Association between group 2 innate lymphoid cells enrichment, nasal polyps and allergy in chronic rhinosinusitis.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) were shown to be involved in the initiation and coordination of Th2-type immune responses in allergic disease animal models. Recently, ILC2s enrichment was noted in chronic rhinosinusitis (CRS) patients; however, the role of ILC2s in coordinating the Th2 response in CRS remains to be elucidated. Here, we characterize the ILC2 compartment in CRS by investigating the correlations between ILC2s, Th2 cells and Th2 cytokines expression in CRS patients. METHODS: We used flow cytometric analysis of sinonasal mucosal tissues of 29 CRS patients and 5 controls to quantify ILC2s and Th2 cells. Messenger RNA expression levels of IL-5, IL-13, IL-25, IL-33, TSLP and GATA3 were determined using qRT-PCR. RESULTS: ILC2s were significantly enriched in nasal polyps (CRSwNP) patients. Multivariate linear regression showed a significant positive association of ILC2 numbers with CRSwNP and allergic CRS and a negative association with the number of previous endoscopic sinus surgeries. Group 2 innate lymphoid cell numbers significantly correlated with Th2 cell frequencies. Messenger RNA expression levels of IL-5 and IL-13 were increased in CRSwNP compared with controls, while mRNA levels of IL-25 and GATA3 were significantly reduced. CONCLUSIONS: Our results characterize the complex interactions between ILC2s and other Th2 response elements in the context of CRS and suggest that ILC2 enrichment occurs in CRSwNP and in allergic CRS patients.

Inflammasome gene expression alterations in Staphylococcus aureus biofilm-associated chronic rhinosinusitis.

BACKGROUND: The role of inflammasomes in chronic inflammation has been the subject of intense research in recent years. Chronic rhinosinusitis (CRS), a persistent inflammatory disease, continues to be investigated hoping that a clearer pathophysiologic description will guide discovery of future treatment modalities. This study investigates the role of inflammasome complexes in CRS patients with Staphylococcus aureus biofilm infection, a key culprit associated with disease severity and recalcitrance. METHODOLOGY: Sinonasal tissue samples were collected from CRS patients with (P+) and without (P-) polyps and controls. S. aureus biofilm status was obtained using fluorescence in situ hybridization and classified as biofilm positive (B+) or negative (B-). RNA was analysed using a Human Inflammasome PCR array, profiling the expression of 84 genes involved in inflammasome function. RESULTS: Sixteen samples were obtained: 5 B+P+, 5 B-P- and 6 controls. Comparing B+P+ vs. controls showed the greatest number of differentially expressed genes. In particular, Absent in Melanoma 2 (AIM2) was consistently and significantly up-regulated in the B+P+ vs. B-P- and controls. In contrast, when comparing the B-P- vs. controls, no genes showed significant changes. CONCLUSION: Our results indicate the involvement of inflammasome complexes and their signalling pathways in CRS patients with polyps and S. aureus biofilms. In particular, AIM2, activated by intracellular double-stranded DNA, is up-regulated in this group, implying that S. aureus may play a role in intracellular triggering of the inflammasome response. Studies with further patient stratification and assessing corresponding protein expression are needed to further characterize the role of inflammasomes in CRS.

Ontogeny of flower parts on naturally growing Iris pumila clones: implications for population differentiation and phenotypic plasticity studies.

Previous studies revealed significant phenotypic plasticity, genetic variability and population differentiation of flower morphometric traits on dwarf bearded iris Iris pumila. Also, study of I. pumila flowering phenology revealed significant impact of habitat type as well as population differentiation for flowering time. Since the flowering time can influence other flower traits, we performed this analysis of flower morphometric traits in three time points during the flower bud ontogenic development in two habitat types (open vs. shaded). Analysis revealed that for most of the traits greater trait values were recorded for open habitat but only on latter time points. For most of the analyzed traits direction of differences in bud stage was the opposite to the direction of differences in mature flower stage detected in previous studies. However, length of the stem, a trait that showed the greatest variability between habitats and populations and therefore greatest genetic differentiation and phenotypic plasticity, was significantly greater in the samples from the late flowering shaded habitat in all time samples, indicating that in case of this trait different mechanisms were involved. Those findings have implications for design of the future studies on I. pumila.

Paving the way towards an effective treatment for multiple sclerosis: advances in cell therapy.

Multiple sclerosis (MS) is a leading cause of chronic neurological disability in young to middle-aged adults, affecting ~2.5 million people worldwide. Currently, most therapeutics for MS are systemic immunosuppressive or immunomodulatory drugs, but these drugs are unable to halt or reverse the disease and have the potential to cause serious adverse events. Hence, there is an urgent need for the development of next-generation treatments that, alone or in combination, stop the undesired autoimmune response and contribute to the restoration of homeostasis. This review analyzes current MS treatments as well as different cell-based therapies that have been proposed to restore homeostasis in MS patients (tolerogenic dendritic cells, regulatory T cells, mesenchymal stem cells, and vaccination with T cells). Data collected from preclinical studies performed in the experimental autoimmune encephalomyelitis (EAE) model of MS in animals, in vitro cultures of cells from MS patients and the initial results of phase I/II clinical trials are analyzed to better understand which parameters are relevant for obtaining an efficient cell-based therapy for MS.

Caffeine induces metformin anticancer effect on fibrosarcoma in hamsters.

