Protein-treated Mice Research Articles

Visualizing of engineered five-helix bundle protein for blocking SARS-CoV-2 variants in live animals

Variants of Concern (VOCs), and variant under monitoring (VUM) of SARS-CoV-2 have emerged with heightened infectivity, antibody resistance, and vaccine breakthrough. In light of this, it is imperative to establish efficient and streamlined testing models for the prompt detection of newly emerging mutant strains. The 5-Helix Bundle (5-HB) protein was engineered as potent inhibitors predicated on the S2 subunit of the SARS-CoV-2 spike protein, exerting a broad-spectrum inhibitory influence against SARS-CoV-2 infections. Positron emission tomography (PET) quantifications revealed significantly reduced radioactive uptake in infected tissue of 5-HB protein-treated mice. The reduction was much lower than that of untreated infected mice imaged at 4 h (48.6 % reduction), 12 h (45.2 % reduction), and 24 h (36.9 % reduction) (p < 0.01). The newly engineered 5-HB protein features with the capability of binding to conservative pre-fusion spike of SARS-CoV-2 in live animals, highlighting its entry-inhibition capacity for a wide spectrum of SARS-CoV-2 variants.

The Effect of a Compound Protein on Wound Healing and Nutritional Status.

Proteins provide the main building blocks for tissue growth, cell renewal, and repair during wound healing. We aimed to examine the effect of a compound protein on wound healing, nutritional status, and underlying mechanisms. We first performed a preliminary experiment to identify the appropriate wound healing assessment points. In the formal experiment, there were five groups (control group: 8.3750 g/kg/day saline solution; model group: 8.3750 g/kg/day saline solution; whey protein group: 8.3750 g/kg/day whey protein; low-dose compound protein group: 4.1875 g/kg/day compound protein; and high-dose compound protein group: 8.3750 g/kg/day compound protein) with eight rats in each group. At each turning point, we observed the wound healing rate and nutritional status of the different groups of rats. In addition, biochemical assays were used to determine the mechanisms underlying the effects of the compound protein. In the preliminary experiment, the third day after modeling was the turning point between the inflammatory and proliferation phases, and the eighth day was the turning point between the proliferation and remodeling phases. The formal experiment evaluated wound healing condition, inflammatory response, angiogenesis, collagen deposition, and nutritional status. A pathological report showed increased vascularization, collagen deposition, and epithelialization in compound protein-treated groups. Protein-treated mice showed decreased interleukin (IL)-6, IL-8, neutrophils, and lymphocytes and increased IL-10, albumin, prealbumin, total protein levels, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), and vascular endothelial growth factor (VEGF) expressions. All parameters were significant (p < 0.05) compared to the model group. There was a dose-dependent effect of the compound protein. The accelerated wound healing mechanism may be that the compound protein accelerates the whole wound healing process, making wounds transition from the inflammatory phase to the proliferation phase faster, entering the remodeling phase earlier. Administration of a compound protein can accelerate wound healing and improve the nutritional status.

Glycosylation reduces the allergenicity of turbot (Scophthalmus maximus) parvalbumin by regulating digestibility, cellular mediators release and Th1/Th2 immunobalance

With turbot being increasingly consumed, turbot parvalbumin (TPV) allergy has become a pressing problem requiring immediate resolution. Glycosylation treatment not only resulted in cross-link formation but also caused changes in the simulated gastric fluid and simulated intestinal fluid digestion stability of TPV. In addition, KU812 experimentation revealed lower levels of β-hexosaminidase, histamine, tryptase, interleukin 4 (IL-4)/IL-13 in glycated protein-treated mice compared with native PV–treated ones. Glycated TPV exhibited a weaker allergic reaction compared with native TPV. Systemic anaphylaxis resulted in mild anaphylactic responses and reduced temperature, along with significantly increased levels of immunoglobulin E and histamine. Furthermore, glycosylation treatment reduced the release of cellular mediators and cytokines (IL-4/IL-13). Glycation to T-PV decreased allergic responses by downregulating Th2 cytokines, regulated the Th1/Th2 balance and effectively reduce the allergenicity and sensitisation ability of T-PV.

