Recombinant Heat Shock Protein Research Articles

The Evolution of Vaccines Development across Salmonella Serovars among Animal Hosts: A Systematic Review.

Salmonella is a significant zoonotic foodborne pathogen, and the global spread of multidrug-resistant (MDR) strains poses substantial challenges, necessitating alternatives to antibiotics. Among these alternatives, vaccines protect the community against infectious diseases effectively. This review aims to summarize the efficacy of developed Salmonella vaccines evaluated in various animal hosts and highlight key transitions for future vaccine studies. A total of 3221 studies retrieved from Web of Science, Google Scholar, and PubMed/Medline databases between 1970 and 2023 were evaluated. One hundred twenty-seven qualified studies discussed the vaccine efficacy against typhoidal and nontyphoidal serovars, including live-attenuated vaccines, killed inactivated vaccines, outer membrane vesicles, outer membrane complexes, conjugate vaccines, subunit vaccines, and the reverse vaccinology approach in different animal hosts. The most efficacious vaccine antigen candidate found was recombinant heat shock protein (rHsp60) with an incomplete Freund's adjuvant evaluated in a murine model. Overall, bacterial ghost vaccine candidates demonstrated the highest efficacy at 91.25% (95% CI = 83.69-96.67), followed by the reverse vaccinology approach at 83.46% (95% CI = 68.21-94.1) across animal hosts. More than 70% of vaccine studies showed significant production of immune responses, including humoral and cellular, against Salmonella infection. Collectively, the use of innovative methods rather than traditional approaches for the development of new effective vaccines is crucial and warrants in-depth studies.

PSLBI-15 Effects of heat shock protein 70 on cell viability and protective capacity of serum against histone cytotoxicity

Abstract Bovine respiratory disease (BRD) is the most significant health problem associated with newly received cattle in the beef industry. Serum concentrations of extracellular histones increase in a variety of diseases and histones are known to be cytotoxic to mammalian cells. Work in our lab indicated that failure to protect against cytotoxic effects of extracellular histones may be a component of severe cases of respiratory disease in cattle. The mechanisms or proteins that neutralize cytotoxic effects of extracellular histones are not fully elucidated, but C-reactive protein and complement activity have been associated with a reduced cytotoxic effect of extracellular histones. Work in the literature suggests that heat shock protein 70 (HSP70) concentrations in the serum is increased in disease and may directly activate complement. Unpublished observations in our lab indicated that HSP70 potentially interacts with histones to exacerbate cytotoxicity leading to our hypothesis that increased concentrations of HSP70 will decrease the protective capacity against histone cytotoxicity. Therefore, the objectives of the current study were to evaluate serum concentrations of HSP70 in weaned bulls entering the feedlot and the effects of increasing HSP70 concentrations on histone cytotoxicity in vitro. Weaned seedstock bulls (n = 130) composed of various breeds were fed using the GrowSafe System at the Tucumcari Bull Test for 122 d. At receiving, blood samples were obtained via coccygeal venipuncture, centrifuged, and serum immediately frozen on dry ice. Serum was analyzed for complement activity (CH50 assay) and protective capacity against extracellular histones using our novel histone toxicity assay. The ratio value of histone toxicity to CH50 was used to separate a subset of bulls as protective (n = 10) and non-protective (n = 10). Concentrations of HSP70 were quantified utilizing a commercially available ELISA kit. Data were analyzed using the GLM procedure of SAS. Serum concentrations of HSP70 were similar (P > 0.10) for protective and non-protective bulls. To test the responses to increased concentrations of HSP70 to mimic disease, protective and non-protective serum was tested against histones alone and in combination with 0.5 or 40 ng/mL recombinant bovine HSP70. Complement activity from protective and non-protective bulls was not influenced (P > 0.10) in the presence and absence of recombinant bovine HSP70. Exogenous HSP70 at 0.5 or 40 ng/mL did not influence protective capacity in non-protective animals (P = 0.95). However, HSP70 at 0.5 ng/mL did not (P = 0.18) reduce protective capacity and reduced protective capacity (P < 0.01) at 40 ng/mL in serum from bulls that were assigned to the protective group. Results indicate that weaned bulls entering the feedlot display varied capacity to protect against histone toxicity and increased concentrations of HSP70 can reduce the protective capacity indicating that circulating HSP70 may play a role in bovine respiratory disease pathogenesis.

DIPG-92. IMMUNE ACTIVATION STUDIES WITH THE NEOANTIGEN-BASED PEPTIDE VACCINE RHSC-DIPGVAX DEMONSTRATE ANTIBODY-MEDIATED RESPONSES AND EPITOPE SPREADING IN PEDIATRIC PATIENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG)

Abstract BACKGROUND Approximately 80% of DIPGs carry gain-of-function lysine-to-methionine (K27M) mutations in histone H3 proteins that frequently co-occur with other protein aberrations, including p53, ACVR1, and PDGFRA. Because the DIPG microenvironment has few infiltrating immune cells, resolution of negative regulatory mechanisms through checkpoint blockade may be necessary to promote vaccine-induced anti-tumor responses. Accordingly, rHSC-DIPGVax is a neoantigen-based peptide vaccine combined with checkpoint inhibitors currently under investigation in clinical trial, NCT04943848. The vaccine contains 16 neoantigen peptides, QS-21 adjuvant, and recombinant heat shock protein, Hsc70. In this study, we investigate vaccine-induced antibody-mediated immunity and the phenomenon of epitope spreading, an extension of the immune response to secondary epitopes not directly targeted by the therapy, thus promoting distinct, individualized responses. METHODS rHSC-DIPGVax is administered every two weeks to patients with DIPG and blood is collected pre-treatment and prior to each dose. To investigate vaccine-induced immunoreactivity to neoantigens targeted by rHSC-DIPGVax, DIPG lysates resolved by two-dimensional gel electrophoresis are immunoblotted with pre- and post-vaccine patient plasma containing antibodies against vaccine antigens. Proteins are immunoprecipitated (IP) from DIPG lysates, followed by mass spectrometry (MS) identification and validation. RESULTS In eight subjects to date, vaccination with rHSC-DIPGVax consistently induces IgM and IgG antibodies against proteins directly targeted by the vaccine, including strong immunoreactivity to ACVR1. Furthermore, antibodies against several secondary proteins are also detected in post-vaccination plasma. Immunoblotting and IP-MS analysis identified several distinct candidates of epitope spreading, including the tumor antigens CEP290, DNAH3, HRNR, KDM6B, LAMA5, and TRPA1. CONCLUSIONS rHSC-DIPGVax induces antibody-mediated tumor-specific responses to DIPG neoantigens, including direct vaccine targets and secondary targets via intra- and inter-molecular epitope spreading. We also present an experimental protocol to investigate vaccine-induced immune activation. Our ongoing studies aim to validate epitope spreading candidates and determine the important biological correlates between antibody responses and clinical outcomes.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis induces the death of mouse lymphocytes in a mechanism dependent on Toll-like receptor 4 and tumor necrosis factor.

Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1.

Cell-Permeable HSP70 Protects Neurons and Astrocytes Against Cell Death in the Rotenone-Induced and Familial Models of Parkinson’s Disease

Heat shock protein 70 (HSP70) is activated under stress response. Its involvement in cell protection, including energy metabolism and quality control makes it a promising pharmacological target. A strategy to increase HSP70 levels inside the cells is the application of recombinant HSP70. However, cell permeability and functionality of these exogenously applied proteins inside the cells is still disputable. Here, using fluorescence- labeled HSP70, we have studied permeability and distribution of HSP70 inside primary neurons and astrocytes, and how exogenous HSP70 changes mitochondrial metabolism and mitophagy. We have found that exogenous recombinant HSP70 can penetrate the neurons and astrocytes and distributes in mitochondria, lysosomes and in lesser degree in the endoplasmic reticulum. HSP70 increases mitochondrial membrane potential in control neurons and astrocytes, and in fibroblasts of patients with familial Parkinson´s disease (PD) with PINK1 and LRRK2 mutations. Increased mitochondrial membrane potential was associated with higher mitochondrial ROS production and activation of mitophagy. Importantly, preincubation of the cells with HSP70 protected neurons and astrocytes against cell death in a toxic model of PD induced by rotenone, and in the PINK1 and LRRK2 PD human fibroblasts. Thus, exogenous recombinant HSP70 is cell permeable, and acts as endogenous HSP70 protecting cells in the case of toxic model and familial forms of Parkinson’s Disease.

Heat Shock Protein 22 Attenuates Nerve Injury-induced Neuropathic Pain Via Improving Mitochondrial Biogenesis and Reducing Oxidative Stress Mediated By Spinal AMPK/PGC-1α Pathway in Male Rats.

Heat shock protein 22 (hsp22) plays a significant role in mitochondrial biogenesis and redox balance. Moreover, it's well accepted that the impairment of mitochondrial biogenesis and redox imbalance contributes to the progress of neuropathic pain. However, there is no available evidence indicating that hsp22 can ameliorate mechanical allodynia and thermal hyperalgesia, sustain mitochondrial biogenesis and redox balance in rats with neuropathic pain. In this study, pain behavioral test, western blotting, immunofluorescence staining, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Dihydroethidium staining are applied to confirm the role of hsp22 in a male rat model of spared nerve injury (SNI). Our results indicate that hsp22 was significantly decreased in spinal neurons post SNI. Moreover, it was found that intrathecal injection (i.t.) with recombinant heat shock protein 22 protein (rhsp22) ameliorated mechanical allodynia and thermal hyperalgesia, facilitated nuclear respiratory factor 1 (NRF1)/ mitochondrial transcription factor A (TFAM)-dependent mitochondrial biogenesis, decreased the level of reactive oxygen species (ROS), and suppressed oxidative stress via activation of spinal adenosine 5'monophosphate-activated protein kinase (AMPK)/ peroxisome proliferative activated receptor γ coactivator 1α (PGC-1α) pathway in male rats with SNI. Furthermore, it was also demonstrated that AMPK antagonist (compound C, CC) or PGC-1α siRNA reversed the improved mechanical allodynia and thermal hyperalgesia, mitochondrial biogenesis, oxidative stress, and the decreased ROS induced by rhsp22 in male rats with SNI. These results revealed that hsp22 alleviated mechanical allodynia and thermal hyperalgesia, improved the impairment of NRF1/TFAM-dependent mitochondrial biogenesis, down-regulated the level of ROS, and mitigated oxidative stress through stimulating the spinal AMPK/PGC-1α pathway in male rats with SNI.

Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus

Human noroviruses (HuNoVs) are the major foodborne pathogens that cause non-bacterial gastroenteritis globally. Conventional RT-qPCR is prone to counting free RNA in the sample, resulting in an inflated infectious virus titer. Porcine gastric mucin (PGM), broadly used as the capture unit of in situ capture RT-qPCR (ISC-RT-qPCR), can precisely capture intact norovirus without adsorbing free RNA. However, PGM is a complex mixture that is difficult to obtain, and the concentration of histo-blood group antigens in it is unclear, hampering its widespread use. This study introduced a newly discovered capture unit, recombinant oyster heat shock protein 70 (roHSP70), to replace PGM. The roHSP70-mediated ISC-RT-qPCR (roHSP-ISC-RT-qPCR) were developed and compared with the PGM-mediated ISC-RT-qPCR (PGM-ISC-RT-qPCR) and conventional column-based RNA extraction RT-qPCR (CBE-RT-qPCR). The results showed that roHSP70 can capture various genotypes of HuNoVs. The smaller concentration of roHSP70 (20.0 μg/mL) captured HuNoVs to a similar extent compared to PGM (1.0 mg/mL). In addition, roHSP-ISC-RT-qPCR can accurately detect the amount of authentic intact virus particles in heat-treated Tulane virus (TV) samples with an accuracy comparable to that of TCID50. Interestingly, roHSP-ISC-RT-qPCR gave lower titers than CBE-RT-qPCR when measuring cell-cultured TV with high titers. However, when measuring lower-titer TV in artificially contaminated oysters, roHSP-ISC-RT-qPCR instead gave significantly higher titers than CBE-RT-qPCR. Overall, roHSP-ISC-RT-qPCR had a similar excellent performance as the PGM-ISC-RT-qPCR in detecting intact norovirus in samples. Moreover, roHSP70 has broader application scenarios than PGM due to its single-identified component and easier accessibility.

Gene cloning and characterization of a novel recombinant 40-kDa heat shock protein from Mesobacillus persicus B48.

The present study reports the recognition and characterization of the gene encoding the co-chaperone DnaJ in the halophilic strain Mesobacillus persicus B48. The new extracted gene was sequenced and cloned in E. coli, followed by protein purification using a C-terminal His-tag. The stability and function of the recombinant DnaJ protein under salt and pH stress conditions were evaluated. SDS-PAGE revealed a band on nearly 40-kDa region. The homology model structure of new DnaJ demonstrated 56% similarity to the same protein from Streptococcus pneumonia. Fluorescence spectra indicated several hydrophobic residues located on the protein surface, which is consistent with the misfolded polypeptide recognition function of DnaJ. Spectroscopic results showed 56% higher carbonic anhydrase activity in the presence of the recombinant DnaJ homolog compared to its absence. In addition, salt resistance experiments showed that the survival of recombinant E. coli+DnaJ was 2.1 times more than control cells in 0.5M NaCl. Furthermore, the number of recombinant E. coli BL21+DnaJ colonies was 7.7 times that of the control colonies in pH 8.5. Based on the results, DnaJ from the M. persicus can potentially be employed for improving the functional features of enzymes and other proteins in various applications.

Exogenous recombinant Hsp70 attenuates sevoflurane anesthesia-induced cognitive dysfunction in aged mice.

Postoperative cognitive dysfunction (POCD) is a severe postoperative neurological sequela in elderly patients, and there is currently no standard treatment for POCD. In this study, whether recombinant human heat shock protein 70 (rHsp70) could alleviate sevoflurane-induced cognitive impairment in aged mice is investigated. To determine the prophylactic effect of rHsp70 in sevoflurane-induced cognitive dysfunction, aged mice were pretreated with different concentrations of rHsp70 (29.4, 58.8, and 117.6μg/kg; intranasal injected; N=12) every day for 1 week; then, 3% sevoflurane was utilized to anesthetize the aged mice. Cognitive function, neurotoxicity, and serum and hippocampal Hsp70 levels in aged mice undergoing sevoflurane anesthesia were assessed by the Morris water maze test and enzyme-linked immunosorbent assay. The effects of rHsp70 on inflammatory response were assessed by proinflammatory cytokine production and nuclear factor-κB (NF-κB) activation assays. We found that aged mice exposed to sevoflurane showed reduced learning and memory ability and reduced Hsp70 expression, which were both restored by rHsp70 pretreatment. RHsp70 also reversed sevoflurane-induced up-regulated Bax and Bcl-2 expression and interleukin-1, IL-6, and monocyte chemoattractant protein-1 overproduction. Finally, rHsp70 pretreatment suppressed sevoflurane-induced NF-κB activation. Our study indicated that rHsp70 was sufficient to suppress sevoflurane-induced cognitive decline and neurotoxicity. Our important finding warrants further study on the clinical application of rHsp70 in elderly patients undergoing anesthesia.

Crocin inhibits KBTBD7 to prevent excessive inflammation and cardiac dysfunction following myocardial infarction.

Myocardial infarction (MI) refers to myocardial ischemic necrosis that is caused by coronary artery disease. Notably, crocin has protective effects on the heart. The present study aimed to i)investigate the protective effect of crocin, an active ingredient in Gardeniajasminoides Ellis and Crocussativus L., on myocardial ischemia and ii)to verify the interaction between crocin and kelch repeat and BTB domain‑containing protein 7 (KBTBD7), which is a novel member of the BTB‑kelch protein family. In the present study, the left anterior descending coronary artery was ligated to establish a myocardial ischemia‑reperfusion injury (MIRI) model in rats and the protective effect of crocin on rat myocardial tissue was observed. The levels of the inflammatory cytokines, interleukin (IL)‑1β, IL‑6 and tumor necrosis factor α (TNFα), in the sham, MI model, MI + crocin (100mg/kg) and MI + crocin (200mg/kg) groups were compared in the rat myocardial tissue. The TUNEL assay was used to detect apoptosis of myocardial cells. In addition, RAW264.7 cells were stimulated with the inflammatory factors recombinant mouse high mobility group box 1 (rmHMGB1) and recombinant mouse heat shock protein 60 (rmHSP60). The inhibitory effect of crocin on inflammatory cytokine levels was observed using ELISA. Western blotting was used to detect the inhibitory effect of crocin on KBTBD7. The inhibitory effect of KBTBD7 knockdown on MAPK and nuclear factor (NF)‑κB signaling pathways was also analyzed. The expression levels of IL‑1β, IL‑6 and TNFα were significantly decreased in the crocin‑treated groups compared with in the model group. Crocin significantly reduced the apoptosis of myocardial cells and significantly inhibited the release of inflammatory cytokines induced by rmHMGB1 and rmHSP60. KBTBD7 was determined to be a target of crocin. Knockdown of KBTBD7 significantly inhibited p38 and NF‑κB signaling pathways. Furthermore, the results demonstrated that KBTBD7 knockdown significantly reduced the production of inflammatory cytokines induced by rmHMGB1 and rmHSP60. KBTBD7 knockdown also significantly reduced p38 and NF‑κB signaling in the rmHMGB1‑ and rmHSP60‑treated groups. The present study demonstrated the potential protective effect of crocin on MIRI in rats. The underlying mechanism may be through direct inhibition of KBTBD7, thereby inhibiting excessive inflammatory responses and myocardial cell apoptosis following myocardial infarction.

Heat shock protein amplification improves cerebellar myelination in the Npc1nih mouse model.

Niemann-Pick disease type C (NPC) is a rare prematurely fatal lysosomal lipid storage disease with limited therapeutic options. The prominent neuropathological hallmarks include hypomyelination and cerebellar atrophy. We previously demonstrated the efficacy of recombinant human heat shock protein 70 (rhHSP70) in preclinical models of the disease. It reduced glycosphingolipid levels in the central nervous system (CNS), improving cerebellar myelination and improved behavioural phenotypes in Npc1nih (Npc1-/-) mice. Furthermore, treatment with arimoclomol, a well-characterised HSP amplifier, attenuated lysosomal storage in NPC patient fibroblasts and improved neurological symptoms in Npc1-/- mice. Taken together, these findings prompted the investigation of the effects of HSP amplification on CNS myelination. We administered bimoclomol daily or rhHSP70 6 times per week to Npc1-/- (BALB/cNctr-Npc1m1N/J, also named Npc1nih) mice by intraperitoneal injection from P7 through P34 to investigate the impact on CNS myelination. The Src-kinase inhibitor saracatinib was administered with/without bimoclomol twice daily to explore the contribution of Fyn kinase to bimoclomol's effects. Treatment with either bimoclomol or rhHSP70 improved myelination and increased the numbers of mature oligodendrocytes (OLs) as well as the ratio of active-to-inactive forms of phosphorylated Fyn kinase in the cerebellum of Npc1-/- mice. Additionally, treatment with bimoclomol preserved cerebellar weight, an effect that was abrogated when co-administered with saracatinib, an inhibitor of Fyn kinase. Bimoclomol-treated mice also exhibited increased numbers of immature OLs within the cortex. These data increase our understanding of the mechanisms by which HSP70 regulates myelination and provide further support for the clinical development of HSP-amplifying therapies in the treatment of NPC. Funding for this study was provided by Orphazyme A/S (Copenhagen, Denmark) and a Pathfinder Award from The Wellcome Trust.

Excessive HSP70/TLR2 activation leads to remodeling of the tumor immune microenvironment to resist chemotherapy sensitivity of mFOLFOX in colorectal cancer

For locally advanced colorectal cancer (CRC), neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision or complete mesocolic excision is the standard therapeutic strategy, which is key to patient survival. Involvement of the tumor immune microenvironment is a factor that regulates tumor progression and sensitivity to nCRT in CRC. In this study, we aimed to identify the effect of heat-shock protein 70 (HSP70)/toll-like receptor-2 (TLR-2) on mFOLFOX sensitization for CRC. A total of 22 patients with advanced CRC who had received neoadjuvant mFOLFOX were enrolled and classified into the mFOLFOX-insensitive or -sensitive group, according to the tumor regression grade. The abundance of immune infiltrates was significantly higher in the post-operative pathological specimens of the mFOLFOX-insensitive group, as compared to those of the mFOLFOX-sensitive group. After transcriptome sequencing, differentially expressed genes between the two groups were annotated to inflammatory and immune responses using Gene Ontology (GO) analysis, and the TLR signaling pathway was analyzed using Kyoto Encyclopedia of Genes and Genomes pathway analysis. Significantly higher expression levels of HSP60, HSP70, HSP90, and TLR-2 in the mFOLFOX-insensitive group were detected using immunofluorescence assays. TIMER2.0 platform was introduced to further narrow the scope of HSP70 (HSPA6 or HSPA7) and TLR-2, which exhibited positive correlations with dendritic cells, Tregs, or CD4+ T cells and negative correlations with CD3+ or CD8+ T cells, implying that HSP70/TLR-2 activation mediates immunosuppressive cells to counteract CD8+ T cells, which may be a novel target of CRC treatment. A promising synergistic effect of mFOLFOX combined with a TLR-2 inhibitor was observed in vivo in mouse allograft models, which could be partly rescued by recombinant HSP70 protein. Immunohistochemical staining of allografts and immunofluorescence assays of clinical specimens corroborated the regulatory effects of the immune microenvironment. In summary, HSP70/TLR-2 activation can regulate the tumor immune microenvironment of CRC and further remodel its sensitivity to mFOLFOX. However, the specific mechanisms remain unclear and require further investigation. This study is expected to provide a new direction for the clinical treatment of CRC.

Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes

AimsVascular dysfunction is a clinical hallmark of diabetes. While various pathways drive vascular alterations in diabetes, many gaps persist in understanding this process. Heat-shock protein 70 (HSP70) has a long-recognized role in diabetes, but the contributions of HSP70 to the diabetic vasculature remain largely unknown. Main methodsWe determined the systemic and local (aorta) levels of HSP70 in control (CTL) and streptozotocin (STZ)-induced diabetic rats. Functional studies were conducted in a wire myograph in the presence or absence of a pharmacological inhibitor for HSP70 (VER155008). Calcium (Ca2+) dynamics was indirectly evaluated as a function of change in force development in vehicle and VER-treated vessels, as well as in the presence of inhibitors for voltage-dependent and -independent plasmalemmal Ca2+ channels. Furthermore, mimicking the extracellular diabetic environment, we exposed aortic rings to serum from CTL and STZ-induced animals, which contains higher levels of HSP70, as well as to purified recombinant HSP70. Then, we performed functional studies following the modulation of Toll-like receptor 4 (TLR4) and its co-adaptor MD2, which interact with HSP70. Key findingsHSP70 plays a dual role in diabetes-induced vascular dysfunction: intracellular (i)HSP70 affects Ca2+ handling mechanisms, and extracellular (e)HSP70 modulates the TLR4-MD2 complex. SignificanceThese newly discovered roles of HSP70 push forward the field of vascular biology and open research avenues for other diseased states associated with altered vascular responses.

The Effects of H2S and Recombinant Human Hsp70 on Inflammation Induced by SARS and Other Agents In Vitro and In Vivo.

The ongoing epidemic caused by SARS-CoV-2 infection led to the search for fundamentally new ways and means to combat inflammation and other pathologies caused by this virus. Using a cellular model of lipopolysaccharide (LPS)-induced sepsis (human promonocytes), we showed that both a hydrogen sulfide donor (sodium thiosulfate, STS) and a recombinant Heat shock protein 70 (rHsp70) effectively block all major inflammatory mediators when administrated before and after LPS challenge. The protective anti-inflammatory effect of rHsp70 and H2S was also confirmed in vivo using various animal models of pneumonia. Specifically, it was found that rHsp70 injections prevented the development of the acute respiratory distress syndrome in highly pathogenic pneumonia in mice, increased animal survival, and reduced the number of Programmed death-1 (PD-1)-positive T-lymphocytes in peripheral blood. Based on our model experiments we developed a combined two-phase therapeutic approach for the treatment of COVID-19 patients. This procedure includes the inhalation of hot helium–oxygen mixtures for induction of endogenous Hsp70 in the first phase and STS inhalation in the second phase. The use of this approach has yielded positive results in COVID-19 patients, reducing the area of lung lesions, restoring parameters of innate immunity and T-cell immune response against coronavirus infection, and preventing the development of pulmonary fibrosis and immune exhaustion syndrome.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp).

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

Influence of the Recombinant Heat Shock Protein 90β (HSP90β) on the Wound Healing Rate in Mice

Influence of the Recombinant Heat Shock Protein 90β (HSP90β) on the Wound Healing Rate in Mice

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.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of ~65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN–γ, TNF–α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70.

Heat shock proteins (HSPs) play an essential role in protecting proteins from denaturation and are implicated in diverse pathophysiological conditions like cardiovascular diseases, cancer, infections, and neurodegenerative diseases. Scientific evidence indicates that if HSP expression falls below a certain level, cells become sensitive to oxidative damage that accelerates protein aggregation diseases. On the other hand, persistently enhanced levels of HSP can lead to inflammatory and oncogenic changes. To date, although techniques for measuring HSPs exist, these assays are limited for use in specific sample types or are time consuming. Therefore, in the present study, we developed a single-molecule assay digital ELISA technology (Single Molecule Array—SIMOA) for the measurement of HSPs, which is time effective and can be adapted to measure multiple analytes simultaneously from a single sample. This technique combines two distinct HSP-specific antibodies that recognize different epitopes on the HSP molecule. A recombinant human HSP protein was used as the standard material. The assay performance characteristics were evaluated by repeated testing of samples spiked with HSP peptide at different levels. The limit of detection was 0.16 and 2 ng/mL for HSP27 and HSP70, respectively. The inter- and intra-assay coefficients of variation were less than 20% in all tested conditions for both HSPs. The HSP levels assayed after serial dilution of samples portrayed dilutional linearity (on average 109%, R2 = 0.998, p < 0.001, for HSP27 and 93%, R2 = 0.994, p < 0.001, for HSP70). A high linear response was also demonstrated with admixtures of plasma exhibiting relatively very low and high levels of HSP70 (R2 = 0.982, p < 0.001). Analyte spike recovery varied between 57% and 95%. Moreover, the relative HSP values obtained using Western blotting correlated significantly with HSP values obtained with the newly developed SIMOA assay (r = 0.815, p < 0.001 and r = 0.895, p < 0.001 for HSP70 and HSP27, respectively), indicating that our method is reliable. In conclusion, the assay demonstrates analytical performance for the accurate assessment of HSPs in various sample types and offers the advantage of a huge range of dilution linearity, indicating that samples with HSP concentration highly above the calibration range can be diluted into range without affecting the precision of the assay.