bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous susceptibility of human melanoma clones to monocyte cytotoxicity: Role of ICAM‐1 defined by antibody blocking and gene transfer
Authors: Jonjic Nives , Alberti Saverio , Bernasconi Sergio , Peri Giuseppe , Jilek Petr , Anichini Andrea , Parmiani Giorgio , Mantovani Alberto
Published: March 2007

Five clones derived from the same human malignant melanoma lesion were studied for their susceptibility to killing by human monocytes activated by exposure to interferon (IFN)‐γ and lipopolysaccharide. Melanoma clones were heterogeneous in their susceptibility to human monocyte cytotoxicity, with one clone (2/21) exhibiting extremely low levels of lysis. The different levels of susceptibility to monocyte cytotoxicity were not accounted for by susceptibility or resistance to monokines [tumor necrosis factor (TNF), interleukin (IL)‐1 and IL‐6] because: (a) these effector molecules had little (TNF) or no (IL‐1 and IL‐6) cytolytic activity under these conditions; and (b) anti‐TNF antibodies had marginal effects on cytotoxicity. Monocytes bound less to resistant than to susceptible melanoma cells. Monocyte‐resistant 2/21 melanoma cells expressed substantially lower levels of ICAM‐1 and VLA‐4 than susceptible cells. Anti‐CD18 and, to a lesser extent, anti‐ICAM‐1 mAb inhibited binding and cytotoxicity of human monocytes on malignant melanoma whereas anti‐VLA‐4 had no inhibitory action. Transfection of the ICAM‐1 gene under the control of a constitutive promotor resulted in high levels of expression of ICAM‐1 in 2/21 melanoma cells and, concomitantly, in augmented susceptibility to activated monocyte cytotoxicity. The augmented killing of ICAM‐1‐transfected 2/21 cells was inhibited by anti‐ICAM‐1 mAb. These results demonstrate that the CD18‐ICAM‐1 adhesion pathway can play an important role in the expression of human monocyte cytotoxicity on melanoma target cells and that heterogeneity in expression of ICAM‐1 can underlie differences in susceptibility to tumoricidal activity.
The induction of resting B cell differentiation does not require T cell contact
Authors: Julius Michael , Haughn Lori
Published: March 2007

A T cell clone as well as immediately ex vivo CD4+ lymph node T cells are shown to support the differentiation of co‐cultured resting B cells in the absence of T cell‐B cell contact. Antibodies specific for class II products of the major histocompatibility complex inhibit the transactiration of resting but not activated B cells. This differential inhibition pattern indicates that the responses obtained from resting B cell populations are not due to their contamination with B cell blasts. Further, supernatants prepared from an activated T cell clone induce resting B cell differentiation. Two lines of evidence suggest that the activity contained in these supernatants can be attributed to interleukin (IL)‐5. Activity is neutralized by monoclonal anti‐IL‐5; and both recombinant and affinity‐purified IL‐5 induce the differentiation of immediately ex vivo resting and activated B cells with comparable efficiency. Taken together, these results demonstrate that contact with T cells does not provide prerequisite signals for the induction of resting B cell differentiation.
Liver of MRL/lpr mice contain interleukin‐4‐producing lymphocytes and accessory cells that support the proliferation of Th2 helper T lymphocyte clones
Authors: Magilavy Daniel B. , Foys Kathleen M. , Gajewski Thomas F.
Published: March 2007

Hepatic nonparenchymal cells (NPC) from mice of nonautoimmune strains support the proliferation of only Th1 and not Th2 helper T lymphocyte (HTL) clones. Because of the multiple systemic and liver‐specific immune defects in the autoimmune MRL/lpr mouse strain, we have explored the possibility that hepatic accessory cells from MRL/lpr mice are capable of stimulating the proliferation of Th2 HTL. We report here that hepatic NPC from MRL/lpr and C3H/lpr female mice older than 8 weeks, in contrast to hepatic NPC from MRL/++ and C3H/HeN strains, are able to support in vitro the proliferation of both Th1 and Th2 CD4 clones. Additionally, hepatic lymphocytes (HL) from MRL/lpr mice can be stimulated to produce interleukin (IL)‐4 to a much higher degree than HL from the nonautoimmune strains. These results suggest that the activation of Th2 cells by hepatic NPC and production of IL‐4 by HL may contribute to the immunologic aberrations in the MRL/lpr mouse strain.
Phenotypic changes accompanying positive selection of CD4+CD8+ thymocytes
Authors: Swat Wojciech , Dessing Mark , Baron Agnes , Kisielow Pawel , von Boehmer Harald
Published: March 2007

We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the αβ T cell receptor (TcRhigh) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co‐receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I‐restricted transgenic TcR develop in vitro exclusively into CD4−CD8+ cells while small CD4+CD8+ TcRhigh cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR‐MHC ligand interactions in the generation of mature αβ T cells.
Characterization of nuclear protein binding to the interferon‐γ promoter in quiescent and activated human T cells
Authors: Brown David A. , Kondo Kimi L. , Wong Shen‐Wu , Diamond Don J.
Published: March 2007