Eliminating Fc N-Linked Glycosylation and Its Impact on Dosing Consideration for a Transferrin Receptor Antibody-Erythropoietin Fusion Protein in Mice.

Erythropoietin (EPO), a hematopoietic growth factor and a promising therapy for Alzheimer's disease, has low permeability across the blood-brain barrier. The transferrin receptor antibody fused to EPO (TfRMAb-EPO) is a chimeric monoclonal antibody that ferries EPO into the brain via the transvascular route. However, TfRMAbs have Fc-effector function-related adverse effects including reticulocyte suppression. To overcome this, we recently developed an effectorless TfRMAb-EPO fusion protein, designated TfRMAb-N292G-EPO, by eliminating the Fc N-linked glycosylation site at position 292 of the antibody heavy chain. The mutant fusion protein showed enhanced plasma clearance and dramatically reduced plasma concentrations compared with the wild-type (WT) nonmutant fusion protein. This increased clearance of the aglycosylated TfRMAb is expected to increase the injection dose of the mutant fusion protein. To provide a basis for future therapeutic uses of this IgG-neurotrophin fusion protein, the current study aimed to characterize the pharmacokinetic profile of this effectorless TfRMAb-N292G-EPO at different doses following different routes of administration in the mouse. Adult C57BL/6J male mice were injected with a single dose (3, 6, 9, or 20 mg/kg; n = 3-6 per dose) of TfRMAb-N292G-EPO through either the subcutaneous (SQ) or intraperitoneal (IP) route. TfRMAb-N292G-EPO plasma concentrations were determined using an enzyme-linked immunosorbent assay. Mice were sacrificed 24 h after injection, and terminal blood was used for a complete blood count. Brain concentrations in the WT- and mutant fusion protein-treated mice were compared. We observed stark differences in the plasma pharmacokinetics of TfRMAb-N292G-EPO between the IP and SQ routes of administration. Dose escalation from 3 to 20 mg/kg increased the plasma Cmax only 3.5-fold for the SQ route, compared with a 35-fold increase for the IP route. The plasma Cmax was 15.0 ± 2.0, 21.3 ± 4.1, 21.3 ± 6.4, and 52.8 ± 27.9 ng/mL following SQ injection and 288 ± 47, 389 ± 154, 633 ± 194, and 10,066 ± 7059 ng/mL following IP injection for 3, 6, 9, and 20 mg/kg doses, respectively. The plasma Cmax following the SQ route was therefore 19- to 190-fold lower than that following the IP route. This finding is consistent with a 31-fold higher apparent clearance following the SQ route compared with the IP route at the highest dose administered. The brain concentrations in the mice treated with a 3 mg/kg dose of the mutant fusion protein were lower than those in the nonmutant WT-treated mice. No reticulocyte suppression was observed at the 3 mg/kg SQ dose of TfRMAb-N292G-EPO. However, reticulocyte suppression increased with an increase in dose and area under the plasma concentration-time curve (AUC) for both the IP and SQ routes. Overall, elimination of Fc N-linked glycosylation, to mitigate TfRMAb effector function side effects, has a profound effect on the plasma exposure of TfRMAb-N292G-EPO at therapeutic as well as high doses (3-20 mg/kg). This effect is more pronounced following SQ injection. The low plasma concentrations of the mutant fusion protein following a 3 mg/kg dose resulted in negligible brain uptake. The beneficial rescue of reticulocyte reduction by the N292G mutation is a function of AUC and is negated at high doses of the N292G mutant.

Toxicity Induced by a Bispecific T Cell-Redirecting Protein Is Mediated by Both T Cells and Myeloid Cells in Immunocompetent Mice.

