bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic peptide immunogens eliciting antibodies to Plasmodium falciparum sporozoite and merozoite surface antigens in H-2b and H-2k mice.
Authors: Rzepczyk C M , Csurhes P A , Lord R , Matile H
Published: December 2022

Peptides representing conserved (MSA2/1A and MSA2/1B) and variant (MSA2/2, MSA2/6 and MSA2/7) regions of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum (FCQ-27/PNG isolate) were coupled to either peptide NP(NANP)5NA or peptide C(NANP)6 both of which contained the core sequence (NANP)n. The coupling was done via the N-terminus of one peptide and a cysteine residue on either terminus of the other. BL/10 (H-2b) and B10.BR (H-2k) mice were immunized with these MSA2-(NANP)n conjugates. The mice were also immunized with the unconjugated MSA2 peptides and with NP(NANP)5NA and C(NANP)6. Antibody responses were evaluated by 1) ELISA, in which the MSA2 peptides and C(NANP)6 were used as Ag; 2) immunofluorescence assays (IFAT) against intact sporozoites and merozoites; and 3) immunoblotting experiments against solubilized P. falciparum blood stage proteins. High titer antibodies to (NANP)n were elicited in both BL/10 and B10.BR mice after immunization with all the conjugates except MSA2/7-(NANP)n which gave only a very limited response in B10.BR mice. These antibodies recognized unfixed sporozoites. The conjugates also elicited antibodies to MSA2 as shown by ELISA, IFAT, and immunoblotting except for mice immunized with MSA2/1B-(NANP)n where an anti-MSA2 response was only detectable by immunoblotting. Immunization with unconjugated MSA2 peptides showed that MSA2/2 was immunogenic in both BL/10 and BR.10 mice, with MSA2/6 and MSA2/7 being immunogenic only in BL/10 mice. The antibodies elicited recognized both merozoites and the MSA2 protein. However, the antibody titers were lower overall than those seen when these peptides were used in the conjugated form. No anti-MSA2 antibodies were detected after immunization with MSA2/1A and MSA2/1B. Immunization of mice with the peptide NP(NANP)5NA produced antibodies in BL/10 (H-2b) mice only, and the immunogenicity of this preparation was poor. In contrast, C(NANP)6 produced a strong antibody response in both mouse strains. The antibodies elicited by NP(NANP)5NA and C(NANP)6 recognised sporozoites in IFAT. The MSA2 peptides studied (or their derivatives) were previously shown to be recognized by human T cells. Their immunogenic potential shows promise in that complex anti-P. falciparum responses can be elicited with simple synthetic immunogens based on these peptides.
The molecular configuration and ultrastructural locations of an IgG Fc binding site in human colonic epithelium.
Authors: Kobayashi K , Hamada Y , Blaser M J , Brown W R
Published: December 2022

Previously, we discovered a binding site for the Fc region of IgG in human small intestinal and colonic mucosa. The binding site (Fc gamma IBS) appeared to be primarily associated with goblet cells, to consist of greater than 200,000 Da and 78,000 Da components, and to be distinct from leukocyte FcR. In the present work, we used mAb made to colonocyte IgG-binding material to more accurately define the molecular structure and cellular locations of the Fc gamma IBS. In immunoblot and fast protein liquid chromatography analysis, the mAb revealed that the Fc gamma IBS consists of a 110,000- to 140,000-Da component in addition to the two components previously recognized. The greater than 200,000 component may be the critical component for IgG binding, inasmuch as mAb to it but not to the other two components inhibited binding of IgG to colonic sections in vitro. Used in immunoelectron microscopy, the mAb documented that the Fc gamma IBS is present in the endoplasmic reticulum of goblet cells, in the cytoplasmic matrix separating secretory granules of goblet cells, and within the granules themselves; occasionally it has the appearance of being secreted into the intestinal lumen with mucus. The Fc gamma IBS could not be solubilized from colonocyte homogenates by three different detergents, which suggests that it exists in complex with cytoskeletal elements. We speculate that the Fc gamma IBS aids in immunologic protection of the intestine by facilitating interaction between intestinal mucus and antigenic material in the lumen.
Inhibition of epidermal Langerhans cell function by low dose ultraviolet B radiation. Ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells.
Authors: Tang A , Udey M C
Published: December 2022

