Prediction of serum anti-HSP27 antibody titers changes using a light gradient boosting machine (LightGBM) technique.

Previous studies have proposed that heat shock proteins 27 (HSP27) and its anti-HSP27 antibody titers may play a crucial role in several diseases including cardiovascular disease. However, available studies has been used simple analytical methods. This study aimed to determine the factors that associate serum anti-HSP27 antibody titers using ensemble machine learning methods and to demonstrate the magnitude and direction of the predictors using PFI and SHAP methods. The study employed Python 3 to apply various machine learning models, including LightGBM, CatBoost, XGBoost, AdaBoost, SVR, MLP, and MLR. The best models were selected using model evaluation metrics during the K-Fold cross-validation strategy. The LightGBM model (with RMSE: 0.1900 ± 0.0124; MAE: 0.1471 ± 0.0044; MAPE: 0.8027 ± 0.064 as the mean ± sd) and the SHAP method revealed that several factors, including pro-oxidant-antioxidant balance (PAB), physical activity level (PAL), platelet distribution width, mid-upper arm circumference, systolic blood pressure, age, red cell distribution width, waist-to-hip ratio, neutrophils to lymphocytes ratio, platelet count, serum glucose, serum cholesterol, red blood cells were associated with anti-HSP27, respectively. The study found that PAB and PAL were strongly associated with serum anti-HSP27 antibody titers, indicating a direct and indirect relationship, respectively. These findings can help improve our understanding of the factors that determine anti-HSP27 antibody titers and their potential role in disease development.

HSP-27 and HSP-70 negatively regulate protective defence responses from macrophages during mycobacterial infection.

Mycobacterium tuberculosis attenuates many defence responses from alveolar macrophages to create a niche at sites of infection in the human lung. Levels of Heat Shock Proteins have been reported to increase many folds in the serum of active TB patients than in latently infected individuals. Here we investigated the regulation of key defence responses by HSPs during mycobacterial infection. We show that infection of macrophages with M. bovis BCG induces higher expression of HSP-27 and HSP-70. Inhibiting HSP-27 and HSP-70 prior to mycobacterial infection leads to a significant decrease in mycobacterial growth inside macrophages. Further, inhibiting HSPs resulted in a significant increase in intracellular oxidative burst levels. This was accompanied by an increase in the levels of T cell activation molecules CD40 and IL-12 receptor and a concomitant decrease in the levels of T cell inhibitory molecules PD-L1 and IL-10 receptor. Furthermore, inhibiting HSPs significantly increased the expression of key proteins in the autophagy pathway along with increased activation of pro-inflammatory promoting transcription factors NF-κB and p-CREB. Interestingly, we also show that both HSP-27 and HSP-70 are associated with anti-apoptotic proteins Bcl-2 and Beclin-1. These results point towards a suppressive role for host HSP-27 and HSP-70 during mycobacterial infection.

Are IgG antibodies to heat shock proteins HSP27 and HSP60 useful markers in endometrial cancer and cervical cancer?

OBJECTIVES: Heat shock proteins are overexpressed in many human malignancies. The role of heat shock proteins as a therapeutic target in cancer as well as their association with drug resistance were widely documented. The aim of this study was to evaluate the concentration of IgG class HSP27 and HSP60 antibodies in serum of patients with endometrial and cervical cancer, as well as to analyse the variability of concentrations of the examined antibodies depending on the cancer stage. MATERIAL AND METHODS: The study included 59 women with adenocarcinoma of the endometrium and 36 women with cervical cancer, the control group consisted of 54 healthy women. The concentrations of IgG class antibodies against the tested heat shock proteins were determined by an immunoenzymatic assay (ELISA) using commercial assays. RESULTS: In both endometrial and cervical cancer, the serum concentration of IgG anti-HSP27 antibody was significantly higher than in the healthy control group. The concentration of IgG anti-HSP60 antibody in endometrial cancer, cervical cancer and healthy control was similar. The median IgG anti-HSP27 antibody serum concentration of endometrial cancer patients was not correlated with FIGO-stage. In cervical cancer inverse correlation between concentration of this antibody and FIGO stage was observed. The median IgG anti-HSP60 antibody concentration in serum of endometrial cancer patients was lower in FIGO stage I and II compared to FIGO stage IV and in FIGO stage IA compared to FIGO stage IB. Concentrations of examined antibodies correlated positively with each other, both in the group of women with cancer and in the group of healthy women. The strongest correlations were found in the group of patients with endometrial cancer. CONCLUSIONS: Concentration of anti-HSP27 antibody could help in detection of cervical and endometrial cancer. We need to look for the cut-off point in large cohort studies. Anti-HSP27 and anti-HSP60 antibodies should be further evaluated for their potential usage as biomarkers in cervical and endometrial cancer as they shown some correlation with stage of disease.

