Response In Young Mice Research Articles

Pleiotrophin modulates acute and long-term LPS-induced neuroinflammatory responses and hippocampal neurogenesis

The hippocampus is one of the most vulnerable regions affected in disorders characterized by overt neuroinflammation such as neurodegenerative diseases. Pleiotrophin (PTN) is a neurotrophic factor that modulates acute neuroinflammation in different contexts. PTN is found highly upregulated in the brain in different chronic disorders characterized by neuroinflammation, suggesting an important role in the modulation of sustained neuroinflammation. To test this hypothesis, we studied the acute and long-term effects of a single lipopolysaccharide (LPS; 5mg/kg) administration in Ptn+/+ and Ptn-/- mice, and in mice with Ptn-overexpression (Ptn-Tg). Endogenous PTN levels proportionally modulate LPS-induced increase in TNF-α plasma levels one hour after treatment. In the dentate gyrus (DG) of the hippocampus, a lower percentage of DCX+ cells were detected in saline-treated Ptn-/- mice compared to Ptn+/+ mice, suggesting a crucial role of PTN in the maintenance of hippocampal neuronal progenitors. The data show that PTN overexpression tends to potentiate acute microglial responses in the DG 16hours after LPS treatment. Remarkably, a significant increase in the number of neuronal progenitors together with astrogliosis was detected 10 months after a single injection of LPS treatment in wild type mice. However, these LPS-induced long-term effects were prevented in Ptn-/- and Ptn-Tg mice, suggesting that PTN modulates LPS-induced long-term neurogenesis changes and astrocytic response in the hippocampus. The data presented here suggest that endogenous PTN levels are crucial in the regulation of acute LPS-induced systemic and hippocampal microglial responses in young mice. Furthermore, our findings provide evidence of the key role of PTN in the regulation of long-term LPS effects on astrocytic response and neurogenesis in the hippocampus.

Spontaneous p53 activation in middle-aged C57BL/6 mice mitigates the lifespan-extending adaptive response induced by low-dose ionizing radiation

Understanding the biological effects of low-dose (<100 mGy) ionizing radiation (LDR) is technically challenging. We investigated age-dependent LDR effects using adaptive response experiments in young (7-to 12-week-old) and middle-aged (40-to 62-week-old) C57BL/6 mice. Compared with 3 Gy irradiation, 0.02 Gy preirradiation followed by 3 Gy irradiation prolonged life in young mice but not middle-aged mice. Preirradiation also suppressed irradiation-induced 53BP1 repair foci in the small intestines, splenic apoptosis, and p53 activity in young mice but not middle-aged mice. Young p53+/− C57BL/6 mice did not show these adaptive responses, indicating that insufficient p53 function in young mice mitigated the adaptive responses. Interestingly, p53 activation in middle-aged mice spontaneously became approximately 4.5-fold greater than that in young mice, possibly masking LDR stresses. Furthermore, adaptive responses in young mice, but not in middle-aged mice, suppressed some senescence-associated secretory phenotype (SASP) factors (IL-6, CCL2, CCL5, CXCL1). Thus, LDR-induced adaptive responses associated with specific SASP factors may be attenuated by a combination of reduced DNA damage sensor/transducer function and chronic p53 activation in middle-aged mice.

Neutrophilic Pleuritis Is a Severe Complication of Klebsiella pneumoniae Pneumonia in Old Mice.

The pathomechanisms underlying the frequently observed fatal outcome of Klebsiella pneumoniae pneumonia in elderly patients are understudied. In this study, we examined the early antibacterial immune response in young mice (age 2-3 mo) as compared with old mice (age 18-19 mo) postinfection with K. pneumoniae. Old mice exhibited significantly higher bacterial loads in lungs and bacteremia as early as 24 h postinfection compared with young mice, with neutrophilic pleuritis nearly exclusively developing in old but not young mice. Moreover, we observed heavily increased cytokine responses in lungs and pleural spaces along with increased mortality in old mice. Mechanistically, Nlrp3 inflammasome activation and caspase-1-dependent IL-1β secretion contributed to the observed hyperinflammation, which decreased upon caspase-1 inhibitor treatment of K. pneumoniae-infected old mice. Irradiated old mice transplanted with the bone marrow of young mice did not show hyperinflammation or early bacteremia in response to K. pneumoniae. Collectively, the accentuated lung pathology observed in K. pneumoniae-infected old mice appears to be due to regulatory defects of the bone marrow but not the lung, while involving dysregulated activation of the Nlrp3/caspase-1/IL-1β axis.