Immunosuppressive effects of low levels of ultraviolet B (UVB) radiation on cutaneous immune responses have been attributed to deleterious effects of UVB radiation on epidermal Langerhans cells (LC). To determine how UVB radiation modulates LC function we examined the effect of in vitro UVB exposure on LC accessory cell activity and surface phenotype. Exposure of BALB/c murine epidermal cells to low dose (less than or equal to 200 J/m2) UVB radiation in vitro inhibited their ability to support the mitogenic response of unstimulated, accessory cell-depleted splenic T cells to anti-CD3 mAb. LC accessory cell activity was also inhibited when LC were exposed to UVB radiation in situ, although several-fold higher doses of UVB radiation were required to achieve complete inhibition of LC function. This dose-dependent inhibition was mediated through a direct effect on LC that could not be reversed by IL-1 or IL-6 alone or in combination, or granulocyte-macrophage-CSF. TNF-alpha did not inhibit LC accessory cell function in this assay and anti-TNF-alpha neutralizing antibodies did not reverse the inhibitory effects of UVB radiation. UVB irradiated LC failed to participate in the anti-CD3-dependent clustering that normally occurs between T cells and LC during the proliferative response of murine T cells to anti-CD3 mAb, suggesting that UV radiation may interfere with accessory cell function by preventing intercellular adhesion. Two-color flow cytometric studies revealed low levels of the ICAM-1 on freshly isolated LC and some keratinocytes. ICAM-1 expression on LC increased 15 to 20-fold within the first 24 h in vitro and continued to increase during a 72-h culture period. The integrin LFA-1 was not identified on freshly isolated or cultured LC but was detected on responding T cells. Prior exposure of LC to UVB radiation (50 or 100 J/m2) inhibited the increase in ICAM-1 expression that normally occurs in vitro by up to 70% whereas surface levels of class II MHC Ag, CD45 and Fc-gamma receptors were not affected. Blocking studies revealed that anti-CD3 induced T cell proliferation and T cell-LC cluster formation was inhibited by both anti-LFA-1 and anti-ICAM-1 mAb suggesting that ICAM-1 expressed on LC must bind to LFA-1 on T cells to facilitate proliferative responses of T cells to anti-CD3 mAb. We conclude that the in vitro inhibitory effects of low dose UVB radiation on LC accessory function may result because UVB radiation prevents upregulation of ICAM-1 expression by LC in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytokines in chronic inflammatory arthritis. VI. Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha.
Authors: Alvaro-Gracia J M , Zvaifler N J , Brown C B , Kaushansky K , Firestein G S
Published: December 2022

Granulocyte-macrophage CSF (GM-CSF) is a potent stimulator of macrophages and neutrophils and is produced by rheumatoid arthritis (RA) synovium. We now report studies that identify some of the synovial cells and cytokines responsible for local GM-CSF production and gene expression in RA. GM-CSF was assayed by ELISA in supernatants from cultured RA fibroblast-like synoviocytes stimulated with various cytokines (IL-1 beta, TNF-alpha, macrophage-CSF, IFN-gamma, IL-6, and TGF-beta). Immunoreactive GM-CSF was detected in IL-1 beta and TNF-alpha-stimulated cultures, but not in cells cultured in medium or stimulated with any of the other cytokines. IL-1 and TNF-alpha had a synergistic effect on GM-CSF production. GM-CSF gene expression by fibroblast-like synoviocytes was analyzed by ribonuclease protection assay, Northern blot analysis, and in situ hybridization. Both IL-1 beta and TNF-alpha induced GM-CSF mRNA accumulation, with a maximum effect after 4 h of stimulation. We then studied GM-CSF production by macrophage-like synoviocytes (MLS) isolated from fresh synovial specimens by flow microfluorimetry. Fresh MLS spontaneously secreted the cytokine and exogenous IL-1 beta or TNF-alpha had no effect. After 1 wk in culture, additional stimulation with IL-1 beta or TNF-alpha was required for GM-CSF production. Finally, in situ hybridization performed on freshly isolated subpopulations of synovial cells, identified GM-CSF RNA transcripts in MLS.
Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.
Authors: Rao N , Ferguson D J , Lee S F , Telen M J
Published: December 2022

