Loss of oral infectivity of tissue cysts of Toxoplasma gondii RH strain to outbred swiss webster mice

We have developed a method for obtaining cysts of Toxoplasma gondii RH strain. Outbred Swiss Webster mice were infected subcutaneously (s.c.) or intraperitoneally (i.p.) with 10⁵ tachyzoites and given sulfadiazine 400 mg l⁻¹ + NaHCO₃ 10 g l⁻¹ in drinking water from day 1 to day 15 post-infection (p.i.). None of the mice infected i.p. survived, compared with 50% of the mice infected s.c. Cysts were detectable in the brain on day 45 p.i., and had ultrastructural features consistent with those of bradyzoites. However, these cysts were incapable of infecting mice via the oral route. In addition, immunofluorescence studies showed the persistence of P36 protein expression, indicating that the conversion to bradyzoites was incomplete.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

Expression of heat shock protein in host macrophages correlates with a protective potential against infection with Leishmania major in mice

We studied the relationship between the expression of 65-kDa heat shock protein (HSP65) and resistance of mice to infection with Leishmania major (L. major), an obligate intracellular protozoan. C57BL/6 (B6) mice, a strain genetically resistant to L. major infection, expressed high level of HSP65 in their peritoneal and draining lymph node macrophages after infection, whereas susceptible BALB/c mice expressed only slightly at the early stage of infection. This protein was not expressed in the parasite itself and macrophages of non-infected mice. We examined the role of T cells in the expression of HSP65 by using SCID mice grafted with the fetal thymus from B6 or BALB/c mice (B6-TG or BALB-TG mice, respectively). Either BALB-TG or B6-TG mice were reconstituted with T cells derived from each grafted fetal thymus. B6-TG mice were markedly resistant against infection with L. major, compared with BALB-TG mice. Furthermore, the HSP65 expression in macrophages of thymus-grafted mice was similar to that of the thymus-donor type. That is, B6-TG mice expressed a high level of this protein, whereas BALB-TG mice did in lower level than B6-TG mice. These results show that T cells are necessary to express HSP65 and the expression correlates with a protective potential of T cells against infection with L. major.     

Bicyclol: a novel antihepatitis drug with hepatic heat shock protein 27/70-inducing activity and cytoprotective effects in mice

Heat shock proteins (HSPs) are the best-known endogenous factors that protect against cell injury under various pathological conditions and that can be induced by various physical, chemical, and biological stressors. New research seeks to discover a compound that is clinically safe and can induce the accumulation of HSPs in patients. This paper reports that the oral administration of three doses of bicyclol, a novel antihepatitis drug, induced hepatic HSP27 and HSP70 expression in a time- and dose-dependent manner, and that bicyclol treatment stimulated heat shock factor 1 (HSF1) activation in mice. The inducing effects of bicyclol on HSP27, HSP70 and HSF1 were all blocked by quercetin, an inhibitor of HSP biosynthesis. The cytoprotective effect of HSP27/70 induced by bicyclol against hepatotoxicity of acetaminophen (AP) was assessed in mice. The prior administration of bicyclol markedly suppressed AP-induced liver injury as indicated by the reduction in the elevation of serum alanine aminotransferase and aspartate aminotransferase, in liver necrosis, in the release of cytochrome c and apoptosis-inducing factor from mitochondria, as well as in hepatic deoxyribonucleic acid fragmentation in mice. However, all the above actions of bicyclol against AP-induced mouse liver injuries were significantly attenuated by quercetin. This is the first report to show that bicyclol induces hepatic HSP27/70 expression via activation of HSF1 and that the cytoprotective action of bicyclol against liver injury is mediated by its induction of HSP27/70. These results provide new evidence for elucidating the mechanism of the hepatoprotective action of bicyclol in animals and patients.     

Potential roles of hepatic heat shock protein 25 and 70i in protection of mice against acetaminophen-induced liver injury

The aim of the present study was to assess the contribution of the level of expression of heat shock protein 25 (HSP25), 60 (HSP60), 70 (HSC70) and 70i (HSP70i) in mouse livers after a lethal dose of acetaminophen (APAP) to their survival. We examined changes in survival ratio, plasma APAP level and alanine aminotransferase (ALT) activity, and hepatic reduced glutathione (GSH), HSP25, HSP60, HSC70 and HSP70i levels following treatment of mice with APAP (500 mg/kg, p.o.). The plasma APAP level increased rapidly, and reached a maximum 0.5 h after APAP treatment. Hepatic GSH decreased rapidly, and was almost completely depleted 1 h after APAP treatment. Plasma ALT activity, an index of liver injury, significantly increased from 3 h onwards after APAP treatment. The survival ratios 9 h, 24 h and 48 h after APAP treatment were 96%, 38% and 36%, respectively. We found a remarkable difference in the patterns of hepatic HSP25 and HSP70i induction in mice that survived after APAP treatment. HSP70i levels increased from 1 h onwards after APAP treatment in a time-dependent manner, and reached a maximum at 9 h. In contrast, HSP25 could be detected just 24 h after APAP treatment, and maximal accumulation was observed at 48 h. Other HSPs examined were unchanged. Notably, the survival ratio dropped by only 2% after HSP25 expression. Recently, a novel role for HSP25 as an anti-inflammatory factor was suggested. We have already shown that 48-h treatment with APAP induces severe centrilobular necrosis with inflammatory cell infiltration in mouse livers. Taken together, the level of expression of hepatic HSP25 may be a crucial determinant of the fate of mice exposed to APAP insult.     

Immunological enhancement action of endotoxin-free tilapia heat shock protein 70 against Streptococcus iniae

The immunological effects of heat shock proteins (HSPs) had been found in humans and mice, but scarce data of endotoxin-free Hsp70 were reported in tilapia. In the current study, we reported that tHsp70 alone and antigen-tHsp70 compound increased the proliferations of lymphocytes and macrophages, significantly increased the NO release and phagocytotic ability of macrophages (p < 0.05), and enhanced the levels of immune-related genes in lymphocytes and macrophages in a dose- and/or time-dependent manner. On the other hand, tHsp70 not only helped to reduce the proliferation inhibitions induced by the ECP treatment, but also assisted antigens to enhance the vaccine-induced protection against Streptococcus iniae (p < 0.05). We described, for the first time, a critical role of endotoxin-free tHsp70 on activation of tilapia lymphocytes and macrophages post S. iniae exposure and its up-regulation effects on vaccine-induced protection. Our research highlights the immunological enhancement action of Hsp70 in teleost immunity.     

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.     

Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice

Chronic infection with the blood fluke Schistosoma japonicum is associated with both liver cirrhosis and liver cancer. Previously, heat shock protein 47, a collagen-specific molecular chaperone, was shown to play a critical role in the maturation of procollagen. However, less is known about the role of heat shock protein 47 in S. japonicum-induced hepatic fibrosis. We therefore investigated the expression of heat shock protein 47 in S. japonicum-induced liver fibrosis and attempted to determine whether inhibition of heat shock protein 47 could have beneficial effects on fibrosis in vitro and in vivo. In this study, we found that the expression of heat shock protein 47 was significantly increased in patients with Schistosoma-induced fibrosis, as well as in rodent models. Immunohistochemistry revealed heat shock protein 47-positive cells were found in the periphery of egg granulomas. Administration of heat shock protein 47-targeted short hairpin (sh)RNA remarkably reduced heat shock protein 47 expression and collagen deposition in NIH3T3 cells and liver tissue of S. japonicum-infected mice. Life-table analysis revealed a dose-dependent prolongation of survival rates with the treatment of heat shock protein 47-shRNA in murine fibrosis models. Moreover, serum alanine aminotransferase and aspartate transaminase activity, splenomegaly, spleen weight index and portal hypertension were also measured, which showed improvement with the anti-fibrosis treatment. The fibrosis-related parameters assessed were expressions of Col1a1, Col3a1, TGF-β1, CTGF, IL-13, IL-17, MMP-9, TIMP-1 and PAI-1 in the liver. This study demonstrated that heat shock protein 47-targeted shRNA directly reduced collagen production of mouse liver fibrosis associated with S. japonicum. We conclude that heat shock protein 47 plays an essential role in S. japonicum-induced hepatic fibrosis in mice and may be a potential target for ameliorating the hepatic fibrosis caused by this parasite.     

A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance

Summary Escherichia coli cells carrying the dnaK756 mutation, were inactivated at 52°C faster than control cells. This suggests that the intact dnaK gene product plays a role in protecting the cell from lethal damage at 52°C. The effect of the dnaK mutation on induced thermotolerance was examined. Prior heat shock at 42°C greatly lowered the subsequent inactivation rate in both mutant and control cells. This result suggests that, although produced in large amounts in response to thermal stress, mutation in the DnaK protein has little or no effect on induced thermotolerance.     

Evidence for a role of autoantibodies to heat shock protein 60, 70, and 90 in patients with dermatitis herpetiformis

Heat shock proteins (Hsp) are highly conserved immunomodulatory molecules upregulated when cells are exposed to stressful stimuli, such as inflammation. Their involvement in various autoimmune diseases, including autoimmune bullous diseases and celiac disease, has been increasingly recognized. To further study the role of Hsp in autoimmune bullous diseases, we have investigated for the first time the humoral autoimmune response to Hsp40, Hsp60, Hsp70, and Hsp90 in patients with dermatitis herpetiformis (DH; n = 26), bullous pemphigoid (BP; n = 23), and pemphigus vulgaris (PV; n = 16), the first representing a cutaneous manifestation of celiac disease. While in patients with active BP and PV, serum levels of autoantibodies against these Hsp did not differ from the corresponding age- and gender-matched healthy controls (n = 9–14); circulating autoantibodies against Hsp60, Hsp70, and Hsp90 were found to be increased at the active disease stage of DH. Further analysis of this latter patient subgroup showed that these anti-Hsp autoantibodies decreased in parallel with serum autoantibodies against epidermal and tissue transglutaminase during remission of skin lesions following a gluten-free diet, revealing significantly positive correlations. Although further studies on larger groups of patients will be needed to confirm the present data, our results support the notion that autoantibodies against Hsp60, Hsp70, and Hsp90 deserve attention in the study of the mechanisms that promote the development and maintenance of DH and possibly also the underlying celiac disease as well as potential novel disease biomarkers.     

The detection and role of heat shock protein 70 in various nondisease conditions and disease conditions: a literature review

As an intracellular polypeptide, heat shock protein 70 (HSP70) can be exposed on the plasma membrane and/or released into the circulation. However, the role of HSP70 in various nondisease and disease conditions remains unknown. Quantitative methods for the detection of HSP70 have been used in clinical studies, revealing that an increase in circulating HSP70 is associated with various types of exercise, elderly patients presenting with inflammation, mobile phones, inflammation, sepsis, chronic obstructive pulmonary disease, asthma, carotid intima-media thickness, glutamine-treated ill patients, mortality, diabetes mellitus, active chronic glomerulonephritis, and cancers. Circulating HSP70 decreases with age in humans and in obstructive sleep apnea, arteriosclerosis, atrial fibrillation (AF) following coronary artery bypass surgery, nonalcoholic fatty liver disease, moderate-to-severe alcoholic fatty liver disease, hepatic steatosis, and Helicobacter pylori infection. In conclusion, quantitative methods can be used to detect HSP70, particularly in determining circulating HSP70 levels, using more convenient and rapid screening methods. Studies have shown that changes in HSP70 are associated with various nondisease and disease conditions; thus, HSP70 might be a novel potential biomarker reflecting various nondisease conditions and also the severity of disease conditions. However, the reliability and accuracy, as well as the underlying mechanism, of this relationship remain poorly understood, and large-sample clinical research must be performed to verify the role.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0618-8) contains supplementary material, which is available to authorized users.     

MAPKAP kinase 2 is activated by heat shock and TNF-α: In vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade

The activation of MAPKAP kinase 2 was investigated under heat-shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor-α (TNF-α). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10–15 min after stimulation either by heat shock or TNF-α. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF-α treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.     

Involvement of calcium in the differential induction of heat shock protein 70 by heat shock protein 90 inhibitors, geldanamycin and radicicol, in human non-small cell lung cancer H460 cells

Both geldanamycin (GA) and radicicol (RA) are HSP90 binding agents that possess antitumour activities. Although the in vitro data indicated that the inhibitory constant of RA is much bigger than that of GA, the in vivo data on drug efficacy might reveal different results. We have recently shown that treatment with GA induces a heat-shock response and that calcium mobilization may be involved in the process. By using induction of HSP70 as the endpoint assay, we found changes in upstream signaling mediators, including HSF1 and calcium mobilization, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with GA and RA. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, are all involved in the HSP70 induction process triggered by the drugs. However, only GA, but not RA, can provoke a rapid calcium mobilization and thereby result in an instant induction of HSP70. Furthermore, the rapid calcium influx, followed by instant HSP induction, could be achieved in GA- or RA-treated cells placed in a medium containing excessive calcium while the response was completely abolished in cells depleted of calcium. Taken together, our findings suggest that differential calcium signaling may account for the differential induction of HSP and the action of GA and RA.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.     

Induction of a heat shock gene (hsp 70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue

Elevation of body temperature by 2–3°C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.     

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r² = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.