Recombinant Heat Shock Protein Research Articles

Heat shock protein 70 (HSP70) promotes air exposure tolerance of Litopenaeus vannamei by preventing hemocyte apoptosis

Brief pretreatment of cold shock at 13 °C for 3 min proved to be an inducer of heat shock protein 70 (HSP70) and improved stress tolerance as a molecular chaperone. With the improvement of air exposure tolerance, HSP70 in shrimp hemocytes was upregulated in mRNA and protein levels after cold shock. Both HSP70 RNA interference (RNAi) gene knockdown and recombinant HSP70 (rHSP70) injection were successfully established in order to investigate the role of HSP70 in response to air exposure stress. Shrimp receiving rHSP70 showed an improved survival rate (80%) with no significant difference (p > 0.05) compared to cold shock treated shrimp (control, 90%) under air exposure, but the survival rate of HSP70-knockdown shrimp was significantly lower (62%, p < 0.05). Reactive oxygen species (ROS) content, relative expression of cytochrome c, caspase-3 activity, and apoptosis rate in hemocytes of HSP70 enriched shrimp (i.e., cold shock and rHSP70 injection) were significantly lower (p < 0.05) than HSP70-knockdown shrimp. Results suggested that HSP70 could be induced by cold shock and contributed to improve the tolerance of shrimp suffering air exposure by blocking the apoptosis pathway through scavenging intracellular ROS, inhibiting cytochrome c expression, inhibiting release from mitochondria, and inactivating caspase-3. This work updates the understanding of cold shock mechanism in water-free transportation of aquatic animals.

Targeted Delivery of Recombinant Heat Shock Protein 27 to Cardiomyocytes Promotes Recovery from Myocardial Infarction.

Ischemic heart disease, especially myocardial infarction (MI), is the leading cause of death worldwide. Apoptotic mechanisms are thought to play a significant role in cardiomyocyte death after MI. Increased production of heat shock proteins (Hsps) in cardiomyocytes is a normal response to promote tolerance and to reduce cell damage. Hsp27 is considered to be a therapeutic option for the treatment of ischemic heart disease due to its protective effects on hypoxia-induced apoptosis. Despite its antiapoptotic effects, the lack of strategies to deliver Hsp27 to the heart tissue in vivo limits its clinical applicability. In this study, we utilized an antibody against the angiotensin II type 1 (AT1) receptor, which is expressed immediately after ischemia/reperfusion in the heart of MI rats. To achieve cardiomyocyte-targeted Hsp27 delivery after ischemia/reperfusion, we employed the immunoglobulin-binding dimer ZZ, a modified domain of protein A, in conjunction with the AT1 receptor antibody. Using the AT1 receptor antibody, we achieved systemic delivery of ZZ-TAT-GFP fusion protein into the heart of MI rats. This approach enabled selective delivery of Hsp27 to cardiomyocytes, rescued cells from apoptosis, reduced the area of fibrosis, and improved cardiac function in the rat MI model, thus suggesting its applicability as a cardiomyocyte-targeted protein delivery system to inhibit apoptosis induced by ischemic injury.

Phosphorylation Enhances Recombinant Heat Shock Protein 27 Performs as Pharmacological Preconditioning to Protect against Lung Ischemia Reperfusion Injury

We previously reported that the evidence for a protective role of the innate immune system in lung ischemic preconditioning (IPC), which is mediated a MyD88-independent pathway through TRIF, leading to NF-κB activation also heat shock proteins (HSPs) and protection against lung ischemia-reperfusion (I/R) injury (LIRI). We therefore hypothesized that exogenous HSPs would provide pharmacological preconditioning (PPC). In the present study, we examined whether phosphorylated recombinant HSP27 (prHSP27), which recombinant HSP27 phosphorylated by MAPKAP kinase 2 in vitro, affected PPC to protect against LIRI. C57BL/6 mice received prHSP27(2.5 g/kg) or vehicle 30 minutes prior to 60 minutes of ischemia of their left lungs, followed by 180 minutes of reperfusion or IPC with six cycles of 5 minutes ischemia and 5 minutes reperfusion prior to I/R. Response to injury was quantified by tissue MPO activity, vascular permeability, and leukocyte and inflammatory mediator accumulation in BAL expression. Pretreatment of mice with prHSP27 resulted in the development of a significant smaller LIRI, which quantified by vascular permeability (P=.005), MPO activity (P=.001), and BAL components when compared with vehicle treated lungs. In the prHSP27 treated group, there was no activation of MyD88, TRIF, or NF-κB before I/R as in the IPC group, however a strong activation of HSP-27 was showing before I/R as in the IPC group. In this study, we demonstrated that prHSP27 migrates into lung tissue and maintains its activation. It suggests that prHSP27 may be a useful therapeutic agent to protect against LIRI as PPC.

Synthesis, characterization and bio-functionalization of magnetic nanoparticles to improve the diagnosis of tuberculosis

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of : +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g−1. For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH2) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

Subcutaneous injection of recombinant heat shock protein 70 ameliorates atopic dermatitis skin lesions in a mouse model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease and sometimes is a tough challenge for physicians. We previously reported that in Th2 environment, the production and secretion of thymic stromal lymphopoietin (TSLP) from human keratinocytes was inhibited by recombinant heat shock protein 70 (rHSP70). The present study assessed the therapeutic effectiveness of rHSP70 in a mouse model of AD. An experimental model of AD was reproduced by systemic sensitization and local epicutaneous challenge with ovalbumin (OVA). Treatment of rHSP70 was performed by subcutaneous administration. The levels of OVA-specific IgE, as well as cytokines, were detected by ELISA. Skin samples from patch areas were also taken for histologic examination. Injection of rHSP70 improved the histologic picture by reducing the thickness of epidermis and allergic inflammation. Skin sonography revealed rHSP70 ameliorated skin remodeling. rHSP70 also significantly decreased the protein expression of TSLP of skin from patch areas. Furthermore, in ex vivo studies also showed group of rHSP70 treatment decreased IL-13, RANTES, MIP-1β and increased IFN-γ secreted from splenocytes stimulated with OVA. The rHSP70 intervention in the mouse model of AD reduced the skin expression of TSLP and attenuated the clinical appearance of OVA-induced AD mice. The effect was achieved by suppressed Th2 immune response in injected skin tissue and enhanced systemic Th1 immune response. These results suggest that rHSP70 have potential as a promising protein for the treatment of AD.

Effects of the Hydrogen Sulfide Donor GYY4137 and HSP70 Protein on the Activation of SH-SY5Y Cells by Lipopolysaccharide

The effects of exogenous recombinant human heat shock protein Hsp70 and hydrogen sulfide donor GYY4137 on the mechanisms of endocytosis of lipopolysaccharide (LPS) by human neuroblastoma cells SH-SY5Ywas studied. Hsp70 and GYY4137 have been shown to significantly reduce LPS-induced production of inflammatory mediators by SH-SY5Y cells, including reactive oxygen species, nitric oxide, TNFα, IL-1β, and IL-6. Both the recombinant protein Hsp70 and the hydrogen sulfide donor GYY4137 exhibited significant protective effects; however, the combined use of these agents did not lead to a cumulative effect. It has been shown that pinocytosis, as well as clathrin-, caveolin-, tubulin- and receptor-dependent endocytosis were involved in protecting the cells by both the hydrogen sulfide donor and Hsp70 from LPS-induced production of reactive oxygen species and NO.

Exogenous HSP70 and Signaling Pathways Involved in the Inhibition of LPS-Induced Neurotoxicity of Neuroblastoma Cells

Neuroinflammation plays a key role in the pathogenesis of neurodegenerative diseases. Microglial cells are the main immune cells of the central nervous system. On exposure to lipopolysaccharides (LPS, components of the cell wall of Gram-negative enterobacteria), microglia is activated to produce reactive oxygen species (ROS), cytokines, and inflammatory mediators, which may cause neuron death. Exogenous recombinant human heat shock protein 70 (HSP70) was tested for effect on the activation of human microglial and neuroblastoma cells in response to LPS from Escherichia coli. Experiments included cell cultivation separately and transferring the conditioned medium from A-172 microglial cells to SK-N-SH neuroblastoma cells to simulate the effect of microglia treated with LPS and/or HSP70. The levels of ROS, TNFα, and apoptosis in LPS-treated cells were estimated in the presence or absence of HSP70. HSP70 was found to reduce the LPS-induced ROS generation, TNFα production, apoptosis, and necrosis, in both separate cell cultures and neuroblastoma cells grown in the conditioned medium from microglial cells. Signaling pathways involving protein kinases p38MAPK, JNK, and PI3K were demonstrated to play an important role in HSP70-mediated protection of microglial and neuroblastoma cells from LPS-induced apoptosis and ROS production.

Effect of boar semen supplementation with recombinant heat shock proteins during summer

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

Evaluation of immuno-modulating effect of recombinant heat shock protein 40 of Brucella abortus in mice.

The present study was aimed to evaluate the immuno-modulatory effect of Brucella-specific recombinant HSP40 (rDnaJ) when co-immunized with Brucella rOmp22 in mice. For this, dnaJ of Brucella abortus was cloned, expressed in E. coli, and purified to homogeneity using Ni-NTA agarose columns. Three groups of mice (n = 6 in each group) were used in the study. The control group was immunized with rOmp22 alone, while group 1 mice were injected subcutaneously with rOmp22 along with conventional adjuvants (FCA, FIA), and group 2 mice with rOmp22 mixed with rDnaJ. IgG isotype (IgG1 and IgG2a) response to rOmp22 immunization was evaluated by enzyme-linked immunosorbent assay which was found to be directed towards the cell-mediated arm of immune system (CMI) in group 2 mice in which rOmp22 was co-immunized with rDnaJ. Expression profiling of IL-4 and IL-12 was checked in all the groups by qRT PCR. IL12 mRNA was up-regulated to a greater extent in group2 mice, suggesting that the CMI arm of immune system was stimulated. Hence, it was concluded that CMI response against rOmp22 is stimulated to a greater extent in mice when co-immunized with Brucella rDnaJ.

Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies

PurposeAnti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice.MethodsHeat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice.ResultsrmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70.ConclusionThe results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins.

A novel MtHSP70-FPR1 fusion protein enhances cytotoxic T lymphocyte responses to cervical cancer cells by activating human monocyte-derived dendritic cells via the p38 MAPK signaling pathway

ObjectiveTo evaluate the potential effects of recombinant mycobacterium tuberculosis heat shock protein 70-formyl peptide receptor 1 (MtHSP70-FPR1) fusion protein on human monocyte-derived dendritic cell (moDC) maturation; cytotoxic T lymphocyte (CTL) responses to cervical cancer (CC) cells; and the roles of the p38 MAPK, ERK, and JNK pathways in its transition. MethodsMonocytes were positively selected with a MACS column with antiCD14 antibody-conjugated microbeads from umbilical cord blood. MoDCs were stimulated with MtHSP70-FPR1, MtHSP70, a mix of MtHSP70 and FPR1, FPR1, or phosphate buffer solution (PBS) as control. Flow cytometry was used to analyze the surface molecule expression of moDCs and IFN-γ-producing CD8+ T cells. T cell proliferation was assessed using [3][H]-thymidine assays. The cytotoxicity of moDC-activated T cells against CC cells was evaluated by MTT assays. Cytokine production was determined by enzyme-linked immunosorbent assay. Western blotting was used to investigate protein expression. ResultsCompared with MtHSP70, MtHSP70 + FPR1, FPR1, or PBS-mediated moDCs, MtHSP70-FPR1-pulsed moDCs expressed higher levels of CD80, CD86, CD83, HLA-DR, and CCR7; secreted more IL-12p70, TNF-ɑ and IL-1β; and elicited stronger CTL priming and proliferation, resulting in an effective, HLA-I-dependent killing effect on CC cells. The p38 MAPK, ERK, and JNK pathways were all activated in MtHSP70-FPR1-mediated moDC maturation, but the p38 MAPK pathway played a vital role. ConclusionsThe excellent capability of MtHSP70-FPR1 fusion protein to induce phenotypical and functional maturation of moDCs and CC-specific CTL responses partly illustrates the potential clinical benefits of DC-based immunotherapy for CC.

Prospects of developing a prophylactic vaccine against human lymphatic filariasis – evaluation of protection in non-human primates

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130–180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

Phosphorylated recombinant HSP27 protects the brain and attenuates blood-brain barrier disruption following stroke in mice receiving intravenous tissue-plasminogen activator.

Loss of integrity of the blood-brain barrier (BBB) in ischemic stroke victims initiates a devastating cascade of events causing brain damage. Maintaining the BBB is important to preserve brain function in ischemic stroke. Unfortunately, recombinant tissue plasminogen activator (tPA), the only effective fibrinolytic treatment at the acute stage of ischemic stroke, also injures the BBB and increases the risk of brain edema and secondary hemorrhagic transformation. Thus, it is important to identify compounds that maintain BBB integrity in the face of ischemic injury in patients with stroke. We previously demonstrated that intravenously injected phosphorylated recombinant heat shock protein 27 (prHSP27) protects the brains of mice with transient middle cerebral artery occlusion (tMCAO), an animal stroke-model. Here, we determined whether prHSP27, in addition to attenuating brain injury, also decreases BBB damage in hyperglycemic tMCAO mice that had received tPA. After induction of hyperglycemia and tMCAO, we examined 4 treatment groups: 1) bovine serum albumin (BSA), 2) prHSP27, 3) tPA, 4) tPA plus prHSP27. We examined the effects of prHSP27 by comparing the BSA and prHSP27 groups and the tPA and tPA plus prHSP27 groups. Twenty-four hours after injection, prHSP27 reduced infarct volume, brain swelling, neurological deficits, the loss of microvessel proteins and endothelial cell walls, and mortality. It also reduced the rates of hemorrhagic transformation, extravasation of endogenous IgG, and MMP-9 activity, signs of BBB damage. Therefore, prHSP27 injection attenuated brain damage and preserved the BBB in tPA-injected, hyperglycemic tMCAO experimental stroke-model mice, in which the BBB is even more severely damaged than in simple tMCAO mice. The attenuation of brain damage and BBB disruption in the presence of tPA suggests the effectiveness of prHSP27 and tPA as a combination therapy. prHSP27 may be a novel therapeutic agent for ischemic stroke patients whose BBBs are injured following tPA injections.

Recombinant Hsp33 and OmpC protein can serve as promising divalent vaccine with protection against Vibrio anguillarum and Edwardsiella tarda in flounder (Paralichthys olivaceus)

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder (Paralichthys olivaceus). In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda, respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda, specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8β and IgM in spleen, head-kidney, and hindgut, RPS of 70 ± 3.45% against V. anguillarum and 60 ± 1.48% against E. tarda, respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ± 3.73% against E. tarda; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ± 1.27% against V. anguillarm; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder.

The Effect of Beta-Amyloid Peptides and Main Stress Protein HSP70 on Human SH-SY5Y Neuroblastoma Proteome

The accumulation and aggregation of β-amyloids are major molecular events underlying the progression of Alzheimer's disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aβ and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aβ42 and isomerized Asp7 (isoAβ42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aβ-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aβ peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aβ peptides.

Recombinant human heat shock protein 27 can inhibit ultraviolet B‐induced differentiation in pterygial‐derived fibroblast

PurposeTo investigate the effect of recombinant human heat shock protein 27 (rhHSP27) on the differentiation of human pterygial‐derived fibroblast.MethodsHuman conjunctival and pterygial fibroblasts were isolated and cultured from specimens of normal conjunctiva and pterygium head, which were donated during pterygium excision. Cultured conjunctival and pterygial fibroblasts were exposed to 20 mJ/cm2 of ultraviolet‐B (UVB) irradiation with and without 4 ug/mL rhHSP27 pre‐treatment. Western blot analyses of α‐smooth muscle actin (α‐SMA), a marker for myofibroblast, were performed in each group and quantified using Image Gauge 4.0 software. The ratios of α‐SMA / GAPDH and phosphorylated HSP27 / GAPDH were compared.ResultsThe α‐SMA level in Pterygial fibroblasts showed 2.85 ± 0.11 fold greater than that of normal conjunctival fibroblasts (P &lt; 0.001). The level of α‐SMA was increased by 1.92 ± 0.02 times (P &lt; 0.001), when normal conjunctival fibroblasts were exposed to UVB irradiation without rhHSP27 pre‐treatment. However, when normal conjunctival fibroblasts were exposed to UVB irradiation with rhHSP27 pre‐treatment, the fibroblasts demonstrated 0.82 ± 0.04 fold smaller α‐SMA level than control did (P = 0.04).ConclusionsThe rhHSP27 revealed inhibitory effects on the UVB irradiation‐induced differentiation from normal conjunctival fibroblasts to pterygial myofibroblast. The rhHSP27 can be a preventive treatment against pterygium growth.

Abstract 4609: Agenus’ next generation cancer vaccine platforms

Abstract Most cancer cells carry mutations unique to the patient’s individual tumor and shared biochemical signatures that are not present in healthy cells. Agenus has three vaccine platforms designed to treat cancers based on the unique needs of a given patient. Our vaccine platforms are designed to educate the patient’s immune system to recognize tumor-specific aberrations, or neo-antigens, and mount an anti-tumor immune response. Agenus’ Prophage™ vaccine platform is an individualized vaccine made from the patient’s own tumor tissue. Heat shock proteins (gp-96) that naturally chaperone and bind tumor-derived peptides are extracted from the patient’s tumor and constitute the vaccine. Some of these peptides are neo-antigens. Agenus has completed Phase 2 clinical trials with Prophage™ vaccine in newly diagnosed glioblastoma (ndGBM), and has previously reported that there was improved progression-free and overall survival with Prophage™ vaccine compared to standard of care. Agenus’ AutoSynVax™ vaccines are uniquely designed and manufactured for each patient based on NGS profiling of the patient’s tumor from a biopsy. Leveraging the Agenus Immunogenic Mutation (AIM™) workflow, we are able to generate a synthesis-ready blueprint for an optimal immunogenic and personalized neo-antigen vaccine. The AIM™ platform provides a robust and efficient approach to computational vaccinology designed to deliver a set of likely immunogenic peptides, agnostic to vaccine format, followed by generation of a format-specific blueprint ready for vaccine synthesis and manufacture. The synthesized neo-epitopes are complexed to recombinant heat shock protein 70 (HSC70) and are administered along with our QS-21 Stimulon® adjuvant. HSC70 is known to transport epitopes and play a role in displaying them to T cells. While the first two of Agenus’ vaccine platforms are highly individualized, our PhosphoSynVax™ vaccine is an off-the-shelf vaccine format targeting a novel class of tumor neo-antigens arising from post translational modifications (PTMs). Due to dysregulated cell signaling pathways in cancer, self-peptides can be aberrantly phosphorylated, a number of which are subsequently presented on HLA molecules. Using mass-spectrometry, we have identified a library of over a thousand HLA phospholigands. Many of these are tumor specific and found in multiple patients across multiple indications, enabling pre-manufacture of PhosphoSynVax™ vaccines for ready use. Upon testing the HSP plus synthetic peptide vaccine format in murine models, we have demonstrated effective tumor control in a therapeutic setting and also effective immune memory in a long-term prophylactic setting. Given Agenus’ diverse portfolio we have the opportunity to combine our immune education strategies with immunomodulatory antibodies to increase therapeutic efficacy. Citation Format: Mohamed Uduman, Mithun Khattar, Bishnu Joshi, Antoine Tanne, Benjamin Morin, Armen Karapetyan, Elise Drouin, Sandra Craig, Paisley Myers, Erin Jeffery, Nicholas Wilson, Amy Yang, Victor H. Engelhard, Mark Cobbold, Donald F. Hunt, Dennis Underwood, Shiwen Lin, Mark Findeis, Jeffrey Raizer, John Goldberg, Jennifer S. Buell, Robert Stein, Daniel L. Levey, John Castle. Agenus’ next generation cancer vaccine platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2017-4609

Development of a colloidal gold immunochromatographic strip based on HSP70 for the rapid detection of Echinococcus granulosus in sheep

Echinococcus granulosus is the causative pathogen of cystic echinococcosis, a serious disease endangering human and animal health. In this study, an immunochromatographic strip was developed based on the recombinant protein Heat shock protein 70 (HSP70) for the serological detection of E. granulosus. The protocol completes within 20min requiring no specialized equipment or chemical reagents, while specificity tests confirmed no cross-reactivity with positive serum of Fasciola hepatica, Haemonchus contortus, Peste des petits ruminants virus (PPRV) and Foot-and-mouth disease virus (FMDV). The strips remained stable after storage at 4°C for up to 8 months. Both immunochromatographic strip and ELISA tests were applied to detect E. granulosus antibody in a total of 728 serum samples obtained from slaughter houses in Zhejiang Province. Our data revealed positive rates of 2.61 and 1.65% by immunochromatographic strip and ELISA methods, respectively. The immunochromatographic strip test developed in this study provides a simple, specific and rapid method of E. granulosus antibody detection and infected sheep monitoring.

Application of antibodies to recombinant heat shock protein 70 in immunohistochemical diagnosis of mycobacterium avium subspecies paratuberculosis in tissues of naturally infected cattle

BackgroundDetection of Mycobacterium avium subspecies paratuberculosis (MAP) infection is key to the control of Johne’s disease. Immunohistochemistry is one of the methods of detection of MAP infection in tissues. However, unavailability of commercial antibodies that can detect the organism is a limiting factor for the use of immunohistochemistry. This study was aimed at developing an immunohistochemistry method to diagnose MAP in infected tissues using antibodies against MAP recombinant heat shock protein 70kd.ResultsMAP Heat shock protein 70 gene was amplified and cloned into an expression vector, Champion pET-SUMO, then expressed in E coli, purified and used to produce polyclonal rabbit antibodies against the Heat shock protein. Immunohistochemistry was performed in 35 MAP infected tissues with anti-HSP70 polyclonal antibodies. All 35 MAP infected tissues were positive for MAP within macrophages, epithelioid cells and giant cells either in clumps or singly as individual bacilli. No positive staining was seen in the three uninfected normal tissues and in MAP infected tissues where primary antibodies were substituted with PBS or pre-immune serum from the same rabbit.ConclusionAnti-HSP70 produced in this study offers an opportunity for improved diagnosis, screening of MAP in animal tissues and in studies on the pathogenesis of MAP

Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100μM) for 24h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.