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.
Tolerance susceptibility of newly generating memory B cells.
Authors: Linton P J , Rudie A , Klinman N R
Published: December 2022

Newly generating memory B cells rapidly accumulate somatic mutations that can alter their Ag-combining sites and potentially engender recognition of self determinants. To investigate the possibility that, during their emergence secondary B cells pass through a window of tolerance susceptibility, we have examined the in vitro generation of memory B cells in the presence or absence of tolerogen. The findings indicate that, before antigenic stimulation, precursors to memory B cells are resistant to tolerance induction. However, 2 to 7 days after T cell-dependent antigenic stimulation, newly emerging hapten-specific secondary B cells can be inactivated by the presence of hapten on a carrier not recognized by available Th cells. This inactivation can be blocked by the presence of free hapten and can be competed by the presence of immunogen. Inactivation of newly generating secondary B cells appears less specific than the tolerance induction of immature neonatal or bone marrow B cells because inactivation can be accomplished by cross-reactive determinants. Interestingly, the presence of tolerogen after primary stimulation did not preclude the generation of cells responsive to a third in vitro stimulation. Therefore, whereas newly emerging memory B cells are highly susceptible to inactivation, the progression of the clones of progenitors to memory B cells appears resistant to tolerance induction.
The influence of avidity on signaling murine B lymphocytes with monoclonal anti-IgM antibodies. Effects of B cell proliferation versus growth inhibition (tolerance) of an immature B cell lymphoma.
Authors: Udhayakumar V , Kumar L , Subbarao B
Published: December 2022

We have investigated the effect of the avidity of a monoclonal anti-mouse IgM antibody (mAb) on its ability to activate normal B cells vs its capacity to inhibit the growth of an immature B lymphoma, BKS-2. A panel of seven mAb with specificities for four different domains of mu-heavy chain were selected and their relative avidities measured. Of the seven mAb studied, four antibodies (b7-6, Ak11, AK15, and DS1) were found to have relatively higher avidities than three others (AK17, 331.12, and Bet-2). Among them, the mAb b7-6 was highly efficient in inducing proliferation of normal B cells, whereas all others (including the remaining three high avidity mAb) were inefficient in inducing B cell proliferation. This failure cannot be simply related to the differences in their fine specificity because all the mAb became highly stimulatory after coupling to Sepharose beads. With one exception (DS1), avidity was a better predictor of the growth-stimulatory capacity when the antibodies were immobilized. Taken together, these findings suggested that although high affinity was important, there were other factors that might influence the mitogenic potential of a soluble anti-mu mAb. On the other hand, all anti-mu mAb irrespective of their avidity, fine specificity, and isotype were capable of effectively inhibiting the growth of BKS-2 cells. Furthermore, there was a direct correlation between the avidity and the dose of the mAb required to cause half-maximal inhibition of BKS-2 growth. The F(ab) fragments of high avidity mAb b7-6 failed to inhibit the growth of BKS-2 cells, which indicates that a minimal level of cross-linking of the membrane Ig receptor is necessary for causing growth inhibition in BKS-2 cells. Although there was no significant difference in the tolerogenic potential of soluble vs immobilized anti-mu mAb in directly inactivating BKS-2 cells, their growth-inhibitory capacity was remarkably different in the presence of rIL-5. Thus, rIL-5 was effective in partially overcoming the tolerogenic effect of soluble but not immobilized anti-mu mAb. Unlike IL-5, LPS rescued BKS-2 cells from the inhibitory effect of both soluble and immobilized anti-mu antibodies. Altogether, these results suggested that the ligand binding requisites for tolerance induction were less stringent than those for B cell activation. Furthermore, a high affinity interaction between membrane Ig receptor and its ligand appeared to generate a dominant negative signal that was not reversed by lymphokines.
Genetic analysis of diabetes in the nonobese diabetic mouse. I. MHC and T cell receptor beta gene expression.
Authors: Livingstone A , Edwards C T , Shizuru J A , Fathman C G
Published: December 2022