Bispecific T cell engagers have demonstrated clinical efficacy; however, their use can be accompanied by severe toxicity. Mechanistic understanding of these toxicities is limited by a lack of suitable immunocompetent preclinical models. In this study, we describe an immunocompetent mouse tumor model that exhibits bispecific T cell engager-induced toxicity and recapitulates key features similar to those in human cytokine release syndrome. In this study, toxicity occurred between the second and fourth injections of an NK Group 2D bispecific T cell engager protein. Symptoms were transient, peaking 3-4 h after treatment and resolving by 8 h. Mice developed weight loss, elevated plasma cytokines, a significant reduction in spleen white pulp, and lymphocyte infiltration in the liver. Systemic cellular immune changes also occurred; notably, an increase in CD8+ T cell activation, an increase in myeloid cells in the blood, and a population of Ly-6Cint monocytes (CD11b+Ly-6G-F4/80-) emerged in the liver and spleens of bispecific protein-treated mice. IFN-γ was primarily produced by CD8+ T cells in the spleen and was required for the observed changes in both T cell and myeloid populations. Rag deficiency, IFN-γ deficiency, or depletion of either CD4+ or CD8+ T cells prevented toxicity, whereas perforin deficiency, GM-CSF deficiency, or modulation of the myeloid population through clodronate-mediated depletion showed a partial abrogation of toxicity. Together, these findings reveal that T cell activation by a bispecific T cell engager leads to changes in the host myeloid cell population, both of which contribute to treatment induced toxicity in immunocompetent mice.

In Vivo Efficacy of Anti-CD20-Interferon-Gamma Fusion Protein Against Syngeneic B Cell Lymphoma Is Mediated By Natural Killer Cells

Background: The interferons, including IFNα/IFNβ (type I) and IFNγ (type II) are essential mediators of anti-cancer immunity. To achieve efficient targeting of IFNs to tumor sites, we have developed antibody (Ab)-IFN fusion protein technology. We previously reported the antigen-specific targeting of IFNα to CD20+ target cells with efficient inhibition of proliferation, induction of apoptosis, and in vivo tumor eradiation dependent upon IFNα receptors on the tumor cell surface (Xuan et al, Blood, 2010). A fusion protein targeting human CD20 (anti-CD20-IFNα) exhibited stronger direct anti-proliferative effects, complement-dependent cytotoxicity (CDC), Ab-dependent cell-mediated cytotoxicity (ADCC), and in vivo potency against B-cell lymphoma xenograft models compared to the parent Ab rituximab (Timmerman et al, Blood 2015). Based on these results, a phase I, first-in-human, dose-escalation trial of anti-CD20-IFNα for B cell non-Hodgkin lymphoma is now underway (NCT02519270). Given the distinct properties of IFNγ from type I IFNs, including upregulation of antigen presentation, control of immune cell trafficking, and activation of T cells, NK cells, and macrophages, we hypothesized that Ab-targeted IFNγ may have anti-tumor effects mechanistically-distinct from those of Ab-IFNα fusions. We now report on the construction and characterization of anti-CD20 fusions containing IFNγ. Methods: The VH and VL regions from antibody 2B8 recognizing human CD20 were engineered in recombinant form with mouse IgG2a constant regions, and fused at the C-terminus with mIFNγ, yielding anti-hCD20-mIFNγ. Tumor cell proliferation in vitro was measured by [3H]-thymidine incorporation, ADCC by LDH release using mouse splenocyte effectors, CDC by propidium iodide (PI) exclusion, and in vivo tumor growth assessed using the huCD20-expressing syngeneic mouse B cell lymphoma 38C13-huCD20. Tumor-infiltrating lymphocytes were measured by flow cytometry. Results: Anti-hCD20-mIFNγ displayed potent IFNγ bioactivity comparable to free recombinant mIFNγ, and suppressed the in vitro proliferation of 38C13-huCD20 lymphoma cells by up to 70% (at 1 nM), though not as potently as anti-hCD20-mIFNα, which inhibited proliferation by 98% (Figure 1). Anti-hCD20-mIFNγ showed enhanced ADCC against lymphoma cells compared with the unfused, parent antibody (16-20% at E:T ratio of 20:1, versus 9-12%, respectively, p=0.0024)(Figure 2), while CDC was identical to unfused antibody. In vivo efficacy was demonstrated in mice bearing established subcutaneous 38C13-huCD20 tumors, with systemic (i.v.) injection of 100 μg anti-hCD20-mIFNγ fusion protein on days 5, 6, 7, or 5, 6, 7, 9 after tumor inoculation resulting in cure of approximately 70-80% of mice in repeated experiments. In contrast, therapy with equimolar doses of unfused, native anti-hCD20 Ab resulted in no cures. Mechanistic studies in anti-hCD20-mIFNγ fusion protein-treated mice showed that depletion of natural killer (NK) cells (using anti-asialo-GM1) significantly abrogated tumor clearance (p=0.01), while depletion of macrophages (clodronate liposomes) had lesser, borderline effects (p= 0.05)(Figure 2), and depletion of complement (cobra venom factor) or T cells (CD4+ or CD8+) had no significant effects on tumor eradication. Subcutaneous mouse B cell lymphomas treated with intratumoral injections of anti-hCD20-mIFNγ displayed increased tumor-infiltrating CD8+ T cells (mean 20.6% versus 5% in PBS-treated controls, p=0.008), and CD4+ T cells (mean 15.3% versus 6.6%). Conclusions: Anti-hCD20-mIFNγ fusion protein has in vitro and in vivo efficacy in a syngeneic, immunocompetent model of B cell lymphoma, with NK cells and possibly macrophages implicated in the mechanism(s) of tumor eradication. Ab-targeted mIFNγ can also promote infiltration of immune cells into the tumor microenvironment. These findings may suggest a novel approach for the immunotherapy of B cell lymphomas and other cancers. Disclosures Vasuthasawat: Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Trinh:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Morrison:Qwixel therapeutics LLC: Other: stake;which receives some funding through UCLA. Timmerman:ImmunGene: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support, Research Funding; Merck: Research Funding; Kite, A Gilead Company: Consultancy, Honoraria, Other: travel support, Research Funding; Spectrum Pharmaceuticals: Research Funding.

