bluuebunny/crossref_metadata_2025 huggingface dataset:
Four CD45/P56lck‐associated phosphorproteins (pp29‐pp32) undergo alterations in human T cell activation
Authors: Schraven Burkhart , Schirren Albrecht , Kirchgessner Henning , Siebert Brigitte , Meuer Stefan C.
Published: March 2007

In human T lymphocytes a functional complex is formed between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and a phospho‐protein, pp32, a possible common substrate. Here we demonstrate that the previously described pp32 protein is composed of two distinct molecules (pp29 and pp32) in both resting human T lymphocytes and continuously proliferating T lymphoma lines. Importantly, T lymphocyte activation employing CD2 monoclonal antibodies (mAb), CD3 mAb or phorbol 12, 13 dibutyrate results in loss of pp29 and pp32 from the CD45/p56lck molecular complex and concomitant association of two distinct phosphoproteins with different molecular weights (pp30 and pp31). These events appear to be unrelated to clonal T cell growth but rather depend on receptor‐mediated differentiation signals. Reprecipitation experiments employing an antiserum directed at a consensus sequence of GTP‐binding proteins suggest that all four pp29‐pp32 molecules might represent proteins with GTP‐binding properties. Biochemical analysis of pp29‐pp32 employing V8‐protease digestion indicates that they differ in low‐molecular weight fragments of 8, 5, 4.5, 4 and 3 kDa, respectively.
Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18°C) inhibit the class I MHC processing pathway
Authors: Harding Clifford V.
Published: March 2007

While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation. We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class II MHC‐restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I MHC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH4Cl), cycloheximide, and hypothermia (18°C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.
Expression of the complement regulatory proteins CD21, CD55 and CD59 on Burkitt lymphoma lines: their role in sensitivity to human serum‐mediated lysis
Authors: Kuraya Mikio , Klein George , Klein Eva , Yefenof Eitan
Published: March 2007

On a panel of nine human B cell lines we showed that the expression of the complement regulatory factors complement receptor type 2 (CR2; CD21), decay‐accelerating factor, (DAF; CD55) and homologous restriction factor (HRF20, CD59) is not correlated. All lines expressed DAF, six lines carried detectable amounts of CR2 and three carried HRF20. Upon incubation in human serum, under conditions which allowed the activation of complement through the alternative pathway, the CR2‐carrying lines bound C3 fragments and two of them (Ramos and one of its two sublines) were damaged. These two lines had the lowest DAF expression, less than 50% of the cells reacted with the IA10 monoclonal antibody.

By modulating the expression of the complement regulatory molecules, the lytic sensitivity of the B cell lines could be altered. Blockade of DAF on the HRF20−, CR2+ lines with the specific monoclonal antibodies increased their sensitivity to lysis by human serum. With the DAF− and HRF20+ cells significant lytic effect was obtained only when they were pretreated with both of the specific antibodies.

Interferon‐γ or tumor necrosis factor‐α treatment elevated the amount of CR2 on the low‐CR2 expressor line (Ramos/HR1K) which thereafter bound higher amounts of C3 fragments and was lysed when incubated in human serum. This line had relatively low DAF level and lacked HRF20. The cytokine treatment did not alter the expression of these molecules.

The CR2+ Ramos and the CR2− Rael cells were treated with 5‐azacytidine which induced HRF20 and increased DAF expression. In parallel with this change Ramos cells became resistant to C‐mediated lysis.

The experiments with the panel of human B cell lines showed thus that cytolysis through activation of complement in homologous serum can be regulated at several steps by cell surface molecules. While expression of CR2 was required for C3 fixation, DAF and HRF20 inhibited lysis. By independent modulation of the quantities of these molecules, cells acquired or lost their sensitivity.
Regulation of interleukin‐2 responses by phosphatidic acid
Authors: Cano Eva , Angeles Muñoz‐Fernández M. , Fresno Manuel
Published: March 2007

Interleukin‐2 (IL‐2) plays a central role in the immune system by regulating the proliferation and differentiation of T lymphocytes. However, the molecular mechanism of the signal transduction through the IL‐2 receptor is poorly understood. We have studied the role of phosphatidic acid (PA) on IL‐2 signal transduction using cloned T lymphocytes. IL‐2 stimulated a transient increase in the PA concentration in resting CTLL‐2 cells prelabeled with [3H]palmitic acid. This effect was detected as early as 1 min after IL‐2 addition and peaked at 5 min. IL‐2 similarly increased phospholipase D activity in intact CTLL‐2 cells, as inferred by phosphatidylethanol production. By contrast, IL‐2 did not affect [3H]palmitic acid‐labeled diacylglycerol levels. Furthermore, exogenous addition of several natural or synthetic PA to T cells mimicked IL‐2 activity. Thus, PA were able to induce DNA synthesis on CTLL‐2 cells, although this effect was only 10%–20% of that observed with IL‐2. PA showed a synergistic effect with low doses of IL‐2. In addition, PA was able to induce c‐myc RNA transcription in CTLL‐2 cells as well as IL‐2 receptor (CD25) expression on the cell membrane with equal potency as saturating doses of IL‐2. It is likely that IL‐2‐induced PA accumulation is a consequence of phospholipase D activation. This hypothesis is further supported by the fact that the addition of exogenous phospholipase D but not phosphatidylinositol‐specific phospholipase C also reproduced the IL‐2 or PA effects mentioned above. In summary, our results suggest a role of phospholipase D activation and PA formation as second messengers of IL‐2 activity.
The calcium‐binding proteins MRP8 and MRP14 form a membrane‐associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions
Authors: Bhardwaj Ranjit S. , Zotz Claudia , Roth Johannes , Goebeler Matthias , Mahnke Karsten , Falk Matthias , Meinardus‐Hager Georg , Sorg Clemens , Zwadlo‐Klarwasser Gabriele
Published: March 2007

