bluuebunny/crossref_metadata_2025 huggingface dataset:
Members of the novel WC1 gene family are differentially expressed on subsets of bovine CD4-CD8- gamma delta T lymphocytes.
Authors: Wijngaard P L , MacHugh N D , Metzelaar M J , Romberg S , Bensaid A , Pepin L , Davis W C , Clevers H C
Published: December 2022

CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.
Treatment of established lung metastases with tumor-infiltrating lymphocytes derived from a poorly immunogenic tumor engineered to secrete human TNF-alpha.
Authors: Marincola F M , Ettinghausen S , Cohen P A , Cheshire L B , Restifo N P , Mulé J J , Rosenberg S A
Published: December 2022

The growth of a poorly immunogenic methylcholanthrene (MCA)-induced murine (m) sarcoma genetically engineered to secrete human (h) TNF-alpha (MCA-102-hTNF) was studied. MCA-102-hTNF tumor cells were implanted in animals bearing three- or 7-day pulmonary metastases established with the parental line MCA-102-WT (wild type). This model approximates the clinical situation in which patients with metastatic cancer would be vaccinated with autologous tumor genetically modified to stimulate the host immune response. Reduction in the number of pulmonary metastases was occasionally seen but was not consistently reproducible. Other cytokine-producing tumors had either no effect on distant pulmonary metastases (mIL-4, IFN-gamma) or a mild, inconclusive effect similar to hTNF-alpha (mTNF-alpha). Significant growth inhibition of MCA-102-hTNF was noted in animals bearing pulmonary metastases. This inhibition was: 1) tumor specific (regression occurred only in animals bearing pulmonary metastases from the same parental line), 2) TNF specific (it was inhibited by in vivo administration of anti hTNF mAbs), 3) dependent on cellular immunity (immune-depletion with anti-CD4 or CD8 mAbs permitted growth). Tumor-infiltrating lymphocytes (TIL) could not be grown from MCA-102-WT or MCA-102-hTNF tumors nor from MCA-102-WT subcutaneous implants in mice bearing MCA-102-WT pulmonary metastases. However, TIL could be grown from hTNF-secreting tumors implanted in mice bearing MCA-102-WT metastases. These TIL were therapeutic against established lung metastases from the parental tumor in adoptive immunotherapy models. These studies suggest a strategy for using gene modified tumors for the therapy of established cancer.
Neutralization of IL-10 up-regulates nitric oxide production and protects susceptible mice from challenge with Candida albicans.
Authors: Romani L , Puccetti P , Mencacci A , Cenci E , Spaccapelo R , Tonnetti L , Grohmann U , Bistoni F
Published: December 2022

In contrast to several inbred strains of mice that develop Th1-associated anticandidal protection, DBA/2 mice are highly susceptible to systemic infection with Candida albicans cells of the attenuated variant PCA-2, and fatal outcome is observed in concurrence with sustained CD4+ cell production in vitro of IL-4 and IL-10. These Th2 cytokines were previously shown to inhibit nitric oxide (NO) production and yeast killing by activated macrophage cultures. We now show that macrophages from DBA/2 mice, either intact or infected with PCA-2, have lower capacity than resistant strains to synthesize NO in response to IFN-gamma. However, when treated with anti-IL-10 Abs at the time of infection, DBA/2 mice survived challenge and displayed increased production of NO in vitro after IFN-gamma activation. Cure was associated with the onset of footpad responses and durable protection, and higher frequencies of IFN-gamma-secreting cells were found in splenic CD4+ lymphocytes that expressed lower levels of IL-4 and IL-10 mRNA. Therefore, in DBA/2 mice, IL-10 contributes significantly to the selection of a Th2 response and lethality after PCA-2 challenge. An IL-10-induced defect in the activation and/or expansion of IFN-gamma-producing Th1 cells, IL-10 suppression of yeast killing, and the relative inability of DBA/2 macrophages to produce adequate levels of candidacidal NO may all contribute to the abnormal susceptibility of these mice to candidiasis.
Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.
Authors: Bennett C F , Condon T P , Grimm S , Chan H , Chiang M Y
Published: December 2022

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.
Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis.
Authors: Sempowski G D , Beckmann M P , Derdak S , Phipps R P
Published: December 2022