Backcross nonobese diabetic (NOD) ((NOD x SWr)F1 x NOD) mice (108 females and 105 males) were typed for MHC, TCR V beta, and monitored for 350 days for the onset of diabetes. The presence of "antipolar" antibodies in the sera and the occurrence of insulitis was examined in a proportion of these backcross mice. There was no difference in the incidence of diabetes in mice heterozygous for TCR V beta b/a vs those homozygous for TCR V beta b/b. Among the 17 diabetics (all female) detected in this backcross, 14/17 were H-2nod/nod but 3/17 were H-2nod/q. This supports a previous observation suggesting that the MHC-linked diabetogenic gene originally thought to be recessive may rather be dominant but have a low penetrance in the heterozygous state. Antipolar autoantibodies were found in both female and male backcross mice, and were similarly distributed in diabetic and nondiabetic mice. There appeared to be no correlation between the level of these auto-antibodies and development of diabetes. The incidence and severity of insulitis was linked to MHC but no influence of TCR genes on insulitis nor an association between insulitis and antipolar antibodies could be demonstrated in this study. Further analyses of H-2nod/nod intercross mice homozygous for TCR V beta a or TCR V beta b are currently underway.
Expression of src family genes during monocytic differentiation of HL-60 cells.
Authors: Katagiri K , Katagiri T , Koyama Y , Morikawa M , Yamamoto T , Yoshida T
Published: December 2022

It has been reported that src family protein-tyrosine kinases were expressed specifically in a certain lineage or differentiation stage of hematopoietic cells. To understand the molecular basis for differentiation and function of monocyte/macrophage, we investigated the expressions of src family genes by the HL-60 cells stimulated with differentiation-inducing agents. TPA and vitamin D3 (D3) were used as stimulants for monocytic development, since each agent has been known to induce phenotypically specific differentiation of HL-60 cells. The fyn, fgr, and lyn genes were characteristically expressed concomitantly with phenotypic changes and expressions of nuclear proto-oncogenes, whereas src, lck, hck, and yes genes were not. In TPA-induced differentiation of HL-60 cells, both fyn and lyn genes, but not fgr gene, were expressed. In contrast, both fgr and lyn genes, but not fyn gene, were expressed in D3-induced differentiation of the cells. The independent and characteristic expressions of these genes were observed in the further advanced differentiation of HL-60 cells induced by TPA plus D3 or D3 plus human transforming growth factor-beta 1. The granulocytic differentiation of the cells treated with retinoic acid was accompanied by intense expression of fgr, but weak or no expression of lyn and fyn gene. These data indicate that each protein-tyrosine kinase encoded by src family genes may play distinct roles in development and/or functions of monocyte/macrophage-lineage cells.
The complete exon-intron structure of a human complement component C4A gene. DNA sequences, polymorphism, and linkage to the 21-hydroxylase gene.
Authors: Yu C Y
Published: December 2022

The human complement component C4A and C4B genes are located within the class III region of the MHC. The polymorphic C4 genes are highly complex including variations in class (isotype), size, and number of genes. The DNA sequence for a C4A gene has been determined, except for a large intron of 6 to 7 kb long. The C4A gene consists of 41 exons encoding a transcript for a precursor protein of 1744 amino acid residues. Several structural and functional aspects of C4 have been located to individual exons. The active site of the anaphylatoxin C4a matches to a splice junction. Some unique properties of C4, such as, the alpha-gamma-chain junction, the tyrosine sulfation sites, and the post-secretory metalloprotease cleavage site, are encoded by a single exon. Comparison of human C4 with published data for mouse C4, human C3 and rat alpha 2 macroglobulin genes revealed that these evolutionary-related genes share very similar exon-intron structures. Altogether 20 polymorphic sites in human C4 have been detected by various techniques. Presumably, these polymorphic residues account for the functional, structural, and serologic variations observed among the various allotypes. A PvuII restriction length polymorphism has been detected within the region of DNA coding for C4a. The intergenic region between C4 and the neighboring 21-hydroxylase gene, CYP21, is approximately 3028 bp in size.
Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II.
Authors: Salmon J E , Brogle N L , Edberg J C , Kimberly R P
Published: December 2022

Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions.
The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.
Authors: Smith A M , Benjamin D C
Published: December 2022

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.
Direct identification of the putative surface IgM receptor-associated molecule encoded by murine B cell-specific mb-1 gene.
Authors: Matsuo T , Kimoto M , Sakaguchi N
Published: December 2022