Monocytes/macrophages expressing an epitope recognized by a monoclonal antibody 27E10 are present in acute but are absent in chronic inflammatory disorders. This report shows that the 27E10 antigen is formed by noncovalent association of the two Ca2+‐binding proteins MRP8 and MRP14 which belong to the S100 protein family. Identification has been confirmed immunochemically, by matrix‐assisted UV‐laser desorption/ionization spectrometry and by partial amino acid sequencing. Surface expression of the MRP8/MRP14 complex on a subset of monocytes is reported for the first time and shown to be up‐regulated in a Ca2+‐dependent manner. The 27E10 surface‐positive monocytes isolated by cell separation techniques release high amounts of tumor necrosis factor‐α and interleukin‐1β in contrast to their 27E10 surface‐negative counterparts thus emphasizing their role in inflammation.
Species‐specific restriction of complement by HRF20 (CD59) generated by cDNA transfection
Authors: Takizawa Hisao , Okada Hidechika , Takahashi Kazuhiro , Murakami Tomoko , Okada Noriko
Published: March 2007

The 20‐kDa homologous restriction factor (HRF20, CD59) is a phosphatidyl inositol‐anchored membrane glycoprotein that inhibits the formation of human complement membrane attack complexes. The cDNA of HRF20 was transfected into Chinese hamster ovary (CHO) cells resulting in expression of human HRF20 protein on the cell surface anchored via glycosylphosphatidyl inositol. The transfected CHO cells were resistant to human complement‐mediated cell killing. However, the cells remained sensitive to rat and guinea pig complement. Therefore, species specificity between HRF20 and complement is maintained in HRF20 generated on the CHO cells following transfection with HRF20 cDNA.
Distinct in vivo functions of two macrophage subpopulations as evidenced by studies using macrophage‐deficient op/op mouse
Authors: Wiktor‐Jedrzejczak Wieslaw , Szperl Malgorzata , Urbanowska Elzbieta , Ansari Aftab A.
Published: March 2007

The op/op mice totally lack macrophage growth factor colony‐stimulating factor (CSF)‐1 and thus, by definition are completely depleted of CSF‐1‐dependent functions of the macrophage cell lineage. Moreover, they possess a severe and generalized macrophage deficiency. However, residual macrophages of these mice should still have normal CSF‐1‐independent functions. Studies designed to elucidate this issue have revealed that op/op mice are capable of normal in vivo phagocytic function and demonstrate normal humoral and cellular response postimmunization with sheep red blood cells. However, release of monokines such as tumor necrosis factor and granulocyte CSF following administration of endotoxin is severely impaired in op/op mice as compared with littermate controls. These studies suggest that the CSF‐1‐dependent macrophage population (absent in the op/op mouse) is primarily responsible for regulatory functions of these cells mediated by monokines, while the CSF‐1‐independent macrophage population (present in the op/op mouse) is primarily responsible for the classical macrophage functions in immunity such as phagocytosis, antigen processing and presentation.
Anti‐human interleukin‐6 receptor antibody inhibits human myeloma growth in vivo
Authors: Suzuki Hiroshi , Yasukawa Kiyoshi , Saito Takashi , Goitsuka Ryo , Hasegawa Atsuhiko , Ohsugi Yoshiyuki , Taga Tetsuya , Kishimoto Tadamitsu
Published: March 2007

Myeloma is one of the interleukin (IL)‐6‐related diseases to which abnormal expression of IL‐6 has been reported to be linked. We examined the in vivo inhibitory effect of anti‐human IL‐6 receptor (IL‐6R) antibody on human myeloma cell growth in mice. SCID mice were subcutaneously inoculated with solid tumor of the myeloma cell line S6B45 in which human IL‐6 was acting as an autocrine growth factor. Ten intraperitoneal administrations of 100 μg of the anti‐human IL‐6R antibody PM1 at 48‐h intervals strongly inhibited the growth of S6B45 cells when the administration started 24 h after tumor inoculation. The tumor growth inhibition in vivo was also observed by administration of the anti‐human IL‐6 antibody MH166 using the same procedure as for PM1. The inhibitory effect of PM1 was not significant when the administration started 5 or more days after tumor inoculation. This work indicates that anti‐human IL‐6R antibody, as well as anti‐human IL‐6 antibody inhibits human myeloma growth in vivo, and provides an animal model for testing the therapeutic value of agents such as antibodies to human IL‐6, IL‐6R and gp130, an IL‐6R‐associated signal transducer, in the treatment of human myelomas.
Strong serum inhibition of specific IgE correlated to competing IgG4, revealed by a new methodology in subjects from a S. mansoni endemic area
Authors: Rihet Pascal , Demeure Christian E. , Dessein Alain J. , Bourgois Alain
Published: March 2007