The purpose of this study was to determine whether or not membrane-bound and soluble forms of IL-4 receptors are expressed by isolated subsets of murine lung fibroblasts and to evaluate the potential functional consequences of IL-4 receptor triggering. Recent studies demonstrate that IL-4-synthesizing Th2 cells and mast cells are present in increased numbers in the lung during inflammation and fibrosis, suggesting that IL-4 may play a regulatory role in these events. We hypothesize that pulmonary fibroblasts and subsets thereof are intimately involved in this inflammatory response and that IL-4 is an active player in stimulating fibroblast collagen synthesis and hyperproliferation, creating a fibrotic environment in the lung. The fibroblast subsets used in these experiments differ not only in surface expression of the thymocyte-1 (Thy-1) Ag, but also in function and morphology. We now report the novel finding that IL-4 receptors are present at discordant levels on Thy-1+ and Thy-1- lung fibroblasts. IL-4R level and affinity were analyzed using a monoclonal anti-IL-4R Ab and equilibrium binding analysis with 125I-labeled IL-4. Reverse transcriptase PCR demonstrated the presence of mRNA for membrane-bound and soluble IL-4R. Lung fibroblast subsets secrete soluble IL-4R protein at dramatically different levels, as detected by an ELISA. Thy-1+ and Thy-1- lung fibroblasts were treated with IL-4 to determine whether this cytokine was profibrotic. Thy-1+ fibroblasts responded to IL-4 by proliferating and up-regulating collagen production. In contrast, Thy-1- fibroblasts proliferate to a lesser degree than Thy-1+ fibroblasts and were not stimulated to secrete increased levels of collagen. Overall, these results suggest that elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis.
Receptors and transduction pathways for monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similarities and differences with MCP-1.
Authors: Sozzani S , Zhou D , Locati M , Rieppi M , Proost P , Magazin M , Vita N , van Damme J , Mantovani A
Published: December 2022

MCP-2 and MCP-3 are recently identified members of the Cys-Cys chemokine family with high sequence similarity with MCP-1 (62% and 71%, respectively). The present study was aimed at defining receptor usage and signal transduction pathways of MCP-2 and MCP-3 in human monocytes in comparison with MCP-1. MCP-2 and MCP-3 induced migration of monocytes with a typical bell-shaped curve and maximal response at 10 and 50 ng/ml, respectively. The maximal response elicited by MCP-2 and MCP-3 was lower (approximately 60%) than that of MCP-1. Pertussis toxin (PTox) inhibited the chemotactic activity of MCP-3 and MCP-1 (IC50 = 6.2 and 4.4 ng/ml, respectively), whereas cholera toxin (CTox) had little effect on these two chemokines (IC50 > 1000 ng/ml). In contrast, MCP-2-induced chemotaxis was blocked by CTox (IC50 = 75 ng/ml) and relatively unaffected by PTox. MCP-3 and MCP-1 induced a rapid increase in intracellular Ca2+ concentration, whereas MCP-2, in the range of concentrations active on chemotaxis, did not. MCP-1-, MCP-2-, and MCP-3-induced chemotactic responses were blocked by C-I, a serine/threonine kinase inhibitor, and by genistein, a tyrosine kinase inhibitor, with the MCP-2 response being more sensitive than those induced by MCP-1 and MCP-3. MCP-1 and MCP-3 rapidly induced arachidonic acid release whereas MCP-2 was ineffective. MCP-1 and MCP-3 cross-desensitized with each other in terms of Ca2+ transients and displaced with a comparable efficiency labeled MCP-1 from human monocytes. On the other hand, MCP-2 did not cross-desensitize with MCP-1 and MCP-3 and only partially (20%) displaced labeled MCP-1. Thus, in spite of high sequence similarity, MCP-2 differed considerably from MCP-1 and MCP-3 in terms of sensitivity to CTox and PTox, arachidonate and calcium mobilization, and capacity to compete for labeled MCP-1.
An amino-terminal fragment of human lipopolysaccharide-binding protein retains lipid A binding but not CD14-stimulatory activity.
Authors: Theofan G , Horwitz A H , Williams R E , Liu P S , Chan I , Birr C , Carroll S F , Mészáros K , Parent J B , Kasler H
Published: December 2022

LPS-binding protein (LBP) mediates the pro-inflammatory effects of bacterial LPS by enhancing LPS-induced cytokine production by monocytic cells. LBP binds specifically to LPS to generate a complex that interacts with the CD14 receptor on the surface of responsive cells. To identify the biologically active regions of the protein responsible for mediating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP25). Both forms of LBP bound to LPS with the same affinity, and similarly inhibited LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP25 were compared for their ability to mediate CD14-dependent LPS effects on cells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP25 had no significant activity in these assays. Similarly, rLBP was able to mediate LPS-induced TNF production by human PBMC whereas rLBP25 was essentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably located in the C-terminal region of the protein. Thus, the LPS-binding activity of LBP can be separated from the CD14-stimulatory activity, suggesting these activities are mediated by structural elements residing in different regions of the protein.
Differential mycobacterial 65-kDa heat shock protein T cell epitope recognition after adjuvant arthritis-inducing or protective immunization protocols.
Authors: Anderton S M , van der Zee R , Noordzij A , van Eden W
Published: December 2022

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis (Mt) in mineral oil induces adjuvant arthritis (AA), associated with T cell responses to residues 180-188 of the mycobacterial 65-kDa heat shock protein (hsp65). Preimmunization with hsp65 protects rats against AA and other forms of arthritis. Several explanations for these protective effects have been proposed, including enhanced responsiveness to protective epitopes in hsp65, down-regulation of T cell responses to the 180-188 epitope, and activation of self-hsp60-reactive T cells. To assess the potential of these hypotheses, we analyzed hsp65 T cell epitopes recognized after immunization of Lewis rats with Mt or hsp65. Here we identify nine RT1.B1-restricted T cell epitopes in hsp65. Mt immunization induced T cell responses in which the 180-188 epitope was dominant, whereas hsp65 immunization resulted in a co-dominance of this and two further epitopes, 216-225 and 226-235. Two minor epitopes were recognized after hsp65 but not Mt immunization. These results indicate that hsp65 preimmunization does not down-regulate responses to the AA-associated epitope, but does enhance responses to several hsp65 epitopes that are minor or absent after the AA-inducing immunization protocol. Cross-reactive T cell recognition of hsp65 and rat hsp60 was limited to a single epitope (256-265), recognized after hsp65 immunization, but poorly recognized after Mt immunization. This study provides the necessary basis for elucidating the T cell events involved in the protective effects of hsp65 preimmunization.
In vivo activation of naive T cells by antigen-presenting B cells.
Authors: Morris S C , Lees A , Finkelman F D
Published: December 2022

In vivo experiments were performed to determine if the cross-linking of mlg on antigen-presenting B cells could induce them to present Ag to naive T cells in a stimulatory rather than a tolerogenic fashion. Mice were injected with a foreign mAb to a B cell Ag (Fc epsilon RII or CR1), and/or a self-anti-IgD mAb. Injection of either mAb alone failed to induce an Ab response; however, simultaneous injection of the foreign anti-B cell mAb plus the self-anti-IgD mAb stimulated a large response. Inasmuch as injection of the anti-IgD mAb should not have facilitated transfer of the foreign mAb to dendritic cells, this observation suggests that cross-linking of B cell mlg can induce B cells to acquire the ability to present Ag to naive T cells in an activating manner. Furthermore, when injected with anti-IgD mAb, both foreign anti-B cell mAbs were more potent in inducing Ab responses in this system than were isotype-matched control mAbs, consistent with the hypothesis that T cells were activated by Ag-presenting B cells. Results of dose-response and cell transfer studies, however, suggested that stringent cross-linking of B cell mlg on large numbers of B cells is required for B cell Ag presentation to induce T cell activation rather than tolerance. Therefore, these observations suggest that professional APCs usually are required to activate naive T cells, and that B cell Ag presentation can only activate naive T cells under unusual circumstances.
TGF-beta mediates natural suppressor activity of IL-2-activated lymphocytes.
Authors: Yamamoto H , Hirayama M , Genyea C , Kaplan J
Published: December 2022

In addition to generating cells with non-MHC-restricted cytotoxic activity that is characteristic of lymphokine-activated killer (LAK) cells, in vitro cultures of lymphocytes with relatively high concentrations of IL-2 generate cells that simultaneously exhibit two distinct types of suppressor activities: veto, the ability of cells to specifically suppress generation of allo-CTL against their own histocompatibility Ags; and natural suppression, the ability of these same cells to nonspecifically suppress the generation of allo-CTL against both their own and unrelated cell surface Ags. In contrast to veto, which is known to require cell-cell contact between veto-active cells and precursors of CTL, natural suppression is known to be mediated by soluble factors. To identify and characterize suppressor factors that might mediate the natural suppressor activity of IL-2-activated lymphocytes, murine spleen cells were cultured with 1000 U/ml IL-2, and, after varying periods of incubation, their LAK cytolytic activity and natural suppressor activity was determined and cell supernatants were collected and tested for their effects on mixed lymphocyte culture-induced generation of allo-CTL. Like the IL-2-activated lymphocytes themselves, supernatants of these cells nonspecifically inhibited mixed lymphocyte culture-induced generation of allo-CTL. Rabbit anti-TGF-beta specifically neutralized the suppressive effects of both LAK cell supernatants and the IL-2-activated lymphocytes themselves. These findings indicate that TGF-beta is the primary mediator of the natural suppressor activity of IL-2-activated lymphocytes.
Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.
Authors: Dick L R , Aldrich C , Jameson S C , Moomaw C R , Pramanik B C , Doyle C K , DeMartino G N , Bevan M J , Forman J M , Slaughter C A
Published: December 2022

The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.
Preferential VH gene usage in rabbit Ig-secreting heterohybridomas.
Authors: Raman C , Spieker-Polet H , Yam P C , Knight K L
Published: December 2022

B cells from leukemic rabbits preferentially use a single VH gene, VH1, in their VDJ gene rearrangements. To determine whether Ig-secreting B cells from normal rabbits also preferentially use VH1, we generated rabbit X mouse heterohybridomas that stably secreted rabbit Ig that expressed VHa allotypic specificities and analyzed the VH genes used in their VDJ gene rearrangements. We cloned the VDJ genes from nine heterohybridomas, and by comparing the restriction map of the DNA immediately 5' of the translational start site of these VDJ genes to that of the same region 5' of VH1, we showed that eight of the nine heterohybridoma clones use VH1. Comparison of the nucleotide sequences of the eight VH1-using VDJ genes with the nucleotide sequence of germ-line VH1 showed that each of them had somatically diversified. The diversified regions included clustered nucleotide changes and codon insertions and deletions, such as would be expected if the diversification process involved somatic gene conversion. We searched our database of germ-line VH genes for genes that could serve as donors for the gene conversion events, and we identified potential VH donor genes for five regions of diversification. One of these regions of diversification spanned at least 132 bp and included a codon insertion as well as 15 nucleotide changes. The data confirm that Ab diversity may be generated by a somatic gene conversion-like mechanism. The results directly demonstrate that Ig-secreting B cells from normal rabbits preferentially use VH1 in their VDJ gene rearrangements.
Use of global amino acid replacements to define the requirements for MHC binding and T cell recognition of moth cytochrome c (93-103).
Authors: Reay P A , Kantor R M , Davis M M
Published: December 2022

Substitution with all naturally occurring L-amino acids at each of 11 residues of the IEk-restricted month cytochrome c (93-103) epitope has allowed us to analyze the requirements for MHC binding and T cell recognition to a level of definition not previously possible. Substitutions at only three positions systematically affect MHC binding and three others appear to be the major TCR contacts. Interestingly, changing residues involved in MHC binding can ablate T cell recognition without altering MHC association. Additionally, residue identity at two positions that do not appear critical for MHC binding, nor to be involved in specific T cell contact, nonetheless dramatically affect T cell responses. This suggests that peptides differing only slightly in sequence can have significantly altered conformations within the class II MHC binding groove. We have also developed a simple scoring program that uses the binding data to quantitate how well a given peptide fits the MCC motif. All strongly immunogenic IEk-restricted epitopes score highly (> or = 0.70, where 1.0 is perfect concordance), and only 3% of all potential nonameric peptides in the two main protein sequence databases have scores greater than 0.70. This indicates that the global amino acid replacement approach using a single peptide is an efficient means of deriving binding motifs for a given class II MHC molecule, and should aid in the identification of novel T cell epitopes.
Contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge.
Authors: VanCott J L , Brim T A , Lunney J K , Saif L J
Published: December 2022

Two antigenically related porcine coronaviruses, transmissible gastroenteritis virus (TGEV) which infects primarily the intestinal tract and causes severe diarrhea, and porcine respiratory coronavirus (PRCV) which infects the respiratory tract and causes subclinical or mild respiratory infections, presented a unique opportunity to study the interrelationship of gut-(GALT) and bronchus-associated lymphoid tissues (BALT) and their contribution to protective immunity against TGEV infection. Pigs were inoculated oral-nasally with TGEV or with PRCV at eleven days of age and challenged 24 days later with TGEV. All pigs initially given TGEV developed diarrhea and were completely protected against disease upon challenge. In contrast, pigs given PRCV had no clinical disease and shed virus in nasal secretions only; after challenge, 5 of 12 pigs developed diarrhea. Virus-specific IgG and IgA Ab-secreting cells (ASC) were enumerated by ELISPOT in the mesenteric and bronchial lymph nodes, spleens, and gut lamina propria at challenge and various post challenge days. Before challenge, in pigs exposed to TGEV, IgA-ASC in the duodenum and jejunum constituted the major ASC response. Conversely, PRCV-exposed pigs had mainly IgG-ASC in bronchial lymph nodes, with low ASC responses in the gut. After challenge, numbers of IgG-ASC increased rapidly in the gut lamina propria and mesenteric lymph nodes of only PRCV-primed pigs. Our results suggest that virus-specific IgG-ASC precursors derived in BALT of PRCV-primed pigs may migrate to the gut in response to TGEV challenge and contribute to the partial protection observed. The presence of IgA-ASC in the gut lamina propria of TGEV-primed pigs at the time of challenge correlated with complete protection against TGEV challenge. Thus a dichotomy exists in the BALT and GALT ASC responses; immunization via BALT induced a systemic type of response (IgG-ASC) and provided imperfect protection against an enteric pathogen, whereas immunization via GALT induced IgA-ASC and provided complete protection.
Pulmonary epithelial cells facilitate TNF-alpha-induced neutrophil chemotaxis. A role for cytokine networking.
Authors: Smart S J , Casale T B
Published: December 2022

TNF-alpha and neutrophils are postulated to play important roles in several inflammatory lung diseases. Thus, we examined TNF-alpha-induced neutrophil migration through polycarbonate filters and human pulmonary type II-like epithelial (A549) cells cultured as monolayers on these filters. TNF-alpha induced both dose- and time-dependent migration of neutrophils across both barriers, but migration across A549 cells was much greater than that across naked filters. Migration of neutrophils across both barriers was completely inhibited by anti-TNF-alpha. Furthermore, supernatants of TNF-alpha (10(-9) M)-stimulated A549 monolayers induced threefold greater migration of neutrophils across naked filters than 10(-9) M TNF-alpha itself, suggesting release of soluble chemotactic factor(s). Pretreatment of A549 monolayers with actinomycin D inhibited both the production of soluble chemotactic factors and TNF-alpha-induced transcellular migration. Supernatants from TNF-alpha-stimulated A549 cells contained significant concentrations of IL-8, and coincubation of these supernatants with anti-IL-8 decreased supernatant-induced chemotaxis. Finally, coincubation of TNF-alpha with anti-IL-8 during transmigration experiments partially inhibited neutrophil migration through A549 monolayers. Therefore, pulmonary type II epithelial cells facilitate TNF-alpha-induced transcellular migration of neutrophils through the production of soluble protein synthesis-dependent chemotactic factors, including IL-8. These data support the concepts of cytokine networking in inflammatory cell recruitment and an active role for pulmonary epithelium in lung inflammation.
Autoreactive epitope profiles of the proliferating cell nuclear antigen define two classes of autoantibodies.
Authors: Brand S R , Bernstein R M , Mathews M B
Published: December 2022

The proliferating cell nuclear antigen (PCNA) is a conserved protein required for cellular DNA replication. PCNA was first recognized using serum from the autoimmune disease SLE. To analyze the regions on PCNA that confer autoantibody binding, we modified the cDNA encoding full-length PCNA to generate a series of amino terminal, carboxyl terminal and internally deleted constructs, which were transcribed and then translated in vitro using the wheat germ cell-free translation system. An immunoprecipitation assay was used to study the ability of these mutated forms of PCNA to bind anti-PCNA Abs from patients with SLE. Eight of the ten sera studied required a protein of nearly full length for binding: antigenicity was abrogated by removal of 39 amino acids from the amino terminus, by various internal deletions, or by the removal of 15 (but not 11) amino acids from the carboxyl terminus. The remaining two sera exhibited an Ab-binding specificity to the carboxyl-terminally truncated proteins similar to that of the majority of anti-PCNA sera, but their specificity was different in the amino terminus: these sera were able to recognize PCNA lacking over 40% of sequence in the amino terminus, but they did not bind proteins with short internal deletions in that region. Thus, these epitopes appear to be conformational and distinguish two classes of autoimmune sera.
Chemically selected subclones of the CEM cell line demonstrate resistance to HIV-1 infection resulting from a selective loss of NF-kappa B DNA binding proteins.
Authors: Qian J , Bours V , Manischewitz J , Blackburn R , Siebenlist U , Golding H
Published: December 2022

To delineate cellular genes that are required for optimal HIV-1 infection, CEM cells were subjected to treatment with the chemical mutagen ethylmethanesulfonate (EMS) and subclones were selected based on their increased resistance to HIV-1 infection and reduced syncytium formation, despite relatively normal CD4 expression (20,000 to 25,000 receptors/cell). Two subclones with this phenotype demonstrated a diminished capacity of HIV-1 long terminal repeat-chloramphenicol acetyl transferase expression either after treatment with the protein kinase C activator PMA, or through Tat-mediated transactivation. In this study, we show that the cellular levels of the NF-kappa B DNA binding proteins (but not AP1 or SP1) are markedly reduced in these cell mutants both at the mRNA and protein levels, resulting in reduced nuclear localization of p50/p65 after PMA induction or treatment with the lymphokine TNF-alpha. Transient reconstitution with a plasmid expressing p50 resulted in partial recovery of PMA-inducible LTR-chloramphenicol acetyl transferase expression. These data suggest that, at least in the CEM T cell line, a selective reduction in the NF-kappa B DNA binding proteins is sufficient to curtail HIV-1 infection.
Regulatory effects of prostaglandin E2 on the growth and differentiation of human B lymphocytes activated through their CD40 antigen.
Authors: Garrone P , Galibert L , Rousset F , Fu S M , Banchereau J
Published: December 2022

