bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of Exon I of SMAD7 in Mice Results in Altered B Cell Responses
Authors: Li Ronggui , Rosendahl Alexander , Brodin Greger , Cheng Alec M. , Ahgren Aive , Sundquist Christina , Kulkarni Sarang , Pawson Tony , Heldin Carl-Henrik , Heuchel Rainer L.
Published: April 2014

The members of the TGF-β superfamily, i.e., TGF-β isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-β superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-β signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-β signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-β signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system’s response to TGF-β.
Phosphatidylinositol 3-Kinase/Akt Positively Regulates Fas (CD95)-Mediated Apoptosis in Epidermal Cl41 Cells
Authors: Lu Bin , Wang Liying , Stehlik Christian , Medan Djordje , Huang Chuanshu , Hu Shuiying , Chen Fei , Shi Xianglin , Rojanasakul Yon
Published: April 2014

Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Δp85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor.
Helicobacter pyloriCagA-Dependent Macrophage Migration Inhibitory Factor Produced by Gastric Epithelial Cells Binds to CD74 and Stimulates Procarcinogenic Events
Authors: Beswick Ellen J. , Pinchuk Irina V. , Suarez Giovanni , Sierra Johanna C. , Reyes Victor E.
Published: April 2014

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinflammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF binding to CD74 on GEC was confirmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA− strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA− strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric epithelium.
Central Role for MyD88 in the Responses of Microglia to Pathogen-Associated Molecular Patterns
Authors: Esen Nilufer , Kielian Tammy
Published: April 2014

Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-α, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways.
The TCR Cβ FG Loop Regulates αβ T Cell Development
Authors: Touma Maki , Chang Hsiu-Ching , Sasada Tetsuro , Handley Maris , Clayton Linda K. , Reinherz Ellis L.
Published: April 2014

The TCRβ chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an αβ TCR cavity against which CD3εγ may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15αβ TCR transfectants expressing a FG loop-deleted chain (βΔFG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type β-chain (βwt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3εγ heterodimer and the β-chain in TCR complexes containing the βΔFG variant. To further investigate the biologic role of the Cβ FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2−/− mice using bone marrow cells from βwt or βΔFG transgenic C57BL/6 (B6) mice. Both βwt and βΔFG precursor cells generate thymocytes representative of all maturational stages. However, βΔFG-expressing thymocytes dominate during subsequent development, resulting in an excess of βΔFG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique Cβ FG loop appendage primarily controls αβ T cell development through selection processes.
A Role for MAPK in Feedback Inhibition ofTcrbRecombination
Authors: Jackson Annette M. , Krangel Michael S.
Published: April 2014

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4−CD8− double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vβ to DJβ rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vβ gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vβ chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vβ chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vβ segment accessibility and normal feedback inhibition of the Vβ to DJβ rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.
Growth, Differentiation, and Malignant Transformation of Pre-B Cells Mediated by Inducible Activation of v-Abl Oncogene
Authors: Jacobsen Elizabeth A. , Ananieva Olga , Brown Matthew L. , Chang Yung
Published: April 2014

The nonreceptor tyrosine kinase, encoded by the v-Abl oncogene of Abelson murine leukemia virus induces transformation of progenitor B cells. The v-Abl oncogene promotes cell cycle progression and inhibits pre-B cell differentiation. The temperature-sensitive form of Abelson murine leukemia virus offers a reversible model to study the role of v-Abl in regulating growth and differentiation. Inactivation of v-Abl elevates p27 and Foxo3a levels and activates NF-κB/Rel, which leads to G1 arrest and induction of Ig L chain gene rearrangement, respectively. In turn, v-Abl reactivation reduces p27 and Foxo3a levels, thus permitting G1-arrested cells to reenter the cell cycle. However, the cell lines derived from SCID mice that are defective in the catalytic subunit of DNA-dependent protein kinase retain elevated levels of p27 and Foxo3a proteins despite reactivation of v-Abl. Consequently, these cells are locked in the G1 phase for an extended period of time. The few cells that manage to bypass the G1 arrest become tumorigenic and fail to undergo pre-B cell differentiation induced by v-Abl inactivation. Deregulation of p27, Foxo3a, c-myc, and NF-κB/Rel was found to be associated with the malignant transformation of SCID temperature-sensitive form of Abelson murine leukemia virus pre-B cells.
Identification of a Candidate Regulatory Element within the 5′ Flanking Region of the MouseIghLocus Defined by Pro-B Cell-Specific Hypersensitivity Associated with Binding of PU.1, Pax5, and E2A
Authors: Pawlitzky Inka , Angeles Christina V. , Siegel Andrea M. , Stanton Michelle L. , Riblet Roy , Brodeur Peter H.
Published: April 2014