Heat shock protein 27 immune complex altered signaling and transport (ICAST): Novel mechanisms of attenuating inflammation.

Blood levels of heat shock protein (HSP27) and natural IgG auto-antibodies to HSP27 (AAbs) are higher in healthy controls compared to cardiovascular disease patients. Vaccination of mice with recombinant HSP25 (rHSP25, murine ortholog of human rHSP27) increased AAb levels, attenuated atherogenesis and reduced plaque inflammation and cholesterol content. We sought to determine if the HSP27 immune complex (IC) altered MΦ inflammation signaling (Toll Like Receptor 4; TLR4), and scavenger receptors involved in cholesterol uptake (SR-AI, CD-36). Combining a validated polyclonal IgG anti-HSP27 antibody (PAb) with rHSP27 enhanced binding to THP-1 MΦ cell membranes and activation of NF-κB signaling via TLR4, competing away LPS and effecting an anti-inflammatory cytokine profile. Similarly, adding the PAb with rHSP27 enhanced binding to SR-AI and CD-36, as well as lowered oxLDL binding in HEK293 cells separately transfected with SR-AI and CD-36, or THP-1 MΦ. Finally, the PAb enhanced the uptake and internalization of rHSP27 in THP-1 MΦ. Thus, the HSP27 IC potentiates HSP27 cell membrane signaling with receptors involved in modulating inflammation and cholesterol uptake, as well as HSP27 internalization. Going forward, we are focusing on the development of HSP27 Immune Complex Altered Signaling and Transport (ICAST) as a means of modulating inflammation.

Effect of high-dose vitamin D supplementation on antibody titers to heat shock protein 27 in adolescent girls.

Background Although vitamin D deficiency is associated with several inflammatory conditions, there have been few studies on the effects of vitamin D supplementation on markers of oxidative stress (OS) and inflammation. The aim of the current study was to evaluate the effects of high-dose vitamin D supplementation on heat shock protein 27 antibody (anti-Hsp27) titers in adolescent girls. Methods Five hundred and fifty adolescent girls received vitamin D3 at a dose of 50,000 IU/week for 9 weeks. Demographic, clinical and biochemical markers including serum fasting blood glucose (FBG), lipid profile and anti-Hsp27 titers as well as hematological parameters including white blood cell (WBC) count and red blood cell (RBC) distribution width (RDW) were determined in all the subjects at baseline and at the end of the study. Results Serum vitamin D significantly increased from 6.4 (4.2-9.6) ng/mL to 35.6 (25.8-47.5) ng/mL (p < 0.001) following the intervention. Furthermore, serum anti-Hsp27 titers were significantly lower after the 9-week vitamin D administration period (0.22 [0.12-0.33] optical density [OD] vs. 0.19 [0.11-0.31] OD; p = 0.002). A significant correlation was found between serum anti-Hsp27 and RDW (r = 0.13, p = 0.037). The reduction in RDW values after intervention was particularly evident in subjects with the greatest increase in serum vitamin D levels. Conclusions High-dose vitamin D supplementation was found to reduce antibody titers to Hsp27. Further randomized placebo-controlled trials are warranted to determine the long-term effect of vitamin D administration on the inflammatory process especially that associated with chronic disease.

Prognostic Factors Associating with Pro-oxidant-antioxidant Balance; Neutrophils to Lymphocytes Ratio, Vitamin D, Heat Shock Protein 27, and Red Cell Distribution Width.

