Immunological responses and anti-tumor effects of HPV16/18 L1-L2-E7 multiepitope fusion construct along with curcumin and nanocurcumin in C57BL/6 mouse model.

AIMS: Human papillomavirus (HPV) L1, L2 and E7 proteins were used as target antigens for development of preventive and therapeutic vaccines. Moreover, linkage of antigens to heat shock proteins (HSPs) could enhance the potency of vaccines. Curcumin and nanocurcumin compounds were suggested as the chemopreventive and chemotherapeutic agents against cancer. In this study, two multiepitope DNA and peptide-based vaccine constructs (L1-L2-E7 and HSP70-L1-L2-E7) were used along with curcumin and nanocurcumin to evaluate immune responses, and protective/therapeutic effects in tumor mouse model. MAIN METHODS: At first, the multiepitope L1-L2-E7 and HSP70-L1-L2-E7 fusion genes were subcloned in eukaryotic and prokaryotic expression vectors. The recombinant multiepitope peptides were generated in E. coli strain. Then, the cytotoxic effects of curcumin and nanocurcumin were evaluated on HEK-293 T non-cancerous and C3 cancerous cells. Finally, mice vaccination was performed using different regimens. Curcumin and nanocurcumin compounds were administered alone or along with different vaccine constructs. KEY FINDINGS: Our data indicated that the use of nanocurcumin along with the multiepitope HSP70-L1-L2-E7 vaccine construct could completely protect mice against HPV-related C3 tumor cells, and eradicate tumors in a therapeutic test. Furthermore, nanocurcumin showed higher protection than curcumin alone. Generally, curcumin and nanocurcumin compounds could reduce tumor growth synergistically with the multiepitope vaccine constructs, but they did not influence the immune responses in different regimens. SIGNIFICANCE: These data demonstrated that the designed multiepitope vaccine constructs along with curcumin and nanocurcumin can be used as a promising method for HPV vaccine development.

Exogenous Hsp70 attenuates nitroglycerin-induced migraine-like symptoms in mice.

Although the connection between heat shock protein 70 (HSP70) and vestibular migraine is not clear, HSP70 is neuroprotective in other scenarios. This study aimed to investigate the potential of exogenous HSP70 for treating migraine-like symptoms in a mouse model of nitroglycerin (NTG)-induced migraine. HSP70 levels were assessed in patients with vestibular migraine and healthy individuals by ELISA. Migraine was induced in mice by NTG, and HSP70 expression was examined in the trigeminal nucleus caudalis (TNC) tissue of mice treated with NTG and NTG together with exogenous HSP70. The effects of exogenous HSP70 on migraine-like symptoms were assessed through behavioral assays. Finally, the impact of HSP70 on oxidative stress and NF-κB signaling in mice with migraine was investigated. Serum HSP70 in patients with vestibular migraine was significantly lower than that of healthy individuals. NTG administration significantly suppressed HSP70 expression in mouse TNC tissue, which was reversed by exogenous HSP70. HSP70 alleviated NTG-induced mechanical hypersensitivity, light aversion, and anxiety-like behavior. Finally, exogenous HSP70 suppressed NTG-induced oxidative stress and NF-κB signaling. Our study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.NEW & NOTEWORTHY The study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.

Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice.

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund's adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro.

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Plasma exosomes protect against cerebral ischemia/reperfusion injury via exosomal HSP70 mediated suppression of ROS.

AIMS: Ischemic stroke is the leading cause of severe disability and death worldwide. As the pathogenesis of stroke has not been clearly elucidated and the ability of current therapeutic drugs on crossing the blood-brain barrier (BBB) is extremely low, there is no effective strategy to treat stroke. We aim at investigating the specific advantages of using plasma exosomes (Pla-Exo) for targeting ischemic brain and exploring its underlying mechanism in neuroprotection. MAIN METHODS: Pla-Exo was obtained by a gradient ultracentrifugation of fresh plasma. The quantification of penetrated Pla-Exo through BBB was investigated in vitro BBB model, furthermore, the effects of Pla-Exo and exosomal HSP70 on cerebral ischemia/reperfusion injury were evaluated. KEY FINDINGS: Pla-Exo enhanced BBB crossing by specific interaction between Pla-Exo inherited heat shock protein 70 (HSP70) and endothelial Toll-like receptor 4 (TLR4). As expected, Pla-Exo increased HSP70 expression in the ischemic region through the transfer of HSP70, and led to HSP70 mediated suppression of ROS, thus alleviating cerebral ischemia/reperfusion (I/R) injury by attenuating the deterioration of BBB and preventing mitochondria damage. SIGNIFICANCE: These findings indicated that Pla-Exo can provide protection against ischemia-reperfusion injury via the regulation of HSP70 and it should be further studied as a potential candidate for protection against ischemic injury.

Vaccine Potential of a Recombinant Bivalent Fusion Protein LcrV-HSP70 Against Plague and Yersiniosis.

To counteract the deadly pathogens, i.e., Y. pestis, Y. enetrocolitica, and Y. pseudotuberculosis, we prepared a recombinant DNA construct lcrV-hsp70 encoding the bivalent fusion protein LcrV-HSP70. The lcrV gene of Y. pestis and hsp70 domain II DNA fragment of M. tuberculosis were amplified by PCR. The lcrV amplicon was first ligated in the pET vector using NcoI and BamHI restriction sites. Just downstream to the lcrV gene, the hsp70 domain II was ligated using BamHI and Hind III restriction sites. The in-frame and the orientation of cloned lcrV-hsp70 were checked by restriction analysis and nucleotide sequencing. The recombinant bivalent fusion protein LcrV-HSP70 was expressed in E. coli and purified by affinity chromatography. The vaccine potential of LcrV-HSP70 fusion protein was evaluated in formulation with alum. BALB/c mice were vaccinated, and the humoral and cellular immune responses were studied. The fusion protein LcrV-HSP70 induced a strong and significant humoral immune response in comparison to control animals. We also observed a significant difference in the expression levels of IFN-γ and TNF-α in LcrV-HSP70-immunized mice in comparison to control, HSP70, and LcrV groups. To test the protective efficacy of the LcrV-HSP70 fusion protein against plague and Yersiniosis, the vaccinated mice were challenged with Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis separately. The bivalent fusion protein LcrV-HSP70 imparted 100% protection against the plague. In the case of Yersiniosis, on day 2 post challenge, there was a significant reduction in the number of CFU of Y. enterocolitica and Y. pseudotuberculosis in the blood (CFU/ml) and the spleen (CFU/g) of vaccinated animals in comparison to the LcrV, HSP70, and control group animals.

Superior adjuvanticity of the genetically fused D1 domain of Neisseria meningitides Ag473 lipoprotein among three Toll-like receptor ligands.

Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways.

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-1ß 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.

Heat shock protein 70 from Litopenaeus vannamei (LvHSP70) is involved in the innate immune response against white spot syndrome virus (WSSV) infection.

White spot syndrome (WSS) caused by white spot syndrome virus (WSSV) is a severe infectious disease in shrimp aquaculture. To find effective therapeutics to control WSSV, it is indispensable to understand the innate immune responses of shrimp to WSSV infection. Previous report demonstrated that the Litopenaeus vannamei heat shock protein 70 (LvHSP70) could induce shrimp innate immunity against bacterial infection. Herein, we further investigate the role of LvHSP70 in anti-WSSV infection. The temporal expression of LvHSP70 was significantly upregulated 2.5- and 1.5-fold at 6 and 24 h post systemic WSSV infection suggesting that the LvHSP70 was a WSSV responsive gene. The recombinant protein of LvHSP70 (rLvHSP70) was produced in an Escherichia coli system and its effect in protection against WSSV infection was investigated. Intramuscularly injection of juvenile shrimp with 1 nmol of rLvHSP70 could significantly prolong 50% mortality of WSSV-infected shrimp from 3 days to 5 days as compared to the control group injected with bovine serum albumin (BSA). Consistently, the injection of rLvHSP70 resulted in 24-fold, 20-fold and 100-fold decrease in the viral copy number after 6, 12 and 24 h post injection, respectively, compared to the control shrimp injected with BSA. Interestingly, it was found that the rLvHSP70 enhanced the expression of the key gene in the prophenoloxidase (proPO) activating system, LvproPO, but reduced the expression of Lvcaspase2 and LvIAP in WSSV-infected shrimp. These results suggested that the LvHSP70 is an important molecule involved in antiviral defense in shrimp presumably via modulating the proPO system and apoptosis.

NK Cell Induced T Cell Anergy Depends on GRAIL Expression.

NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.

The Effect of Human HSP70 Administration on a Mouse Model of Alzheimer's Disease Strongly Depends on Transgenicity and Age.

In humans, heat shock protein 70 is a key component of the machinery that protects neuronal cells from various stress conditions and whose production significantly declines during aging. Herein, we investigated the protective effect of sub-chronic intranasal administration of human Hsp70 on the state of neurons in the temporal cortex and areas of the hippocampus of old transgenic (Tg) 5XFAD mice (11-13 months), representing a late-onset model of hereditary Alzheimer's disease. Quantitative analysis of the various neuronal pathologies between the two groups (Tg versus nTg) revealed maximal levels of abnormalities in the brains of aged Tg mice. Importantly, intranasal application of HSP70 had profound beneficial effects on neuron morphology in the temporal cortex and hippocampal regions when applied to the aged Tg mice but not in the case of age-matched, non-transgenic, littermate animals. Furthermore, the effect of HSP70 administration on neurons in the hippocampus and temporal cortex differed characteristically between the groups. Using RNA-Seq, we identified a lot of differentially expressed genes in the hippocampus of old Tg mice compared with those of nTg mice. Most importantly, we observed HSP70-induced upregulation of multiple genes participating in antigen processing and presentation especially the members of major histocompatibility complex (class I and II) in the brains of old 5XFAD Tg animals, suggesting that Hsp70 executes its beneficial role via activation of adaptive immunity. Overall, our data enable to conclude that Hsp70 treatment may be a safe and effective therapeutic application against Alzheimer-type neuropathologies manifested at the late stages of the disease.

Vaccination with three tandem repeats of M2 extracellular domain fused to Leismania major HSP70 protects mice against influenza A virus challenge.

Influenza viruses are globally important respiratory pathogens with high degree of morbidity and mortality during annual epidemics. Influenza A vaccination has been challenged due to genetic evolution. M2 protein is the surface antigen of the virus and facilitates virus entry into the host cells. N-terminal 24 amino acid residues that constitute the extracellular domain of M2 protein (M2e) show remarkable conservation among all subtypes of influenza A viruses. The aim of the present study was to investigate the effects of using HSP70 as an adjuvant fused to three tandem repeats of M2e (3M2e) to enhance the immune responses against influenza A challenge. The mice were immunized three times by intradermal inoculations of 3M2e alone or in combination with Alum adjuvant or in fused form to HSP70. The specific immune responses were evaluated by measuring the serum antibody titers, lymphocyte proliferation, as well as Th1 and Th2 cytokines. The results showed that, although 3M2e with no adjuvant could induce secretion of specific antibodies, significantly higher humoral immune responses were stimulated in combination with Alum adjuvant (p < 0.05). Moreover, analysis of specific immune responses revealed that the 3M2e-HSP70 chimer protein mainly stimulated IgG2a and IFN-γ responses indicating aTh1 bias which shows the ability of HSP as a powerful adjuvant for activation of cellular immune responses. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IL-4 production and lymphocyte proliferation (P ˂ 0.001) compared with mice vaccinated with 3M2e alone or supplemented with Alum, suggesting a mixture of Th1 and Th2 type cellular immune response with a Th1 bias. The findings of this study indicated that 3M2e-HSP70 enhances humoral and cellular immune responses and improves immune protection against influenza challenge in mice. Thus, it has the potential to be used as a promising vaccine candidate.

Topical heat shock protein 70 prevents imiquimod-induced psoriasis-like inflammation in mice.

Psoriasis is a chronic inflammatory skin disease with systemic manifestations and potential genetic etiology. The newest treatments utilize antibodies against one of several cytokines known to underlie the inflammatory signaling molecules that produce the skin and systemic symptoms. However, these agents must be regularly injected, and they may compromise the normal responses of the immune system. Furthermore, they do not address the causes of the abnormal immunoregulatory responses of the disease because the etiology is not yet completely understood. In this short-term treatment study, the potential anti-inflammatory activity of an alfalfa-derived Hsp70-containing skin cream (aHsp70) was tested on imiquimod (IMQ)-induced psoriasis-like lesions in wild-type mice. Treatment of the mice with the aHsp70 skin cream simultaneously with the imiquimod application mitigated the induction of psoriatic-like lesions and correlated with altered expression of various skin cytokines.

HSP70 Facilitates Memory Consolidation of Fear Conditioning through MAPK Pathway in the Hippocampus.

Heat shock proteins of the 70-kDa (HSP70) family are cytoprotective molecular chaperones that are present in neuronal cells and can be induced by a variety of homeostatically stressful situations (not only proteostatic insults), but also by synaptic activity, including learning tasks. Physiological stimuli that induce long-term memory formation are also capable of stimulating the synthesis of HSP70 through the activation of heat shock transcription factor-1 (HSF1). In this study, we investigated the influence of HSP70 on fear memory consolidation and MAPK activity. Male rats were trained in contextual fear conditioning task and HSP70 content was analyzed by western blot in the hippocampus at different time points. We observed rapid and transient elevations in HSP70 60 min following training. Double immunofluorescence with GFAP and HSP72 revealed that astrocytes were not the site for HSP72 induction by CFC training. HSP72 distribution markedly surrounded synapses between Shaffer collateral and CA1 pyramidal cells. Infusion of recombinant HSP70 (hspa1a) into the dorsal hippocampus immediately after training facilitated memory consolidation and enhanced ERK activity while decreasing the activated forms of JNK and p38 in the hippocampus. Blocking endogenous extracellular HSP70 through the administration of specific antibody did not produce any further effect on memory consolidation when applied immediately after training, suggesting that it is indeed acting intracellularly. Induction of HSP70 after fear conditioning is fast and can act as a signaling molecule, modulating MAPK downstream signaling during memory consolidation in the hippocampus, which is crucial for fear memory formation.

Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice.

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 µg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4-6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.

Molecular Mechanisms Underlying Neuroprotective Effect of Intranasal Administration of Human Hsp70 in Mouse Model of Alzheimer's Disease.

Heat shock protein 70, encoded by the HSPA1A gene in humans, is a key component of the machinery that protects neuronal cells from various stress conditions and whose production significantly declines during the course of aging and as a result of several neurodegenerative diseases. Herein, we investigated whether sub-chronic intranasal administration of exogenous Hsp70 (eHsp70) exerts a neuroprotective effect on the temporal cortex and areas of the hippocampus in transgenic 5XFAD mice, a model of Alzheimer's disease. The quantitative analysis of neuronal pathologies in the compared groups, transgenic (Tg) versus non-transgenic (nTg), revealed high level of abnormalities in the brains of transgenic mice. Treatment with human recombinant Hsp70 had profound rejuvenation effect on both neuronal morphology and functional state in the temporal cortex and hippocampal regions in transgenic mice. Hsp70 administration had a smaller, but still significant, effect on the functional state of neurons in non-transgenic mice as well. Using deep sequencing, we identified multiple differentially expressed genes (DEGs) in the hippocampus of transgenic and non-transgenic mice. Furthermore, this analysis demonstrated that eHsp70 administration strongly modulates the spectrum of DEGs in transgenic animals, reverting to a pattern similar to that observed in non-transgenic age-matched mice, which included upregulation of genes responsible for amine transport, transmission of nerve impulses and other pathways that are impaired in 5XFAD mice. Overall, our data indicate that Hsp70 treatment may be an effective therapeutic against old age diseases of the Alzheimer's type.

Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production.

BACKGROUND: Heat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90). METHODS: rCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA. RESULTS: Both proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1ß, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90. CONCLUSION: This study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

HSP70-based anti-cancer immunotherapy.

Heat shock protein 70, (Hsp70) constitutes a powerful system of cytoprotection in all organisms studied to date. Exerting such activity, Hsp70 rescues cancer cells from antitumor therapy, posing a great challenge for oncologists. In contrast to its protective action, Hsp70 was found to be released from cancer cells, prompting cytotoxic lymphocytes to target and kill the tumor. A great number of vaccines have been developed on the basis of the ability of Hsp70 to present tumor antigen or to elevate the sensitivity of cancer cells to cytotoxic lymphocytes. In this commentary, we consider novel data on the employment of pure Hsp70 in the therapy of glioma and melanoma malignancies. We show that intratumorally delivered Hsp70 penetrates cancer cells and pulls its intracellular analog outside of the cell. This displacement may activate cells, constituting both innate and adaptive immunity. In vivo delivery of Hsp70 was found to inhibit tumor growth and to extend survival. The technology of intratumoral injection of pure Hsp70 passed through preclinical trials and was investigated in clinics for children with brain cancer; the results show the safety and feasibility of a new approach.

70-kDa heat shock protein coated magnetic nanocarriers as a nanovaccine for induction of anti-tumor immune response in experimental glioma.

Nanovaccines based on superparamagnetic iron oxide nanoparticles (SPIONs) provide a novel approach to induce the humoral and cell-based immune system to fight cancer. Herein, we increased the immunostimulatory capacity of SPIONs by coating them with recombinant heat shock protein 70 (Hsp70) which is known to chaperone antigenic peptides. After binding, Hsp70-SPIONs deliver immunogenic peptides from tumor lysates to dendritiс cells (DCs) and thus stimulate a tumor-specific, CD8+ cytotoxic T cell response. We could show that binding activity of Hsp70-SPIONs to the substrate-binding domain (SBD) is highly dependent on the ATPase activity of its nucleotide-binding domain NBD), as shown by (31)P NMR spectroscopy. Immunization of C6 glioma-bearing rats with DCs pulsed with Hsp70-SPIONs and tumor lysates resulted in a delayed tumor progression (as measured by MRI) and an increased overall survival. In parallel an increased IFNγ secretion were detected in the serum of these animals and immunohistological analysis of subsequent cryosections of the glioma revealed an enhanced infiltration of memory CD45RO+ and cytotoxic CD8+ T cells. Taken together the study demonstrates that magnetic nanocarriers such as SPIONs coated with Hsp70 can be applied as a platform for boosting anti-cancer immune responses.

The Fate of Exogenous Human HSP70 Introduced into Animal Cells by Different Means.

Over the last decade, it has become evident that in mammals, including humans, heat shock protein 70 (HSP70), apart from its intracellular localization, is found in extracellular space, where it may execute various protective functions. Furthermore, the upregulation of HSP70 family members can be beneficial in the prevention and treatment of various human neurodegenerative diseases and cancer. Here, we demonstrate that recombinant human HSP70 after intranasal administration can penetrate various brain regions of mice in its native form and subsequently undergo rapid degradation. It was also shown that labeled HSP70 added to culture medium of different human and mouse cell lines enters the cells with strikingly different kinetics, which positively correlates with the basic levels of membrane bound Toll-like receptors (TLR) that are characteristic of these cell lines. HSP70 administration does not significantly modulate the level of TLR expression at the protein or RNA level. The degradation of the introduced recombinant HSP70 after entering the cells is likely proteasome-dependent and varies significantly depending on the cells type and origin. These results should be considered when developing HSP70-based therapies.