Nuclear protein binding to the human interferon‐γ (IFN‐γ) promoter was investigated to determine the structural basis for the control of gene expression during T cell activation. DNase I footprinting of gel‐shift complexes demonstrated that proteins bind to two downstream (−124 to −114 and −36 to −30) and one upstream (−534 to −486) element in the IFN‐γ gene promoter. Treatment of human peripheral blood lymphocytes or continuous T cell tumors with phorbol 12‐myristate 13‐acetate (PMA) plus phytohemagglutinin or calcium ionophore results in a pattern of response that is similar when using either the upstream or downstream elements. Upon induction of T cells, the lower mobility gel‐shift band disappears. Yet the equivalent band which is also present in non‐T cells is unperturbed after PMA + calcium ionophore treatment. The higher mobility band which is modified upon induction is restricted to the T cell lineage. Upstream and downstream elements share similar protein‐binding motifs as indicated by the homology of footprinted sequences, the similarity of protein‐binding patterns, and the ability of these elements to compete against each other in gel‐shift protein‐binding assays. Protein binding to the downstream elements appears to be interactive, since both sites are required for complex formation. When either of the two downstream elements is disrupted by site‐directed mutagenesis, the higher mobility gel‐shift band is diminished by an amount that is consistent with the reduction in reporter (chloramphenicol acetyltransferase) gene expression. Therefore, proteins in the ubiquitous gel‐shift band appear to be associated with the inactive state of IFN‐γ, while the modified band is closely associated with the positive regulation of IFN‐γ gene expression.
Macrophage‐induced cytotoxicity of N‐methyl‐D‐aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines
Authors: Piani Daniela , Spranger Matthias , Frei Karl , Schaffner Andreas , Fontana Adriano
Published: March 2007

The co‐localization of activated macrophages and damaged neurons observed in brain injury and degenerative brain diseases may hint to macrophage‐induced neuronal cytotoxicity. Recently, macrophages have been found to secrete neurotoxic molecules such as radical oxygen intermediates and glutamate, the latter interacting with N‐methyl‐D‐aspartate (NMDA) receptors. As shown in the present study, brain macrophages termed microglial cells co‐cultured with differentiated cerebellar neurons excert potent neurotoxic effects. Neurotoxicity is unlikely to be due to cytokines since tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 and interferon (IFN)‐α/IFN‐β/IFN‐γ had no such effects. In contrast, when treating neurons with H2O2 or oxygen radical‐generating systems cytotoxicity was induced. Furthermore, microglia were found to produce O and H2O2 when triggered with phorbol 12‐myristate 13‐acetate. However, in co‐cultures of neurons and microglia, oxygen‐radical scavengers catalase and superoxide dismutase, failed to protect neurons from microglia‐induced killing. Moreover, when using undifferentiated neurons which are susceptible to H2O2 but not to NMDA receptor‐dependent killing, microglia did not destroy the neurons. Thus, the amount of reactive oxygen intermediates produced by microglia in co‐culture do not reach the critical concentrations required for neurotoxicity. As dibenzocyclohepteneimide, an antagonist to NMDA receptors neutralized neurotoxicity in microglia‐neuronal co‐cultures, excitatory amino acids released by microglia are suggested to compose the major determinant of neurotoxicity.
Netwhat?
Authors: Etlinger Howard M. , Gray David
Published: March 2007

The hallmark of an antibody response is considered to be its specificity for the immunogen. Nonetheless, most antibody elicited by an immunogen has been reported to be unreactive with it. We have evaluated specificity by characterizing the primary antibody response of mice to heat‐aggregated human γ‐globulin with four approaches: (a) quantification of antibody‐producing cells; (b) hybridoma analysis; (c) in situ antigen binding and (d) analysis of secreted antibody. The results show that the nonspecific component of this response is negligible. These observations suggest that such a component is not a basic feature of the antibody response and it is discussed that nonantigen‐specific antibody may arise from a variety of causes many of which are artifactual.
CD4‐class II major histocompatibility complex interaction does not enhance killing by a class I‐restricted CD4+CD8+ cytotoxic T cell clone
Authors: McAdam Alexander J. , Blieden Timothy M. , Frelinger John G. , Lord Edith M.
Published: March 2007

As unusual tumor‐specific cytotoxic T lymphocyte (CTL) clone was isolated which expressed both CD4 and CD8 molecules. The target cells for this CTL can be induced to express either class I major histocompatibility complex (MHC) alone (with dimethylsulfoxide) or both class I and class II MHC (with interferon‐γ). Lysis of the tumor target depends on expression of class I MHC molecules, but does not require expression of class II MHC molecules. Furthermore, the lysis of target cells expressing both class I and class II is inhibited only by antibodies to class I (Kd), and not by antibodies to class II, demonstrating that the T cell receptor is class I restricted. We have used this CTL to assess the role of the interaction between CD4 and class II MHC in the absence of a class II‐restricted T cell receptor. Our data indicate that CD4‐class II interaction does not contribute to recognition by T cells in the absence of binding of the T cell receptor to class II molecules.
Langerhans cells of the human skin possess high‐affinity 12(S)‐hydroxyeicosa tetraenoic acid receptors
Authors: Arenberger Petr , Kemény Lajos , Rupec Rudolf , Bieber Thomas , Ruzicka Thomas
Published: March 2007

