RESUMO
Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Complexo CD3/genética , Complexo CD3/imunologia , Técnicas de Introdução de Genes , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/citologia , Linfócitos T/imunologia , Timócitos/citologia , Timócitos/imunologiaRESUMO
We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.
Assuntos
Anticorpos Biespecíficos , Doenças Autoimunes/tratamento farmacológico , Fator Ativador de Células B/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fator Ativador de Células B/imunologia , Feminino , Células HEK293 , Células HT29 , Humanos , Interleucina-17/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Fator Ativador de Células B/metabolismo , Tolerância Imunológica , Interleucina-33/imunologia , Animais , Autoantígenos/administração & dosagem , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Células Dendríticas , Modelos Animais de Doenças , Humanos , Imunoglobulina M/imunologia , Interleucina-33/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos , Cultura Primária de Células , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Autoimmune manifestations are common in human immunodeficiency virus (HIV) patients. However, the autoantibody spectrum associated with HIV infection and the impact of antiretroviral therapy (ART) remains to be determined. The plasma autoantibody spectrum for HIV patients was characterized by protein microarrays containing 83 autoantigens and confirmed by enzyme-linked immunosorbent assay (ELISA). Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry and their effects on autoantibodies production were determined by B cell ELISpot. Higher levels of autoantibody and higher prevalence of elevated autoantibodies were observed in ART-naive HIV patients compared to healthy subjects and HIV patients on ART. The highest frequency of CD33(+)CD11b(+)HLA-DR(+) cells was observed in ART-naive HIV patients and was associated with the quantity of elevated autoantibodies. In addition, CD33(+)CD11b(+)HLA-DR(+) cells other than Tregs or MDSCs boost the B cell response in a dose-dependent manner by in vitro assay. In summary, HIV infection leads to elevation of autoantibodies while ART suppresses the autoimmune manifestation by decreasing CD33(+)CD11b(+)HLA-DR(+) cells in vivo.The roles of CD33(+)CD11b(+)HLA-DR(+) cells on disease progression in HIV patients needs further assessment.
Assuntos
Antirretrovirais , Autoanticorpos/sangue , Autoimunidade , Infecções por HIV , Linfócitos T Reguladores/imunologia , Antirretrovirais/administração & dosagem , Antirretrovirais/imunologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Antígeno CD11b/análise , Progressão da Doença , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Antígenos HLA-DR/análise , Humanos , Prognóstico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análiseRESUMO
UNLABELLED: Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. IMPORTANCE: There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by not only nAbs but also non-nAbs. Our results are significant in developing novel immune-based approaches to restore the functions of nAbs and non-nAbs in the circulation of HIV-1-infected individuals to specifically target and clear HIV-1 virions and infected cells. Our data also provide new insights into the mechanisms by which HIV-1 virions and infected cells escape Ab-mediated immunity and could aid in the design and/or development of therapeutic HIV-1 vaccines. In addition, a combination of antiretroviral therapy with RCA blockage, provirus activators, and therapeutic vaccines may represent a novel approach to eliminate HIV-1 reservoirs, i.e., the infected cells harboring replication-competent proviruses and residual viremia.
Assuntos
Anticorpos Neutralizantes/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD59/imunologia , Proteínas do Sistema Complemento/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Provírus/imunologia , Vírion/imunologia , Latência Viral/imunologia , Feminino , Humanos , MasculinoRESUMO
Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD59/imunologia , Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Provírus/imunologia , Adolescente , Adulto , Fármacos Anti-HIV/administração & dosagem , Linhagem Celular , Feminino , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1ß treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE: Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.
Assuntos
Macrófagos/virologia , Vaccinia virus/fisiologia , Vacínia/virologia , Replicação Viral , Animais , Células Cultivadas , Humanos , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Monócitos/virologia , Vacínia/metabolismoRESUMO
UNLABELLED: The pathogenesis of accelerated liver damage in subjects coinfected with hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) remains largely unknown. Recent studies suggest that ongoing chronic liver inflammation is responsible for the liver injury in HCV-infected patients. We aimed to determine whether HIV-1 coinfection altered intrahepatic inflammatory profiles in HCV infection, thereby hastening liver damage. We used a real-time RT-PCR-based array to comparatively analyze intrahepatic inflammation gene profiles in liver biopsy specimens from HCV-infected (nâ=â16), HCV/HIV-1-coinfected (nâ=â8) and uninfected (nâ=â8) individuals. We then used human hepatocytes to study the molecular mechanisms underlying alternations of the inflammatory profiles. Compared with uninfected individuals, HCV infection and HCV/HIV-1 coinfection markedly altered expression of 59.5% and 50.0% of 84 inflammation-related genes tested, respectively. Among these genes affected, HCV infection up-regulated the expression of 24 genes and down-regulated the expression of 26 genes, whereas HCV/HIV-1 coinfection up-regulated the expression of 21 genes and down-regulated the expression of 21 genes. Compared with HCV infection, HCV/HIV-1 coinfection did not dramatically affect intrahepatic gene expression profiles of cytokines and their receptors, but profoundly altered expression of several chemokine genes including up-regulation of the CXCR3-associated chemokines. Human hepatocytes produced these chemokines in response to virus-related microbial translocation, viral protein stimulation, and antiviral immune responses. CONCLUSIONS: HIV-1 coinfection profoundly alters intrahepatic chemokine but not cytokine profiles in HCV-infected subjects. The altered chemokines may orchestrate the tissue-specific and cell-selective trafficking of immune cells and autoimmunity to accelerate liver disease in HCV/HIV-1 coinfection.