The Igh locus is controlled by cis-acting elements, including Eμ and the 3′ IgH regulatory region which flank the C region genes within the well-studied 3′ part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the VH gene array that extends to the 5′ end of the locus, the 5′ border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal VH segments and scanned 46 kb of the immediate 5′ flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5′ VH gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5′ of the VH gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5′ flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating V(D)J recombination.
The Fas-Associated Death Domain Protein Is Required in Apoptosis and TLR-Induced Proliferative Responses in B Cells
Authors: Imtiyaz Hongxia Z. , Rosenberg Stephen , Zhang Yuhang , Rahman Ziaur S. M. , Hou Ying-Ju , Manser Tim , Zhang Jianke
Published: April 2014

The Fas-associated death domain protein (FADD)/Mort1 is a signaling adaptor protein which mediates the activation of caspase 8 during death receptor-induced apoptosis. Disruption of FADD in germ cells results in death receptor-independent embryonic lethality in mice. Previous studies indicated that in addition to its function in apoptosis, FADD is also required in peripheral T cell homeostasis and TCR-induced proliferative responses. In this report, we generated B cell-specific FADD-deficient mice and showed that deletion of FADD at the pro-B cell stage had minor effects on B cell development in the bone marrow, and resulted in increased splenic and lymph node B cell numbers and decreased peritoneal B1 cell numbers. As in T cells, a FADD deficiency inhibited Fas-induced apoptosis in B cells. However, B cell-proliferative responses induced by stimulation of the BCR and CD40 using anti-IgM or anti-CD40 Abs were unaffected by the absence of FADD. Further analyses revealed that FADD-deficient B cells were defective in proliferative responses induced by treatments with dsRNA and LPS which stimulate TLR3 and TLR4, respectively. Therefore, in addition to its apoptotic function, FADD also plays a role in TLR3- and TLR4-induced proliferative responses in B cells.
Precursor B Cell Receptor Signaling Activity Can Be Uncoupled from Surface Expression
Authors: Guloglu F. Betul , Roman Christopher A. J.
Published: April 2014

Signals from the precursor BCR (preBCR) cause proliferation and differentiation of progenitor (pro-) B cells into pre-B cells. Given the very low amounts of surface preBCRs and the demonstrated cell autonomy of preBCR signaling, we examined the possible occurrence of preBCR signal propagation from intracellular membranes such as the endoplasmic reticulum (ER) and the trans-Golgi network (TGN) in transformed and primary pro-B cells. PreBCRs composed of normal Ig μ or truncated Dμ heavy chains (HCs) were redirected to intracellular sites via localization sequences appended to the HC cytoplasmic tail. PreBCR complexes retained in the TGN or shunted from the TGN to lysosomes were as or 50% as active as the corresponding wild-type preBCRs in directing preBCR-dependent events, including CD2 and CD22 expression and proliferation in primary pro-B cells. This occurred despite their low to undetectable surface expression in transformed cells, which otherwise allowed significant surface accumulation of wild-type preBCRs. In contrast, ER-retained preBCRs were inactive. These results suggest that preBCR signaling is remarkably tolerant of dramatic changes in its subcellular distribution within post-ER compartments and support the possibility that the preBCR can activate signaling pathways in the TGN as well as the plasma membrane.
Evidence for a Domain-Swapped CD4 Dimer as the Coreceptor for Binding to Class II MHC
Authors: Maekawa Akiko , Schmidt Bryan , Fazekas de St. Groth Barbara , Sanejouand Yves-Henri , Hogg Philip J.
Published: April 2014

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4’s coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.
Development and Selection of Edited B Cells in B6.56R Mice
Authors: Sekiguchi Debora R. , Yunk Lenka , Gary David , Charan Deepshikha , Srivastava Bhaskar , Allman David , Weigert Martin G. , Prak Eline T. Luning
Published: April 2014

Tolerance to dsDNA is broken in mice with a high-affinity anti-DNA H chain transgene, 56R, on the C57BL/6 background (B6.56R). B6.56R produce more anti-dsDNA Abs than BALBc.56R. To investigate how anti-DNA Abs are regulated on the B6 background, phenotypic and genetic studies were performed. B6.56R have reduced numbers of B cells and phenotypically altered B cell subsets, including relative increases in the proportions of IgM-negative bone marrow B cells, cells with a marginal zone phenotype, and cells with a transitional T3 phenotype. The peripheral B cell repertoire in B6.56R is restricted: most B cells express the 56R H chain and use a similar, limited subset of editor L chains. DNA binding is more common in B6.56R because the repertoire is shifted toward L chains that are more permissive for DNA binding. H chain editing is also observed and is increased in spontaneous as compared with LPS hybridomas. A subset of spontaneous hybridomas appears to lack H chain expression.
Immunologic Consequences ofFrancisella tularensisLive Vaccine Strain Infection: Role of the Innate Immune Response in Infection and Immunity
Authors: Cole Leah E. , Elkins Karen L. , Michalek Suzanne M. , Qureshi Nilofer , Eaton Linda J. , Rallabhandi Prasad , Cuesta Natalia , Vogel Stefanie N.
Published: April 2014

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >106 organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 μg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.
HIV-1 Adapts to a Retrocyclin with Cationic Amino Acid Substitutions That Reduce Fusion Efficiency of gp41
Authors: Cole Amy L. , Yang Otto O. , Warren Andrew D. , Waring Alan J. , Lehrer Robert I. , Cole Alexander M.
Published: April 2014

Retrocyclin (RC)-101 is a cationic θ-defensin that inhibits HIV-1 entry. Passaging HIV-1BAL under selective pressure by this cyclic minidefensin resulted in only a 5- to 10-fold decrease in viral susceptibility to RC-101. Emergent viral isolates had three amino acid substitutions in their envelope glycoprotein. One was in a CD4-binding region of gp120, and the others were in the heptad repeat (HR) domains of gp41 (HR1 and HR2). Each mutation replaced an electroneutral or electronegative residue with one that was positively charged. These mutations were evaluated either alone or in combination in a single-round viral entry assay. Although the mutation in gp120 did not affect viral entry, the mutation in HR1 of gp41 conferred relative resistance to RC-101. Interestingly, the envelope with the HR2 mutation was less efficient and became codependent on the presence of RC-101 for entry. The adaptive response of HIV-1 to this cationic host defense peptide resembles the responses of bacteria that modulate their surface or membrane charge to evade analogous host defense peptides. These findings also suggest that interactions between θ-defensins and gp41 may contribute to the ability of these cyclic minidefensins to prevent HIV-1 entry into target cells.
PU.1 and a TTTAAA Element in the MyeloidDefensin-1Promoter Create an Operational TATA Box That Can Impose Cell Specificity onto TFIID Function
Authors: Yaneva Mariana , Kippenberger Serena , Wang Nan , Su Qin , McGarvey Margaret , Nazarian Arpi , Lacomis Lynne , Erdjument-Bromage Hediye , Tempst Paul
Published: April 2014

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein (“D1BP”), acting through, respectively, proximal (−22/−19) and distal (−62/−59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)α/GABPβ. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking “TTTAAA” element (−32/−27), a “weak” but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a “strong” TATA box (“TATAAA”) eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.
F4/80+ Alternatively Activated Macrophages Control CD4+ T Cell Hyporesponsiveness at Sites Peripheral to Filarial Infection
Authors: Taylor Matthew D. , Harris Anjanette , Nair Meera G. , Maizels Rick M. , Allen Judith E.
Published: April 2014

Both T cells and APC have been strongly implicated in the immune suppression observed during filarial nematode infections, but their relative roles are poorly understood, particularly in regard to timing and locality of action. Using Litomosoides sigmodontis infection of susceptible BALB/c mice, we have studied the progression of filarial immunosuppression leading to patent infection with blood microfilaremia. Patent infection is associated with decreased immune responsiveness in the draining thoracic lymph nodes (tLN) and intrinsically hyporesponsive CD4+ T cells at the infection site. We now show that we are able to separate, both in time and space, different suppressive mechanisms and cell populations that contribute to filarial hyporesponsiveness. L. sigmodontis infection recruited a F4/80+ population of alternatively activated macrophages that potently inhibited Ag-specific CD4+ T cell proliferative responses even in the presence of competent naive APC. T cell responsiveness was partially restored by neutralizing TGF-β, but not by blocking IL-10 or CTLA-4 signaling. During prepatent infection, the macrophage population was restricted to the infection site. However, once infection became patent with systemic release of microfilariae, the suppressive macrophage activity extended peripherally into the tLN. In contrast, the hyporesponsive CD4+ T cell phenotype remained localized at the infection site, and the tLN CD4+ T cell population recovered full Ag responsiveness in the absence of suppressive macrophages. Filarial immunosuppression, therefore, evolves over time at sites increasingly distal to infection, and the mechanisms of filarial down-regulation are dependent on proximity to the infection site.
Epitopes Derived by Incidental Translational Frameshifting Give Rise to a Protective CTL Response
Authors: Zook Matthew B. , Howard Michael T. , Sinnathamby Gomathinayagam , Atkins John F. , Eisenlohr Laurence C.
Published: April 2014

Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.
Identification of HLA-A*0201-Presented T Cell Epitopes Derived from the Oncofetal Antigen-Immature Laminin Receptor Protein in Patients with Hematological Malignancies
Authors: Siegel Sandra , Wagner Andreas , Friedrichs Birte , Wendeler Anneke , Wendel Lena , Kabelitz Dieter , Steinmann Jörg , Barsoum Adel , Coggin Joseph , Rohrer James , Dreger Peter , Schmitz Norbert , Zeis Matthias
Published: April 2014

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies.
Mature Hepatocyte Growth Factor/Scatter Factor on the Surface of Human Granulocytes Is Released by a Mechanism Involving Activated Factor Xa
Authors: Ohnishi Tomokazu , Kakimoto Kyoko , Bandow Kenjiro , Lowenstein Charles J. , Daikuhara Yasushi , Matsuguchi Tetsuya
Published: April 2014

Serum hepatocyte growth factor (HGF) is rapidly increased in patients suffering from various tissue injuries including arterial occlusive diseases. However, the cellular sources of the HGF increase remain largely unknown. In the present study, we showed that bioactive mature HGF is constitutively present on the surface of granulocytes in human peripheral blood. Exogenously added 125I-labeled iodo-HGF efficiently bound to granulocyte surface, whereas only a scarce amount of HGF mRNA was detected in granulocytes, indicating that the mature HGF on granulocytes is likely to be derived from other cell types. Interestingly, treatment of granulocytes with human serum rapidly induced the release of the cell surface-associated HGF. In vivo, thromboplastin injection into mice increased HGF release from transplanted human granulocytes, which was inhibited by the pretreatment with DX9065a, a specific inhibitor of factor Xa. Furthermore, DX9065a also inhibited the serum-induced HGF release from human granulocytes in vitro, suggesting that the HGF-releasing factor(s) in serum is associated with factor Xa activation. Thus, human granulocytes may function as a transporter of HGF in the peripheral blood, releasing HGF at the injured sites caused by blood coagulation, where HGF may promote tissue repair.
Sequencing, Expression, and Functional Analyses Support the Candidacy of Ncf2 in Susceptibility to Salmonella Typhimurium Infection in Wild-Derived Mice
Authors: Sancho-Shimizu Vanessa , Malo Danielle
Published: April 2014

