bluuebunny/crossref_metadata_2025 huggingface dataset:
Curcumin (Diferuloylmethane) Inhibits Receptor Activator of NF-κB Ligand-Induced NF-κB Activation in Osteoclast Precursors and Suppresses Osteoclastogenesis
Authors: Bharti Alok C. , Takada Yasunari , Aggarwal Bharat B.
Published: April 2014

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-κB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-κB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-κB activation. Curcumin inhibited the pathway leading from activation of IκBα kinase and IκBα phosphorylation to IκBα degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-κB was also confirmed independently, as RANKL failed to activate NF-κB in cells stably transfected with a dominant-negative form of IκBα and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-κB, and treatment with curcumin inhibits both the NF-κB activation and osteoclastogenesis induced by RANKL.
Conversion of CTLA-4 from Inhibitor to Activator of T Cells with a Bispecific Tandem Single-Chain Fv Ligand
Authors: Madrenas Joaquín , Chau Luan A. , Teft Wendy A. , Wu Paul W. , Jussif Jason , Kasaian Marion , Carreno Beatriz M. , Ling Vincent
Published: April 2014

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors’ native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.
IL-4 Confers NK Stimulatory Capacity to Murine Dendritic Cells: A Signaling Pathway Involving KARAP/DAP12-Triggering Receptor Expressed on Myeloid Cell 2 Molecules
Authors: Terme Magali , Tomasello Elena , Maruyama Koji , Crépineau Florent , Chaput Nathalie , Flament Caroline , Marolleau Jean-Pierre , Angevin Eric , Wagner Erwin F. , Salomon Benoı̂t , Lemonnier François A. , Wakasugi Hiro , Colonna Marco , Vivier Eric , Zitvogel Laurence
Published: April 2014

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-α, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DCGM/IL-4) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DCGM/IL-4 capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.
Autoreactive T Cells in Healthy Individuals
Authors: Danke Nancy A. , Koelle David M. , Yee Cassian , Beheray Sucheta , Kwok William W.
Published: April 2014

The presence of autoreactive CD4+ T cells in the peripheral blood of healthy human subjects was investigated after removal of CD4+CD25+ regulatory T cells (Treg). CD4+ T cells that were directed against the type 1 diabetes-associated autoantigen glutamic acid decarboxylase 65, the melanocyte differentiation Ag tyrosinase, and the cancer/testis tumor Ag NY-ESO-1 were readily derived from PBMC of healthy individuals. These autoreactive T cells could be visualized, using Ag-specific class II tetramer reagents, in the peripheral blood of most individuals examined. Addition of CD4+CD25+ Treg back to the CD4+CD25− population suppressed the expansion of the autoreactive T cells. Autoreactive T cells were cloned based on tetramer binding, and expressed characteristic activation markers upon self-Ag stimulation. These results show that autoreactive T cells are present in most healthy individuals and that Treg likely play an important role of keeping these autoreactive T cells in check.
Induction offoxP3+ Regulatory T Cells in the Periphery of T Cell Receptor Transgenic Mice Tolerized to Transplants
Authors: Cobbold Stephen P. , Castejon Raquel , Adams Elizabeth , Zelenika Diana , Graca Luis , Humm Susan , Waldmann Herman
Published: April 2014

Transplantation tolerance can be induced in mice by grafting under the cover of nondepleting CD4 plus CD8 or CD154 mAbs. This tolerance is donor Ag specific and depends on a population of CD4+ regulatory T cells that, as yet, remain poorly defined in terms of their specificity, origin, and phenotype. Blocking of the Ag-specific response in vitro with an anti-CD4 mAb allowed T cells from monospecific female TCR-transgenic mice against the male Ag Dby, presented by H-2Ek, to express high levels of foxP3 mRNA. foxP3 induction was dependent on TGF-β. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-β. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-γ and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)+ CD25+ T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF-β-dependent mechanism, and come to accumulate in tolerated grafts.
Stimulation by Soluble CD70 Promotes Strong Primary and Secondary CD8+ Cytotoxic T Cell Responses In Vivo
Authors: Rowley Tania F. , Al-Shamkhani Aymen
Published: April 2014

