Response In Mouse Model Research Articles

Abstract 725: In vivo imaging and quantitative proteomics to study responsiveness to EGFR targeted therapy.

Abstract Lung cancer is the leading cause of cancer related mortality in the United States and is the most common cancer worldwide. Somatic mutations in the kinase domain of EGFR such as in-frame deletion of LREA amino acids in exon 19 and L858R point mutation in exon 21 largely account for 15-30% of lung adenocarcinoma. Although lung cancer patients carrying either of these mutations respond dramatically to EGFR-directed tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib, acquired resistance to these drugs has been the main obstacle in treating these patients for the long term. Secondary mutation at the gatekeeper residue of the kinase domain (T790M) of EGFR accounts for drug resistance in about 50% of patients. We have used doxycycline inducible mutant EGFR transgenic mice to model human lung adenocarcinoma that is sensitive and resistant to TKIs. Our overall goal is to examine the treatment response to EGFR-directed TKIs in lung adenocarcinoma harboring various EGFR mutants. Our approach is aimed to identify imaging surrogates and proteomics profile of treatment response in mouse models of mutant EGFR-driven lung tumorigenesis. In this study, we have used transgenic mice that express exon 19 deletion mutant, L858R mutant, and L858R/T790M double mutant in type II pneumocytes upon doxycycline induction. Tumor burden in these mice was monitored by serial MRI imaging. Each group of mice with significant tumor burden was treated with erlotinib, a reversible EGFR inhibitor, BIBW-2992, an irreversible EGFR/ERBB2 dual inhibitor, or a combination of BIBW-2992 and cetuximab, an antibody directed against EGFR followed by 2[18F]Fluoro-2-deoxy-d-glucose (FDG)-PET imaging to assess any tumor regression. We observed a significant reduction of 18FDG uptake by tumors after the treatment with erlotinib in L858R mutant mice. However, in L858R/T790M mutant mice, no significant reduction of 18FDG uptake was observed upon treatment with erlotinib and BIBW-2992 had only mild effect. We are currently designing rational combination treatments based on the response in 18FDG-PET imaging. Finally, we are performing global phosphoproteomic analysis using quantitative mass spectrometry and immunohistochemistry with phospho-specific antibodies on tumor tissue before and after various treatments and correlating with the response in PET imaging. Results from these experiments will help us identify potential biomarkers (imaging and proteomic) of treatment response in lung adenocarcinoma driven by both TKI-sensitizing and resistant mutants of EGFR. Citation Format: Abhilash Venugopalan, Niu Gang, Jinxia Guo, Tapan Maity, Xu Zhang, Constance Cultraro, Xiaoyuan Chen, Udayan Guha. In vivo imaging and quantitative proteomics to study responsiveness to EGFR targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2013-725

Abstract 692: Histone deacetylase 11 is an epigenetic regulator of CD8+ T-cell effector function and memory formation.

