bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid and Delayed p42/p44 Mitogen-Activated Protein Kinase Activation by Nitric Oxide: The Role of Cyclic GMP and Tyrosine Phosphatase Inhibition
Authors: Callsen Dagmar , Pfeilschifter Josef , Brüne Bernhard 
Published: December 2022

Abstract
The exposure of rat mesangial cells to cytokines promoted activation of the p42/p44 mitogen-activated protein kinase (MAPK). We identified a rapid and delayed phase of MAPK activation with distinctive activity increases at 5 to 15 min and 15 to 24 h. Rapid and late MAPK activation were attenuated by the redox-modulating agent N-acetylcysteine. Specifically, late-phase activation coincided with endogenous nitric oxide (NO) generation and in turn was suppressed by the NO synthase-blocking compounds diphenyliodonium or nitroarginine methyl ester. By using NO-liberating agents such as S-nitrosoglutathione and 3-morpholinosydnonimine, we investigated intermediary signaling elements of NO in promoting MAPK activation. Early and transient activation at 5 min was suppressed by the soluble guanylyl cyclase-blocking agent 1H-(1,2,4)-oxdiazolo-(4,3-α)-6-bromoquinoxazin-1-one (NS 2028) and, moreover, was mimicked by the lipophilic cyclic GMP (cGMP) analogue 8-bromo-cGMP. In contrast, NO-mediated activation achieved within hours was unrelated to cGMP signaling. Late and persistent MAPK activation, induced by NO donors or endogenously generated NO, was found in association with inhibition of phosphatase activity. In vitro dephosphorylation of activated and immunoprecipitated p42/p44 by cytosolic phosphatases was sensitive to the readdition of NO and was found to be inhibited in cytosol of S-nitrosoglutathione-stimulated cells. Also, cells that had been exposed to cytokines for 24 h revealed a blocked phosphatase activity, which was successfully attenuated by the NO synthase inhibitor nitroarginine methyl ester and, therefore, was NO mediated. Conclusively, NO affects p42/p44 MAPK in rat mesangial cells twofold: rapid activation is cGMP mediated, whereas late activation is transmitted via inhibition of tyrosine dephosphorylation.
Genetic Immunization Generates Cellular and Humoral Immune Responses Against the Nonstructural Proteins of the Hepatitis C Virus in a Murine Model
Authors: Encke Jens , zu Putlitz Jasper , Geissler Michael , Wands Jack R. 
Published: December 2022

Abstract
Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.
IL-12 Is an Effective Adjuvant for Induction of Mucosal Immunity
Authors: Boyaka Prosper N. , Marinaro Mariarosaria , Jackson Raymond J. , Menon Satish , Kiyono Hiroshi , Jirillo Emilio , McGhee Jerry R. 
Published: December 2022

AbstractWe addressed the effects of two cytokines, IL-6 and IL-12, derived from APCs, for the development of mucosal IgA Ab responses following their nasal delivery with the protein vaccine tetanus toxoid (TT). Mice treated nasally with IL-6 and TT showed higher TT-specific serum IgG (mainly IgG1 and IgG2b) Ab responses than did control mice, but exhibited no IgE and negligible secretory IgA (S-IgA) Ab responses. In contrast, IL-12 administered nasally with TT not only induced sharp increases in TT-specific serum IgG (mainly IgG1 and IgG2b) and IgA, but also elevated mucosal S-IgA Ab responses. Coadministration of IL-6 and IL-12 with TT did not enhance the mucosal or serum Ab responses over those seen with IL-12 alone. TT-specific CD4+ T cells from mice given TT with IL-6 or IL-12 produced higher levels of IFN-γ, IL-6, and IL-10 than did those from control mice, but only negligible levels of IL-4 and IL-5. In summary, both intranasal IL-6 and IL-12 induced serum Abs that protected mice from systemic challenge with TT, whereas only IL-12 induced mucosal S-IgA Ab responses. The significance of IL-12-induced Th1-type responses for regulation of both mucosal and systemic immunity is discussed.
Follicular Exclusion and Rapid Elimination of Hen Egg Lysozyme Autoantigen-Binding B Cells Are Dependent on Competitor B Cells, But Not on T Cells
Authors: Schmidt Kerstin N. , Cyster Jason G. 
Published: December 2022