We have studied the effects of prostaglandin E2 (PGE2) on the growth and differentiation of human tonsillar B lymphocytes cultured in the CD40 system with or without IL-4 or IL-10. PGE2 (10(-9) to 10(-6) M) enhanced proliferation of B cells activated through their CD40 Ag, but not their Ig secretion. PGE2 further potentiated both IL-4- and IL-10-induced B cell growth as determined by [3H]TdR uptake and cellular enumeration. The IL-10-induced IgM, IgG, and IgA secretion was enhanced twofold to fourfold after addition of PGE2, whereas IL-4-induced IgG and IgE secretion was inhibited. The IgE production was particularly sensitive as an approximately 90% inhibition was obtained for 10(-7) M PGE2. In addition, PGE2 inhibited IgE production by naive surface IgD+ B cells cultured in the CD40 system, suggesting that PGE2 may interact with mechanisms involved in IgE switching. PGE2 displayed similar effects on cytokine-induced proliferation and Ig secretion of B cells activated by anti-CD40 Abs used in a soluble form. Finally, the PGE2 effects were mimicked by agents increasing cAMP, indicating that the PGE2 activities are likely to depend on the activation of the cAMP pathway. Altogether, the present data indicate that PGE2 stimulates human CD40-activated B cell growth, but differently modulates cytokine-induced differentiation. Thus, in microenvironments supporting the development of an immune response, the secretion of PGE2 by competent cells such as macrophages may participate in the regulation of the humoral response.
Failure to demonstrate long-lived MHC saturation both in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides.
Authors: Ishioka G Y , Adorini L , Guery J C , Gaeta F C , LaFond R , Alexander J , Powell M F , Sette A , Grey H M
Published: December 2022

Peptides that bind with high affinity to class II MHC molecules can inhibit T cell activation both in vitro and in vivo. Thus, they have been suggested as potential therapeutic agents for MHC-associated autoimmune diseases. We have constructed nonnatural peptides with high affinity for certain disease-associated MHC alleles. More specifically, a particular peptide, designated as CY-760.50, was found to have a high binding affinity for DR1, slow dissociation kinetics after binding to MHC, and prolonged stability in human serum. However, when the ability of this peptide to block peptide presentation to an influenza hemagglutinin 307-319 peptide-specific, DR1-restricted T cell clone was examined, it was found that MHC blockade could only be achieved when high concentrations of peptide were present along with Ag in the fluid phase. Thus, pretreatment of APC with MHC class II blocker, followed by removal of unbound blocker, did not result in saturation of MHC molecules, because practically immediate reacquisition of Ag-presenting capacity was observed after removal of fluid phase blocker. The pharmacokinetic behavior and the duration of blocking activity of CY-760.50 were also examined in vivo, taking advantage of the fact that the mouse MHC class II molecule I-Ab also bound CY-760.50 with high affinity. CY-760.50 administered i.v. to C57BL/6 mice was rapidly cleared from the circulation and virtually undetectable in the serum 10 min after injection. This fast clearance rate was paralleled by a similarly short duration of the MHC blockade effect. These in vivo results have implications concerning the biology of peptide-MHC interactions, and suggest that MHC blockade may not be feasible as a therapeutic approach unless effective concentrations of inhibitor can be maintained over extended periods of time in the extracellular fluids.
Up-regulation of c-myc induces the gene expression of the murine homologues of p34cdc2 and cyclin-dependent kinase-2 in T lymphocytes.
Authors: Kim Y H , Buchholz M A , Chrest F J , Nordin A A
Published: December 2022

The expression and/or up-regulation of several early T cell activation genes is dependent on signals transmitted through the interaction of IL-2 and IL-2R well before entry of the cells into S phase. In these studies, murine G0 T cells activated by immobilized anti-CD3 and subsequently blocked in late G1 expressed normal surface levels and mRNA for IL-2R alpha, IL-2R beta, and transferrin receptor (TfR). However, there was no expression of p34cdc2, and cyclin-dependent kinase (cdk)-2 was not up-regulated even in the presence of exogenous rIL-2. In addition the accumulation of c-myc-specific mRNA and protein was significantly reduced. Pretreatment of G0 T cells with c-myc antisense oligonucleotide effectively reduced the level of specific c-myc protein induced by activation of the cells by immobilized anti-CD3. The presence of antisense c-myc oligonucleotide inhibited the expression of cdc2 and cdk2 without affecting the expression of IL-2R alpha and blocked the activated T cells in the G1 phase. Together these studies demonstrate that c-myc regulates the expression of these cdk and suggest a role for c-myc in the G1/S transition.
Immunological mimicry between N-acetyl-beta-D-glucosamine and cytokeratin peptides. Evidence for a microbially driven anti-keratin antibody response.
Authors: Shikhman A R , Cunningham M W
Published: December 2022

We discovered recently that a subset of mouse anti-streptococcal mAbs cross-reacted with N-acetyl-beta-D-glucosamine (GlcNAc) and certain cytoskeletal proteins, and recognized both carbohydrate and peptide antigenic determinants. To further study the nature and biologic significance of immunologic mimicry between carbohydrate and peptide Ags, eight human hybridomas secreting anti-GlcNAc mAbs were produced by in vitro stimulation of PBL with streptococcal peptidoglycan-polysaccharide complexes and pokeweed mitogen. All human anti-GlcNAc mAbs described in this study were shown to express marked cross-reactivity with keratin from human skin in the ELISA and Western immunoblot. Mapping of the mAbs with overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 revealed that human anti-GlcNAc mAbs recognized specific cytokeratin decapeptides. Four human anti-GlcNAc mAbs recognized a single cytokeratin decapeptide whereas two mAbs reacted with several individual peptide epitopes in different fragments of cytokeratin 14. In addition, two mAbs, 1.C8 and 9.B12, reacted with multiple cytokeratin decapeptides, predominantly in the head domain of the molecule, and their reactivity correlated with positive binding of the mAbs to cytokeratin 14 in the Western immunoblot and with positive staining of human epidermis in the indirect immunofluorescent assay. Finally, we demonstrated that Abs to keratin and synthetic keratin decapeptides were induced in BALB/c mice immunized with GlcNAc-BSA but not with BSA, suggesting that the anti-keratin Ab response in vivo may be driven by nonkeratin Ags containing terminal O-linked GlcNAc.
A mutation of the mu transmembrane that disrupts endoplasmic reticulum retention. Effects on association with accessory proteins and signal transduction.
Authors: Stevens T L , Blum J H , Foy S P , Matsuuchi L , DeFranco A L
Published: December 2022

The mu heavy chain has an unusually high content of hydroxyl-containing amino acids in its membrane-spanning region. We have examined the involvement of two of these hydrophilic residues in endoplasmic reticulum (ER) retention, interactions with Ig-alpha/Ig-beta, and transmembrane signaling. Neighboring tyrosine and serine residues were mutated to either phenylalanine and alanine (mutant YS/FA) or valine and valine (mutant YS/VV). Membrane Ig (mIgM) molecules containing these mutant mu chains were expressed on the surface of transfected B lymphoma cells. Anti-Ig-induced signaling by the YS/FA mutant mIgM was equivalent to wild-type (wt) mIgM, whereas signaling by the YS/VV mutant mIgM was notably diminished. Association between mutant YS/VV mIgM and Ig-alpha/Ig-beta was detectable but reduced in comparison to YS/FA or wt mIgM. Signaling by YS/VV mutant mIgM appeared to involve Ig-alpha/Ig-beta, because these proteins were tyrosine phosphorylated on receptor cross-linking. When YS/VV and wt mu chains were cotransfected with light chains into nonlymphoid cells, mutant mIgM was expressed at the cell surface in the absence of Ig-alpha/Ig-beta, whereas wt mIgM was not. These data suggest that the mutated residues contribute to ER retention and directly or indirectly to association with Ig-alpha/Ig-beta. Moreover, ER retention can be disrupted without preventing functional association with Ig-alpha/Ig-beta. In addition, these data indicate that the hydroxyl groups of the mutated residues are not required for functional association between mu and Ig-alpha/Ig-beta because their removal did not reduce the ability of the YS/FA mutant mIgM to associate with accessory proteins or to participate in signal transduction.
The origin of the primate Mhc-DRB genes and allelic lineages as deduced from the study of prosimians.
Authors: Figueroa F , O'hUigin C , Tichy H , Klein J
Published: December 2022

MHC class II genes of the DRB family were partially sequenced from 10 individuals representing six species of prosimians: Galago senegalensis, G. moholi, Otolemur garnetti, Loris tardigradus, Petterus (Lemur) fulvus, and Lemur catta. Altogether, 41 different genes were discerned, all distinct from genes identified previously. Comparative analysis of the sequences has led to the following conclusions. First, the DRB loci present in human populations diverged from one another before the divergence of prosimian and anthropoid primates. Second, major allelic lineages of the DRB1 locus, such as DRB1*03 (DRB1*13) and DRB1*04, were established more than 85 million years ago. Third, the DRB6 gene was inactivated before the separation of prosimians and anthropoids, and has remained a pseudogene for more than 85 million years. Fourth, the primate DRB region is structurally and functionally unstable. In Lemur catta, for example, all DRB genes have apparently been lost and their function taken over by DOB and/or DPB genes. DRB genes are, however, present in a related species, Petterus (Lemur) fulvus. Fifth, the prosimian DRB3 genes are all inactive; their function seems to have been taken over by new genes. Sixth, several of the prosimian DRB genes and pseudogenes have recently been duplicated. In Otolemur garnetti, for example, one chromosome carries at least three copies of the DRB3 pseudogene.
Effects of human class I transgenes on Toxoplasma gondii cyst formation.
Authors: Brown C R , David C S , Khare S J , McLeod R
Published: December 2022