BACKGROUND: Several chronic diseases are mediated by oxidative stress. Oxidative stress affects cell morphology and function and is associated with alterations in the serum protein component. In the current study, we analyzed four individual prognostic factors associating with serum Pro-Oxidant-Antioxidant Balance (PAB): neutrophil to lymphocyte ratio (NLR), Vitamin D, anti-heat shock protein 27 (anti-hsp27) antibody titer, and red blood cell distribution width (RDW) to evaluate them as the potential prognostic markers. In the current study, we attempted to investigate the relationship between serum PAB, RDW, NLR, serum vitamin D and anti-hsp27 concentration. METHODS: A total of 852 participants (438 males and 414 females) aged 47.64 ± 7.77 years were recruited in a cross-sectional study based on the Mashhad stroke and heart atherosclerotic disorders (MASHAD) cohort study data. Hematological parameters, and vitamin D, PAB and anti-hsp27 antibody titers were measured using the Sysmex auto analyzer system and enzyme-linked immune sorbent assay (ELISA), respectively. RESULTS: The results showed a significant correlation between Vitamin D and anti-hsp27 antibody titers (r = -0.13 and p <0.001) as well as between RDW and serum PAB (r = 0.120 and p <0.001). Moreover, we found that serum PAB was positively associated with serum anti hsp27 antibody titers. The results showed increasing 1 unit of serum vitamin D can cause 3% decreases in anti hsp 27 values (OR = 0.97; CI 95% (0.96-0.99); p = 0.004). While this association was not significant for RDW, NLR and PAB (p >0.05) we found a significant association between serum PAB and serum anti hsp-27 antibody titers. Subjects with PAB levels 36.31-82.63 had a higher risk (1.83 fold) of having an increased anti-hsp27 antibody titers in comparison to the reference group (PAB level <36.31) (OR = 1.83 (95% CI = 1.33-2.52), p <0.001). CONCLUSION: The present study shows that serum vitamin D can be associated with reduction in inflammatory status probably by decreasing levels of serum anti-hsp27 antibody titers, reduction in oxidative stress and therefore may reduce the risk of cardiovascular diseases. Anti-hsp27 antibody titers are associated with oxidative stress through the serum PAB, therefore these factors may be of prognostic values in detecting oxidative stress and risk of atherosclerosis. The evaluation of these factors in a larger population may help further confirm these findings.

Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses.

To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.

Role of mechanical and thermal damage in pericapsular inflammatory response to injectable silicone in a rabbit model.

Silicone is used widely for tissue augmentation in humans. However, late complications, such as delayed inflammation and capsular contracture, remain uncharacterized, despite their importance. In the present study, we aimed to determine whether mechanical and thermal damage induce capsular inflammation around a foreign body, and elucidate the biological mechanism underlying this phenomenon. We injected silicone into the subcutaneous layer of the skin of New Zealand white rabbits. The rabbits were divided into two groups: the control group received no treatment; in the experimental group, external force was applied near the injection silicone using high-intensity focused ultrasound (HIFU). Tissues near the injected silicone were harvested from both groups on Days 4, 7, and 30 after HIFU treatment for comparative analysis. Visual and histological examinations showed clearly increased inflammation in the experimental group compared with that in the control group. Furthermore, capsular tissue from the experimental group displayed markedly increased collagen production. Immunofluorescence revealed marked activation of macrophages in the early stages of inflammation (Days 4 and 7 after HIFU treatment), which decreased on Day 30. Assessment of cytokine activation showed significantly increased expression of heat shock protein (HSP)27, HSP60, HSP70, toll-like receptor (TLR)2, TLR4, and interleukin-8 in the experimental group. The expression of transforming growth factor-ß1 did not increase significantly in the experimental group. In conclusion, damage to tissues around the injected silicone induced capsular inflammation. Macrophages and damage-associated molecular pattern molecules were involved in the early stages of inflammation. HSP release activated TLRs, which subsequently activated innate immunity and induced the inflammatory response.

Biophysical analyses and functional implications of the interaction between Heat Shock Protein 27 and antibodies to HSP27.

Heat Shock Protein 27 (HSP27) is a small molecular chaperone that reduces the development of atherosclerosis by lowering plasma cholesterol levels as well as inflammation. Human studies show an inverse correlation between atherosclerotic burden and HSP27 expression, and are supported by murine models in which augmenting HSP27 levels curbs experimental atherogenesis. Natural HSP27 auto-antibodies (AAb) are found in human plasma, however their role in modulating the athero-protective effects of HSP27 is unknown. The purpose of this study is to characterize the biophysical interaction between human recombinant HSP27 and AAb. A validated polyclonal anti-HSP27 IgG antibody (PAb) was used to mimic natural AAb. Homology modeling and secondary structure prediction tools facilitated the design of HSP27 truncation and phosphorylation mutants. Secondary structural changes were identified using Circular Dichroism (CD) and Dynamic Light Scattering (DLS). Similar to prior structural investigations of HSP27, there was a predominance of α-helical content in the N-terminal truncation and dephosphorylation ("AA") mutants. The α-crystallin domain (ACD) predominantly consists of ß-strands, with the addition of the N-terminal increasing helical content and the C-terminal maintaining ß structure. With increasing ratios of PAb to HSP27 ß structure abundance and particle size increased, with a similar trend observed with the N-terminus, C-terminus and ACD peptides but an opposite trend with the phosphorylation peptides. Taken together, these studies provide insights into the interaction of HSP27 and its AAb that ultimately may aid in optimizing the design of HSP27 peptidomimetics with anti-atherogenic potential.

Significance of serum antibodies against HPV E7, Hsp27, Hsp20 and Hp91 in Iranian HPV-exposed women.

BACKGROUND: Among different types of human papillomavirus (HPV), types 16 and 18 were known to be high-risk agents causing mainly cervical cancer. Up to now, the potential of HPV E7 protein has been proved as a diagnostic marker of cervical cancer. Moreover, the levels of anti-heat shock protein (Hsp) and anti-high mobility group box-1 (HMGB1) antibodies in cancer patients have been useful in tumor diagnosis. The goal of the present study was to determine the efficiency of the potential serologic markers including HPV E7, Hsp20, Hsp27 proteins and Hp91 peptide in Iranian HPV-exposed women, for the first time. METHODS: At first, the recombinant HPV E7, Hsp20 and Hsp27 proteins were expressed in E. coli system, and purified by affinity chromatography under native conditions. Then, antibody responses were detected against the recombinant proteins as well as Hp91 peptide as potential markers in 49 Iranian women who were seropositive for HPV-16 and 18 L1 capsids (i.e., HPV-exposed women) and 49 controls using indirect ELISA. RESULTS: Our data indicated that the seroreactivities of women exposed to HPV16, HPV18 and both of them against the recombinant E7, Hsp20, Hsp27 proteins and Hp91 peptide were significantly higher than those in control group (p < 0.05 for HPV16 or HPV18; p < 0.01 for both of them versus all markers). HPV-exposed women with high antibody responses to HPV-16 and 18 L1 capsids as a commercial biomarker had significant seroreactivity to HPV-16 and 18 E7 and Hsp27 (p < 0.05). The recombinant E7 and Hsp27 proteins showed higher efficiency than Hsp20 and Hp91 for detection of individuals exposed to HPV infections (p < 0.05). CONCLUSION: Generally, the levels of serum E7 and Hsp27 were increased in HPV-16 and 18 L1- seropositive women suggesting their potential value as a diagnostic marker for HPV infections.

HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

HSP27 immunization reinforces AII amacrine cell and synapse damage induced by S100 in an autoimmune glaucoma model.

Previous studies have revealed a loss of retinal ganglion cells (RGCs) and optic nerve fibers after immunization with the S100B protein. Addition of heat shock protein 27 (HSP27) also leads to a decrease of RGCs. Our present aim has been to analyze various retinal cell types after immunization with S100B or S100B + HSP27 (S100 + HSP). After 28 days, retinas were processed for immunohistology and Western blot. RGCs, immunostained for NeuN, were significantly decreased in the S100 and the S100 + HSP groups. Significantly fewer ChAT+ cells were noted in both groups, whereas parvalbumin+ cells were only affected in the S100 + HSP group. Western blot results also revealed fewer ChAT signals in both immunized groups. No changes were noted with regard to PKCα+ rod bipolar cells, whereas a significant loss of recoverin+ cone bipolar cells was observed in both groups via immunohistology and Western blot. The presynaptic marker Bassoon and the postsynaptic marker PSD95 were significantly reduced in the S100 + HSP group. Opsin+ and rhodopsin+ photoreceptors revealed no changes in either group. Thus, the inner retinal layers are affected by immunization. However, the combination of S100 and HSP27 has a stronger additive effect on the retinal synapses and AII amacrine cells.

The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch.

Heat shock protein 27 (HSP27), functioning as a stress induced protective protein, has been reported to participate in various biological processes, including apoptosis, thermal protection, and virus infection. In this study, a HSP27-like gene from the seawater fish sea perch, designated as LjHSP27, was characterized. The 1361 bp full-length cDNA of LjHSP27 encoded a 221 amino acid protein containing a conserved α-crystallin domain, two variable amino- and carboxy-terminal extensions, a WD/EPF motif, two serine phosphorylation sites, and two putative actin binding regions. Phylogenetic analysis showed that LjHSP27 shared the closest genetic relationship with HSP27 of the Asian seabass Lates calcarifer. LjHSP27 mRNA was ubiquitously expressed in all tissues examined, but significantly up-regulated in spleen and kidney and down-regulated in brain post red spotted grouper nervous necrosis virus (RGNNV) infection. In vitro, LjHSP27 transcript was remarkably reduced post RGNNV infection, but rapidly increased after polyinosinic-polycytidylic acid treatment. Up-regulation and down-regulation of LjHSP27 inhibited and promoted RGNNV replication in cultured LJB cells, respectively. Luciferase assay indicated that LjHSP27 could enhance the promoter activities of zebrafish interferon (IFN)1 and IFN3, suggesting its potential role in innate immune responses. Moreover, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, as indicated by the up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes, while KNK437 caused down-regulation of LjHSP27 dramatically led to opposite results, suggesting that LjHSP27 might exert its anti-RGNNV activities by regulating the apoptosis signaling pathway. Our results would provide a new insight into the underlying molecular mechanism of HSP and RGNNV interaction.

Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity.

Novel vaccine modalities have been designed to improve the efficiency of vaccines against HIV infections. In this way, the HIV-1 Nef protein has been known as an attractive antigenic candidate in therapeutic vaccine development. Moreover, the endogenous adjuvants such as heat shock proteins (HSPs) and high mobility group box 1 protein (HMGB1) have been suggested effectively to induce antigen-specific humoral and cellular immune responses. In this study, different Nef DNA and protein constructs were produced in eukaryotic and prokaryotic expression systems, and their immunostimulatory properties were evaluated using small heat shock protein 27 (Hsp27) and the HMGB1-derived peptide (Hp91) in a mouse model. Generally, our results indicated that the Hsp27-Nef fusion DNA or protein could significantly elicit higher humoral and cellular immune responses than Nef DNA or protein, respectively. Analysis of the immune responses demonstrated that the Hsp27-Nef fusion protein, and also the mixture of Nef and Hp91 significantly enhanced the Nef-specific T cell responses. Indeed, these regimens induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies. The immunostimulatory properties of Freund's adjuvant were significantly less than Hsp27 and Hp91 peptide in various immunization strategies. These findings showed that the use of Hsp27 and Hp91 in protein strategy could improve HIV-1 Nef-specific B- and T-cell immune responses, and also represent a promising HIV-1 vaccine candidate in future.

Effect of Crocin on Antibody Titers to Heat-Shock Protein 27 in Subjects with Metabolic Syndrome.

BACKGROUND: Recent studies have shown that antibody titers to heat shock protein 27 (anti-Hsp27) and serum hs-CRP concentrations are elevated in patients with MetS, and may be associated with an increased risk of cardiovascular disease. Crocin is a natural carotenoid with cardio protective effects. OBJECTIVE: Because of the previous evidence for the beneficial effects of saffron in patients with MetS, this study investigated the effect of supplementation with crocin, the active ingredient of saffron, on serum anti-Hsp27 and hs-CRP in patients with MetS. DESIGN: Sixty subjects with metabolic syndrome were randomized to receive crocin (n=30, 15 mg twice a day) or placebo (n=30, twice a day) for a duration of eight weeks. At the end of study, serum anti-Hsp27 and hs-CRP concentrations were measured and compared between the groups. RESULTS: Serum anti-Hsp27 titers fell by 13% (p>0.05) in the crocin group but it rose in the placebo group by 22% (p>0.05). The magnitude of change in serum anti-Hsp27 titers was not significantly different between the study groups (p = 0.28). In the crocin group, serum anti-Hsp27 changes had a borderline negative correlation with glucose (r= -0.35, p=0.06) and a positive correlation with waist circumference (r=0.39, p=0.035). Serum hs-CRP levels were significantly reduced in both groups but these reductions were not significantly different between the study groups (p = 0.31). CONCLUSION: There was no significant effect of crocin on serum anti-Hsp27 titers in subjects with MetS, but this needs further confirmation in larger-scale trials.

Relationship between serum anti-heat shock protein 27 antibody levels and obesity.

BACKGROUND: Heat shock protein 27 (HSP27) is an intracellular molecular chaperone that is expressed at high levels following the exposure of cells to environmental stressors such as heat, toxins, and free radicals. High levels of HSP antigens and antibody titers have been reported in several conditions including cardiovascular disease and cancers. We measured serum anti-HSP27 antibody levels in 993 subjects and assessed the associations between serum anti-HSP27 antibody levels and demographic characteristics including coronary risk factors. METHODS: A total of 993 subjects were recruited as part of the Mashhad Stroke and Heart Atherosclerotic Disorders (MASHAD) cohort study. Demographic, clinical, and biochemical parameters and serum anti-HSP27 antibody titers were determined in all the subjects. RESULTS: Serum anti-HSP27 antibody levels increased with increasing age in men. No significant differences in levels were detected between men and women. Serum anti-HSP27 antibody levels were significantly higher in obese subjects than in nonobese subjects (P=0.046); however, no significant influence of smoking status was observed. Moreover, serum anti-HSP27 antibody titers were positively associated with age, body mass index, waist/hip ratio, the presence of diabetes mellitus, nonsmoking habit, serum triglycerides, cholesterol, and high-sensitivity c-reactive protein. CONCLUSION: We have found that serum anti-HSP27 antibody titers are related to several cardiovascular risk factors, necessitating further studies on the value of this emerging marker for risk stratification.

Course of serum autoantibodies in patients after acute angle-closure glaucoma attack.

BACKGROUND: The aim of our investigation was to analyze the autoantibody -reactivities of patients after acute angle-closure glaucoma (AACG) by means of a protein microarray approach to identify intraocular pressure(IOP)-dependent antibodies. METHODS: Collected sera from different study time points (AACG n = 6, 0, 2, 4 and 12 weeks) and control group (CTRL n = 11, 0 and 12 weeks) were analyzed. Protein-microarrays were incubated with sera, and occurring immunoreactivities were visualized with fluorescence labeled secondary antibodies. To detect changes, spot intensities were digitized and compared with statistical techniques. RESULTS: Three autoantibodies with significant level-alteration in the time course of the survey could be identified. Immunoreactivities to heat shock 27-kDa protein (HSP27), tubulin-tyrosine ligase-like protein 12 (TTLL12), and neuron-specific enolase (NSE) show an increasing linear trend from week 0 up to week 12 with a positive correlation coefficient (P ≤ 0.05, r ≥ 0.4). In the CTRL- group, no significant alterations could be detected in corresponding autoantibody-level. Analysis of variance revealed significant changes of antibody-level between certain time points (anti-HSP27 antibody [week 0 vs. 2], anti-TTLL12 antibody [week 0 vs. 12], and anti-NSE antibody [week 4 vs. 12] [P ≤ 0.05, respectively]) in AACG group. CONCLUSIONS: With this autoantibodies profiling approach, we were able to detect autoimmune reactivities in sera of patients without former indication for glaucomatous damage after rise of IOP due to AACG attack. After further validation in subsequent studies, this autoantibodies could give further insights into the pathogenesis of glaucoma and could possibly help to understand the effect of IOP on glaucomatous optic neuropathy.

Oxidized Low-Density Lipoprotein (OxLDL)-Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let-7c Is Integral to the Effect.

BACKGROUND: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation. METHODS AND RESULTS: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1ß and IL-6, and increased transforming growth factor-ß and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease.

BACKGROUND & AIMS: The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy. METHODS: We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy. RESULTS: IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors. CONCLUSIONS: A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

In Vivo Suppression of Heat Shock Protein (HSP)27 and HSP70 Accelerates DMBA-Induced Skin Carcinogenesis by Inducing Antigenic Unresponsiveness to the Initiating Carcinogenic Chemical.

Heat shock proteins (HSPs) are constitutively expressed in murine skin. HSP27 is present in the epidermis, and HSP70 can be found in both the epidermis and dermis. The purpose of this study was to investigate the role of these proteins in cutaneous chemical carcinogenesis and to determine whether their effects on cell-mediated immune function were a contributing factor. In vivo inhibition of HSP27 and HSP70 produced a reduction in the T cell-mediated immune response to 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene in C3H/HeN mice and resulted in a state of Ag-specific tolerance. When mice were pretreated with anti-HSP27 and anti-HSP70 Abs in vivo prior to subjecting them to a standard two-stage DMBA/12-O-tetradecanoylphorbol-13-acetate cutaneous carcinogenesis protocol, the percentage of mice with tumors was much greater (p < 0.05) in anti-HSP27- and HSP70-pretreated animals compared with mice pretreated with control Ab. Similar results were obtained when the data were evaluated as the cumulative number of tumors per group. Mice pretreated with HSP27 and HSP70 Abs developed more H-ras mutations and fewer DMBA-specific cytotoxic T lymphocytes. These findings indicate that in mice HSP27 and HSP70 play a key role in the induction of cell-mediated immunity to carcinogenic polyaromatic hydrocarbons. Bolstering the immune response to carcinogenic polyaromatic hydrocarbons may be an effective method for prevention of the tumors that they produce.