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Papers by li you
Infection and Immunity, 2007
Little is known about the immune distribution and localization of antigen-specific T cells in muc... more Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In ext...
Infection and Immunity, 2008
ABSTRACT Little is known about the immune distribution and localization of antigen-specific T cel... more ABSTRACT Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection.
Horttechnology, Feb 1, 2011
We investigated a practical method for immobilizing liquid spawn of oyster mushroom (Pleurotus os... more We investigated a practical method for immobilizing liquid spawn of oyster mushroom (Pleurotus ostreatus) to prolong the storage time and provide convenient transportation of liquid spawn of edible mushrooms. The method was based on the mycelial pellets of liquid spawn adsorbed in carriers. Selected carriers were similar to cultivation substrates, and the best carrier was a mixture of cottonseed hull, corn core, and wheat bran with a ratio of 4.5:4.5:1 by weight. Immobilized spawn were prepared by mixing the pellets from liquid spawn with carriers using a ratio of 1:8 by weight. Within the first 15 days of storage at 20–25 °C, the immobilized spawn grew strongly, respiration intensity and cellulase activities rose rapidly, and the count and brightness of the isozyme bands of esterase, peroxidase, and polyphenol oxidase increased remarkably as well. From days 30 to 60, the cellulase activities fell and the brightness of the peroxidase and polyphenol oxidase bands gradually decreased, whereas the respiration intensity and the band count of esterase and peroxidase remained constant. After 60 days, the cultivated characteristics of the immobilized spawn were same as the fresh conventional solid cottonseed hull spawn. The results showed that immobilized spawn on the basis of the mycelial pellets of liquid spawn adsorbed in carrier can be used to extend the storage time and simplify transportation of liquid spawn of edible mushroom.
Applied Microbiology and Biotechnology, Feb 1, 2007
The Journal of infectious diseases, Jan 15, 2004
Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vg... more Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of human immunodeficiency virus-infected humans can be generated during effective antiretroviral treatment. Here, simian immunodeficiency virus (SIV)mac-infected macaques previously exposed to bacille Calmette-Guerin (BCG) were reinfected with BCG, were treated either with tenofovir or tenofovir plus indinavir, and were assessed for the development of Vgamma2Vdelta2+ T cell responses during active BCG coinfection. A restored capacity of Vgamma2Vdelta2+ T cells to undergo major expansions and pulmonary migration during active BCG coinfection was detected after simultaneous BCG reinfection and treatment with tenofovir of the SIVmac-infected macaques. Interestingly, a restored expansion of Vgamma2Vdelta2+ T cells in the SIVmac/BCG-coinfected macaques was detectable, even though antiretroviral treatment was initiated 1 mo...
Journal of virology, 2003
Adaptive immune responses of gammadelta T cells during active mycobacterial coinfection of human ... more Adaptive immune responses of gammadelta T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vgamma2Vdelta2(+) T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vgamma2Vdelta2(+) T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vgamma2Vdelta2(+) T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vgamma2Vdelta2(+) T-cell responses was associated with profound CD4(+) T-cell deficiency and subsequent development of SIVmac-related tubercul...
Science, 2002
To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model ... more To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model was used to follow Vγ2Vδ2 + T cell responses to mycobacterial infections. These phosphoantigen-specific γδ T cells displayed major expansion during Mycobacterium bovis Bacille Calmette-Guérin (BCG) infection and a clear memory-type response after BCG reinfection. Primary and recall expansions of Vγ2Vδ2 + T cells were also seen during Mycobacterium tuberculosis infection of naı̈ve and BCG-vaccinated macaques, respectively. This capacity to rapidly expand coincided with a clearance of BCG bacteremia and immunity to fatal tuberculosis in BCG-vaccinated macaques. Thus, Vγ2Vδ2 + T cells may contribute to adaptive immunity to mycobacterial infections.
Proceedings of the ICE - Structures and Buildings, 2003
ABSTRACT
PLoS Pathogens, 2013
Dominant Vc2Vd2 T-cell subset exist only in primates, and recognize phosphoantigen from selected ... more Dominant Vc2Vd2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vc2Vd2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vc2Vd2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vc2Vd2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vc2Vd2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNc, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vc2Vd2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vc2Vd2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vc2Vd2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the cd T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vc2Vd2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.
PLoS ONE, 2012
Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tube... more Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific ab and cd T effector cells, and developed severe lung tuberculosis(TB) and ''secondary'' Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNc-producing cd T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vc2Vd2 T cells in lung with severe TB and in liver/kidney without apparent TB. Methodology/Principal Findings: We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vc2Vd2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vc2Vd2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vc2Vd2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vc2Vd2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine. Conclusion: We were the first to demonstrate clonal immune responses of mycobacterium-specific Vc2Vd2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vc2Vd2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vc2Vd2 T cells.
PLoS ONE, 2013
Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are... more Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.
Journal of Virology, 2004
The immune mechanisms associated with the evolution from latent to clinically active mycobacteria... more The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood. Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation. In the present study, SIVmac-infected monkeys that developed clinically quiescent mycobacterial infection after BCG inoculation were followed prospectively for the reactivation of the BCG and the development of SIV-related tuberculosis-like disease. The development of clinically latent BCG coinfection in these SIVmac-infected monkeys was characterized by a change from high to undetectable levels of bacterial organisms, with or without measurable BCG mRNA expression in lymph node cells. The reactivation of clinically latent BCG coinfection and develo...
Journal of Virology, 2003
Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunode... more Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vγ2Vδ2 + T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vγ2Vδ2 + T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vγ2Vδ2 + T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vγ2Vδ2 + T-cell responses was associated with profound CD4 + T-cell deficiency and subsequent development of SIVmac-related tuberculosis-like disease in the coinfected monkeys. ...
The Journal of Infectious Diseases, 2008
The immune mechanisms by which early host-mycobacterium interaction leads to the development of s... more The immune mechanisms by which early host-mycobacterium interaction leads to the development of severe tuberculosis (TB) remain poorly characterized in humans. Here, we demonstrate that severe TB in juvenile rhesus monkeys down-regulated many genes in the blood but up-regulated selected genes constituting gene networks of Th17 and Th1 responses, T cell activation and migration, and inflammation and chemoattractants in the pulmonary and lymphoid compartments. Overexpression (450-2740-fold) of 13 genes encoding inflammatory cytokines and receptors (IL-22, CCL27, MIP-1alpha, IP-10, CCR4, CCR5, and CXCR3), immune dysfunctional receptors and ligands (PD1 and PDL2), and immune activation elements (IL-3, IFN-beta, TIM1, and TLR2) was seen in tissues, with low antigen-specific cellular responses. Thus, severe TB in macaques features unbalanced up-regulation of immune-gene networks without proportional increases in antigen-specific cellular responses.
The Journal of Infectious Diseases, 2004
Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vg... more Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of human immunodeficiency virus-infected humans can be generated during effective antiretroviral treatment. Here, simian immunodeficiency virus (SIV)mac-infected macaques previously exposed to bacille Calmette-Guerin (BCG) were reinfected with BCG, were treated either with tenofovir or tenofovir plus indinavir, and were assessed for the development of Vgamma2Vdelta2+ T cell responses during active BCG coinfection. A restored capacity of Vgamma2Vdelta2+ T cells to undergo major expansions and pulmonary migration during active BCG coinfection was detected after simultaneous BCG reinfection and treatment with tenofovir of the SIVmac-infected macaques. Interestingly, a restored expansion of Vgamma2Vdelta2+ T cells in the SIVmac/BCG-coinfected macaques was detectable, even though antiretroviral treatment was initiated 1 month after BCG reinfection. Importantly, the restored expansion of Vgamma2Vdelta2+ T cells coincided with increases in numbers of purified protein derivative-specific interferon- gamma -producing CD4+ T cells and increases in the magnitude of their proliferative responses. In contrast, the SIVmac-infected control macaques exhibited diminished responses of Vgamma2Vdelta2+ T cells and mycobacterium-specific CD4+ T cells during active BCG coinfection. Our results suggest that the development of adaptive immune responses of phosphoantigen-specific Vgamma2Vdelta2+ T cells during active mycobacterium/HIV coinfection requires control of viral infection and immune competence of peptide-specific CD4+ T cells.
The Journal of Infectious Diseases, 2007
Gene networks of protective lymphocytes after immune activation with live attenuated vaccines rem... more Gene networks of protective lymphocytes after immune activation with live attenuated vaccines remain poorly characterized. Because Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine can confer protection against fatal forms of tuberculosis in humans and monkeys, we made use of macaque models to optimally study immune gene networks after BCG vaccination/infection. We first established and validated a large-scale real-time quantitation system and then used it to measure expression levels of 138 immune genes after BCG vaccination/infection of rhesus macaques. Systemic BCG vaccination induced up to 600-fold increases in expression of 78 immune genes among the 138 genes tested at the time when BCG-elicited T cell responses and immunity were apparent. These up-regulated transcripts constituted multiple gene networks that were linked to various aspects of immune function. Surprisingly, the up-regulation of most of these immune genes in the gene networks occurred at 1 week and was sustained at > or = 6 weeks after BCG vaccination/infection. Although early activation of immune gene networks was an immune correlate of anti-BCG immunity, prolonged up-regulation of these networks coincided with the development of vaccine-elicited T cell responses after BCG vaccination/infection. These findings provide molecular evidence suggesting that the BCG-induced gene networks may represent global transcriptomes and proteomes underlying the development of T cell responses and, ultimately, immunity to mycobacteria.
The Journal of Immunology, 2010
Clonal responses of Mycobacterium tuberculosis-specific CD4(+) or CD8(+) T effector cells produci... more Clonal responses of Mycobacterium tuberculosis-specific CD4(+) or CD8(+) T effector cells producing antituberculosis cytokine IFN-γ in the context of immune protection against tuberculosis remain poorly characterized in humans. Utilizing decade-long TCR expertise, we previously developed a useful method to isolate clonotypic TCR sequences from Ag-specific IFN-γ-producing T cells and to specifically measure clonotypic TCR frequencies in the T cell pool. In this study, we investigated TCR Vβ repertoires/CDR3 usage, clonal expansion or dominance, and pulmonary trafficking or accumulation for purified protein deritative (PPD)-specific T effector cells producing IFN-γ during bacillus Calmette-Guérin (BCG) vaccination and subsequent M. tuberculosis challenge of macaques. We found that while PPD-specific CD4(+) and CD8(+) T effector clones employed diverse TCR Vβ repertoires, 30-33% of IFN-γ(+)CD4(+) T cell clones from three M. tuberculosis-infected macaques expressed TCR bearing a conserved residue leucine in CDR3. Many Ag-specific IFN-γ(+) CD4(+) and few CD8(+) T effector cells emerged as dominant clones during mycobacterial infections and underwent major recall expansion after pulmonary M. tuberculosis infection of BCG-vaccinated macaques. PPD-specific T cell clones readily trafficked to the airway or lung after BCG vaccination or M. tuberculosis infection, and some of them continuously accumulated in lungs during M. tuberculosis infection even after they became undetectable in the circulation. Importantly, remarkable recall expansion and pulmonary accumulation of T effector cells coincided with BCG-induced protection against tuberculosis. Thus, rapid clonal expansion and pulmonary accumulation of Ag-specific T effector cells appear to be one of the immune mechanisms underlying immunity against tuberculosis.
Journal of Immunological Methods, 2006
Despite recent advances in measuring cellular immune responses, the quantitation of antigen-speci... more Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based realtime quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vh families and 13 Jh segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNg-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10 À 5 to 10 À 6 antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by z 2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNg-producing T cell clones using as few as 2 Â 10 6 PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.
International Journal of Medicinal Mushrooms, 2012
Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster... more Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.
Infection and Immunity, 2008
lesions. Finally, V␥2V␦2 T cells accumulated in tissues appeared to possess cytokine production f... more lesions. Finally, V␥2V␦2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the ␥␦ T cells present within granulomas. Thus, clonally expanded V␥2V␦2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection.
Infection and Immunity, 2007
Little is known about the immune distribution and localization of antigen-specific T cells in muc... more Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In ext...
Infection and Immunity, 2008
ABSTRACT Little is known about the immune distribution and localization of antigen-specific T cel... more ABSTRACT Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection.
Horttechnology, Feb 1, 2011
We investigated a practical method for immobilizing liquid spawn of oyster mushroom (Pleurotus os... more We investigated a practical method for immobilizing liquid spawn of oyster mushroom (Pleurotus ostreatus) to prolong the storage time and provide convenient transportation of liquid spawn of edible mushrooms. The method was based on the mycelial pellets of liquid spawn adsorbed in carriers. Selected carriers were similar to cultivation substrates, and the best carrier was a mixture of cottonseed hull, corn core, and wheat bran with a ratio of 4.5:4.5:1 by weight. Immobilized spawn were prepared by mixing the pellets from liquid spawn with carriers using a ratio of 1:8 by weight. Within the first 15 days of storage at 20–25 °C, the immobilized spawn grew strongly, respiration intensity and cellulase activities rose rapidly, and the count and brightness of the isozyme bands of esterase, peroxidase, and polyphenol oxidase increased remarkably as well. From days 30 to 60, the cellulase activities fell and the brightness of the peroxidase and polyphenol oxidase bands gradually decreased, whereas the respiration intensity and the band count of esterase and peroxidase remained constant. After 60 days, the cultivated characteristics of the immobilized spawn were same as the fresh conventional solid cottonseed hull spawn. The results showed that immobilized spawn on the basis of the mycelial pellets of liquid spawn adsorbed in carrier can be used to extend the storage time and simplify transportation of liquid spawn of edible mushroom.
Applied Microbiology and Biotechnology, Feb 1, 2007
The Journal of infectious diseases, Jan 15, 2004
Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vg... more Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of human immunodeficiency virus-infected humans can be generated during effective antiretroviral treatment. Here, simian immunodeficiency virus (SIV)mac-infected macaques previously exposed to bacille Calmette-Guerin (BCG) were reinfected with BCG, were treated either with tenofovir or tenofovir plus indinavir, and were assessed for the development of Vgamma2Vdelta2+ T cell responses during active BCG coinfection. A restored capacity of Vgamma2Vdelta2+ T cells to undergo major expansions and pulmonary migration during active BCG coinfection was detected after simultaneous BCG reinfection and treatment with tenofovir of the SIVmac-infected macaques. Interestingly, a restored expansion of Vgamma2Vdelta2+ T cells in the SIVmac/BCG-coinfected macaques was detectable, even though antiretroviral treatment was initiated 1 mo...
Journal of virology, 2003
Adaptive immune responses of gammadelta T cells during active mycobacterial coinfection of human ... more Adaptive immune responses of gammadelta T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vgamma2Vdelta2(+) T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vgamma2Vdelta2(+) T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vgamma2Vdelta2(+) T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vgamma2Vdelta2(+) T-cell responses was associated with profound CD4(+) T-cell deficiency and subsequent development of SIVmac-related tubercul...
Science, 2002
To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model ... more To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model was used to follow Vγ2Vδ2 + T cell responses to mycobacterial infections. These phosphoantigen-specific γδ T cells displayed major expansion during Mycobacterium bovis Bacille Calmette-Guérin (BCG) infection and a clear memory-type response after BCG reinfection. Primary and recall expansions of Vγ2Vδ2 + T cells were also seen during Mycobacterium tuberculosis infection of naı̈ve and BCG-vaccinated macaques, respectively. This capacity to rapidly expand coincided with a clearance of BCG bacteremia and immunity to fatal tuberculosis in BCG-vaccinated macaques. Thus, Vγ2Vδ2 + T cells may contribute to adaptive immunity to mycobacterial infections.
Proceedings of the ICE - Structures and Buildings, 2003
ABSTRACT
PLoS Pathogens, 2013
Dominant Vc2Vd2 T-cell subset exist only in primates, and recognize phosphoantigen from selected ... more Dominant Vc2Vd2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vc2Vd2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vc2Vd2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vc2Vd2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vc2Vd2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNc, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vc2Vd2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vc2Vd2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vc2Vd2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the cd T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vc2Vd2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.
PLoS ONE, 2012
Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tube... more Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific ab and cd T effector cells, and developed severe lung tuberculosis(TB) and ''secondary'' Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNc-producing cd T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vc2Vd2 T cells in lung with severe TB and in liver/kidney without apparent TB. Methodology/Principal Findings: We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vc2Vd2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vc2Vd2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vc2Vd2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vc2Vd2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine. Conclusion: We were the first to demonstrate clonal immune responses of mycobacterium-specific Vc2Vd2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vc2Vd2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vc2Vd2 T cells.
PLoS ONE, 2013
Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are... more Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.
Journal of Virology, 2004
The immune mechanisms associated with the evolution from latent to clinically active mycobacteria... more The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood. Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation. In the present study, SIVmac-infected monkeys that developed clinically quiescent mycobacterial infection after BCG inoculation were followed prospectively for the reactivation of the BCG and the development of SIV-related tuberculosis-like disease. The development of clinically latent BCG coinfection in these SIVmac-infected monkeys was characterized by a change from high to undetectable levels of bacterial organisms, with or without measurable BCG mRNA expression in lymph node cells. The reactivation of clinically latent BCG coinfection and develo...
Journal of Virology, 2003
Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunode... more Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vγ2Vδ2 + T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vγ2Vδ2 + T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vγ2Vδ2 + T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vγ2Vδ2 + T-cell responses was associated with profound CD4 + T-cell deficiency and subsequent development of SIVmac-related tuberculosis-like disease in the coinfected monkeys. ...
The Journal of Infectious Diseases, 2008
The immune mechanisms by which early host-mycobacterium interaction leads to the development of s... more The immune mechanisms by which early host-mycobacterium interaction leads to the development of severe tuberculosis (TB) remain poorly characterized in humans. Here, we demonstrate that severe TB in juvenile rhesus monkeys down-regulated many genes in the blood but up-regulated selected genes constituting gene networks of Th17 and Th1 responses, T cell activation and migration, and inflammation and chemoattractants in the pulmonary and lymphoid compartments. Overexpression (450-2740-fold) of 13 genes encoding inflammatory cytokines and receptors (IL-22, CCL27, MIP-1alpha, IP-10, CCR4, CCR5, and CXCR3), immune dysfunctional receptors and ligands (PD1 and PDL2), and immune activation elements (IL-3, IFN-beta, TIM1, and TLR2) was seen in tissues, with low antigen-specific cellular responses. Thus, severe TB in macaques features unbalanced up-regulation of immune-gene networks without proportional increases in antigen-specific cellular responses.
The Journal of Infectious Diseases, 2004
Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vg... more Vgamma2Vdelta2+ T cells play a role in antimicrobial responses. It is unknown whether adaptive Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of human immunodeficiency virus-infected humans can be generated during effective antiretroviral treatment. Here, simian immunodeficiency virus (SIV)mac-infected macaques previously exposed to bacille Calmette-Guerin (BCG) were reinfected with BCG, were treated either with tenofovir or tenofovir plus indinavir, and were assessed for the development of Vgamma2Vdelta2+ T cell responses during active BCG coinfection. A restored capacity of Vgamma2Vdelta2+ T cells to undergo major expansions and pulmonary migration during active BCG coinfection was detected after simultaneous BCG reinfection and treatment with tenofovir of the SIVmac-infected macaques. Interestingly, a restored expansion of Vgamma2Vdelta2+ T cells in the SIVmac/BCG-coinfected macaques was detectable, even though antiretroviral treatment was initiated 1 month after BCG reinfection. Importantly, the restored expansion of Vgamma2Vdelta2+ T cells coincided with increases in numbers of purified protein derivative-specific interferon- gamma -producing CD4+ T cells and increases in the magnitude of their proliferative responses. In contrast, the SIVmac-infected control macaques exhibited diminished responses of Vgamma2Vdelta2+ T cells and mycobacterium-specific CD4+ T cells during active BCG coinfection. Our results suggest that the development of adaptive immune responses of phosphoantigen-specific Vgamma2Vdelta2+ T cells during active mycobacterium/HIV coinfection requires control of viral infection and immune competence of peptide-specific CD4+ T cells.
The Journal of Infectious Diseases, 2007
Gene networks of protective lymphocytes after immune activation with live attenuated vaccines rem... more Gene networks of protective lymphocytes after immune activation with live attenuated vaccines remain poorly characterized. Because Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine can confer protection against fatal forms of tuberculosis in humans and monkeys, we made use of macaque models to optimally study immune gene networks after BCG vaccination/infection. We first established and validated a large-scale real-time quantitation system and then used it to measure expression levels of 138 immune genes after BCG vaccination/infection of rhesus macaques. Systemic BCG vaccination induced up to 600-fold increases in expression of 78 immune genes among the 138 genes tested at the time when BCG-elicited T cell responses and immunity were apparent. These up-regulated transcripts constituted multiple gene networks that were linked to various aspects of immune function. Surprisingly, the up-regulation of most of these immune genes in the gene networks occurred at 1 week and was sustained at > or = 6 weeks after BCG vaccination/infection. Although early activation of immune gene networks was an immune correlate of anti-BCG immunity, prolonged up-regulation of these networks coincided with the development of vaccine-elicited T cell responses after BCG vaccination/infection. These findings provide molecular evidence suggesting that the BCG-induced gene networks may represent global transcriptomes and proteomes underlying the development of T cell responses and, ultimately, immunity to mycobacteria.
The Journal of Immunology, 2010
Clonal responses of Mycobacterium tuberculosis-specific CD4(+) or CD8(+) T effector cells produci... more Clonal responses of Mycobacterium tuberculosis-specific CD4(+) or CD8(+) T effector cells producing antituberculosis cytokine IFN-γ in the context of immune protection against tuberculosis remain poorly characterized in humans. Utilizing decade-long TCR expertise, we previously developed a useful method to isolate clonotypic TCR sequences from Ag-specific IFN-γ-producing T cells and to specifically measure clonotypic TCR frequencies in the T cell pool. In this study, we investigated TCR Vβ repertoires/CDR3 usage, clonal expansion or dominance, and pulmonary trafficking or accumulation for purified protein deritative (PPD)-specific T effector cells producing IFN-γ during bacillus Calmette-Guérin (BCG) vaccination and subsequent M. tuberculosis challenge of macaques. We found that while PPD-specific CD4(+) and CD8(+) T effector clones employed diverse TCR Vβ repertoires, 30-33% of IFN-γ(+)CD4(+) T cell clones from three M. tuberculosis-infected macaques expressed TCR bearing a conserved residue leucine in CDR3. Many Ag-specific IFN-γ(+) CD4(+) and few CD8(+) T effector cells emerged as dominant clones during mycobacterial infections and underwent major recall expansion after pulmonary M. tuberculosis infection of BCG-vaccinated macaques. PPD-specific T cell clones readily trafficked to the airway or lung after BCG vaccination or M. tuberculosis infection, and some of them continuously accumulated in lungs during M. tuberculosis infection even after they became undetectable in the circulation. Importantly, remarkable recall expansion and pulmonary accumulation of T effector cells coincided with BCG-induced protection against tuberculosis. Thus, rapid clonal expansion and pulmonary accumulation of Ag-specific T effector cells appear to be one of the immune mechanisms underlying immunity against tuberculosis.
Journal of Immunological Methods, 2006
Despite recent advances in measuring cellular immune responses, the quantitation of antigen-speci... more Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based realtime quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vh families and 13 Jh segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNg-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10 À 5 to 10 À 6 antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by z 2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNg-producing T cell clones using as few as 2 Â 10 6 PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.
International Journal of Medicinal Mushrooms, 2012
Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster... more Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.
Infection and Immunity, 2008
lesions. Finally, V␥2V␦2 T cells accumulated in tissues appeared to possess cytokine production f... more lesions. Finally, V␥2V␦2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the ␥␦ T cells present within granulomas. Thus, clonally expanded V␥2V␦2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection.