bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor necrosis factor‐mediated cell lysis in vitro: relationship to cAMP accumulation and guanine nucleotide‐binding proteins
Authors: Branellec Didier , De Cremoux Patricia , Barreau Patricia , Calvo Fabien , Chouaib Salem
Published: March 2007

We have investigated the modulation of tumor necrosis factor (TNF)‐mediated tumor cell lysis by cAMP. Among a panel of human breast tumor cell lines, MCF7 and MDA MB 231 were shown to be, respectively, sensitive and resistant to TNF‐mediated cell lysis in vitro. 125I‐labeled TNF‐binding experiments demonstrated that both cell lines bind TNF, indicating that the differential sensitivity to TNF was not related to TNF receptor expression. To study the relationship between TNF‐mediated cell lysis and cAMP accumulation, cAMP measurement was performed following TNF treatment. Our data show that TNF alone did not induce an enhancement of intracellular cAMP accumulation either in the TNF‐sensitive or in the TNF‐resistant cell line. Experiments in which cells were exposed to forskolin revealed that this cAMP elevating drug was efficient in enhancing the sensitivity to TNF of MCF7 cell line. This potentiating effect of forskolin was maximal for suboptimal concentrations of TNF (10 ng/ml), reaching up to 100% when forskolin was added at 100 μM. However, costimulation with forskolin of either MDA MB 231 or a TNF‐resistant MCF7 clone (MCF7‐R‐A1) did not induce any reversal of resistance to TNF.

We further assessed the interaction of TNF with transmembrane signalling and the possible involvement of guanine nucleotide‐binding proteins (G‐proteins). Bacterial toxin‐catalyzed ADP ribosylation of MCF7 and MDA MB 231 membranes was, therefore, performed. Using cholera toxin, we demonstrate that TNF treatment did not quantitatively alter the activity of stimulatory G‐proteins either in MCF7 or MDA MB 231 cell line. In contrast, pertussis toxin‐catalyzed ADP ribosylation experiments suggest a functional coupling of TNF receptors to a 40‐kDa pertussis toxin‐sensitive G‐protein in the TNF‐sensitive MCF78 cell line but not in the TNF‐resistant MDA MB 231 cell line. Taken together, these data indicate that cAMP might play a role in TNF‐mediated cell lysis and are in support of the involvement of a pertussis toxin‐sensitive G‐protein in TNF‐mediated MCF7 cells lysis.
Interferon‐γ regulates expression of a novel keratin classe I gene
Authors: Flohr Thomas , Buwitt Ute , Bonnekoh Bernd , Decker Thomas , Böttger Erik C.
Published: March 2007

Interferon (IFN)‐γ has been implicated in the pathogenesis of several autoimmune disorders and inflammatory skin diseases. To identify novel mediators involved in the IFN‐γ response we have used differential hybridization of a cDNA library prepared from IFN‐γ‐treated HeLa cells to isolate a gene that is induced following treatment with IFN‐γ. We report here the molecular cloning and characterization of a cDNA detecting a 1.6‐kb mRNA that accumulated in response to IFN‐γ but not in response to IFN‐α or IFN‐β. The gene is regulated by IFN‐γ in human cell lines of epithelial origin. The mRNA encodes a predicted protein of 432 amino acids and the primary structure of the protein demonstrates that it is a novel member of developmentally regulated keratin class I genes.
Polymorphism of human immunoglobulin VH4 germ‐line genes
Authors: Weng Nan‐Ping , Snyder James G. , Yu‐Lee Li‐Yuan , Marcus Donald M.
Published: March 2007

The human immunoglobulin VH4 gene family is thought to contain approximately 10 germ‐line genes and to exhibit little polymorphism. We report here an analysis of VH4 germ‐line genes that were amplified from DNA of two unrelated individuals. Ten unique (non‐repetitive) sequences were obtained from individual A and 11 from individual B. Nine of these sequences represent new germ‐line genes, and 8/9 exhibit only 89%–96% similarity to genes identified previously. Subsets of VH4 genes displayed distinctive nucleotide motifs that account for most of the differences between them. This observation suggests that diversity in the VH4 gene family arose from the acquisition of blocks of nucleotides, rather than by accumulation of point mutations. These nucleotide blocks could have been acquired by gene conversion or by homologous recombination. All of the VH4 genes have a potential N‐linked glycosylation site at Asn 60, and some genes encode a second site at Asn 52. The VH4 gene family is larger and more polymorphic than appreciated previously. Immunoglobulin gene polymorphism may make a significant contribution to hereditary variations in the immune response and to the genetic predisposition to autoimmune diseases.
A single mRNA encodes the α150 and α80/70 forms of the α subunit of VLA4
Authors: Rubio Miguel , Nueda Arsenio , Vara Alicia , Lopez Angel L. Corbí
Published: March 2007