Abstract
In mice with a diverse B cell repertoire, hen egg lysozyme (HEL) autoantigen-binding B cells are excluded from follicles and eliminated in 3 days. To explore the roles of competitor B cells and of T cells in this mechanism of self-tolerance, HEL-specific B cells were transferred into mice containing HEL and deficient in endogenous B cells (μMT), T cells (TCR−/−), or B and T cells (RAG1−/−). Previous studies suggested a dual requirement for B cell receptor (BCR) engagement and competition in HEL autoantigen-binding B cell elimination, but interpretation of these experiments has been confounded by the possible failure to independently regulate autoantigen concentration and competitor B cell frequency. In experiments in this study, we have fixed one variable, HEL concentration, while varying the second, the presence or absence of other B cells. By this approach, we find that follicular exclusion and rapid elimination of autoreactive B cells require BCR engagement plus competition with other B cells, rather than BCR engagement alone. We also find, by transfers into T cell-deficient mice, that T cells are not required for this peripheral tolerance mechanism. Unexpectedly, in mice lacking both T cells and competitor B cells (RAG1−/−), transferred HEL-binding cells survive less well than in mice just lacking competitor B cells. These results suggest T cells can enhance autoreactive B cell survival. Enhanced survival of autoreactive B cells, due to the presence of T cells and the lack of competitor B cells, might contribute to the elevated frequency of autoimmunity in B cell-deficient individuals.
Clathrin-Coated Pit-Associated Proteins Are Required for Alveolar Macrophage Phagocytosis
Authors: Perry Douglas G. , Daugherty Gena L. , Martin William J. 
Published: December 2022

Abstract
During phagocytosis, phagocytic receptors and membrane material must be inserted in the pseudopod membrane as it extends over the phagocytic target. This may require a clathrin-mediated recycling mechanism similar to that postulated for leading edge formation during cell migration. To investigate this possibility, liposomes were used to deliver to intact rat alveolar macrophages (AMs): 1) Abs to clathrin, clathrin adaptor AP-2, and hsc70, and 2) amantadine. Phagocytosis was assayed by fluorometric and colorimetric techniques. Liposome-delivered Abs to clathrin and AP-2 inhibited AM phagocytosis of zymosan-coated, fluorescent liposomes from 16.3 ± 0.3 to 5.8 ± 0.3, and 10.1 ± 0.9 to 4.8 ± 0.2 liposomes/cell (p &lt; 0.01). Similarly, liposome-delivered Ab to clathrin also inhibited AM phagocytosis of IgG-opsonized RBCs from 11.7 ± 1.7 to 3.8 ± 0.7 RBCs/cell (p &lt; 0.01). Amantadine, which blocks the budding of clathrin-coated vesicles, inhibited phagocytosis from 13.8 ± 0.8 to 5.7 ± 0.6 (p &lt; 0.01). Ab blockade of hsc70, which catalyzes clathrin turnover, also inhibited phagocytosis from 9.1 ± 0.5 to 4.3 ± 0.2 (p &lt; 0.01). These findings suggest that clathrin-mediated receptor/membrane recycling is required for phagocytosis.
Activation-Induced Resistance of Human Macrophages to HIV-1 Infection In Vitro
Authors: Zybarth Gabriele , Reiling Norbert , Schmidtmayerova Helena , Sherry Barbara , Bukrinsky Michael 
Published: December 2022

Abstract
Cells of the monocyte/macrophage lineage are the first targets of HIV-1 in patients and also serve as reservoirs for the virus during the course of infection. We investigated the effects of cell activation on early events of HIV-1 infection of monocyte-derived macrophages. Addition of LPS, a potent stimulator of macrophages, at the time of infection stimulated entry of HIV-1 into monocyte-derived macrophages, as judged by accumulation of early products of RT, but inhibited the synthesis of late RT products and strongly repressed nuclear import of the viral DNA, resulting in protection from infection. This effect was mediated by the CD14 receptor and involved activation of the p38 mitogen-activated protein kinase pathway. Disruption of this signaling pathway using a specific inhibitor of the p38 mitogen-activated protein kinase (SB203580) restored HIV-1 infection in the presence of LPS. These results suggest a novel view of the role of macrophage activation in anti-HIV responses of the immune system.
Interaction Between α5β1 Integrin and Secreted Fibronectin Is Involved in Macrophage Differentiation of Human HL-60 Myeloid Leukemia Cells
Authors: Laouar Amale , Collart Frank R. , Chubb Cynthia B. H. , Xie Bei , Huberman Eliezer 
Published: December 2022