A method allowing the immunopurification of human IgE from small volumes of sera with a yield close to 100% (mean = 97.8%; SEM = 0.7) has been developed. The immunopurification eluates were cleared of other class antibodies that could compete with IgE in specific assays. Immunopurification of IgE followed by specific IgE enzyme‐linked immunosorbent assay (ELISA) (IMMEL) was then applied to sera of 160 individuals from an area endemic for Schistosoma mansoni. In comparison with radioimmunosorbent test (RAST) and ELISA performed on unfractionated sera, IMMEL provided the highest specific IgE signals. Furthermore, the best correlations between the specific IgE levels and either the specific basophil histamine release levels (r = 0.84; p<10−4) or the anti‐S. mansoni skin test values (r = 0.45; p = 10−4) were obtained with IMMEL.

Measurement of anti‐S. mansoni IgE levels in immunopurified fractions and in unfractionated sera of these 160 individuals revealed a strong serum inhibition (geometric means of 98.6% and 96.8% for the adult worms and the larvae, respectively) of the specific IgE reactivity in ELISA. This inhibition was correlated with the anti‐adult worm and anti‐larval IgG4 levels (r = 0.65; p < 10−4) and r = 0.58;p < 10−4, respectively). In contrast, this inhibition did not correlate with the specific IgG1, IgG2, IgG3 and IgM levels. Furthermore, the level of specific IgG4 was clearly lower than that of specific IgG1, suggesting that the major contribution of IgG4 in the competition effect is not due to higher levels but rather to a specificity spectrum close to that of the specific IgE. These results support the idea that a specific function of IgG4 in serum might be to control antigen recognition by IgE and consequently, to regulate anaphylactic reactions and IgE‐mediated immunity.
Identification of a source of biologically active CD40 ligand
Authors: Armitage Richard J. , Sato Timothy A. , Macduff Brian M. , Clifford Ky N. , Alpert Alan R. , Smith Craig A. , Fanslow William C.
Published: March 2007

We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin‐labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface‐expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high‐affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4‐stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti‐IgM‐activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane‐bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.
Molecular analysis of a pro‐T cell clone transformed by Abelson‐murine leukemia virus, displaying progressive γδ T cell receptor gene rearrangement and surface expression
Authors: Heuze Françoise , Pardoll Drew , Diez Elisabeth , Ezine Sophie , Jotereau Francine
Published: March 2007

We present a molecular analysis of T cell differentiation in a set of clones derived from in vitro Abelson murine leukemia virus (A‐MuLV) infection of fetal liver cells. The parental clone had partial rearrangement of the β and γ loci and spontaneously displayed progressive rearrangement of Vγ genes during in vitro culture. Further differentiation of these clones leading to δ gene rearrangement and CD4 expression, then CD8, CD3 and T cell receptor γδ chain surface expression was obtained after intrathymic transfer followed by in vitro co‐culture with thymic tissue. These A‐MuLV clones, therefore, appear to represent a powerful model sytem for studying the early molecular events of T cell development at the clonal level.
Characterization of molecular components associated with surface immunoglobulin M in human B lymphocytes: Presence of tyrosine and serine/threonine protein kinases
Authors: Leprince Corinne , Draves Kevin E. , Ledbetter Jeffrey A. , Torres Raul M. , Clark Edward A.
Published: March 2007

To characterize the signal transduction through the antigen receptor (AgR) on human B lymphocytes, we analyzed its association with other molecular components. The surface IgM (sIgM) complex isolated in digitonin contains two surface expressed polypeptides ‐ the previously described Igα and Igβ proteins ‐ covalently linked to each other in a 48/39‐kDa heterodimer. We show herein that the human sIgM complex isolated from the Burkitt's lymphoma cell line, Ramos, or from dense tonsillar B cells contains additional molecules −160 kDa and 75 kDa in size — and enzymatic activities able to phosphorylate on tyrosine as well as serine/threonine residues the 39−, 48−, 75− and 160‐kDa polypeptides. By specific immunoprecipitation with antibodies to src‐family kinases, we consistently detected p56lyn in the sIgM complex. In the Ramos cell line, both p56lck and p59fyn activity were also observed, although to a much lesser extent than p56lyn. These kinases are associated with sIgM before cell stimulation. As shown by two‐dimensional electrophoresis, they interact in a tight complex with multimeric forms of the Igα and Igβ components. The kinases are active in vitro but must be highly regulated in vivo: Western blotting with anti‐phosphotyrosine antibodies revealed that stimulation of the AgR on viable B cells increased detectable phosphotyrosine residues on the components present in the sIgM complex. Based on these phosphorylation changes, the 39−, 48−, 75− and 160‐kDa molecules are likely to be functionally active elements in an IgM complex crucial for the transduction of the antigenic signal.
Nitric oxide mediates intestinal pathology in graft‐vs.‐host disease
Authors: Garside Paul , Hutton Angela K. , Severn Alison , Liew Foo Y. , Mc Mowat Allan I.
Published: March 2007

We have investigated the involvement of nitric oxide (NO) in intestinal graft‐vs.‐host reaction (GvHR) in mice. Treatment of mice with L‐NG‐monomethyl arginine (L‐NMMA), a specific inhibitor of NO synthesis, abolished the mucosal pathology of intestinal GvHR and reduced the associated lymphocytic infiltration of the epithelium. L‐NMMA had no effect on splenomegaly in GvHR, nor did it interfere with the growth of an undifferentiated crypt stem cell line, or the production of tumor necrosis factor‐α by activated macrophages in vitro. In contrast, L‐NMMA inhibited the enhanced activity of natural killer (NK) cells which occurs in GvHR. We conclude that a NO‐dependent mechanism is essential for intestinal immunopathology in GvHR and that this may reflect a role for NO in NK cell function.
Attenuation of autoimmune disease and lymphocyte accumulation in MRL/lpr mice by treatment with anti‐Vβ8 antibodies
Authors: De Alborán Ignacio M. , Gonzalo José A. , Kroemer Guido , Leonardo Esther , Marcos Miguel A. R. , Carlos Martínez‐A.
Published: March 2007

MRL‐MP‐lpr/lpr mice are afflicted by a severe systemic autoimmune disease that is aggravated by the lpr mutation resulting in the accumulation of phenotypically abnormal lpr cells (CD3+CD4−CD8−) in all lymphoid issues including hyperplastic lymph nodes. Given that products of the T cell receptor Vβ8 gene family are overrepresented among lpr cells, different schedules aimed at selectively decreasing the frequency of lpr cells were designed. First, continuous administration of the monoclonal antibody F23.1 (specific for Vβ8 products) resulted in a significant depletion of Vβ8+ cells and prevented the manifestation of lymph accumulation at the same time as it reduced the serological, clinical, and histopathological signs of autoimmune disease. Along the same line, administration of either F23.1 or two different anti‐F23.1 anti‐idiotypic antibodies to MRL/Mp‐lpr/lpr mothers elicited, in the offspring, the production of antibodies sharing a recurrent idiotype with F23.1 and resulted in long‐term amelioration of autoimmunity and lymphadenopathy. Thus, a strategy aimed at specifically reducing the frequency of a subset of lpr cells proved successful in mitigating the autoimmune process.
The specificity and efficiency of endogenous peptides in the induction of HLA class I α chain refolding
Authors: Tanigaki Nobuyuki
Published: March 2007

The specificity and efficiency of endogenous peptides in the HLA class I binding have been investigated by the use of a simple procedure that is based on the serological detection of the folding of HLA class I α chains that is induced by the binding to specific peptides in the presence of β2 microglobulin. HLA class I proteins were solubilized with a nonionic detergent from cultured HLA homozygous B lymphoblastoid cells and dissociated by alkaline denaturation. The resulting unfolded α chains were isolated by gel filtration at a neutral pH. The unfolded α chains showed a high refolding capacity and specificity when tested in the presence of an excess β2 microglobulin against endogenous peptides extracted by alkaline or acid treatment from cultured B lymphoblastoid cells of various HLA class I phenotypes. Cells of identical or overlapping HLA phenotypes clearly showed shared peptides, whereas such peptide sharing was rarely, if at all, seen between cells of non‐overlapping HLA phenotypes. The efficiency of endogenous peptides in the induction of refolding was high; at an estimated concentration of 0.2 μM or less, a strong refolding effect was observed.
Presence of CD8α‐CD8β‐positive TcR γ/δ thymocytes in the fetal murine thymus and their in vitro expansion with interleukin‐7
Authors: Leclercq Georges , De Smedt Magda , Plum Jean
Published: March 2007

Several groups have described that a low percentage of in vitro cultured T cell receptor (TcR) γ/δ cells express CD8. Contrary to TcR α/β cells, however, CD8 on these TcR γ/δ cells was shown to be a CD8α homodimer. We describe here that addition of interleukin‐7 (IL‐7) to a short‐term in vitro culture of fetal day 14 thymic lobes in an organ culture system or of fetal day 18 fetal thymocytes in cell suspension yields CD8β‐positive TcR γ/δ cells. This is not the result of IL‐7‐induced expression of CD8β on previously CD8β‐negative cells. It is due to IL‐7‐induced expansion of CD8α‐CD8β‐positive TcR γ/δ cells which are shown to be present in the starting fetal thymocyte cell population.
Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus‐1‐infected mothers
Authors: Cheynier Rémi , Langlade‐Demoyen Pierre , Marescot Marie‐Rose , Blanche Stephane , Blondin Gilles , Wain‐Hobson Simon , Griscelli Claude , Vilmer Etienne , Plata Fernando
Published: March 2007

Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV‐1‐infected mothers. These CTL killed HLA‐matched HIV‐1‐infected H9 target cells or doubly transfected P815‐A2‐env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA‐A1/A3 or HLA‐A2, respectively. HIV‐1‐specific CTL were detected early after birth (>2 months) and remained present during the asymptomatic phase of the infection. As in HIV‐1‐infected adults, HIV‐specific CTL declined with disease progression. Surprisingly, HIV‐1‐specific CTL were detected in the PBMC of three children who subsequently became seronegative.
Human monoclonal striational autoantibodies isolated from thymic B lymphocytes of patients with myasthenia gravis use VH and VL gene segments associated with the autoimmune repertoire
Authors: Victor Kimberly D. , Pascual Virginia , Williams Carol L. , Lennon Vanda A. , Capra J. Donald
Published: March 2007

The production of autoantibodies reactive with components of skeletal muscle is characteristic of myasthenia gravis (MG) and is strongly associated with the presence of thymic epithelial tumors in patients with MG. The nucleotide sequences of the heavy and light chain variable regions (VH and VL) of three human monoclonal striated muscle antibodies (StrAb) isolated from thymic B lymphocyte lines from two patients with MG and thymoma were analyzed. The VH and VL gene segments used by these anti‐striated muscle antibodies appear to be derived from the same repertoire of gene segments as have been found in other autoantibodies isolated from patients with a number of different autoimmune diseases. While the IgM StrAb SA‐1A is a direct copy of germ‐line VH and VL gene segments, analysis of the IgG StrAb SA‐4A and SA‐4B, which may be more representative of antibodies found in the serum of MG patients, suggests that the processes of antigenic selection and somatic mutation may contribute to the generation of autoantibodies in MG.
Association of the human invariant chain with H‐2 Db class I molecules
Authors: Cerundolo Vincenzo , Elliott Tim , Elvin John , Bastin Judy , Townsend Alain
Published: March 2007

We describe two proteins of 24 kDa and 33 kDa (p24 and p33) which associate with H‐2 Kb and H‐2 Db molecules, respectively, in human cells transfected with H‐2 Kb and H‐2 Db genes. This association is particularly clear in the mutant cell line T2, in which association of endogenous peptide with newly synthesized class I molecules may not occur (V. Cerundolo et al., Nature 1990. 345: 449). We show that p33 is the 33‐kDa form of the human invariant chain which is resident in the endoplasmic reticulum of T2 cells (P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 1989. LIV: 309). The stability of the invariant chain H‐2 Db complex is critically dependent upon occupation of the class I binding site by peptide ligand. In the absence of peptide, the complex is stable at 4°C whereas following exposure to peptide, the invariant chain dissociates rapidly from H‐2 Db molecules (half‐life of 30 min at 4°C). Although the interaction between the human invariant chain and murine H‐2 Db is unlikely to have any functional significance, the peptide‐induced dissociation of the invariant chain is consistent with a conformational change in H‐2 Db on peptide binding.
Mouse tumor rejection antigens P815A and P815B : Two epitopes carried by a single peptide
Authors: Lethé Bernard , Van den Eynde Benoǐt , Van Pel Aline , Corradin Giampietro , Boon Thierry
Published: March 2007

Mouse mastocytoma P815 expresses several distinct tumor rejection antigens recognized by syngeneic cytolytic T lymphocytes (CTL). Two of these tumor rejection antigens, P815A and P815B, are encoded by gene P1A, the sequence of which was reported previously. Tumor cell variants having lost one or both of these antigens were isolated by in vitro selection with CTL and also by collecting tumor cells that progressed in vivo after escaping a nearly complete immune rejection process. The structure of gene P1A in these antigen‐loss variants was examined. Several A−B− variants presented a complete or partial deletion of the gene. One variant that had lost only antigen A displayed a point mutation in the first exon. Peptides were synthesized that corresponded to the normal sequence located in the region of this point mutation. They sensitized target cells to both anti‐A and anti‐B CTL. The homologous peptide encoded by the mutated gene of the P815 A−B+ variant sensitized cells only to anti‐B CTL. We conclude that anti‐A and anti‐B CTL recognize on the same peptide two distinct epitopes that are affected differently by the mutation.
Enhancers of nonspecific immunity induce nitric oxide synthase: Induction does not correlate with toxicity or adjuvancy
Authors: Palacios Miriam , Knowles Richard G. , Moncada Salvador
Published: March 2007

A range of bacterial products and related synthetic compounds, either alone or in combination, enhance nonspecific resistance to infection and tumors. These compounds, which vary in their other properties such as pyrogenicity, toxicity and adjuvancy, were used to assess the hypothesis that nonspecific resistance is mediated by induction of the L‐arginine: nitric oxide (NO) pathway. The results obtained show that agents which stimulate nonspecific immunity, such as endotoxin, muramyl dipeptide (MDP) and combinations of monophosphoryl lipid A (MPL) with either trehalose dimycolate (TDM) or MDP cause a substantial induction of Ca2+‐independent NO synthase in murine lung. In contrast, agents which do not stimulate nonspecific resistance, such as either MPL or TDM alone or threonyl MDP (ThrMDP), do not induce NO synthase. This difference in the ability of MDP and ThrMDP to induce NO synthase in the lung in vivo was also manifest in peritoneal macrophages in vivo as well as being evident in the > 100‐fold greater potency of MDP in inducing the enzyme in vitro in lung slices. In contrast to the good correlation between induction of NO synthase and induction of nonspecific resistance, no correlation was observed with either the toxic effects of these agents or their adjuvancy.
Mechanisms of resistance to Leishmania aethiopica. I. Interferon‐γ in combination with a cytokine (not tumor necrosis factor‐α) is required, but cannot act alone in the inhibition of intracellular forms of L. aethiopica in THP1 cells
Authors: Mohamed Abdi Muse , Taye Tesema , Akuffo Hannah O.
Published: March 2007