Identification of the signals required for optimal differentiation of naive CD8+ T cells into effector and memory cells is critical for the design of effective vaccines. In this study we demonstrate that CD27 stimulation by soluble CD70 considerably enhances the magnitude and quality of the CD8+ T cell response. Stimulation with soluble CD70 in the presence of Ag significantly enhanced the proliferation of CD8+ T cells and their ability to produce IL-2 and IFN-γ in vitro. Administration of Ag and soluble CD70 resulted in a massive (>300-fold) expansion of Ag-specific CD8+ T cells in vivo, which was due to the enhanced proliferation and survival of activated T cells. In mice that received Ag and soluble CD70, CD8+ T cells developed into effectors with direct ex vivo cytotoxicity. Furthermore, unlike peptide immunization, which resulted in a diminished response after rechallenge, CD27 stimulation during the primary challenge evoked a strong secondary response upon rechallenge with the antigenic peptide. Thus, in addition to increasing the frequency of primed Ag-specific T cells, CD27 signaling during the primary response instills a program of differentiation that allows CD8+ T cells to overcome a state of unresponsiveness. Taken together these results demonstrate that soluble CD70 has potent in vivo adjuvant effects for CD8+ T cell responses.
The Toll-Like Receptor-2/6 Agonist Macrophage-Activating Lipopeptide-2 Cooperates with IFN-γ to Reverse the Th2 Skew in an In Vitro Allergy Model
Authors: Weigt Henning , Muhlradt Peter F. , Larbig Michael , Krug Norbert , Braun Armin
Published: April 2014

Dendritic cells (DC) are the most potent APCs with the capacity to induce, modulate, or shut down immune function. These features make them potentially useful for treating diseases associated with misled immunologic responses. Therefore, it was the aim of this study to reverse the allergen-dependent Th2 reaction responsible for allergic symptoms by modulating DC function. This issue was addressed in an in vitro test system consisting of human monocyte-derived allergen-pulsed DC from allergics cocultured with autologous lymphocytes. A Th2 reaction judged by the amplification of IL-4 and the down-regulation of IFN-γ was induced by pulsing DC with the relevant allergen. To modulate this reaction, the Toll-like receptor 2/6 engaging mycoplasmal lipopetide macrophage-activating lipopeptide 2 kDa was combined with IFN-γ to stimulate allergen-pulsed DC. Such treatment resulted in a 500-fold increase in IFN-γ production in the supernatant of cocultured autologous lymphocytes, while the Th2 marker IL-4 was not affected. This phenomenon was associated with an increase in proliferation and the number of IFN-γ-producing lymphocytes. Phenotype and function of thus treated DC remained stable. These data indicate that a former allergen-dependent Th2 reaction can be reversed toward a Th1-type response by an appropriate treatment of DC.
Glycoprotein 96 Can Chaperone Both MHC Class I- and Class II-Restricted Epitopes for In Vivo Presentation, but Selectively Primes CD8+ T Cell Effector Function
Authors: Doody Amy D. H. , Kovalchin Joseph T. , Mihalyo Marianne A. , Hagymasi Adam T. , Drake Charles G. , Adler Adam J.
Published: April 2014

The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the duel function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8+ T cell effector responses. Although the ability of HSPs to elicit effector CD8 cell responses has been well established, their potential to prime CD4 cell effector responses has been relatively unexplored. In the current study we compared the ability of the endoplasmic reticulum-resident HSP gp96 to prime CD4 vs CD8 cells using TCR transgenic adoptive transfer systems and soluble gp96-peptide complexes. As expected, gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide; however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.
Loading of MHC Class I and II Presentation Pathways by Exogenous Antigens: A Quantitative In Vivo Comparison
Authors: Storni Tazio , Bachmann Martin F.
Published: April 2014

The MHC class I pathway is usually fueled by endogenous Ags, while exogenous Ags reach the MHC class II pathway. Although exogenous epitopes may also enter the MHC class I pathway, quantification of the efficiency of the process has remained a difficult task. In an attempt of such a quantification, we directly compared the amount of exogenous virus-like particles required for induction of cytotoxic T cell responses by cross-priming with the amount of virus-like particles required for induction of Th cell responses by the conventional route of MHC class II loading as an internal standard. Surprisingly, we found that cross-presentation of peptides derived from exogenous Ags on MHC class I molecules is of only marginally lower efficiency (∼1- to 10-fold) than the classical MHC class II pathway in vitro and in vivo. Thus, Ag quantities required for cross-presentation and cross-priming are similar to those required for fueling the MHC class II pathway.
IL-2 Secretion by CD4+ T Cells In Vivo Is Rapid, Transient, and Influenced by TCR-Specific Competition
Authors: Sojka Dorothy K. , Bruniquel Denis , Schwartz Ronald H. , Singh Nevil J.
Published: April 2014

