bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies.
Authors: Parhami-Seren B , Margolies M N
Published: December 2022

We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (< or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.
Human monocytes induce a carcinoma cell line to secrete high amounts of nitric oxide.
Authors: Konur A , Krause S W , Rehli M , Kreutz M , Andreesen R
Published: December 2022

Nitric oxide (NO) is a short-lived pleiotropic mediator with a multitude of biologic functions. The inducible form of NO synthase (iNOS) is responsible for the discontinuous production of high amounts of NO and is important for the cytotoxic capacity of macrophages in rodents, whereas NO production by human macrophages or monocytes (MO) is under debate. Here we report that high amounts of NO are synthesized in cocultures of human MO with the human carcinoma cell line RT4 without further stimulation. Both cell types have to be viable and metabolically active for NO production. However, in contrast to reports by others, we could demonstrate that tumor cells and not MO are the producers of NO by the following findings: 1) NO release was induced in RT4 cells, but not in MO, by diluted supernatants (SN) of RT4/MO cocultures; 2) SN of MO stimulated with tumor cell membrane preparations were sufficient to induce NO release by tumor cells; and 3) NOS mRNA expression could be detected only in tumor cells, not in MO. Separating both cells by a cell-impermeable membrane resulted in NO amounts comparable to those in cocultures with direct cell contact, indicating one or more soluble NO-inducing factors. Considerable amounts of IL-1 beta and TNF-alpha were present in cocultures. IL-1 beta and TNF-alpha, mediators produced by activated MO, in combination induce NO release in RT4 cells. Blocking of TNF-alpha or IL-1 in SN inhibited NO release in RT4 cells. This indicates that IL-1 beta and TNF-alpha play prominent roles in iNOS induction by MO in RT4 tumor cells.
Rat peritoneal mast cells produce IFN-gamma following IL-12 treatment but not in response to IgE-mediated activation.
Authors: Gupta A A , Leal-Berumen I , Croitoru K , Marshall J S
Published: December 2022

Mast cells have been reported to secrete a wide range of immunoregulatory cytokines following IgE-mediated activation and to play an important role in allergic inflammation. We have previously demonstrated that mast cells can also produce certain cytokines following activation with bacterial LPS or prostanoids without preformed mediator release. IL-12 is a potent inducer of IFN-gamma production by T cells and NK cells, and is thought to play a critical role in determining the nature of the local immune response to infection. We here report that highly purified peritoneal mast cells from Brown Norway rats will produce IFN-gamma in response to IL-12 without significant histamine release. IFN-gamma protein was detected by ELISA in supernatants of mast cells cultured with 2 U/ml recombinant mouse IL-12 for between 6 and 24 h. The production of IFN-gamma was dependent on the dose of IL-12 and was significantly inhibited by concurrent treatment with IL-10 or PGE2. Supernatants from IL-12-stimulated mast cells induced MHC class II expression on the mouse epithelial cell line, MODE-K, by an IFN-gamma-dependent mechanism. Peritoneal mast cells cultured following activation with anti-IgE or LPS, under conditions that will induce the production of IL-6, demonstrated no detectable protein production of IFN-gamma. We conclude that mast cells are capable of contributing to the IFN-gamma response to IL-12, but substantial mast cell IFN-gamma production does not occur as a result of IgE-mediated activation. These observations have important implications for the role of the mast cell in local immune regulation.
Endogenous nitric oxide activates prostaglandin F2 alpha production in human microglial cells but not in astrocytes: a study of interactions between eicosanoids, nitric oxide, and superoxide anion (O2-) regulatory pathways.
Authors: Janabi N , Chabrier S , Tardieu M
Published: December 2022

Secretion of the eicosanoids, nitric oxide (NO.) and superoxide anion (O2.-) was evaluated in human embryonic astrocytes and microglia. An inducible form of cyclo-oxygenase (COX 2) was demonstrated in astrocytes and microglia after IL-1 beta plus IFN-gamma stimulation; since 1) large amounts of PGF2 alpha were released; 2) PGF2 alpha secretion required protein synthesis and was blocked by indomethacin; and 3) the response was delayed and persistent. Using the same inducers, astrocytes, but not microglial cells, produced NO. and had an inducible form of nitric oxide synthase. Conversely, microglial cells were induced by IL-1 beta and IFN-gamma to generate superoxide anions (O2.-) through an NADPH oxidase-dependent pathway. We then investigated interactions between these different pathways of synthesis by inhibition experiments. The cytokine-induced production of PGF2 alpha in astrocytes was not affected by exposure to N-omega-monomethyl-L-arginine, which inhibits NO. production, whereas it was reduced by 40% in microglia. Since microglia did not secrete any detectable NO. in their supernatant, intracellular production of NO. could occur in these cells that positively regulated PGF2 alpha production. Exposure to indomethacin, which prevented PGF2 alpha production in both astrocytes and microglia, resulted in a 64% increase in cytokine-induced NO. production by astrocytes and a 70% inhibition of O2.- generation by stimulated microglia. Finally, superoxide dismutase depletion of O2.- in astrocytes and microglia had no effect on PGF2 alpha production in these cells. These results demonstrate that there are important interactions between the pathways of synthesis of inflammatory mediators in glial cells that could unveil additional regulatory mechanisms.
HIV-1 gp120-specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression.
Authors: Baum L L , Cassutt K J , Knigge K , Khattri R , Margolick J , Rinaldo C , Kleeberger C A , Nishanian P , Henrard D R , Phair J
Published: December 2022

