Whole genome transcriptomic reveals heat stroke molecular signatures in humans.

An evolutionary heat shock response (HSR) protects most living species, including humans, from heat-induced macromolecular damage. However, its role in the pathogenesis of heat stroke is unknown. We examined the whole genome transcriptome in peripheral blood mononuclear cells of a cohort of subjects exposed to the same high environmental heat conditions, who developed heat stroke (n = 19) versus those who did not (n = 19). Patients with heat stroke had a mean rectal temperature at admission of 41.7 ± 0.8°C, and eight were in deep coma (Glasgow Coma Score = 3). The transcriptome showed that genes involved in more than half of the entire chaperome were differentially expressed relative to heat stress control. These include the heat shock protein, cochaperone, and chaperonin genes, indicating a robust HSR. Differentially expressed genes also encoded proteins related to unfolded protein response, DNA repair, energy metabolism, oxidative stress, and immunity. The analysis predicted perturbations of the proteome network and energy production. Cooling therapy attenuated these alterations without complete restoration of homeostasis. We validated the significantly expressed genes by a real-time polymerase chain reaction. The findings reveal the molecular signature of heat stroke. They also suggested that a powerful HSR may not be sufficient to protect against heat injury. The overwhelming proteotoxicity and energy failure could play a pathogenic role. KEY POINTS: Most living species, including humans, have inherent heat stress response (HSR) that shields them against heat-induced macromolecular damage. The role of the HSR in subjects exposed to environmental heat who progressed to heat stroke versus those that did not is unknown. Our findings suggest that heat stroke induces a broad and robust HSR of nearly half of the total heat shock proteins, cochaperones, and chaperonin genes. Heat stroke patients exhibited inhibition of genes involved in energy production, including oxidative phosphorylation and ATP production. Significant enrichment of neurodegenerative pathways, including amyloid processing signalling, the Huntington's and Parkinson's disease signalling suggestive of brain proteotoxicity was noted. The data suggests that more than a powerful HSR may be required to protect against heat stroke. Overwhelming proteotoxicity and energy failure might contribute to its pathogenesis.

Transcriptome Changes and Metabolic Outcomes After Bariatric Surgery in Adults With Obesity and Type 2 Diabetes.

Context: Bariatric surgery has been shown to be effective in inducing complete remission of type 2 diabetes in adults with obesity. However, its efficacy in achieving complete diabetes remission remains variable and difficult to predict before surgery. Objective: We aimed to characterize bariatric surgery-induced transcriptome changes associated with diabetes remission and the predictive role of the baseline transcriptome. Methods: We performed a whole-genome microarray in peripheral mononuclear cells at baseline (before surgery) and 2 and 12 months after bariatric surgery in a prospective cohort of 26 adults with obesity and type 2 diabetes. We applied machine learning to the baseline transcriptome to identify genes that predict metabolic outcomes. We validated the microarray expression profile using a real-time polymerase chain reaction. Results: Sixteen patients entered diabetes remission at 12 months and 10 did not. The gene-expression analysis showed similarities and differences between responders and nonresponders. The difference included the expression of critical genes (SKT4, SIRT1, and TNF superfamily), metabolic and signaling pathways (Hippo, Sirtuin, ARE-mediated messenger RNA degradation, MSP-RON, and Huntington), and predicted biological functions (ß-cell growth and proliferation, insulin and glucose metabolism, energy balance, inflammation, and neurodegeneration). Modeling the baseline transcriptome identified 10 genes that could hypothetically predict the metabolic outcome before bariatric surgery. Conclusion: The changes in the transcriptome after bariatric surgery distinguish patients in whom diabetes enters complete remission from those who do not. The baseline transcriptome can contribute to the prediction of bariatric surgery-induced diabetes remission preoperatively.

Classic and exertional heatstroke.

In the past two decades, record-breaking heatwaves have caused an increasing number of heat-related deaths, including heatstroke, globally. Heatstroke is a heat illness characterized by the rapid rise of core body temperature above 40 °C and central nervous system dysfunction. It is categorized as classic when it results from passive exposure to extreme environmental heat and as exertional when it develops during strenuous exercise. Classic heatstroke occurs in epidemic form and contributes to 9-37% of heat-related fatalities during heatwaves. Exertional heatstroke sporadically affects predominantly young and healthy individuals. Under intensive care, mortality reaches 26.5% and 63.2% in exertional and classic heatstroke, respectively. Pathological studies disclose endothelial cell injury, inflammation, widespread thrombosis and bleeding in most organs. Survivors of heatstroke may experience long-term neurological and cardiovascular complications with a persistent risk of death. No specific therapy other than rapid cooling is available. Physiological and morphological factors contribute to the susceptibility to heatstroke. Future research should identify genetic factors that further describe individual heat illness risk and form the basis of precision-based public health response. Prioritizing research towards fundamental mechanism and diagnostic biomarker discovery is crucial for the design of specific management approaches.

Oxidative stress, caloric intake and outcomes of critically ill patients.

BACKGROUND: The aim of this study was to investigate the patterns of oxidative stress in critically ill patients and the association with caloric intake and outcomes. METHODS: In this pre-planned sub-study of the PermiT (Permissive Underfeeding versus Target Enteral Feeding in Adult Critically Ill Patients Trial- ISRCTN68144998), we included patients expected to stay in the ICU for ≥14 days. Serum samples were collected on days 1, 3, 5, 7 and 14 of enrollment. We measured total anti-oxidant capacity (TAC), protein carbonyl concentration (a measure of protein oxidation) and 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (8-OHdG) (a measure of DNA oxidation). We used principal component analysis (PCA) and hierarchical cluster analysis (HCA) to group patients according to oxidative stress. RESULTS: Principal component analysis identified 2 components that were responsible for 79% of the total variance, and cluster analysis grouped patients in three statistically distinct clusters. Majority of patients 78.6% (44/55) were included in cluster 1 with lowest TAC, protein carbonyl and 8-OHdG levels and cluster 2 which accounted for 16.1% (9/55) of patients had the highest levels of TAC and intermediate levels of protein carbonyl levels. Cluster 3 patients 5.4% (3/56) had the highest protein carbonyl levels. Incident renal replacement therapy was highest in cluster 2 (4/8, 50.0%), compared to cluster 1 (4/42, 9.5%) and cluster 3 (1/3, 33.3%, p 0.01). When adjusted to oxidative stress cluster membership, permissive underfeeding was not associated with 90-day mortality (adjusted odds ratio, aOR 1.37, 95% CI 0.36, 5.25, p 0.64) but was associated significantly with lower incident renal replacement therapy (aOR 0.02, 95% CI < 0.001, 0.57, p 0.02). CONCLUSIONS: There are different distinct patterns of oxidative stress in critically ill patients. Incident renal replacement therapy was different among the three clusters. Our data suggest a protective effect of permissive underfeeding on incident renal replacement therapy that may differ by clusters of oxidative stress.

Physical exercise reduces the expression of RANTES and its CCR5 receptor in the adipose tissue of obese humans.

RANTES and its CCR5 receptor trigger inflammation and its progression to insulin resistance in obese. In the present study, we investigated for the first time the effect of physical exercise on the expression of RANTES and CCR5 in obese humans. Fifty-seven adult nondiabetic subjects (17 lean and 40 obese) were enrolled in a 3-month supervised physical exercise. RANTES and CCR5 expressions were measured in PBMCs and subcutaneous adipose tissue before and after exercise. Circulating plasma levels of RANTES were also investigated. There was a significant increase in RANTES and CCR5 expression in the subcutaneous adipose tissue of obese compared to lean. In PBMCs, however, while the levels of RANTES mRNA and protein were comparable between both groups, CCR5 mRNA was downregulated in obese subjects (P < 0.05). Physical exercise significantly reduced the expression of both RANTES and CCR5 (P < 0.05) in the adipose tissue of obese individuals with a concomitant decrease in the levels of the inflammatory markers TNF- α , IL-6, and P-JNK. Circulating RANTES correlated negatively with anti-inflammatory IL-1 ra (P = 0.001) and positively with proinflammatory IP-10 and TBARS levels (P < 0.05). Therefore, physical exercise may provide an effective approach for combating the deleterious effects associated with obesity through RANTES signaling in the adipose tissue.

Therapeutic targeting of B7-H1 in breast cancer.

INTRODUCTION: Breast cancer is the most common form of malignancy occurring in women worldwide. B7-H1 is a co-inhibitory molecule expressed by several types of tumors, including breast cancer. The aberrant expression of B7-H1 in breast cancer cells has been determined, its role in recruiting regulatory T cells into the tumor microenvironment has been elucidated and a strong link to B7-H1 induction in highly proliferative breast cancer has been provided. It has also been demonstrated that doxorubicin, a drug commonly used for breast cancer treatment, downregulates the cell surface expression of B7-H1 and upregulates its nuclear expression, which therefore suggests an anti-apoptotic role of B7-H1 in breast cancer. AREAS COVERED: This review illustrates the various factors involved in the induction of B7-H1 and its role in immune evasion and chemoresistance. It also provides potential therapeutic strategies for targeting B7-H1 in breast cancer. EXPERT OPINION: B7-H1 should be considered as a potential therapeutic target for breast cancer. Indeed, there is increasing evidence for the potential efficacy of B7-H1 blockade in the prevention of immune evasion by cancer cells. Additionally, B7-H1 targeting can be used in conjunction with other therapeutic modalities for improved efficacy and reduced toxicity. We expect that B7-H1 blockade in combination with other therapeutics will be a prime therapeutic strategy in the future.

Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule.

INTRODUCTION: B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. METHODS: In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. RESULTS: Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. CONCLUSIONS: Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

Identification of a novel peptide derived from the M-phase phosphoprotein 11 (MPP11) leukemic antigen recognized by human CD8+ cytotoxic T lymphocytes.

BACKGROUND AND OBJECTIVES: There is an urgent need for the development of leukemia-targeted immunotherapeutic approaches using defined leukemia-associated antigens that are preferentially expressed by most leukemia subtypes and absent or minimally expressed in vital tissues. M-phase phosphoprotein 11 protein (MPP11) is extensively overexpressed in leukemic cells and therefore is considered an attractive target for leukemia T cell therapy. We sought to identify potential CD8+ cytotoxic T lymphocytes that specifically recognised peptides derived from the MPP11 antigen. METHODS: A computer-based epitope prediction program SYFPEITHI, was used to predict peptides from the MPP11 protein that bind to the most common HLA- A*0201 molecule. Peptide binding capacity to the HLA-A*0201 molecule was measured using the T2 TAP-deficient, HLA-A*0201-positive cell line. Dendritic cells were pulsed with peptides and then used to generate CD8+ cytotoxic T lymphocytes (CTL). The CML leukemic cell line K562-A2.1 naturally expressing the MPP11 antigen and engineered to express the HLA-A*0201 molecule was used as the target cell. RESULTS: We have identified a potential HLA-A*0201 binding epitope (STLCQVEPV) named MPP-4 derived from the MPP11 protein which was used to generate a CTL line. Interestingly, this CTL line specifically recognized peptide-loaded target cells in both ELISPOT and cytotoxic assays. Importantly, this CTL line exerted a cytotoxic effect towards the CML leukemic cell line K562-A2.1. CONCLUSION: This is the first study to describe a novel epitope derived from the MPP11 antigen that has been recognized by human CD8+ CTL.

Enhancement of lytic activity of leukemic cells by CD8+ cytotoxic T lymphocytes generated against a WT1 peptide analogue.

The Wilms tumor antigen 1 (WT1) antigen is over-expressed in human leukemias, making it an attractive target for immunotherapy. Most WT1-specific Cytotoxic T Lymphocytes (CTLs) described so far displayed low avidity, limiting its function. To improve the immunogenicity of CTL epitopes, we replaced the first-amino-acid of two known immunogenic WT1-peptides (126 and 187) with a tyrosine. This modification enhances 126Y analogue-binding ability, triggers significant number of IFN-gamma-producing T cells (P = 0.0003), induces CTL that cross-react with the wild-type peptide, exerts a significant lytic activity against peptide-loaded-targets (P = 0.0006) and HLA-A0201-matched-leukemic cells (P = 0.0014). These data support peptide modification as a feasible approach for the development of a leukemia-vaccine.

The Wilms' tumor antigen is a novel target for human CD4+ regulatory T cells: implications for immunotherapy.

Compelling evidences indicate a key role for regulatory T cells (T(reg)) on the host response to cancer. The Wilms' tumor antigen (WT1) is overexpressed in several human leukemias and thus considered as promising target for development of leukemia vaccine. However, recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T(regs). We have generated T-cell lines and clones that specifically recognized a WT1-84 (RYFKLSHLQMHSRKH) peptide in an HLA-DRB1*0402-restricted manner. Importantly, they recognized HLA-DRB1*04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a T helper 2 cytokine profile, had a CD4(+)CD25(+)Foxp3(+)GITR(+)CD127(-) T(reg) phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell contact. Priming of alloreactive T cells in the presence of T(regs) strongly inhibited the expansion of natural killer (NK), NK T, and CD8(+) T cells and had an inhibitory effect on NK/NK T cytotoxic activity but not on CD8(+) T cells. Furthermore, priming of T cells with the WT1-126 HLA-A0201-restricted peptide in the presence of T(regs) strongly inhibited the induction of anti-WT1-126 CD8(+) CTL responses as evidenced by both very low cytotoxic activity and IFN-gamma production. Moreover, these T(reg) clones specifically produced granzyme B and selectively induced apoptosis in WT1-84-pulsed autologous antigen-presenting cells but not in apoptotic-resistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 interleukin-5(+)/granzyme B(+)/Foxp3(+) CD4(+) T(regs) in five of eight HLA-DR4(+) acute myeloid leukemia patients. Collectively, our in vitro and in vivo findings strongly suggest important implications for the clinical manipulation of T(regs) in cancer patients.

Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: a novel method for identifying potential contact allergens.

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83(+) cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model.

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration.

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a(+) LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a(+) LCs, CD83(+) mature dendritic cells (DCs) and CCR7(+) LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a(+)CD83(+) LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a(+) cells. About 50% of skin emigrating CD1a(+) LCs were CD83(-) (immature) but all were CCR7(+). Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.

The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors.

B7-H1 molecule increases the apoptosis of tumor-reactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells), but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%). Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade III-negative (P = .012), estrogen receptor-negative (P = .036), and progesterone receptor-negative (P = .040) patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042), histologic grade III (P = .015), positivity of Her2/neu status (P = .019), and severe tumor lymphocyte infiltration (P = .001). Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule.

Assessment of contact allergens by dissociation of irritant and sensitizing properties.

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose-response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.

A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens.

Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.