Following exposure to promastigotes of various Leishmania species, mononuclear cells from non‐exposed as well as potentially exposed individuals produced a cytokine response which inhibited intracellular forms of Leishmania aethiopica in a permissive monocytic cell line (THP1). Interferon‐γ (IFN‐γ), was one of the cytokines responsible for this anti‐leishmanial effect. IFN‐γ was necessary for inhibition but could not act on its own in inhibiting L. aethiopica. Tumor necrosis factor‐α seemed not to be involved in the anti‐L. aethiopica effect. The observed effects were in the absence of endotoxin. The results suggest that the mechanisms of killing of L. aethiopica in human cells may differ from those responsible for inhibition of other Leishmania parasites (such as Leishmania major) in mouse macrophages. Furthermore, that potentially relevant responses to Leishmania antigens may exist in normal individuals.
Processing and presentation of intact hen egg‐white lysozyme by dendritic cells
Authors: De Bruijn Marloes L. H. , Nieland John D. , Harding Clifford V. , Melief Cornelis J. M.
Published: March 2007

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen‐presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H‐2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg‐white lysozyme (HEL) protein to present to an I‐Ak‐restricted T hybridoma cell line, specific for HEL peptide 46–61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide‐induced B cell blasts for effective presentation. Uptake of 125I‐labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H‐2k dendritic cells are capable of processing and presenting intact HEL protein.
Dependency on interleukin‐1 of primary human in vitro T cell responses to soluble antigens
Authors: Plebanski Magdalena , Elson Chris J. , Billington W. David
Published: March 2007

The role of interleukin (IL)‐1 in antigen‐specific activation of naive human T cells has been examined. Primary human T cell proliferative responses to the soluble antigen keyhole limpet hemocyanin (KLH) were decreased by neutralizing antisera to IL‐1α (25 ± 7 % standard error) and IL‐1β (56 ± 6 % standard error). Inhibition by both antisera in a primary culture was usually additive. Recombinant IL‐1α and recombinant IL‐1β could both re‐establish responses in cultures blocked by neutralizing anti‐IL‐1β Interestingly, the susceptibility of KLH‐stimulated T cell responses to inhibition by neutralizing anti‐IL‐1 sera decreased with time in culture. This observation suggested that T cell responses may become less IL‐1 dependent as T cells become activated or primed. In support of this notion, secondary T cell responses to purified protein derivative from Mycobacterium tuberculosis (PPD) were markedly less affected by the addition of comparable amounts of the neutralizing anti‐IL‐1 sera. These results demonstrate that IL‐1 is one of the main co‐stimulators for primary T cell activation and suggest a different requirement for IL‐1 in the activation of naive compared to memory human T cells.
Identification of a surface protein (p100) associated with two glycosyl‐phosphatidylinositol‐linked molecules (Thy‐1 and ThB) by natural anti‐lymphocyte autoantibodies
Authors: Lehuen Agnès , Monteiro Renato C. , Kearney John F.
Published: March 2007

Our previous study of natural autoantibodies showed that anti‐lymphocyte antibodies are frequently produced by perinatal B cells from normal strains of mice. One‐third of these monoclonal antibodies (mAb) recognized similar epitopes on the surface of thymocytes. In the present report, we have characterized the molecule recognized by three of these mAb (D10, G7, 22). These mAb identified a 100‐kDa protein (p100) on the surface of thymocytes. This protein resolved into 70‐kDa polypeptide chains under reducing conditions. Inhibition experiments as well as antibody immunoprecipitations in the presence of mild detergents revealed non‐covalent association of the p100 with Thy‐1 and ThB. A similar multimolecular complex was identified following chemical cross‐linking of thymocyte surface proteins. Analysis of several Thy‐1‐defective mutant cells lines, and thymocytes treated with phosphatidylinositol‐specific phospholipase C (PI‐PLC) showed that the expression of p100 was strongly influenced by Thy‐1 molecule. The p100 was resistant to PI‐PLC treatment and was not released into the supernatant as was the case for Thy‐1 and ThB molecules. These data lead us to propose that the p100 is a transmembrane protein, the expression of which in the plasma membrane is dependent on the association or presence of Thy‐1 molecule.
Mast cells contribute to fibrin deposition in reverse passive Arthus reaction in mouse skin
Authors: Ramos Bernard F. , Zhang Yan , Jakschik Barbara A.
Published: March 2007