The Ab-dependent cell-mediated cytotoxicity (ADCC) activity of anti-gp120 Abs in serum from four groups of HIV-1-positive individuals in the Multicenter AIDS Cohort Study was evaluated at several time points over a 10-yr period. HIV-1-positive individuals who progressed to AIDS within 3 yr of seroconversion (rapid progressors) were compared with seroconverters who did not progress to AIDS within 6 yr (nonrapid progressors) and individuals who were seropositive when they entered the study and did not progress to AIDS within 9-10 yr (nonprogressors). At the visit closest to AIDS, rapid progressors had significantly lower titers of Abs that mediate ADCC against HIV-1 gp120 than those of nonrapid progressors at corresponding visits or those of nonprogressors at any visit. Nonprogressors generally had high titers of ADCC Abs at all visits. Differences between ADCC titers of rapid progressors and nonrapid progressors or nonprogressors remained when longitudinal changes within individuals were compared. Among seroconverters who were nonrapid progressors, those with low or declining ADCC titers lost significantly more CD4+ cells during the study than those whose ADCC titers were stable or increasing, even though both groups had similar serum virion RNA levels. This demonstrates that high titers of Abs that mediate ADCC correlate with a successful host defense against AIDS.
Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance.
Authors: Elson C O , Beagley K W , Sharmanov A T , Fujihashi K , Kiyono H , Tennyson G S , Cong Y , Black C A , Ridwan B W , McGhee J R
Published: December 2022

We report here a murine model for experimental chronic colitis where administration of trinitrobenzene sulfonic acid (TNBS) in 50% ethanol induced inflammation of large intestine in susceptible (C3H/HeJ and BALB/c) but not resistant (C57BL/6 and DBA/2) mouse strains. We queried whether mucosal trinitrophenyl (TNP)-specific B cell responses were induced in mice with TNBS-induced colitis, and if induction of tolerance to TNBS by oral administration of this hapten protected mice from development of colitis. Isotypes and subclasses of polyclonal and TNP-specific Ab-forming cells (AFC) were assessed in mucosal and peripheral lymphoid tissues of C3H/HeJ mice with TNBS-induced colitis. Increased numbers of IgA- and IgG-secreting cells were found in the inflamed colon lamina propria. Inflamed colonic tissue also contained high frequencies of IgG anti-TNP AFC (predominantly of IgG1, IgG2a, and IgG2b subclasses); however, anti-TNP responses in noninflamed mucosal tissues of mice with colitis exhibited dominant IgA and IgM with low IgG anti-TNP responses. CD4+ T cells stimulated with TNP-splenocytes produced more IFN-gamma and less IL-4, suggesting a Th1-type response. Oral administration of TNBS before induction of colitis markedly decreased mucosal anti-TNP responses and completely inhibited anti-TNP IgG2a and IgG2b responses. Control mice did not show inhibition of anti-TNP AFC responses or TNBS-induced colitis. Intracolonic sensitization of susceptible C3H/HeJ mice with TNBS induces a localized IgG anti-TNP B cell response in the inflamed tissue, whereas prior oral administration of TNBS results in unresponsiveness to this agent and protects mice from development of TNBS-induced colitis.
Autoantigenic epitopes on hY5 Ro RNA are distinct from regions bound by the 60-kDa Ro and La proteins.
Authors: Granger D , Tremblay A , Boulanger C , Chabot B , Ménard H A , Boire G
Published: December 2022

We recently reported the identification in human anti-Ro serum of Abs specifically immunoprecipitating deproteinized hY5 RNA. In the present report, we characterized the epitopes recognized by anti-hY5 RNA Abs. Using deletion and site-directed mutagenesis of hY5 cDNA and in vitro transcribed RNAs with intact and 3'-shortened ends, we have defined two conformational antigenic determinants distinct from the regions known to bind Ro and La proteins. One of these epitopes (epitope A) is present in the middle portion of hY5 RNA and is dependent on the presence of a four-nucleotide sequence (AACC at position 58-61) that may form a single-stranded loop. Deleting these four nucleotides or modifying the stem structures proximal or distal to this loop abolishes recognition of the mutated RNAs by Abs. The second epitope (epitope B) requires the presence of another four-nucleotide sequence (CUUG at position 74-77) in between the Ro and La binding sites. Deleting this CUUG sequence or modifying nucleotides on the 5' side of the stem structure below the Ro60 binding site severely compromises the interaction with Abs. Since Abs to deproteinized hY RNAs are restricted to anti-hY5 RNA and target determinants not involved in interactions with known hY5 RNA-binding proteins, human RohY5 particles may play a direct role in the immunization process, leading to the production of anti-hY5 RNA autoantibodies.
A model for induction of T cell-independent humoral immunity in response to polysaccharide antigens.
Authors: Snapper C M , Mond J J
Published: December 2022

