Heat shock protein production and immunity and altered fetal development in diabetic pregnant rats

We evaluated associations between the concentrations of heat shock proteins (hsp60 and hsp70) and their respective antibodies, alterations in maternal reproductive performance, and fetal malformations in pregnant rats with hyperglycemia. Mild diabetes (MD) or severe diabetes (SD) was induced in Sprague-Dawley rats prior to mating; non-treated non-diabetic rats (ND) served as controls. On day 21 of pregnancy, maternal blood was analyzed for hsp60 and hsp70 and their antibodies; and fetuses were weighed and analyzed for congenital malformations. Hsp and anti-hsp levels were correlated with blood glucose levels during gestation. There was a positive correlation between hsp60 and hsp70 levels and the total number of malformations (R∈=∈0.5908, P∈=∈0.0024; R∈=∈0.4877, P∈=∈0.0134, respectively) and the number of malformations per fetus (R∈=∈0.6103, P∈=∈0.0015; R∈=∈0.4875, P∈=∈0.0134, respectively). The anti-hsp60 IgG concentration was correlated with the number of malformations per fetus (R∈=∈0.3887, P∈=∈0.0451) and the anti-hsp70 IgG level correlated with the total number of malformations (R∈=∈0.3999, P∈=∈0.0387). Moreover, both hsp and anti-hsp antibodies showed negative correlations with fetal weight. The results suggest that there is a relationship between hsp60 and hsp70 levels and their respective antibodies and alterations in maternal reproductive performance and impaired fetal development and growth in pregnancies associated with diabetes. © 2012 Cell Stress Society International.

Induction of the 72 kDa heat shock protein by glucose ingestion in black pregnant women

Obese Black women are at increased risk for development of gestational diabetes mellitus and have worse perinatal outcomes than do obese women of other ethnicities. Since hsp72 has been associated with the regulation of obesity-induced insulin resistance, we evaluated associations between glucose ingestion, hsp72 release and insulin production in Black pregnant women. Specifically, the effect of a 50-g glucose challenge test (GCT) on heat shock protein and insulin levels in the circulation 1 h later was evaluated. Hsp27 and hsp60 levels remained unchanged. In contrast, serum levels of hsp72 markedly increased after glucose ingestion (p = 0.0054). Further analysis revealed that this increase was limited to women who were not obese (body mass index <30). Insulin levels pre-GCT were positively correlated with body mass index (p = 0.0189). Median insulin concentrations also increased post GCT in non-obese women but remained almost unchanged in obese women. Post-GCT serum hsp72 concentrations were inversely correlated with post GCT insulin concentrations (p = 0.0111). These observations suggest that glucose intake during gestation in Black women rapidly leads to an elevation in circulating hsp72 only in non-obese Black women. The release of hsp72 may regulate the extent of insulin production in response to a glucose challenge and, thereby, protect the mother and/or fetus from development of hyperglycemia, hyperinsulinemia, and/or immune system alterations. © 2013 Cell Stress Society International.

High levels of heat shock protein 70 are associated with pro-inflammatory cytokines and may differentiate early- from late-onset preeclampsia

Preeclampsia (PE), a specific syndrome of pregnancy, can be classified into early and late onset, depending on whether clinical manifestations occur before or after 34 weeks' gestation. We determined whether plasma concentrations of Hsp60 and Hsp70 were related to circulating cytokine levels, as well as kidney and liver functions, in early- and late-onset PE. Two hundred and thirty-seven preeclamptic women (95 with early- and 142 with late-onset PE) were evaluated. Plasma levels of Hsp60, Hsp70, and their specific antibodies, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-10, IL-12, and soluble TNF-α-receptor I (sTNFRI) concentrations, were determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of Hsp70, TNF-α, IL-1β, IL-12, and sTNFRI were significantly elevated in patients with early-onset PE compared with women with late-onset PE; IL-10 levels were significantly lower in the early-onset PE group. Concentrations of urea, uric acid, proteinuria, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were also significantly higher in early-onset PE. The percentage of infants with intrauterine growth restriction was also significantly higher in women with early-onset PE. There were positive correlations between Hsp70 levels and TNF-α, TNFRI, IL-1β, IL-12, GOT, GPT, LDH, and uric acid concentrations in early-onset PE group. Thus, early-onset PE was associated with greater maternal and fetal impairment. There are differences in pathophysiology between early- and late-onset PE, highlighting by the difference in Hsp70 levels. © 2013 Elsevier B.V. All rights reserved.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U. S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

A randomized, blinded, multicenter trial of lipid-associated amphotericin B alone versus in combination with an antibody-based inhibitor of heat shock protein 90 in patients with invasive candidiasis

BACKGROUND: Mycograb (NeuTec Pharma) is a human recombinant monoclonal antibody against heat shock protein 90 that, in laboratory studies, was revealed to have synergy with amphotericin B against a broad spectrum of Candida species. METHODS: A double-blind, randomized study was conducted to determine whether lipid-associated amphotericin B plus Mycograb was superior to amphotericin B plus placebo in patients with culture-confirmed invasive candidiasis. Patients received a lipid-associated formulation of amphotericin B plus a 5-day course of Mycograb or placebo, having been stratified on the basis of Candida species (Candida albicans vs. non-albicans species of Candida). Inclusion criteria included clinical evidence of active infection at trial entry plus growth of Candida species on culture of a specimen from a clinically significant site within 3 days after initiation of study treatment. The primary efficacy variable was overall response to treatment (clinical and mycological resolution) by day 10. RESULTS: Of the 139 patients enrolled from Europe and the United States, 117 were included in the modified intention-to-treat population. A complete overall response by day 10 was obtained for 29 (48%) of 61 patients in the amphotericin B group, compared with 47 (84%) of 56 patients in the Mycograb combination therapy group (odds ratio [OR], 5.8; 95% confidence interval [CI], 2.41-13.79; P<.001). The following efficacy criteria were also met: clinical response (52% vs. 86%; OR, 5.4; 95% CI, 2.21-13.39; P<.001), mycological response (54% vs. 89%; OR, 7.1; 95% CI, 2.64-18.94; P<.001), Candida-attributable mortality (18% vs. 4%; OR, 0.2; 95% CI, 0.04-0.80; P = .025), and rate of culture-confirmed clearance of the infection (hazard ratio, 2.3; 95% CI, 1.4-3.8; P = .001). Mycograb was well tolerated. CONCLUSIONS: Mycograb plus lipid-associated amphotericin B produced significant clinical and culture-confirmed improvement in outcome for patients with invasive candidiasis.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve

Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.

Experimental pneumococcal meningitis causes central nervous system pathology without inducing the 72-kd heat shock protein

We examined whether experimental pneumococcal meningitis induced the 72-kd heat shock protein (HSP72), a sensitive marker of neuronal stress in other models of central nervous system (CNS) injury. Brain injury was characterized by vasculitis, cerebritis, and abscess formation in the cortex of infected animals. The extent of these changes correlated with the size of the inoculum (P less than 0.003) and with pathophysiologic parameters of disease severity, i.e., cerebrospinal fluid (CSF) lactate (r = 0.61, P less than 0.0001) and CSF glucose concentrations (r = -0.55, P less than 0.0001). Despite the presence of numerous cortical regions having morphologic evidence of injury, HSP72 was not detected in most animals. When present, only rare neurons were HSP72 positive. Western blot analysis of brain samples confirmed the paucity of HSP72 induction. The lack of neuronal HSP72 expression in this model suggests that at least some of the events leading to neuronal injury in meningitis are unique, when compared with CNS diseases associated with HSP72 induction.

Immunohistochemical evaluation of heat shock protein expression in normal canine nerve and peripheral nerve sheath tumours

Abnormal expression of heat shock proteins (HSPs) has been observed in many human neoplasms and such expression has prognostic, predictive and therapeutic implications. The aim of this study was to evaluate immunohistochemically the expression of HSP 27, HSP 32 and HSP 90 in normal canine peripheral nerves and in four benign and 15 malignant canine peripheral nerve sheath tumours (PNSTs). In normal nerve, all of the HSPs were detected in axons, epineurial fibroblasts and scattered Schwann cell bodies. Cytoplasmic expression of HSP 27 was more widespread and intense in benign PNSTs compared with malignant PNSTs (P <0.05). Widespread and intense nuclear expression of HSP 32 was also associated with benign tumours (P <0.01), while high HSP 90 immunoreactivity was detected in all tumours, suggesting that HSP 90 might represent a new therapeutic target.

Increased expression of heat shock protein 90 in keratinocytes and mast cells in patients with psoriasis.

BACKGROUND Psoriasis is a chronic inflammatory skin disease and various stress factors mediate inflammation. Heat shock protein (HSP) 90 plays an important role in cell survival; cytokine signaling, such as interleukin-17 receptor signaling; and immune responses. OBJECTIVE We sought to elucidate protein expression and distribution of HSP90 in psoriasis. METHODS HSP90 expression and its cellular source were analyzed on normal-appearing, nonlesional, lesional, and ustekinumab-treated psoriatic skin using immunohistochemistry and double immunofluorescence. RESULTS HSP90α, the inducible isoform of HSP90, was significantly up-regulated in epidermal keratinocytes and mast cells of lesional skin and down-regulated after ustekinumab therapy. LIMITATIONS There was a limited sample size. CONCLUSIONS HSP90 from keratinocytes and mast cells is a key regulator of psoriatic inflammation and HSP90 inhibitors may represent a novel therapeutic approach to the disease.

Expression analysis of heat shock protein 90 (HSP90) and Her2 in colon carcinoma.

PURPOSE The molecular chaperone heat shock protein 90 (HSP90) plays an important role in several types of tumors also participating in the modulation of the activity of receptor tyrosine kinases activity such as members of the Her family. We evaluated the significance of HSP90 and Her2 expression in colon cancer. METHODS HSP90 and Her2 expression was determined by immunohistochemistry and by fluorescence in situ hybridization (FISH) on 355 primary resected colon carcinomas. Results were correlated with pathologic features (Union for International Cancer Control (UICC) pTNM category, tumor localisation, tumor differentiation), additional molecular genetic characteristics (BRAF, KRAS mutational status, mismatch repair genes (MMR)), and survival. RESULTS HSP90 immunoreactivity was observed in various degrees. Fifty-one cases (14 %) were positive for Her2 (score 2+ and 3+) with 16/43 cases with Her2 2+ staining pattern showing amplification of Her2 determined by FISH. There was a significant correlation between high HSP90 expression and Her2 overexpression (p = 0.011). High HSP90 expression was associated with earlier tumor stages (p = 0.019), absence of lymph node (p = 0.006), and absence of distant metastases (p = 0.001). Patients with high tumoral HSP90 levels had a better survival (p = 0.032), but this was not independent from other prognostic relevant pathologic parameters. Her2 expression was not associated with any of the investigated histopathological, molecular, or clinical parameters. CONCLUSIONS High HSP90 levels are reflecting lower malignant potential in colon cancer. Her2 positivity can be observed in a small number of cases. Targeting HSP90 and/or Her2 may be an alternative therapeutic approach in colon cancer in a subset of patients.