The secretion of IL-2 is a critical and early landmark in the activation program of CD4+ T cells in vitro, but the lack of sensitive assays has limited its application for studying T cell activation in vivo. Using a mouse cytokine capture assay we were able to detect the rapid secretion of IL-2 after an in vivo stimulus by 1–2 h in naive T cells and as early as 30 min in memory T cells. Maximal secretion was achieved within 1–2 h for memory cells or 6–8 h for naive T cells. Surprisingly IL-2 production terminated quickly in vivo and secretion was undetectable by 20–24 h in either cell type. We further demonstrated that this short duration of secretion can be influenced by cellular competition between Ag-specific CD4+ T cells. The consequences of competition were mimicked by reducing the strength of the antigenic stimulus. These data argue that early competition between T cells influences both the eventual frequency of IL-2 producers in the population and also the duration of their secretion, potentially by altering the strength or duration of the stimulus available to each T cell.
Genetic Makeup of theDRRegion in Rhesus Macaques: Gene Content, Transcripts, and Pseudogenes
Authors: de Groot Nanine , Doxiadis Gaby G. , de Groot Natasja G. , Otting Nel , Heijmans Corrine , Rouweler Annemiek J. M. , Bontrop Ronald E.
Published: April 2014

In the human population, five major HLA-DRB haplotypes have been identified, whereas the situation in rhesus macaques (Macaca mulatta) is radically different. At least 30 Mamu-DRB region configurations, displaying polymorphism with regard to number and combination of DRB loci present per haplotype, have been characterized. Until now, Mamu-DRB region genes have been studied mainly by genomic sequencing of polymorphic exon 2 segments. However, relatively little is known about the expression status of these genes. To understand which exon 2 segments may represent functional genes, full-length cDNA analyses of -DRA and -DRB were initiated. In the course of the study, 11 cDRA alleles were identified, representing four distinct gene products. Amino acid replacements are confined to the leader peptide and cytoplasmatic tail, whereas residues of the α1 domain involved in peptide binding, are conserved between humans, chimpanzees, and rhesus macaques. Furthermore, from the 11 Mamu-DRB region configurations present in this panel, 28 cDRB alleles were isolated, constituting 12 distinct cDRA/cDRB configurations. Evidence is presented that a single configuration expresses maximally up to three -DRB genes. For some exon 2 DRB sequences, the corresponding transcripts could not be detected, rendering such alleles as probable pseudogenes. The full-length cDRA and cDRB sequences are necessary to construct Mhc class II tetramers, as well as transfectant cell lines. As the rhesus macaque is an important animal model in AIDS vaccine studies, the information provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian human immunodeficiency virus-infected rhesus macaques.
Fusion of Two Malaria Vaccine Candidate Antigens Enhances Product Yield, Immunogenicity, and Antibody-Mediated Inhibition of Parasite Growth In Vitro
Authors: Pan Weiqing , Huang Daqing , Zhang Qingfeng , Qu Li , Zhang Dongmei , Zhang Xiaoli , Xue Xiangyang , Qian Feng
Published: April 2014

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and ∼1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1–19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1–19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.
Structures of HLA-A*1101 Complexed with Immunodominant Nonamer and Decamer HIV-1 Epitopes Clearly Reveal the Presence of a Middle, Secondary Anchor Residue
Authors: Li Lenong , Bouvier Marlene
Published: April 2014

HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313–321) (AIFQSSMTK) and decamer Nef(73–82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.
Toll-Like Receptor 4 Dependence of Innate and Adaptive Immunity to Salmonella: Importance of the Kupffer Cell Network
Authors: Vazquez-Torres Andrés , Vallance Bruce A. , Bergman Molly A. , Finlay B. Brett , Cookson Brad T. , Jones-Carson Jessica , Fang Ferric C.
Published: April 2014