We provide a model for induction of T cell-independent, polysaccharide-specific Ig secretion in response to bacterial challenge. Two predominant pathways are defined that require the concerted action of multivalent membrane Ig cross-linking by the polysaccharide Ag with 1) various B cell-activating moieties contained within the bacterial pathogen and/or 2) cytokines, such as IFN-gamma and granulocyte-macrophage colony-stimulating factor produced by NK cells and macrophages, that become activated in a T cell-independent manner during bacterial infection.
Regulation of CTL recognition by receptors for HLA-DR molecules.
Authors: Nakajima H , Takiguchi M
Published: December 2022

NK cells and some T cells have receptors for MHC class I molecules that negatively regulate the recognition of these cells. In the present study, we show alpha beta CTL clones carrying the receptors for HLA-DR molecules that inhibit this cytotoxicity. The cytotoxicity of two CTL clones possessing NK activity was inhibited by the surface expression of HLA-DR and HLA class I molecules on the target cells. The inhibitory effect of DR molecules on the cytotoxicity of the CTL clones was confirmed by using T2 transfectants expressing HLA-DR3. These results suggest that receptors for both HLA-DR and HLA class I molecules negatively regulate the recognition of T cells.
Effect of HLA-DM transfection on hen egg lysozyme presentation by T2.Ak cells.
Authors: Albert L J , Ghumman B , Watts T H
Published: December 2022

T2 cells have a large homozygous deletion in the MHC II region. Transfection of MHC class II genes into T2 cells allows presentation of peptide but not native protein Ags. This defect in protein presentation has been attributed to the lack of HLA-DM, an MHC class II-related protein that facilitates the release of an invariant chain peptide (CLIP) intermediate from nascent MHC class II proteins within the endocytic compartment of APC. Here, we show that Ak molecules within isolated late endosome fractions of T1.Ak (wild-type) vs T2.Ak (HLA-DM-deficient) bind biotin-HEL46-61 at comparable levels, consistent with previous observations that Ak molecules on T2 cells are not predominantly occupied with CLIP. However, Ak molecules in the late endosomes of T2.Ak fail to present peptide to a T hybrid, whereas the late endosomes from T1.Ak have no such defect. Transfection of HLA-DM A and B into T2.Ak partially restores protein Ag presentation by T2.Ak cells. These data suggest that HLA-DM can play a role in Ag presentation in addition to its role in CLIP release. However, even after DM transfection there remains a 10-fold difference in the dose-response curve for hen egg lysozyme presentation by T1.Ak vs T2.Ak/DM cells. In addition, HLA-DM transfection fails to restore presentation by late endosome fractions. The failure to fully restore Ag presentation in T2.Ak cells by DM transfection suggests that another gene product, required for efficient Ag presentation, may be absent from the late endosomes of T2.
TCR recognition of the Hb(64-76)/I-Ek determinant: single conservative amino acid changes in the complementarity-determining region 3 dramatically alter antigen fine specificity.
Authors: Hsu B L , Donermeyer D L , Allen P M
Published: December 2022

To identify the structural basis of Ag fine specificity, TCR sequences from a panel of Hb(64-76)/I-Ek-specific T cells were compared and found to be restricted in variable (V) gene usage, predominantly using BV1 or BV15 and AV4 or AV10 genes. TCRA and TCRB junctional sequences were extremely diverse. No conservation of length or position was found, which distinguishes this response from others, but correlates with the range of fine specificities that these T cells display. A remarkable subtlety in the recognition of Hb(64-76) was revealed from the study of the response to the D73 variant of Hb(64-76), which contains a conservative change in an MHC anchor residue not affecting the binding affinity to I-Ek. To one group of T cells this determinant was non-cross-reacting with Hb(64-76), whereas another recognized both Ags. Interesting, they all used a different constellation of TCRBV genes than that found in Hb(64-76) recognition. To limit the variability in the anti-Hb(64-76) TCR repertoire, transgenic mice expressing a fixed TCRB rearrangement from a Hb(64-76)-specific T cell were used. In Hb(64-76)-specific TCR from these mice, the endogenous alpha-chains pairing with the transgenic beta-chain were highly restricted in their AV gene usage. A comparison of two pairs of closely related T cells of these endogenous TCR variants, one differing by a single, conservative substitution in the complementarity-determining region 3 and the other containing a positional switch of two amino acids, revealed dramatically different fine specificities. Overall, these findings highlight the exquisite sensitivity of the TCR- peptide/MHC interaction to subtle alterations in any of the components.
Species-dependent post-translational modification and position 2 allelism: effects on streptococcal superantigen SSA structure and V beta specificity.
Authors: Stevens K R , Van M , Lamphear J G , Orkiszewski R S , Ballard K D , Cook R G , Rich R R
Published: December 2022