VLA4 is a cell surface heterodimer (α4β1, CD49d/CD29) which belongs to the integrin family and is involved in cell‐extracellular matrix interactions, as well as in intercellular adhesion. Unlike other integrin α subunits, the α subunit of VLA4 (α4) can appear as a 150‐kDa polypeptide (α150), or cleaved into two non‐covalently associated polypeptides (α70–80). The relative proportion of each form is highly variable among different cells and is dependent on the state of cellular activation. The α4 cleavage site has recently been shown to occur between residues Arg558‐Ser559. We report the isolation of genomic clones encoding the α4 subunit, the location of the cleavage site‐encoding nucleotides to a specific exon and the determination of the exon‐intron organization around the cleavage site. Comparison with the gpIIb (CD41) and the αx (p150,95α, CD11c) genes revealed a similar genomic structure in this region, with exons of similar length separated by introns of identical phase. The structure of the α4 mRNA in cells expressing the alternative forms of α4 has been analyzed by means of reverse transcription‐polymerase chain reaction. Our results indicate that the exon encoding the cleavage site is present in α4 mRNA molecules from cells expressing either the α150 or the α70–80 form on the cell surface. Moreover, the structure of the α4 mRNA around the cleavage site does not change during the switch towards the α70–80 form that takes place upon lymphocyte activation. Therefore, both forms of α4 arise from a common mRNA, the α150 form contains the cleavage sequence and the α70–80 form must be generated by a post‐translational proteolytic event.
γδ T cells and the immune response in visceral leishmaniasis
Authors: Raziuddin Syed , Telmasani Abdul Wahab , El‐Awad M. El‐Hag , Al‐Amari Omar , Al‐Janadi Mansour
Published: March 2007

Visceral leishmaniasis (VL) caused by Leishmania donovani, a protozoan parasite, is a disease of high morbidity associated with hepatosplenomegaly, hypergammaglobulinemia, fever and death. One of the immunological hallmarks of VL is a remarkable increase in serum immunoglobulin levels as a result of polyclonal B cell activation. This study demonstrated that T lymphocytes expressing the T cell receptors (TcR) γδ in association with CD3 molecules are increased in circulation of patients with VL. A large proportions of TcR γδ‐bearing T cells had CD4+CD8− phenotype, and expressed CD25, CD38, CD71 and HLA‐DR activation antigens. Furthermore, we demonstrated wide functional differences in TcR γδ and TcR αβ T cells in their proliferative response, secretion of interleukin‐2 (IL‐2), B cell growth factor (BCGF) and B cell differentiation factor (BCDF). It was of interest that the TcR γδ T cells from patients with VL could be expanded by in vitro culture with human recombinant IL‐2. Although these TcR γδ cells secreted diminished levels of IL‐2, they produced highly augmented levels of both BCGF and BCDF, suggesting that secretion of these lymphokines in these T cell subsets is regulated independently. The relative increases in the CD4+ CDw29+ TcR γδ T cell subsets and their secretion of highly elevated levels of BCGF and BCDF largely accounted for the humoral immune system abnormality and hypergammaglobulinemia found in this disease. These observations may help to explain that TcR γδ T cells might be functional in vivo and are involved in immunological mechanisms of pathogenesis in VL.
Sublytic complement attack protects tumor cells from lytic doses of antibody and complement
Authors: Reiter Yoram , Ciobotarin Adina , Fishelson Zvi
Published: March 2007

Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL‐60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called “complement‐induced protection”. C7‐ and C8‐deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement‐induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on “protected” and control cells during complement activation. The “protected” K562 and HL‐60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The “induced protection” is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.
Kinetics of chicken embryonic thymocyte development in ovo and in organ culture
Authors: Davidson Natalie J. , Chen Chen‐lo H. , Boyd Richard L.
Published: March 2007

Thymocyte development was monitored in an embryonic thymus organ culture system to establish a model in the chicken in which the functional nature of the thymic microenvironment could be assessed. Thymus lobes were removed from 10‐day‐old embryos and cultured for 2–10 days. Cell yield increased to a maximum in 4–8 days of culture with a corresponding decrease in average cell size. An initial thymocyte population of predominantly CD3−CD4−CD8− cells gave rise to all CD3/CD4/CD8‐defined subpopulations in vitro, maintaining high levels of CD3−CD4−CD8+ and CD3+CD4−CD8+ cells and a low representation of CD3−CD4+CD8−, CD3+CD4+CD8−, CD3−CD4+CD8− and CD3+CD4+CD8+ thymocytes. This is the first observation of a CD3−CD4+CD8− population in the chicken. Developmental kinetics of CD3+ cells were similar to that in the embryo, suggesting that the in vitro environment is sufficient to promote and maintain thymocyte maturation. Thymocytes of both the γδ and αβ T cell receptor (TcR) lineages developed in that order, confirming in ovo data and the lineage potential of the first wave of thymocyte precursors. One unusual finding was a relative accumulation of γδ TcR+ thymocytes in culture, incorporating all CD4/CD8 subsets, including a previously undetected population, CD4+CD8−. This may indicate a favorable developmental environment or simply a lack of normal cellular emigration. A detailed comparison with T cell development in the embryo demonstrated that the chicken thymus organ culture system reflects thymic events in ovo during a limited time period and thus should prove useful in the identification of functionally relevant thymic molecules.
Immunity after treatment of human schistosomiasis: association between IgE antibodies to adult worm antigens and resistance to reinfection
Authors: Dunne David W. , Butterworth Anthony E. , Fulford Anthony J. C. , Curtis Kariuki H. , Langley Jane G. , Ouma John H. , Capron Andre , Pierce Raymond J. , Sturrock Robert F.
Published: March 2007

Previous studies in school children have demonstrated the slow development with age of resistance to reinfection after chemotherapy of Schistosoma mansoni infections, and have indicated that inappropriate (“blocking”) antibody responses prevent the expression of immunity in young children. The present study was designed to investigate further the nature of the protective responses, by serological studies on a group of 151 S. mansoni‐infected individuals resident in an endemic areain Machakos District, Kenya. Antibody levels against various antigens in blood samples before treatment were related to intensity of previous infections; antibodies in blood samples taken 6 months after treatment were related to cumulative reinfection rates over the following 30 months.