Immunogenicity and Safety of Sinopharm Covid-19 Vaccine in Young Mice

Background and Aim: Vaccines to prevent SARS-CoV-2 infection may be considered a promising way for reduction of the pandemic. Many different vaccines have become obtainable for use in many countries. The present study aims to evaluate the immune response and the safety of Sinopharm COVID-19 vaccine on 14 days old mice. Materials and Methods:Our experimental study was performed on two weeks old mice, selected by random allocation. The mice were divided into three groups of 12. Group one received asingle dose of 0.5 ml Sinopharm COVID-19 vaccine, group two received two doses of 0.5 ml Sinopharm COVID-19 vaccine, and group three (control) received two doses of 0.5 ml of 0.9 % NaCl. Results: Our study shows that Sinopharm COVID-19 vaccine is safe and induces good immunity in young mice. Conclusions: The Sinopharm COVID-19 vaccine was safe and immunogenic in 14 days old mice. The two doses of Sinopharm COVID-19 vaccine elicit a safe antibody response in young mice. Further post-marketing toxicity studies are required to assess potential hazards for children to evaluate the histopathological characteristics.

Abstract IA22: Pharmacologic and physiologic processes that affect response to immunotherapy

Abstract Immune checkpoint blockade therapy has revolutionized the treatment of many cancer types and has proven effective for ~10-20% of breast cancer patients in early clinical trials. For patients with triple-negative breast cancer, these responses represent an improvement over standard therapeutic strategies, offering hope that improving efficacy of immunotherapies will save patient lives. However, the reasons for resistance or lack of response to these therapies in breast cancer are unknown. First, we made preclinical observations that provide rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anticancer treatment. Using various preclinical breast cancer models, we showed that selective CDK4/6 inhibitors cause tumor regression by promoting antitumor immune responses. Genome-wide expression data from patient tumors in the NeoPalAna trial revealed similar antitumor immune signatures. The antitumor immunity occurs through processes that affect both tumor cells and the immune microenvironment. Hence, disease regression was further enhanced using a combination therapy regimen comprising a CDK4/6 inhibitor with immune checkpoint blockade. Second, we found that age-related changes to immune function present limitations to the application of immunotherapy in our triple-negative breast cancer (TNBC) preclinical models. While TNBC incidence is similar in young and old patients, it is not known whether age impacts response to immunotherapy in breast cancer. In fact, while over 50% of patients are over 65 years of age at diagnosis, elderly individuals are often excluded from clinical trials for various reasons. We found that aged mice (&amp;gt;12 months, corresponding to ~65-year old humans) had significantly reduced ability to respond to either anti-PD-L1 or anti-CTLA4 compared to young counterparts (8-10 weeks, corresponding to ~25-year old humans). Using high-content flow cytometric analyses and RNA-seq, we identified cellular and molecular mechanisms that correlate with response in young mice that are attenuated in old mice. Many of the differentially expressed genes that defined response in the young cohorts and resistance in the older cohorts were also predictors of response to immunotherapy in cancer patients. Our data suggested novel combination therapies that, when combined with checkpoint blockade, improved responses and survival in the old TNBC cohort. By studying pharmacologic and physiologic processes that improve responses, we hope to suggest new treatment strategies that ensure that breast cancer patients see the most benefit possible from checkpoint blockade therapy. Citation Format: Sandra S. McAllister. Pharmacologic and physiologic processes that affect response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA22.

Abstract 1737: Efficacy of immunotherapy is attenuated with age in triple-negative breast cancer

