[The Major Human Stress Protein Hsp70 as a Factor of Protein Homeostasis and a Cytokine-Like Regulator].

Heat shock proteins (HSPs) are important factors of protein homeostasis and possess chaperone properties, providing for a folding and intracellular transport of proteins and facilitating the recovery or utilization of proteins partly denatured on exposure to various stress factors. Proteins of the Hsp70 family are the most universal molecular chaperones and interact with the greatest number of protein substrates. Several proteins of the Hsp70 family are released into the extracellular space, where they play an important role in intercellular communications and act as alarmins, or "danger signals," to modulate the immune response. The secreted Hsp70 can additionally act as an effective neuroprotector, increasing the survival of neurons in various proteinopathies, as has been demonstrated in Alzheimer's and Parkinson's disease models. In this regard, recombinant Hsp70 and inducers of endogenous Hsp70 synthesis may be considered as candidate therapeutics with immune-modulating and neuroprotective properties.Heat shock proteins (HSPs) are important factors of protein homeostasis and possess chaperone properties, providing for a folding and intracellular transport of proteins and facilitating the recovery or utilization of proteins partly denatured on exposure to various stress factors. Proteins of the Hsp70 family are the most universal molecular chaperones and interact with the greatest number of protein substrates. Several proteins of the Hsp70 family are released into the extracellular space, where they play an important role in intercellular communications and act as alarmins, or "danger signals," to modulate the immune response. The secreted Hsp70 can additionally act as an effective neuroprotector, increasing the survival of neurons in various proteinopathies, as has been demonstrated in Alzheimer's and Parkinson's disease models. In this regard, recombinant Hsp70 and inducers of endogenous Hsp70 synthesis may be considered as candidate therapeutics with immune-modulating and neuroprotective properties.

A novel nanoparticle containing neuritin peptide with grp170 induces a CTL response to inhibit tumor growth.

Malignant glioma is among the most challenging of all cancers to treat successfully. Despite recent advances in surgery, radiotherapy and chemotherapy, current treatment regimens have only a marginal impact on patient survival. In this study, we constructed a novel nanoparticle containing neuritin peptide with grp170. The nanoparticle could elicit a neuritin-specific cytotoxic T lymphocyte response to lyse glioma cells in vitro. In addition, the nanoparticle could inhibit tumor growth and improve the lifespan of tumor-bearing mice in vivo. Taken together, the results demonstrated that the nanoparticle can inhibit tumor growth and represents a promising therapy for glioma.

Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration.

PURPOSE: To measure the cytoprotective effects of rhHsp70 against oxidative stress and study its cellular uptake, intracellular and intraocular distribution in the retinal pigment epithelium. METHODS: Human retinal pigment epithelial cells (ARPE-19) were pre-treated with rhHsp70 for 24 h, 48 h, and 72 h before being exposed to 1.25 mM hydrogen peroxide. Non-treated cells served as control. We analysed interleukin 6 secretion, cell viability, and cytolysis. Uptake and intracellular distribution of fluorescently labelled rhHsp70 were investigated with flow cytometry and confocal microscopy, respectively. Ocular distribution of radioactively labelled rhHsp70 was followed ex vivo in porcine eyes by micro SPECT/CT. RESULTS: After exposure to hydrogen peroxide, IL-6 secretion decreased by 35-39% when ARPE-19 cells were pre-treated with rhHsp70. Cell viability increased by 17-32%, and cell lysis, measured by the release of lactate dehydrogenase, decreased by 6-43%. ARPE-19 cells endocytosed rhHsp70 added to the culture medium and the protein was localized in late endosomes and lysosomes. Following intravitreal injection into isolated porcine eyes, we found 20% rhHsp70 in the RPE. CONCLUSIONS: Recombinant hHsp70 protein offers protection against oxidative stress. RPE cells take up the exogenously delivered rhHsp70 and localize it in late endosomes and lysosomes. This work provides the basis for a therapeutic strategy to target aggregate-associated neurodegeneration in AMD.

Enhanced bleaching treatment: opportunities for immune-assisted melanocyte suicide in vitiligo.

Depigmentation in vitiligo occurs by progressive loss of melanocytes from the basal layer of the skin, and can be psychologically devastating to patients. T cell-mediated autoimmunity explains the progressive nature of this disease. Rather than being confronted with periods of rapid depigmentation and bouts of repigmentation, patients with long-standing, treatment-resistant vitiligo can undergo depigmentation treatment. The objective is to remove residual pigmentation to achieve a cosmetically acceptable result--that of skin with a uniform appearance. In the United States, only the use of mono-benzyl ether of hydroquinone (MBEH) is approved for this purpose. However, satisfactory results can take time to appear, and there is a risk of repigmentation. MBEH induces necrotic melanocyte death followed by a cytotoxic T-cell response to remaining, distant melanocytes. As cytotoxic T-cell responses are instrumental to depigmentation, we propose that combining MBEH with immune adjuvant therapies will accelerate immune-mediated melanocyte destruction to achieve faster, more definitive depigmentation than with MBEH alone. As Toll-like Receptor (TLR) agonists--imiquimod, CpG, and Heat Shock Protein 70 (HSP 70)--all support powerful Th1 responses, we propose that using MBEH in combination with these agents can achieve superior depigmentation results for vitiligo patients.

[Immunoregulatory properties of Hsp70]. / Immunoregulacyjne wlasciwosci bialek Hsp70.

Heat shock proteins 70 (Hsp70) play an important role in maintaining cellular homeostasis. As molecular chaperones, Hsp70 are responsible for proper folding of newly synthesized polypeptides and refolding of misfolded and aggregated proteins. Hsp70 are involved in cellular transport and participate in signal transduction processes. The presence of Hsp70 in the extracellular space is associated with development of various pathological conditions, including autoimmune and cancer diseases. Recent data suggest anti-inflammatory property of Hsp70 confirmed in both in vitro and animal models. This paper summarizes recent data regarding immunoregulatory properties of Hsp70, and presents promising results of the studies concerning the application of heat shock proteins in treatment of chronic inflammatory diseases.

The 70 kDa heat shock protein protects against experimental traumatic brain injury.

Traumatic brain injury (TBI) causes disruption of the blood brain barrier (BBB) leading to hemorrhage which can complicate an already catastrophic illness. Matrix metalloproteinases (MMPs) involved in the breakdown of the extracellular matrix may lead to brain hemorrhage. We explore the contribution of the 70 kDa heat shock protein (Hsp70) to outcome and brain hemorrhage in a model of TBI. Male, wildtype (Wt), Hsp70 knockout (Ko) and transgenic (Tg) mice were subjected to TBI using controlled cortical impact (CCI). Motor function, brain hemorrhage and lesion size were assessed at 3, 7 and 14 days. Brains were evaluated for the effects of Hsp70 on MMPs. In Hsp70 Tg mice, CCI led to smaller brain lesions, decreased hemorrhage and reduced expression and activation of MMPs compared to Wt. CCI also significantly decreased right-biased swings and corner turns in the Hsp70 Tg mice. Conversely, Hsp70 Ko mice had significantly increased lesion size, worsened brain hemorrhage and increased expression and activation of MMPs with worsened behavioral outcomes compared to Wt. Hsp70 is protective in experimental TBI. To our knowledge, this is the direct demonstration of brain protection by Hsp70 in a TBI model. Our data demonstrate a new mechanism linking TBI-induced hemorrhage and neuronal injury to the suppression of MMPs by Hsp70, and support the development of Hsp70 enhancing strategies for the treatment of TBI.

HSP70 modified response against HPV based tumor.

BACKGROUND AND OBJECTIVES: DNA immunization is quite inventive vaccination strategies that engage the direct introduction of plasmid DNA encoding the desired antigen into the host. DNA vaccines expand strong protective responses against tumors. The desired target E7 oncogene products represent a target of choice for the therapeutic vaccination. The efficacy of vaccination is limited and it is often necessary to enhance the immune response by using adjuvant in order to achieve the desired responses. Numerous approaches have been applied to boost the effectiveness, such as the fusion or co-administration of cytokine and co-stimulatory molecules gene. Heat-shock protein 70 a family of chaperone proteins makes possible delivery of non-covalently bound peptide to MHC I molecules and influences peptide-specific CTL responses and cure treated individuals. HSP70 have been proposed as the affective adjuvant and expected to act as an appropriate substitute of both cytokine and co-stimulatory genes. MATERIALS AND METHODS: In the current study, the impact of HSP70 co-delivery and HPV-E7 boosting on cellular immune responses and protection has been investigated by intramuscular injection of mixed DNA constructs. RESULTS: Our results reveal that the target DNA vaccine can influence an E7-specific CTL response, which is imperative in the lysis of infected tumor cells, compared to negative control (p < 0.05). Additionally, treatment of tumor-bearing mice with pcDNA/E7 + HSP70 plasmid generates stronger immune responses and decreased significantly tumor sizes. Immunization with HSP-based vaccine with special target immunogene can induce potent and specific anti-tumor or anti-viral immune responses. CONCLUSIONS: Co-administration of pcDNA/E7 + HSP70 plasmid was immunologically more effective than pcDNA/E7 alone. It was concluded that all the characteristics observed during our investigation demonstrate the potent adjuvant activities of HSP70 and could be an efficient approach to persuade dramatically E7-specific immune responses as future cervical cancer vaccine.  

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite.

OBJECTIVES: Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. METHODS: We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. RESULTS: The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. CONCLUSION: This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Inflammation and damage-associated molecular patterns in major psychiatric disorders.

BACKGROUND: Emerging evidence indicates that inflammation plays an important role as a mechanism underlying mental disorders. However, most of the research on inflammatory mechanisms focuses on serum levels of interleukins and very few studies have investigated molecules that initiate and expand innate immune pathways such as damage-associated molecular patterns (DAMPs). OBJECTIVES: This study investigated the levels of DAMPs among patients diagnosed with major depressive disorder (MDD), bipolar disorder (BD) I and II, schizophrenia (SCZ), and generalized anxiety disorder (GAD). We quantified serum levels of heat shock proteins (HSPs) 70 and 60 and of S100 calcium-binding protein B (S100B). METHODS: Serum levels of HSP70, HSP60, and S100B were assessed in a sample of participants with psychiatric disorders (n = 191) and a control group (CT) (n = 59) using enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum HSP70 concentrations were significantly higher in the MDD group compared to the CT, SCZ, and BD groups. The GAD group had higher concentrations of HSP70 than the SCZ group. Exploring associations with medications, lithium (p = 0.003) and clozapine (p = 0.028) were associated with lower HSP70 levels. Approximately 64% of the sample had DAMPs levels below the limits of detection indicated by the respective ELISA kit. CONCLUSION: This was the first study to assess DAMPs levels in a transdiagnostic sample. Our preliminary findings suggest that HSP70 may be associated with MDD pathophysiology. Medications such as lithium and clozapine were associated with lower HSP70 levels in BD and SCZ groups, respectively. Therefore, it is worth mentioning that all participants were medicated and many psychotropic drugs exert an anti-inflammatory effect, possibly reducing the signs of inflammation.

Liposome-templated gold nanoparticles for precisely temperature-controlled photothermal therapy based on heat shock protein expression.

Mild temperature photothermal therapy is gaining more and more attention due to high safety, high specificity and moderate efficacy. However, the therapeutical outcome of mild photothermal therapy is limited due to the overexpression of heat shock proteins (HSPs). Therefore, the precise management of HSP expression is the key to improvement of mild temperature photothermal therapy. However, the correlation between HSP expression and photothermal temperature in vivo is still unclear. To precisely control the photothermal temperature by managing the HSP expression, we quantified the HSP expression at different photothermal temperatures after irradiation on liposome-templated gold nanoparticles, which have high photostability, high photothermal conversion efficiency and low temperature fluctuation (smaller than 1 â„ƒ). We found that the expression of HSP70 was least at 47 â„ƒ, which was the optimal temperature for HSP management. We chose to co-administrate HSP70 inhibitor during 47 â„ƒ photothermal therapy, leading to greatly enhanced tumor inhibition. Our precise temperature-controlled photothermal therapy based on HSP expression offers a new strategy for clinical tumor photothermal therapy.

Inducible Heat Shock Protein 70 Levels in Patients and the mdx Mouse Affirm Regulation during Skeletal Muscle Regeneration in Muscular Dystrophy.

BACKGROUND: Stress-inducible heat shock protein 70 (HSP70) is both a protective chaperone involved in protein homeostasis and an immune regulator. In both capacities, HSP70 has been implicated in muscle disorders, yet with fragmented and differing results. In this study we aimed to compare results obtained in the mouse model for the severest form of muscular dystrophy (MD) equivalent to Duchenne MD, termed the mdx mouse, with results obtained in human MD. METHODS: Skeletal muscle and serum samples were obtained from 11 healthy controls, 11 fully characterized patients diagnosed with Becker MD and limb girdle MD (LGMD), and six muscle disease controls. In addition, muscle extracts were prepared from tibialis anterior of mdx and control mice at ages 4, 8 and 12 weeks. The HSP70 levels were quantified using RT-PCR, western blotting and protein arrays, and localized in muscle tissue sections using double immunofluorescence. RESULTS: We found selective and significant 2.2-fold upregulation of HSP70 protein in mdx tibialis muscle at the earliest disease phase only. In LGMD and Becker MD patients, HSP70 protein levels were not significantly different from those of healthy muscle and serum. HSP70 was localized to regenerating muscle fibers both in mouse and human MD skeletal muscle tissues. Toll-like receptor (TLR) 2 and TLR4 expression was moderately increased on the sarcolemma in MD muscle, yet protein levels were not significantly different from normal controls. CONCLUSIONS: HSP70 upregulation in MD appears disease stage-dependent, marking the phase of most active muscle regeneration in the mdx mouse. We postulate that well-timed supportive therapeutic interventions with HSP70 agonists could potentially improve muscle tissue's regenerative capacities in MD, attenuating loss of muscle mass while we await gene therapies to become more widely available.

Mortalin inhibition in experimental Parkinson's disease.

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinson's disease. In the present work a rat model of Parkinson's disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinson's disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6-hydroxydopamine rat model of Parkinson's disease. The electrophysiological effects of MKT-077, a rhodamine-123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham-operated, and 6-hydroxydopamine-denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6-hydroxydopamine-treated rats in comparison with sham-operated animals. MKT-077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT-077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinson's disease.

[Research properties of the exogenous recombinant human heat shock protein HSP70 on test rodents].

Our research with Sprague-Dawley rats demonstrates protective properties of recombinant human heat shock protein 70 kDa (exogenous rhHSP70) as a prevent therapy agent for gram-positive sepsis. In this study we investigate acute toxicity of rhHSP70 on CD-1 mice and demonstrate very low dangerous of the substance.

[Protective and antiedema effect of heat shock HSP70 protein in hemorrhagic stroke model in vitro].

An in vitro model of hemorrhagic stroke in live olfactory cortex slices under long-term influence of autoblood has been used and the development of edema in the samples has been studied with simultaneous monitoring of bioelectric activity of the nervous cells. Protection of the nervous cells in the olfactory cortex slices of the spontaneously hypertensive (SHR) rats from consequences of the hemorrhagic stroke was achieved by incubating brain slices for 20 min in glass vials with 1 ml of incubation solution containing heat shock protein HSP70 at a concentration of 10 mg/ml. Then the incubation medium was replaced by 3 ml of autoblood, the action of which on the nervous cells modeled the hemorrhagic stroke. After 360-min incubation in autoblood, the slices were extracted, placed in a perfusion chamber, and washed from autoblood in a flow of pure incubation solution. Then the amplitudes of separate components of the focal potentials (FPs) evoked by electrostimulation of the slices were measured and their changes analyzed. A comparison of the FP amplitudes after the action of HSP70 and autoblood to those in control group of slices showed the degree of injury and the possibility of recovery. The antiedema effects of HSP70 on the hemorrhagic stroke model was evaluated by weighing brain slices with and without the preincubation with protein, and after the subsequent exposure in autoblood. The slices were weighed on a torsion balance. The difference in weights before and after the exposure in autoblood characterized the extent of swelling and edema development in brain slices. It was established that HSP70 produced a pronounced protective antiedema effect on the slices kept in autoblood.

Exogenous recombinant Hsp70 mediates neuroprotection after photothrombotic stroke.

Ischaemic stroke is an acute interruption of the blood supply to the brain, which leads to rapid irreversible damage to nerve tissue. Ischaemic stroke is accompanied by the development of neuroinflammation and neurodegeneration observed around the affected brain area. Heat shock protein 70 (Hsp70) facilitates cell survival under a variety of different stress conditions. Hsp70 may be secreted from cells and exhibits cytoprotective activity. This activity most likely occurs by decreasing the levels of several proinflammatory cytokines through interaction with a few receptors specific to the innate immune system. Herein, we demonstrated that intranasal administration of recombinant human Hsp70 shows a significant twofold decrease in the volume of local ischaemia induced by photothrombosis in the mouse prefrontal brain cortex. Our results revealed that intranasal injections of recombinant Hsp70 decreased the apoptosis level in the ischaemic penumbra, stimulated axonogenesis and increased the number of neurons producing synaptophysin. Similarly, in the isolated crayfish stretch receptor, consisting of a single sensory neuron surrounded by the glial envelope, exogenous Hsp70 significantly decreased photoinduced apoptosis and necrosis of glial cells. The obtained data enable one to consider human recombinant Hsp70 as a promising compound that could be translated from the bench into clinical therapies.

Heat shock protein 70 expression protects against sepsis-associated cardiomyopathy by inhibiting autophagy.

OBJECTIVES: Increasing evidence suggests that heat shock protein 70 (Hsp70) has a protective effect in sepsis-induced cardiomyopathy; however, the protective mechanism remains unclear. METHODS: Previous studies have also implicated autophagy in sepsis-induced cardiomyopathy. The aim of the current study was to reveal the protective mechanisms of Hsp70 in sepsis-induced cardiomyopathy using a cecal ligation and puncture (CLP) rat sepsis model. The roles of Hsp70 and autophagy in sepsis-induced cardiomyopathy were investigated by pretreating rats with the Hsp70 inhibitor quercetin or the autophagy inhibitor 3-methyladenine (3-Ma) before CLP. We also investigated the protective mechanisms of Hsp70 and the relationship between Hsp70 and autophagy in vitro by stimulating H9c2 cells with lipopolysaccharide (LPS) to simulate sepsis. RESULTS: The result show that inhibition of Hsp70 promoted sepsis-induced death in rats, while inhibition of autophagy inhibited sepsis-induced death. These results suggested that both Hsp70 and autophagy were involved in sepsis-induced cardiomyopathy. Overexpression of Hsp70 in H9c2 myocardial cells in vitro suppressed LPS-induced apoptosis, while inhibition of autophagy with 3-Ma also decreased LPS-induced H9c2 cell apoptosis, suggesting that the protective effect of Hsp70 in sepsis-induced cardiomyopathy was related to autophagy regulation. CONCLUSION: Overall, these results suggested that Hsp70 protected against sepsis-induced cardiac impairment by attenuating sepsis-induced autophagy.

Exogenous protein Hsp70/Hsc70 can penetrate into brain structures and attenuate the severity of chemically-induced seizures.

Heat shock protein 70 kDa (Hsp70) possesses a remarkable neuroprotective activity and the results of recent studies demonstrated its efficacy in the attenuation of epileptic seizures. The aim of this study was to explore the effects of a pure Hsp70/Hsc70 preparation delivered to the brain regions involved in generalized seizures induced in rats by intracerebroventricular microinjections of NMDA or systemic injections of pentylenetetrazole. Purified Hsp70/Hsc70 was administered (intracerebroventricular) 2 h before the induction of seizures. Compared to the vehicle-treated control animals, Hsp70/Hsc70-pretreated rats demonstrated reduced severity of NMDA- and pentylenetetrazole-induced seizures. To identify the brain structures potentially implicated in the Hsp70/Hsc70-mediated anticonvulsant effect, we analysed the localization of a fluorescently-labelled chaperone in the brain. Labelled Hsp70/Hsc70 was found in neurons and terminals of the limbic seizure complex of the brain and was co-localized in these regions with NMDA receptors, synaptophysin and the GABA-synthesizing enzyme, L-glutamic acid decarboxylase 67. An immunoprecipitation assay confirmed interactions between Hsp70 and both synaptophysin and L-glutamic acid decarboxylase 67 in brain tissue. We suggest that the anticonvulsant effect of exogenous Hsp70/Hsc70 is not only based on its protective capacity but is also related to its ability to modulate GABA neurotransmission, which in turn contributes to the maintenance of the excitatory-inhibitory balance of the CNS.

Tumor antigen delivered by Salmonella III secretion protein fused with heat shock protein 70 induces protection and eradication against murine melanoma.

Attenuated Salmonella typhimurium possess the ability to stimulate innate immune responses and preferentially allocate within the solid tumor. These two main characteristics make attenuated Salmonella one of the most attractive vehicles for development of vaccine and also targeted cancer therapies. However, location of Salmonella prevents the process of antigen presentation. Salmonella Type III secretion system can be utilized to circumvent this problem because this system secretes the protein it encoded outside the cells. Heat shock protein 70 (Hsp70) is referred to as an "immunochaperone" for its capacity to elicit tumor-specific adaptive immune responses in the form of Hsp70-TAA (tumor associated antigen) complex. Hsp70 facilitates the cross-presentation of exogenous antigens through its receptor on antigen-presenting cells and therefore activates an antigen-specific cytotoxic T lymphocyte (CTL) response, which can directly contribute to potent anti-tumor immunity. Here, we designed a novel therapeutic vaccine utilizing the type III secretion system and Hsp70 to deliver and present the tumor-specific antigen. This live recombinant bacteria vaccine, when administrated orally, successfully broke the immune tolerance, induced a specific CTL response against tumor cells, and therefore revealed protective and therapeutic effects against generation and growth of B16F10 melanoma in C57BL/6J mice.

[Effect of the recombinant human heat shock protein HSP70 on the biochemical properties of blood in a model of endotoxic shock in rats].

The protective effects of 70-kDa heat shock proteins (HSP70) were studied following the intravenous administration in rats with endotoxic shock. The antitoxic effects of both bovine HSP70 (bHSP70) and human recombinant HSP70 (hrHSP70) were compared. The preventive uptake of HSP70 decreased the toxic influence of the E. coll endotoxin on the rats' bodies and significantly increased the survival of the animals during the experiment.

[Therapeutic effects of B and T lymphocyte attenuator extracellular domain and heat shock protein 70 antigen peptide on cervical cancer in mouse model].

OBJECTIVE: To investigate the synergistic therapy effects of B and T lymphocyte attenuator (BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. METHODS: (1) Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA, HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3.1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 + pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL)2 and interferon-γ (IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: (1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2.83 ± 0.35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66 ± 0.25 (P < 0.05). While HVEM mRNA expression did not change significantly (P > 0.05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P < 0.05); while the difference of HVEM expression was not statistically significant (57.2 vs 49.3, P > 0.05). (2) The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P < 0.05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3.12 ± 0.71, 3.20 ± 0.62) expression but also reduced transforming growth factor-ß (TGF-ß), Foxp3 and IL-10 expression (0.25 ± 0.03, 0.19 ± 0.03, 0.31 ± 0.04; P < 0.05). It also promoted CD8(+) T lymphocyte infiltration (52 ± 6)/high power field, cytotoxicity (65.5 ± 2.4)%, proliferation (15.0 × 10³ cpm) and cytokine IL-2, IFN-γ secretion (824 ± 51), (1096 ± 112) pg/ml, which were all significantly higher than other groups (P < 0.05). CONCLUSION: The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex, which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.