IgE against an adult‐worm antigen preparation correlated positively with age andnegatively with reinfection. In contrast, IgE antibodies against other life‐cycle stages showed either no relationship or the reverse correlation. Furthermore, antibodies of other isotypes against adult‐worm antigens showed no correlations withreinfection.

The correlation with IgE could be demonstrated for different preparations of adult worms, including a periodate‐treated preparation presumptively depleted of carbohydrate epitopes. For both the intact and the periodate‐treated preparations, multiple regression analysis of the results for children ≤16 years old demonstrated an IgEeffect after allowing for age, although this effect was not observed in a previouslystudied group of school children. Western blot analysis of the adult‐worm preparation revealed a limited set of antigens recognized by IgE, among which an antigen of 22 kDa was prominent. The qualitative presence of IgE against this antigen could also beshown to be related to a lack of subsequent reinfection.
Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E
Authors: Bennett Karen , Levine Timothy , Ellis Joanna S. , Peanasky Robert J. , Michael Samloff I. , Kay John , Chain Benjamin M.
Published: March 2007

Proteolytic degradation (processing) of antigen by antigen‐presenting cells is a major regulatory step in the activation of a T lymphocyte immune response. However, the enzymes responsible for antigen processing remain largely undefined. In this study we show that cathepsin E, and not the ubiquitous lysosomal cathepsin D, is the major aspartic proteinase in a murine antigen‐presenting cell line, A20. Thisenzyme is localized to a non‐lysosomal compartment of the endosomal system in these cells. Functional studies using a highly specific inhibitor of cathepsin E show that this enzyme is essential for the processing of ovalbumin by this cell line. Thus, cathepsin E, whose function was hitherto unknown, may play a major role in antigen processing.
Cloning and sequencing of the cDNA encoding the human homologue of the murine immunoglobulin‐associated protein B29
Authors: Müller Brigitte , Cooper Laura , Terhorst Cox
Published: March 2007

Membrane‐bound immunoglobulins (Ig) on the surface of murine B cells are noncovalently associated with a heterodimeric protein complex of MB‐1 and B29 (alsocalled Ig‐α and Ig‐β). The Ig‐associated proteins are predicted to regulate the assembly and transport of the Ig complex tothe cell surface and to couple membrane‐bound Ig to intracellular signal transduction pathways. Wehave isolated and sequenced a full‐length cDNA clone encoding the human homologue of the B29 protein. The predicted amino acid sequence was compared to its murine counterpart, to MB‐1 and to the human T cell receptor (TcR)‐associated CD3 proteins. The alignment of the human B29 protein with its murine counterpart revealed 90%homology in the C‐terminal portion comprisingthe cytoplasmic tails, the transmembrane regions and the adjacent 26 amino acids of the extracellular regions. Only 59% homology was found in the rest of the Ig‐like extracellular domains. The high degree of conservation observed for the C‐terminal amino acids suggested that thesedomains of the proteins play important functional roles for the Ig complex. Indicative of this was the conservation of the antigen receptor tail motif D‐(X)7−E/D‐(X)2−Y‐(X)2−L‐(X)7−Y‐(X)2−L/I which is thought to be a component of signal transduction pathways. This motif is also found in the human and murine MB‐1 proteins as well as in the TcR‐associated CD3 molecules. Further regions of homologybetween B29, MB‐1 and the CD3 proteins included extracellular residues which were predicted to maintain the Ig‐like structure, and hydrophilic residues within the transmembrane regions which may be utilized during the intracellular assembly and transport of the oligomeric Ig/MB‐1/B29 or TcR/CD3 complexes. Thus the similarities found between B29, MB‐1 and the CD3 proteins suggest conserved functions for both the Ig‐ and TcR‐associated proteins.
A viral peptide can mimic an endogenous peptide for allorecognition of a major histocompatibility complex class I product
Authors: Guimezanes Annick , Schumacher Ton N. M. , Ploegh Hidde L. , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

Alloreactive class I‐restricted T cells may recognize the class I structure alone, in association with a specific peptide, or with any stabilizing peptide. We havetested the role of endogenous peptides in the recognition of H2Kb molecules by two alloreactive cytolytic T lymphocyte (CTL) clones using the mutanttumor lineRMA‐S, which expresses its surface H‐2b molecules devoid of peptides and is not lysed by these two CTL clones. Empty H‐2b molecules on RMA‐S cells can be stabilized by binding exogenously added peptides. H‐2Kb−specific recognition of the RMA‐S cells by one of the CTL clones was restored by endogenous peptide extracts which only minimally stabilized H2Kb on the surface of RMA‐S cells, indicating the requirement for a specific peptide on a limited number of H2Kb molecules. In addition, one out of three peptides which greatly enhance the expression of H2Kb, the nucleoprotein peptide 52–59 from vesicular stomatitis virus (VSV), was also able to restore the lysis of RMA‐S cells by the clone. The recognition of a common motif by an alloreactive clone (H‐2k anti‐H‐2Kb) and virus‐specific Kb−restricted clones suggests that both H‐2k and H‐2b thymic environments allow selection of T cells capable of recognizing H2Kb+VSV and that tolerance to self, as would be the case in the (H‐2k x H‐2b)F1 mice, would partially delete the repertoire of antiviral T cells.
Lipopolysaccharide induces up‐regulation of CD14 molecule on monocytes in human whole blood
Authors: Marchant Arnaud , Duchow Jochen , Delville Jean‐Pierre , Goldman Michel
Published: March 2007