The B cell-specific mb-1 gene was recently reported to encode a putative surface glycoprotein with CD3-like structural properties. Hombach et al. suggested and presented evidence to show that this mb-1 gene encodes the 34-kDa membrane glycoprotein (B34 or IgM-alpha) associated with IgMR molecule. To identify the mb-1 gene product directly in B cells, affinity-purified MB-1-specific antibody was prepared by immunization of rabbits with synthetic MB-1 oligopeptide. Immunoprecipitation in combination with two-dimensional diagonal gel electrophoresis analysis revealed that this antibody detected a B cell-specific surface glycoprotein that is very similar to the IgM-alpha (B34) protein described by Hombach et al. However, MB-1 protein exists usually as the monomeric form on the surface of B cells, in contrast to IgM-alpha, which was detected as the dimeric (IgM-alpha/IgM-alpha or IgM-alpha/Ig-beta) protein. We also found that MB-1 protein is already expressed on the sIgM- pre-B cell lymphoma, which might suggest an alternative functional role of this B cell-specific MB-1 protein in B cell differentiation. The molecular identity of MB-1 protein and IgM-alpha (B34) is discussed.
Major component of Ra-reactive factor, a complement-activating bactericidal protein, in mouse serum.
Authors: Ihara S , Takahashi A , Hatsuse H , Sumitomo K , Doi K , Kawakami M
Published: December 2022

A complement-activating bactericidal protein, Ra-reactive factor was isolated from mouse serum by an affinity method. The m.w. of the isolated RaRF estimated by glycerol density gradient sedimentation (around 300,000) was the same as that of the active material in mouse serum. As evidenced by gel filtration, the intact RaRF was decomposed into high (higher than 200,000) and low (50,000 to 200,000) m.w. components by treatment with 10% acetonitrile. SDS- and acid/urea-PAGE demonstrated that the high m.w. component was completely dissociated into equimolar quantities of two kinds of 28 kDa polypeptides, P28a and P28b, under reducing conditions, indicating that the association of these polypeptides was stabilized by disulfide bonds. The ability to bind specifically to the Ra determinant was retained in the high m.w. component, although the complement-activating potency was lost. The amino acid compositions of P28a and P28b polypeptides were compared with those of related serum proteins. The P28a and P28b polypeptides were found to have the highest homology to rat mannan-binding protein and mouse and human C1q subcomponent of complement.
Characterization of the human complement receptor 2 (CR2, CD21) promoter reveals sequences shared with regulatory regions of other developmentally restricted B cell proteins.
Authors: Rayhel E J , Dehoff M H , Holers V M
Published: December 2022

Expression of human complement receptor 2 (CR2, CD21, C3d,g/EBV receptor) is developmentally restricted on human B lymphocytes to cells of the late-pre and mature stages. CR2 is also a member of the genetically linked regulators of complement activation family found on human chromosome 1q32. Regulators of complement activation proteins are variably expressed in plasma, on cell membranes, and in nonvascular extracellular fluid sites. To begin to understand the mechanisms that control both tissue specific and B cell developmental restriction of CR2 expression, we have cloned and characterized the CR2 promoter upstream of a single apparent transcriptional initiation site. Within this region are sequences with significant similarity to previously characterized TATA, SP1, AP-2, AP-1-like, and Ig enhancer E motif DNA protein binding sites, in addition to direct and inverted repeats. Significant regions of identity are also found between CR2 promoter sequences and those of the CD23 promoter, another protein whose expression is developmentally restricted on B cells. The CR2 promoter will direct transcription of the reporter gene chloramphenicol acyltransferase when transiently transfected into the human Raji B cell line. Therefore, we have identified the promoter for a human B cell protein whose expression is developmentally restricted. Further analysis of this region and the transcriptional regulation of CR2 gene expression should lead to significant insights into the molecular mechanisms by which B cells mature and are activated.
Kinetics of T cell activation in acute and chronic forms of murine graft-versus-host disease.
Authors: Via C S
Published: December 2022