Mammalian cells recognize LPS from Gram-negative bacteria via the Toll-like receptor 4 (TLR4) complex. During experimental Salmonella infection, C3H/HeJ mice carrying a dominant-negative mutation in TLR4 exhibited delayed chemokine production, impaired NO generation, and attenuated cellular immune responses. However, dramatically enhanced bacterial growth within the Kupffer cell network before the recruitment of inflammatory cells appeared to be primarily responsible for the early demise of Salmonella-infected TLR4-deficient mice. LPS-TLR4 signaling plays an essential role in the generation of both innate and adaptive immune responses throughout the course of infection with Gram-negative bacteria. Alternative pattern-recognition receptors cannot completely compensate for the loss of TLR4, and compensation occurs at the expense of an increased microbial burden.
Induction of HIV Immunity in the Genital Tract After Intranasal Delivery of a MVA Vector: Enhanced Immunogenicity After DNA Prime-Modified Vaccinia Virus Ankara Boost Immunization Schedule
Authors: Gherardi M. Magdalena , Pérez-Jiménez Eva , Nájera José Luis , Esteban Mariano
Published: April 2014

Vaccines intended to prevent mucosal transmission of HIV should be able to induce multiple immune effectors in the host including Abs and cell-mediated immune responses at mucosal sites. The aim of this study was to characterize and to enhance the immunogenicity of a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 Env IIIB Ag (MVAenv) inoculated in BALB/c mice by mucosal routes. Intravaginal inoculation of MVAenv was not immunogenic, whereas intranasally it induced a significant immune response to the HIV Ag. Intranasal codelivery of MVAenv plus cholera toxin (CT) significantly enhanced the cellular and humoral immune response against Env in the spleen and genitorectal draining lymph nodes, respectively. Heterologous DNAenv prime-MVAenv boost by intranasal immunization, together with CT, produced a cellular immune response in the spleen 10-fold superior to that in the absence of CT. A key finding of these studies was that both MVAenv/MVAenv and DNAenv/MVAenv schemes, plus CT, induced a specific mucosal CD8+ T cell response in genital tissue and draining lymph nodes. In addition, both immunizations also generated systemic Abs, and more importantly, mucosal IgA and IgG Abs in vaginal washings. Specific secretion of β-chemokines was also generated by both immunizations, with a stronger response in mice immunized by the DNA-CT/MVA-CT regimen. Our findings are of relevance in the area of vaccine development and support the optimization of protocols of immunization based on MVA as vaccine vectors to induce mucosal immune responses against HIV.
Immunological Characteristics Associated with the Protective Efficacy of Antibodies to Ricin
Authors: Maddaloni Massimo , Cooke Corrie , Wilkinson Royce , Stout Audrey V. , Eng Leta , Pincus Seth H.
Published: April 2014

A/B toxins, produced by bacteria and plants, are among the deadliest molecules known. The B chain binds the cell, whereas the A chain exerts the toxic effect. Both anti-A chain and anti-B chain Abs can neutralize toxins in vivo and in vitro. B chain Abs block binding of the toxin to the cell. It is not known how anti-A chain Abs function. Working with ricin toxin, we demonstrate that immunization with A chain induces greater protection than immunization with B chain. A panel of mAbs, binding to A chain, B chain, or both chains, has been produced and characterized. Immunologic characteristics evaluated include isotype, relative avidity, and epitope specificity. The ability to inhibit ricin enzymatic or cell binding activity was studied, as was the ability to block ricin-mediated cellular cytotoxicity on human and murine cell lines. Finally, the in vivo protective efficacy of the Abs in mice was studied. The Ab providing the greatest in vivo protective efficacy was directed against the A chain. It had the greatest relative avidity and the greatest ability to block enzymatic function and neutralize cytotoxicity. Interestingly, we also obtained an anti-A chain Ab that bound with high avidity, blocked enzymatic activity, did not neutralize cytotoxicity, and actually enhanced the in vivo toxicity of ricin. Anti-A chain Abs with moderate avidity had no in vivo effect, nor did any anti-B chain Abs.
Mycobacterium tuberculosis-Induced CXCR4 and Chemokine Expression Leads to Preferential X4 HIV-1 Replication in Human Macrophages
Authors: Hoshino Yoshihiko , Tse Doris B. , Rochford Gemma , Prabhakar Savita , Hoshino Satomi , Chitkara Nishay , Kuwabara Kenichi , Ching Elbert , Raju Bindu , Gold Jeffrey A. , Borkowsky William , Rom William N. , Pine Richard , Weiden Michael
Published: April 2014

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1β), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1α). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4, using HIV-1, but not of CCR5, using HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.
IgA Class Switch Occurs in the Organized Nasopharynx- and Gut-Associated Lymphoid Tissue, but Not in the Diffuse Lamina Propria of Airways and Gut
Authors: Shikina Takashi , Hiroi Takachika , Iwatani Kohichi , Jang Myoung Ho , Fukuyama Satoshi , Tamura Manabu , Kubo Takeshi , Ishikawa Hiromichi , Kiyono Hiroshi
Published: April 2014

Secretory IgA plays a crucial role in the host immune response as a first line of defense. A recent demonstration of in situ IgA class switching in intestinal lamina propria provided an opportunity to reconsider the model for the homing of IgA-committed B cells characterized by distinctive trafficking patterns to effector sites. Those effector sites depend on the organized mucosa-associated lymphoid tissues as their site of induction. In this report we show the preferential presence of IgM+B220+ and IgA+B220+ cells belonging to pre- and post-IgA isotype class-switched cells in the organized mucosa-associated lymphoid tissues, such as nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer’s patches, and the defect of those populations in the diffuse effector tissues, such as the nasal passage and intestinal lamina propria. Consistent with these findings, the expressions of a series of IgA isotype class switch recombination-related molecules, including activation-induced cytidine deaminase, Iα-Cμ circle transcripts, and Iα-Cμ circle transcripts, were selectively detected in these organized mucosa-associated lymphoid structures, but not in the diffuse mucosal effector sites. Taken together, these findings suggest that IgA isotype class switching occurs only in the organized mucosa-associated lymphoid organs (e.g., nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer’s patches), but not in the diffuse effector tissues of the upper respiratory and gastrointestinal tracts.
Cellular and Humoral Immunity against Vaccinia Virus Infection of Mice
Authors: Xu Rong , Johnson Aaron J. , Liggitt Denny , Bevan Michael J.
Published: April 2014

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8+ T cells vs that of CD4+ T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-γ responses in CD4+ and CD8+ T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4+ T cells or B cells in IgH−/− mice, but was not sensitive to CD8+ T cell depletion alone. However, a role for CD8+ T cells in primary protection was demonstrated in MHC class II−/− mice, where depleting CD8+ T cells lead to increase severity of disease. Unlike control MHC class II−/− mice, the group depleted of CD8+ T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8+ T cells can mediate protective memory. These results collectively show that both CD4+ and CD8+ T cell-mediated immunity can contribute to protection against VV infection. However, CD4+ T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8+ T cells can contribute to protection against disease.
Augmentation of Chemotherapy-Induced Cytokine Production by Expression of the Platelet-Activating Factor Receptor in a Human Epithelial Carcinoma Cell Line
Authors: Darst Marc , Al-Hassani Mohammed , Li Tao , Yi Qiaofang , Travers John M. , Lewis Davina A. , Travers Jeffrey B.
Published: April 2014

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-α induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-κB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFκB pathway using a super-repressor IκBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-κB-dependent process.
Intracellular Bacterial Infection-Induced IFN-γ Is Critically but Not Solely Dependent on Toll-Like Receptor 4-Myeloid Differentiation Factor 88-IFN-αβ-STAT1 Signaling
Authors: Rothfuchs Antonio Gigliotti , Trumstedt Christian , Wigzell Hans , Rottenberg Martin E.
Published: April 2014

Infection of murine bone marrow-derived macrophages (BMMφ) with Chlamydia pneumoniae induces IFN-αβ-dependent IFN-γ secretion that leads to control of the intracellular bacterial growth. Enhanced growth of C. pneumoniae in Toll-like receptor (TLR) 4−/− and myeloid differentiation factor (MyD) 88−/− (but not TLR2−/−, TLR6−/−, or TLR9−/−) BMMφ is shown in this study. Reduced accumulation of IFN-α and IFN-γ mRNA was also observed in TLR4−/−- and MyD88−/−-infected cells. IL-1R and IL-18R signaling did not account for differences between MyD88−/− and wild-type BMMφ. Surprisingly, infection-induced NF-κB activation as well as TNF-α, IL-1, or IL-6 mRNA expression were all normal in TLR4−/− and MyD88−/− cells. Phosphorylation of the transcription factor STAT1 during bacterial infection is IFN-αβ dependent, and necessary for increased IFN-γ mRNA accumulation and chlamydial growth control. Signaling through common cytokine receptor γ-chain and RNA-dependent protein kinase both mediated IFN-αβ-dependent enhancement of IFN-γ mRNA levels. Accumulation of IFN-γ mRNA and control of C. pneumoniae growth required NF-κB activation. Such NF-κB activation was independent of IFN-αβ, STAT1, and RNA-dependent protein kinase. In summary, C. pneumoniae-induced IFN-γ expression in BMMφ is controlled by a TLR4-MyD88-IFN-αβ-STAT1-dependent pathway, as well as by a TLR4-independent pathway leading to NF-κB activation.
Attenuation of Experimental Autoimmune Encephalomyelitis and Nonimmune Demyelination by IFN-β plus Vitamin B12: Treatment to Modify Notch-1/Sonic Hedgehog Balance
Authors: Mastronardi Fabrizio G. , Min Weixian , Wang Huimin , Winer Shawn , Dosch Michael , Boggs Joan M. , Moscarello Mario A.
Published: April 2014

Interferon-β is a mainstay therapy of demyelinating diseases, but its effects are incomplete in human multiple sclerosis and several of its animal models. In this study, we demonstrate dramatic improvements of clinical, histological, and laboratory parameters in in vivo mouse models of demyelinating disease through combination therapy with IFN-β plus vitamin B12 cyanocobalamin {B12CN) in nonautoimmune primary demyelinating ND4 (DM20) transgenics, and in acute and chronic experimental autoimmune encephalomyelitis in SJL mice. Clinical improvement (p values <0.0001) was paralleled by near normal motor function, reduced astrocytosis, and reduced demyelination. IFN-β plus B12CN enhanced in vivo and in vitro oligodendrocyte maturation. In vivo and in vitro altered expression patterns of reduced Notch-1 and enhanced expression of sonic hedgehog and its receptor were consistent with oligodendrocyte maturation and remyelination. IFN-β-B12CN combination therapy may be promising for the treatment of multiple sclerosis.
Type I Regulatory T Cells Specific for Desmoglein 3 Are More Frequently Detected in Healthy Individuals than in Patients with Pemphigus Vulgaris
Authors: Veldman Christian , Höhne Annette , Dieckmann Detlef , Schuler Gerold , Hertl Michael
Published: April 2014

Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies against desmoglein 3 (Dsg3) that are presumably regulated by Th cells. The aim of this study was to identify Dsg3-specific T regulatory (Tr) cells that may help to maintain and restore natural tolerance against Dsg3. Dsg3-responsive IL-10-secreting Tr1 cells were isolated by MACS cytokine secretion assay from healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, but were only rarely detected in PV patients. The Dsg3-specific Tr1 cells secreted IL-10, TGF-β, and IL-5 upon Ag stimulation, proliferated in response to IL-2 but not to Dsg3 or mitogenic stimuli, and inhibited the proliferative response of Dsg3- and tetanus toxoid-responsive Th clones in an Ag-specific (Dsg3) and cell number-dependent manner. Moreover, their inhibitory effect was blocked by Ab against IL-10, TGF-β, and by paraformaldehyde fixation. These observations strongly suggest that 1) Dsg3-responsive Tr1 cells predominate in healthy individuals, 2) their growth requires the presence of IL-2, and 3) they exert their Dsg3-dependent inhibitory function by the secretion of IL-10 and TGF-β. Because autoaggressive T cells responsive to identical epitopes of Dsg3 were recently found both in PV patients and healthy individuals, the identified Tr1 cells may be critically involved in the maintenance and restoration of tolerance against Dsg3.
Advantages and Disadvantages of Cytidine Deamination
Authors: Cascalho Marilia
Published: April 2014

Cytidine deamination of nucleic acids underlies diversification of Ig genes and inhibition of retroviral infection, and thus, it would appear to be vital to host defense. The host defense properties of cytidine deamination require two distinct but homologous cytidine deaminases—activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G. Although cytidine deamination has clear benefits, it might well have biological costs. Uncontrolled cytidine deamination might generate misfolded polypeptides, dominant-negative proteins, or mutations in tumor suppressor genes, and thus contribute to tumor formation. How cytidine deaminases target a given nucleic acid substrate at specific sequences is not understood, and what protects cells from uncontrolled mutagenesis is not known. In this paper, I shall review the functions and regulation of activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G, and speculate about the basis for site specificity vis-à-vis generalized mutagenesis.
Cutting Edge: IL-2 Is Critically Required for the In Vitro Activation of CD4+CD25+ T Cell Suppressor Function
Authors: Thornton Angela M. , Donovan Erin E. , Piccirillo Ciriaco A. , Shevach Ethan M.
Published: April 2014

CD4+CD25+ T cells are potent immunoregulatory cells that suppress TCR-induced proliferation of CD4 and CD8 T cells in vitro by a cell contact-dependent mechanism. Addition of IL-2 or anti-CD28 abrogates CD4+CD25+-mediated suppression of proliferation and has been assumed to “break suppression.” We examined IL-2 mRNA by quantitative PCR in cocultures of mouse CD4+CD25+ and CD4+CD25− T cells. Although IL-2 gene transcription was inhibited in the presence or absence of exogenous IL-2, the addition of anti-CD28 stimulated endogenous IL-2 production. Surprisingly, transcription of IL-2 mRNA was also restored in the cocultures in the presence of anti-IL-2. These results are most compatible with a model in which CD4+CD25+ T cells do not suppress the initial activation of CD4+CD25− T cells, but mediate their suppressive effects following production of IL-2 by the responder cells resulting in both the expansion of the CD4+CD25+ T cells and the induction of their suppressor function.
Cutting Edge: Hemokinin Has Substance P-Like Function and Expression in Inflammation
Authors: Metwali Ahmed , Blum Arthur M. , Elliott David E. , Setiawan Tommy , Weinstock Joel V.
Published: April 2014

Substance P (SP) belongs to the tachykinin family of molecules. SP, cleaved from preprotachykinin A, is a neuropeptide and a proinflammatory leukocyte product. SP engages neurokinin 1 receptor (NK-1R) to stimulate cells. Hemokinin (HK) is another tachykinin that binds NK-1R. HK comes from preprotachykinin C, which is distinct from preprotachykinin A. We determined whether HK functions like SP at inflammatory sites. Preprotachykinin C mRNA was in murine schistosome granulomas and intestinal lamina propria mononuclear cells. Granuloma T cells and macrophages expressed preprotachykinin C mRNA. HK bound granuloma T cell NK-1R with high affinity. SP and HK stimulated IFN-γ production with equal potency. NK-1R antagonist blocked the effect of SP and HK on IFN-γ secretion. Thus, both HK and SP are expressed at sites of chronic inflammation and share cell origin, receptor, and immunoregulatory function. Two distinct but functionally overlapping tachykinins govern inflammation through NK-1R at sites of chronic inflammation.
Essential Role of Bystander Cytotoxic CD122+CD8+ T Cells for the Antitumor Immunity Induced in the Liver of Mice by α-Galactosylceramide
Authors: Nakagawa Ryusuke , Inui Takuo , Nagafune Ikuko , Tazunoki Yoshiko , Motoki Kazuhiro , Yamauchi Akira , Hirashima Mitsuomi , Habu Yoshiko , Nakashima Hiroyuki , Seki Shuhji
Published: April 2014

We recently reported that NK cells and CD8+ T cells contribute to the antimetastatic effect in the liver induced by α-galactosylceramide (α-GalCer). In the present study, we further investigated how CD8+ T cells contribute to the antimetastatic effect induced by α-GalCer. The injection of anti-CD8 Ab into mice 3 days before α-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-γ production nor did it reduce the NK activity of liver mononuclear cells after α-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after α-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8+ T cells did. CD122+CD8+ T cells in the liver increased after α-GalCer injection, and antitumor cytotoxicity of CD8+ T cells in the liver gradually increased until day 6. These CD8+ T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122+CD8+ T cells. The critical, but bystander role of CD122+CD8+ T cells was further confirmed by adoptive transfer experiments into CD8+ T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/α-GalCer injection for the mice to generate CD8+ T cells that can reject s.c. rechallenged B16 cells. These findings suggest that α-GalCer activates bystander antitumor CD122+CD8+ T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8+ CTLs.
Fatal Acute Lymphoblastic Leukemia in Mice Transgenic for B Cell-Restricted bcl-xL and c-myc
Authors: Swanson Penelope J. , Kuslak Sheri L. , Fang Wei , Tze Lina , Gaffney Patrick , Selby Scott , Hippen Keli L. , Nunez Gabriel , Sidman Charles L. , Behrens Timothy W.
Published: April 2014

Expression of the c-myc gene is frequently dysregulated in malignant tumors and translocations of c-myc into the Ig H chain locus are associated with Burkitt’s-type lymphoma. There is indirect evidence that bcl-x, an anti-apoptotic member of the bcl-2 gene family, may also contribute to a variety of B lymphoid tumors. In this study, we show that mice transgenic for both B cell-restricted c-myc and bcl-xL developed aggressive, acute leukemias expressing early B lineage and stem cell surface markers. Of interest, the tumor cells proliferated and differentiated down the B cell developmental pathway following in vitro treatment with IL-7. Analysis of sorted leukemic cells from spleen indicated constitutive expression of sterile μ and κ transcripts in combination with evidence for D-JH DNA rearrangements. Several B cell-specific genes were either not expressed or were expressed at low levels in primary tumor cells and were induced following culture with IL-7. IL-7 also increased V-Jκ and V-DJH rearrangements. These data demonstrate oncogenic synergy between c-myc and bcl-xL in a new mouse model for acute lymphoblastic leukemia. Tumors in these animals target an early stage in B cell development characterized by the expression of both B lineage and stem cell genes.
Linker for Activation of B Cells: A Functional Equivalent of a Mutant Linker for Activation of T Cells Deficient in Phospholipase C-γ1 Binding
Authors: Janssen Erin , Zhu Minghua , Craven Brandon , Zhang Weiguo
Published: April 2014

Adaptor proteins have important functions in coupling stimulation through immunoreceptors with downstream events. The adaptor linker for activation of B cells (LAB)/non-T cell activation linker (NTAL) is expressed in various immune cell types and has a similar domain structure as linker for activation of T cells (LAT). In this study we generated a LAB transgenic mouse to compare the functional differences between LAB and LAT. A LAB transgene expressed in LAT-deficient T cells was able to restore T cell development. However, these mice developed severe organomegaly with disorganized lymphoid tissues. Lymphocytes from these transgenic mice were hyperactivated, and T cells produced large amounts of type II cytokines. In addition, these activities appeared to be uncoupled from the TCR. An examination of the signaling capabilities of these T cells revealed that LAB resembled a LAT molecule unable to bind phospholipase C-γ1.
Toll-Like Receptor 9 Signaling Activates NF-κB through IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein in Dendritic Cells
Authors: Tsujimura Hideki , Tamura Tomohiko , Kong Hee Jeong , Nishiyama Akira , Ishii Ken J. , Klinman Dennis M. , Ozato Keiko
Published: April 2014

Unmethylated CpG DNA binds to the Toll-like receptor 9 (TLR9) and activates NF-κB to induce cytokine genes in dendritic cells (DCs). IFN regulatory factor (IRF)-8/IFN consensus sequence binding protein is a transcription factor important for development and activation of DCs. We found that DCs from IRF-8−/− mice were unresponsive to CpG and failed to induce TNF-α and IL-6, targets of NF-κB. Revealing a signaling defect selective for CpG, these cytokines were robustly induced in IRF-8−/− DCs in response to LPS that signals through TLR4. IRF-8−/− DCs expressed TLR9, adaptor myeloid differentiation factor 88, and other signaling molecules, but CpG failed to activate NF-κB in −/− cells. This was due to the selective inability of −/− DCs to activate I-κB kinase αβ, the kinases required for NF-κB in response to CpG. IRF-8 reintroduction fully restored CpG activation of NF-κB and cytokine induction in −/− DCs. Together, TLR signals that activate NF-κB are diverse among different TLRs, and TLR9 signaling uniquely depends on IRF-8 in DCs.
Infection-Induced Expansion of a MHC Class Ib-Dependent Intestinal Intraepithelial γδ T Cell Subset
Authors: Davies Adrian , Lopez-Briones Sergio , Ong Helena , O’Neil-Marshall Cynthia , Lemonnier François A. , Nagaraju Kanneboyina , Metcalf Eleanor S. , Soloski Mark J.
Published: April 2014

Salmonella species invade the host via the intestinal epithelium. Hence, intestinal intraepithelial lymphocytes (iIELs) are potentially the first element of the immune system to encounter Salmonella during infection. In this study, we demonstrate, in a mouse model, the expansion of a CD8αβ+CD94−TCRγδ+ T cell subset within the iIEL population in response to oral infection with virulent or avirulent Salmonella. This population can be detected 3 days following infection, represents up to 15% of the TCRγδ+ iIELs, and is dependent on the MHC class Ib molecule T23 (Qa-1). Qa-1 is expressed by intestinal epithelial cells and thus accessible for iIEL recognition. Such cells may play a role in the early immune response to Salmonella.