We examined by flow cytometry the expression of lipopolysaccharide (LPS) receptor CD14 molecule on monocytes after addition of LPS to human whole blood. Within 30 min LPSinduced an increase in monocyte CD14 expression, peaking between 1 and 3 h andfollowedby a slow decrease. Maximal increase in anti‐CD14 monoclonal antibody binding sites was estimated as twofold the basal value. This effect, already observed with very low concentrations of LPS (10 pg/ml), was dose dependent. Protein synthesis was not involved in the CD14 hyperexpression since it was not influenced by co‐incubation with cycloheximide. Finally, LPS‐induced up‐regulation of monocyte CD14 wasassociated with an increased binding of fluoresceinated LPS. We conclude that LPS in whole blood up‐regulates the expression of its own CD14 receptor on monocytes, a phenomenon that could be relevant to the pathogenesis of gram‐negative sepsis.
A V region‐connected autoreactive subfraction of normal human serum immunoglobulin G
Authors: Dietrich Gilles , Kaveri Srinivas‐Venkatesh , Kazatchkine Michel D.
Published: March 2007

Mouse and human natural IgM autoantibodies have been shown to be polyreactive and “connected” through V region‐dependent interactions. In the present study, we have identified a connected subfraction of normal human serum IgG by using affinity chromatography of F(ab′)2 fragments of pooled IgG (IVIg) or of IgG from a single donor on Sepharose‐bound F(ab′)2 fragments of the same source of IgG. The connected fraction of IgG exhibited a high content of autoantibodies directed against a wide panel of evolutionarily conserved self antigens and of self antigens that may be targets of autoantibodies in autoimmune diseases. Connected IgG also contained higher amounts of antibodies directed against commonly encountered microbial antigens than unfractionated IgG. The connected fraction did not, however, differ from unchromatographed IgG nor from non‐connected IgG in its content of antibodies to vaccinal antigens and to distant foreign antigens. Thus, in humans as in mice, connectivity is a prominent feature of autoantibodies. Our observations are suggestive of a tight control by IgG of the expressed autoreactive repertoire in healthy individuals and strengthen the concept that the therapeutic infusion of pooled normal IgG (IVIg) may be effective in autoimmune diseases by bringing to patients normal regulatory components of the immunoglobulin network.
Analysis of immunoglobulin variable region genes from human IgG anti‐DNA hybridomas
Authors: Winkler Thomas H. , Fehr Holger , Kalden Joachim R.
Published: March 2007

The molecular mechanisms leading to anti‐double‐stranded (ds)DNA antibody production in systemic lupus erythematosus (SLE) are poorly understood. We describe here the immunoglobulin variable region genes of six human hybridomas secreting IgG anti‐dsDNA antibodies derived from three SLE patients. The monoclonal IgG anti‐dsDNA antibodies have been shown to be of high affinity and no multireactivity was observed (Winkler et al., Clin. Exp. Immunol., 1991. 85: 379). The comparison of the variable region genes expressed in the hybridomas with known germ‐line genes as well as with the germ‐line counterparts from one patient shows that the VH and VL sequences are somatically mutated. The pattern and extent of the observed somatic mutations are suggestive for an antigen‐driven selection of at least four of these B cell clones. Several VH and VL genes used by the hybridomas were found to be expressed in the natural antibody repertoire, in the restricted fetal repertoire and in B cell malignancies expressing the CD5 antigen.
Interleukin‐4‐mediated tumor suppression in nude mice involves interferon‐γ
Authors: Platzer Cornelia , Richter Günther , Überla Klaus , Hock Hanno , Diamantstein Tibor , Blankenstein Thomas
Published: March 2007

The molecular events during the anti‐tumor response induced by interleukin (IL)‐4 were investigated by quantitative polymerase chain reaction. The growth of Chinese hamster ovary cells transfected to produce IL‐4 (CHO.T1) was strongly suppressed when cells were injected intraperitoneally into nude mice and this suppression was accompanied by the rapid accumulation of activated macro‐phages. Peritoneal cells from such mice were analyzed for mRNA induced by IL‐4. Correlating with a high local IL‐4 concentration, several transcripts were found to be up‐regulated during the early phase of the anti‐tumor response [IL‐4 receptor, IL‐5, tumor necrosis factor (TNF) and interferon (IFN)‐γ]. The functional relevance of the elevated mRNA levels was analyzed by injection of CHO.T1 cells together with anti‐cytokine monoclonal antibodies (mAb). In contrast to anti‐IL‐5 and anti‐TNF mAb, an anti‐IFN‐γ mAb interfered with the anti‐tumor response demonstrating the involvement of IFN‐γ during the IL‐4‐induced tumor suppression. Tumor growth in anti‐IFN‐γ mAb‐treated animals was significantly delayed in comparison to anti‐IL‐4 mAb‐treated mice, suggesting that IFN‐γ‐independent effector cells may also be involved.
GMP‐140 (P‐selectin/CD62) binds to chronically stimulated but not resting CD4+ T lymphocytes and regulates their production of proinflammatory cytokines
Authors: Damle Nitin K. , Klussman Kerry , Dietsch Mary T. , Aruffo Alejandro , Mohagheghpour Nahid
Published: March 2007

