Integrative biology : quantitative biosciences from nano to macro, Jan 22, 2016
Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associat... more Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associated with diabetes, emphysema, and aging. Importantly, these conditions are also marked by abnormal subendothelial matrix crosslinking (stiffness). Yet, whether and how abnormal matrix stiffness contributes to leukocyte-endothelial adhesion remains poorly understood. Using a co-culture of human monocytic cells and human microvascular endothelial cells (ECs) grown on matrices of tunable stiffness, we demonstrate that matrix stiffness exerts biphasic control over monocyte-EC adhesion, with both matrix softening and stiffening eliciting a two-fold increase in this adhesive interaction. This preferential endothelial adhesivity on softer and stiffer matrices was consistent with a significant increase in α-actinin-4-associated endothelial ICAM-1 clustering, a key determinant of monocyte-EC adhesion. Further, the enhanced ICAM-1 clustering on soft and stiff matrices correlated strongly with an in...
Page 1. Chapter 4 Aging and the Effect of Calorie Restriction in Rhesus Monkeys Ilhem Messaoudi, ... more Page 1. Chapter 4 Aging and the Effect of Calorie Restriction in Rhesus Monkeys Ilhem Messaoudi, Jennifer E. Young, Ricki J. Colman, April M. Handy, George S. Roth, Donald K. Ingram, and Julie A. Mattison 4.1 Overview of CR Studies in Rhesus Macaques ...
Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics o... more Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at ... more T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.
Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families ... more Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families are remarkably tightly regulated between birth and advanced adulthood. By contrast, individual elderly humans and C3H.SW and B10.BR aged mice display drastic disruption in such regulation. It was suggested that the alterations in the murine aged T cell compartment were due to age-related clonal T cell expansions (TCE). Here, we studied the kinetics of homeostatic dysregulation of T cell populations in aged C57BL/6 (B6) mice. Using mAb staining, we show that the percentages of alphabeta+CD8+ or CD4+ T cells bearing different TCRVbeta elements remain virtually constant in mice up to 12 mo of age. In 22-mo-old mice, however, there is a dramatic disturbance of this pattern owing to the emergence of CD8+ TCE. Expanded T cells did not show any obvious bias in Vbeta usage and were derived in all cases examined thus far from a single clone. TCE appeared later in life, compared with B cell clonal expansions. However, and in contrast to those detected in humans, TCE were frequently unstable disappearing within 2-4 mo, with other TCE appearing within the same time frame. Additional studies carried on thymic T cells, thymectomized mice, and young T transferred cells into Rag1-/- mice suggest that the clonal expansions occur in the periphery and that their onset is accelerated by decreased thymic output and/or function(s).
We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self... more We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to Modified Vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure to ethanol and then again after 7 months of 22 h/day of "open-access" drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVA-specific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.
Ebolaviruses pose significant public health problems due to their high lethality, unpredictable e... more Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity.
Integrative biology : quantitative biosciences from nano to macro, Jan 22, 2016
Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associat... more Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associated with diabetes, emphysema, and aging. Importantly, these conditions are also marked by abnormal subendothelial matrix crosslinking (stiffness). Yet, whether and how abnormal matrix stiffness contributes to leukocyte-endothelial adhesion remains poorly understood. Using a co-culture of human monocytic cells and human microvascular endothelial cells (ECs) grown on matrices of tunable stiffness, we demonstrate that matrix stiffness exerts biphasic control over monocyte-EC adhesion, with both matrix softening and stiffening eliciting a two-fold increase in this adhesive interaction. This preferential endothelial adhesivity on softer and stiffer matrices was consistent with a significant increase in α-actinin-4-associated endothelial ICAM-1 clustering, a key determinant of monocyte-EC adhesion. Further, the enhanced ICAM-1 clustering on soft and stiff matrices correlated strongly with an in...
Page 1. Chapter 4 Aging and the Effect of Calorie Restriction in Rhesus Monkeys Ilhem Messaoudi, ... more Page 1. Chapter 4 Aging and the Effect of Calorie Restriction in Rhesus Monkeys Ilhem Messaoudi, Jennifer E. Young, Ricki J. Colman, April M. Handy, George S. Roth, Donald K. Ingram, and Julie A. Mattison 4.1 Overview of CR Studies in Rhesus Macaques ...
Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics o... more Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at ... more T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.
Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families ... more Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families are remarkably tightly regulated between birth and advanced adulthood. By contrast, individual elderly humans and C3H.SW and B10.BR aged mice display drastic disruption in such regulation. It was suggested that the alterations in the murine aged T cell compartment were due to age-related clonal T cell expansions (TCE). Here, we studied the kinetics of homeostatic dysregulation of T cell populations in aged C57BL/6 (B6) mice. Using mAb staining, we show that the percentages of alphabeta+CD8+ or CD4+ T cells bearing different TCRVbeta elements remain virtually constant in mice up to 12 mo of age. In 22-mo-old mice, however, there is a dramatic disturbance of this pattern owing to the emergence of CD8+ TCE. Expanded T cells did not show any obvious bias in Vbeta usage and were derived in all cases examined thus far from a single clone. TCE appeared later in life, compared with B cell clonal expansions. However, and in contrast to those detected in humans, TCE were frequently unstable disappearing within 2-4 mo, with other TCE appearing within the same time frame. Additional studies carried on thymic T cells, thymectomized mice, and young T transferred cells into Rag1-/- mice suggest that the clonal expansions occur in the periphery and that their onset is accelerated by decreased thymic output and/or function(s).
We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self... more We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to Modified Vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure to ethanol and then again after 7 months of 22 h/day of "open-access" drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVA-specific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.
Ebolaviruses pose significant public health problems due to their high lethality, unpredictable e... more Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity.
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