Exogenous heat shock proteins HSPA1A and HSPB1 regulate TNF-α, IL-1ß and IL-10 secretion from monocytic cells.

Endogenous molecules, such as heat shock proteins (HSP), can function as danger signals when released into the extracellular environment in response to cell stress, where they elicit an immune response such as cytokine secretion. There has also been some suggestion that contamination of exogenous HSPs with lipopolysaccharide (LPS) may be responsible for these effects. This study investigates the effects of exogenous HSPA1A and HSPB1 on the activation of immune cells and the resulting secretion of cytokines, which are involved in inflammatory responses. To address whether exogenous HSPs can directly activate cytokine secretion, naïve U937 cells, differentiated U937 cells and peripheral blood mononuclear cells (PBMCs) were treated with either exogenously applied HSPA1A or HSPB1 and then secreted IL-1ß, TNF-α and IL-10 were measured by ELISA. Both HSPs were able to induce a dose-dependent increase in IL-10 secretion from naïve U937 cells and dose-dependent IL-1ß, TNF-α and IL-10 secretion were also observed in differentiated U937 cells and PBMCs. We also observed that CD14 affects the secretion levels of IL-1ß, TNF-α and IL-10 from cells in response to exogenous HSP treatment. In addition, HSPA1A and HSPB1 were shown to interact with CD14, CD36 and CD11b extracellular receptor proteins. Several approaches used in this study indicate that HSP-induced cytokine secretion is largely independent of any contaminating LPS in the samples.

Primary Colorectal Cells Culture as a Translational Research Model.

Preclinical studies are an essential stage for any pharmacological agent hoping to make its way into clinical trials. An ideal preclinical model that can accurately predict clinical response does not exist and the best that the scientific community have at the moment is to select the most relevant study model pertaining to the disease of interest from those available, which includes: cell lines, animal models, and even in-silico methodology. Currently, there is a huge gap between preclinical and clinical trial results, indicating that there is much room for improvement in developing a better model to bridge the translational gap.

Re-examining HSPC1 inhibitors.

HSPC1 is a critical protein in cancer development and progression, including colorectal cancer (CRC). However, clinical trial data reporting the effectiveness of HSPC1 inhibitors on several cancer types has not been as successful as predicted. Furthermore, some N-terminal inhibitors appear to be much more successful than others despite similar underlying mechanisms. This study involved the application of three N-terminal HSPC1 inhibitors, 17-DMAG, NVP-AUY922 and NVP-HSP990 on CRC cells. The effects on client protein levels over time were examined. HSPC1 inhibitors were also applied in combination with chemotherapeutic agents commonly used in CRC treatment (5-fluorouracil, oxaliplatin and irinotecan). As HSPA1A and HSPB1 have anti-apoptotic activity, gene-silencing techniques were employed to investigate the significance of these proteins in HSPC1 inhibitor and chemotherapeutic agent resistance. When comparing the action of the three HSPC1 inhibitors, there are distinct differences in the time course of important client protein degradation events. The differences between HSPC1 inhibitors were also reflected in combination treatment-17-DMAG was more effective compared with NVP-AUY922 in potentiating the cytotoxic effects of 5-fluorouracil, oxaliplatin and irinotecan. This study concludes that there are distinct differences between N-terminal HSPC1 inhibitors, despite their common mode of action. Although treatment with each of the inhibitors results in significant induction of the anti-apoptotic proteins HSPA1A and HSPB1, sensitivity to HSPC1 inhibitors is not improved by gene silencing of HSPA1A or HSPB1. HSPC1 inhibitors potentiate the cytotoxic effects of chemotherapeutic agents in CRC, and this approach is readily available to enter clinical trials. From a translational point of view, there may be great variability in sensitivity to the inhibitors between individual patients.

Measuring Hsp72 (HSPA1A) by indirect sandwich ELISA.

The enzyme-linked immunosorbent assay (ELISA) is an immunological technique which is used to determine the presence or quantity of an antigen within a sample. ELISAs rely on the use of at least one antibody (Ab) specific for the antigen being measured. This antibody is covalently linked to an enzyme which is detected through the use of an enzymatic substrate, which can be colorimetric, fluorogenic, or chemiluminescent. The ELISA for Hsp72 described here is a typical indirect sandwich ELISA, which can be used for measuring Hsp72 from cellular/tissue extracts, tissue culture supernatant, and serum. Typically, a 96-well ELISA plate is coated with a specific antibody which captures Hsp72 from the sample, and another antibody specific for a different Hsp72 epitope is used to detect Hsp72. An enzyme-labelled species-specific antibody conjugate is then applied which is consequently detected using a colorimetric enzyme substrate. The quantity of Hsp72 present in the samples is interpolated using a standard curve of known amounts of pure Hsp72.