Epidemiologic and molecular population genetic analyses support a role for superantigens (SAg) in the pathogenesis of severe staphylococcal and streptococcal infections. To investigate how variations in SAg structure influence immunomodulatory activity, we examined the biochemical and functional properties of two allelic variants of streptococcal SAg SSA that differ at position 2. Mass spectrometry revealed both recombinant (Escherichia coli) and native (Streptococcus pyogenes) SSA allelic variants to have significantly larger molecular masses than predicted by primary sequence alone and provided evidence that the proteins were modified by the addition of biochemical moieties, a phenomenon that has not been described for related SAg. Furthermore, the molecular masses of native and recombinant SSA were not the same; SSA was differentially post-translationally modified by the two bacterial genera. The substitution of E. coli-dependent processing for that of S. pyogenes altered both protease digestion and V beta specificity, suggesting that recombinant SAg from E. coli may not accurately represent the native toxin. In addition, the observation that SSA allelic variants differed in V beta specificity supports a role for position 2 in SSA-TCR interactions. That SSA position 2 contributes to V beta specificity could not have been predicted from functional or crystallographic studies of other SAg and suggests that SSA may adopt unique interactions with TCR and/or MHC class II molecules. Determining the structural basis for these differences should offer additional clues to the manner in which SAg exert their effects on the immune system during infection and may allow the designing of SAg mutants with specific quantitative and qualitative immunomodulatory properties.
Bacteria in the bloodstream are trapped in the liver and killed by immigrating neutrophils.
Authors: Gregory S H , Sagnimeni A J , Wing E J
Published: December 2022

The critical role of the liver in the resolution of systemic bacterial infections is well documented. In the case of Listeria monocytogenes, approximately 60% of bacteria inoculated i.v. into mice are recovered in the liver at 10 min after infection. Here we report that the Listeria recovered at 10 min were distributed equally among the hepatocyte and nonparenchymal liver cell populations. The majority (>/= 75%) of these organisms were bound extracellularly as judged by their sensitivity to gentamicin. In contrast, >/= 93% of Listeria recovered in the liver at 6 h were located within hepatocytes. The listerial burden of the liver decreased 0.5 to 1.0 log, between 10 min and 6 h after infection. This decrease correlated with a sevenfold increase in the percentage of neutrophils that constituted the nonparenchymal cell population. Mice rendered neutrophil deficient by pretreatment with anti-granulocyte (RB6-8C5) mAb exhibited a significant increase (>300%) rather than a decrease in liver Listeria and a marked increase in hepatocyte damage. Similarly, neutrophil-deficient mice exhibited a reduced capacity to eliminate Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus that were cleared by the liver and bound extracellularly to hepatocytes and nonparenchymal cells. These findings document the crucial role of immigrating neutrophils in nonspecific host defenses to systemic bacterial infections expressed within the liver.
L-selectin (CD62L) blockade does not impair peritoneal neutrophil emigration or subcutaneous host defense to bacteria in rabbits.
Authors: Sharar S R , Chapman N N , Flaherty L C , Harlan J M , Tedder T F , Winn R K
Published: December 2022

Neutrophil (PMN) recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. We explored the role of L-selectin (CD62L) in leukocyte emigration following instillation of bacteria into the peritoneum or s.c. skin in rabbits. Pretreatment with blocking mAb against L-selectin (LAM1.3) reduced peritoneal PMN emigration 4 h after i.p. inoculation with 10(10) CFU of Escherichia coli by only 17% compared with animals receiving a nonblocking L-selectin mAb (LAM1.14). Peritoneal PMNs from saline-treated rabbits demonstrated a complete absence of L-selectin, whereas those from LAM1.3-treated animals retained 43% of their baseline L-selectin expression. This suggests that L-selectin shedding is not a requisite event for PMN emigration under these conditions. In rabbits given s.c. inoculations with either Staphylococcus aureus or E coli, pretreatment with mAb LAM1.3 did not significantly impair PMN emigration at 24 h, nor increase the incidence, size, or associated mortality of resulting abscesses at 7 days compared with animals receiving nonblocking mAb LAM1.14. We conclude that: 1) mAb blockade of L-selectin in vivo only modestly affects acute, E. coli-induced peritoneal PMN emigration; and 2) L-selectin blockade does not increase infectious sequelae associated with s.c. bacterial inoculation. These findings of only mildly reduced PMN emigration into the peritoneum and no alteration in s.c. host defense differ from those reported with L-selectin blockade under other, nonbacterial inflammatory conditions, and suggest that redundant selectin-mediated mechanisms (P- and E-selectin) are sufficient for normal PMN emigration in response to bacterial stimulation.
I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasone-induced apoptosis.
Authors: Van Snick J , Houssiau F , Proost P , Van Damme J , Renauld J C
Published: December 2022

We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.
The baboon erythrocyte complement receptor is a glycophosphatidylinositol-linked protein encoded by a homologue of the human CR1-like genetic element.
Authors: Birmingham D J , Logar C M , Shen X P , Chen W
Published: December 2022