A recessive Salmonella Typhimurium susceptibility locus (immunity to Typhimurium (Ity3) was reported previously on distal mouse chromosome 1 using a cross between C57BL/6J and wild-derived MOLF/Ei mice. This quantitative trait locus is located in a genomic region spanning 84 Mb, rich in candidate genes for which a role in host resistance to Salmonella infection is either known or can be envisioned. In this study, we report the evaluation of neutrophil cytosolic factor 2 (Ncf2) as a candidate Salmonella susceptibility gene for Ity3. Ncf2 encodes p67phox, a subunit of the multiprotein enzyme complex NADPH oxidase, known to be responsible for the generation of superoxides. Congenic mice carrying the Ity3 region from MOLF/Ei, B6.MOLF-Ity/Ity3 were more susceptible to infection compared with control mice heterozygous at Ity3, B6.MOLF-Ity/Ity3MOLF/B6, confirming the existence of a recessive Salmonella susceptibility locus on distal chromosome 1. Spleen Ncf2 expression levels were lower in infected congenic mice homozygous for the MOLF/Ei allele at Ity3 compared with mice heterozygous at Ity3. C57BL/6J and MOLF/Ei Ncf2 sequence comparisons revealed one nonconservative amino acid change (R394Q) in the functional and highly conserved Phox and Bem1 domain of the protein. Functional analysis revealed that the MOLF/Ei allele had reduced PMA- and Salmonella-induced superoxide induction as compared with their wild-type counterparts ex vivo. The R394Q substitution seems to occur on an amino acid involved in electrostatic interactions with p40phox, crucial in its activation. Moreover, a human mutation in the corresponding R395W, resulting in chronic granulatomous disease, is known to lead to reduced superoxide levels. These results support the candidacy of Ncf2 as the gene underlying Ity3.
Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D
Authors: Hartshorn Kevan L. , White Mitchell R. , Tecle Tesfaldet , Holmskov Uffe , Crouch Erika C.
Published: April 2014

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.
Maintenance of HIV-Specific CD4+ T Cell Help Distinguishes HIV-2 from HIV-1 Infection
Authors: Duvall Melody G. , Jaye Assan , Dong Tao , Brenchley Jason M. , Alabi Abraham S. , Jeffries David J. , van der Sande Marianne , Togun Toyin O. , McConkey Samuel J. , Douek Daniel C. , McMichael Andrew J. , Whittle Hilton C. , Koup Richard A. , Rowland-Jones Sarah L.
Published: April 2014

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57−), and more frequently produced IFN-γ or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-γ+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.
STAT1 Regulates IFN-αβ- and IFN-γ-Dependent Control of Infection withChlamydia pneumoniaeby Nonhemopoietic Cells
Authors: Rothfuchs Antonio Gigliotti , Trumstedt Christian , Mattei Fabrizio , Schiavoni Giovanna , Hidmark Åsa , Wigzell Hans , Rottenberg Martín E.
Published: April 2014

STAT1 mediates signaling in response to IFN-α, -β, and -γ, cytokines required for protective immunity against several viral, bacterial, and eukaryotic pathogens. The protective role of STAT1 in the control of intranasal infection with the obligate intracellular bacterium Chlamydia pneumoniae was analyzed. IFN-γ−/− or IFN-γ receptor (R)−/− mice were highly susceptible to infection with C. pneumoniae. We found that STAT1−/− mice were even more susceptible to C. pneumoniae than IFN-γ−/− or IFN-γR−/− mice. Phosphorylation of STAT1 was detected in the lungs of C. pneumoniae-infected wild-type, IFN-γR−/−, and IFN-αβR−/− mice, but not in mice lacking both IFN-αβR and IFN-γR. In line with this, IFN-αβR−/−/IFN-γR−/− mice showed increased susceptibility to infection compared with IFN-γR−/− mice. However, C. pneumoniae-infected IFN-αβR−/− or IFN regulatory factor 3−/− mice showed no increased susceptibility and similar IFN-γ expression compared with wild-type mice. CD4+ or CD8+ cells released IFN-γ in vivo and conferred protection against C. pneumoniae in a STAT1-independent manner. In contrast, STAT1 mediated a nonredundant protective role of nonhemopoietic cells but not of hemopoietic cells. Nonhemopoietic cells accounted for the expression of STAT1-mediated indoleamine 2, 3-dioxygenase and the p47 GTPase LRG-47, but not inducible NO synthase mRNA. In summary, we demonstrate that STAT1 mediates a cooperative effect of IFN-αβ and IFN-γ on nonhemopoietic cells, resulting in protection against C. pneumoniae.
Leukocyte Recruitment to the Inflamed Glomerulus: A Critical Role for Platelet-Derived P-Selectin in the Absence of Rolling
Authors: Kuligowski Michael P. , Kitching A. Richard , Hickey Michael J.
Published: April 2014

The renal glomerulus is one of the few sites within the microvasculature in which leukocyte recruitment occurs in capillaries. However, due to the difficulty of directly visualizing the glomerulus, the mechanisms of leukocyte recruitment to glomerular capillaries are poorly understood. To overcome this, we rendered murine kidneys hydronephrotic to allow the visualization of the functional glomerular microvasculature during an inflammatory response. These experiments demonstrated that following infusion of anti-glomerular basement membrane (GBM) Ab, leukocytes became adherent in glomerular capillaries via a process of immediate arrest, without undergoing prior detectable rolling. However, despite the absence of rolling, this recruitment involved nonredundant roles for the P-selectin/P-selectin glycoprotein ligand-1 and β2 integrin/ICAM-1 pathways, suggesting that a novel form of the multistep leukocyte adhesion cascade occurs in these vessels. Anti-GBM Ab also increased glomerular P-selectin expression and induced a P-selectin-independent increase in platelet accumulation. Moreover, platelet depletion prevented both the increase in glomerular P-selectin, and the leukocyte recruitment induced by anti-GBM Ab. Furthermore, depletion of neutrophils and platelets also prevented the increase in urinary protein excretion induced by anti-GBM Ab, indicating that their accumulation in glomeruli contributed to the development of renal injury. Finally, infusion of wild-type platelets into P-selectin-deficient mice restored the ability of glomeruli in these mice to support leukocyte adhesion. Together, these data indicate that anti-GBM Ab-induced leukocyte adhesion in glomeruli occurs via a novel pathway involving a nonrolling interaction mediated by platelet-derived P-selectin.
Antifibrotic Activity of an Inhibitor of Group IIA Secretory Phospholipase A2 in Young Spontaneously Hypertensive Rats
Authors: Levick Scott , Loch David , Rolfe Barbara , Reid Robert C. , Fairlie David P. , Taylor Stephen M. , Brown Lindsay
Published: April 2014

The development of fibrosis in the chronically hypertensive heart is associated with infiltration of inflammatory cells and cardiac hypertrophy. In this study, an inhibitor of the proinflammatory enzyme, group IIA human secretory phospholipase A2 (sPLA2-IIA), has been found to prevent collagen deposition as an important component of cardiovascular remodeling in a rat model of developing chronic hypertension. Daily treatment of young male spontaneously hypertensive rats (SHR) with an sPLA2-IIA inhibitor (KH064, 5-(4-benzyloxyphenyl)-4S-(phenyl-heptanoylamino)-pentanoic acid, 5 mg/kg/day p.o.) prevented increases in the content of perivascular (SHR 20.6 ± 0.9%, n = 5; SHR+KH064 14.0 ± 1.2%, n = 5) and interstitial (SHR 7.9 ± 0.3%, n = 6; SHR+KH064 5.4 ± 0.7%, n = 6) collagen in the left ventricle of rat hearts, but did not affect numbers of infiltrating monocytes/macrophages, left ventricular hypertrophy (SHR 2.88 ± 0.08, n = 12; SHR+KH064 3.09 ± 0.08 mg/g body weight, n = 9), increased systolic blood pressure, or thoracic aortic responses. This selective antifibrotic activity suggests that sPLA2-IIA may have an important but specific role in cardiac fibrosis, and that its inhibitors could be useful in dissecting molecular pathways leading to fibrotic conditions.
Different Stabilities of the Structurally Related Receptors for IgE and IgG on the Cell Surface Are Determined by Length of the Stalk Region in Their α-Chains
Authors: Kubota Toshiyuki , Mukai Kaori , Minegishi Yoshiyuki , Karasuyama Hajime
Published: April 2014