Analysis of heat-shock protein localisation using flow cytometry.

The use of flow cytometry in heat-shock protein (HSP) research is increasing rapidly due to the high sensitivity and versatility of the technique. The method allows the simultaneous analysis of multiple proteins within numerous cell types in a heterogeneous sample, providing advantages over alternative techniques, such as ELISA and Western blotting. As a result, flow cytometry is becoming the leading technique used in this area of research. The current chapter describes the methodology for preparing samples for this technique and outlines two protocols for the analysis of surface- and intracellular-localised HSPs.

Antipsychotic medication and oxidative cell stress: a systematic review.

OBJECTIVE: To look at (1) the association between antipsychotics and cell stress, (2) whether first-generation antipsychotics may show different effects than second-generation antipsychotics, and (3) whether recommendations can be made regarding medication. DATA SOURCES: We conducted a systematic review of 5 databases for all articles published until December 31, 2007: PubMed, Ovid MEDLINE, EMBASE, PsycINFO, and EBM Reviews. Under specific headings (eg, "heat shock proteins" and "oxidative stress"), a systematic search of these databases included such terms as HSP70 and homocysteine, and specific search strings were constructed. No limits were placed on the year or language of publication. References from pertinent articles or books were retrieved. STUDY SELECTION: We included 42 articles of human studies from 2,387 references originally retrieved. We included only articles that (1) were quantitative; (2) referred only to human tissue, in vivo, or in vitro; (3) stated what tissue was examined; (4) identified what metabolites were measured; and (5) had references. DATA EXTRACTION: All articles were assessed by 2 authors, which ensured that the inclusion criteria were met. The selected studies were too heterogeneous to be combined for any useful meta-analysis. Three authors, therefore, independently interpreted the data, using specified criteria to judge whether each study showed a beneficial, detrimental, or no effect on the markers measured. DATA SYNTHESIS: The analysis revealed no conclusive association with direct or indirect markers of oxidative cell stress and antipsychotics. For every reviewed antipsychotic, we revealed differing research results showing a beneficial, detrimental, or no effect. This was true for in vivo as well as in vitro studies. CONCLUSIONS: It remains unclear whether antipsychotics increase or reduce cell stress. Claims of neuroprotective properties of antipsychotics seem premature.

Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation.

INTRODUCTION: Monocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers. METHODS: CCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function. RESULTS: CCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals. CONCLUSIONS: CCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.

Heat Shock Protein translocation induced by membrane fluidization increases tumor-cell sensitivity to chemotherapeutic drugs.

Treatment of chronic lymphocytic leukemia (CLL) remains a challenge due to the frequency of drug resistance amongst patients. Improving the delivery of chemotherapeutic agents while reducing the expression of anti-apoptotic Heat Shock Proteins (HSPs) within the cancer cells may facilitate in overcoming this drug resistance. We demonstrate for the first time that sub-lethal doses of chemotherapeutic agents can be combined with membrane fluidizing treatments to produce a significant increase in drug efficacy and apoptosis in vitro. We show that fluidizers result in a transient decrease in intracellular HSPs, resulting in increased tumor-cell sensitivity and a membrane-associated induction of HSP gene expression.

Differential heat shock protein localization in chronic lymphocytic leukemia.

Mechanisms behind carcinogenesis and resistance of tumor cells to treatment regimes remain elusive. The major stress proteins Hsp72, Hsp90, and Hsp27 are credible candidates to provide this resistance, as their overexpression in many cancer types is well documented. In addition to being present inside tumor cells, where they confer resistance to apoptosis, Hsp72, in particular, is presented externally, embedded in the cell membrane of cancer cells. This study aimed to investigate the localization of Hsp72, Hsp90, and Hsp27 in leukocytes from patients with CLL and age-matched control subjects. CLL patients were found to express significantly higher levels of iHsp90 (CLL=2463 MFI; control=748 MFI) and iHsp27 (CLL=2190 MFI; control=1031 MFI) in lymphocytes than that expressed by lymphocytes from control subjects. Furthermore, expression of iHsp90 was shown to be related to stage of disease, and expression of iHsp27 correlated with levels of active caspase-3. Patients were found to express very high levels or very low levels of sHsp72 and iHsp72 in CD5(+)/CD19(+) cells, although surface and intracellular datasets did not correlate. Levels of extracellular Hsp72 circulating in the serum were found to correlate with internal levels of Hsp72 and were also found to be significantly lower in patients receiving corticosteroid treatment than in patients not receiving corticosteroid treatment. Finally, analysis of the number of circulating Tregs revealed significantly elevated numbers in CLL patients compared with control subjects.

Vitamin B(12) and redox homeostasis: cob(II)alamin reacts with superoxide at rates approaching superoxide dismutase (SOD).

We report a kinetic study of the reaction between superoxide and an important intracellular form of vitamin B(12), cob(II)alamin. Superoxide is implicated in the pathophysiology of many inflammatory diseases, whereas vitamin B(12) derivatives are often beneficial in their treatment. We found that cob(II)alamin reacts with superoxide at rates approaching those of superoxide dismutase itself, suggesting a probable mechanism by which vitamin B(12) protects against chronic inflammation and modulates redox homeostasis.

Detection of human blood by immunoassay for applications in forensic analysis.

The detection and confirmation of bloodstains as being human in origin is important in crime scene investigations. There are a number of blood detection methods currently available. The aim of this work was to develop an assay capable of detecting the presence of human blood from both liquid blood samples and dried bloodstains. A simple, direct competitive ELISA was developed utilising a polyclonal antibody against human IgG. Once optimised, the ELISA was found to be specific for human IgG, with no cross-reaction observed with pig, sheep, cow, goat, horse and rabbit IgG. The assay was also found to be sensitive, with a detection limit of 0.1 microg/mL. This compares favourably with leading blood detection methods. The assay was able to confirm the presence of human blood in blood mixtures, in stains on a variety of surfaces and also gave positive results with bloodstains that were up to 1 year old. The assay was simple to use, rapid and highly reproducible. The ELISA performance makes it suitable for development as a kit to rival currently used methods for the routine detection of human blood at crime scenes. Further applications of the anti-human IgG antibody are reported, including immunodot assays and a sandwich ELISA format. The methods described here are simple, reliable assays for the identification of human blood and are presented as viable alternatives to existing techniques for blood detection.

A novel role for vitamin B(12): Cobalamins are intracellular antioxidants in vitro.

Oxidative stress is a feature of many chronic inflammatory diseases. Such diseases are associated with up-regulation of a vitamin B(12) (cobalamin) blood transport protein and its membrane receptor, suggesting a link between cobalamin and the cellular response to inflammation. The ability of cobalamin to regulate inflammatory cytokines suggests that it may have antioxidative properties. Here we show that cobalamins, including the novel thiolatocobalamins N-acetyl-l-cysteinylcobalamin and glutathionylcobalamin, are remarkably effective antioxidants in vitro. We also show that thiolatocobalamins have superior efficacy compared with other cobalamin forms, other cobalamins in combination with N-acetyl-l-cysteine (NAC) or glutathione (GSH), and NAC or GSH alone. Pretreatment of Sk-Hep-1 cells with thiolatocobalamins afforded robust protection (>90% cell survival) against exposure to 30 microM concentrations of the pro-oxidants homocysteine and hydrogen peroxide. The compounds inhibited intracellular peroxide production, maintained intracellular glutathione levels, and prevented apoptotic and necrotic cell death. Moreover, thiolatocobalamins are remarkably nontoxic in vitro at supraphysiological concentrations (>2 mM). Our results demonstrate that thiolatocobalamins act as powerful but benign antioxidants at pharmacological concentrations. Because inflammatory oxidative stress is a component of many conditions, including atherosclerosis, dementia, and trauma, their utility in treating such disorders merits further investigation.

Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells.

Osteoprotegerin (OPG) is a major regulator of osteoclastogenesis, bone resorption and vascular calcification. OPG is produced by various cell types including mesenchymally derived cells, in particular, osteoblastic cells. Here we show OPG production by osteoblastic cells was stimulated by platelet-derived growth factor (PDGF) in two human osteosarcoma cell lines (MG63, Saos-2), a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (hMSC) by 152%, 197%, 113% and 45% respectively over 24 h. OPG was measured in the cell culture medium by immunoassay. PDGF isoforms AA, BB and AB show similar stimulation of OPG production. Message for OPG was also increased similarly to the increased secretion into the culture medium. Using specific inhibitors of cell signalling we demonstrate that PDGF acts through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NF-kB or JNK. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process.

Sensing danger--Hsp72 and HMGB1 as candidate signals.

Molecules that behave as danger signals are produced when the body is perceived to be under attack, and they alert the immune system to the problem. The immune system can then mount an appropriate response. Two molecules that have received attention as potential danger signals are heat shock protein 72 (Hsp72) and high mobility group box 1 (HMGB1), which are intracellular proteins but are released when cells are under stress, in particular, when necrosis occurs. This review considers the similarities between these two molecules and then contrasts their mechanism of action and problems that can arise when they are overpresented in the extracellular environment. It is proposed that Hsp72 and HMGB1 are members of a suite of danger molecules that provide a fingerprint of the threat, or stressor, to tissue or organism integrity.

Measuring the secretion of heat shock proteins from cells.

Heat shock proteins have been shown to be secreted from a number of cell types. Necrotic cells release heat shock proteins in a passive manner, whereas we, and others, have shown that viable cells secrete Hsp70 and Hsp60 through an active mechanism involving lysosomal vesicles and lipid rafts. This release of Hsp70 and Hsp60 is regulated, for example by being increased by elevated temperature. This article outlines procedures, using Hsp70 as the example, to: ensure the status of cells (viable, apoptotic or necrotic); identify the heat shock protein secreted; and quantify the secreted protein. Hsp70 has previously been quantified by ELISA, but newer methods are now being adopted, such as BIAcore and bead-based assays for use by FACS. These methods have the advantages of being more sensitive and requiring less sample than ELISA. The BIAcore has the potential to analyse Hsp70 ligands and provide affinity constants. The FACS bead assay system can be used to run multiplex assays.

Birth defects and anti-heat shock protein 70 antibodies in early pregnancy.

Abstract It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223-406) microg/mL compared to 150 (88-207) microg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.

The development of immunoassays to identify and quantify species source of gum arabic.

Gum arabic from Acacia senegal is commonly used as an additive in foodstuffs. Adulteration of gum arabic by other gums is a potential problem for reasons of safety and quality. This study aimed to develop and evaluate the use of enzyme-linked immunosorbent assays (ELISAs) for the detection of potential adulterants of gum arabic. Indirect competitive ELISAs (IC-ELISAs) were developed using the monoclonal antibodies SY CC7 (A. senegal), SY HH3 (Acacia seyal), and SY J1A1 (Combretum erythrophyllum). All IC-ELISAs had a working range of 0.005-10 mg/mL. The antibodies used were tested using the IC-ELISAs for cross-reactivity with other Acacia species and other gums. The antibodies were very specific for their respective antigens. Significant cross-reactivity was found for SY CC7 (between A. senegal and A. melliferae) and SY J1A1 (between C. erythrophyllum and A. seyal). The IC-ELISA was adapted further to test confectionery samples for the presence of gum arabic, which was successful, although recovery rates were reduced. Both IC- and plate trapped antigen ELISA (PTA-ELISA) formats were able to distinguish an adulterated sample of gum arabic when blended with either A. seyal or C. erythrophyllum. The PTA-ELISA was more sensitive for A. seyal than the IC-ELISA, but both were equally sensitive for C. erythrophyllum. The results suggest that the antibodies SY CC7, SY HH3, and SY J1A1 could be used in combination with each other for the detection of potential adulterants of A. senegal and the detection of gum arabic in foodstuffs.

Hsp70 release from peripheral blood mononuclear cells.

There are an increasing number of studies reporting the presence of Hsps in human serum. We have investigated the release of Hsp70 into blood and culture medium from peripheral blood mononuclear cells (PBMCs), and whether this release is due to cell damage or active secretion from the cells. Intact Hsp70 was released from cells within whole blood and from purified PBMCs under normal culture conditions. Hsp70 release was rapid (0.1 ng/10(6) cells/h) over the first 2 h of culture and continued at a reduced rate up to 24 h (<0.025 ng/10(6) cells/h). Using viable cell counts and lactate dehydrogenase release we were able to confirm that the release of Hsp70 was not due to cellular damage. Hsp70 release was inhibited by monensin, methyl-beta-cyclodextrin, and methylamine, but not by brefeldin A. These data suggest that Hsp70 is released from cells via a non-classical pathway, possibly involving lysosomal lipid rafts.

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor.

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 microg ml(-1) using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42 degrees C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.

Transcobalamin polymorphism and serum holo-transcobalamin in relation to Alzheimer's disease.

Isoforms of the vitamin B(12) carrier protein transcobalamin (TC) might influence its cellular availability and contribute to the association between disrupted single-carbon metabolism and Alzheimer's disease (AD). We therefore investigated the relationships between the TC 776C>G (Pro259Arg) genetic polymorphism, total serum cobalamin and holo-TC levels, and disease onset in 70 patients with clinically diagnosed AD and 74 healthy elderly controls. TC 776C>G polymorphism was also determined for 94 histopathologically confirmed AD patients and 107 controls. Serum holo-TC levels were significantly higher in TC 776C homozygotes (p = 0.04). Kaplan-Meier survival functions differed between homozygous genotypes (Cox's F-Test F(42, 46) = 2.1; p = 0.008) and between 776C homozygotes and heterozygotes (Cox's F test F(46, 108) = 1.7; p = 0.02). Proportionately fewer TC 776C homozygotes appear to develop AD at any given age, but this will require confirmation in a longitudinal study.