GMP‐140 (P‐selectin), a 140‐kDa granular membrane glycoprotein localized to the α granules of platelets and the Weibel‐Palade bodies of endothelial cells, is thought to play an important role in adhesive interactions predominantly between granulocytes, platelets and vascular endothelial cells during inflammation. Although GMP‐140 binds to granulocytes, its binding to lymphocytes has not been demonstrated. Using genetically engineered IgG Cγ1 fusion protein of the extracellular domains of GMP‐140, we demonstrate that GMP‐140 binds to chronically antigen (Ag)‐stimulated CD4+ T cells. Freshly isolated CD4+ T cells did not bind GMP‐140, but priming and subsequent stimulation with alloantigen induced and gradually increased expression of GMP‐140‐reactive structures on their surface. T cells isolated from rheumatoid synovial fluids also exhibited strong binding to GMP‐140. The binding of GMP‐140 to primed T cells is not influenced by preactivation with phorbol 12‐myristate 13‐acetate, is almost completely abolished by pretreatment of T cells with neuraminidase or trypsin, and is also strongly inhibited by EDTA, the soluble sulfated glycans dextran sulfate, fucoidan, and heparin, but not by chondroitin sulfates. In spite of its strong binding to Ag‐primed T cells, GMP‐140 did not modulate the proliferative responses of these cells to various stimuli. However, GMP‐140 in conjunction with anti‐T cell receptor αβ monoclonal antibodies augmented the production of granulocyte‐macrophage colony‐stimulating factor GM‐CSF and inhibited the production of interleukin‐8 by Ag‐primed T cells without influencing their tumor necrosis factor‐α production. These results suggest that GMP‐140 binds to chronically stimulated CD4+ T cells and differentially modulates their production of proinflammatory cytokines. The ability of Ag‐primed T cells to bind GMP‐140 may facilitate interactions with activated platelets and endothelial cells affecting the course of inflammation.
Molecular basis for a severe case of leukocyte adhesion deficiency
Authors: Corbí Angel L. , Vara Alicia , Ursa Angeles , Rodriguez Mari Cruz García , Fontán Gumersindo , Sánchez‐Madrid Francisco
Published: March 2007

The leukocyte integrins LFA‐1, Mac‐1 and p150,95 (CD11a/CD18, CD11b/CD18, CD11c/CD18) mediate crucial leukocyte adhesive functions in immune and inflammatory reactions. Leukocyte adhesion deficiency (LAD) disease is caused by the defective expression of these adhesion molecules on leukocytes, and is characterized by recurrent infections and impaired pus formation due to the blockade of leukocyte migration into inflamed tissues. LAD is originated by heterogeneous mutations affecting the CD18 gene and, based on the severity of the deficiency, two phenotypes (severe and moderate) have been defined. Biochemical and genetic studies have allowed the classification of five different types of LAD. We have identified a type V LAD patient (severe phenotype, and normal size and levels of both CD18 precursor and CD18 mRNA), and determined its molecular basis. Reverse transcription‐polymerase chain reaction and cloning and sequencing of CD18 cDNA derived from this patient revealed three silent mutations and a missense mutation that leads to the substitution of glycine at position 169 for an arginine. Analysis of patient‐derived cDNA clones revealed the concomitant presence of aberrant splicing within the 5' region of the CD18 gene. The description of an identical mutation at residue 169 in an unrelated severe LAD patient raises the possibility that severe LAD type V is caused by a unique genetic defect.
Phenotypic characterization of KU812, a cell line identified as an immature human basophilic leukocyte
Authors: Blom Thomas , Huang Ranyang , Aveskogh Maria , Nilsson Kenneth , Hellman Lars
Published: March 2007

The knowledge about the differentiation of basophilic leukocytes is fragmentary. This report discusses a detailed phenotypic characterization of molecular markers for hematopoietic differentiation in a basophilic leukemia cell line, KU812. The expression of markers for lymphoid, erythroid, neutrophil, eosinophil, monocytic, megakaryocytic, mast cell and basophil differentiation was analyzed at the mRNA level by Northern blots in the KU812 cells, and for reference, in a panel of human cell lines representative of the different hematopoietic differentiation lineages. KU812 was found to express a number of mast cell and basophil‐related proteins, i.e. mast cell tryptase, mast cell carboxypeptidase A, high‐affinity immunoglobulin (IgE) receptor α and γ chains and the core protein for heparin and chondroitin sulphate synthesis. We found no expression of a number of monocyte/‐macrophage or neutrophil leukocyte markers except for lysozyme. From earlier studies, it has been shown that lysozyme is not expressed in murine mucosal mast cell lines. This finding, together with the expression of the mast cell carboxypeptidase in KU812 might distunguish the phenotype of this cell line from that typical of mucosal mast cell lines in rodents. We found a low level of expression of the eosinophil and basophil marker, major basic protein, which might indicate a relationship between basophils and eosinophils. No expression is, however, detected with the eosinophil‐specific markers eosinophil cationic protein, eosinophil‐derived neurotoxin or eosinophil peroxidase.

We also report an extensive screening for inducers of basophilic differentiation of the KU812 cells. The most efficient protocol of induction included serum starvation which led to a dramatic increase in a number of markers specific for mast cells and basophils such as tryptase, carboxypeptidase A and the heparin core protein.

Finally, diisopropylfluorophosphate analysis of total protein extracts from KU812 show four labeled protein bands with sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis, indicating that this cell line expresses at least three previously undescribed serine proteases of which one or more could be a potential basophil‐specific marker(s).
Naturally occurring ABO antibodies: Long‐term stable, individually distinct anti‐A IgG spectrotypes
Authors: Rieben Robert , Frauenfelder Agnes , Nydegger Urs E.
Published: March 2007

Spectrotypes of naturally occurring IgG antibodies to histo‐blood group substance A were analyzed by isoelectric focusing and a newly developed, non‐radioactive affinity immunoblotting assay. Specificity was tested by preabsorption of sera on red blood cells and on four different types of blood group substance A. Clonotype‐banding patterns of sera from 18 different blood donors were individually distinct, most of them showing an oligoclonal pattern with less than 15 visible bands. To analyze the influence of IgG subclass composition on spectrotype patterns, IgG subclasses were measured by ABO enzyme‐linked immunosorbent assay (ELISA) and were shown to be mostly IgG1 and IgG2. Anti‐A IgG1 spectrotypes could be detected with four sera and were also individually distinct. A series of six and seven serum samples, respectively, from two donors was used to analyze the time‐related changes of anti‐A IgG spectrotypes. Banding pattern of both individuals remained stable during the whole observation period of 167 and 82 days, respectively, and no differences between follow‐up samples could be detected. This long‐term stability of individual anti‐A IgG spectrotypes is backed by constant serum levels of anti‐A IgG and ‐IgM antibodies during the observation period as measured by ABO ELISA.
Altered patterns of T cell migration through lymph nodes and skin following antigen challenge
Authors: Mackay Charles R. , Marston Wendy , Dudler Lisbeth
Published: March 2007

Antigen challenge has profound effects on a regional lymph node (LN); it leads to an increase in blood flow to the node, and a marked increase in lymphocyte output through the efferent lymphatics. We used the isolated LN model developed in the sheep to see if antigen challenge in a LN resembled inflammation in peripheral tissues. Following stimulation with an antigen (purified protein derivative of tuberculin), lymphocyte output from the LN showed the typical periods of “lymphocyte shutdown” and “recruitment”. The shutdown phase, when cell numbers in efferent lymph dropped by ∼80%, affected almost exclusively the naive‐type (adhesion10, L‐selectin+) T cell population. The large increase in T cell traffic through the node during the recuitment phase was mostly due to CD4+ memory‐type T cells and, moreover, the majority of these T cells were L‐selectin−, indicating that these cells were crossing from the blood by a molecular mechanism other than L‐selectin interaction with its ligand, the “lymph node vascular addressin” (MECA‐79). Examination of LN high endothelial venules revealed the presence of vascular cell adhesion molecule‐1 (VCAM‐1), an endothelial adhesion molecule which has been reported to bind preferentially memory‐type T cells in inflammatory lesions. Within the skin, antigen challenge also induced the rapid expression of VCAM‐1 on vascular endothelium. It was purely memory‐type T cells (β1−, L‐selectin+/−) that collected in lymph draining from this tissue. However within chronically inflamed skin, the MECA‐79 determinant appeared on vascular endothelium, and a small proportion of T cells draining from chronically inflamed skin were of naive‐type. The present results illustrate that there are similarities in the cellular and molecular events that characterize antigen stimulation of a LN and inflammation in a peripheral tissue.
Immunosuppression induced by nitric oxide and its inhibition by interleukin‐4
Authors: Al‐Ramadi Basel K. , Meissler Joseph J. , Huang Duan , Eisenstein Toby K.
Published: March 2007

Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non‐Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens. Suppression of antibody responses is mediated by macrophage (Mϕ)‐released soluble factors, and is completely reversed by treatment with interleukin (IL)‐4. The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL‐4 abrogates suppression. Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235‐immunized mice. NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes. Further, immunosuppression was reversed by NG‐monomethyl‐L‐arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L‐arginine. Treatment with IL‐4, or anti‐interferon (IFN)‐γ monoclonal antibody (mAb), also abrogated suppression. Optimal reversal of suppression was observed only when NMLA, IL‐4, or anti‐IFN‐γ mAb, was added at day 0 of the 5‐day plaque‐forming cell assay. Treatment with either IL‐4 or anti‐IFN‐γ mAb also lead to a sharp inhibition of NO production by immune spleen cells. Moreover, the addition of IL‐4 to splenic adherent Mϕ inhibited their ability to generate NO. Our data characterize an immunoregulatory pathway, involving IFN‐γ and NO, by which Mϕ mediate immunosuppression and identify IL‐4 as a potent inhibitor of this pathway.
Molecular cloning and exon‐intron mapping of the gene encoding human transmembrane secretory component (the poly‐Ig receptor)
Authors: Krajči Peter , Kvale Dag , Taskén Kjetil , Brandtzaeg Per
Published: March 2007

Secretory component (SC or the poly‐Ig receptor) plays a crucial role in mucosal immunity by translocating polymeric IgA and IgM through secretory epithelial cells into external body fluids. Labeled restriction fragments from human SC cDNA were used to screen a human genomic leukocyte library. Three overlapping clones, spanning a total of 19 kb of the human SC gene, including 3 kb of the 5′ flanking region, were characterized. The putative TATA box candidate, preceded by a CAAT‐like box, was found 329 nucleotides upstream of the first exon. Altogether 11 exons covering the entire coding region were identified. The exon size ranged from 59 to 657 nucleotides and exon‐intron junctions followed known consensus sequences. Three of the five extracellular Ig‐related domains (D1, D4 and D5) were confined to one exon each (E3, E5 and E6), whereas D2 and D3 were encoded by the same exon (E4). The latter exon corresponds to that involved in alternate splicing of rabbit SC. The membrane‐spanning segment was confined to part of one exon (E8). The cytoplasmic tail was encoded by four exons (E8–E11), whose boundaries encompassed fairly well the structural determinants proposed to be responsible for intracellular sorting of SC in the rabbit. The polymorphic restriction site reported earlier for Pvu II was localized to the third intron.
HLA class I and II molecules present influenza virus antigens with different kinetics
Authors: Kuijpers Karel C. , Van Kemenade Folkert J. , Hooibrink Berend , Neefjes Jacques J. , Lucas Cees J. , Van Lier René A. W. , Miedema Frank
Published: March 2007

Human leukocyte antigen (HLA) class I and class II molecules differ with respect to their intracellular pathways and the compartments where they associate with processed antigen. To study possible consequences of these differences for the kinetics of antigen presentation by HLA class I and class II molecules, we analyzed changes in the concentrations of free intracellular calcium ions in influenza virus‐specific T cell clones after recognition of specific antigen/HLA complexes. HLA class II‐restricted viral antigen presentation by Epstein‐Barr virus‐transformed B lymphoblastoid cell lines (B‐LCL) to CD4+ T cell clones started within 1 h and showed little variability, irrespective of antigen specificity or restriction element tested. In contrast, kinetics of viral antigen presentation by HLA class I molecules to CD8+ T cell clones were slower and differed for three antigen/HLA class I complexes tested. While B‐LCL presented antigen by HLA‐A2 and by HLA‐B37 after at least 2 h, they only started to present antigen in the context of HLA‐B7 after more than 4 h. This difference in kinetics did not correlate with differences in bulk transport rates of HLA‐A2, HLA‐B37, and HLA‐B7, but seemed greatly influenced by differential rates of peptide generation. Brefeldin A treatment of B‐LCL showed for both HLA class I and class II that de novo synthesized HLA molecules were involved in antigen presentation. Thus, differences between intracellular pathways of HLA class I and class II molecules may result in different kinetics of antigen presentation.
Inhibition of human immunodeficiency virus infection in human colon epithelial cells by recombinant interferon‐γ
Authors: Yahi Nouara , Baghdiguian Stephen , Bolmont Christine , Fantini Jacques
Published: March 2007

Pretreatment of human colon epithelial cells HT29 by recombinant gamma interferon (IFN)‐γ was found to protect the cells from infection with various isolates of human immunodeficiency virus (HIV)‐1 and HIV‐2, as assessed by co‐cultivation with human T lymphoblastoid cells and gene amplification by polymerase chain reaction technique. Additionally, IFN‐γ induced a dose‐dependent inhibition of HIV‐1 and HIV‐2 production in chronically infected HT29 cells. In situ hybridization studies demonstrated that IFN‐treated cells were still able to synthesize viral messenger ribonucleic acid. However, the expression of the p24 product of the gag gene was markedly decreased after IFN treatment as demonstrated by radio‐immunoprecipitation assay. Taken together, these data suggested that the cytokine acted at the post‐translationnal level by inhibiting the processing of structural viral proteins. It is concluded from this study that IFN‐γ has a potent anti‐HIV effect on epithelial gastrointestinal cells.
Modulation of restricted class II T cell responses by peptides derived from self class II molecule
Authors: Li Wan‐Fen , Fan Ming‐Dau , Pan Ching‐Biau , Gefter M. L. , Lai Ming‐Zong
Published: March 2007

We have explored the possibility of using peptides derived from a major histocompatibility complex (MHC) class II (I‐Ab) molecule to modulate I‐Ab‐restricted T cell responses. Six peptides spanning the polymorphic regions of I‐Ab were analyzed for competitive binding to the I‐Ab molecule, and for efficacies in blocking I‐Ab‐specific T cell response. Only PB1 (residues 75‐91 of β chain) bound the I‐Ab molecule with high affinity. When these MHC‐derived peptides were administered simultaneously with antigen, PB1 effectively inhibited I‐Ab‐restricted T cell responses as well as another peptide PB2 (residues 59‐78 of β chain). PB2 inhibited specific T cell response only when it was administered simultaneously with antigen. Since PB2 is a weak binder of I‐Ab, an additional mechanism must account for its inhibitory activity. Both PB1 and PB2 peptides elicited specific T cell responses, indicating that these peptides were not tolerogenic in syngeneic mice. However, the induction of T cells in response to PB1 and PB2 did not increase reactivity to I‐Ab. MHC class II‐derived peptides thus can be used to regulate T cell responses without the risk of autoreactivity.
The importance of cross‐linking in the homotypic aggregation of lymphocytes induced by anti‐leukosialin (CD43) antibodies
Authors: Cyster Jason G. , Williams Alan F.
Published: March 2007

Leukosialin (CD43) is a major glycoprotein of T lymphocytes which has an extracellular domain of 45 nm in length that is heavily O‐glycosylated. Monoclonal antibodies (mAb) to the extracellular domain of human leukosialin induce aggregation of T lymphocytes, monocytes and some cell lines that express leukosialin. The aggregation was reported in one case to be inducible by Fab fragments. In the present study, nine mAb specific for rat leukosialin were tested as inducers of thymocyte aggregation and all were effective. The level of aggregation was reduced by metabolic and cytoskeletal inhibitors, by removal of divalent cations and by reducing the temperature from 37°C to 4°C. The aggregation produced by mAb specific for certain epitopes was less sensitive to these inhibitors than aggregation induced by mAb to other epitopes. To examine the requirement for cross‐linking, Fab fragments of four of the antibodies were tested and found to be inactive except for those derived from the OX75 mAb. However, OX75 Fab showed a tendency to dimerize, and monomeric OX75 Fab obtained directly after gel filtration was unable to induce aggregation. Thus induction of rat thymocyte aggregation by anti‐leukosialin antibodies requires bivalent cross‐linking and maximal aggregation is dependent on energy and an, intact cytoskeleton. Mechanisms of antibody‐induced aggregation are considered and it is proposed that in the case of leukosialin the antibodies may cross‐link cells to overcome inherent repulsion between them and that subsequently other adhesion molecules complete the clustering process.
Activated human T cells express a ligand for the human B cell‐associated antigen CD40 which participates in T cell‐dependent activation of B lymphocytes
Authors: Lane Peter , Traunecker André , Hubele Sabine , Inui Seiji , Lanzavecchia Antonio , Gray David
Published: March 2007

To identify the ligand for the B cell‐associated antigen CD40, we constructed a chimeric immunoglobulin molecule where the extracellular portion of the CD40 protein replaced the normal immunoglobulin variable region. No binding was detected on resting peripheral blood T cells. However, following T cell activation with phorbol esters and ionomycin, the chimeric protein bound specifically to activated human T cells and precipitated a 35‐kDa protein from such cells. The induction of the CD40 ligand was detectable on the cell surface after 1 h, with maximal expression after 8 h of stimulation. The T cells expressing CD40 ligand were predominantly CD4 positive, although a proportion of CD8‐positive cells also expressed the protein. There was no particular correlation with CD45 phenotype. Finally, we found that soluble CD40 inhibited T‐dependent B cell proliferation. The results are discussed in the context of cognate interactions between B and T cells.
Pore‐forming protein in individual cytotoxic T lymphocytes: the effect of senescence provides a probe for understanding the lytic mechanism
Authors: Horvath Judith A. , Mostowski Howard S. , Okumura Ko , Bloom Eda T.
Published: March 2007

The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell‐mediated cytolysis. The effect of age on the generation of pore‐forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme‐linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC‐stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp‐producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age‐related decrease in lytic activities (∼2‐fold vs. ∼7.4‐fold, respectively). Time‐course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age‐related difference in the proportion of Pfp+ cells was observed in MLC‐stimulated lymph node cells despite a large and significant difference in lytic activity (∼ 6.5‐fold). Purified CD8+ T cells demonstrated a large age‐related difference in CTL activity (∼3–11‐fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be ∼ 12‐fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.
The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides
Authors: Partidos Charalambos , Stanley Carolynne , Steward Michael
Published: March 2007

CBA and TO mice were immunized with chimeric peptide immunogens consisting of B cell (residues 404–414) and T cell (residues 288–302) epitopes from the F protein of measles virus. The chimeras were co‐linearly synthesized to contain one or two copies of the T cell epitope linked to one or two copies of the B cell epitope via a glycine glycine spacer. Two orientations were synthesized such that the T cell epitope(s) were located at either the amino or carboxyl terminus of the B cell epitope(s). The levels of antibody induced following immunization with the chimeras were assessed by enzyme‐linked immunosorbent assay using microtiter plates coated with either the homologous chimera or the B cell epitope sequence. The affinities of the anti‐chimera antibodies for the B cell epitope were assessed by a fluid‐phase double‐isotope radioimmunoassay. All the chimeras induced good antibody responses in both strains of mice with specificity for the B cell epitope. Chimeras containing two copies of the T cell epitope induced antibodies with higher affinity for the B cell epitope than did chimeras containing one copy of the T cell epitope or two copies of the B cell epitope. Furthermore, the amino terminal location of the T cell epitope in relation to the B cell epitope in the chimera induced higher affinity anti‐B cell antibody than did the reverse orientation. These results suggest that orientation of epitopes and amino acid composition of chimeric peptides affect antigen processing and presentation to T cells which govern both the specificity and affinity of antibody produced. Thus, for the production of synthetic peptide immunogens with vaccine potential, attention needs to be given to the number and orientation of the component epitopes required to produce highest affinity antibody.