A variant of the high affinity IgE receptor FcεRI, which is composed of α- and γ-chains without the β-chain, is expressed on human APC, such as dendritic cells, and has been suggested to facilitate Ag uptake through IgE and hence to facilitate Ag presentation to T cells. The level of FcεRI on these cells is correlated with the serum IgE concentration, suggesting IgE mediates the up-regulation of the αγ2-type FcεRI. The IgE-mediated FcεRI up-regulation on mast cells and basophils has been shown to enhance the ability of these cells to release chemical mediators and cytokines that are responsible for allergic inflammatory reactions. Here, to elucidate the mechanism controlling FcεRI expression, we compared two structurally related Ig receptors, human FcεRI and FcγRIIIA, which carry different α-chains but the same γ-chains. The half-life of FcεRI on the cell surface was short unless it bound IgE, whereas FcγRIIIA was stably expressed without IgG binding. Shuffling of the non Ig-binding portions of the FcεRIα and FcγRIIIAα chains revealed that the stalk region was critical in determining the difference in their stability and ligand-induced up-regulation. Unexpectedly, analyses with added or deleted amino acids in the stalk region strongly suggested that the length rather than the amino acid sequence of the stalk region was of major importance in determining the different stabilities of FcεRI and FcγRIIIA on the cell surface. This finding provides new insights into the mechanism regulating surface FcεRI expression.
Decreased Capacity of Immune Cells to Cause Tissue Injury Mediates Kidney Ischemic Preconditioning
Authors: Burne-Taney Melissa J. , Liu Manchang , Baldwin William M. , Racusen Lorraine , Rabb Hamid
Published: April 2014

Ischemic preconditioning (IP) is a well-established phenomenon, and the underlying mechanisms of IP are thought to involve adaptive changes within the injured tissue. Because one of the main functions of immune cells is to harbor memory, we hypothesized that circulating immune cells could mediate IP by responding to an initial ischemia reperfusion injury (IRI) and then mediate decreased injury after a second IRI event. C57BL/6 mice underwent 30 min of bilateral renal clamping or sham operation. At 5 days after ischemia, purified leukocytes from spleen were adoptively transferred into T cell-deficient (nu/nu) mice. After 1 wk, these mice underwent 30 min of renal IRI. The nu/nu mice receiving leukocytes from ischemic wild-type mice had significantly reduced renal injury compared with nu/nu mice receiving leukocytes from sham-operated, wild-type mice. Infiltration of neutrophil and macrophage in postischemic kidney did not correlate with the protection. No difference in kidney C3d or IgG deposition was detected between groups. Given that inducible NO synthase (iNOS) has been implicated in IP, leukocytes from ischemic or sham-operated, iNOS-deficient mice were transferred into nu/nu mice. Effects similar to those of wild-type transfer of ischemic leukocytes were demonstrated; thus, iNOS was not mediating the IP effect of leukocytes. This is the first evidence that immune cells are primed after renal IRI and thereby lose the capacity to cause kidney injury during a second episode of IRI. This finding may also be relevant for elucidating the mechanisms underlying cross-talk between injured kidney and distant organs.
Identification of Small Heat Shock Protein B8 (HSP22) as a Novel TLR4 Ligand and Potential Involvement in the Pathogenesis of Rheumatoid Arthritis
Authors: Roelofs Mieke F. , Boelens Wilbert C. , Joosten Leo A. B. , Abdollahi-Roodsaz Shahla , Geurts Jeroen , Wunderink Liza U. , Schreurs B. Willem , van den Berg Wim B. , Radstake Timothy R. D. J.
Published: April 2014

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant αA crystallin, αB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both αA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP αA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.