The arachidonic acid metabolite 12‐hydroxyeicosatetraenoic acid (12‐HETE) is the main eicosanoid formed by epidermal cells and is assumed to play an important role in skin physiology and pathophysiology. Our aim was to find out whether epidermal Langerhans cells possess specific receptors for 12‐HETE which would mediate the effects of this eicosanoid in their skin microenvironment. By radioligand binding studies on isolated human Langerhans cells, we could identify specific binding sites for 12(S)‐HETE. The analysis of binding data revealed a single class of binding sites with a Kd of 3.32 ± 0.45 nM and a Bmax of 691 000 ± 58 000 receptors per cell. The binding was saturable, readily reversible, and specific for 12(S)‐HETE. The receptor is likely to mediate the potent chemotactic response of human Langerhans cells towards 12(S)‐HETE, which we previously described. Our results strongly suggest that extremely low concentrations of 12‐HETE which is formed by epidermal keratinocytes may dramatically influence the biology of Langerhans cells by receptor‐mediated effects.
Improved survival from potentially lethal graft‐vs.‐host disease by donor pretreatment with a recipient‐specific blood transfusion. I. Requirements for induction and specificity of the effect
Authors: Knulst André C. , Bril‐Bazuin Clara , Tibbe G. John M. , Benner Robbert
Published: March 2007

Pretreatment of prospective donors of hemopoietic cells with a single recipientspecific blood transfusion can significantly decrease the morbidity and mortality of graft‐vs.‐host disease (GVHD) in lethally irradiated, allogeneically reconstituted mice. This beneficial effect of donor pretreatment could be demonstrated in donor‐recipient strain combinations that were H‐2 + non‐H‐2, H‐2, or only class II‐disparate, but not in the class I‐disparate C57BL‐B6.C‐H‐2bm1 strain combination. The effect was proportional to the amount of recipient‐strain blood used for transfusion. Donor transfusion with a single dose of 1 ml recipient‐specific whole blood resulted in minimum GVHD, lower doses being less or not effective. The interval between donor pretreatment and the use of their hemopoietic cells for reconstitution appeared to be important. The best survival was found at an interval of 4 days. Multiple transfusion was not more effective than a single one. We compared the effectiveness of whole blood and irradiated spleen cells for donor pretreatment. Both protocols have been shown previously to suppress anti‐recipient delayed‐type hypersensitivity. It appeared that the blood transfusion protocol was superior to the spleen cell protocol. The beneficial effect appeared to be recipient specific, since a third‐party blood transfusion did not improve GVHD. We found that the beneficial effect of donor blood transfusion was due to suppression of the anti‐host immune response. The donor blood transfusion was able to induce bystander suppression to alloantigens that were not used for the induction of suppression, provided they were co‐expressed with the specific alloantigens by the recipients. This also indicates that, although the induction of suppression is specific, the ultimate suppressive effect is nonspecific.
Effects of the thymic microenvironment on the response of thymocytes to stimulation
Authors: Moore Nel C. , Jenkinson Eric J. , Owen John J. T.
Published: March 2007

We show that, in vitro, the response of thymocytes to certain stimuli, and their survival largely depend on the nature of the culture environment, i.e. whether thymocytes are stimulated within intact thymus lobes or in cell suspension. Exposure of isolated thymocytes to 12‐O‐tetra‐decanoylphorbol 13‐acetate (TPA) + ionomycin rapidly abolishes the expression of recombination‐activating gene‐1 (RAG‐1) mRNA (3 h), down‐regulates CD4 surface antigen expression (3 h), and enhances apoptosis (24 h). On the other hand, when thymocytes are cultured in intact lobes, TPA plus ionomycin down‐regulate rather than abolish RAG‐1 mRNA expression (3 h), have little effect on CD4 expression even following 24‐h exposure, and only marginally induce apoptosis (24 h). Differences between the culture systems are less pronounced in reponse to anti‐CD3 antibodies. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. In addition, it has been suggested that termination of RAG‐1 expression can be linked to thymocyte selection processes. We found that the down‐regulation of RAG‐1 expression was not dependent on the induction of apoptosis, supporting a proposed link with positive selection.
Genetics of nonspecific immunity: I. Bidirectional selective breeding of lines of mice endowed with maximal or minimal inflammatory responsiveness
Authors: Ibanez Olga M. , Stiffel Claude , Ribeiro Orlando G. , Cabrera Wafa K. , Massa Solange , De Franco Marcelo , Sant'Anna Osvaldo A. , Decreusefond Claude , Mouton Denise , Siqueira Maria , Biozzi Guido
Published: March 2007

The genetic regulation of acute inflammatory reaction (AIR) was studied by the method of bidirectional selective breeding, used to produce a line of mice giving the maximal and a line of mice giving the minimal inflammatory reaction (AIR max and AIR min, respectively). The AIR was triggered by subcutaneous injection of a neutral substrate (suspension of polyacrylamide microbeads), and measured by the leukocyte and serum protein accumulation in the exudate. The two parameters are positively correlated and present a normal frequency distribution. The highly genetically heterogeneous foundation population was produced by the equipoised intercrossing of eight inbred strains of mice, and selective breeding carried out by assortative matings of extreme phenotypes. The response to selection in 11 consecutive generations was highly asymmetrical: a marked AIR increase in the AIR max and no change in the AIR min line occurred. The mean value of realized heritability in the AIR max line was 0.26 and 0.18 for cell and protein concentrations, respectively. The response to selection must have resulted from the interaction of seven to nine independent gene loci endowed with additive effects. The lack of response to selection of the AIR min line is discussed. The large inter‐line difference opens new possibilities for studying the biochemistry and molecular genetics of inflammation, and also for investigating the beneficial or detrimental effect of inflammatory responses.
Evidence for oligoclonality of T cell receptor δ chain transcripts expressed in rheumatoid arthritis patients
Authors: Olive Colleen , Gatenby Paul A. , Serjeantson Susan W.
Published: March 2007

The inflamed synovium of rheumatoid arthritis (RA) patients contains γ/δ T cells which express predominantly T cell receptor (TcR) variable (Vδ1 and Vδ2 chains. Such T cells may contribute to the pathogenesis of RA. To assess the extent of clonality among these T cell populations we sequenced the junctional regions of rearranged TcR Vδ1‐Cδ and Vδ2‐Cδ chain cDNA, after using the polymerase chain reaction (PCR) to amplify TcR δ chain transcripts isolated from synovial membrane mononuclear cells (SMC) of five RA patients. The sequences of these δ chain transcripts were compared with those found in peripheral blood mononuclear cells (PBMC) of the same patients and in PBMC of four healthy controls. In contrast to control PBMC, Vδ1 chain cDNA derived from PBMC of three patients showed a strong bias towards usage of the same V‐joining (J) combination and junctional region sequences, although the specific sequences were unique in each patient. However, oligoclonality of the Vδ1 chain was less marked in SMC of two of these patients and absent in SMC of the other patients. For Vδ2, oligoclonality was detected in PBMC of two patients. In SMC of a single patient, a dominant Vδ2 transcript was detected that utilized the Jδ2 segment, which was rarely expressed in the normal TcR repertoire. These results indicate in vivo clonal expansion of Vδ1 and Vδ2‐expressing γ/δ T cells in the peripheral blood of RA patients and a synovial T cell infiltrate which consists largely of polyclonally expanded γ/δ T cells, but shows clonal dominance in some patients. Our data strongly support a role for Vδ1+ and Vδ2+ γ/β T cells in the pathogenesis of RA, and, although the nature of the antigen(s) recognized by these cells remains elusive, this report suggests the potential involvement of antigen(s) specific for the V region and V‐J junction.
Lipopolysaccharide induces human interleukin‐1 receptor antagonist and interleukin‐1 production in the same cell
Authors: Andersson Jan , Björk Lars , Dinarello Charles A. , Towbin Harry , Andersson Ulf
Published: March 2007

A new member of the interleukin‐1 (IL‐1) family has recently been described. Human IL‐1 receptor antagonist (IL‐1ra) is structurally related to IL‐1α and IL‐1β but binds to IL‐1 receptors on various target cells without demonstrable agonist activity. Understanding the mechanisms of regulation of IL‐1ra production may clarify the biology of this unique cytokine as well as elucidate its possible role as a natural anti‐inflammatory protein. The effects of lipopolysaccharide (LPS) on IL‐1α, IL‐1β and IL‐1ra production was studied at a single‐cell level by use of cytokine‐specific antibodies and indirect immunofluorescence technique. The peak synthesis of IL‐1ra and IL‐1α/β occurred in peripheral blood monocytes obtained from healthy blood donors within 4 and 6 h of cell stimulation, respectively. By double‐staining procedure all IL‐1ra‐positive cells were also IL‐1α and/or β positive. Thus, endotoxin induced simultaneous synthesis of the IL‐1 gene family in the same cells. Only monocytes contributed to the production of IL‐1α, β and IL‐1ra during the 96 h of cell culture. The maximum number of IL‐1ra‐producing monocytes was 48 ± 16% as compared to peak production of IL‐1α and β which occurred in 75 ± 9% and 80 ± 12% (p < 0.001), respectively, of all peripheral blood monocytes. The incidence of IL‐1α and β‐containing cells was not only significantly higher but also occurred for a longer time period, 72 h as compared to 24 h for IL‐1ra localized in the Golgi organelle. However, IL‐1ra‐containing cells with a diffuse cytoplasmic appearance were also evident (20%–30%) at a later stage, 12 to 72 h after stimulation. Blocking IL‐1 surface receptors by addition of exogenous recombinant IL‐1β before stimulation could not inhibit the diffuse cytosolic localization. This indicates that the „late”︁ staining pattern did not reflect IL‐1ra being secreted and internalized after binding to extracellular receptors. Thus, perhaps IL‐1ra modulates IL‐1 effector mechanisms by receptor interactions both inside and outside the cell.
T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38‐kDa antigen in patients with active disease
Authors: Vordermeier Hans‐M. , Harris David P. , Friscia Giuseppe , Román Eulogia , Surcel Heljä‐Marja , Moreno Carlos , Pasvol Geoffrey , Ivanyi Juraj
Published: March 2007

It is generally accepted that both host protection and pathogenic reactions in tuberculosis are mediated by T lymphocytes. However, little is known about the structures and discreet functions of epitopes stimulating the immune response. In this study, proliferative responses of blood T lymphocytes to synthetic peptides derived from the sequence of the 38‐kDa antigen from Mycobacterium tuberculosis have been investigated in 41 healthy individuals and in 36 patients with active tuberculosis. Of the healthy purified protein derivative (PPD)‐positive donors, 90% responded to a permissively recognized peptide, 38.G (residues 350–359), located at the carboxy terminus of the molecule. Four other permissively recognized epitopes of this molecule (38.A, 38.I, 38.E, 38.K) were stimulatory for more then 50% of healthy PPD‐positive individuals. Patients with lymphatic tuberculosis responded to these peptides in a similar manner. In contrast, we observed a selective anergy to stimulation with peptide 38.G in the majority of patients with pulmonary (11% responders) and nonlymphatic extrapulmonary tuberculosis (25% responders). The lack of responsiveness to 38.G was epitope specific since the degree of responsiveness to the other four permissively recognized peptide epitopes was similar for patients and PPD‐positive controls. Using the PEPSCAN technology and truncated peptides, the core epitope of 38.G was localized to a peptide 10 amino acids long (HFQPLPPAVV). This minimal structure was capable of inducing a proliferative response in all healthy 38.G responders tested. The mechanisms influencing this epitope‐specific anergy in patients could give new insights into the immunopathogenesis of tuberculosis.
Expression of major histocompatibility complex class I antigens at low levels in the thymus induces T cell tolerance via a non‐deletional mechanism
Authors: Husbands Sandra D. , Schönrich Gunther , Arnold Bernd , Chandler Phillip R. , Simpson Elizabeth , Philpott Karen L. , Tomlinson Peter , O'Reilly Lorraine , Cooke Anne , Mellor Andrew L.
Published: March 2007

Transgenic CBA (H‐2k haplotype) mice expressing the H‐2 Kb major histocompatibility complex (MHC) class I gene under control of transcriptional promoter elements from a milk protein gene display high‐level H‐2 Kb transcription in lactating mammary glands and low‐level transcription in skin and thymus of male and virgin female transgenic mice. However, H‐2 Kb antigen could be detected only in lactating mammary gland epithelial cells by immunohistological methods. All transgenic mice are tolerant of H‐2 Kb since they fail to reject skin grafts from mice expressing H‐2 Kb molecules. Furthermore, anti‐H‐2 Kb cytotoxic responses could not be generated using responder T cells from transgenic mice but T cells from the same mice proliferated, in the presence of interleukin‐2, in response to stimulator cells expressing H‐2 Kb. Tolerance to H‐2 Kb is induced in the thymus since CBA mice grafted with thymus tissue from transgenic mice fail to reject H‐2 Kb disparate skin grafts. However, experiments with double‐transgenic mice also expressing a T cell receptor with anti‐H‐2 Kb specificity reveal that tolerance induction is not brought about by elimination of thymocytes bearing H‐2 Kb‐reactive receptors. Instead, a non‐deletional mechanism which results in down‐modulation of both CD8 and T cell receptor expression in peripheral T cells correlates with the induction of tolerance in these mice. These data reveal that extremely low levels of self‐antigen expression in the thymus are sufficient to induce tolerance via non‐deletional mechanisms.
Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide deletion in the HRF20 (CD59) gene
Authors: Motoyama Noboru , Okada Noriko , Yamashina Manabu , Okada Hidechika
Published: March 2007

HRF20 (CD59) is a membrane glycoprotein which protects cells from the membrane attack reaction of homologous complement. A patient who is completely deficient in HRF20 expression and is suffering from paroxysmal nocturnal hemoglobinuria (PNH) was studied. His parents are cousins and both have decreased HRF20 expression, suggesting that the deficiency is genetic. We established a cultured cell line (NCU1) which is HRF20 deficient from the patient's lymphocytes by Epstein‐Barr‐virus (EBV) infection. Northern blot analysis revealed HRF20 mRNA signals, indicating that HRF20 mRNA were transcribed. HRF20 cDNA was amplified by the polymerase chain reaction (PCR) method. Sequencing of the cDNA from the NCU1 showed two single‐base deletions at amino acid 16 and 96 from the N terminus of the mature protein. Deletion in the genomic DNA of peripheral blood lymphocytes was confirmed by the DNA sequence of an HRF20 open reading frame containing amino acid 16. Furthermore, the patient's parents and sister possessed both intact and deleted genomic HRF20 DNA while his brother's DNA was intact. These findings demonstrate that the HRF20 deficiency was genomic in origin, and that complete deletion was brought about by a homozygous abnormality in the HRF20 gene. The base deletion caused a codon frame shift resulting in failure to produce intact HRF20 protein in the patient.
α1‐Acid glycoprotein potentiates lipopolysaccharide‐induced secretion of interleukin‐1 β, interleukin‐6 and tumor necrosis factor‐α by human monocytes and alveolar and peritoneal macrophages
Authors: Boutten Anne , Dehoux Monique , Deschenes Marianne , Rouzeau Jean‐Denis , Bories Phuong Nhi , Durand Geneviève
Published: March 2007

Although the physiological role of α1‐acid glycoprotein (AGP), an acute‐phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)‐1β, IL‐6 and tumor necrosis factor (TNF)‐α by human monocytes, macrophages and the monocytic THP‐1 cell line. AGP significantly enhanced (2‐ to 7‐fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum‐free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250–500 μg/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS‐induced IL‐1β, IL‐6 or TNF‐α secretion. AGP acts as a co‐inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo‐AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS‐induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.
Assessment of major histocompatibility complex class I interaction with Epstein‐Barr virus and human immunodeficiency virus peptides by elevation of membrane H‐2 and HLA in peptide loading‐deficient cells
Authors: Stuber György , Modrow Susanne , Höglund Petter , Franksson Lars , Elvin John , Wolf Hans , Kärre Klas , Klein George
Published: March 2007

Earlier findings indicate that peptides can affect the expression of major histocompatibility complex (MHC) class I molecules on the surface of cells with defective peptide loading mechanism. We have used peptide induced increase of class I antigen expression to assess peptide interaction with MHC class I molecules. A panel of 41 overlapping synthetic peptides derived from the human immunodeficiency virus‐1 (HIV‐1) gag protein and 33 nonoverlapping peptides from Epstein‐Barr virus (EBV) proteins EBNA‐1, 2, 3, 4, 5, 6, LMP, BZLF2, BILF2, BSLF2, BALF4 and BcLF1 was assessed for the ability to enhance the expression of HLA‐A2.1, H‐2Db, Kb and Dd on the murine RMA‐S and human 721.174/T2 (.174/T2) lines by indirect immunofluorescence. Considering doubling of the fluorescence intensity in the peptide‐treated samples as positivity, 6 of 39 HIV and 1 of 32 EBV peptides were found to bind to A2.1, 6 of 39 HIV gag and 7 of 16 EBV peptides to Db, 8 of 39 HIV gag and 5 of 16 EBV peptides to Kb and 2 of 39 HIV gag and 1 of 17 EBV peptides to Dd. The sensitivity of the method is comparable to the in vitro class I assembly assay with conformation‐dependent monoclonal antibody and is more discriminating than the solid‐phase assay. Due to its simplicity this method can also serve for testing large peptide panels for binding capacity to various class I molecules. Moreover, the method provides information about the relevance of in vitro tests for class I assembly in living cells.
Self tolerance to human A and B histo‐blood group antigens exists at the B cell level and cannot be broken by potent polyclonal B cell activation in vitro
Authors: Rieben Robert , Tucci Alessandra , Nydegger Urs E. , Zubler Rudolf H.
Published: March 2007

It is generally considered that tolerance to self antigens is less complete in B than in T lymphocytes. However, B cell tolerance through either functional inactivation (anergy) or clonal deletion has been demonstrated in transgenic mice. In the present study, we investigated whether B cells specific for self A/B histo‐blood group antigens can be detected in normal humans. It is a key feature of the ABO system that all normal individuals make natural antibodies against those A or B carbohydrates which are not present in their organism. To detect B cells by the limiting dilution approach we used a specific enzyme‐linked immunosorbent assay for the quantitation of anti‐A/B antibodies, and a culture system in which polyclonal B cell activation occurs through cell contact with EL4 thymoma cells. As was reported for other B cell studies, we frequently detected „polyreactive”︁ immunoglobulin (Ig)M (but not IgG) with apparent autoreactivity but of uncertain significance regarding physiologic conditions. However, A‐ or B‐specific B cell responses occurred with selective patterns in agreement with classical blood group serology in 14 individuals with A, B, AB or 0 blood group phenotypes: 1/11 600 B cells made anti‐allo A/B IgM and 1/26 500 B cells such as IgG, while only 1/104 000 B cells apparently made anti‐self A/B IgM and 1/350 000 B cells such as IgG. This shows self tolerance at the B cell level. Since anergy of B cells can frequently be broken by polyclonal B cell activation in vitro, and EL4 cells are potent B cell stimulators, the present results argue for either a highly resistant anergic state or for clonal deletion of self‐A/B histo‐bloos group‐specific human B cells.
A versatile method to produce antibodies to human T cell receptor Vβ segments: frequency determination of human Vβ2+ T cells that react with toxic‐shock syndrome toxin‐1
Authors: Romagné François , Besnardeau Lydia , Malissen Bernard
Published: March 2007

Human Vβ (hVβ) regions have been expressed in the context of mouse T cell receptor (TcR)‐CD3 complexes, and subsequently used to raise hVβ‐specific monoclonal antibodies (mAb). The method of expression of hVβ outlined in this report contrasts in its versatility with the one reported by Choi et al. (Proc. Natl. Acad. Sci. USA 1991. 88: 8357). For instance, we have applied it successfully to the construction of mouse T cell transfectants expressing hVβ1, hVβ2, hVβ3, hVβ8, hVβ9, hVβ13.5, hVβ19, hVβ21, and hVβ22 gene segments. mAb against the hVβ2 and hVβ19 regions have been raised by using these transfectants as immunogens in mice. Here, we illustrate the application of the anti‐hVβ mAb to the measurement of human T cells that react with the staphylococcal toxic‐shock syndrome toxin‐1.
The B29 and mb‐1 polypeptides are differentially expressed during human B cell differentiation
Authors: Mason David Y. , Van Noesel Carel J. M. , Cordell Jacqueline L. , Marieke Comans‐Bitter W. , Micklem Kingsley , Tse Albert G. D. , Van Lier René A. W. , Van Dongen4 Jacques J. M.
Published: March 2007

Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb‐1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37‐kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti‐B29 antibodies was compared with those of antibodies to the mb‐1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb‐1 whereas B29 was expressed in only 13 cases. Most of these B29‐positive ALL expressed immunoglobulin μ heavy chain in their cytoplasm (pre‐B ALL). In lymphoid tissue sections, anti‐B29‐labeled B cell follicles in a similar fashion to anti‐mb‐1, with the striking exception that plasma cells were unreactive for B29, but positive for mb‐1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb‐1, and ceases before the terminal plasmacyte phase.
Differentiation‐specific expression of a novel G protein‐coupled receptor from Burkitt's lymphoma
Authors: Dobner Thomas , Wolf Ingrid , Emrich Thomas , Lipp Martin
Published: March 2007

Deregulation of the proto‐oncogene MYC by specific chromosomal translocations has been shown to be essential but not sufficient for the development of Burkitt's lymphoma (BL). To identify other genes which either mark important steps in tumorigenesis or which reflect the cellular differentiation state of BL cells we have compared tumor cells to immortalized lymphoblastoid B cells by subtractive hybridization. We have identified a complementary DNA clone which encodes a novel member of the superfamily of GTP‐binding (G) protein‐coupled receptors, designated BLR1. The corresponding mRNA is expressed in BL and lymphatic tissues but not in other cell lines either of the B cell lineage or of other hematopoietic or non‐hematopoietic origin. This exclusive expression of BLR1 and the oncogenic potential of this receptor class supports the hypothesis that BLR1 exerts a regulatory function in BL lymphomagenesis and/or B cell differentiation. Moreover, the protein sequence is highly related to that of receptors for the cytokine interleukin (IL)‐8 and other neutrophil chemoattractants. We conclude that BLR1 may represent a potential candidate involved in the process of physiologic trafficking, cell‐cell interactions, and activation of mature B lymphocytes in lymphatic tissues.
Proliferation and differentiation of human CD5+ and CD5− B cell subsets activated through their antigen receptors or CD40 antigens
Authors: Defrance Thierry , Vanbervliet Béatrice , Durand Isabelle , Briolay Jérǒme , Banchereau Jacques
Published: March 2007

The pan‐T cell antigen CD5 has been shown to delineate two different mouse B cell subsets, originating from distinct progenitors. In man, on average, 30% of the tonsillar B cell pool expresses this antigen. In the present report, a detailed comparison of the CD5+ and CD5− B cell response to cytokines, following activation via surface immunoglobulins (sIg) or CD40 antigen, was undertaken. CD5+ B cells were positively selected by panning or by sorting from tonsils. Two‐color immunofluorescence analysis performed on tonsillar B cell populations showed that CD5+ B cells displayed most of the phenotypic features of mantle zone B cells. CD5+ B cells could be stimulated for DNA synthesis by mitogenic concentrations of Staphylococcus aureus, Cowan I strain (SAC), insolubilized anti‐IgM antibodies, immobilized anti‐CD40 antibodies and phorbol 12‐myristate 13‐acetate (PMA). The growth‐response of small dense CD5− B cells to these T cell‐independent mitogens was comparable to that of CD5+ B cells, whereas the low‐density, in vivo‐activated, CD5− B cells were only marginally stimulated by Ig‐cross‐linking agents and PMA. Following ligation of sIg, both B cell subsets proliferated essentially in response to interleukin (IL)‐2 and IL‐4. When used in co‐stimulation with immobilized anti‐CD40 antibodies, IL‐4 promoted growth of CD5+ and CD5− B cells, whereas IL‐2 displayed only moderate stimulatory effects. CD5+ and CD5− B cells differentiated into Ig‐secreting cells when they were co‐cultured with SAC or cross‐linked anti‐CD40 antibodies and IL‐2. However, IgM constituted the major component of the Ig response of CD5+ B cells, whereas high levels of IgG were secreted by CD5− B cells.
Expression and biological activity of interleukin‐1 receptors in mouse γ/δ thymocytes during ontogeny
Authors: Liberman Isabelle , Sarthou Pierre , Cazenave Pierre‐André , Rueff‐Juy Dominique
Published: March 2007

We have recently shown that the response of mouse thymocytes to interleukin (IL)‐1 + IL‐2 was maximal at birth and that the responding cells displayed a CD4−CD8− T cell receptor (TcR) γ/δ+ phenotype. Unexpectedly, despite their high proportion of γ/δ+ cells, fetal thymocyte populations responded only weakly to IL‐1 + IL‐2. In this report, we demonstrate that the discrepancy between the day 17.5 fetal and newborn sensitivities to the combined action of IL‐1 and IL‐2 is a consequence of the different patterns of high‐affinity IL‐1 receptor (IL‐1R) expression displayed by these two cell subsets. Actually, high‐ and low‐affinity IL‐1R are found in TcR γ/δ+ newborn cells and, in contrast, only low‐affinity IL‐1R are detectable in day 17.5 fetal cells. Our binding and functional studies strongly support the hypothesis that high‐affinity IL‐1R on the one hand, and low‐affinity ones on the other hand, are involved in the response to IL‐1 + IL‐2 of newborn and day 17.5 fetal thymocytes, respectively. In addition, the high‐affinity IL‐1R appear to be far more efficient than the low‐affinity receptors in promoting IL‐2 responsiveness of thymocytes.
Integrins and other adhesion molecules on lymphocytes from synovial fluid and peripheral blood of rheumatoid arthritis patients
Authors: Takahashi Hiroki , Söderström Kalle , Nilsson Ethel , Kiessling Rolf , Patarroyo Manuel
Published: March 2007

Cell and matrix adhesion of lymphocytes participates in homing, migration and accumulation of these cells in inflamed tissues as well as in the generation of immune and inflammatory responses. In inflamed joints of rheumatoid arthritis (RA) patients, lymphocytes accumulate in the synovial membrane and the synovial fluid. In the present study we have analyzed the expression of integrins and other adhesion molecules in synovial fluid lymphocytes (RA‐SFL) and paired peripheral blood lymphocytes (RA‐PBL) from 21 RA patients by immunofluorescence flow cytometry. We have also investigated the expression of these adhesion molecules on peripheral blood lymphocytes obtained from 13 sex‐ and age‐matched healthy controls (CO‐PBL). RA‐SFL, which consisted mostly of T cells, showed higher expression of the integrin subunits β1 (CD29), VLA‐1α, −3α, −4α, −5α and −6α when compared to RA‐PBL. In turn, RA‐PBL showed lower expression of these molecules than CO‐PBL. The expression of the immunoglobulin‐related molecules CD2, CD54 (ICAM‐1) and CD58 (LFA‐3) was higher on RA‐SFL when compared to RA‐PBL or CO‐PBL, and similar results were obtained with the β2 integrin subunits CD11a and CD18. In contrast, L‐selectin (LECAM‐1) and ICAM‐2 were expressed at much lower levels on RA‐SFL than on RA‐PBL or CO‐PBL. CD44, a receptor for hyaluronic acid and collagen, was expressed by most RA‐SFL, RA‐PBL and CO‐PBL cells but at higher density on RA‐SFL. The results indicate that RA‐SFL express a distinct array of adhesion molecules, similar to the one of memory T lymphocytes. This characteristic phenotype may contribute to the lymphocytic infiltration of the synovium and to the pathogenesis of RA.
Signal transduction for interleukin‐3‐dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase C
Authors: Krieger Martin , Tscharner Vinzenz Von , Dahinden Clemens A.
Published: March 2007

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin‐3 (IL‐3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL‐3 in these differentiated, non‐dividing blood leukocytes. IL‐3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL‐3 pretreatment, indicating that the priming effect of IL‐3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a‐induced histamine release nor IL‐3‐dependent LTC4 formation in contrast to the IgE receptor‐dependent basophil response. Activation of protein kinase C (PKC) by phorbol‐12‐myristate‐13‐acetate (PMA) induced histamine release without leukotriene formation. PMA‐treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL‐3 effect on C5a‐induced LTC4 synthesis. Only the C5a signal but not the IL‐3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG‐50864 and herbimycin A, were both very efficient blockers of IL‐3‐dependent lipid mediator formation whereas C5a‐induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine‐sensitive serine/threonine kinase. To the contrary, IL‐3‐dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.
Expression and role of interleukin‐2 receptor β chain on CD4−CD8+ T cell receptor αβ+ cells
Authors: Takeuchi Yumiko , Tanaka Toshiyuki , Hamamura Keisuke , Sugimoto Tsuneaki , Miyasaka Masayuki , Yagita Hideo , Okumura Ko
Published: March 2007

Using anti‐murine interleukin‐2 receptor β chain (IL‐2Rβ) monoclonal antibody (mAb), we have examined the expression of IL‐2Rβ on murine thymocyte subpopulations. We found that it was constitutively expressed on 1 %‐4% of thymocytes in an almost mutually exclusive fashion with IL‐2Rα. The expression of IL‐2Rβ is developmentally regulated. While it is expressed mainly on T cell receptor βδ+ (TcRγδ+) cells during fetal age, the major subpopulation expressing IL‐2Rβ in adult mouse shifts to CD4−CD8−TcR αβ+ thymocytes. A considerable portion of CD4−CD8− TcR αβ+ cells in other organs, including spleen, bone marrow and liver, was also found to express IL‐2Rβ. In fetal thymus organ culture, the above thymocyte subset was induced to expand in response to exogeneous IL‐2, and the expansion was inhibited by addition of anti‐IL‐2Rβ mAb, suggesting that IL‐2Rβ is functional in this subpopulation. However, in vivo blockade of the IL‐2/IL‐2R pathway with the mAb did not exert any effects on the appearance of CD4−CD8− TcR αβ+ cells both in the thymus and the periphery. This indicates that the development of CD4−CD8− TcR αβ+ cells is not solely controlled by IL‐2 but also by other complex elements.
CD59 molecule: A second ligand for CD2 in T cell adhesion
Authors: Deckert Marcel , Kubar Joanna , Zoccola Didier , Bernard‐Pomier Ghislaine , Angelisova Pavla , Horejsi Vaclav , Bernard Alain
Published: March 2007

The T cell surface molecule CD2 forms, with its counter‐receptor CD58 (LFA‐3), a powerful adhesion/activation pathway. There is some evidence that CD2 might bind more than a single ligand. Chinese hamster ovary cells (CHO) expressing human CD59 after cDNA transfection (CD59+CHO) form rosettes with human T cells; these rosettes are inhibited in a dose‐dependent fashion by the CD59 monoclonal antibody (mAb) H19 and by the CD2 mAb O275 known to block natural rosettes, but not by the CD2R mAb D66, a poor rosette blocker. CD2+CHO transfectants form rosettes with human erythrocytes; these rosettes are inhibited by the CD59 mAb H19 in addition to CD2 mAb O275 and CD58 mAb; murine thymoma cells expressing human CD2 form rosettes with CD59+CHO cells that again are blocked by CD59 H19 and by CD2 O275 mAb. In a marked contrast with H19, two others CD59 mAb, YTH 53.1 and MEM 43, which react with a different epitope on CD59, led to a 50% −70% increase of the number of cells forming rosettes. In addition to rosette experiments, the binding of 125I‐labeled CD59 molecules to CD2+CHO cells was specifically inhibited by unlabeled CD59 molecule and CD2 mAb. Furthermore, the binding of CD59 molecules to resting T cells induced expression of CD2R epitopes. These results directly show that CD2 binds CD59 and that subtle molecular changes occur upon binding.