Abstract
We examined the role of fibronectin (FN) and FN-binding integrins in macrophage differentiation. Increased FN and α5β1 integrin gene expression was observed in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or macrophage-CSF-treated blood monocytes before the manifestation of macrophage markers. After treatment of HL-60 cells and monocytes, newly synthesized FN was released and deposited on the dishes. An HL-60 cell variant, HL-525, which is deficient in the protein kinase Cβ (PKC-β) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Transfecting HL-525 cells with a PKC-β expression plasmid restored PMA-induced FN gene expression and macrophage differentiation. Untreated HL-525 cells (which have a high level of the α5β1 integrin) incubated on FN differentiated into macrophages. The percentage of cells having a macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525 cells, or by either PMA or macrophage-CSF in monocytes was reduced in the presence of mAbs to FN and α5β1 integrin. The integrin-signaling nonreceptor tyrosine kinase, p72Syk, was activated in PMA-treated HL-60 and FN-treated HL-525 cells. We suggest that macrophage differentiation involves the activation of PKC-β and expression of extracellular matrix proteins such as FN and the corresponding integrins, α5β1 integrin in particular. The stimulated cells, through the integrins, attach to substrates by binding to the deposited FN. This attachment, in turn, may through integrin signaling activate nonreceptor tyrosine kinases, including p72Syk, and later lead to expression of other genes involved in evoking the macrophage phenotype.
Role of the 75-kDa TNF Receptor in TNF-Induced Activation of Neutrophil Respiratory Burst
Authors: Dri Pietro , Haas Elvira , Cramer Rita , Menegazzi Renzo , Gasparini Chiara , Martinelli Roberta , Scheurich Peter , Patriarca Pierluigi 
Published: December 2022

AbstractThe exclusive role of the 55-kDa TNF receptor (TNF-R55) as the signaling receptor in TNF-induced activation of respiratory burst by human polymorphonuclear leukocytes residing on biologic surfaces has been inferred from results obtained with receptor-specific monoclonal and polyclonal Abs. In this work, we confirm this assumption by a more direct approach, i.e., by using receptor-specific TNF mutants (p55TNF and p75TNF) and, as a novel contribution, we show that cooperation of the 75-kDa TNF receptor (TNF-R75) is required for a full blown response to the cytokine. This conclusion stems from three sets of data: 1) none of the TNF-R55-specific agonists used, i.e., mAbs or p55TNF, induced a respiratory burst comparable with that induced by TNF; 2) selective down-modulation of TNF-R75 resulted in a diminished response to TNF but not to TNF-R55-specific agonists or to the chemotactic peptide FMLP; and 3) mAbs that either block or stabilize binding of TNF to TNF-R75 inhibited the response to the cytokine, suggesting that cooperation requires not only TNF binding to the receptor but also an appropriate dissociability from it. The inhibitory effect of the Abs increased as the cytokine concentrations decreased, indicating that cooperation by TNF-R75 becomes more relevant at low TNF doses. Such a cooperation does not seem to rely on the activation of a TNF-R75-linked signaling pathway independent of TNF-R55, since the response to p55TNF and p75TNF given in combination was not higher than the response to p55TNF alone. The possible mechanisms of cooperation are discussed.
Peripheral Tolerance to Class I Mismatched Renal Allografts in Miniature Swine: Donor Antigen-Activated Peripheral Blood Lymphocytes from Tolerant Swine Inhibit Antidonor CTL Reactivity
Authors: Ierino Francesco L. , Yamada Kazuhiko , Hatch Tevis , Rembert James , Sachs David H. 
Published: December 2022

AbstractStudies utilizing partially inbred miniature swine have demonstrated that a short course of cyclosporin A leads to indefinite survival of two haplotype class I mismatched renal allografts. In the present study, we have examined peripheral regulatory mechanisms that may be involved in maintenance of tolerance by coculturing PBL from long-term tolerant animals with naive recipient-matched PBL in cell-mediated lympholysis assays. We show that PBL from tolerant animals, primed in vitro with donor Ag, suppress antidonor CTL reactivity by naive recipient-matched PBL. Suppression was not observed when PBL from naive animals, primed with donor-matched PBL, were cocultured with PBL from a second naive animal, nor did PBL from either tolerant or naive recipient-matched control animals, primed with third-party Ag, suppress the generation of anti-third-party CTL by a second naive animal. The suppression was cell dose-dependent, radiation-sensitive, required cell-to-cell contact not reversed by the provision of exogenous IL-2, and associated with lower levels of IL-2R expression on the suppressive effector group (particularly the CD8 single positive cells) when compared with the control effector group. These data indicate an association between the presence of peripheral regulatory cells demonstrable in vitro and the maintenance of tolerance to renal allografts.
Activated Eosinophils Are the Major Source of Th2-Associated Cytokines in the Schistosome Granuloma
Authors: Rumbley Catherine A. , Sugaya Hiroko , Zekavat S. Ali , El Refaei Mohamed , Perrin Peter J. , Phillips S. Michael 
Published: December 2022