MHC class I genes are important in the regulation of cyst number in mice perorally infected with Toxoplasma gondii. Since human MHC class I molecules may be capable of functioning as restriction elements in mice, to determine whether class I genes also control outcome of T. gondii infection for humans, a transgenic mouse model was developed using human class I genes in mice. Human HLA-B27 and Cw3 transgenic mice, with the cyst-susceptible B10 mouse background, were tested for resistance to cyst formation after peroral infection with the Me49 strain of T. gondii. Introduction of the B27 gene into susceptible mice made them even more susceptible to T. gondii brain cyst formation. In contrast, HLA-DB27 transgenic mice that have a substitution of the alpha 3 portion of the human class I molecule with the alpha 3 portion of the mouse Kd molecule had the same number of brain cysts as susceptible B10 control mice when perorally infected. Similarly, HLA-Cw3 transgenic mice had the same number of brain cysts as the susceptible B10 controls. These results indicate that the interaction between CD8 on mouse T cells and the human class I molecule in transgenic mice does not always occur, and sometimes may hinder a normal response. Furthermore, B27 and Cw3 transgenes did not generate resistance to cyst formation in infection by T. gondii.
Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library.
Authors: Ames R S , Tornetta M A , Jones C S , Tsui P
Published: December 2022

A panel of mAbs against the activated complement component C5a was obtained from a filamentous phage M13-Fab display library generated from mice immunized with human rC5a. Fabs isolated from the library after iterative selection vs rC5a bound to both rC5a and purified C5. To isolate Fabs specific for neoepitopes expressed on C5a but not on the native complement component C5 the library was rescreened in a competitive manner. The phage Fab library was first incubated with immobilized C5 to deplete C5 reactive Fabs. The C5 nonadherent phage were then incubated with immobilized rC5a in the presence of soluble C5. Bound phage were eluted and subjected to two additional cycles of subtraction with immobilized C5 and selection with immobilized rC5a in the presence of soluble C5. After three cycles of this competitive biopanning four Fabs reactive with rC5a were isolated. Two bound preferentially with and neutralized C5a. Competitive biopanning of phage display libraries may increase the probability of identification of Abs of the desired specificity.
Proteinase 3. A neutrophil proteinase with activity on platelets.
Authors: Renesto P , Halbwachs-Mecarelli L , Nusbaum P , Lesavre P , Chignard M
Published: December 2022

Purified proteinase 3 (PR3) devoid of any elastase (HLE) and cathepsin G (Cat.G) contaminants, was prepared from azurophilic granules of human polymorphonuclear neutrophils by using a novel procedure. Although unable to induce platelet activation (up to 25 micrograms/ml) by itself, PR3 at a concentration as low as 2.5 micrograms/ml enhanced the platelet response to a concomitantly added threshold concentration of Cat.G, a recognized platelet agonist. In the presence of 10 micrograms/ml PR3, aggregation and degranulation of platelets induced by Cat.G were 43.2 +/- 5.9% and 27.1 +/- 1.9% as compared with 5.5 +/- 2.9% and 4.2 +/- 1.5% (n = 4) for Cat.G alone. This enhancing effect by PR3 was also observed with collagen and a cyclic endoperoxide analogue, and was inhibited by eglin C and elafin, two PR3 inhibitors. Associated with the removal of activity by a anti-PR3 mAb and the lack of effect of the secretory leukocyte proteinase inhibitor, these data demonstrated that the effect is specifically related to the enzymatic activity of PR3. It is hypothesized that this mechanism could play a role in the polymorphonuclear neutrophil-mediated platelet activation, an event already known to be dependent on Cat.G and HLE. This is supported by the fact that the association of PR3 and HLE, at concentrations ineffective by themselves, was able to potentiate Cat.G-induced platelet activation.
Characterization of a new functional TCR J delta segment in humans. Evidence for a marked conservation of J delta sequences between humans, mice, and sheep.
Authors: Davodeau F , Peyrat M A , Hallet M M , Vié H , Bonneville M
Published: December 2022

Through analysis of TCR delta-chain cDNA derived from human gamma delta T cell clones and polyclonal gamma delta T cell lines, we isolated a novel functional J delta gene segment (termed J delta 4) whose genomic fragment has been mapped within the TCR-delta locus between J delta 2 and J delta 1. Frequency of J delta 4 use was estimated among adult gamma delta PBL by using V delta 1, V delta 2, and V delta 5 genes. In all cases, this new J element was used at a low, albeit significant frequency, close to that of J delta 2. Finally, like human J delta 1 and J delta 2, which show a high degree of homology with their counterparts in the mouse and sheep, but unlike other J gamma, J beta, or J alpha elements, J delta 4 turned out to be highly homologous to a recently described ovine J delta. These results suggest the existence of strong selective pressures, possibly linked to an Ag-driven process, leading to specific conservation of J delta sequences among these three species.