The human erythrocyte CA receptor (E-CR) is the type 1 complement receptor (CR1), the most common form of which is a 220,000 Mr integral membrane glycoprotein composed of 30 short consensus repeats (SCRs). The E-CR of many nonhuman primates is a smaller receptor of unknown genetic origin. Recently, we identified a chimp cDNA, termed CR1b, which represented transcription of a homologue of the human genetic element, CR1-like. The purpose of this study was to identify CR1b in the baboon and, if present, determine whether it encodes the 65,000 Mr baboon E-CR. Baboon bone marrow cDNA was amplified by PCR using primers specific for the signal peptide-encoding region of human CR1 and the 3' region of chimp CR1b. This amplification yielded a CR1b sequence predicted to encode seven SCRs followed by a hydrophobic region, with an N terminus homologous to the N terminus of baboon E-CR. Expression of baboon CR1b yielded a membrane protein that reacted with an anti-CR1 mAb, was identical in size to baboon E-CR, and, like baboon E-CR, could bind baboon C3 linked to activated thiol-Sepharose (C3i-ATS), but not human C3i-ATS. Phosphatidylinositol-specific phospholipase C (PIPLC) released CR1b from Chinese hamster ovary cells and E-CR from baboon erythrocytes, demonstrating that both of these proteins are glycophosphatidylinositol linked to the membrane. Thus, the data indicate that baboon CR1b, a homologue of the human CR1-like genetic element, encodes a glycophosphatidylinositol-linked protein that is the baboon E-CR.
Regulation of inducible nitric oxide synthase expression in IFN-gamma-treated murine macrophages cultured under hypoxic conditions.
Authors: Melillo G , Taylor L S , Brooks A , Cox G W , Varesio L
Published: December 2022

We recently reported that a hypoxia-responsive element mediates a novel pathway of transcriptional activation of the inducible nitric oxide synthase (iNOS) promoter in murine macrophages treated with IFN-gamma plus hypoxia (1% O2). In this study, we investigated the expression of NOS activity and the regulation of NOS induction in IFN-gamma treated ANA-1 murine macrophages or thioglycollate-elicited peritoneal macrophages cultured under hypoxic conditions. We found that murine macrophages stimulated with IFN-gamma plus hypoxia, despite a significant accumulation of iNOS mRNA, did not release nitrite into culture supernatant. However, cytosol from macrophages treated with IFN-gamma plus hypoxia contained significant levels of iNOS protein and enzymatic activity. Experiments in which cells were treated with IFN-gamma plus hypoxia and then cultured in normoxic conditions (20% O2) demonstrated that reoxygenation was required to achieve detectable accumulation of nitrite in the culture supernatant. Furthermore, we demonstrated that IL-4 inhibited IFN-gamma plus hypoxia-dependent induction of iNOS mRNA expression, iNOS protein, and enzymatic activity. Experiments in which ANA-1 macrophages were transfected transiently with the full-length iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene demonstrated that IL-4 also down-regulated the IFN-gamma plus hypoxia-induced activation of the iNOS promoter. These data establish that hypoxia is a costimulus with IFN-gamma for the induction of iNOS activity in ANA-1 macrophages as well as in murine peritoneal macrophages, and they provide the first evidence that IL-4 inhibits hypoxia-inducible gene expression. In addition, our results suggest that hypoxia, which occurs in many pathologic conditions, may play an important role in the activation of murine macrophages.
A novel action of IL-13: induction of diminished monocyte glucocorticoid receptor-binding affinity.
Authors: Spahn J D , Szefler S J , Surs W , Doherty D E , Nimmagadda S R , Leung D Y
Published: December 2022

We have recently demonstrated that the combination of IL-2 and IL-4 blunts T cell responses to glucocorticoids in steroid resistant (SR) asthma by reducing glucocorticoid receptor (GCR)-binding affinity. Since immune activation appears to be involved in the acquisition of steroid resistance, we sought to identify whether other cytokines could also induce diminished GCR-binding affinity. In the current report, utilizing a [3H]dexamethasone radioligand-binding assay and Scatchard analysis, we found that IL-13, a cytokine with similar actions as IL-4, could induce diminished GCR binding-affinity (GCR Kd = 34.4 +/- 2.3 nM with IL-13 vs Kd = 8.8 +/- 0.7 nM for unstimulated control cells; p < 0.001) in PBMC from normal subjects. In contrast, PBMC incubated with IL-1, IL-3, IL-5, IL-7, IL-8, IL-12, or granulocyte-macrophage-CSF had no effect on GCR-binding affinity; and no additive effect to the decreased GCR-binding affinity was noted when IL-13 was cocultured with IL-2 or IL-4. The cell target of IL-13-induced GCR effects was studied and found to reside in the non-T cell population; specifically, the monocyte fraction. To determine the functional significance of the decreased GCR-binding affinity, monocytes were pretreated with and without IL-1 3 prior to stimulation with LPS and hydrocortisone. IL-13 pretreatment of monocytes significantly diminished (p = 0.005) the suppressive effects of hydrocortisone on LPS-induced IL-6 production. IL-13, by virtue of its ability to induce diminished GCR-binding affinity, may contribute to impaired GC responsiveness during inflammatory illnesses.
The natural history of disease expression in CD4 and CD8 gene-deleted New Zealand black (NZB) mice.
Authors: Chen S Y , Takeoka Y , Ansari A A , Boyd R , Klinman D M , Gershwin M E
Published: December 2022