Assuntos
Quimiocinas/metabolismo , Coinfecção/virologia , Infecções por HIV/complicações , Infecções por HIV/virologia , Hepatite C/complicações , Mediadores da Inflamação/metabolismo , Fígado/patologia , Adolescente , Adulto , Idoso , Quimiocinas/genética , Estudos de Coortes , Coinfecção/genética , Demografia , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Hepatite C/genética , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR3/metabolismo , Adulto JovemRESUMO
Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs.
Assuntos
Leucócitos/virologia , Microdomínios da Membrana/metabolismo , Receptores Virais/biossíntese , Vaccinia virus/fisiologia , Tropismo Viral , Ligação Viral , Células Cultivadas , HumanosRESUMO
Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⺠T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA), but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA) binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA)-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA)-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.
Assuntos
Antígenos CD/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , HIV-1/imunologia , Neisseria gonorrhoeae/metabolismo , Linfócitos T Citotóxicos/imunologia , Imunidade Adaptativa , Proliferação de Células , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Regulação para Baixo , Fímbrias Bacterianas/fisiologia , Humanos , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Neisseria gonorrhoeae/fisiologia , Especificidade da Espécie , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/virologia , Antígeno CD83RESUMO
UNLABELLED: Several enveloped viruses including human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), Ebola virus, vaccinia virus, and influenza virus have been found to incorporate host regulators of complement activation (RCA) into their viral envelopes and, as a result, escape antibody-dependent complement-mediated lysis (ADCML). Hepatitis C virus (HCV) is an enveloped virus of the family Flaviviridae and incorporates more than 10 host lipoproteins. Patients chronically infected with HCV develop high-titer and crossreactive neutralizing antibodies (nAbs), yet fail to clear the virus, raising the possibility that HCV may also use the similar strategy of RCA incorporation to escape ADCML. The current study was therefore undertaken to determine whether HCV virions incorporate biologically functional CD59, a key member of RCA. Our experiments provided several lines of evidence demonstrating that CD59 was associated with the external membrane of HCV particles derived from either Huh7.5.1 cells or plasma samples from HCV-infected patients. First, HCV particles were captured by CD59-specific Abs. Second, CD59 was detected in purified HCV particles by immunoblot analysis and in the cell-free supernatant from HCV-infected Huh7.5.1 cells, but not from uninfected or adenovirus serotype 5 (Ad5) (a nonenveloped cytolytic virus)-infected Huh7.5.1 cells by enzyme-linked immunosorbent assay. Last, abrogation of CD59 function with its blockers increased the sensitivity of HCV virions to ADCML, resulting in a significant reduction of HCV infectivity. Additionally, direct addition of CD59 blockers into plasma samples from HCV-infected patients increased autologous virolysis. CONCLUSION: Our study, for the first time, demonstrates that CD59 is incorporated into both cell line-derived and plasma primary HCV virions at levels that protect against ADCML. This is also the first report to show that direct addition of RCA blockers into plasma from HCV-infected patients renders endogenous plasma virions sensitive to ADCML.
Assuntos
Antígenos CD59/metabolismo , Ativação do Complemento , Hepacivirus/imunologia , Imunidade Humoral , Vírion/imunologia , Linhagem Celular Tumoral , Feminino , Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Vírion/metabolismoRESUMO
The vast majority of people living with human immunodeficiency virus type 1 (HIV-1) have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG) neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP), and an increase in the resistance at threshold (R(Th)). Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5) activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs) of DRGs. However, Tat-mediated actions on the rheobase and R(Th) were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons.