AbstractEosinophils are a numerically dominant cell population within the schistosome granuloma. These granuloma eosinophils can produce a variety of cytokines, including IL-2, IL-4, IL-5, and IFN-γ. Therefore, eosinophils may play a key role in the determination of the unique cytokine microenvironment within the granuloma milieu. These studies investigated the potential role of eosinophils in the regulation of granuloma immunopathology. We have characterized spleen- and granuloma-derived eosinophils based on cellular activation and cytokine production during the development of murine schistosomiasis. Based on the criteria of hypodensity and CD69 expression, granuloma eosinophils were highly activated and very homogeneous at 7 and 11 wk postinfection. Splenic eosinophils were also activated at 7 wk postinfection, but were much more heterogeneous than their granuloma counterparts. By 11 wk postinfection, few hypodense splenic eosinophils were observed. Eosinophils represented the majority of cytokine-producing cells in the granuloma and were a dominant source of IL-4. Eosinophils also produced IL-2, IL-5, and IFN-γ, using the criteria of mRNA in situ hybridization and intracellular cytokine staining by FACS. Granuloma eosinophil activation and cytokine production were greatest at the time of maximum granuloma formation, i.e., 10–12 wk after initial cercarial exposure. Therefore, locally activated eosinophils, not Th2 lymphocytes, produce the majority of Th2 cytokines in the granuloma milieu and may be important determinators of immunopathology in schistosomiasis.
IL-10 Gene Knockout Mice Show Enhanced Th1-Like Protective Immunity and Absent Granuloma Formation FollowingChlamydia trachomatisLung Infection
Authors: Yang Xi , Gartner John , Zhu Lihua , Wang Shuhe , Brunham Robert C. 
Published: December 2022

AbstractWe previously reported that higher IL-10 production is correlated with lower IFN-γ production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-γ production and significant elevation of IL-12 and TNF-α production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.
A Comparison of C3a and C5a-Mediated Stable Adhesion of Rolling Eosinophils in Postcapillary Venules and Transendothelial Migration In Vitro and In Vivo
Authors: DiScipio Richard G. , Daffern Pamela J. , Jagels Mark A. , Broide David H. , Sriramarao P. 
Published: December 2022

AbstractThe comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1β-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in αMβ2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of αMβ2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-α4 and anti-β2 integrin mAbs. In vitro, both C3a (but not C3adesArg) and C5a (including C5adesArg)-dependent transmigration of eosinophils across IL-1β-stimulated endothelial monolayer was mediated by α4β1 and αMβ2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.
IFN-γ Induces Endothelial Cells to Proliferate and to Invade the Extracellular Matrix in Response to the HIV-1 Tat Protein: Implications for AIDS-Kaposi’s Sarcoma Pathogenesis
Authors: Fiorelli Valeria , Barillari Giovanni , Toschi Elena , Sgadari Cecilia , Monini Paolo , Stürzl Michael , Ensoli Barbara 
Published: December 2022

AbstractPrevious studies indicated that the Tat protein of HIV functions as a progression factor in Kaposi’s sarcoma (KS), an angioproliferative disease common and aggressive in HIV-1-infected individuals (AIDS-KS). In particular, Tat that is released by infected cells stimulates the growth and invasion of spindle cells of endothelial origin derived from KS lesions (KS cells). Other work suggested that inflammatory cytokines may act as initiating factors in KS since they induce normal endothelial cells to acquire the same phenotype and functional features of KS cells, including the responsiveness to Tat. In this study, we show that among the inflammatory cytokines increased in AIDS-KS lesions, IFN-γ alone is sufficient to induce endothelial cells to proliferate and to invade the extracellular matrix in response to Tat. This is because IFN-γ up-regulates the expression and activity of the receptors for Tat identified as the integrins α5β1 and αvβ3. These results suggest that, by triggering Tat effects, IFN-γ plays a major role in AIDS-KS pathogenesis.
CTLA-4 (CD152) Inhibits the Specific Lysis Mediated by Human Cytolytic T Lymphocytes in a Clonally Distributed Fashion
Authors: Saverino Daniele , Tenca Claudya , Zarcone Daniela , Merlo Andrea , Megiovanni Anna M. , Valle Maria T. , Manca Fabrizio , Grossi Carlo E. , Ciccone Ermanno 
Published: December 2022

AbstractSince the functional outcome of effector T lymphocytes depends on a balance between activatory and inhibitory receptors, we studied the ability of CTLA-4 (CD152) to inhibit the cytolytic function of CTL. In 22 TCRαβ+CD3+8+ CTL clones, activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb was inhibited by anti-CD152 mAb in a redirected killing assay. In eight clones inhibition was &gt;40%, in 10 it ranged between 20–40%, and in four it was &lt;20%. This suggests the existence of a clonal heterogeneity as well as for the ability of CTLA-4 to inhibit CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further this contention, we used an experimental model based upon Ag-specific CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected B lymphocytes, but are unable to lyse allogeneic EBV-infected B cell lines, were used in a cytolytic assay in which anti-CD152 mAb or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In this system, at variance from the redirected killing assay, cross-linking of surface molecules by mAb does not occur. Thus, addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86 mAb to the assay should result in a blockade of receptor/ligand interactions. As a consequence, inhibition of a negative signal, such as that delivered via CD152, should enhance lysis. A &gt;40% increment of target cell lysis was achieved in three of eight clones studied. Since it is not equally shared by all CTL clones, this feature also appears to be clonally distributed.