CD4 and CD8 gene-deleted New Zealand black (NZB) mice and, as controls, B6.CD4 -/-, B6.CD8 -/-, NZB, and B6 wild-type (wt) mice were studied for phenotypic and immunologic parameters to determine the contribution of CD4 and CD8 cell lineages in NZB mice. These studies suggest surprisingly that a number of abnormalities are not due to either CD4+ or CD8+ cell lineages but rather are most likely due to non-CD4+ and -CD8+ cell lineages and/or background genes. Such abnormalities include altered thymic architecture, decreased staining of MITS 33+ medullary thymocytes, and an increased frequency of splenic IgM secretory cells. In contrast, deletion of either CD4+ or CD8+ cells appears to differentially influence immunologic function. Deletion of CD8+ cells did not influence titers of spontaneously occurring anti-erythrocyte or anti-DNA autoantibodies. interestingly, 50% of NZB.CD4 -/- mice contained levels of anti-erythrocyte IgG and anti-ssDNA IgM autoantibodies; even without detectable CD4+ cells. Such deletion of CD4+ cells, while leading to marked decreases in the prototype cytokines that characterize Th1 and Th2 subsets in B6 mice, led to a marked increase in IFN-gamma and a moderate increase in IL-4 mRNA levels in NZB.CD4 -/- mice. These data suggest that whereas non-CD4+ and -CD8+ cell lineages and NZB background genes have a marked influence in the development of autoimmune abnormalities, CD4+ cells appear to play a major role in influencing the cytokine environment, whereas CD8+ cells appear to play a minor role.
Anti-IL-8 autoantibodies in alveolar fluid from patients with the adult respiratory distress syndrome.
Authors: Kurdowska A , Miller E J , Noble J M , Baughman R P , Matthay M A , Brelsford W G , Cohen A B
Published: December 2022

IL-8 is a potent neutrophil attractant and activator. IL-8 has been reported to be involved in the pathogenesis of several diseases, including rheumatoid arthritis, sepsis, psoriasis, and the adult respiratory distress syndrome (ARDS). Our previous studies demonstrated that high concentrations of IL-8 were present in alveolar fluids from patients with ARDS and were associated with increased mortality. In this study we report that a major portion of IL-8 in bronchoalveolar fluids from patients with ARDS is associated with anti-IL-8 autoantibody (anti-IL-8:IL-8 complexes). Free autoantibodies that recognize IL-8 were also detected in these fluids. Next, we examined the properties of anti-IL-8 autoantibodies present in lung fluids from ARDS patients and compared them with autoantibodies from normal plasma and arthritic synovial fluids. The anti-IL-8 autoantibody was polyclonal, and IgG3 and IgG4 were the primary IgG subclasses. Anti-IL-8:IL-8 complexes consisted of one IgG and one IL-8 molecule. In addition, anti-IL-8 autoantibody bound IL-8 with a high affinity (approximately 10(-12) M) and inhibited IL-8 interaction with its specific receptors on neutrophils. The results suggest that anti-IL-8 autoantibodies may regulate IL-8 activity.
Backcross analysis of genes linked to autoantibody production in New Zealand White mice.
Authors: Vyse T J , Morel L , Tanner F J , Wakeland E K , Kotzin B L
Published: December 2022

F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice are genetically predisposed to develop a lupus-like autoimmune disease characterized by IgG autoantibody production and an immune complex glomerulonephritis. Genes from both parental strains contribute to autoimmunity in the F1 animal. NZW mice produce mostly non-pathogenic autoantibodies to ssDNA and histones as their major autoimmune trait. We studied the genetics of this trait in order to gain insight into the NZW contribution to F1 disease. Genome-wide mapping of (NZW x BALB/c)F1 x NZW backcross mice showed that four NZW non-MHC loci on chromosomes 1, 11, 16, and 19 were linked with IgG autoantibody production. Another NZW locus on chromosome 14 appeared to be selectively linked with IgG anti-histone Abs. In this backcross, contributions from the nonautoimmune BALB/c strain were also apparent. Heterozygosity for the BALB/c MHC (H2d) was linked with IgG autoantibody production. This influence of H2d is therefore similar to that seen in (NZW x NZB)F1 mice, in which heterozygosity for H2d enhances autoantibody production and disease. Surprisingly, two non-MHC BALB/c loci were linked with IgM autoantibody levels, whereas no NZW loci had such an effect. Neither of these two loci have been previously linked with autoimmunity in lupus-prone mice. These data show that autoantibody production in NZW mice is a polygenic trait that is influenced by contributions from MHC and non-MHC genes. The results also support the hypothesis that NZW genes act to class-switch the autoantibody response, an effect that appears to contribute to disease in (NZB x NZW)F1 mice.
Activation of the Ras signaling pathway by the CD40 receptor.
Authors: Gulbins E , Brenner B , Schlottmann K , Koppenhoefer U , Linderkamp O , Coggeshall K M , Lang F
Published: December 2022

The CD40 receptor is an important molecule regulating B lymphocyte proliferation, maturation, Ab class switching, and cell survival. In the present study, we identified signal transduction events triggered by cross-linking the CD40 receptor. Stimulation of Daudi B cells with anti-CD40 resulted in activation of p21ras, an important switch point in the regulation of cell growth and differentiation. Ras activation correlated with a stimulation of Rac1 and MEK-1 as well as tyrosine phosphorylation of phosphatidylinositol 3-kinase. Inhibition of endogenous Ras by transfection of transdominant inhibitory Ras prevented tyrosine phosphorylation or stimulation of phosphatidylinositol 3-kinase, Rac1, or MEK-1 upon CD40 receptor triggering, proving an activation of the Ras pathway by CD40. Ras activation was partially inhibited by either herbimycin A or calphostin pretreatment and completely inhibited by preincubation with a combination of both inhibitors, indicating a synergistic role for protein tyrosine kinases and diglycerides in Ras activation after CD40 stimulation. In support of a role for diglycerides, we detected a 30 +/- 5% decrease of cellular phosphatidylcholine content, correlating with a threefold increase of diacylglycerol synthesis induced by CD40. Supporting a role for protein tyrosine kinase, we measured a five- to eightfold stimulation of p56lyn and p58blk kinase activity. These results suggest the activation of the Ras pathway via an additive function of src kinases and phospholipases that may be important in the mediation of biologic effects after CD40 receptor engagement.
Expression of trKB neurotrophin receptor during T cell development. Role of brain derived neurotrophic factor in immature thymocyte survival.
Authors: Maroder M , Bellavia D , Meco D , Napolitano M , Stigliano A , Alesse E , Vacca A , Giannini G , Frati L , Gulino A , Screpanti I
Published: December 2022

The relationships between the nervous and the immune systems raise the question of whether neurotrophic factors, in addition to the regulation of neural cell ontogeny, may influence lymphocyte development. We report in this work that the pattern of neurotrophin receptor expression depends on the developmental stage of T cells. The presence of nerve growth factor receptor trkA could not be detected in any of the thymocyte subsets, whereas brain-derived neurotrophic factor (BDNF) receptor trkB was expressed in all thymocyte subpopulations. Interestingly, both trkB mRNA and protein expression inversely correlated with the maturation stage and the differentiation potential of thymocytes, being more expressed in CD4-8- immature thymocytes and progressively declining in CD8+ and CD4+ single-positive and CD4+8+ more mature thymocytes. The developmentally regulated expression of trkB is further shown by the inhibition or enhancement of trkB expression induced by signals that either trigger or impair the transition from immature to more differentiated stages of the thymocyte developmental pathway. Signals generated following the interaction of BDNF with trkB receptor resulted in the stimulation of trkB autophosphorylation and in the up-regulation of the expression of the c-fos gene in CD4-8- cells and enhanced thymocyte survival. Finally, BDNF is expressed in thymic stroma and is further up-regulated by signals generated by the thymocyte/stromal cell interaction. These data suggest that BDNF may be a novel survival factor for thymocyte precursors and support the presence of developmentally regulated feedback mechanisms based on autocrine/paracrine neurotrophin/receptor interactions that may be involved in the thymocyte differentiation process.
Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.
Authors: Rheinnecker M , Hardt C , Ilag L L , Kufer P , Gruber R , Hoess A , Lupas A , Rottenberger C , Plückthun A , Pack P
Published: December 2022

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.
A highly variable region in members of the streptococcal M protein family binds the human complement regulator C4BP.
Authors: Johnsson E , Thern A , Dahlbäck B , Hedén L O , Wikström M , Lindahl G
Published: December 2022

Strains of Streptococcus pyogenes express one or more molecules that are members of the M protein family, a group of surface proteins implicated in virulence. A characteristic property of the molecules in this family is the presence of a highly variable N-terminal region, whose function is unknown. Here we show that human C4b-binding protein (C4BP), a regulatory component of the complement system, binds to the highly variable region of many members of the M protein family. Chimeric molecules, in which the N-terminal regions of four different C4BP-binding proteins were combined with the C-terminal part of the non-binding M5 protein, had intact C4BP-binding ability, as judged by binding assays and Scatchard analysis with highly purified molecules. Moreover, work with the C4BP-binding Arp4 protein showed that an N-terminal 52-residue fragment retained binding ability, and that a 21-residue synthetic peptide derived from the variable region completely inhibited the binding of C4BP. Computer-assisted analysis of the four C4BP-binding regions studied here (45-66 amino acid residues) indicated that they lack residue identities that could explain their ability to bind the same ligand, but differ from the nonbinding M5 protein in their lower propensity to form a coiled-coil. Thus, the variable C4BP-binding regions have an extraordinary capacity for sequence variation, while retaining the ability to bind C4BP. These data indicate that an important function of the variable region in members of the M protein family is to bind a host protein that down-regulates the complement system.
Multiple melanoma-associated epitopes recognized by HLA-A3-restricted CTLs and shared by melanomas but not melanocytes.
Authors: Mazzocchi A , Storkus W J , Traversari C , Tarsini P , Maeurer M J , Rivoltini L , Vegetti C , Belli F , Anichini A , Parmiani G , Castelli C
Published: December 2022