Induction of graft-vs-host disease (GVHD) in the parent-into-F1 model is dependent on the presence of T cells in the donor inoculum. Although in vivo activation of donor T cells in response to F1 alloantigens is thought to be critical to GVHD induction, direct evidence of activated donor T cells has been lacking in this model. In the present study, spleen cells from acute or chronic GVHD mice were studied for evidence of T cell activation at multiple intervals early after GVHD induction. Spleen cells from both acute and chronic GVHD mice exhibited striking elevations in spontaneous proliferation and IL-2 production, which were maximal 24 to 48 h after GVHD induction. Persistent lower levels of spontaneous in vitro activity were observed for spleen cells from mice tested 7 to 9 days after GVHD induction. In both forms of GVHD, increased spontaneous proliferation and IL-2 production were dependent on the presence of donor CD4+ T cells. These results strongly support the presence of activated donor T cells in vivo. Furthermore, these data imply that despite the significant differences in outcome, acute and chronic GVHD share a common early event.
Studies on the induction of anterior chamber-associated immune deviation (ACAID). 1. Evidence that an antigen-specific, ACAID-inducing, cell-associated signal exists in the peripheral blood.
Authors: Wilbanks G A , Streilein J W
Published: December 2022

The anterior chamber of the eye is an immunologically privileged site. Recent evidence indicates that this privilege is an actively acquired immune state in which a unique form of systemic immune deviation exists, anterior chamber-associated immune deviation (ACAID). ACAID is characterized in part by the generation of Ag-specific splenic T lymphocytes that mediate suppression of induction and expression of delayed hypersensitivity and that suppress production of C-fixing antibodies. Delayed hypersensitivity and C fixation are usually associated with extensive nonspecific inflammation and innocent bystander tissue injury. It is, therefore, believed that ACAID represents physiologic adaptations of the immune system that mitigate wanton destruction of the anatomically delicate visual axis, thereby preserving sight, while at the same time providing selective immune protection. As a means of examining the potential contribution of the eye to ACAID induction, we have studied the immune properties of blood harvested from mice that had received BSA into the anterior chamber 48 h earlier. We found that an Ag-specific "signal" is present in the blood of these mice; when blood was transfused into naive syngeneic mice, regulatory cell populations were induced and the recipients were unable to display BSA-specific delayed hypersensitivity. Further analysis of this signal revealed it to be associated with the leukocyte fraction, but not with either the plasma or RBC components of whole blood. Among leukocytes, the suppression-inducing activity correlated positively with cells bearing the mature macrophage marker F4/80 and negatively with cells bearing Thy-1, surface Ig, and class II MHC molecules. These findings are discussed with regard to the potential intraocular sources of the ACAID-inducing signal and the possible mode of action of this factor.
Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1.
Authors: Zhang L , English D , Andersen B R
Published: December 2022

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.
IFN-gamma reverses IL-4 inhibition of fetal thymus growth in organ culture.
Authors: Plum J , De Smedt M , Billiau A , Heremans H , Leclercq G , Tison B
Published: December 2022

IL-4 is known to inhibit the growth and differentiation of 14-day-old fetal mouse thymus in organ culture. Here we report that IFN-gamma reverses this IL-4-mediated growth inhibition. Thymus lobes from 14-day-old fetuses were cultured for 12 days in medium containing 100 IU/ml rIL-4 either in the absence or presence of rIFN-gamma (100 to 1000 IU/ml). After culture, the cell yields and the absolute numbers and frequencies of the major subpopulations according the coordinate expression of CD4 and CD8 were estimated. IL-4 treatment alone was found to result in a seven-fold decrease in cell yield and an almost complete absence of the CD4+CD8+ subpopulation. Addition of IFN-gamma reversed IL-4-mediated inhibition in a dose-dependent fashion, with an optimal dose ranging from 200 to 500 IU/ml. IFN-gamma exerted this effect only when added within the first 48 h of initiating the culture. The specificity of the reversal effect was ascertained by neutralization of the effect by a neutralizing anti-IFN-gamma mAb and by lack of activity of human IFN-gamma. In the absence of IL-4, IFN-gamma had a growth-promoting effect as evident from a threefold increase in cell numbers.
Nonrandom selection of T cell specificities in anti-HLA-DR responses. Sequence motifs of the responder HLA-DR allele influence T cell recruitment.
Authors: Weyand C M , Hicok K C , Goronzy J J
Published: December 2022

