolysaccharide (LPS) stimulation, the percentages of IgM+ B cells increased dramatically (from 32%‐64%). The majority (91%‐97%) of the IgM+ B cells express x chains, but a very small percentage (3.1%‐5.0%) express λ. A similarly high x/λ ratio was found in 418 hybridomas prepared from these LPS‐stimulated B cells (388 x+ and 30 λ+). Thus, the high x/λ ratio characteristic of the mouse peripheral B cell repertoire is already evident in the antigen‐independent transition from pre‐B to B cells.
Peptide loading of empty major histocompatibility complex molecules on RMA‐S cells allows the induction of primary cytotoxic T lymphocyte responses
Authors: De Bruijn Marloes L. H. , Schumacher Ton N. M. , Nieland John D. , Ploegh Hidde L. , Martin Kast W. , Melief Cornelis J. M.
Published: March 2007

The antigen processing‐defective mutant cell line RMA‐S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide‐loaded RMA‐S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell‐independent fashion. This was shown for an H‐2Kb‐binding peptide of Sendai virus nucleoprotein and an H‐2Db‐binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA‐S cells. Primary Sendai peptide‐induced CTL lyse both peptide‐loaded and virus‐infected cells. Pre‐culture of RMA‐S cells at low temperature (22°–26°C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide‐specific CTL responses induced by peptide‐loaded RMA‐S cells are CD4+ cell‐ and MHC class II+ cell‐independent. CTL response induction is blocked by the presence of anti‐CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA‐S cells with peptide and show 2.5‐fold more peptide bound per RMA‐S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA‐S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA‐S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.
Cells producing antibodies specific for myelin basic protein region 70–89 are predominant in cerebrospinal fluid from patients with multiple sclerosis
Authors: Martino Gianvito , Olsson Tomas , Fredrikson Sten , Hojeberg Bo , Kostulas Vasilios , Grimaldi Luigi M. E. , Link Hans
Published: March 2007

Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1–20, 70–89, 108–126, or 157–166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70–89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG‐secreting cells on average) than in patients with OND (1/2083 IgG‐secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70–89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti‐MBP 70–89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown.
Cross‐linking of surface IgM activates NF‐xB in B lymphocyte
Authors: Rooney John W. , Dubois Patrice M. , Sibley Carol Hopkins
Published: March 2007

In B lymphocytes, cross‐linking of surface IgM activates changes in both the cell cycle and differentiation. In normal B cells and B cell tumors, many stimuli induce the activation of NF‐xB and its translocation from the cytoplasm to the nucleus. In this study we sought to determine if cross‐linking of surface IgM led to the activation of NF‐xB. Our results show that activation of B cells by cross‐linking anti‐IgM antibodies activated NF‐xB in the murine B lymphoid cell lines 70Z/3 and M12, and in the dense fraction of splenic cells. The activation of NF‐xB required optimal doses of anti‐IgM antibodies and took 5 to 10 min to reach maximal levels. Cross‐linking of IgM has also been shown to activate protein kinases including protein kinase C (PKC). To test wether PKC activation was required for NF‐xB translocation, we treated 70Z/3 cells for 18 h with phorbol 12‐myristate 13‐acetate, a procedure which depletes these cells of functional PKC. This treatment did not abrogate the nuclear translocation of NF‐xB following anti‐IgM cross‐linking. These results indicate that the nuclear translocation of NF‐xB is rapidly induced by surface IgM cross‐linking and that this activation appears to use a pathway which does not require PKC.
Polarized expression of major histocompatibility complex class I molecules in human endometrial and endocervical epithelial cells
Authors: Van Eijkeren Marion A. , Peters Peter J. , Geuze Hans J.
Published: March 2007

The localization of major histocompatibility complex (MHC) class I molecules in human endometrial and endocervical epithelial cells was studied with an immunogold technique on ultrathin cryosections. At the cell surface, MHC class I molecules are delivered solely to the basolateral plasma membrane in human epithelia. Therefore, only the basolateral domain of epithelial cells is capable of presenting antigen to MHC class I‐restricted cytotoxic T cells.