The activation of the clotting system is an important process during inflammation to contain the injury and initiate tissue repair. In the present study, we investigated the effect of mast cells on fibrin deposition in reverse passive Arthus reaction in mast cell‐deficient WBB6F1‐W/WV(W/WV) and control WBB6F1‐+/+(+/+) mice, that were given 125I‐labeled fibrogen intravenousty. An antibody dose‐dependent increase in radioactivity was observed in the challenged skin sites. Sequential water and urea extractions characterized the radioiodinated fibrinogen derivatives present in the tissue. The radioactivity found in the various fractions of the stimulated samples from +/+ was 2–10‐fold higher than that in specimens from W/WV mice. The greatest difference was observed in the urea‐insoluble pellet (cross‐linked fibrin and its early degradation products). Reconstitution of W/WV mice with mast cells augmented the response to levels similar to those in +/+ mice. Pretreatment with the antihistamine pyrilamine blocked the accumulation of 125I‐labeled fibrinogen and its derivatives by approximately 70% in +/+ but not in W/WV mice. Inhibition of leukotriene synthesis by A‐63162 markedly decreased the accumulation of iodinated fibrinogen in both +/+ and W/WV mice. The data suggest that mast cells and their vasoactive mediator histamine contribute to the exudation of clotting factors, which results in fibrin deposition and that mast cells also enhance fibrin cross‐linkage.
Anti‐insulin and regulatory anti‐idiotypic antibodies use the same germ‐line VHIX gene
Authors: Thomas James W.
Published: March 2007

In humans and in BALB/c mice, immune responses to the hormone insulin use evolutionarily related VHV (human) and VHIX (murine) gene families. To determine if these structural relationships include regulatory elements, BALB/c mice were pretreated with autologous immunoglobulin G (IgG) monoclonal antibodies (mAb) that recognize shared idiotopes on human anti‐insulin antibodies and the subsequent immune response to human insulin assessed. One mAb, Id227, was found to augment and accelerate the insulin response by inducing a human idiotype that is expressed on both insulin‐binding and non‐insulin‐binding BALB/c antibodies. Analysis of VH gene utilization by Id227 shows that it expresses a VHIX gene similar to that of anti‐insulin mAb 125, but the anti‐Id has no anti‐insulin activity. Using DNA amplification, four germ‐line VHIX genes were isolated from BALB/c liver DNA and sequence analysis shows that the anti‐insulin and anti‐Id are derived from the same germ‐line gene. Consistent with its role as a regulatory idiotype, IgG Id227 entirely preserves germ‐line sequence in the complementray determining regions and contains only three mutations in framework regions. These studies show that both structural and regulatory features of immune responses to conserved self antigens extend beyond species boundaries.
Biochemical evidence of the physical association of the majority of CD3 δ chains with the accessory/co‐receptor molecules CD4 and CD8 on nonactivated T lymphocytes
Authors: Suzuki Satoru , Kupsch Jörg , Eichmann Klaus , Saizawa Mitsuyoshi K.
Published: March 2007

The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. Enhanced surface iodination was achieved by a water‐soluble derivative of the Bolton‐Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co‐precipitate a 28‐30‐kDa band identical in mobility to the δ chain of the CD3 complex. Components corresponding in mobility to the ε and γ chains of the CD3 complex are also co‐precipitated but to a much lesser extent. The identity of the co‐precipitated 28‐30‐kDa material with the CD3 δ chain was ascertained by two‐dimensional nonreducing/reducing SDS‐PAGE, by two‐dimensional non‐equilibrium pH gradient electrophoresis/SDS‐PAGE and by one‐dimensional peptide mapping with three different proteases. The co‐precipitated 28‐30‐kDa material was identical to the CD3 δ chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 δ co‐precipitated with anti‐CD4 and anti‐CD8 add up to those in anti‐Vβ precipitates and to an average of 90% of those in anti‐CD3ε precipitates. We conclude that the majority of CD3 δ chains are associated with the accessory/co‐receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen‐specific recognition by the T cell receptor.
The microbicidal activity of interferon‐γ‐treated macrophages against Trypanosoma cruzi involves an L‐arginine‐dependent, nitrogen oxide‐mediated mechanism inhibitable by interleukin‐10 and transforming growth factor‐β
Authors: Gazzinelli Ricardo T. , Oswald Isabel P. , Hieny Sara , James Stephanie L. , Sher Alan
Published: March 2007

The present study was carried out to determine the effector mechanism of anti‐Trypanosoma cruzi activity by interferon (IFN)‐γ plus lipopolysaccharide (LPS)‐treated macrophages. A macrophage cell line (IC‐21) that failed to mount an appreciable oxidative burst was nevertheless found able to control T. cruzi growth after exposure to IFN‐γ alone or IFN‐γ plus LPS. Moreover, microbicidal functions of both inflammatory macrophages and IC‐21 against T. cruzi was found to be inhibited in the presence of NG‐monomethyl‐L‐arginine (NGMMA), a competitive inhibitor of L‐arginine. Addition of supplemental L‐arginine to the culture overcame the capacity of NGMMA to block activated macrophage anti‐T. cruzi functions. The ability of NGMMA to reverse both parasite growth inhibition and killing by IFN‐γ plus LPS‐activated macrophages was found to correlate with the suppression of nitrite accumulation in the culture supernatants. Together, these results implicate the L‐arginine‐dependent production of nitric oxide in T. cruzi killing by activated macrophages. We also tested the ability of interleukin(IL)‐10 and transforming growth factor (TGF)‐β, to block regulation of T. cruzi growth in this system. Both IL‐10 and TGF‐β inhibited anti‐parasite function by IFN‐γ‐activated macrophages, with an optimal dose of 100 units/ml and 0.5 ng/ml, respectively. Moreover, when used in combination, suboptimal doses of IL‐10 and TGF‐β were found to produce a synergistic inhibitory effect in the regulation of T. cruzi growth. The ability of IL‐10 and TGF‐β to suppress microbicidal function was also positively correlated with inhibition of nitrite generation in macrophage culture supernatants. These results predict an in vivo role for IL‐10 and TGF‐β in promoting parasite survival in the face of the host cell‐mediated immune response.
A trypsin‐like serine protease activity on activated human B cells and various B cell lines
Authors: Biró Anna , Sármay Gabriella , Rozsnyay Zoltán , Klein Eva , Gergely János
Published: March 2007