Abstract Cytotoxic T-cells play a vital role in immune mediated tumor responses. Indeed, adoptive transfer of CD8+ T-cells can result in regression of tumor in refractory, progressive metastatic melanoma in a minority of patients (Chapuis, Thompson et al. 2012). In adoptive transfer recipients, clinical response correlates with the persistence of T-cells. Central memory CD8+ T-cells (Tcm) are a population of long-lived T-cells with an enhanced replicative capacity. Resultantly, adoptive transfers of Tcm populations confer a superior anti-melanoma response in mouse models (Klebanoff, Gattinoni et al. 2005). To improve upon the efficacy of T-cell adoptive immunotherapy a more extensive understanding of the mechanisms regulating the formation of central memory cells as well as the effector function and proliferative capacity of CD8+ T-cells is needed. Here we report that the newest discovered histone deacetylase (HDAC), HDAC11, is a negative regulator of Eomes, a transcription factor controlling memory formation and effector phenotype in CD8+ T-cells (Pearce, Mullen et al. 2003; Intlekofer, Takemoto et al. 2005). T-cells from HDAC11 knockout (HDAC11KO) mice demonstrate a robust proliferative capacity upon stimulation when compared to wild-type CD8+ T-cells. Additionally, these cells favor a more robust inflammatory phenotype, producing significantly higher levels of INF-γ, IL-2 and TNF. Analysis of the thymus, spleen and lymph nodes from HDAC11KO mice demonstrates no gross differences in overall percentages or numbers of CD4+ or CD8+ T-cells, nor double positive or double negative thymocytes. However, further examination reveals a significantly increased percentage of Tcm cells in the CD8+ compartment. To further understand these phenotypic differences, we investigated the expression and acetylation of the transcription factor Eomes message. Significantly higher levels of Eomes, both at the basal state and post stimulation, are expressed in HDAC11KO CD8+ T-cells. Additionally, increased levels of acetylated histone 3 at the Eomes promoter, a marker for transcriptional activation, are seen. Finally, in vivo, adoptive transfer of HDAC11KO T-cells results in significantly decreased tumor progression in mice with established melanoma tumors. These results demonstrate HDAC11 as a negative regulator of Eomes expression and, resultantly, a negative regulator of central memory formation and effector function in CD8+ T-cells. Therefore, HDAC11 represents a novel therapeutic target for augmenting the efficacy of T-cell adoptive immunotherapy. Citation Format: David M. Woods, Karrune Woan, Fengdong Cheng, Hong Wei Wang, Eva Sahakian, John Powers, Jennifer Rock-Klotz, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo Sotomayor. Histone deacetylase 11 is an epigenetic regulator of CD8+ T-cell effector function and memory formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 692. doi:10.1158/1538-7445.AM2013-692

Interleukin‐21 in chronic inflammatory diseases

Interleukin-21 (IL-21), a cytokine produced by various subsets of activated CD4+ T cells, regulates multiple innate and adaptive immune responses. Indeed, IL-21 controls the proliferation and function of CD4+ and CD8+ T lymphocytes, drives the differentiation of B cells into memory cells and Ig-secreting plasma cells, enhances the activity of natural killer cells and negatively regulates the differentiation and activity of regulatory T cells. Moroever, IL-21 can stimulate nonimmune cells to synthesize various inflammatory molecules. Excessive production of IL-21 has been described in many human chronic inflammatory disorders and there is evidence that blockade of IL-21 helps attenuate detrimental responses in mouse models of immune-mediated diseases. In this article we briefly review data supporting the pathogenic role of IL-21 in immune-inflammatory pathologies and discuss the benefits and risks of IL-21 neutralization in patients with such diseases.

Genomic responses in mouse models poorly mimic human inflammatory diseases

A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

Systemic delivery of a TLR7 agonist in combination with radiation primes durable antitumor immune responses in mouse models of lymphoma

AbstractPassive immunotherapy with monoclonal antibodies has improved outcome for patients with B-cell malignancies, although many still relapse and little progress has been made with T-cell malignancies. Novel treatment approaches are clearly required in this disease setting. There has been much recent interest in developing therapeutic approaches to enhance antitumor immune responses using novel immunomodulatory agents in combination with standard of care treatments. Here we report that intravenous administration of the Toll-like receptor 7 (TLR7) agonist, R848 in combination with radiation therapy (RT), leads to the longstanding clearance of tumor in T- and B-cell lymphoma bearing mice. In combination, TLR7/RT therapy leads to the expansion of tumor antigen-specific CD8+ T cells and improved survival. Furthermore, those mice that achieve long-term clearance of tumor after TLR7/RT therapy are protected from subsequent tumor rechallenge by the generation of a tumor-specific memory immune response. Our findings demonstrate the potential for enhancing the efficacy of conventional cytotoxic anticancer therapy through combination with a systemically administered TLR7 agonist to improve antitumor immune responses and provide durable remissions.

The Toll-IL-1R member Tir8 modulates post-transplant kidney ischemia/reperfusion injury by inhibiting resident leukocyte expansion

Event Abstract Back to Event The Toll-IL-1R member Tir8 modulates post-transplant kidney ischemia/reperfusion injury by inhibiting resident leukocyte expansion Paola Cassis1, Sistiana Aiello1*, Nadia Azzollini1, Samantha Solini1, Giuseppe Remuzzi1 and Marina Noris1 1 IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Italy Ischemia/reperfusion injury (IRI), occurring after transplantation, activates TLRs/IL-1R on inflammatory leukocytes amplifying adaptive immune response. In a mouse model of renal artery clamp deficiency of Tir8, a IL-1R/TLR negative regulator, aggravated post-ischemic acute renal failure in association with intrarenal leukocyte accumulation. We investigated the role of TIR8 in modulating post-transplant IRI and inflammatory response in mouse model of syngeneic kidney transplant. Donor and recipient C57BL/6 mice were congenic for CD45 leukocyte antigen to allow discriminating between intragraft recipient and donor leukocytes. Wild-type (wt-group n=5) or Tir8-/- (Tir8-/--group, n=5) donor kidneys were exposed to 30 min cold ischemia before transplant. Transient impairment of graft function was found at 1day post-transplant in wt-group (BUN: 1day 53±19, 30day: 27±3mg/dl). At variance the Tir8-/--group showed irreversible severe graft dysfunction (BUN: 1day 95±23, 30day: 67±23 mg/dl, P<0.05 vs wt-group). Both at 10 and 30 days post-transplant lower numbers (P<0.05) of intragraft leukocytes were found in the wt-group (20±9/field, 61±18/field, respectively) vs the Tir8-/--group (104±17/field, 117±24/field, respectively). At 10 days the majority of leukocytes were of donor origin both in the wt and the Tir8-/- groups (79±8%, 78±2%, respectively). At 30days, in the wt-group the majority of intragraft leukocytes were of recipient origin (63±6%) while 63±2% of intragraft leukocytes were of donor origin in the Tir8-/--group. These results indicate that TIR8 modulates post-transplant IRI possibly by suppressing expansion and activation of resident leukocytes within the kidney graft. Keywords: Ischemia, TLRs (Toll-like receptors), Kidney Transplantation, Dendritic Cells, congenic mice Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Cassis P, Aiello S, Azzollini N, Solini S, Remuzzi G and Noris M (2013). The Toll-IL-1R member Tir8 modulates post-transplant kidney ischemia/reperfusion injury by inhibiting resident leukocyte expansion. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00691 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sistiana Aiello, IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy, sistiana.aiello@marionegri.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Paola Cassis Sistiana Aiello Nadia Azzollini Samantha Solini Giuseppe Remuzzi Marina Noris Google Paola Cassis Sistiana Aiello Nadia Azzollini Samantha Solini Giuseppe Remuzzi Marina Noris Google Scholar Paola Cassis Sistiana Aiello Nadia Azzollini Samantha Solini Giuseppe Remuzzi Marina Noris PubMed Paola Cassis Sistiana Aiello Nadia Azzollini Samantha Solini Giuseppe Remuzzi Marina Noris Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Immune responses of B. malayi thioredoxin (TRX) and venom allergen homologue (VAH) chimeric multiple antigen for lymphatic filariasis

Although multiple vaccine strategy for lymphatic filariasis has provided tremendous hope, the choice of antigens used in combination has determined its success in the previous studies. Multiple antigens comprising key vaccine candidates from different life cycle stages would provide a promising strategy if the antigenic combination is chosen by careful screening. In order to analyze one such combination, we have used a chimeric construct carrying the well studied B. malayi antigens thioredoxin (BmTRX) and venom allergen homologue (BmVAH) as a fusion protein (TV) and evaluated its immune responses in mice model. The efficacy of fusion protein vaccine was explored in comparison with the single antigen vaccines and their cocktail. In mice, TV induced significantly high antibody titer of 1,28,000 compared to cocktail vaccine TRX+VAH (50,000) and single antigen vaccine TRX (16,000) or VAH (50,000). Furthermore, TV elicited higher level of cellular proliferative response together with elevated levels of IFN-γ, IL-4 and IL-5 indicating a Th1/Th2 balanced response. The isotype antibody profile showed significantly high level of IgG1 and IgG2b confirming the balanced response elicited by TV. Immunization with TV antigen induced high levels of both humoral and cellular immune responses compared to either cocktail or antigen given alone. The result suggests that TV is highly immunogenic in mice and hence the combination needs to be evaluated for its prophylactic potential.

Of Timescales, Animal Models, and Human Disease: The 50th Anniversary of C. elegans as a Biological Model

Of Timescales, Animal Models, and Human Disease: The 50th Anniversary of C. elegans as a Biological Model

Modified protocol for in vivo imaging of wild-type mouse retina with customized miniature spectral domain optical coherence tomography (SD-OCT) device

This protocol outlines and evaluates a modified scanning procedure for a customized spectral domain optical coherence tomography (SD-OCT) imaging apparatus within the wild-type C57Bl/6 mouse posterior segment. This modified protocol allows for the capture of a 50 degree field of view spanning 3 mm by 3 mm perimeter with the optic disc as the central point. By utilizing this scanning protocol a more reliable measurement of retinal thickness can be achieved outside the fluctuating region of the optic disc. This protocol, when applied to this high resolution device, enables non-invasive in vivo histological imaging and biometric assessment of the various layers of the rodent posterior segment within a 20 – 30 min procedural time-frame. This protocol could establish a standardized method for evaluating morphological changes, with this commercial SDOCT device, when assessing longitudinal disease pathophysiology and treatment response in mouse models for future vision science research.

Dietary abscisic acid ameliorates influenza-virus-associated disease and pulmonary immunopathology through a PPARγ-dependent mechanism

The anti-inflammatory phytohormone abscisic acid (ABA) modulates immune and inflammatory responses in mouse models of colitis and obesity. ABA has been identified as a ligand of lanthionine synthetase C-like 2, a novel therapeutic target upstream of the peroxisome proliferator-activated receptor γ (PPARγ) pathway. The goal of this study was to investigate the immune modulatory mechanisms underlying the anti-inflammatory efficacy of ABA against influenza-associated pulmonary inflammation. Wild-type (WT) and conditional knockout mice with defective PPARγ expression in lung epithelial and hematopoietic cells (cKO) treated orally with or without ABA (100 mg/kg diet) were challenged with influenza A/Udorn (H3N2) to assess ABA's impact in disease, lung lesions and gene expression. Dietary ABA ameliorated disease activity and lung inflammatory pathology, accelerated recovery and increased survival in WT mice. ABA suppressed leukocyte infiltration and monocyte chemotactic protein 1 mRNA expression in WT mice through PPARγ since this effect was abrogated in cKO mice. ABA ameliorated disease when administered therapeutically on the same day of the infection to WT but not mice lacking PPARγ in myeloid cells. We also show that ABA's greater impact is between days 7 and 10 postchallenge when it regulates the expression of genes involved in resolution, like 5-lipoxygenase and other members of the 5-lipoxygenase pathway. Furthermore, ABA significantly increased the expression of the immunoregulatory cytokine interleukin-10 in WT mice. Our results show that ABA, given preventively or therapeutically, ameliorates influenza-virus-induced pathology by activating PPARγ in pulmonary immune cells, suppressing initial proinflammatory responses and promoting resolution.

Correction: Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

Correction: Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

Background and AimsThe egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy.Methodology/Principal FindingsEffect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes.ConclusionsMinor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.

Neuroprotective effects of bee venom by suppression of neuroinflammatory responses in mouse model of Parkinson’s disease: role of CD4+CD25+Foxp3+ regulatory T cells. (119.2)

Abstract In the present study, we examined whether bee venom (BV) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the MPTP-induced Parkinson’s disease (PD) mice model. Treatment of BV prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This nueroprotection afforded by bee venom was associated with the suppression of microglia and inhibition of recruitment of CD4+ T cells. Additionally, CD4+CD25+Foxp3+ Treg cells were significantly increased in vivo and in vitro after treatment of BV. Therefore, we carefully propose that neuroprotection of BV might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation.

Oncolytic Virus and Anti–4-1BB Combination Therapy Elicits Strong Antitumor Immunity against Established Cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.

Optimization of the codon strengthened the human rotavirus VP6 antigen's serum immune responses and protective responses in mice model

To observe the serum immune responses and protection in mice model of the recombinant adenovirus vector mediated human rotavirus VP6 gene expression through coden optimization (rvAdVP6(o)) in comparison with the wild type (rvAdVP6). 6-8 week female BALB/c mice were randomly grouped and immunized three times intranasally with 10(8) TCID50 rvAdVP6(o) and rvAdVP6, respectively, then detect the serum IgG level against rotavirus induced by rvAdVP6(o) and rvAdVP6. The amount of sheding viral antigens in feces was detectd after mice rotavirus was taked orally. The serum IgG level against rotavirus induced by rvAdVP6(o) was higher than that of rvAdVP6 after three times of immunization. The immunized mice shed lower amount of viral antigens in feces as compared with the rvAdVP6. The recombinant adenovirus which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger serum immune and protective responses against the challenge of the rotavirus than the wild type (rvAdVP6) at the same immunizing dosage.

Implications of nasopharynx‐associated lymphoid tissue (NALT) in the development of allergic responses in an allergic rhinitis mouse model

Nasopharynx-associated lymphoid tissue (NALT) serves as an important inductive site for mucosal immunity in the upper respiratory tract. Despite its importance in the mucosal immune system, little is known regarding the role of NALT in airway allergic immune responses. We aimed to elucidate the role of NALT in the induction of upper airway allergic responses in a mouse model. Inhibitor of DNA binding/differentiation 2 (Id2)(-/-) and Id2(+/-) mice was exposed to the ovalbumin (OVA)-induced allergic rhinitis model, because the former resulted in the NALT deficiency. The allergic parameters, such as allergic symptoms, serum OVA-specific immunoglobulin E (IgE) levels, eosinophil infiltration, and cytokine profiles in the nasal mucosa, were compared between Id2(-/-) and Id2(+/-) groups. NALT-null, Id2(-/-) mice displayed significantly lower allergic responses compared with Id2(+/-) mice, as demonstrated by lower levels of allergic symptoms, serum OVA-specific IgE, eosinophilic infiltration, and local Th2 cytokine transcriptions. To determine which of two factors, that is, the absence of NALT or the alteration of immunocompetent cell populations caused by the Id2 deficiency, has a larger effect on the attenuated allergic immune responses in Id2(-/-) mice, lethally irradiated Id2(-/-) mice were engrafted with C57BL/6 wild-type bone marrow cells and showed still significantly lower allergic immune responses compared with equally treated Id2(+/-) mice. In addition, IgE class switch recombination-associated molecules, such as ε immunoglobulin heavy-chain germline gene transcript, ε mRNA, and activation-induced cytidine deaminase mRNA, were detected in NALT from OVA-sensitized wild-type mice. These results show the critical role of NALT for the induction of allergic responses in the upper airway at least in part by means of class switching to IgE in situ.

CD205 antigen targeting combined with dendritic cell recruitment factors and antigen-linked CD40L activation primes and expands significant antigen-specific antibody and CD4+ T cell responses following DNA vaccination of outbred animals