Abstract 113: Recombinant Tafazzin Enzyme Replacement Therapy Rescues Metabolic and Functional Defects in a Mouse Model of Barth Syndrome

Barth syndrome (BTHS) is an X-linked recessive disease where patients most commonly die from cardiomyopathy-induced heart failure before middle age. BTHS is caused by mutations in the tafazzin (TAZ) gene, resulting in defective TAZ protein. TAZ is an enzyme that generates mature cardiolipin (CL) from monolysocardiolipin (MLCL) in the mitochondrial membrane, a reaction essential for normal mitochondrial function. Current therapies can only treat the symptoms of BTHS. In this study, we propose an enzyme replacement therapy for BTHS which utilizes recombinant human TAZ fused to a cell penetrating peptide (hTAZ-CTP) to facilitate tissue uptake. The efficacy of this protein was tested in vitro on C2C12 TAZ-knockout (TAZ-KO) skeletal myoblasts and in vivo on a myocardial-specific TAZ conditional knockout mouse, modelling the cardiomyopathy associated with BTHS. In vitro tests of TAZ-KO cells, using oxygen consumption rate as a measure of mitochondrial activity, showed treatment of the cells with hTAZ-CTP effected a partial rescue of the fatty acid oxidation capabilities of the TAZ-KO cells. In vivo tests showed that BTHS mice display increasing septal wall thickness over time, an effect halted upon treatment with hTAZ-CTP. Pressure-volume (PV) loop analysis indicated that heart function, impaired in the vehicle-treated BTHS mouse, was similar between treated mice and normal mice. The ratio of MLCL/CL, a direct measure of TAZ enzymatic activity, was measured in heart mitochondria isolated from BTHS and control mice after treatment. The vehicle treated BTHS mouse showed the high MLCL/CL ratio typical of BTHS patients, whereas the MLCL/CL ratios in protein-treated mice matched the much lower ratio of the control mice. Similarly, oxygen consumption rate measurements of these isolated heart mitochondria demonstrated partial rescue by hTAZ-CTP treatment. Coupled with the lack of toxicity observed in the liver, spleen, kidney, and heart due to hTAZ-CTP injection, these results indicate that TAZ enzyme replacement therapy has great potential as a future treatment for BTHS.

Therapeutic effects of the rhSOD2-Hirudin fusion protein on bleomycin-induced pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and high mortality, posing a major threat to human health. Increased levels of inflammatory cytokines, reactive oxygen species and coagulation cascade have been extensively reported in IPF. We previously fused Hirudin and human manganese superoxide dismutase (hSOD2) to generate a dual-feature fusion protein, denoted as rhSOD2-Hirudin fusion protein. In this study, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Hydroxyproline (HYP) assays were used to investigate the effects of rhSOD2-Hirudin protein on thrombin-induced fibroblast proliferation and collagen accumulation in vitro. Subsequently, the mice model of pulmonary fibrosis induced by bleomycin was used for evaluating the anti-inflammatory and anti-fibrotic effects of rhSOD2-Hirudin protein in vivo. Results showed that rhSOD2-Hirudin protein could inhibit the proliferation of fibroblasts and reduce the HYP production in vitro by inhibiting the activity of thrombin. In vivo experiments showed that lung inflammation and fibrosis were significantly decreased in rhSOD2-Hirudin protein-treated mice. Furthermore, rhSOD2-Hirudin protein treatment reduced profibrotic protein and gene expression while reducing the number of inflammatory cells in the lung. In conclusion, rhSOD2-Hirudin protein can effectively attenuate pulmonary fibrosis in vitro and in vivo, mainly by inhibiting the activity of thrombin meanwhile increasing SOD2 levels prevent cells from being damaged by reactive oxygen species, thereby mitigating IPF progression. This study provided important information on the feasibility and efficacy of rhSOD2-Hirudin protein as a novel therapeutic agent for IPF.

Abstract 16604: Recombinant Tafazzin Enzyme Replacement Therapy Rescues Metabolic and Functional Defects in a Mouse Model of Barth Syndrome

Barth syndrome (BTHS) is an X-linked autosomal recessive disease caused by mutations in the tafazzin (TAZ) gene. These mutations lead to hypotonia, cyclic neutropenia, 3-methyglutaconic aciduria, and the most common cause of mortality in patients, cardiomyopathy. The TAZ gene encodes an enzyme that generates mature cardiolipin (CL) from monolysocardiolipin (MLCL)in the mitochondrial membrane, which is essential for normal mitochondrial morphology and function. Currently, there is no effective therapy for BTHS. In this study, we test an enzyme replacement therapy for BTHS in myocardial-specific TAZ conditional knockout mice which model the cardiomyopathy of BTHS. Serial echocardiograms showed that the BTHS mice developed increased septal wall thickness at 5 weeks of age. The BTHS mice were treated for 12 weeks with either vehicle or recombinant human TAZ fused to a cell penetrating peptide (hTAZ-CTP) to facilitate tissue uptake. During the treatment period, septal wall thickness in the vehicle-treated mouse continued to increase, whereas it remained stable in those treated with hTAZ-CTP . Pressure-volume (PV) loop analysis indicated that heart function was impaired in the vehicle-treated BTHS mouse while the protein-treated mice were shown to have PV loops similar to normal mice. The ratio of MLCL/CL, a direct measure of TAZ enzymatic activity, was measured in heart mitochondria isolated from BTHS and control floxed allele mice after treatment. The MLCL/CL ratio in the vehicle treated BTHS mouse was 20.4, consistent with a BTHS phenotype, whereas the MLCL/CL ratios in protein-treated and control mice were 0.01, indicating that injection of hTAZ-CTP successfully rescued the function of TAZ in BTHS mice. Histological observation of liver, spleen, kidney, and heart did not show any sign of toxicity for hTAZ-CTP injection. These results indicate that TAZ enzyme replacement therapy has great potential as a future treatment for BTHS.

Rac1 regulates bacterial toxin-induced thrombin generation.

