bluuebunny/crossref_metadata_2025 huggingface dataset:
TNF translationally modulates the expression of G1 protein alpha(i2) subunits in human polymorphonuclear leukocytes.
Authors: Scherzer J A , Lin Y , McLeish K R , Klein J B
Published: December 2022

Priming of polymorphonuclear leukocyte responses to chemoattractants by TNF plays an important role in host defenses and inflammatory responses. TNF-induced priming is associated with an 80% increase in the membrane density of G alpha(i2) protein that is coupled to chemoattractant receptors. The present study examines the hypothesis that TNF stimulates increased synthesis of alpha(i2). Within 10 min of addition, TNF stimulated a significant increase in total cellular G alpha(i2), as determined by pertussis toxin-catalyzed ADP ribosylation, which was blocked by the translation inhibitor cycloheximide. Immunoprecipitation of biosynthetically labeled alpha(i2) showed that TNF increased alpha(i2) synthesis by about 20% at 10 min. Nuclear run-ons showed no change in alpha(i2) mRNA synthesis in TNF-treated cells; however, steady state alpha(i2) mRNA levels were reduced following a 10-min exposure to TNF. Pretreatment with cycloheximide prevented the TNF-induced reduction in steady state alpha(i2) mRNA levels. Therefore, TNF stimulates alpha(i2) protein synthesis and mRNA degradation in the same time frame as priming. The increased alpha(i2) synthesis results from increased translation, not transcription, of alpha(i2) mRNA. Simultaneous G alpha(i2) protein synthesis and mRNA degradation provide a mechanism by which TNF priming is associated with a rapid, self-limiting increase in G protein expression.
Granulocyte-macrophage colony-stimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils.
Authors: Sung K L , Li Y , Elices M , Gang J , Sriramarao P , Broide D H
Published: December 2022

As very late antigen-4 (VLA-4) can exist in different functional states, we have sought to determine whether a cytokine expressed by inflamed endothelium (i.e., granulocyte-macrophage CSF (GM-CSF)) could regulate the functional state of VLA-4 expressed by eosinophils. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation force, 0.022 +/- 0.003 mdynes). In contrast, individual eosinophils bound to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes), TNF-stimulated endothelium (0.62 +/- 0.05 mdynes), or IL-4-stimulated endothelium (0.11 +/- 0.01 mdynes) with increased avidity as assessed by separation force. Eosinophil binding to IL-4-stimulated endothelium was significantly inhibited by neutralizing Abs to either vascular cell adhesion molecule (VCAM) or VLA-4. The strength of eosinophil adhesion to VCAM (0.31 +/- 0.02 mdynes) or to connecting segment-1 (CS-1) (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but was less than the strength of eosinophil adhesion to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes). After incubating eosinophils for 30 min with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84 and 54%, respectively, compared with that of controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterations in VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by confocal microscopy). These studies suggest that endothelial-derived cytokines such as GM-CSF have the potential to alter the functional state of eosinophil-expressed VLA-4 from a low affinity state to a high affinity state.
Structural and affinity studies of IgM polyreactive natural autoantibodies.
Authors: Diaw L , Magnac C , Pritsch O , Buckle M , Alzari P M , Dighiero G
Published: December 2022

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.
Successful formation of a chimeric human thymus allograft following transplantation of cultured postnatal human thymus.
Authors: Markert M L , Kostyu D D , Ward F E , McLaughlin T M , Watson T J , Buckley R H , Schiff S E , Ungerleider R M , Gaynor J W , Oldham K T , Mahaffey S M , Ballow M , Driscoll D A , Hale L P , Haynes B F
Published: December 2022

Transplantation of cultured postnatal human thymus was performed in a patient with complete DiGeorge syndrome. Biopsy of the graft 3 mo after implantation revealed normal CD1+ thymocytes in thymic cortical epithelial regions and CD1- thymocytes in thymic medullary epithelial regions, respectively. HLA analysis of graft thymocyte and thymic microenvironment components demonstrated that developing thymocytes and thymic macrophages were recipient derived, while thymic epithelial components were of donor origin. The patient, who initially had no T cells and had profoundly defective T cell function, developed normal T cell responses to mitogens and Ags, tolerance to donor in a mixed lymphocyte reaction, and normal Ab titers after tetanus toxoid and pneumovax immunization. Thus, transplantation of cultured postnatal human thymic tissue in humans can form functional chimeric thymic tissue, and may provide a strategy to reconstitute the peripheral T cell pool in select congenital and acquired immune deficiency syndromes.
Characterization of B cells expressing recombination activating genes in germinal centers of immunized mouse lymph nodes.
Authors: Hikida M , Mori M , Kawabata T , Takai T , Ohmori H
Published: December 2022

Products of recombination activating genes (RAG-1 and RAG-2) involved in the rearrangement of Ig genes have been shown to be expressed only in immature stages of B cells. However, we have recently found that RAG genes were re-expressed in mature mouse B cells activated in vitro and in germinal centers (GCs) of immunized mouse lymph nodes (LNs). Here, we report that RAG transcripts and their proteins were expressed in parallel with the formation of GCs in popliteal LNs from mice immunized in the hind footpads. Immunocytochemical analysis revealed that RAG+ B cells were localized within GCs and were present as apoptotic tingible body cells. RAG expression is not considered a nonspecific result of apoptosis, since apoptotic B cells generated by surface Ig-engagement did not express RAG genes. These results suggest a novel role of RAG products in the differentiation of B cells in GCs.
MHC class I-associated peptides produced from endogenous gene products with vastly different efficiencies.
Authors: Anton L C , Yewdell J W , Bennink J R
Published: December 2022

We compared the efficiency of generating antigenic peptides from various polypeptide contexts expressed by recombinant vaccinia viruses. These included full-length influenza virus nucleoprotein (NP(1-498)), two truncated forms, and cytosolic and endoplasmic reticulum-targeted minimal peptides. Two peptides were studied, NP(50-57) (Kk-restricted) and NP(147-155) (Kd-restricted). The efficiency of peptide generation was measured in cytotoxicity assays by determining 1) the kinetics of presentation following infection using brefeldin A to block additional presentation and 2) the concentration of anti-class I mAbs required to block presentation. The two determinants behaved similarly, being presented most efficiently from minigene products, with intermediate efficiency from fragments, and least efficiently from NP(1-498). Direct quantitation of HPLC-purified peptides supported the validity of these simple methods to roughly estimate the efficiency of class I Ag presentation. It also surprisingly revealed that 60- to 90-fold more NP(50-57) than NP(147-155) peptide was present in cells expressing NP(1-498) or a rapidly degraded fragment (for NP(1-498), 1800 peptides/cell of NP(50-57) vs 30 peptides/cell of NP(147-155)). By contrast, nearly identical (and much greater) amounts of peptides were recovered from cells expressing minigene products (55,000 copies of either peptide/cell). These findings demonstrate 1) that immunodominant peptides from the same protein can be generated with vastly different efficiencies, and 2) that cytosolic or endoplasmic reticulum-targeted minigene products are presented far more efficiently than longer polypeptides.
The common gamma-chain of cytokine receptors regulates intrathymic T cell development at multiple stages.
Authors: He Y W , Nakajima H , Leonard W J , Adkins B , Malek T R
Published: December 2022

Signaling through the common gamma chain (gamma c), a subunit of the receptors for IL-2, -4, -7, -9, and -15, is critical for lymphocyte development, with the IL-7/IL-7R representing one important interaction. To investigate the stages of intrathymic T cell development that are dependent on gamma c and to determine whether gamma c controls T cell development solely as a component of the IL-7R, intrathymic T cell development was compared in IL-7R alpha-deficient mice and anti-gamma c-treated chimeric mice reconstituted with bone marrow and purified pro-T cells. In the presence of anti-gamma c, each of four phenotypically distinguishable stages of CD4- CD8- thymocytes failed to reconstitute T cell development, suggesting that each of these subsets of pro-T cells required gamma c for their differentiation and/or growth. Reconstitution of anti-gamma c-treated chimeric mice with bone marrow from IL-7R alpha-deficient mice indicated that IL-7R only partially contributed to intrathymic T cell development. Furthermore, when compared with IL-7R-deficient mice, anti-gamma c chimeric and gamma c-deficient mice exhibited a distinct phenotypic pattern of pro-T cell development. Collectively, these results indicate that several gamma c-sharing cytokines may contribute to T cell development in the thymus and suggest that one of these cytokines may be novel.
Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death.
Authors: Hurtado J C , Kim Y J , Kwon B S
Published: December 2022

Previously, we and others showed that signals relayed through the murine T cell Ag 4-1BB enhance primary T cell responses, and that blocking the interaction of 4-1BB with its ligand results in decreased responses to polyclonal activators and to alloantigens. Because 4-1BB expression is induced following primary stimulation, we investigated the role of signaling through this molecule in the reactivation of proliferating T cells. To this end, preactivated, 4-1BB-expressing T cells were restimulated in the presence of plate-immobilized mAbs directed against 4-1BB or the prototypic costimulatory molecule CD28. In this work, we show that in the presence of either signal, T cells respond to TCR cross-linking with strong proliferative responses and cytokine production; moreover, our findings indicate that T cell proliferation partially correlates with surface 4-1BB expression. In addition, our results suggest that Ab-mediated costimulatory signals can act independently of potential accessory B7-CD28/CTLA-4 (cytotoxic T lymphocyte Ag-4) interactions. Importantly, the characteristic DNA fragmentation and apoptotic cell death observed after TCR re-engagement are inhibited comparably in the presence of either 4-1BB or CD28 signaling.
Induction of IL-15 messenger RNA and protein in human blood-derived dendritic cells: a role for IL-15 in attraction of T cells.
Authors: Jonuleit H , Wiedemann K , Müller G , Degwert J , Hoppe U , Knop J , Enk A H
Published: December 2022

IL-15 is a pleiotropic cytokine with IL-2-like functions. As IL-15 was shown to be mitogenic for T cells, we wondered whether human blood-derived dendritic cells (DC), as the primary stimulators of T cell responses, are able to produce IL-15. To test our hypothesis, DC were grown under serum-free conditions from human peripheral blood using granulocyte-macrophage CSF and IL-4. Cultures were assayed for IL-15 mRNA production at various times by semiquantitative reverse transcription-PCR. Low baseline signals were detected from days 0 to 5 of culture. A significant increase was detected from days 5 to 9 of the culture. When DC were further enriched by immunomagnetic beads to >98% purity as determined by CD83 staining, IL-15 mRNA signals were exclusively found in the CD83+ fraction. This increase in mRNA signals was paralleled by IL-15 protein release from days 9 to 12 as detected by CTLL-2 assay and ELISA. In addition, protein levels were increased >10-fold by adding paramagnetic beads to the cultures, thereby inducing phagocytic activity. Furthermore, DC supernatants were tested for chemokinetic and chemotactic activities for T cells in a checkerboard filter assay. It was shown that supernatants express chemokinetic and chemotactic activity for T cells. This activity was blocked almost completely by addition of an anti-IL-15 mAb. Our data show that human blood DC contain IL-15 mRNA and produce functional protein that is induced in culture. Protein release is triggered by phagocytic activity. Furthermore, DC-derived IL-15 has chemotactic and chemokinetic activities for T cells, suggesting a role for IL-15 as an attractant of T cells during the initial DC/T cell interaction.
Roles of IL-4 and IFN-gamma in stabilizing the T helper cell type 1 and 2 phenotype.
Authors: Nakamura T , Lee R K , Nam S Y , Podack E R , Bottomly K , Flavell R A
Published: December 2022

It has previously been reported that Th1 CD4 T cell populations can be converted to IL-4 producers, whereas Th2 populations are refractory to IL-12-mediated IFN-gamma production. We have recently shown that CD30 is a marker for the IL-4 response and have therefore used CD30 here to study Th1 and Th2 commitment. We show that Th2 cell populations normally show a stable phenotype and fail to respond to IL-12 because of endogenous IL-4 production. IFN-gamma abrogates this antagonistic effect of IL-4 and permits the conversion of Th2 populations to IFN-gamma producers by IL-12. In the complete absence of IL-4, however, IFN-gamma is not required for this transformation, and Th1 cells generated by IL-12 become committed to the Th1 pathway and lose the ability to respond to IL-4. Thus, the balance between local IL-4 and IFN-gamma in an immune response is a key factor in determining the outcome of the CD4 effector T cell response.
Presentation on class II MHC molecules of endogenous lysozyme targeted to the endocytic pathway.
Authors: Parra-López C A , Lindner R , Vidavsky I , Gross M , Unanue E R
Published: December 2022

We compared the processing and presentation of the model Ag, hen-egg white lysozyme (HEL) expressed in C3.F6 APC as a fusion protein to three different acid hydrolases: cathepsin D, to an unglycosylated form of cathepsin D, and to pepsinogen. As expected from the biology of mannose 6-phosphate (Man-6-P)-containing enzyme, cathepsin D-HEL was delivered to the endosomal/lysosomal system. In contrast, the unglycosylated cathepsin D-HEL was retained in ER/Golgi and some was found in lysosomes. Most of pepsinogen-HEL was rapidly secreted from the APC. All transfectants presented HEL epitopes to T cell hybridomas. Regardless of the main route of traffic of the proteins, the strong I-Ak binding epitope HEL 48-62 was well presented by all. The biochemical forms of this epitope were identical for all. Three other epitopes of HEL that bind I-Ak with less affinity were processed equally well by unglycosylated cathepsin D-HEL and HEL-Ld. The glycosylated cathepsin D-HEL was less efficient in generating the 114-129 epitope. Pepsinogen-HEL was the less efficient of all transfectants in presenting these subdominant epitopes. Soluble cathepsin D-HEL recovered from culture supernatant was strongly immunogenic when added to C3.F6. The uptake was inhibited by free Man-6-P, indicating that the surface Man-6-P receptor can effectively deliver proteins to the class II MHC system.
Induction of alloantigen-specific T cell tolerance through the treatment of human T lymphocytes with wortmannin.
Authors: Taub D D , Murphy W J , Asai O , Fenton R G , Peltz G , Key M L , Turcovski-Corrales S , Longo D L
Published: December 2022

Signaling through the CD28 molecule on T cells by its natural ligand, B7, on APCs has recently been shown to require the presence of an active phosphatidylinositol 3-kinase pathway to mediate some of its costimulatory activities (1-7). Using the phosphatidylinositol 3-kinase inhibitor, wortmannin (WN) (8), on human and murine T cells, we have inhibited B7-1-mediated T cell activation and induced Ag-specific tolerance. The addition of WN and/or the B7-1 antagonist, CTLA4Ig, to primary human T cell cultures stimulated with B7-1-transfected allogeneic melanoma cell lines inhibited the generation of alloantigen-specific proliferative and cytolytic responses in vitro. Subsequent examination of these WN- and CTLA4Ig-treated primary T cell cultures revealed that these lymphocyte populations were tolerized to rechallenge with the priming alloantigens in secondary cultures in the absence of additional inhibitor(s). However, reactivity to a third party allogeneic stimulator remained intact. This WN-induced tolerance was reversed by the addition of high dose IL-2, but not IL-4 or IL-7, to the primary cultures, indicating that T cell anergy, not deletion, was responsible for this phenomenon. In vivo studies using a murine graft-vs-host disease (GVHD) model demonstrated that WN treatment of allogeneic donor lymphocytes in vitro failed to generate a significant GVHD in irradiated mouse recipients compared with control allogeneic donor lymphocytes. These findings suggest potentially novel therapeutic strategies for the prevention of GVHD.
Transcriptional regulation of platelet-activating factor receptor gene in B lymphoblastoid Ramos cells by TGF-beta.
Authors: Yang H H , Pang J H , Hung R Y , Chau L Y
Published: December 2022

The expression of platelet-activating factor (PAF) receptor gene was up-regulated in a time- and dose-dependent manner in a B cell line (Ramos) following exposure to TGF-beta 2. The TGF-beta 2-induced increment of PAF receptor mRNA was at least partly due to an increase in transcriptional rate as demonstrated by nuclear run-off experiments. Transient transfection of cells with PAF receptor transcript I gene promoter fused to a luciferase reporter gene revealed that the TGF-beta-responsive element (T beta RE) lies between the sequence from -44 to -17 relative to the transcriptional start site. Insertion of the T beta RE upstream of the unresponsive minimal thymidine kinase promoter conferred the TGF-beta-inducibility. Gel mobility shift assay demonstrated the specific binding of nuclear factors to the T beta RE. The T beta RE binding activity was gradually increased and reached a maximum at 3 h and subsequently returned to basal level at 5 h in cells following TGF-beta 2-treatment. Concomitant treatment of cells with cycloheximide abolished the increases in both T beta RE-binding activity and expression of PAF receptor mRNA, indicating that de novo protein synthesis is required to exert TGF-beta 2 effect. Methylation interference analysis revealed that the T beta RE-binding protein recognized a purine-rich sequence, 5'-GGGGTG-3'. Point mutations of the consecutive guanine nucleotides significantly reduced the DNA-binding activity and the TGF-beta-induced promoter activity. Collectively, these results clearly demonstrate that a T beta RE proximal to the transcriptional initiation site of the human PAF receptor transcript I gene mediates the up-regulation of PAF receptor gene expression in Ramos cells by TGF-beta 2.
Alloreactive T cell recognition of MHC class I molecules: the T cell receptor interacts with limited regions of the MHC class I long alpha helices.
Authors: Smith K D , Lutz C T
Published: December 2022

T cells recognize MHC-bound peptide, suggesting that the TCR contacts surface MHC residues adjacent to bound peptide, but the extent of MHC contact is not known. T cells also may recognize peptide-induced conformational changes, and alloreactive T cells may recognize surface MHC structures in addition to or independent of bound peptide. Alloreactive T cells are not intentionally biased to recognize particular MHC-bound peptides and should reveal general constraints for TCR binding. To map TCR binding sites, we tested 60 HLA-B7 site-specific mutations with 12 alloreactive CTL clones that express different TCRs. The alloreactive CTL clones recognize solvent-accessible residues that cluster between positions 62 to 80 and 150 to 170. Thus, TCRs contact largely overlapping MHC structures in the alpha1 and alpha2 domain long alpha helices. The dimensions and location of this site are consistent with recently reported crystallographic studies of two TCR/peptide-MHC class I complexes. In contrast to TCR, Abs recognize multiple discrete epitopes that encircle the peptide binding groove and potentially encompass the entire surface of the MHC molecule. Our data suggest that TCRs dock with a common discrete MHC site and that recent crystallographic models are likely to be generally applicable to T cell recognition of peptide-MHC class I complexes.
CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice.
Authors: Chatenoud L , Primo J , Bach J F
Published: December 2022

Low doses of the CD3 mAb 145 2C11 restored self tolerance to beta cell Ags in adult overtly diabetic NOD mice. Within 2 to 4 wk after treatment, complete and permanent remission of diabetes was observed. Autoreactive T cells were not deleted in CD3 Ab-protected animals as evidenced first, by the persistence of peripheral insulitis and, second, by the capacity of spleen cells from CD3 Ab-treated mice to transfer diabetes to adult irradiated syngeneic recipients. Moreover, the conferred tolerance was reproducibly reversed by a single injection of cyclophosphamide. For 5 to 7 wk after treatment, IFN-gamma production by stimulated spleen cells was significantly decreased in treated animals. One unique feature was that the CD3 Ab-induced tolerance ensued only from treatment of overtly diabetic NOD mice. Durable protection was exclusively observed when treating mice with recent onset disease (14-20 wk old). At variance with this finding, treatment of 4- and 8-wk-old mice was without effect, and complete but transient protection followed the treatment of 12-wk-old NOD mice. The tolerogenic properties of 145 2C11 did not depend on its mitogenic capacity, since nonmitogenic F(ab')2 fragments also appeared potent at inducing durable remission in overtly diabetic NOD, although nonmitogenic CD3 F(ab')2 fragments could mediate T cell signaling, as evidenced by cytokine gene transcription (IL-2, IFN-gamma, IL-4, and IL-10) assessed by PCR on splenocytes from treated mice. A concomitant cyclosporine treatment abrogated the CD3 mAb-induced protection, further pointing to the crucial role of T cell signaling in the effect observed.
Studies in Isohemagglutination
Authors: Parr Leland W.
Published: December 2022

Summary Of Results

Data for Armenians, Arabic Moslems and Arabic Christians are given making it possible to assign these groups their racial indices. It is to be noted that Arabic Moslems and Arabic Christians, speaking the same language, living the same life, and locally thought to be the same people, yield significantly different values.

The value of this method of approach to racial relationships is questioned.

Preliminary data are presented for the Druzes and the Metwallis, both of which groups derive from the Moslems. Further work will have to be done before these groups can be either identified with or set definitely apart from the Moslem Arab.

A new family in which both father and mother belong to group AB is presented. No evidence is offered from this family or from one other, where one parent is AB, which will enable us to choose the three multiple allelomorphs theory over that of two independent pairs of factors hypothesis, but the accumulating evidence favors the former.
Studies in Isohemagglutination
Authors: Wiener Alexander S. , Lederer Max , Polayes S. H.
Published: December 2022

Of all the theories on the heredity of blood groups that have been presented up to the present time, Bernstein's theory is most satisfactory. Apparent exceptions to this theory are due to: (a) faulty technique; (b) failure to test a sufficient number of people; (c) errors in computation; (d) failure to study a homogeneous group, and (e) selection of a subgroup for study which is not representative of the entire group.
Quantitative Studies on the Action of Compound Hemolysins
Authors: Gahl Rudolf
Published: December 2022

A quantitative study of the characteristic curves defined in preceding papers (1, 2 and 3) led to the conclusion that the complexities of these curves as they are experimentally obtained can be removed and the curves so simplified that they assume forms which are in harmony with the views proposed by Svante Arrhenius on the reaction between hemolytic amboceptor and complement by introducing the assumption into the calculation that neither heat inactivated hemolytic amboceptor serum is free from complement nor complement serum from natural amboceptor.

Geometrical methods for introducing suitable corrections were developed for this purpose.

The application of such methods permits the quantitative estimation of the contaminations.

Heat inactivated hemolytic amboceptor serum may, when undiluted, contain complement in quantities not much lower in order of magnitude than the complement serum. Its effect may nevertheless not be noticeable on the Wassermann reaction as long as potent amboceptor serum is used which permits a correspondingly high dilution.

Application of the law of chemical mass action makes the construction of theoretical systems of characteristic curves possible. Only the exponents of the mass law equations determine the shape of these curves. Such curve systems were calculated under various assumptions regarding the chemical combination between amboceptor and complement and found to present typical pictures characteristic of each reaction.

It was shown that when the corrections above referred to are applied to the curves derived from the law of chemical mass action in the opposite direction complex curves result which agree with those experimentally obtained within the limits of experimental errors except in the region of complement deviation which the theory is not designed to cover.

Rational systems for expressing strength of amboceptor and complement were proposed.

Conclusions were drawn regarding the relative molar concentrations of amboceptor and complement.

The assumption of a stoichiometric chemical reaction between amboceptor and complement explains all observations. The stipulation of an enzymatic nature of one or both does not seem justified.

Summing up the results of all four papers on this subject, we might state that while Manwaring rightfully pointed out that Arrhenius' mathematical representation of the reaction between hemolytic amboceptor, complement and red blood cells is not in agreement with the experimental facts, we find it in agreement with them so far as only the reaction between amboceptor and complement is concerned. The constants which Arrhenius used in his equations are as all constants obtained by experiment subject to revision by later investigators.
The Skin Reaction in the Sensitized Guinea Pig
Authors: Ramsdell Susan Griffith
Published: December 2022

Guinea pigs actively sensitized and skin-tested, using trypan blue as a technical aid, gave reactions to the antigen-serum in dilutions as high as 1:10,000. Passive sensitization of the skin of a normal animal could be demonstrated as early as the sixth day after treatment of the actively sensitized animal, and was regularly more consistent than the test for passive sensitization by shock. Whether the antibody responsible for the skin reaction is a separate one from the anaphylactic antibody or merely an extension of its manifestation is not clear.
Creating CTL Targets with Epitope-Linked β2-Microglobulin Constructs
Authors: Uger Robert A. , Barber Brian H.
Published: December 2022

Eliciting a strong CTL response is dependent upon displaying suitably high levels of specific class I MHC/peptide complexes at the cell surface. In an effort to enhance the presentation of defined CTL target structures, two unique peptide-linked β2-microglobulin (β2m) molecules were constructed. The first, designated NP(366–374)-L8-hβ2m, links the carboxyl terminus of the H-2Db-restricted influenza nucleoprotein (NP) epitope NP366–374 to the amino terminus of hβ2m through an eight-amino acid glycine/serine linker. The second molecule, designated NP(147–155)-L12-hβ2m, similarly couples the H-2Kd-restricted influenza NP epitope NP147–155 to hβ2m via a 12-residue polypeptide linker. Transfection of the NP(366–374)-L8-hβ2m vector into H-2b-expressing cell lines sensitized these cells for lysis by NP366–374-specific CTLs. Free NP peptide could not be detected when class I bound peptides were acid-extracted from the surface of NP(366–374)-L8-hβ2m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to hβ2m and not by a degradation by-product. CTL target structure formation was also achieved by an exogenous presentation pathway. H-2d-expressing target cells were sensitized for lysis when pulsed with NP(147–155)-L12-hβ2m protein derived from an Escherichia coli cell lysate. The effect of recombinant NP(147–155)-L12-hβ2m was inhibited by competitor wild-type hβ2m, indicating that the active peptide-hβ2m fusion protein remained intact. The observation that β2m with covalently attached peptide can effectively create CTL target structures in vitro offers new possibilities for the in vivo induction of epitope-specific CTL responses by either DNA immunization or injection of the purified epitope-linked β2m.
Amastigote Surface Proteins ofTrypanosoma cruziAre Targets for CD8+ CTL
Authors: Low Hoi Pang , Santos Maria A. M. , Wizel Benjamin , Tarleton Rick L.
Published: December 2022

Amastigotes of Trypanosoma cruzi express surface proteins that, when released into the host cell cytoplasm, are processed and presented on the surface of infected cells in the context of MHC class I molecules to be recognized by CD8+ CTL. To further understand the role of CTL in T. cruzi infection, we used the available MHC class I peptide binding motifs to identify potential CTL target epitopes in two recently described T. cruzi amastigote surface proteins, ASP-1 and ASP-2. The predicted amino acid sequences of ASP-1 and ASP-2 were screened for H-2b allele-specific class I peptide motifs, and four peptides (PA11, PA12, PA13, and PA14) and six peptides (PA5, PA6, PA7, PA8, PA9, and PA10) were synthesized from ASP-1 and ASP-2, respectively. The majority of the peptides bound to some degree to H-2b class I MHC molecules, and six of 10 of the peptides stimulated spleen cells from T. cruzi-infected mice to lyse target cells sensitized with the homologous peptides. Short term T cell lines specific for three of these peptides also lysed T. cruzi-infected target cells. These results demonstrate that ASP-1 and ASP-2 are targets of in vivo generated CTLs and that this CTL response induced by T. cruzi infection is parasite and peptide specific, MHC restricted, and CD8 dependent.
High Dose Intravenous Immunoglobulin Does Not Affect Complement-Bacteria Interactions
Authors: Wagner Eric , Platt Jeffrey L. , Frank2 Michael M.
Published: December 2022

Pooled IgG preparations for i.v. use (IVIg) have been shown to possess anticomplementary activity in autoimmune and systemic inflammatory diseases. Both in vitro and in vivo, IVIg is a preferential acceptor of activated C4 and C3, thus diverting complement activation from the target surface. We explored the effect of IVIg on complement-bacteria interactions in an attempt both to determine the safety of IVIg preparations in relation to natural immunity to bacteria and to extend our knowledge of the physiologic mechanism of action of IVIg. Using both complement-sensitive and complement-resistant bacterial strains, we investigated the effect of IVIg on C3 binding to bacterial surfaces. In all cases, whether complement could be directly activated by bacteria through the classical or the alternative pathway, IVIg had no effect on the amount of C3 bound to bacteria. In addition, IVIg did not inhibit complement-dependent bacterial lysis. Interestingly, increasing concentrations of IVIg induced an increase in C1q binding, suggesting the presence of low affinity complement-fixing antibacterial Abs in certain preparations. Using serum samples from patients treated with IVIg, complement binding to and lysis of complement-sensitive bacterial strains were not modified as compared with normal controls and pretreatment samples, although a decrease in C3 binding to sensitized human erythrocytes was observed. Our data suggest that IVIg does not affect direct complement-bacteria interactions, although it is a potent agent to use for diversion of complement activation on sensitized target surfaces.
Differential Regulation of IL-4 and IL-13 Secretion by Human Basophils: Their Relationship to Histamine Release in Mixed Leukocyte Cultures
Authors: Redrup Angela C. , Howard Brian P. , MacGlashan Donald W. , Kagey-Sobotka Anne , Lichtenstein Lawrence M. , Schroeder John T.
Published: December 2022

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.
Primary γδ Cell Clones Can Be Defined Phenotypically and Functionally as Th1/Th2 Cells and Illustrate the Association of CD4 with Th2 Differentiation
Authors: Wen Li , Barber Domingo F. , Pao William , Wong F. Susan , Owen Michael J. , Hayday Adrian
Published: December 2022

The division of CD4+ αβ T cells into Th1 and Th2 subsets has become an established and important paradigm. The respective activities of these subsets appear to have profound effects on the course of infectious and autoimmune diseases. It is believed that specific programs of differentiation induce the commitment of an uncommitted Th0 precursor cell to Th1 or Th2. A component of these programs is hypothesized to be the nature of MHC-peptide antigen presentation to the αβ T cell. It has heretofore remained uncertain whether a Th1/Th2 classification likewise defines, at the clonal level, γδ T cells. Such cells do not, as a general rule, express either CD4 or CD8αβ, and they do not commonly recognize peptide-MHC. In this report, γδ cell clones are described that conform strikingly to the Th1/Th2 classification, both by cytokine expression and by functional activities of the clones in vitro and in vivo. Provocatively, both the γδ cell clones and primary γδ cells in vivo showed a strong association of the Th2 phenotype with CD4 expression. These results are discussed with regard to the immunoregulatory role that is increasingly emerging for γδ cells.
p38 Mitogen-Activated Protein Kinase Activation Is Required for Human Neutrophil Function Triggered by TNF-α or FMLP Stimulation
Authors: Zu You-Li , Qi Jiafan , Gilchrist Annette , Fernandez Gustavo A. , Vazquez-Abad Dolores , Kreutzer Donald L. , Huang Chi-Kuang , Sha’afi Ramadan I.
Published: December 2022

Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-α triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-α-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-α, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-α- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.
The Secreted Hepatitis B Precore Antigen Can Modulate the Immune Response to the Nucleocapsid: A Mechanism for Persistence
Authors: Milich David R. , Chen Margaret K. , Hughes Janice L. , Jones Joyce E.
Published: December 2022

The hepatitis B precore Ag (HBeAg) is a secreted nonparticulate version of the viral nucleocapsid hepatitis B core Ag (HBcAg), and its function is unknown. A proportion of HBeAg-specific Th cells evade deletion/anergy in HBeAg-transgenic (Tg) mice and mediate anti-HBe “autoantibody” (autoAb) production after in vivo activation with the appropriate Th cell peptide. This model system was used to determine how secretory HBeAg may effect deletion of Th cells in the periphery. For this purpose, HBeAg-Tg mice were bred with Fas and Fas ligand (FasL)-defective lpr/lpr and gld/gld mutant mice. Fas-FasL interactions mediate activation-induced apoptosis in the periphery. In HBeAg-Tg/+ mice, high-titrated anti-HBe autoAb was produced that was exclusively composed of the IgG1 isotype (i.e., Th2-like profile). In contrast, HBeAg-Tg/lpr and HBeAg-Tg/gld mice produced significantly less anti-HBe autoAb, and the IgG isotype patterns were broadened to include IgG2a, IgG2b and IgG3 as well as IgG1 (i.e., mixed Th1/Th2-like profile). These results suggest that HBeAg-specific Th1 cells are preferentially depleted by Fas-FasL-mediated interactions. The effect of circulating HBeAg on HBcAg-specific Th1 cells was also examined by transferring HBe/HBcAg-specific Th cells into dual HBeAg- and HBcAg-expressing Tg recipient mice. The presence of serum HBeAg ablated the expected Th1-mediated anti-HBc Ab response and shifted it toward a Th2 phenotype. These results suggest that in the context of a hepatitis B viral infection, circulating HBeAg has the potential to preferentially deplete inflammatory HBeAg- and HBcAg-specific Th1 cells that are necessary for viral clearance, thereby promoting hepatitis B virus persistence.
Long-Lived Th2 Clones Specific for Seasonal and Perennial Allergens Can Be Detected in Blood and Skin by Their TCR-Hypervariable Regions
Authors: Bohle Barbara , Schwihla Herwig , Hu Huai-Zhong , Friedl-Hajek Roswitha , Sowka Slawomir , Ferreira Fátima , Breiteneder Heimo , Bruijnzeel-Koomen Carla A. F. M. , de Weger Roel A. , Mudde Geert C. , Ebner Christof , Van Reijsen Frank C.
Published: December 2022

We investigated the longevity of allergen-specific Th cells derived from patients suffering from either allergic rhinitis or atopic dermatitis. T cell clones (TCC) specific for seasonal and perennial allergens were raised. To determine whether these TCC were long-lived in vivo, PBMC and allergen-specific polyclonal T cell lines, collected and established inside a period of up to 4 years, were screened for the TCC of interest. For this purpose, a T cell tracing protocol was established in which oligonucleotides specific for the TCR β-chain hypervariable junctional region were used as tools to identify each particular TCC. Seven pollen-specific TCC and two house dust mite-specific TCC, with a Th2-like cytokine production pattern in vitro, were demonstrated to be long-lived memory T cells in vivo. Specificity of the tracing protocol was ascertained by TCR sequence analysis. We conclude that allergen-specific TCC can persist for years, evidence for which can be monitored in blood, but also in the target organ of the allergic disorder. The data indicate that in vitro-characterized, allergen-specific, long-lived TCC may well reflect a repertoire of T lymphocytes of pathogenetic importance in vivo.
Inside the Crawling T Cell: Leukocyte Function-Associated Antigen-1 Cross-Linking Is Associated with Microtubule-Directed Translocation of Protein Kinase C Isoenzymes β(I) and δ
Authors: Volkov Yuri , Long Aideen , Kelleher Dermot
Published: December 2022

T cells activated via integrin receptors can polarize and start crawling locomotion with repeated cycles of cytoskeletal reassembly processes, many of which depend on phosphorylation. We demonstrate that protein kinase C (PKC) activation represents an essential event in induction of active T cell motility. We find that in crawling T cells triggered via cross-linking of integrin LFA-1 two PKC isoenzymes, β(I) and δ, are targeted to the cytoskeleton with specific localization corresponding to the microtubule-organizing center (MTOC) and microtubules, as detected by immunocytochemistry and immunoblotting. Clustering of LFA-1 associated with its signaling function also occurs at the membrane sites adjacent to the MTOC. We further show that cells of a PKC-β-deficient clone derived from parental PKC-β-expressing T cell line can neither crawl nor develop a polarized microtubule array upon integrin cross-linking. However, their adhesion and formation of actin-based pseudopodia remain unaffected. Our data demonstrate the critical importance of the microtubule cytoskeleton in T cell locomotion and suggest a novel microtubule-directed intracellular signaling pathway mediated by integrins and involving two distinctive PKC isoforms.
Importance of B7-1-Expressing Host Antigen-Presenting Cells for the Eradication of B7-2 Transfected P815 Tumor Cells
Authors: La Motte Ross N. , Sharpe Arlene H. , Bluestone Jeffrey A. , Mokyr Margalit B.
Published: December 2022

We have previously shown that B7-2 (CD86)-transfected P815 tumor cells elicit tumor-eradicating immunity that leads to the regression of the B7-2+ P815 tumor after transient growth in normal DBA/2 mice. Here, we show that both the B7-2 and B7-1 (CD80) molecules contribute to the eradication of B7-2+ P815 tumors as treatment of the mice with both anti-B7-2 and anti-B7-1 mAb was required to prevent B7-2+ P815 tumor regression. The cells that expressed the B7-1 molecule following inoculation of B7-2+ P815 tumor cells into normal mice were not the tumor cells but rather host APCs including MAC-1+ cells present in the draining lymph nodes. Moreover, B7-1-expressing host APCs were found to be important for the rejection of B7-2+ P815 tumors as anti-B7-2 mAb alone, which was ineffective in preventing B7-2+ P815 tumor rejection by normal wild-type mice, was effective in preventing B7-2+ P815 tumor rejection by mice in which the B7-1 gene was disrupted. Finally, consistent with the importance of B7-1-expressing host APCs for the generation of tumor-eradicating immunity against B7-2+ P815 tumor cells, CD4+ T cells (not only CD8+ T cells) were found to participate in tumor-eradicating immunity against B7-2+ P815 tumor cells. Thus, in addition to eliciting tumor-eradicating immunity directly, B7-2+ P815 tumor cells elicit tumor-eradicating immunity indirectly through B7-1-expressing host APCs that present tumor-associated Ags to CD4+ T cells.
Differential Effects of Peptide Diversity and Stromal Cell Type in Positive and Negative Selection in the Thymus
Authors: Anderson Graham , Partington Katharine M. , Jenkinson Eric J.
Published: December 2022

Thymocyte positive selection results in maturation to the single-positive stage, while negative selection results in death by apoptosis. Although kinetic analyses indicate only 3–5% of CD4+8+ cells reach the single-positive stage, the balance of positive and negative selection and the nature and quantity of cells mediating maximal negative selection are uncertain. Here, using a system where the number and type of stromal cells and thymocytes can be controlled, we investigated the maturation of CD4+8+ thymocytes in the presence or absence of thymic epithelium and dendritic cells (DC) from wild-type (wt) and H-2M−/− mice expressing different peptide arrays. We find that titration of wt DC into reaggregates of wt epithelium has a dramatic effect on the number of CD4+ cells generated, with 1% DC causing a maximal 80% reduction. Moreover, while addition of 1% wt DC into cultures of H-2M−/− epithelium causes a 90% reduction in CD4+ cells, no effect was observed when similar numbers of wt thymic epithelium were added. Collectively, these data provide the first accurate indication of the quantity and quality of stromal cells required for maximal negative selection in the thymus, demonstrate the importance of peptide diversity in T cell selection, and highlight a large degree of overlap between positive and negative selection events.