Dendritic cell antigen targeting primes robust immune responses in mouse models. Optimizing this immunization strategy in the actual hosts that require protection will advance development of efficacious contemporary vaccines. In a proof-of-concept study, we tested the immunogenicity of a single, low dose of a novel multi-component DNA construct expressing a CD205-targeted antigen fused to a CD40L minimal functional domain for linked DC activation. The DNA construct was formulated with DNA-encoded Flt3L and GM-CSF for DC recruitment and the formulation was evaluated in MHC class II-matched calves. Immunization of the calves with the CD205 antigen-targeting construct mixed with the cytokine constructs induced significant IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and antibody responses detectable within one week post-immunization. CD205 antigen-targeting significantly expanded IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and IgG antibody responses three weeks post-immunization. Nineteen weeks post-priming, the IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and the IgG titers were waning, but they remained significant. Following boosting at nineteen weeks post-immunization, the immune responses primed by the CD205-targeted antigen underwent rapid recall and the mean response tripled within one week post-boost. Comparative analysis of the immune responses observed one week post-priming versus the responses detected one week post-boost revealed that the average number of the IFN-γ-secreting CD4(+) T-cells observed in the calves immunized with the CD205 antigen targeting construct increased five-fold, the mean CD4(+) T-cell proliferation increased three-fold, whereas the mean IgG antibody titer increased two hundred-fold. These promising outcomes support testing the protective efficacy of CD205-targeted antigens in the calf model.

Neuroregenerative Mechanisms of Allopregnanolone in Alzheimer’s Disease

The proliferative pool and regenerative potential of neural stem cells diminishes with age, a phenomenon that may be exacerbated in prodromal and mild Alzheimer’s disease (AD) brains. In parallel, the neuroactive progesterone metabolite, allopregnanolone (APα), along with a host of other factors, is decreased in the AD brain. Results of preclinical analyses demonstrate that APα is a potent inducer of neural progenitor proliferation of both rodent and human derived neural progenitor cells in vitro. In vivo, APα significantly increased neurogenesis within the subgranular zone of the dentate gyrus and subventricular zone of the 3xTgAD mouse model. Functionally, APα reversed the learning and memory deficits of 3xTgAD mice prior to and following the onset of AD pathology and was comparably efficacious in aged normal mice. In addition to inducing regenerative responses in mouse models of AD, APα significantly reduced beta-amyloid burden, beta-amyloid binding alcohol dehydrogenase load, and microglial activation. In parallel, APα increased markers of white matter generation and cholesterol homeostasis. Analyses to determine the optimal treatment regimen in the 3xTgAD mouse brain indicated that a treatment regimen of APα once per week was optimal for both inducing neurogenesis and reducing AD pathology. Pharmacokinetic analyses indicated that APα is rapidly increased in both plasma and brain following a single dose. APα is most efficacious when administered once per week which will contribute to its margin of safety. Further, analyses in both animals and humans have provided parameters for safe APα dosage exposure in humans. From a translational perspective, APα is a small molecular weight, blood brain barrier penetrant molecule with substantial preclinical efficacy data as a potential Alzheimer’s therapeutic with existing safety data in animals and humans. To our knowledge, APα is the only small molecule that both promotes neural progenitor regeneration in brain and simultaneously reduces AD pathology burden.

A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1

Suppression of immune responses is necessary to limit damage to host tissue during inflammation, but it can be detrimental in specific immune responses, such as sepsis and antitumor immunity. Recently, immature myeloid cells have been implicated in the suppression of immune responses in mouse models of cancer, infectious disease, bone marrow transplantation, and autoimmune disease. Here, we report the identification of a subset of mature human neutrophils (CD11cbright/CD62Ldim/CD11bbright/CD16bright) as what we believe to be a unique circulating population of myeloid cells, capable of suppressing human T cell proliferation. These cells were observed in humans in vivo during acute systemic inflammation induced by endotoxin challenge or by severe injury. Local release of hydrogen peroxide from the neutrophils into the immunological synapse between the neutrophils and T cells mediated the suppression of T cell proliferation and required neutrophil expression of the integrin Mac-1 (αMβ2). Our data demonstrate that suppression of T cell function can be accomplished by a subset of human neutrophils that can be systemically induced in response to acute inflammation. Identification of the pivotal role of neutrophil Mac-1 and ROS in this process provides a potential target for modulating immune responses in humans.

The Contribution of NT‐gp96 as an Adjuvant for Increasing HPV16 E7‐Specific Immunity in C57BL /6 Mouse Model

To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-γ response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity.