bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the fetal and adult functional B cell repertoires by analysis of VH gene family expression.
Authors: Jeong H D , Teale J M
Published: June 2004

The functional B cell repertoire in BALB/c mice was assessed at various stages in ontogeny. This was done by analyzing VH gene family expression using the sensitive technique of in situ hybridization. The B cell repertoire was probed with the mitogen, LPS, and the antigen DNP. DNP was chosen because B cells responsive to this hapten appear very early in ontogeny. The APCs that developed after stimulation with LPS or DNP were analyzed for VH gene expression by in situ hybridization of individual cells using radiolabeled VH gene family probes. The results indicated that VH gene expression in fetal B cells after stimulation was distinct from adult B cells in that there was a biased expression of D proximal families. The results indicated that this bias was associated with developmental age and not a given differentiation stage in the B cell lineage. In addition, stimulation of fetal B cells with DNP resulted in a large increase in expression of member(s) of VH 36-60, suggesting that the early appearance of DNP-responsive B cells is not strictly correlated with preferential rearrangement of D proximal families, VH 7183 and VH Q52. However, the results suggested that a large proportion of pre-B cells that preferentially rearrange D proximal families early in ontogeny become part of the functional developing repertoire.
Phenotypic variation in the response to the human immunodeficiency virus among derivatives of the CEM T and WIL-2 B cell lines.
Authors: Chaffee S , Leeds J M , Matthews T J , Weinhold K J , Skinner M , Bolognesi D P , Hershfield M S
Published: June 2004

Derivatives of the CEM T and WIL-2 B cell lines showed striking diversity in their responses to the HTLV-IIIB strain of the human immunodeficiency virus (HIV). Several stable phenotypic patterns could be defined, based on whether cells were permissive (P+, P-) for virus production, were sensitive or insensitive to cytopathic effects after infection by free virus (C+, C-), and whether they underwent fusion on contact with virus-infected cells (F+, F-). Although expression of CD4 was essential for infection by HTLV-IIIB, very low levels were sufficient for productive infection of WIL-2 derivatives. Conversely, some CEM T cell lines that expressed ample CD4, and which were able to bind virus gp120 and undergo fusion, did not support productive infection by free virus. One nonpermissive, CD4+ derivative of CEM could bind gp120 but failed to undergo fusion, suggesting an alteration in some membrane protein other than CD4 that is essential for virus entry and HIV-induced cell fusion. The AA2 derivative of the WIL-2 cell line is also described, which is remarkably permissive for HIV replication and exquisitely sensitive to virus cytopathic effect. The panel of related cell lines with different host-virus phenotypes could be useful for more precisely defining steps in the infectious cycle of HIV, and for identifying host cell genes and gene products that determine the outcome of HIV infection.
CD4+ T cells are required for development of a murine retrovirus-induced immunodeficiency syndrome (MAIDS).
Authors: Yetter R A , Buller R M , Lee J S , Elkins K L , Mosier D E , Fredrickson T N , Morse H C
Published: June 2004

Mice depleted in vivo of CD4+ Th cells by treatment with mAb GK1.5 were found to be resistant to the lymphoproliferative/immunodeficiency disease (MAIDS) induced in intact mice by infection with the mixture of LP-BM5 murine leukemia viruses. Depleted mice did not develop lymphadenopathy or splenomegaly, had normal serum IgM levels, normal CTL responses to alloantigens, and were able to generate PFC responses to Th-independent antigens even though frequencies of virus-producing spleen cells were comparable in depleted and intact mice. Depletion of CD4+ Th cells after infection resulted in a reversal of many abnormalities exhibited by infected controls; spleen weights, serum IgM levels, and allogeneic CTL responses of treated mice were comparable to those of uninfected controls. These results demonstrate that dysfunction of CD4+ Th cells is central to the induction and progression of both T and B cell abnormalities in MAIDS.
Tumor necrosis factor/cachectin-induced intravascular fibrin formation in meth A fibrosarcomas.
Authors: Nawroth P , Handley D , Matsueda G , De Waal R , Gerlach H , Blohm D , Stern D
Published: June 2004

Recent studies have indicated that TNF can promote activation of the coagulation mechanism by modulating coagulant properties of endothelial cells. In this report, we demonstrate that infusion of low concentrations of TNF (3 micrograms/animal) into mice bearing meth A fibrosarcomas leads to localized fibrin deposition with formation of occlusive intravascular thrombi in close association with the endothelial cell surface. Studies with 125I-fibrinogen showed tenfold enhanced accumulation of radioactivity in tumor within 2 h after TNF infusion. Western blots of tumor extracts subjected to SDS-PAGE and visualized with a fibrin-specific mAb indicated that fibrin forms in the tumor after the TNF infusion. Electron microscopic studies demonstrated fibrin strands, based on the characteristic 21-nm periodicity, which appeared to be adherent to the endothelial cell surface. Further ultrastructural studies indicated that fibrin formation, first evident within 30 min of the TNF infusion, led to occlusive thrombi limited to the tumor vascular bed (i.e., not in the normal mouse vasculature) within 2 h and was associated with an 80% reduction in tumor perfusion based on studies with Evans blue. In view of previous work concerning TNF induction of endothelial cell procoagulant activity, the hypothesis that tumor cell products prime the response of endothelium to this cytokine was tested. Supernatants of cultured meth A fibrosarcomas obtained serum-free conditions, which had no intrinsic procoagulant activity, considerably enhanced tissue factor induction in endothelium in response to submaximal concentrations of TNF. The factor(s) in the tumor-conditioned medium appeared to be distinct from IL-1, fibroblast growth factor, IFN-gamma, TNF, endotoxin, TGF-alpha, and TGF-beta. These studies delineate a novel model of localized clot formation in which thrombosis is initiated by a pathophysiologic mediator, TNF, and provides an opportunity to examine mechanisms in the microenvironment directing clot formation to the tumor vascular bed.
Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance.
Authors: Sykes M , Sheard M A , Sachs D H
Published: June 2004

The ability of normal recipient-type lymphocytes to break tolerance in long-term allogenic radiation chimeras has been investigated. Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic T cell-depleted (TCD) bone marrow (BM) has previously been shown to lead to mixed chimerism and permanent, specific tolerance to donor and host alloantigen (3-5). If allogeneic T cells are not depleted from the reconstituting inoculum, complete allogeneic chimerism results; however, no clinical evidence for GVHD is observed, presumably due to the protective effect provided by syngeneic TCD BM. This model has now been used to study the effects of allogenic T cells administered in reconstituting BM inocula on stability of long-term tolerance. We have attempted to break tolerance in long-term chimeras originally reconstituted with TCD or non-TCD BM by challenging them with inocula containing normal, nontolerant recipient strain lymphocytes. tolerance was broken with remarkable ease in recipients of mixed marrow inocula in which both original BM components were TCD. In contrast, tolerance in chimeras originally reconstituted with non-TCD allogeneic BM was not affected by such inocula. Susceptibility to loss of chimerism and tolerance was not related to initial levels of chimerism per se, but rather to T cell depletion of allogeneic BM, since chimeras reconstituted with TCD allogeneic BM alone (mean level of allogeneic chimerism 98%) were as susceptible as mixed chimeras to the tolerance-breaking effects of such inocula. The possible contribution of GVH reactivity to this resistance was investigated using an F1 into parent strain combination. In these animals, the use of non-TCD F1 BM inocula for reconstitution did not lead to resistance to the tolerance-breaking effects of recipient strain splenocytes. These results suggest that the ability of T cells in allogeneic BM inocula to confer resistance to late graft failure may be related to their graft-vs.-host reactivity, even in situations in which they do not cause clinical GVHD. These findings may have relevance to the mechanism whereby T cell depletion of allogeneic BM leads to an increased incidence of late graft failure in clinical BM transplantation situations.
In vivo stimulation and restoration of the immune response by the noninflammatory fragment 163-171 of human interleukin 1 beta.
Authors: Boraschi D , Nencioni L , Villa L , Censini S , Bossù P , Ghiara P , Presentini R , Perin F , Frasca D , Doria G
Published: June 2004

The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 beta protein. In fact, both IL-1 beta and the 163-171 fragment stimulated the immune response of normal mice and restored immune reactivities of immunocompromised animals. In addition, the synthetic IL-1 peptide was as efficient as the entire protein in inducing tumor rejection and radioprotection. On the other hand, the 163-171 fragment did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 beta molecule in vivo: hypoferremia, hypoglycemia, hyperinsulinemia, increase in circulating corticosterone, SAA and fibrinogen, decrease in hepatic drug-metabolizing enzymes. Furthermore, at variance with IL-1 beta, the 163-171 peptide did not show the toxic effects causing shock and death in adrenalectomized mice. Thus, these results confirm our previous in vitro observations that functional domains are identifiable within the multipotent cytokine IL-1 beta, and demonstrate the biological relevance of this finding in a variety of in vivo systems. The identification of a selectively active fragment of a cytokine may thus represent a significant step towards a better directed and more rational immunotherapeutic approach.
Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.
Authors: Mercolino T J , Arnold L W , Hawkins L A , Haughton G
Published: June 2004

We have found that, in the peritoneums of normal adult mice, 5-15% of lymphocytes bind a fluorescent liposome probe. In ontogeny, cells with this specificity were shown to appear by 8 d after birth, and increase to the adult frequency by 2-3 wk. Some older mice contain an expanded population of these cells. We have shown that liposome binding occurs by cell surface IgM recognizing the common membrane phospholipid, phosphatidyl choline (PtC). Virtually all of these PtC-specific cells bear the cell surface marker Ly-1. Our results indicate that roughly 1 in 10 peritoneal Ly-1+ B cells has this single specificity. We have found that the precursors to all the cells that form plaques on protease-treated autologous erythrocytes (BrMRBC) are included in the PtC-specific population and can be isolated by FACS. We believe this is the first report of sorting large numbers of B cells with a single antigen specificity from normal, unimmunized animals. This method will allow for in vitro and in vivo studies of differentiative and proliferative properties of Ly-1+ B cells, which may help define their role in development and disease.
Immunological responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE.
Authors: Haba S , Nisonoff A
Published: June 2004

We have previously shown that adult A/J mice produce high titers of anti-IgE with isotypic or idiotypic specificities in response to challenge with a conjugate of KLH with syngeneic monoclonal IgE. Thus, B cells that can synthesize anti-IgE are present in the mice. Adult mice are unresponsive to unconjugated IgE in CFA, suggesting that tolerance exists at the level of T cells. The present study shows that neonatal mice produce anti-IgE antibodies in response to unconjugated IgE in CFA, but that this capacity is lost after the age of 2-3 wk. The loss of responsiveness corresponds closely with the appearance of detectable IgE in serum, suggesting that the IgE may induce tolerance. The affinities of anti-IgE antibodies produced by neonatal mice fall in the range of values obtained with KLH-IgE in adult mice. Tolerance to unconjugated IgE in CFA can be induced in neonatal mice by administration of IgE in saline. In addition, the tolerant state can be induced by adoptive transfer of spleen cells from adult mice. The time-dependent acquisition of tolerance provides a useful model for studying mechanisms of tolerance and autoimmunity.
Transforming growth factor beta 1 selectively regulates early murine hematopoietic progenitors and inhibits the growth of IL-3-dependent myeloid leukemia cell lines.
Authors: Keller J R , Mantel C , Sing G K , Ellingsworth L R , Ruscetti S K , Ruscetti F W
Published: June 2004

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.
Aberrant production of leukotriene C4 by macrophages from autoimmune-prone mice.
Authors: Santoro T J , Morris D H , Murphy R C , Baker R C
Published: June 2004

Eicosanoids have been implicated in the pathogenesis of autoimmune diseases. In this study, peritoneal macrophages from autoimmune-prone mice were examined for their capacity to produce proinflammatory 5-lipoxygenase metabolites. The results indicate that enhanced production of leukotriene C4 is a common feature of murine autoimmunity and suggest further that aberrations in 5-lipoxygenase activity may play a role in the development of lupus.
Long-term residence of a graft is an insufficient stimulus for the induction of tolerance. Investigating the role of cyclosporine in class I-disparate heart grafts in the rat.
Authors: Lim S M , White D J
Published: June 2004

This study demonstrates that the induction of tolerance is possible across a class I only antigenic barrier that fails to produce heart graft rejection. However, the long-term residence alone of such a graft per se, does not necessarily lead to the establishment of systemic tolerance in the recipient. The important finding in this study with regard to the biology of allograft tolerance, is that while the class I antigen provides the stimulus, its presence alone is not sufficient for the induction of tolerance; indeed, the action of the Cyclosporine A (CyA) is a necessary adjunct to its induction.
Surface expression of only gamma delta and/or alpha beta T cell receptor heterodimers by cells with four (alpha, beta, gamma, delta) functional receptor chains.
Authors: Saito T , Hochstenbach F , Marusic-Galesic S , Kruisbeek A M , Brenner M , Germain R N
Published: June 2004

Surface expression of TCR dimers by cells synthesizing three or four distinct types of receptor chains was analyzed. Cells containing intact gamma, alpha, and beta chains had only gamma delta dimers on the cell surface. In human PEER cells, addition of a functional alpha chain led to the loss of gamma delta dimer expression and expression of only alpha beta dimers. This result was not due to transcriptional down-regulation of the gamma or delta loci. In murine cells expressing all four chains, both gamma delta and alpha beta dimers could be demonstrated on a single cell. No other chain combinations (alpha gamma, alpha delta, beta gamma, or beta delta) were detected. Thus, there is stringent control of assembly and/or transport of TCR heterodimers, such that functional receptors consist only of alpha beta and gamma delta pairs, and no additional repertoire diversity is generated by cross pairing.
Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. A possible mechanism of foam cell formation.
Authors: Griffith R L , Virella G T , Stevenson H C , Lopes-Virella M F
Published: June 2004

Human macrophages play a key role in atherogenesis and are believed to be the progenitors of the cholesteryl ester (CE)-laden foam cells present in early atherosclerotic lesions. Several mechanisms by which macrophages accumulate CE have been recently described. One involves a perturbation in LDL metabolism subsequent to macrophage activation. Thus, we decided to study the effect of macrophage activation by immune complexes on N-LDL metabolism. Initially, LDL-containing immune complexes (LDL-IC) were chosen, since increased plasma levels of these IC have been reported in patients with coronary heart disease. Human macrophages stimulated for 22 h with LDL-IC (250 micrograms/ml) and incubated afterwards for 20 h with 10 micrograms/ml 125I-N-LDL showed a six- and fourfold increase in the accumulation and degradation, respectively, of 125I-N-LDL over the values observed in nonstimulated cells. Scatchard analysis of 125I-N-LDL-specific binding suggests an increase (20-fold) in the number of LDL receptors in macrophages stimulated with LDL-IC. We studied other immune complexes varying in size and antigen composition. Some of the IC were able to stimulate, although to a lesser degree, the uptake of N-LDL by macrophages. Lipoprotein IC are more efficient and have the greatest capacity to increase N-LDL uptake and CE accumulation. We conclude that human macrophage activation by LDL-IC leads to an increase in LDL receptor activity and promotes in vitro foam cell formation.
Early events in human T cell ontogeny. Phenotypic characterization and immunohistologic localization of T cell precursors in early human fetal tissues.
Authors: Haynes B F , Martin M E , Kay H H , Kurtzberg J
Published: June 2004

During early fetal development, T cell precursors home from fetal yolk sac and liver to the epithelial thymic rudiment. From cells that initially colonize the thymus arise mature T cells that populate T cell zones of the peripheral lymphoid system. Whereas colonization of the thymus occurs late in the final third of gestation in the mouse, in birds and humans the thymus is colonized by hematopoietic stem cell precursors during the first third of gestation. Using a large series of early human fetal tissues and a panel of monoclonal antibodies that includes markers of early T cells (CD7, CD45), we have studied the immunohistologic location and differentiation capacity of CD45+, CD7+ cells in human fetal tissues. We found that before T cell precursor colonization of the thymus (7-8 wk of gestation), CD7+ cells were present in yolk sac, neck, upper thorax, and fetal liver, and were concentrated in mesenchyme throughout the upper thorax and neck areas. By 9.5 wk of gestation, CD7+ cells were no longer present in upper thorax mesenchyme but rather were localized in the lymphoid thymus and scattered throughout fetal liver. CD7+, CD2-, CD3-, CD8-, CD4-, WT31- cells in thorax and fetal liver, when stimulated for 10-15 d with T cell-conditioned media and rIL-2, expressed CD2, CD3, CD4, CD8, and WT31 markers of the T cell lineage. Moreover, CD7+ cells isolated from fetal liver contained all cells in this tissue capable of forming CFU-T colonies in vitro. These data demonstrate that T cell precursors in early human fetal tissues can be identified using a mAb against the CD7 antigen. Moreover, the localization of CD7+ T cell precursors to fetal upper thorax and neck areas at 7-8.5 wk of fetal gestation provides strong evidence for a developmentally regulated period in man in which T cell precursors migrate to the epithelial thymic rudiment.
Glycosylation of a VH residue of a monoclonal antibody against alpha (1----6) dextran increases its affinity for antigen.
Authors: Wallick S C , Kabat E A , Morrison S L
Published: June 2004

We have observed that antidextran hybridomas with potential N-linked glycosylation sites in VH have higher affinity for polymeric dextran and for isomaltoheptaose than those lacking potential glycosylation sites. In these studies we have used gene transfection and expression techniques to verify that the carbohydrate addition sites in VH were used. The carbohydrate of the VH region was accessible for binding by the lectin Con A. By ELISA analysis it was demonstrated that the aKa of the antibody for dextran was influenced by the presence of carbohydrate in VH, with the aglycosylated antibody having an aKa 15-fold lower than its untreated counterpart. The aKa for antigen of antibodies that contain carbohydrate only in their constant region was unaffected by lack of carbohydrate. Thus, not only the amino acid sequence of the variable region but also its carbohydrate moieties can determine the magnitude of the antigen-antibody interaction.
Inhibition of human immunodeficiency virus (HIV-1/HTLV-IIIBa-L) replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides.
Authors: Perno C F , Yarchoan R , Cooney D A , Hartman N R , Gartner S , Popovic M , Hao Z , Gerrard T L , Wilson Y A , Johns D G
Published: June 2004

Because of the probable role of HIV-infected monocyte/macrophages in the pathogenesis and progression of AIDS, it is essential that antiretroviral therapy address viral replication in cells of this lineage. Several dideoxynucleosides have been shown to have potent in vitro and, in the case of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), in vivo activity against HIV. However, because these compounds must be phosphorylated (activated) in target cells, and because monocyte/macrophages may have levels of kinases that differ from those in lymphocytes, we investigated the capacity of these drugs to suppress HIV replication in monocyte/macrophages using HIV-1/HTLV-IIIBa-L (a monocytotropic isolate). In the present study, we observed that HTLV-IIIBa-L replication in fresh human peripheral blood monocyte/macrophages was suppressed by each of three dideoxynucleosides: 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyadenosine (ddA). Similar results were observed in 5-d-cultured monocyte/macrophages, although higher concentrations of the drugs were required. We then studied the metabolism of AZT and ddC in such cells. The phosphorylation of ddC to a triphosphate moiety was somewhat decreased in monocyte/macrophages as compared with H9 T cells. On the other hand, the phosphorylation of AZT in monocyte/macrophages was markedly decreased to 25% or less of the level in T cells. However, when we examined the level of the normal endogenous 2'-deoxynucleoside triphosphate pools, which compete with 2',3'-dideoxynucleoside triphosphate for viral reverse transcriptase, we found that the level of 2'-deoxycytidine-triphosphate (dCTP) was six- to eightfold reduced, and that of 2'-deoxythymidine-triphosphate (dTTP) was only a small fraction of that found in T cell lines. These results suggest that the ratio of dideoxynucleoside triphosphate to normal deoxynucleoside triphosphate is a crucial factor in determining the antiviral activity of dideoxynucleosides in HIV target cells, and that the lower levels of dTTP may account for the antiretroviral activity of AZT in the face of inefficient phosphorylation of this compound.
Cytotoxic T lymphocytes from HLA-A2 transgenic mice specific for HLA-A2 expressed on human cells.
Authors: Bernhard E J , Le A X , Barbosa J A , Lacy E , Engelhard V H
Published: June 2004

CTL clones were derived from HLA-A2.1 transgenic mice by immunization with a human cell expressing HLA-A2.1. None of these clones lysed murine transfectants, and only 3 of 23 lysed monkey transfectants expressing HLA-A2. In contrast, all of these clones lysed a wide variety of human cells expressing HLA-A2.1. These results demonstrate the existence of species-specific epitopes on the HLA-A2.1 molecule, and suggest that these epitopes are formed by the association of class I MHC products with one or more endogenous species-specific molecules. These results provide an explanation for the frequently observed failure of HLA class I-specific CTL to recognize these antigens on murine transfectants. These results also suggest that such endogenous proteins may also contribute to the formation of epitopes recognized by allospecific CTL.
Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus.
Authors: Cannon M J , Openshaw P J , Askonas B A
Published: June 2004

We have examined the function of class I MHC-restricted cytotoxic T cells in experimental respiratory syncytial virus (RSV) infection of BALB/c mice by transfer of T cell line MJC-A2 and CTL clone E8a into RSV-infected mice. The T cell line cleared pulmonary RSV infection within 5 d in persistently infected gamma-irradiated mice, but caused acute respiratory disease. This was only seen in infected mice and was often lethal after transfer of greater than 3 x 10(6) CTL. Lower numbers of CTL produced less severe disease but still cleared lung RSV, albeit over a longer time course (up to 10 d). Clearance of lung RSV in immunocompetent mice by the T cell line and CTL clone was again accompanied by acute and sometimes lethal respiratory disease. Bronchoalveolar lavage showed severe lung hemorrhage and frequent neutrophil efflux in mice with CTL-augmented disease.
Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters.
Authors: Lee S H , Crocker P R , Westaby S , Key N , Mason D Y , Gordon S , Weatherall D J
Published: June 2004

Stromal macrophages (M phi) have been localized in situ and isolated within erythroid clusters from human marrow. Stromal M phi arborize in an extensive network uniformly distributed throughout marrow interstitium, and express the phenotype CD4+, CD11a+, CD11c+, CD13+, CD14+, CD16+, CD18+, CD31+, CD32+, FcRI+, HLA-DR+, and CD35-, transferrin receptor-negative, and CD11b (weak). They express endocytic receptor antigens, but show significant differences in myeloid antigen expression compared with freshly harvested or cultured monocytes. Human stromal M phi are therefore specialized mature marrow M phi that are accessible for further investigations in infectious, storage, or hemopoietic disorders.
Regulation of murine MHC class II molecule expression. Identification of A beta residues responsible for allele-specific cell surface expression.
Authors: Buerstedde J M , Pease L R , Nilson A E , Bell M P , Chase C , Buerstedde G , McKean D J
Published: June 2004

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.
Heterogeneity of lymphocyte calcium metabolism is caused by T cell-specific calcium-sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential.
Authors: Ishida Y , Chused T M
Published: June 2004

Calcium management differs in T and B lymphocytes. [Ca2+]i elevation in response to calcium ionophores is up to 10 times greater in T cells than B cells. There is no difference between them in ionophore uptake. T cells, but not B cells, possess a calcium-sensitive potassium channel which produces membrane hyperpolarization at [Ca2+]i above 200 nM. This alters T cell density providing a rapid and easy method of cell separation. In contrast, B cells depolarize when [Ca2+]i is increased. Isolated B cell membrane vesicle ATP-dependent calcium pump activity is higher than T cell vesicles. Membrane depolarization reduces the [Ca2+]i response to ionomycin, most dramatically in T cells because they are hyperpolarized by increased [Ca2+]i. The most likely basis of this behavior is an effect of membrane potential on lymphocyte membrane calcium pump activity. This mechanism provides an explanation of the inhibitory effect of membrane depolarization on T lymphocyte responses.
Extensive deletions and insertions in different MHC supratypes detected by pusled field gel electrophoresis.
Authors: Tokunaga K , Saueracker G , Kay P H , Christiansen F T , Anand R , Dawkins R L
Published: June 2004

The genomic organization of the human MHC was examined in multiple examples of six different supratypes using pulsed field electrophoresis (PFGE) after digestion of genomic DNA with infrequency cutting restriction endonucleases. Differences in restriction fragment length and band intensity were shown to be specific for each supratype. Mapping of the MHC revealed that each supratype contains previously undescribed deletions and insertions between HLA B and DQ regions.
Detection of a gamma interferon-induced protein IP-10 in psoriatic plaques.
Authors: Gottlieb A B , Luster A D , Posnett D N , Carter D M
Published: June 2004

The pathologic features of psoriatic plaques are inflammation and increased epidermal turnover. IP-10, a cytokine the expression of which is induced by gamma-interferon, is a member of a family of soluble mediators with inflammatory and growth-promoting activities. IP-10 protein was detected in keratinocytes and the dermal infiltrate from active psoriatic plaques using an affinity-purified rabbit anti-IP-10 antibody in immunoperoxidase studies. Successful treatment of active plaques decreased IP-10 expression in plaques. These results were corroborated by Northern blot analysis with an IP-10 cDNA probe. We have previously detected activated T cells and HLA-DR keratinocytes in active psoriatic plaques. Since IP-10 is detected in delayed cellular immune responses, the present study further points to the role of ongoing cellular immune responses in the pathogenesis of psoriasis.
Epstein-Barr virus regulates activation and processing of the third component of complement.
Authors: Mold C , Bradt B M , Nemerow G R , Cooper N R
Published: June 2004

Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.
Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen.
Authors: Townsend A , Bastin J , Gould K , Brownlee G , Andrew M , Coupar B , Boyle D , Chan S , Smith G
Published: June 2004

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.
Antibody to mouse interferon alpha/beta abrogates resistance to the multiplication of Friend erythroleukemia cells in the livers of allogeneic mice.
Authors: Gresser I , Maury C , Vignaux F , Haller O , Belardelli F , Tovey M G
Published: June 2004

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously into adult syngeneic DBA/2 or allogeneic C57B1/6 (H-2b) or C3H (H-2k) mice lodge in the liver but only multiply in the liver of syngeneic mice. Our results indicated that endogenous IFN-alpha/beta was a crucial factor in preventing the multiplication of FLC in the liver of adult allogeneic mice. (a) Treatment of allogeneic adult C57B1/6 or C3H mice with polyclonal antibody to mouse IFN-alpha/beta (but not antibody to IFN-gamma) completely abrogated the resistance to the multiplication of FLC in the liver and 87% of tumor-injected, antibody-treated C57B1/6 mice died with extensive tumor involvement of the liver. In contrast, after intravenous inoculation FLC do not multiply at all (or very rarely) in the liver of adult C57B1/6 mice left untreated or treated with a variety of control globulins, and no deaths occurred. (b) 8 h after intravenous inoculation of FLC, poly(A)+ RNA hybridizable with specific DNA probes for mouse IFN-alpha or -beta (but not -gamma) was present in the liver of injected C57B1/6 mice. Using the expression of the Mx protein as an indicator of the presence of IFN-alpha/beta, we showed that Mx+ congenic C57B1/6 mice injected with FLC exhibited a marked increase in the expression of the Mx protein in the liver, spleen, kidney and lung, and this increase was blocked by treatment of mice with antibody to IFN-alpha/beta. The possibility that different host mechanisms are elicited depending on the site of tumor growth in allogeneic mice is discussed. IFN-alpha/beta appears to be of particular importance in determining the resistance of the liver to FLC in allogeneic mice.
Synthesis and release of platelet-activating factor is inhibited by plasma alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin and is stimulated by proteinases.
Authors: Camussi G , Tetta C , Bussolino F , Baglioni C
Published: June 2004

TNF and IL-1 stimulate the synthesis and release of platelet-activating factor (PAF) by neutrophils and vascular endothelial cells. Serum inhibits PAF production even after inactivation of an acetylhydrolase that degrades PAF. Human plasma was fractionated by gel filtration chromatography, and two inhibitory fractions were detected, one containing PAF-acetylhydrolase activity and the other alpha 1-proteinase inhibitor. Low concentrations of this antiproteinase and of human plasma alpha 1-antichymotrypsin inhibited TNF-induced PAF synthesis in neutrophils, macrophages, and vascular endothelial cells. Both antiproteinases also inhibited PAF production stimulated by phagocytosis in macrophages and induced with IL-1 in neutrophils or with TNF in vascular endothelial cells. These results suggest that a proteinase activated on the plasma membrane or secreted by these cells is involved in promoting PAF synthesis. Indeed, addition of elastase to macrophages, neutrophils, and endothelial cells stimulated synthesis and release of PAF much faster than TNF. A similar stimulation was observed in incubations with cathepsin G. To identify a proteinase activated in TNF-treated cells, neutrophils and endothelial cells were incubated with specific chloromethyl ketone inhibitors of elastase and cathepsin G. Synthesis of PAF was significantly inhibited by low concentrations of the cathepsin G inhibitor. The finding that antiproteinases are inhibitory at concentrations 100-fold lower than those present in plasma raises questions as to the ability of TNF and IL-1 to stimulate neutrophils in circulation or endothelial cells to synthesize PAF. We propose that PAF production is limited to zones of close contact between cells, which exclude antiproteinases.
Cytoadherence by Plasmodium falciparum-infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes.
Authors: Magowan C , Wollish W , Anderson L , Leech J
Published: June 2004

Plasmodium falciparum-infected erythrocytes (IRBCs) adhere specifically to venular endothelium and thereby evade spleen-dependent immune mechanisms. We have investigated the molecular basis of cytoadherence. We report here that the capacity for cytoadherence of IRBCs is correlated with the expression of a family of variable proteins on the surface of IRBCs. Essential to these studies was the use of in vitro techniques for modulating the cytoadherence phenotype of cloned parasites. In initial studies, we found culture-adapted parasites to be poorly cytoadherent or noncytoadherent. To select for cytoadherent parasites, we incubated knobbed IRBCs with C32 melanoma cells and cultured the adherent cells. Repeated rounds of selection produced parasites with increased cytoadherence. To select for noncytoadherent parasites, we cultured the cells that did not adhere to C32 melanoma cells. Cytoadherent IRBCs from two different cloned isolates had large (Mr greater than 2.4 x 10(5) radioiodinatable proteins that differed in size between the isolates but had in common the biochemical properties of trypsin sensitivity and insolubility with Triton X-100. The proteins were not detected with uninfected erythrocytes, indicating that they were parasite determined, nor were they detected with IRBCs containing parasites cultured for many months without selection. With continued selection for the cytoadherent phenotype, additional IRBC surface proteins with larger molecular sizes (Mr 2.9 x 10(5) and 3.2 x 10(5] appeared. A sequence of reversible changes in the cytoadherence phenotype of cloned parasites was accompanied by variation in the molecular size of the IRBC surface protein. Increased cytoadherence was correlated with expression of larger proteins and decreased cytoadherence was correlated with expression of smaller proteins; there was no change in the molecular size of two other parasite proteins associated with the IRBC membrane. The results indicate that the expression of this family of proteins is closely linked to the cytoadherence phenotype of the parasites, suggesting that the members of the protein family have a role in mediating cytoadherence between IRBCs and endothelial cells.
Interleukin 4 inhibits the proliferation but not the differentiation of activated human B cells in response to interleukin 2.
Authors: Defrance T , Vanbervliet B , Aubry J P , Banchereau J
Published: June 2004

The combined effect of IL-4 and IL-2 on proliferation of anti-IgM antibody or Staphylococcus aureus strain Cowan I (SAC)-preactivated B cells was investigated. It was observed that in most cases, rIL-2 used at optimal concentration induced higher levels of tritiated thymidine ([3H]TdR) uptake than rIL-4 used at optimal concentration. When rIL-4 and rIL-2 were added together, it was repeatedly found that B cell proliferation induced by rIL-2 was significantly reduced and was, in most cases, comparable with the proliferation induced by rIL-4 alone. Cell cycle studies demonstrated that rIL-4 significantly reduced the number of cells entering S and G2/M phases of the cell cycle upon rIL-2 stimulation. B cell blasts preincubated for 24 or 48 h with rIL-4 displayed a reduced proliferation in response to rIL-2. In contrast, preculture of resting B cells with rIL-4 did not impair their subsequent proliferation in response to rIL-2 plus insolubilized anti-IgM antibody. This suggests that rIL-4 can only exert its inhibitory effect once B cells have received an activation signal. The differentiative activity of rIL-2 measured on B cell blasts preactivated for 2 d with SAC was not altered by rIL-4, which suggests that rIL-4 did not exert its inhibitory activity on rIL-2-induced B cell proliferation by enhancing rIL-2-mediated differentiation. Delayed addition of a neutralizing anti-IL-4 antiserum demonstrated that a period of contact of at least 24 h between IL-4 and B cell blasts was necessary for the development of the antagonistic effect of IL-4 on IL-2-mediated growth of activated B cells. These data demonstrate that IL-4 antagonizes the B cell growth-promoting effect of IL-2 without affecting the differentiation of preactivated B cells in response to IL-2.
Interaction and sequence diversity among T15 VH genes in CBA/J mice.
Authors: Ferguson S E , Rudikoff S , Osborne B A
Published: June 2004

Nucleotide sequences of the four genes composing the T15 heavy chain variable region (VH) family of the CBA/J mouse have been determined. Comparison of these sequences with their published BALB/c and C57BL/10 homologues reveals that nucleotide differences found between given alleles of two strains, i.e., CBA/J and BALB/c, are observed in other family members of the same strain. We suggest that these patterns of sequence variation are most readily explained by gene interaction (conversion). Additionally, the sequence of a CBA/J hybridoma, 6G6, proposed to have been generated by gene conversion, is directly encoded by the CBA/J V11 gene indicating that the putative conversion has occurred meiotically in the germline. These results are consistent with the premise that gene correction is occurring frequently among members of this family and that such processes may contribute significantly to the evolution of Ig variable region genes even in the relatively short time frame of inbred strain derivation.
Dissecting human T cell responses against Bordetella species.
Authors: De Magistris M T , Romano M , Nuti S , Rappuoli R , Tagliabue A
Published: June 2004

To identify the minimal structures that may be important for the creation of a synthetic and/or recombinant vaccine against whooping cough, human T cell clones were obtained against Bordetella antigens. Cloned peripheral blood T lymphocytes from an immune donor were grown in IL-2 and tested for proliferation in response to inactivated Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica) and mutants deficient for the expression of virulence-associated antigens. All the T cell clones obtained were CD4+8- and recognized specifically the Bordetella antigens when presented by autologous B cells. On the basis of the responsiveness to the whole inactivated bacteria, it was possible to cluster the 12 clones obtained into four groups with the following specificity: (1) filamentous hemagglutinin (FHA); (2) B. pertussis-specific antigens; (3) virulence-associated Bordetella-specific antigens; and (4) nonvirulence-associated Bordetella-specific antigens. Using two new B. pertussis deletion mutants, clone 6 (representative of cluster 1) was found to recognize the COOH terminus of FHA. Furthermore, three out of four clones of cluster 3 were specifically stimulated by the soluble 69-kD protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized pertussis toxin (PT), which is believed to be the most important protein to be included in an acellular vaccine. However, when a new generation of clones was obtained using soluble PT as the in vitro stimulus, it was observed that 11 clones of this group recognized this antigen. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria, although T cell memory against PT exists in a donor who had the disease several years ago.