The molecular characterization of melanoma-associated Ags allowed the definition of several HLA class I-presented peptides recognized by T cells. However, no HLA-A3.1-restricted melanoma epitopes have been identified to date. To gain insight into the HLA-A3.1-restricted T cell epitope repertoire of human melanoma, we analyzed the immunologic reactivity of CTLs isolated from tumor-involved or tumor-free lymph nodes in two HLA-A3.1+ melanoma patients. Three CTL lines, clonal or highly oligoclonal in their TCR composition, and two CTL clones were selected for HLA class I-restricted lysis of the autologous tumor and then tested for the recognition of HLA-A3+ and HLA-A3- normal or neoplastic cells of the melanocyte lineage. One CTL recognized a unique HLA-A3.1-restricted Ag expressed only by the autologous tumors, while all the other CTLs defined three HLA-A3.1 epitopes shared by melanomas, but not by melanocytes. Moreover, the epitopes of two CTL lines with different specificity were reconstituted by nonoverlapping fractions of HLA-A3+ melanoma-derived peptides resolved by reverse phase-HPLC, indicating that distinct naturally processed peptides were specifically recognized on melanoma cells in association with HLA-A3.1 molecules. These novel lineage-unrelated HLA-A3.1-restricted melanoma epitopes do not derive from MAGE, BAGE, or GAGE gene families, as evaluated by the COS-7 transfection assay. Our data show that CTLs may recognize HLA-A3.1-class 1 complexes presenting melanoma (but not melanocyte)-associated epitopes that are either unique to a given patient's tumor or that are shared between multiple melanomas.
Tumor necrosis factor-alpha modulates the expression of its p60 receptor and several cytokines in rat tracheal epithelial cells.
Authors: Bader T , Nettesheim P
Published: December 2022

The epithelium of the conducting airways is frequently the target of toxic chemical and microbial agents causing inflammation, hypersecretion, and epithelial necrosis. TNF-alpha is a prototypical inflammatory cytokine released by macrophages and other inflammatory cells. The purpose of this study was to characterize TNF-alpha receptors in fully differentiated rat tracheal epithelial (RTE) cells in culture and to examine the effects of TNF-alpha on this epithelium. We demonstrated the presence of approximately 250 TNF-alpha receptors per RTE cell. Both known receptor types, p60 (TNF-RI, CD 120a) and p80 (TNF-RII, CD 120b), were expressed. The level of p80 mRNA was unaffected by TNF-alpha treatment, whereas p60 mRNA was down-regulated, and soluble TNF-RI was shed from cells within 30 min. Treatment of RTE cultures with TNF-alpha (1000 U) caused no cytotoxicity (as determined by lactate dehydrogenase release). However, TNF-alpha exposure of the cultures induced the expression of several inflammatory mediators, as determined by reverse transcription-PCR and ELISA. Low levels of IFN-gamma mRNA became detectable after 4 h. Increased levels of TNF-alpha mRNA and protein were found, which peaked after 6 h of TNF-alpha treatment, but neither IL-1 alpha nor IL-1 beta was detectable. Calcium-independent nitric oxide synthase transcripts were elevated two- to threefold within 2 to 6 h of TNF-alpha treatment. These findings suggest that the airway epithelium may actively participate in the pathogenesis of airway inflammation through the production of mediators similar to those found in a Th1 response.
Diminished production of prostaglandin E2 by monocytes of newborns is due to altered fatty acid membrane content and reduced cyclooxygenase activity.
Authors: Murphy F J , Reen D J
Published: December 2022

To determine the biochemical basis for reduced PGE2 expression by cord blood monocytes relative to adult peripheral blood monocytes, fatty acid composition of cell membranes together with the amount and activity of enzymes involved in the cyclooxygenase pathway were investigated. Gas chromatographic analysis of mononuclear cell fatty acids showed a significant reduction in long chain fatty acids, including arachidonic acid. Acylase and phospholipase A2 activity, together with lipocortin 1 levels, were similar in cord and adult monocytes. No difference in amounts of the 70-kDa cyclooxygenase type 1 enzyme was observed between the monocyte sources. The 74k-Da cyclooxygenase type 2 enzyme was synthesized de novo upon LPS stimulation in comparable amounts in both cord and adult monocytes, with both monocyte types responding with equal sensitivity to LPS. The ability of microsomes, prepared from cord monocytes, to convert arachidonic acid into eicosanoids of the cyclooxygenase pathway was reduced. In contrast, the ability of acetylated microsomes to convert PGG2 to PGE2 was comparable, suggesting that the deficiency in PGE2 production by cord monocytes is due to reduced activity, specifically at the cyclooxygenase-active site rather than the peroxidase active site. In conclusion, it appears that both reduced fatty acid content and diminished cyclooxygenase activity are responsible for diminished eicosanoid production by cord monocytes.