Ana Marie Landin

A metabotropic glutamate receptor variant functions as a taste receptor

Sensory transduction for many taste stimuli such as sugars, some bitter compounds and amino acids is thought to be mediated via G protein-coupled receptors (GPCRs), although no such receptors that respond to taste stimuli are yet identified. Monosodium L-glutamate (l-MSG), a natural component of many foods, is an important gustatory stimulus believed to signal dietary protein. We describe a GPCR cloned from rat taste buds and functionally expressed in CHO cells. The receptor couples negatively to a cAMP cascade and shows an unusual concentration–response relationship. The similarity of its properties to MSG taste suggests that this receptor is a taste receptor for glutamate.

Aging Down-Regulates the Transcription Factor E2A, Activation-Induced Cytidine Deaminase, and Ig Class Switch in Human B Cells

Elderly humans have compromised humoral and cellular immune responses, which lead to reduced protection to infectious agents and to vaccines. Currently, available vaccines suboptimally protect the elderly population. The capacity to class switch the Ig H chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown in mice that the E2A-encoded transcription factor E47, which regulates many B cell functions, is down-regulated in old splenic B cells. This leads to a reduction in the activation-induced cytidine deaminase (AID), which is known to induce class switch recombination and Ig somatic hypermutation. The old activated murine B cells also have less AID and less switched Abs. We have extended our study here to investigate whether aging also affects Ab production and E47 and AID expression in B cells isolated from the peripheral blood of human subjects (18–86 years). Our results obtained with activated CD19+ B cells show that the expression of E47, AID, and Igγ1 circle transcripts progressively decrease with age. We also show an age-related decline in the percentage of switch memory B cells (IgG+/IgA+), an increase in that of naive B cells (IgG−/IgA−/CD27−) for most individuals, and no decrease in that of IgM memory cells in peripheral blood, consistent with our data on the decrease seen in class switch recombination in vitro. Our results provide a possible molecular mechanism for a B cell intrinsic defect in the humoral immune response with aging and suggest avenues for improvement of vaccine response in elderly humans.

Age effects on B cells and humoral immunity in humans.

Both humoral and cellular immune responses are impaired in aged individuals, leading to decreased vaccine responses. Although T cell defects occur, defects in B cells play a significant role in age-related humoral immune changes. The ability to undergo class switch recombination (CSR), the enzyme for CSR, AID (activation-induced cytidine deaminase) and the transcription factor E47 are all decreased in aged stimulated B cells. We here present an overview of age-related changes in human B cell markers and functions, and also discuss some controversies in the field of B cell aging.

Intrinsic defects in B cell response to seasonal influenza vaccination in elderly humans.

We have evaluated the serum response to seasonal influenza vaccination in subjects of different ages and associated this with the specific B cell response to the vaccine in vitro. Although the serum response has previously been shown to decrease with age, this has largely been associated to decreased T cell functions. Our results show that in response to the vaccine, the specific response of B cells in vitro, as measured by AID (activation-induced cytidine deaminase), the in vivo serum HI (hemagglutination inhibition) response, and the in vivo generation of switch memory B cells are decreased with age, as evaluated in the same subjects. This is the first report to demonstrate that intrinsic B cell defects with age contribute to reduced antibody responses to the influenza vaccine. The level of AID in response to CpG before vaccination can also predict the robustness of the vaccine response. These results could contribute to developing more effective vaccines to protect the elderly as well as identifying those most at risk.

Mechanisms for Decreased Function of B Cells in Aged Mice and Humans

The immune system has been known for some time to be compromised in aged individuals, e.g., both mice and humans, and in both humoral and cellular responses. Our studies have begun to elucidate intrinsic B lymphocyte defects in Ig class switch recombination, activation-induced cytidine deaminase, and E47 transcription factor expression. These defects occur in both mice and humans. Our studies have also shown that tristetraprolin is one of the key players in regulating the decreased E47 mRNA stability in aged B lymphocytes. These and current studies should lead to improvements in B lymphocyte function in aged populations.

Tristetraprolin, a Negative Regulator of mRNA Stability, Is Increased in Old B Cells and Is Involved in the Degradation of E47 mRNA

We have previously shown that the E2A-encoded transcription factor E47, which regulates class switch in splenic B cells, is down-regulated in old B cells, due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by proteins. We have herein looked at the specific proteins responsible for the degradation of the E47 mRNA and found that tristetraprolin (TTP), a physiological regulator of mRNA expression and stability, is involved in the degradation of the E47 mRNA. Although many studies have characterized TTP expression and function in macrophages, monocytes, mast cells, and T cells, little is known about the expression and function of TTP in primary B cells. We show herein that TTP mRNA and protein expression are induced by LPS in B cells from young and old mice, the levels of TTP in old B cells always being higher than those in young B cells. Although TTP mRNA is degraded at a significantly higher rate in old B cells, TTP mRNA expression is higher in old than in young, likely due to its increased transcription. Like in macrophages, TTP protein expression and function in B cells are dependent upon p38 MAPK. We found that there is less phospho-TTP (inactive form), as well as phospho-p38, in old than in young splenic-activated B cells. This is the first report showing that TTP is involved in the degradation of the E47 mRNA and is up-regulated in old B cells.

Unique biomarkers for B-cell function predict the serum response to pandemic H1N1 influenza vaccine

In order to develop predictive markers for a beneficial humoral immune response, we evaluated the in vivo and in vitro response to the pandemic (p)H1N1 vaccine in young and elderly individuals. We measured serum antibody response and associated this with the in vitro B-cell response to the vaccine, measured by activation-induced cytidine deaminase (AID). Both responses decrease with age and are significantly correlated. The percentage of switched memory B cells in blood, both before and after vaccination, is decreased with age. The percentage of switched memory B cells at t0 correlates with the hemagglutination inhibition response and therefore, we suggest that this may be used as a predictive marker for B-cell responsiveness. AID induced by CpG before vaccination also predicts the robustness of the vaccine response. Plasmablasts showed a trend to increase after vaccination in young individuals only. This report establishes molecular biomarkers of response, percentage of switched memory B cells and AID response to CpG, useful for identifying individuals at risk of poor response and also for measuring improvements in vaccines and monitoring optimal humoral responses.

Cytomegalovirus (CMV) seropositivity decreases B cell responses to the influenza vaccine

Cytomegalovirus (CMV)-seropositivity has been shown to have a negative effect on influenza vaccine-specific antibody responses. In this paper, we confirm and extend these results showing for the first time, a negative association between CMV-seropositivity and B cell predictive biomarkers of optimal vaccine responses. These biomarkers are switched memory B cells and AID in CpG-stimulated B cell cultures measured before vaccination which positively correlate with the serum response to the influenza vaccine. We also found that CMV-seropositivity is associated with increased levels of B cell-intrinsic inflammation and these both correlate with lower B cell function. Finally, CMV-seropositivity is associated with decreased percentages of individuals responding to the vaccine in both young and elderly individuals.

A Molecular Mechanism for TNF-α–Mediated Downregulation of B Cell Responses

B cell function with age is decreased in class switch recombination (CSR), activation-induced cytidine deaminase (AID), and stability of E47 mRNA. The latter is regulated, at least in part, by tristetraprolin (TTP), which is increased in aged B cells and also negatively regulates TNF-α. In this study, we investigated whether B cells produce TNF-α, whether this changes with age, and how this affects their function upon stimulation. Our hypothesis is that in aging there is a feedback mechanism of autocrine inflammatory cytokines (TNF-α) that lowers the expression of AID and CSR. Our results showed that unstimulated B cells from old BALB/c mice make significantly more TNF-α mRNA and protein than do B cells from young mice, but after stimulation the old make less than the young; thus, they are refractory to stimulation. The increase in TNF-α made by old B cells is primarily due to follicular, but not minor, subsets of B cells. Incubation of B cells with TNF-α before LPS stimulation decreased both young and old B cell responses. Importantly, B cell function was restored by adding anti–TNF-α Ab to cultured B cells. To address a molecular mechanism, we found that incubation of B cells with TNF-α before LPS stimulation induced TTP, a physiological regulator of mRNA stability of the transcription factor E47, which is crucial for CSR. Finally, anti–TNF-α given in vivo increased B cell function in old, but not in young, follicular B cells. These results suggest new molecular mechanisms that contribute to reduced Ab responses in aging.

Stimulatory Effects of Mesenchymal Stem Cells on cKit+ Cardiac Stem Cells Are Mediated by SDF1/CXCR4 and SCF/cKit Signaling Pathways.

RATIONALE Culture-expanded cells originating from cardiac tissue that express the cell surface receptor cKit are undergoing clinical testing as a cell source for heart failure and congenital heart disease. Although accumulating data support that mesenchymal stem cells (MSCs) enhance the efficacy of cardiac cKit(+) cells (CSCs), the underlying mechanism for this synergistic effect remains incompletely understood. OBJECTIVE To test the hypothesis that MSCs stimulate endogenous CSCs to proliferate, migrate, and differentiate via the SDF1/CXCR4 and stem cell factor/cKit pathways. METHODS AND RESULTS Using genetic lineage-tracing approaches, we show that in the postnatal murine heart, cKit(+) cells proliferate, migrate, and form cardiomyocytes, but not endothelial cells. CSCs exhibit marked chemotactic and proliferative responses when cocultured with MSCs but not with cardiac stromal cells. Antagonism of the CXCR4 pathway with AMD3100 (an SDF1/CXCR4 antagonist) inhibited MSC-induced CSC chemotaxis but stimulated CSC cardiomyogenesis (P<0.0001). Furthermore, MSCs enhanced CSC proliferation via the stem cell factor/cKit and SDF1/CXCR4 pathways (P<0.0001). CONCLUSIONS Together these findings show that MSCs exhibit profound, yet differential, effects on CSC migration, proliferation, and differentiation and suggest a mechanism underlying the improved cardiac regeneration associated with combination therapy using CSCs and MSCs. These findings have important therapeutic implications for cell-based therapy strategies that use mixtures of CSCs and MSCs.