Assuntos
Gânglios Espinais/citologia , Gânglios Espinais/patologia , HIV-1 , Neurônios/efeitos dos fármacos , Dor/patologia , Dor/virologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Capsaicina/farmacologia , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/biossíntese , Quinase 5 Dependente de Ciclina/genética , Masculino , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Dor/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Many pathogenic enveloped viruses, including HIV-1, escape complement-mediated virolysis by incorporating host cell regulators of complement activation into their own viral envelope. The presence of complement regulators including CD59 on the external surface of the viral envelope confers resistance to complement-mediated virolysis, which may explain why human pathogenic viruses such as HIV-1 are not neutralized by complement in human fluids, even in the presence of high Ab titers against the viral surface proteins. In this study, we report the development of a recombinant form of the fourth domain of the bacterial toxin intermedilysin (the recombinant domain 4 of intermedilysin [rILYd4]), a 114 aa protein that inhibits human CD59 function with high affinity and specificity. In the presence of rILYd4, HIV-1 virions derived from either cell lines or peripheral blood mononuclear cells of HIV-1-infected patients became highly sensitive to complement-mediated lysis activated by either anti-HIV-1 gp120 Abs or by viral infection-induced Abs present in the plasma of HIV-1-infected individuals. We also demonstrated that rILYd4 together with serum or plasma from HIV-1-infected patients as a source of anti-HIV-1 Abs and complement did not mediate complement-mediated lysis of either erythrocytes or peripheral blood mononuclear cells. These results indicate that rILYd4 may represent a novel therapeutic agent against HIV-1/AIDS.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Bacteriocinas/imunologia , Antígenos CD59/metabolismo , Proteínas do Sistema Complemento/imunologia , HIV-1/imunologia , Bacteriocinas/farmacologia , Antígenos CD59/imunologia , Ativação do Complemento/imunologia , Citometria de Fluxo , Humanos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Microbial translocation has been recognized as an important factor in monocyte activation and contributing to AIDS pathogenesis with elevated plasma lipopolysaccharide (LPS) levels, as a marker for microbial translocation, seen in advanced HIV disease. Therefore, the current study was undertaken to assess monocyte activation in vitro by LPS and to determine its impact on monocyte phenotype. METHODS: Monocytes from non-HIV-infected donors were analyzed for CD14, CD16, CD69, TNFα, and CCR5 by flow cytometry pre- and post-stimulation with LPS. In-vitro cultures were then set up to expose non-activated and activated monocytes to R5-, X4-, and dual (R5/X4)-tropic viruses; and the amount of HIV present on the cells was assayed. RESULTS: Non-HIV-infected monocytes, after LPS stimulation, were confirmed to have an activated phenotype with increase in CD16 and CD69 surface expressions (p<0.05). The activation phenotype was supported by increase in TNFα production, p<0.05. The activated monocytes had increased surface CCR5 (from 21% to 98%; p=0.05); and were found to have more R5-tropic virus than non-activated monocytes (p<0.05). CONCLUSIONS: Following activation by LPS, non-HIV-infected monocytes were found to have increase in surface CCR5. These activated monocytes, when exposed to R5-tropic virus, were found to have more virus compared to non-activated monocytes. The significance of the findings could lie in explaining how microbial translocation plays a role in HIV progression; and possibly promoting CCR5-directed strategies in treating HIV.
RESUMO
Poxviruses have been extensively used as a promising vehicle to efficiently deliver a variety of antigens in mammalian hosts to induce immune responses against infectious diseases and cancer. Using recombinant vaccinia virus (VV) and canarypox virus (ALVAC) expressing enhanced green fluorescent protein (EGFP) or multiple HIV-1 gene products, we studied the role of four cellular signaling pathways, the phosphoinositide-3-OH kinase (PI3K), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK), in poxvirus-mediated foreign gene expression in mammalian cells. In nonpermissive infection (human monocytes), activation of PI3K, ERK, p38 MAPK, and JNK was observed in both VV and ALVAC and blocking PI3K, p38 MAKP, and JNK pathways with their specific inhibitors significantly reduced viral and vaccine antigen gene expression. Whereas, blocking the ERK pathway had no significant effect. Among these cellular signaling pathways studied, PI3K was the most critical pathway involved in gene expression by VV- or ALVAC-infected monocytes. The important role of PI3K in poxvirus-mediated gene expression was further confirmed in mouse epidermal cells stably transfected with dominant-negative PI3K mutant, as poxvirus-mediated targeted gene expression was significantly decreased in these cells when compared with their parental cells. Signaling pathway activation influenced gene expression at the mRNA level rather than virus binding. In permissive mammalian cells, however, VV DNA copies were also significantly decreased in the absence of normal function of the PI3K pathway. Poxvirus-triggered activation of PI3K pathway could be completely abolished by atazanavir, a new generation of antiretroviral protease inhibitors (PIs). As a consequence, ALVAC-mediated EGFP or HIV-1 gag gene expression in infected primary human monocytes was significantly reduced in the presence of atazanavir. These findings implicate that antiretroviral therapy (ART), also known as highly active antiretroviral therapy (HAART), may negatively impact the efficacy of live poxvirus vector-based vaccines and should be carefully considered when administering such live vaccines to individuals on ART.
Assuntos
Vírus da Varíola dos Canários/imunologia , Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vaccinia virus/imunologia , Animais , Sulfato de Atazanavir , Vírus da Varíola dos Canários/genética , Linhagem Celular , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Imunossupressores/farmacologia , Camundongos , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Poxviruses including canarypox (ALVAC) and vaccinia viruses have, in recent years, received considerable attention as live vectors for the development of vaccines against infectious diseases such as AIDS, malaria, and tuberculosis. However, the cellular targets for viral infection within the human immune system and the consequences of infection for cells involved in the generation of immune responses have not been clearly delineated. Using recombinant enhanced green fluorescence protein (EGFP)-expressing ALVAC and vaccinia viruses, we have focused here on a side-by-side comparison of ALVAC and vaccinia virus tropism for cells from human peripheral blood and bone marrow. Both ALVAC and vaccinia viruses showed a strong bias toward monocyte infection. ALVAC minimally infected CD19(+) B cells and was unable to infect ex vivo NK cells and T lymphocytes, whereas vaccinia virus could infect B lymphocytes and NK cell populations. Vaccinia virus was also able to infect T lymphocytes at low but detectable levels which could be enhanced upon their activation. Both ALVAC and vaccinia viruses could infect immature monocyte-derived dendritic cells (MDDCs), but only ALVAC infection induced their subsequent maturation. Infection in human bone marrow cells showed that ALVAC infection was restricted to a myelomonocytoid cell-specific CD33(+) cell population, while vaccinia virus showed a strong, but not exclusive, preference for these cells.
Assuntos
Células Sanguíneas/virologia , Poxviridae/fisiologia , Anticorpos/imunologia , Células Sanguíneas/metabolismo , Células Cultivadas , Citocinas/biossíntese , DNA/genética , DNA/isolamento & purificação , Citometria de Fluxo , Humanos , Immunoblotting , Especificidade de ÓrgãosRESUMO
A novel and simple method for improving the detection limit of conventional Raman spectra using a micro-Raman system and picoliter volumes is presented. A micro-cavity in a reflecting metal substrate uses various mechanisms that collectively improve the entire Raman spectrum from the sample. A micro-cavity with a radius of several micrometers acts as a very effective device that provides multiple excitation of the sample with the laser and couples the forward-scattered Raman photons toward the collection optics in the back-scattered Raman geometry. One of the important features of the micro-cavity substrate is that it enhances the entire Raman spectrum of the molecules under investigation and maintains the relative intensity ratios of the various Raman bands. This feature of maintaining the overall integrity of the Raman features during signal enhancement makes the micro-cavity substrate ideal for forensic science applications for chemical detection of residual traces and other applications requiring low sample concentrations. The spectra measured in these cavities are also observed to be highly reproducible and reliable. A simple method for fabricating micro-cavity substrates with precise sizes and shapes is described. It is further shown that micro-cavities coated with nanofilms of gold take advantage of both surface-enhanced Raman scattering (SERS) and micro-cavity methods and also significantly improve sample detection limits.
Assuntos
Teste de Materiais/instrumentação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/instrumentação , Manejo de Espécimes/instrumentação , Análise Espectral Raman/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais/métodos , Nanotecnologia/métodos , Tamanho da Partícula , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Análise Espectral Raman/métodosRESUMO
The poxviruses including canarypox (ALVAC) and vaccinia viruses are promising vaccine vectors in humans, but little is known about their biology in human cells. Using recombinant enhanced green fluorescence protein (EGFP)-expressing ALVAC and vaccinia viruses, we have focused here on a side-by-side comparison of ALVAC and vaccinia virus tropism for cells from human peripheral blood and bone marrow. Both ALVAC and vaccinia viruses showed a strong bias towards monocyte infection. ALVAC minimally infected CD19+ B cells and was unable to infect ex vivo NK cells and T lymphocytes, whereas vaccinia virus could infect B lymphocytes and NK cell populations. Vaccinia virus was also able to infect T lymphocytes at low, but detectable levels that could be enhanced upon their activation. The observed preferential infection of ALVAC or vaccinia virus to monocytes was the result of preferential binding to this population, rather than lineage-specific differences in the expression of viral genes. Moreover, the level of CD14 expression on monocytes correlated with their preference to be infected with ALVAC or vaccinia virus. Both ALVAC and vaccinia viruses could infect immature monocyte derived dendritic cells (MDDCs), but only ALVAC infection induced their subsequent maturation. Vaccinia virus, however, showed greater tropism for mature MDDCs compared to ALVAC. Infection in human bone marrow cultures showed that ALVAC infection was restricted to a myelomonocytoid cell-specific CD33(+) cell population, while vaccinia virus showed a strong, but not exclusive, preference for these cells. These findings have implications in terms of choosing optimal pox virus derived vectors as vaccines in terms of reducing clinical reactogenicity and inducing dendritic cell (DC) maturation.
Assuntos
Vírus da Varíola dos Canários/fisiologia , Linhagem da Célula , Monócitos/citologia , Monócitos/virologia , Vaccinia virus/fisiologia , Ligante de CD40/metabolismo , Vírus da Varíola dos Canários/genética , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/virologia , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Fatores de Tempo , Vaccinia virus/genéticaRESUMO
As an in vivo model for studying human MHC (HLA) class I-restricted CTL responses to viral infection, we established a series of HLA Tg mice expressing HLA-A2, -B7 or -B27 human/mouse hybrid genes on a background deficient for H2 class I (Tg HLA(hyb)/H2 class I DKO). To determine whether CTL recognition of influenza A (flu) infection in Tg HLA-A2(hyb)/H2 DKO mice is similar to HLA-A2+ humans, we compared the HLA-A2-restricted Tg mouse and human CD8+ T-cell responses to an immunodominant flu epitope (wild-type [WT] M1 58-66), as well as a variant of this peptide (var. M1 58-66). Similar to HLA-A2+ humans, our results show WT M1 58-66 is likely the dominant CTL epitope recognized in infected Tg HLA-A2(hyb)/H2 DKO mice. Var. M1 58-66 was also recognized by WT peptide-reactive T cells from both HLA-A2+ humans and Tg mice, although slightly less efficiently than WT M1 58-66 in both cases. Reduced variant recognition was shown to be associated with reduced peptide/A2 binding, as well as a more limited repertoire of utilized TCR Vbeta chains. The similar pattern of recognition and cross reaction observed here for the WT and variant M1 58-66 epitopes with HLA-A2 by human and Tg HLA mouse CTLs indicates that A2-dependent events of Ag processing, presentation and recognition are well-conserved between species. These findings demonstrate that this Tg HLA-A2/H2 DKO model will aid identification and development of epitopes as vaccines for numerous viral and tumor antigens for the HLA-A2 supertype.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Animais , Citotoxicidade Imunológica , Citometria de Fluxo , Humanos , Imunização , Influenza Humana/prevenção & controle , Interferon gama/metabolismo , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
BACKGROUND: The immune response against xenografts is vigorous and poorly controlled with conventional immunosuppressants. Therefore, success in xenotransplantation will depend on developing additional approaches such as induction of immunologic unresponsiveness or tolerance. Although classic protocols of neonatal tolerance induction in mice are very tolerogenic in many allogeneic models, they have generally failed in xenogeneic models. The purpose of these studies was to determine whether failure results from an intrinsic property of xenogenic major histocompatibility complex (MHC) molecules themselves or, instead, is caused by some limitation in species-specific molecular interactions distinct from the polymorphic domains of xenogenic MHC molecules. METHODS: Our approach was to test the ability of lymphoid cells from a transgenic (Tg) mouse donor expressing a xeno-MHC class I molecule encoding the polymorphic alpha1/alpha2 for human leukocyte antigen (HLA)-B7 to induce neonatal tolerance in non-Tg syngeneic C57BL/6 recipients. Because the donor and recipient strains are genetically identical (C57BL/6, H-2b) except for Tg human MHC HLA-B7, any species-specific molecular incompatibility in this mouse anti-human class I xeno-combination that could potentially interfere with induction of tolerance has been eliminated. RESULTS: Our results show that HLA-B7 Tg-, but not C57BL/6 syngeneic-, injected neonates were unresponsive as adults to HLA-B7-expressing target cells in vitro and specifically accepted HLA-B7-expressing Tg skin grafts. In addition, neonatal injection of donor cells resulted in peripheral chimerism. CONCLUSIONS: These experiments demonstrate that, as long as species-specific molecular interactions are maintained, recognition of the polymorphic domains of xenogeneic MHC does not represent a barrier to neonatal tolerance induction.