We have studied the trypsin‐like serine protease activity of human tonsillar B lymphocytes. The lysate of the low‐density, in vivo activated B cells as well as the lysate of cells stimulated with anti‐human IgM F(ab')2 show elevated trypsin‐like serine protease activity compared to the resting subset as monitored by the cleavage of Tos‐Gly‐Pro‐Arg‐pNA. The cleavage is sensitive to N‐tosyl‐L‐lysyl‐chloromethyl ketone and benzamidine but not to iodoacetamide. Experiments with intact cells give similar results. The finding that the intact cells hydrolyze the substrate, while their supernatant does not, suggests that the protease activity is cell membrane associated. It is possible that C3 is a substrate of the enzyme since the activated B cells cleave C3, whereas the resting B cells do not, and also C3 inhibits the enzyme‐substrate reaction.

In addition to the ex vivo B cells, we studied the serine protease activity of certain well‐characterized B cell lines. The results show a correlation between the phenotype and the enzyme expression of the cell lines. BL41, an Epstein‐Barr virus (EBV)‐negative Burkitt lymphoma line, with a resting phenotype, has low activity, while its EBV genome‐carrying convertants E95‐A‐BL41, E95‐C‐BL41, EHR‐A‐BL41 and BL41/95 that have the phenotype of activated B cells, have high proteolytic activity. The lymphoblastoid cell line WW‐1‐LCL which has the phenotype of an immunoblast, has the highest serine protease activity.

On the basis of the above data, we suggest that a rather tight correlation exists between the degree of activation and the appearance of serine protease(s) on the surface of human B cells.
Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum μ chain deduced from cDNA sequence
Authors: Fellah Julien S. , Wiles Michael V. , Charlemagne Jacques , Schwager Joseph
Published: March 2007

CDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) μ heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5′‐end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis μ chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl μ chain, consisting of a 1362‐nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3′ direction by a 179‐nucleotide untranslated region and a polyA+ tail. The axolotl Cμ is divided into four typical domains (Cμ1‐Cμ4) and can be aligned with the Xenopus Cμ with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between Cμ1 (59%) and Cμ4 (71%). The C‐terminal 20‐amino acid segment which constitutes the secretory part of the μ chain is stronglyhomologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N‐linked oligosaccharide, the penultimate cysteine and the C‐terminal lysine. The four Cμ domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of μ‐specific residues which are conserved into each of the four Cμ domains argues for a continuous line of evolution of the vertebrate μ chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the Cμ4 sequences.
Modulation of integrin expression during mast cell differentiation
Authors: Ducharme Lori A. , Weis John H.
Published: March 2007

Previously we have reported that rodent mast cells synthesize the mRNA encoding the α and β integrin chains (α4, β1 and β7) of the lymphocyte Peyer's patch adhesion molecule (LPAM)‐1 and LPAM‐2 lymphocyte homing receptors, and that they possess an α4‐containing integrin complex on their cell surface. In this report, we have examined the expression of these integrin chain genes by mature connective tissue mast cells (CTMC) and by bone marrow‐derived mast cells (BMMC) differentiated from bone marrow precursor cells in the presence of interleukin (IL)‐3 and/or the c‐kit ligand (also known as mast cell growth factor and stem cell growth factor). High levels of both the β7 and β1 transcripts were present in mature CTMC while those encoding the α4 chain were absent. Similarly, when BMMC were grown in IL‐3 for 28 days and analyzed for integrin chain transcripts, those specific for the α4 chain were also diminished compared to β7 and β1 transcripts. To compare the expression of these integrin genes during mucosal mast cell and CTMC development, BMMC were derived in the presence of IL‐3 alone, c‐kit alone, or IL‐3/c‐kit together. These experiments indicated that c‐kit inhibited the transcription of the β7 and FcεRI genes while enhancing α4 transcript levels. The enhancement of α4 levels, however, was abrogated with the addition of IL‐3. Similarly, the c‐kit‐induced depression of β7 and Fcε RI transcript levels was overcome by the addition of IL‐3. These data suggest that the integrin complexes synthesized by the mast cells may differ depending upon their path of differentiation and that another α chain integrin may be synthesized to complex with the β7 and/or β1 chains.