Systemic inflammatory response syndrome is associated with severe coagulopathy. The purpose of this study was to examine thrombin generation in systemic inflammation triggered by the endotoxin lipopolysaccharide (LPS) and the exotoxin streptococcal M1 protein. Thrombin generation, lung histology and myeloperoxidase (MPO) activity were determined 6 and 24 h after induction of systemic inflammation. Male C57BL/6 mice received the Rac1 inhibitor NSC23766 prior to challenge with bacterial toxins. LPS and M1 protein challenge increased neutrophil infiltration and caused damage in the lung. Time to peak thrombin formation was increased and peak and total generation of thrombin were decreased in plasma from LPS- and M1 protein-treated mice. Coincubation of samples from mice exposed to bacterial toxins with platelet poor plasma from healthy mice completely reversed the inhibitory effect of LPS and M1 protein on thrombin generation, suggesting that bacterial toxins decreased levels of plasma factors explaining the reduction of thrombin generating capacity of plasma from septic animals. NSC23766 treatment not only decreased LPS- and M1 protein-induced neutrophil accumulation as well as levels of interleukin-6 and CXCL2 in the lung, but also abolished bacterial toxin-induced changes in thrombin generation. For example, NSC23766 increased peak formation by 57% and total thrombin generation by 48% in LPS-treated animals at 6 h. Taken together, our novel findings show that bacterial toxins increase thrombin generation via consumption of plasma factors and that Rac1 signaling plays an important role in thrombin generation in response to bacterial toxins. Thus, targeting Rac1 activity might be a useful way not only to ameliorate pulmonary inflammation, but also inhibit pathological changes in coagulation in bacterial infections.

Role of the NADPH Oxidases in the Subfornical Organ in Angiotensin II–Induced Hypertension

Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22(phox). SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22(phox), Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min(-1) × 2 weeks) was blunted in adenovirus encoding cre-recombinase-treated mice, as detected by dihydroethidium fluorescence. Deletion of p22(phox) in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97 ± 15 versus 154 ± 6 mm Hg, respectively; P=0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22(phox). These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling in hypertension.

Abstract 1578: MTBhsp70 fusion protein targeting mesothelin augments tumor specific T cell responses and prolongs survival in a murine model of ovarian cancer

Abstract Background and Purpose: There is a clear logic to adjuvant immunotherapy to enhance conventional treatment of ovarian cancer. Mesothelin (MSLN) is a surface glycoprotein expressed at high levels by ovarian cancers and is already a clinical target for immunotherapy. We have created a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting MSLN to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is known as a potent immune activator. This fusion protein binds MSLN on tumor cells and activates antigen presenting cells (APCs) through the interaction of MTBhsp70 with CD40 on APCs, thus promoting tumor antigen presentation and cross presentation and adjuvanting antitumor specific CD4 and CD8 T cell responses. We tested the hypothesis that the MSLN targeted MTBhsp70 fusion protein would improve survival and increase antitumor immune responses. Experimental Procedures: The efficacy of the MSLN targeted fusion protein was evaluated in a syngeneic mouse model of papillary ovarian cancer in immune competent FVB/NJ mice. Mice received 4 intraperitoneal (ip) treatments with experimental or control proteins from 7 days post ip injection of BR5FVB ovarian cancer cells. Survival time was compared from the day of tumor inoculation to that of the onset of disease (clinically evident ascites). In immunological studies, mice were sacrificed 26 days after tumor cell inoculation. Splenocytes were stained for CD3, CD4, CD8, CD25 and Foxp3, and examined by flow cytometry. Splenocytes were stimulated with MSLN or Her2/neu peptide, or mitomycin C-treated BR5FVB cells. IFNα-generated CD3+CD8+ T cells and CD3+CD8+ T Cell-surface exposure of CD107a/b were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo antibody depletion experiments using standard methodologies. Results: MSLN targeted MTBhsp70 fusion protein significantly delayed the onset of disease compared to saline (p=0.0025) or an unfused mixture of the component proteins, MTBhsp70 and scFv(MSLN) (p=0.0149). The survival advantage of the fusion protein treatment was abrogated by CD8 T cell depletion in tumor bearing mice. The fusion protein also reduced the proportion of CD4+CD25+Foxp3 Treg cells (p=0.0159) and increased the proportion of CD3+CD8+ T cells (p=0.0079) in the spleen compared to controls. In preliminary studies, significantly greater CD8 T cell responses were demonstrated in the spleen from the majority of fusion protein-treated mice compared to controls. The MSLN targeted fusion protein was also shown to evoke dendritic cell activation as well as antigen presentation and cross presentation in vitro. Conclusion: MSLN targeted MTBhsp70 fusion protein treatment of ovarian cancer significantly enhances survival and slows tumor growth while augmenting anti-tumor antigen responses in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1578. doi:1538-7445.AM2012-1578

Modulation of immune cell proliferation and chemotaxis towards CC chemokine ligand (CCL)-21 and CXC chemokine ligand (CXCL)-12 in undenatured whey protein-treated mice

Whey protein concentrates (WPCs) enhance innate mucosal immunity during early life and have a protective role in some immune disorders. To further elucidate the potential benefits of this protein, the present study investigated the effect of dietary supplementation with WPCs on blood parameters, plasma cytokine profiles, and immune cell proliferation and chemotaxis. A total of 45 male mice were equally distributed into three experimental groups and treated daily for 21 days as follows: group I was a control group that was orally supplemented with distilled water, group II was orally supplemented with undenatured WP (100 mg/kg body weight), and group III was orally supplemented with bovine serum albumin (100 mg/kg body weight). We found that the plasma cytokine levels of interleukin (IL)-1α, IL-1β, IL-10 and tumor necrosis factor-α and the levels of reactive oxygen species, cholesterol, triglycerides and the lipid profile were significantly decreased in the WP-treated group compared to the control group. In contrast, the levels of IL-2, IL-4, IL-7, IL-8 and glutathione were significantly elevated, and consequently, the ability of peripheral blood mononuclear cells to proliferate in response to stimulation with different antigens was significantly increased in the WP-treated group. Moreover, the in vitro chemotaxis of B, T and bone-marrow-derived dendritic cells toward CC chemokine ligand-21 and CXC chemokine ligand-12 was significantly increased, by twofold, in WP-treated mice compared to the control group. Taken together, our data reveal the benefits of WP supplementation in enhancing immune cell proliferation and migration to the secondary lymphoid organs.

P38 Mitogen-activated protein kinase signaling regulates streptococcal M1 protein-induced neutrophil activation and lung injury

M1 serotype of Streptococcus pyogenes can cause STSS and acute lung damage. Herein, the purpose was to define the role of p38 MAPK signaling in M1 protein-induced pulmonary injury. Male C57BL/6 mice were treated with specific p38 MAPK inhibitors (SB 239063 and SKF 86002) prior to M1 protein challenge. Edema, neutrophil infiltration, and CXC chemokines were determined in the lung, 4 h after M1 protein administration. Flow cytometry was used to determine Mac-1 expression. Phosphorylation and activity of p38 MAPK were determined by immunoprecipitation and Western blot. IVM was used to analyze leukocyte-endothelium interactions in the pulmonary microcirculation. M1 protein challenge increased phosphorylation and activity of p38 MAPK in the lung, which was inhibited by SB 239063 and SKF 86002. Inhibition of p38 MAPK activity decreased M1 protein-induced infiltration of neutrophils, edema, and CXC chemokine formation in the lung, as well as Mac-1 up-regulation on neutrophils. IVM showed that p38 MAPK inhibition reduced leukocyte rolling and adhesion in the pulmonary microvasculature of M1 protein-treated mice. Our results indicate that p38 MAPK signaling regulates neutrophil infiltration in acute lung injury induced by streptococcal M1 protein. Moreover, p38 MAPK activity controls CXC chemokine formation in the lung, as well as neutrophil expression of Mac-1 and recruitment in the pulmonary microvasculature. In conclusion, these findings suggest that targeting the p38 MAPK signaling pathway may open new opportunities to protect against lung injury in streptococcal infections.

Paracrine IL-33 Stimulation Enhances Lipopolysaccharide-Mediated Macrophage Activation

BackgroundIL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases.Methodology/Principal FindingsTo elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs.Conclusions/SignificanceOur findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.

Paracrine IL-33 stimulation enhances lipopolysaccharide-mediated macrophage activation (117.30)

Abstract BACKGROUND: IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.

Reevaluating Thyrotropin Receptor-Induced Mouse Models of Graves’ Disease and Ophthalmopathy

We aimed to establish and extend the characterization of murine models of thyroiditis and Graves' ophthalmopathy, induced by transfer of TSH receptor (TSHR) primed T cells. Experiments were performed in a different animal unit but using female BALB/cbyJico mice from the same supplier as previously. We report our findings together with a reevaluation of the earlier studies. In the first experiment, genetic immunization or TSHR fusion protein induced TSHR antibodies in all nine mice. Some of the antibodies functioned as thyroid-stimulating antibodies and/or TSH binding inhibiting Igs with two of seven mice having elevated T4. Thyroiditis and orbital changes were absent. Splenocyte transfer induced no immune response in naive BALB/cbyJico recipients. Subsequently genetic immunization or fusion protein-treated mice were maintained in either local or Brussels conditions (water, chow, and bedding). TSHR antibodies were induced in nine of nine Brussels (with decreased T4 in one of nine) but five of nine local mice. No thyroiditis or orbital changes were induced, but misleading fixation artefacts in extraocular muscles were noted. Nonspecific in vitro stimulation induced more CD-4+/IL-4+ cells in Brussels maintained. TSHR stimulation produced a significant increase in IL-4 secretion in six of nine local but one of seven Brussels mice. Thyroids from many TSHR-treated and control mice contained ectopic thymus. Our results confirm that thyroiditis is required for disease transfer but indicate the heterogeneity in TSHR-induced immune response in an inbred strain. Ectopic thymus can masquerade as thyroiditis, and care is required to avoid muscle artefacts. Because neither animal unit is pathogen free, microbial environment may contribute to determining TSHR-induced responses.

Decreased food intake does not completely account for adiposity reduction after ob protein infusion.

The effects of recombinantly produced ob protein were compared to those of food restriction in normal lean and genetically obese mice. Ob protein infusion into ob/ob mice resulted in large decreases in body and fat-depot weight and food intake that persisted throughout the study. Smaller decreases in body and fat-depot weights were observed in vehicle-treated ob/ob mice that were fed the same amount of food as that consumed by ob protein-treated ob/ob mice (pair feeding). In lean mice, ob protein infusion significantly decreased body and fat-depot weights, while decreasing food intake to a much lesser extent than in ob/ob mice. Pair feeding of lean vehicle-treated mice to the intake of ob protein-treated mice did not reduce body fat-depot weights. The potent weight-, adipose-, and appetite-reducing effects exerted by the ob protein in ob protein-deficient mice (ob/ob) confirm hypotheses generated from early parabiotic studies that suggested the existence of a circulating satiety factor of adipose origin. Pair-feeding studies provide compelling evidence that the ob protein exerts adipose-reducing effects in excess of those induced by reductions in food intake.

Effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness in the NZO mouse.

The effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness was studied in the NZO mouse, a model of non-insulin dependent diabetes and obesity. A single IV injection of 5 ng/g body weight islet activating protein markedly lowered plasma glucose and the glucose response to IP glucose administration, measured 5 days later (mean +/- SEM, plasma glucose levels 0, 10, 30 and 60 min after glucose 6.0 +/- 0.9, 14.6 +/- 1.3, 14.1 +/- 1.3 and 13.2 +/- 1.7 mmol/l in islet activating protein-treated NZO mice versus 12.8 +/- 1.6, 27.8 +/- 3.4, 34.7 +/- 4.1, 39.1 +/- 3.8 mmol/l in carrier-treated NZO mice). There was no difference in fasting plasma insulin levels between islet activating protein and carrier-treated mice. No response of plasma insulin to glucose occurred in the carrier-treated mice, but a highly significant insulin response to glucose was seen in the islet activating protein-treated mice. The in vitro responsiveness of pancreatic islets of islet activating protein-treated NZO mice to glucose was improved, and the inhibitory effect of adrenaline on insulin secretion was reduced. The in vivo hypoglycaemic response to exogenous insulin was not improved by islet activating protein and a demonstrated defect in the insulin sensitivity and responsiveness of glucose utilization by isolated soleus muscle was not reversed by islet activating protein treatment. It is concluded that islet activating protein is highly effective in improving glucose tolerance and insulin secretion in NZO mice, and that the improvement in glucose tolerance occurs without demonstrable improvement in the responsiveness to exogenous insulin or sensitivity of soleus muscle to insulin.