Abstract Age is associated with increasing immune dysfunction that includes significant changes to both the innate and adaptive immune responses. These age-related changes could present limitations to the application of immunotherapy in breast cancer, as over 50% of patients are over 60 years old at diagnosis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, but remains the only subtype that lacks any type of targeted therapy. Early results from clinical trials of anti-PD-1 therapy in metastatic TNBC patients have shown promise, with approximately 20% of patients demonstrating a partial or complete response. While TNBC incidence is similar in young and old patients, differences in disease progression and outcome with age suggest that treatment needs to be tailored to both age as well as breast cancer subtype. It is not known whether age impacts response to immunotherapy, particularly checkpoint blockade, in breast cancer. Using multiple preclinical models of age-dependent TNBC, we found that aged mice had significantly reduced ability to respond to either anti-PD-L1 or anti-CTLA4. Old mice showed decreased numbers of circulating CD4-positive and CD8-positive T cells, as well as NK cells. Furthermore, PD-1 expression was increased on these populations relative to young mice, suggesting increased immune cell exhaustion with age. The old mice also demonstrated increased central and effector memory T cells, but decreased naive T cells compared to young mice, consistent with reports from clinical studies of aged individuals. These findings suggest decreased ability to respond to neoantigens with age. Importantly, using high-content flow cytometric analyses and RNA-seq, we defined cellular and molecular mechanisms that correlate with response in young mice that are attenuated or lacking in old mice. Many of the differentially expressed genes that defined response to anti-CTLA4 in young mice and resistance to treatment in the old mice were also predictors of response to immunotherapy in cancer patients. Our data suggested novel combination therapies that, when combined with checkpoint blockade, improved responses and survival in old mice with TNBC. Our findings should suggest age-stratified treatments to ensure that TNBC patients see the most benefit possible and prevent overtreatment with futile and harsh chemotherapies. Citation Format: Sandra S. McAllister, Jaclyn Sceneay, Tyler Laszewski, Molly DeCristo, Jessalyn Ubellacker, Kristin Wilson, Yuanbo Qin, John Hutchinson. Efficacy of immunotherapy is attenuated with age in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1737.

Live attenuated Leishmania donovani Centrin knock out parasites generate non-inferior protective immune response in aged mice against Visceral Leishmaniasis

Abstract Visceral Leishmaniasis (VL) causes significant mortality. Age appears to be critical in determining the clinical outcome of VL and there is no effective vaccine available against VL for any age group. Previously, we showed that gene deleted live attenuated L. donovani parasites (LdCen−/−) induced a strong protective immune response in young mice. In this study we analyzed LdCen−/− mediated modulation of immune response in aged mice (18 months) and compared to young (2 months) mice. Analysis of innate response in dendritic cells (DCs) from both young and aged mice upon infection with LdCen−/− parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected DCs in vitro. Similarly, parasitized splenic DCs from LdCen−/− infected young and aged mice revealed induction of proinflammatory cytokines and down regulation of anti-inflammatory cytokine genes compared to LdWT infected mice. Furthermore, immunization with LdCen−/− induced higher IgG2a antibodies, lymphoproliferative response and stimulated splenocytes for heightened leishmanicidal activity in young and aged mice. Additionally, upon virulent L. donovani challenge, LdCen−/− immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells leading to significantly reduced parasite burden in the spleen and liver compared to naïve mice. Although, there was no difference in the LdCen−/− induced innate response in DCs between aged and young, the adaptive response in terms of T cell and B cell activation in aged was reduced, which correlated with LdCen−/− induced significant but diminished protective response in aged compared to young mice against VL.

Immunogenicity and protective immunity against otitis media caused by pneumococcus in mice of Hib conjugate vaccine with PsaA protein carrier.

This study evaluated the immunogenicity and protective immunity of a Hemophilus influenzae b (Hib) polysaccharide conjugate vaccine with the pneumococcal surface adhesin A (PsaA) protein carrier in young mice. The Hib polysaccharide was conjugated with the rPsaA protein carrier, which was produced using recombinant DNA technology. A total of 15 young mice aged 3 weeks to 5 weeks were immunized with the conjugate vaccine, and another 15 young mice of the same age were immunized with the licensed Hib-tetanus toxoid (TT) vaccine. Furthermore, the third group of 15 young mice was inoculated with phosphate buffer saline as control. The immunized mice were inoculated with pneumococcus in the middle ear. Results showed that IgG antibody responses against both the PsaA protein and Hib polysaccharide were observed in the Hib-PsaA group. However, no statistical difference was observed in the titer of IgG against the Hib polysaccharide between Hib-PsaA and Hib-TT groups. The elimination rate of pneumococcus and the inflammation of the middle ear showed the effectiveness of protective immunity against otitis media caused by pneumococcus. Our results suggest that the Hib polysaccharide can be successfully conjugated with rPsaA via amide condensation. This new Hib-PsaA conjugate vaccine can induce both anti-PsaA and anti-Hib immune responses in young mice and elicit effective protection against acute otitis media caused by pneumococcus.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice.

The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

Efficacy of prophylactic flavan-3-ol in permanent focal ischemia in 12-mo-old mice.

The consumption of flavan-3-ol-containing foods, including (-)-epicatechin (EC), has been linked to lower incidence of cardiovascular disease and stroke. We previously demonstrated nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) -dependent EC efficacy in reducing stroke-induced deficits in 2-mo-old mice; yet stroke is primarily a disease of the elderly. Because neuroinflammation, oxidative stress, and vascular dysfunction are hallmarks of aging, we tested whether Nrf2 mediates EC efficacy in aging mice through modulation of glial responses and blood brain barrier permeability. First, we compared anastomosis in naïve wild-type and C57BL/6 Nrf2(-/-) mice to identify potential differences in cerebrovascular architecture. Data showed no significant differences in the number of anastomoses or mean intersection points, indicating similar gross vascular physiology. To assess efficacy and mechanisms of protection, wild-type or Nrf2(-/-) mice were administered the minimum effective EC dose established in our previous studies before the permanent distal middle cerebral artery occlusion. Similar to previous results with young mice, 12-mo-old wild types also showed significant reductions in infarct volume (41.01 ± 29.57%) and improved performance in removing adhesive tape relative to vehicle-treated controls, whereas a trend toward protection was observed in Nrf2(-/-). However, EC did not reduce immunoreactivity for the microglia/macrophage marker anti-ionized calcium-binding adapter molecule 1, suggesting that dampened activation/recruitment did not account for EC protection. Furthermore, there were no differences in mouse IgG extravasation or spontaneous hemorrhage between EC-treated groups. These data demonstrate that EC protection occurs independent of microglia/macrophage modulation or blood brain barrier preservation, suggesting that the glial cell responses in young mice are compensatory to another, and potentially novel, protective mechanism.

Aging periosteal progenitor cells have reduced regenerative responsiveness to bone injury and to the anabolic actions of PTH 1-34 treatment

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-Catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-Catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.

Fishing for allergy prevention

Fishing for allergy prevention

Elevated Tumor-Associated Antigen Expression Suppresses Variant Peptide Vaccine Responses

Variant peptide vaccines are used clinically to expand T cells that cross-react with tumor-associated Ags (TAA). To investigate the effects of elevated endogenous TAA expression on variant peptide-induced responses, we used the GP70 TAA model. Although young BALB/c mice display T cell tolerance to the TAA GP70(423-431) (AH1), expression of GP70 and suppression of AH1-specific responses increases with age. We hypothesized that as TAA expression increases, the AH1 cross-reactivity of variant peptide-elicited T cell responses diminishes. Controlling for immunosenescence, we showed that elevated GP70 expression suppressed AH1 cross-reactive responses elicited by two AH1 peptide variants. A variant that elicited almost exclusively AH1 cross-reactive T cells in young mice elicited few or no T cells in aging mice with Ab-detectable GP70 expression. In contrast, a variant that elicited a less AH1 cross-reactive T cell response in young mice successfully expanded AH1 cross-reactive T cells in all aging mice tested. However, these T cells bound the AH1/MHC complex with a relatively short half-life and responded poorly to ex vivo stimulation with the AH1 peptide. Variant peptide vaccine responses were also suppressed when AH1 peptide is administered tolerogenically to young mice before vaccination. Analyses of variant-specific precursor T cells from naive mice with Ab-detectable GP70 expression determined that these T cells expressed PD-1 and had downregulated IL-7Rα expression, suggesting they were anergic or undergoing deletion. Although variant peptide vaccines were less effective as TAA expression increases, data presented in this article also suggest that complementary immunotherapies may induce the expansion of T cells with functional TAA recognition.

Thymic stromal lymphopoietin can restore functional T cell-independent antibody responses in young mice (150.5)

Abstract Unlike adults, young children cannot mount an antibody response to T cell-independent (TI) antigens such as Pneumococcal polysaccharides (PPS) and the basis for this impairment remains elusive. The ontogeny of B cells early in life is IL-7-independent, whereas in adults it is IL-7-dependent. Despite having TI B cell subsets, young wildtype or adult IL-7-/- mice are impaired in anti-PPS response and do not survive Pneumococcal challenge. In contrast, enforced expression of IL-7 restores protective anti-PPS responses in young mice, indicating that IL-7-dependent B cells are critical for TI responses. Thymic stromal lymphopoietin (TSLP) is structurally similar to IL-7 and utilizes IL-7Rα for signaling, therefore it may also contribute to the development of TI B cell responses. Indeed, we found that young TSLP transgenic mice generate an anti-PPS response and protective immunity comparable to that of adult mice. TSLP can be induced upon activation of TLR2 and TLR9. Borrelia hermsii, an agent of relapsing fever, activates TLR2 and TLR9 signaling and induces a protective TI antibody response even in young mice, suggesting that TSLP induced during this infection could play a role in anti-B. hermsii responses. Indeed, IL-7Rα-/- mice (deficient in both IL-7 and TSLP signaling) have a more severe impairment in anti-B.hermsii responses and suffer more prolonged bacteremia than IL-7-/- or TSLPR-/- mice. These data indicate that TSLP can restore functional TI responses in the young.

Development of tumor-associated antigen (TAA)-specific T cell responses in heavily-tolerized mice requires vaccination with peptide variants that target T cells with decreased responsiveness to TAA (131.7)

Abstract Vaccination with peptide variants of a tumor-associated antigen (TAA) can induce T cell responses that cross-react with the TAA. Using the gp70(423-431, AH1) epitope of endogenous MuLV as our model TAA, we have developed peptide variants that, unlike AH1 itself, induce AH1-reactive T cell responses upon vaccination of BALB/c mice. Two of these variants, A5 and 39, induce robust AH1-reactive responses in young mice. These variants were used to vaccinate aging mice, which have increased expression of and tolerance to gp70 protein. Variant-39 consistently induced AH1-specific responses, regardless of levels of gp70 expression in individual mice. Conversely, variant-A5 did not induce AH1-specific responses in most aging mice tested. The inability of variant-A5 to induce AH1-specific responses in individual mice correlated with increased gp70 expression. We hypothesized that variant-39 expands AH1-specific cells that are less reactive to AH1 than those expanded by variant-A5, thus impeding tolerance by peripheral AH1 presentation. Analyzing variant-induced responses in young mice, we determined that T cells expanded by variant-39 indeed have diminished function upon stimulation with AH1 relative to those elicited by variant-A5. These findings suggest that tumor vaccines require TAA variants that target cross-reactive T cell repertoires with low enough reactivity to TAA to avoid tolerance in the periphery, yet sufficient reactivity to provide effective antitumor responses.

An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice

A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.

Deficiency in Expression of the Signaling Protein Sin/Efs Leads to T-Lymphocyte Activation and Mucosal Inflammation

Our studies have concentrated on elucidating the role of the signaling protein Sin in T-lymphocyte function. We have previously shown that Sin overexpression inhibits T-lymphocyte development and activation. Here we show that Sin-deficient mice exhibit exaggerated immune responses characterized by enhanced cytokine secretion and T-cell-dependent antibody production. Excessive T-cell responses in young mice correlate with spontaneous development of inflammatory lesions in different organs of aged Sin(-/-) mice, particularly the small intestine. The intestinal inflammation is characterized by T- and B-cell infiltrates in the lamina propria, which correlate with crypt enlargement and marked villus expansion and/or damage. Similar to the human intestinal inflammatory disorder Crohn's disease (CD), and in contrast to most mouse models of mucosal inflammation, inflammatory lesions in the gastrointestinal tract of Sin(-/-) mice are restricted to the small bowel. Taken together, these results suggest that Sin regulates immune system and T-lymphocyte function and that immune system dysfunction in the absence of Sin may underlie the pathogenesis of tissue-specific inflammation and enteropathies such as CD.

Comparison of genomic damage caused by 5-nitrofurantoin in young and adult mice using the in vivo micronucleus assay

The antibiotic 5-nitrofurantoin (5-NF) has been used widely for the treatment of urosepsis in children during the last 20 years. Recent experimentation suggests the need for reevaluating its genotoxic potential. Because of possible differences in the metabolism and clearance of 5-NF in young and adult animals, we conducted a study to determine whether micronuclei caused by 5-NF were age-related. The in vivo micronucleus (MN) assay was performed on 3- and 8-week-old mice given single intraperitoneal injections of 5, 10, and 50 mg/kg 5-NF. Blood samples from the tail vein were taken before injection (baseline) and at 48, 96, 168, and 336 hr (2 weeks) after the treatment. One thousand reticulocytes were analyzed for micronuclei from each animal. Compared to similar baseline values for young and adult mice, 5-NF caused a significant increase in MN frequency in both age groups. The mean MN frequency in the young animals was higher than in the adult animals for each dose and sampling time. MN frequencies remained significantly elevated in young animals even 2 weeks after exposure to 5-NF. The results of the study confirm the genotoxic potential of 5-NF in young and adult animals, and indicate that young animals are more sensitive to the genotoxic effects of 5-NF than adult mice and that the response in young mice persists for a significantly longer time. These findings may be related to poorly developed mechanisms of xenobiotic detoxification and renal elimination in young animals.

Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice.

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.

Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies

Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1–7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies.