Levels of Small Extracellular Vesicles Containing hERG-1 and Hsp47 as Potential Biomarkers for Cardiovascular Diseases.

The diagnosis of cardiovascular disease (CVD) is still limited. Therefore, this study demonstrates the presence of human ether-a-go-go-related gene 1 (hERG1) and heat shock protein 47 (Hsp47) on the surface of small extracellular vesicles (sEVs) in human peripheral blood and their association with CVD. In this research, 20 individuals with heart failure and 26 participants subjected to cardiac stress tests were enrolled. The associations between hERG1 and/or Hsp47 in sEVs and CVD were established using Western blot, flow cytometry, electron microscopy, ELISA, and nanoparticle tracking analysis. The results show that hERG1 and Hsp47 were present in sEV membranes, extravesicularly exposing the sequences 430AFLLKETEEGPPATE445 for hERG1 and 169ALQSINEWAAQTT- DGKLPEVTKDVERTD196 for Hsp47. In addition, upon exposure to hypoxia, rat primary cardiomyocytes released sEVs into the media, and human cardiomyocytes in culture also released sEVs containing hERG1 (EV-hERG1) and/or Hsp47 (EV-Hsp47). Moreover, the levels of sEVs increased in the blood when cardiac ischemia was induced during the stress test, as well as the concentrations of EV-hERG1 and EV-Hsp47. Additionally, the plasma levels of EV-hERG1 and EV-Hsp47 decreased in patients with decompensated heart failure (DHF). Our data provide the first evidence that hERG1 and Hsp47 are present in the membranes of sEVs derived from the human cardiomyocyte cell line, and also in those isolated from human peripheral blood. Total sEVs, EV-hERG1, and EV-Hsp47 may be explored as biomarkers for heart diseases such as heart failure and cardiac ischemia.

Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats

Abstract Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male SpragueDawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

Resumo O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats / Alterações dos parâmetros bioquímicos séricos e fosforilação da tirosina em rim e fígado de ratos induzidos por estresse crônico

Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male Sprague-Dawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.

Sorghum bicolor SbHSP110 has an elongated shape and is able of protecting against aggregation and replacing human HSPH1/HSP110 in refolding and disaggregation assays.

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.

The small heat shock protein Hsp12.1 has a major role in the stress response and virulence of Cryptococcus gattii.

Cryptococcus gattii is one of the etiological agents of cryptococcosis. To achieve a successful infection, C. gattii cells must overcome the inhospitable host environment and deal with the highly specialized immune system and poor nutrients availability. Inside the host, C. gattii uses a diversified set of tools to maintain homeostasis and establish infection, such as the expression of remarkable and diverse heat shock proteins (Hsps). Grouped by molecular weight, little is known about the Hsp12 subset in pathogenic fungi. In this study, the function of the C. gattii HSP12.1 and HSP12.2 genes was characterized. Both genes were upregulated during murine infection and heat shock. The hsp12.1 Δ null mutant cells were sensitive to plasma membrane and oxidative stressors. Moreover, HSP12 deletion induced C. gattii reactive oxygen species (ROS) accumulation associated with a differential expression pattern of oxidative stress-responsive genes compared to the wild type strain. Apart from these findings, the deletion of the paralog gene HSP12.2 did not lead to any detectable phenotype. Additionally, the double-deletion mutant strain hsp12.1 Δ /hsp12.2 Δ presented a similar phenotype to the single-deletion mutant hsp12.1 Δ, suggesting a minor participation of Hsp12.2 in these processes. Furthermore, HSP12.1 disruption remarkably affected C. gattii virulence and phagocytosis by macrophages in an invertebrate model of infection, demonstrating its importance for C. gattii pathogenicity.

IL-6 secretion of CD4+ T cells stimulated by LC3-positive extracellular vesicles in human epithelial ovarian cancer.

BACKGROUND: Ovarian cancer (OC) as the most fatal gynecological malignancy worldwide, with epithelial ovarian cancer (EOC) being the predominant and most lethal form, poses a serious threat to human health. LC3-positive extracellular vesicles (LC3+ EVs) promote tumorigenesis by educating CD4+ T cells in a murine melanoma model. However, regulation of LC3+ EVs in human EOC remains largely unknown.  METHODS: Differential analysis of Rab8a, Hsp90α and Il6 expression was performed using GEPIA2. The number of LC3+ EVs and the frequency of Heat shock protein 90α+ LC3+ EVs (HSP90α+ LC3+ EVs) in the ascites of EOC patients were tested by flow cytometry. IL-6, IL-10, IFN-γ, IL-4 and TGF-ß were measured by ELISA. CD4+ T cells were isolated from peripheral blood of healthy human donors using MACS magnetic bead technology.  RESULTS: Higher Rab8a, Hsp90a and Il6 expression of cancer tissues compared with normal adjacent tissues in OC were found. The level of IL-6 was positively correlated with LC3+ EVs number, HSP90α+ LC3+ EVs percentage in the ascites, and ROMA index of the patient. In addition, elevated IL-6 production by CD4+ T cells induced by LC3+ EVs was observed, which was suppressed by anti-HSP90α or anti-TLR2.  CONCLUSIONS: LC3+ EVs level and HSP90α+ LC3+ EVs percentage were associated with elevated IL-6 in the ascites of EOC patients. HSP90α on LC3+ EVs from human EOC could stimulate CD4+ T cell production of IL-6 via TLR2.

HSP60-Derived Peptide as an LPS/TLR4 Modulator: An in silico Approach.

As a part of innate immunity mechanisms, the Toll-like receptor (TLR) signaling pathway serves as one of the mainstay lines of defense against pathogenic microorganisms and cell dysfunction. Nevertheless, TLR overactivation induces a systemic proinflammatory environment compromising organ function or causing the patient's death. TLRs modulators, specially those focused for TLR4, remain a promising approach for inflammatory diseases treatment, being peptide-based therapy a trendy approach. Heat shock protein 60 (HSP60) not only plays a pivotal role in the development of several maladies with strong inflammatory components but also HSP60 peptides possess anti-inflammatory properties in TLR4-mediated diseases, such as diabetes, arthritis, and atherosclerosis. The experimental treatment using HSP60 peptides has proven to be protective in preclinical models of the heart by hampering inflammation and modulating the activity of immune cells. Nonetheless, the effect that these peptides may exert directly on cells that express TLR and its role to inhibit overactivation remain elusive. The aim of this study is to evaluate by molecular docking, a 15 amino acid long-HSP60 peptide (Peptide-2) in the lipopolysaccharide (LPS) binding site of TLR4/MD2, finding most Peptide-2 resulting conformations posed into the hydrophobic pocket of MD2. This observation is supported by binding energy obtained for the control antagonist Eritoran, close to those of Peptide-2. This last does not undergo drastic structural changes, moving into a delimited space, and maintaining the same orientation during molecular dynamics simulation. Based on the two computational techniques applied, interaction patterns were defined for Peptide-2. With these results, it is plausible to propose a peptidic approach for TLR4 modulation as a new innovative therapy to the treatment of TLR4-related cardiovascular diseases.

Identification of Leishmania species by next generation sequencing of hsp70 gene.

Leishmaniasis is a major public health problem worldwide. Although next generation sequencing technology has been widely used in the diagnosis of infectious diseases, it has been scarcely applied in identification of Leishmania species. The aim of this study was to compare the efficiency of MinION™ nanopore sequencing and polymerase chain reaction restriction fragment length polymorphism in identifying Leishmania species. Our results showed that the MinION™ sequencer was able to discriminate reference strains and clinical samples with high sensitivity in a cost and time effective manner without the prior need for culture.

Molecular investigations of the effect of thermal manipulation during embryogenesis on muscle heat shock protein 70 and thermotolerance in broiler chickens

The objective of this study was to elucidate the optimum protocol timing of thermal manipulation (TM) during embryogenesis, which underline genetic improvement of muscle thermotolerance acquisition. For the present study, 1,440 fertile eggs were divided randomly and equally into control (37.8 °C with 56% relative humidity) and four thermally manipulated groups (TM1, TM2, TM3, and TM4) subjected to 39 °C for 18 h with 65% relative humidity daily during different embryonic periods. Then, at day 35 post-hatch, all groups were subjected to thermal challenge at 43 °C for 6 h to identify the level of thermotolerance acquisition differences between them. Hsp70 mRNA expression was evaluated by using a relative quantitatively RT-qPCR. Single nucleotide polymorphisms sequence of the Hsp70 gene was evaluated by Sanger's sequencing method. Pectoral and thigh muscles samples were subjected to immunohistochemistry to detect Hsp70. Among TM conditions that were investigated, TM1 (39 °C for 18 h during embryonic days (ED) 7­11) induced a significant improvement in thermotolerance parameters (body temperature and T3 levels) during thermal challenge combined with an increase in the levels of Hsp70 mRNA and its protein with a high stability of nucleotide sequences in both pectoral and thigh muscles. The partial DNA sequence of Hsp70 gene in TM1 was reported, and nucleotide sequences were deposited in NCBI GenBank database with the accession numbers (MK852579) and (MK852580). Thigh muscle thermotolerance acquisition was higher than pectoral muscle during thermal challenge at 43 °C for 6 h. Thus, TM during ED7­11 may improve thermotolerance acquisition without adversely affecting performance.(AU)

Unravelling the Interaction of Piperlongumine with the Nucleotide-Binding Domain of HSP70: A Spectroscopic and In Silico Study.

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van't Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M-1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.

Increased Levels of Plasma Extracellular Heat-Shock Proteins 60 and 70 kDa Characterized Early-Onset Neonatal Sepsis.

Background: Extracellular heat-shock proteins (eHsp) are highly conserved molecules that play an important role in inflammatory diseases and have been quantified in plasma from patients with infectious diseases, including sepsis. There is a constant search for dependable biochemical markers that, in combination with conventional methods, could deliver a prompt and reliable diagnosis of early-onset neonatal sepsis. Objective: We sought to assess the level of eHsp-27, eHsp-60, eHsp-70, and tumor necrosis factor-alpha (TNFα) in plasma of healthy neonates at term and infants with early-onset neonatal sepsis. Methods: This study included 34 newborns that were classified as healthy neonates at term (blood samples from the umbilical cord, n = 23) or infants with early-onset neonatal sepsis (blood samples obtained from umbilical artery by standard sterile procedures before starting a systemic antibiotic intervention, n = 11). All blood samples were centrifuged, and the plasma recovered to determine eHsp-27, eHsp-60, eHsp-70, and TNFα levels by ELISA. Results: Our results indicate that the level of eHsp-27 in healthy neonates at term was 0.045 ± 0.024 pg/ml. This value decreased 2.5-fold in infants with early-onset neonate sepsis (0.019 ± 0.006 pg/ml, p = 0.004). In contrast, the levels of eHsp-60 and eHsp-70 in healthy neonates at term were 13.69 ± 5.3 and 4.03 ± 2.6 pg/ml, respectively. These protein levels increased significantly 1.8- and 1.9-fold in the plasma of infants with early-onset neonatal sepsis (p ≤ 0.001). The level of TNFα in healthy neonates at term was 2.94 ± 0.46 pg/ml, with a 3.0-fold increase in infants with early-onset neonatal sepsis (8.96 ± 0.72 pm/ml, p ≤ 0.001). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of eHsp compared with that of C-reactive protein were 73.3, 60.0, 47.8, and 33.3%, respectively. Conclusion: This study demonstrated a consistent increase of eHsp-60 and eHsp-70 in the plasma of infants diagnosed with early-onset neonatal sepsis. These proteins showed higher sensitivity and specificity than C-reactive protein and blood culture test.

Oral Immunization With a Plant HSP90-SAG1 Fusion Protein Produced in Tobacco Elicits Strong Immune Responses and Reduces Cyst Number and Clinical Signs of Toxoplasmosis in Mice.

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral and cellular immune responses to diverse proteins and peptides. In this study, we explored whether Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1m), from aa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 fused to AtHsp81.2 sequence. When comparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in N. benthamiana leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1m fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1HC was expressed as intact fusion protein, yielding up to 90µg/g of fresh weight. Besides, the AtHsp81.2-SAG1HC mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1m mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1HC-infiltrated fresh leaves (plAtHsp81.2-SAG1HC group), recombinant AtHsp81.2-SAG1HC purified from infiltrated leaves (rAtHsp81.2-SAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2-SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue was also lower in this group compared with the remaining groups. In an immunoblot assay, plant-expressed AtHsp81.2-SAG1HC was shown to react with antibodies present in sera from T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T. gondii can improve the vaccine efficacy, and plant extract can be directly used for vaccination without the need to purify the protein, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.

In silico genomic and proteomic analyses of three heat shock proteins (HSP70, HSP90-a, and HSP90-b) in even-toed ungulates

BACKGROUND Heat shock proteins (HSPs) play important roles in the responses to different environmental stresses. In this study, the genomic and proteomic characteristics of three HSPs (HSP70, HSP90-a and HSP90-b) in five even-toed ungulates (sheep, goats, water buffalo, Zebu cattle and cattle) were analyzed using Multiple sequence alignment, SWISS modeling and phylogenetics analysis tools. RESULTS The bioinformatic analysis revealed that the HSP70 gene in cattle, Zebu cattle, and goat is located on chromosome 23, and is intronless, while in water buffalo and sheep it is located on chromosomes 2 and 20, respectively, and contains two exons linked by one intron. The HSP90-a gene is located on chromosome 21 in cattle, Zebu cattle, and goat, while in water buffalo and sheep it is located on chromosomes 20 and 18, respectively. The HSP90-b gene is located on the same chromosome as the HSP70 gene and contains 12 exons interspersed by 11 introns in all studied animals. In silico Expasy translate tool analysis revealed that HSP70, HSP90-a and HSP90-b encode 641, 733, and 724 amino acids, respectively. The data revealed that goat HSP70 protein has seven variable amino acid residues, while in both sheep and cattle only one such amino acid was detected. CONCLUSIONS This study will be supportive in providing new insights into HSPs for adaptive machinery in these studied animals and selection of target genes for molecular adaptation of livestock

Corticosteroid receptors as a model for the Hsp90•immunophilin-based transport machinery.

Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.

Oral Tolerance Induced by Heat Shock Protein 65-Producing Lactococcus lactis Mitigates Inflammation in Leishmania braziliensis Infection.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders.

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.

Strength training improves insulin resistance and differently affects mitochondria in skeletal muscle and visceral adipose tissue in high-fat fed mice.

AIMS: Strength training (ST) improves insulin resistance and glucose tolerance by yet unknown mechanisms. The aims of this study were to investigate the effects of ST on mitochondrial adaptation in skeletal muscle and adipose tissue, on heat shock protein 72 (Hsp72) in skeletal muscle, and on visceral adipocyte size in mice with high-fat diet (HFD)-induced insulin resistance. MATERIALS AND METHODS: Male Balb/c mice were divided into sedentary control-chow (C-chow), strength trained-chow (ST-chow), sedentary control-HFD (C-HFD) and strength trained-HFD (ST-HFD). Diet was provided for 12 weeks, while ladder climbing ST was performed for the final six weeks of the study at a frequency of three days per week. KEY FINDINGS: Strength training led to increased strength, muscular endurance, and skeletal muscle hypertrophy. Compared to the C-HFD group, mice in the ST-HFD group decreased their whole-body insulin resistance, improved their glucose tolerance, and had higher activation of the insulin pathway in skeletal muscle. ST increased citrate synthase (CS) activity in skeletal muscle, but this increase was blunted in ST-HFD. Conversely, HFD reduced adipose tissue CS activity regardless of training status. Hsp72 content was reduced in C-HFD, but returned to control levels in ST-HFD. Finally, reduced epididymal adipocyte size was observed in ST-HFD. SIGNIFICANCE: These results suggest that the improvement in insulin resistance induced by ST is related to mitochondrial adaptation in skeletal muscle, but not in adipose tissue. Moreover, this improvement might be related to increased skeletal muscle Hsp72 and reduced epididymal adipocyte size.

Amino acid restriction alters survival mechanisms in pancreatic beta cells: possible role of the PI3K/Akt pathway.

BACKGROUND AND AIMS: Malnutrition in the early stages of life may lead to changes in the glycemic metabolism during adulthood, such as pancreatic beta cells dysfunction and failure. Therefore, this study aimed to evaluate the effects of an in vitro amino acid restriction model on the function and viability of pancreatic beta cells. METHODS: Insulin-producing cells (INS-1E) were maintained in control or amino acid restricted culture medium containing 1 × or 0.25 × of amino acids, respectively, for 48 h. RESULTS: Amino acid restricted group showed lower insulin secretion and insulin gene expression, reduced mitochondrial oxygen consumption rate and reactive oxygen species production. Besides, amino acid restricted group also showed higher levels of endoplasmic reticulum stress and apoptosis markers and enhanced Akt phosphorylation. However, even with higher levels of apoptosis markers, amino acid restricted group did not show higher levels of cell death unless the PI3K/Akt pathway was inhibited. CONCLUSION: Amino acid restricted beta cell viability seems to be dependent on the PI3K/Akt pathway.

Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.