The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual's T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.
Characterization of the anti-inflammatory effect of FK-506 on human mast cells.
Authors: de Paulis A , Cirillo R , Ciccarelli A , de Crescenzo G , Oriente A , Marone G
Published: December 2022

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).
Identification and characterization of a new gene family induced during macrophage activation.
Authors: Wynn T A , Nicolet C M , Paulnock D M
Published: December 2022

In this report, we describe the primary structure and regulation of two novel IFN-gamma-inducible genes expressed during the process of macrophage activation. We used the RAW 264.7 cell line to prepare a cDNA library, from which inducible genes were selected by differential hybridization. Two cDNA clones, mag-1 and mag-2 (for macrophage-activation gene-1 and -2), were induced by IFN-gamma treatment in both RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages, but not in the noncytolytic cell line, WEHI-3. A comparison of the nucleotide and deduced amino acid sequences of clones mag-1 and mag-2 with sequences in available data bases revealed no homologs. However, comparison of mag-1 and mag-2 sequences with each other revealed that these genes are homologous, with conserved residues concentrated at the amino terminus. Kinetic analyses revealed similar temporal patterns of induction of mRNA expression for these genes after IFN-gamma treatment. In addition, the genes showed distinct response patterns to the macrophage-activating stimuli IFN-gamma and LPS used either alone or in combination. Analysis of a panel of cell types of various lineages demonstrated that expression of these genes was associated with cellular activation in multiple cell types. As a result of the sequence similarities between these genes, we propose that they define a new family of IFN-gamma-regulated genes in macrophages.
Hydrocortisone inhibits rat basophilic leukemia cell mediator release induced by neutrophil-derived histamine releasing activity as well as by anti-IgE.
Authors: White M V , Igarashi Y , Lundgren J D , Shelhamer J , Kaliner M
Published: December 2022

We determined the ability of hydrocortisone to inhibit rat basophilic leukemia cell mediator release induced by anti-IgE and by neutrophil-derived histamine-releasing activity (HRA-N). Serotonin release induced by HRA-N and anti-IgE was inhibited by 78 +/- 5 and 70 +/- 4%, respectively (IC50 7.5 x 10(-7)M) by hydrocortisone (10(-5)M). HRA-N does not cause arachidonic acid metabolism, however, anti-IgE induced the generation of PGD2 and leukotriene (LT)C4, and the generation of both mediators was inhibited by 10(-5)M hydrocortisone (IC50 = 4.8 x 10(-7)M, and 3.6 x 10(-9)M, respectively). Inhibition required at least 5 to 6 h of hydrocortisone exposure and was maximal after 22 h. The observed effects of hydrocortisone could be reproduced by human recombinant lipocortin-I (5 x 10(-7)M). Hydrocortisone, 10(-5)M, was a less potent inhibitor of calcium ionophore A23187-mediated serotonin release and PGD2 and LTC4 generation (inhibition of 20 +/- 2, 17 +/- 10, and 37 +/- 10%, respectively). Inasmuch as A23187-induced stimulation is not dependent on receptor coupling, the enhanced ability of hydrocortisone to inhibit IgE- and HRA-N-mediated events as compared with A23187 suggests that one possible site of action of hydrocortisone may be interruption of receptor-effector signals. In the presence of arachidonic acid, hydrocortisone-treated cells released as much LTB4 and PGD2 as control cells, however, serotonin release and LTC4 generation were inhibited 50 and 55%, respectively. Thus, these data suggest that hydrocortisone has three possible sites of action: 1) inhibition of phospholipase A2 activity, 2) inhibition of glutathione-s-transferase, and 3) inhibition of serotonin release by a third mechanism, possibly by interrupting the coupling of receptor and effector systems.
Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.
Authors: Chang J F , Thomas C A , Kung J T
Published: December 2022

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.
A covalent dimer of complement C4b serves as a subunit of a novel C5 convertase that involves no C3 derivatives.
Authors: Masaki T , Matsumoto M , Yasuda R , Levine R P , Kitamura H , Seya T
Published: December 2022

A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis.
Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.
Authors: Kuijpers T W , Hakkert B C , Hoogerwerf M , Leeuwenberg J F , Roos D
Published: December 2022

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.