bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.
Authors: Zarrin A A , Fong I , Malkin L , Marsden P A , Berinstein N L 
Published: January 2023

Abstract
Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.
Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry.
Authors: Mordue D G , Sibley L D 
Published: January 2023

Abstract
We have characterized the intracellular fate of Toxoplasma in bone marrow-derived macrophages following two disparate modes of uptake: phagocytosis vs active invasion. The fate of parasite-containing vacuoles was followed by immunofluorescence localization of endogenous endocytic markers to track phagocytic processing in pulse-infected cells. During uptake of both opsonized and dead parasites, host cell plasma membrane MHC class II molecules and FcR were internalized into the phagosome and then gradually lost. Maturation of phagosomes containing Toxoplasma was a rapid, dynamic process of sequential fusion with early endosomes, late endosomes, and lysosomes that was complete within 15 min. Toxoplasma-containing phagosomes were transiently positive for transferrin receptor between 0 and 2.5 min, then contained the cation-independent mannose 6-phosphate receptor between 2.5 and 7.5 min, and finally matured to lysosome-like compartments containing lysosomal membrane glycoprotein 1 and the proton pump, but lacking cation-independent mannose 6-phosphate receptor. Toxoplasma-containing phagosomes also sequentially acquired host proteins that regulate endocytic fusion including rab5, N-ethylmaleimide-sensitive factor, and rab7. In marked contrast, MHC class II molecules and FcR were excluded from the parasitophorous vacuole formed by active parasite invasion. The parasitophorous vacuole also failed to acquire any host compartmental markers or fusion proteins analyzed. Our results indicate that Toxoplasma evades endocytic processing due to an absence of host regulatory proteins necessary to drive endocytic fusion, and that this divergence from normal maturation occurs during the formation of the primary vacuole.
Parasite-driven in vitro human lymphocyte cytotoxicity against autologous infected macrophages from mucosal leishmaniasis.
Authors: Brodskyn C I , Barral A , Boaventura V , Carvalho E , Barral-Netto M 
Published: January 2023

Abstract
Parasite-specific cytotoxicity in human leishmaniasis was evaluated in an autologous system. PBL from cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML) patients were exposed to Leishmania amazonensis-infected autologous macrophages for 7 days and then used as effector cells in a cytotoxic assay using 51Cr-labeled autologous infected macrophages as targets. Results are reported as LU per 10(7) PBMC. Cytotoxic activity is present in ML (9.7 +/- 2.1 LU/10(7) PBMC) but not in CL (1.5 +/- 2.4 LU/10(7) PBMC) patients' lymphocytes, and the differences were highly significant (p &lt; 0.0001). Both CD8+ T cells and NK cells exhibited cytotoxic activity. Addition of rIL-12, but not of IFN-gamma, during the generation of effector cells increased cytotoxic responses against infected macrophages. On the other hand, addition of mAb against human IL-12 or IFN-gamma during the stimulation of PBL significantly decreased the cytotoxic responses. Addition of IL-10 led to diminished cytotoxic responses, whereas the addition of anti-IL-10 did not significantly increase the cytotoxic responses. The observation of parasite-driven autologous cytotoxic responses in patients with ML, the destructive form of leishmaniasis, but not in CL, suggests that this phenomenon is involved in tissue pathology rather than in protection. Understanding the regulation of cytotoxic responses in leishmaniasis may be relevant to strategies aimed at limiting pathologic tissue destruction.
Activation of p38mapk, MKK3, and MKK4 by TNF-alpha in mouse bone marrow-derived macrophages.
Authors: Winston B W , Chan E D , Johnson G L , Riches D W 
Published: January 2023

Abstract
TNF-alpha regulates the expression of many proinflammatory and profibrogenic gene products in macrophages, and hence plays a vital role in controlling the inflammatory response. We have shown previously that exposure of macrophages to TNF-alpha stimulates the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we have investigated the mechanism of activation of the p38mapk by TNF-alpha in mouse bone marrow-derived macrophages. Exposure to TNF-alpha resulted in the activation of p38mapk, as measured by 1) the trans-phosphorylation of recombinant activating transcription factor-2 substrate by immunoprecipitated p38mapk and 2) specific tyrosine phosphorylation of immunoprecipitated p38mapk. In addition, selective ligation of the TNF-alpha receptor CD120a (p55) with human TNF-alpha was sufficient to induce p38mapk activation. Using an in vitro kinase assay with recombinant kinase-inactive p38mapk as substrate in the presence of [gamma-32P]ATP, the upstream kinases MKK3 (mitogen-activated protein kinase kinase 3) and MKK4 were found to be activated in response to TNF-alpha. These findings suggest that TNF-alpha transiently phosphorylates and activates the three members of the MAPK family, namely p42(mapk/erk2), p46 c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38mapk following cross-linking of CD120a (p55), and that MKK3 and MKK4 are capable of phosphorylating p38mapk.
Sensitive responses of leukocytes to lipopolysaccharide require a protein distinct from CD14 at the cell surface.
Authors: Vasselon T , Pironkova R , Detmers P A 
Published: January 2023

Abstract
Responses of leukocytes to low concentrations of LPS require the expression of membrane-associated CD14 (mCD14) on the cell surface; mCD14 is, however, a glycosyl-phosphatidylinositol-anchored protein and, therefore, a poor candidate for transducing signals across the plasma membrane. The role of other cell surface proteins in responses of leukocytes to LPS was tested by measuring IL-6 secretion of cultured human monocytes and adhesion of PMN in response to LPS after treatment of the cells with trypsin. Trypsin abolished leukocyte integrin-mediated adhesion of PMN in response to LPS, but the trypsinized cells remained responsive to the alternate agonists TNF, formyl peptide, and PMA. Similarly, trypsin treatment of monocytes inhibited IL-6 production in response to LPS, but not to formyl peptide or PMA. No change in cell surface expression of mCD14 was observed by cytofluorometry, and no proteolysis of mCD14 was detected by immunoblot analysis. These results suggest that digestion of a cell surface protein distinct from mCD14 must account for the loss of responsiveness to LPS. The uptake by monocytes of [3H]LPS presented in LPS-soluble CD14 complexes was also inhibited by trypsin treatment. Monomeric LPS was taken up superstoichiometrically to mCD14 with a ratio of up to 15:1, and trypsin treatment decreased this uptake by more than half. This suggests that a cell surface protein may function in accepting LPS from mCD14. Together the results suggest that one or more trypsin-sensitive cell surface proteins distinct from CD14 participate in both the uptake of LPS by leukocytes and the initiation of the signaling process.
Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration.
Authors: Tsang Y T , Neelamegham S , Hu Y , Berg E L , Burns A R , Smith C W , Simon S I 
Published: January 2023

Abstract
L-selectin enables capture and rolling of neutrophils on inflamed endothelium. This may facilitate the binding of agonists such as IL-8 and platelet-activating factor (PAF), which signal CD18-mediated firm adhesion and transmigration. Recent studies demonstrate that L-selectin can mediate transmembrane signaling. However, the functional effects of costimulation through agonist and L-selectin require further study. Here, we quantify cell adhesion, motility, and transmigration in response to co-activation through L-selectin and agonist. The surface expression of CD11b/CD18 increased and L-selectin decreased in proportion to the extent of L-selectin cross-linking. A flow cytometric assay was used to measure CD11b/CD18-dependent adhesion to fluorescent beads adsorbed with albumin. Neutrophil adhesion was detected within seconds of adding PAF (20 pM), IL-8 (50 pM), or cross-linking L-selectin. Costimulation through agonist and L-selectin potentiated by up to threefold the rate and extent of bead capture. Stimulation through L-selectin induced membrane ruffling, whereas PAF or IL-8 induced bipolar shape change. L-selectin cross-linking sustained the transient shape change induced by low concentrations (10-50 pM) of agonist. Chemokinesis stimulated by IL-8 was inhibited in the presence of cross-linking L-selectin. This was attributed to enhanced cell spreading following costimulation. Migration across HUVEC monolayers stimulated with IL-1 was also potentiated in the presence of L-selectin cross-linking. We propose that cross-linking of L-selectin and binding of agonist receptors may act synergistically to amplify neutrophil activation and emigration in the inflamed vasculature.
Regulation of anti-HIV-1 activity of RANTES by heparan sulfate proteoglycans.
Authors: Oravecz T , Pall M , Wang J , Roderiquez G , Ditto M , Norcross M A 
Published: January 2023

Abstract
The role of cell surface proteoglycans in CC chemokine-mediated anti-HIV-1 activity in T cells and macrophages was investigated. Enzyme digestion of heparan sulfate (HS), but not chondroitin sulfate, from the surface of PM1(CD26H) cells (a human T cell line selected for high CD26 expression) rendered them resistant to the antiviral effects of RANTES and macrophage-inflammatory protein-1beta at otherwise inhibitory chemokine concentrations. HIV-1 infection of macrophages, however, was inhibited only partially, even at high concentrations of RANTES, and this inhibition was not prevented by HS removal. Flow cytometry revealed that digestion of cell surface proteoglycans, including HS, prevented the binding of RANTES at 10 to 100 nM concentrations to PM1(CD26H) cells. However, the binding of RANTES to activated macrophages occurred only at higher concentrations (100-300 nM) and was mostly chondroitin sulfate, and not HS, dependent. These results support a role for HS in facilitating the interaction of CC chemokines with the cell surface and the consequent inhibition of HIV-1 infection. The absence of HS-dependent binding of RANTES at lower concentrations to macrophages is consistent with the resistance of these cells to the antiviral effects of chemokines.
Cutting Edge: Critical Role of NK1+ T Cells in IL-12-Induced Immune Responses In Vivo
Authors: Kawamura Toshihiko , Takeda Kazuyoshi , Mendiratta Sanjeev K. , Kawamura Hiroki , Van Kaer Luc , Yagita Hideo , Abo Toru , Okumura Ko 
Published: January 2023

Abstract
CD1-dependent NK1+ T cells rapidly produce IL-4 upon stimulation through the TCR. These cells may therefore play an important role in the initiation of Th2 responses. Here, we show that NK1+ T cells constitutively express receptors for IL-12 and IFN-γ, and that IL-12 induces production of perforin in these cells. Moreover, while IL-12 induces high levels of IFN-γ and cytotoxic activity of hepatic or splenic mononuclear cells against tumor cells, this effect of IL-12 is significantly reduced in CD1-deficient mice with impaired NK1+ T cells development. These results indicate that NK1+ T cells play a critical role in IL-12-induced production of IFN-γ to initiate Th1 immune responses and as IL-12-induced cytotoxic effector cells to initiate antitumor immunity.
Nuclear Factor of Activated T Cells (NFAT) as a Molecular Target for 1α,25-Dihydroxyvitamin D3-Mediated Effects
Authors: Takeuchi Atsuko , Reddy G. Satyanarayana , Kobayashi Tadashi , Okano Toshio , Park Jungchan , Sharma Surendra 
Published: January 2023

Abstract
The molecular basis of the immunomodulatory properties of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) remains elusive. We demonstrate here that 1α,25(OH)2D3-mediated suppressive effects on the inducible expression of cytokine genes in human T cells may, in part, be due to diminished activity of the transcription factor NFAT. The vitamin D3 receptor (VDR) and its heterodimeric partner retinoid X receptor α (RXRα) specifically bound to the distal NFAT site in the human IL-2 promoter, and this binding was abolished by mutating unique regions in the NFAT oligonucleotide. In vitro inhibition of NFAT complex formation was noted when VDR-RXRα heterodimers were added to DNA binding reactions containing nuclear extracts from activated B or T cells, whereas in vitro NFκB complex formation was not significantly influenced. Furthermore, 1α,25(OH)2D3 treatment of activated T cells resulted in decreased formation of NFAT complexes detected upon incubation of nuclear extracts from these cells with 32P-labeled probe. Transient expression of both VDR and RXRα, but not of a single component, was capable of inhibiting expression of a NFAT-driven reporter gene in stimulated Jurkat cells in a ligand-dependent manner. These results suggest that NFAT plays a crucial role in 1α,25(OH)2D3-mediated immunosuppressive activity.
Identification of Distinct Regions of 5′ Flanking DNA That Mediate Constitutive, IFN-γ, STAT1, and TGF-β-Regulated Expression of the Class II Transactivator Gene
Authors: Piskurich Janet F. , Wang Ying , Linhoff Michael W. , White Leigh C. , Ting Jenny P.-Y. 
Published: January 2023

Abstract
Class II transactivator (CIITA) is a master regulator required for constitutive and IFN-γ-inducible expression of class II MHC genes. Although the role of CIITA is greatly appreciated, the mechanisms underlying constitutive and IFN-γ-induced expression of CIITA are not understood. The study of CIITA induction is extremely important, but has been fraught with difficulty. This study describes for the first time a large (7-kb) fragment of 5′ flanking sequences that mediates the B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA. This pattern of expression matches the authentic expression of the endogenous gene. Within the 7-kb fragment, sequences that lie between nucleotides −545 and −113 relative to the transcriptional start site are critical for constitutive promoter expression in B cells. In contrast, inducible activation of CIITA by IFN-γ requires sequences contained in an additional 4 kb of upstream DNA. This region mediates an IFN-γ response when linked to either the endogenous CIITA promoter or a heterologous promoter. A role for STAT1 in regulation of the CIITA promoter is shown by the rescue of IFN-γ induction by expression of STAT1 in STAT1-defective U3A cells. TGF-β significantly inhibits IFN-γ-mediated induction of the CIITA promoter in 2fTGH fibroblasts, which indicates that the promoter is a target for TGF-β. This inhibition is achieved by suppression of the basal promoter. This study provides a focal point for understanding the mechanism of B cell-specific, IFN-γ-induced, and TGF-β-suppressed expression of CIITA.
Peptides That Mimic the Group B Streptococcal Type III Capsular Polysaccharide Antigen
Authors: Pincus Seth H. , Smith Michael J. , Jennings Harold J. , Burritt James B. , Glee Pati M. 
Published: January 2023

Abstract
Microbial polysaccharides are notably poor immunogens. We have developed an alternate route for the production of Abs to important carbohydrate epitopes. mAb S9, a protective mAb against the type III capsular polysaccharide of group B streptococci (GBS), was used to select epitope analogues from a peptide display phage library. Depending upon desorption conditions, two populations of phage were identified with displayed sequences of WENWMMGNA and FDTGAFDPDWPA. ELISA results demonstrated that these phage bound to S9 and no other Abs. Phage blocked the binding of S9 to type III GBS, but did not block binding by another anti-GBS mAb. Phage displaying the latter peptide sequence showed greater inhibition. Ab S9 and other monoclonal and polyclonal anti-GBS type III antisera bound the synthetic peptide FDTGAFDPDWPAC. The binding of S9 to GBS was inhibited by the free peptide with an IC50 of 30 μg/ml. The binding of polyclonal anti-GBS antibodies to peptide could be blocked by intact GBS as well as purified capsular polysaccharide. The peptide was conjugated to three different carriers and was used to immunize mice. All mice produced a significant antibody response to GBS and to the purified capsular polysaccharide following a single immunization. These data demonstrate that a peptide mimetic of the GBS capsular polysaccharide is both antigenic and immunogenic. The incorporation of such peptides into vaccine preparations may enhance the efficacy of vaccines in inducing Ab responses to important carbohydrate epitopes.
IL-18 (IFN-γ-Inducing Factor) Regulates Early Cytokine Production in, and Promotes Resolution of, Bacterial Infection in Mice
Authors: Bohn Erwin , Sing Andreas , Zumbihl Robert , Bielfeldt Claudia , Okamura Haruki , Kurimoto Masashi , Heesemann Jürgen , Autenrieth Ingo B. 
Published: January 2023

Abstract
IL-12-induced IFN-γ production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-γ-inducing factor) is produced by macrophages and induces IFN-γ production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-γ production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-γ production was not IL-18 dependent. IL-18-induced IFN-γ production was to a higher degree dependent on IFN-γR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-γ levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-γ production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.
Neutrophils but Not Eosinophils Are Involved in Growth Suppression of IL-4-Secreting Tumors
Authors: Noffz Gabriele , Qin Zhihai , Kopf Manfred , Blankenstein Thomas 
Published: January 2023

Abstract
Local expression of IL-4 by gene-modified tumor cells increases their immunogenicity by inducing an inflammatory response that is dominated by eosinophils. Eosinophils have been implicated as antitumor effector cells because the application of a granulocyte-depleting Ab inhibited rejection of IL-4 transfected tumors. This Ab did not discriminate between eosinophils and neutrophils and, therefore, this experiment could not exclude neutrophils as primary effector cells, whereas eosinophils were innocent bystander cells in IL-4 transfected tumors. We analyzed tumor growth suppression and granulocyte infiltration in IL-5-deficient (IL-5−/−) mice that had a deficiency of eosinophils, using two tumor lines (B16-F10 and MCA205) transfected to secrete IL-4. IL-4-expressing tumors were at least as efficiently rejected in IL-5−/− mice as in wild-type mice, despite an almost complete absence of tumor-infiltrating eosinophils. However, neutrophils were present in undiminished amounts and their depletion partially restored tumor growth. Furthermore, the growth of IL-5-secreting tumors was not impaired in either wild-type or IL-5−/− mice, even though it induced eosinophilia in both mouse strains. These findings demonstrate that eosinophils can be induced in IL-5−/− mice by exogenous IL-5 and argue against a compensatory effect of neutrophils in the absence of eosinophils. We conclude that 1) infiltration of IL-4 transfected tumors by eosinophils is completely IL-5 dependent, 2) eosinophils have no tumoricidal activity, and 3) neutrophils are responsible, at least in part, for tumor suppression.
Helminth Antigens Selectively Differentiate Unsensitized CD45RA+CD4+ Human T Cells in Vitro
Authors: Steel Cathy , Nutman Thomas B. 
Published: January 2023

AbstractHuman filarial helminth infections are characterized by type 2 immune responses to parasite Ag that can persist for the life of the individual; one possible cause for this may be prenatal exposure to the blood-borne microfilarial (Mf) stage of the parasite. To examine the relationship between early exposure to filarial Ag and subsequent immune responsiveness, CD45RA+CD4+ cells from normal unsensitized donors were stimulated in vitro with soluble microfilarial Ag (MfAg) from the filarial parasite Brugia malayi in the presence of APCs. MfAg alone induced proliferation and IFN-γ and IL-5 production in unsensitized CD45RA+CD4+ cells, demonstrating the ability of filarial Ags to prime naive T cells in the absence of exogenous cytokines and dendritic cells. Adding exogenous cytokine(s) (particularly IL-12 and IL-4) during priming was able to alter the MfAg-specific responses of CD45RA+CD4+ cells as well as subsequent responses to Ag. Interestingly, priming solely with MfAg led to enhanced IL-5 production following Ag restimulation, suggesting that MfAg preferentially primes for type 2 responses. These data demonstrate that filarial Ags by themselves can specifically prime CD45RA+CD4+ cells in vitro and do so in such a way as to deviate the immune response.