OBJECTIVE: We investigated the effect of metformin and caffeine on fibrosarcoma in hamsters. MATERIALS AND METHODS: 32 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups, with a minimum of 6 animals per group. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group started peroral treatment with metformin 500 mg/kg daily, the second with caffeine 100 mg/kg daily and the third with a combination of metformin 500 mg/kg and caffeine 100 mg/kg daily, via a gastric probe 3 days before tumor inoculation. After 2 weeks, when the tumors were approximately 2 cm in the control group, all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were pathohistologically (HE) and immunohistochemically (Ki-67, CD 31, COX IV, GLUT-1, iNOS) assessed and the main organs toxicologically analyzed, including the control animals that had received metformin and caffeine. Tumor volume was determined using the formula LxS2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified. Images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances were determined by the Student's t-test. RESULTS: The combination of metformin and caffeine inhibited fibrosarcoma growth in hamsters without toxicity. CONCLUSIONS: Administration of metformin with caffeine might be an effective and safe approach in novel nontoxic adjuvant anticancer treatment.

Effect of metformin on fibrosarcoma in hamsters.

OBJECTIVE: We investigated the effect of metformin on an in vivo solid tumor model of fibrosarcoma in hamsters. MATERIALS AND METHODS: 33 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group (7 animals) started peroral treatment with metformin 500 mg/kg daily via a gastric probe 7 days before tumor inoculation, the second (8 animals) 3 days before inoculation and the third (6 animals) immediately after inoculation. After 2 weeks, when the tumors were approximately 2-3 cm in the control group with tumors (6 hamsters), all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were histologically assessed and the main organs toxicologically analyzed, including 6 control animals that had received metformin without tumor inoculation. Tumor volume was determined using the formula Lx S2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified; images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances of differences in tumor weight, volume, number of Ki-67-positive cells and other parameters were determined by the Student´s t-test. RESULTS: Metformin inhibited fibrosarcoma growth in hamsters without toxicity. The seven-day pretreatment was important for the statistically significant effect. CONCLUSIONS: Administration of metformin as an anti-tumor drug might be an effective and safe therapeutic approach in novel non-toxic therapies for human sarcomas.

Are there good and bad responders to prehospital thrombolysis in the acute phase of myocardial infarction? OPTIMAL study rationale.

The effectiveness of thrombolytics has been clearly demonstrated in more than half the cases in the large cohorts of patients selected for trials during the acute phase of myocardial infarction. At individual level, thrombolysis will clinically either succeed or fail so, for the medical team managing the patient, choice of treatment may be likened to a gamble which in the best of cases (most often) leads to an uncomplicated success and, in the worst of cases, failure worsened by a severe complication. OPTIMAL is a multidisciplinary and multicentre, prospective cohort study associating mobile medical teams and interventional cardiology units to test the hypothesis that the outcome of prehospital thrombolysis does not depend on chance alone but also varies according to demographic, etiological, clinical and logistic factors involved in the occurrence and management of myocardial infarction. The primary objective of this French study, conducted over one year on more than 800 subjects, is to identify the predictors of the results of prehospital thrombolysis from a very early angiographic evaluation. The results for this cohort may be useful for setting up appropriate management strategies for acute myocardial infarction, from the prehospital phase (thrombolysis or not) up to in-hospital orientation of the patients (angiography room or Intensive Care Unit) and to determine the most judicious time for coronary angiography. OPTIMAL is to date the largest prospective serie of prehospital thrombolysis evaluated by an early angiographic control.

Gut-associated lymphoid tissue, gut microbes and susceptibility to experimental autoimmune encephalomyelitis.

Gut microbiota and gut-associated lymphoid tissue have been increasingly appreciated as important players in pathogenesis of various autoimmune diseases, including multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that can be induced with an injection of spinal cord homogenate emulsified in complete Freund's adjuvant in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats. In this study, mesenteric lymph nodes (MLN), Peyer's patches (PP) and gut microbiota were analysed in these two rat strains. There was higher proportion of CD4(+) T cells and regulatory T cells in non-immunised DA rats in comparison to AO rats. Also, DA rat MLN and PP cells were higher producers of pro-inflammatory cytokines interferon-γ and interleukin-17. Finally, microbial analyses showed that uncultivated species of Turicibacter and Atopostipes genus were exclusively present in AO rats, in faeces and intestinal tissue, respectively. Thus, it is clear that in comparison of an EAE-susceptible with an EAE-resistant strain of rats, various discrepancies at the level of gut associated lymphoid tissue, as well as at the level of gut microbiota can be observed. Future studies should determine if the differences have functional significance for EAE pathogenesis.

Activity, but not mRNA expression of gelatinases correlates with susceptibility to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis (MS), inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Clinically manifested EAE can be induced in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats by immunization with spinal cord homogenate (SCH) and complete Freund's adjuvant (CFA). Matrix metalloproteinases (MMP) play important roles in various steps of MS and EAE pathogenesis. Expression of gelatinases MMP2 and MMP9, their activator MMP14 and their inhibitor tissue inhibitor of MMP (TIMP)1 in the CNS of AO and DA rats immunized with SCH+CFA was determined. Expression of mRNA for MMP2, MMP9 and MMP14 was higher and expression of TIMP1 mRNA was lower in AO rats. However, gelatinase activity in spinal cords was higher in samples obtained from DA rats. Further, while there was no strain difference in MMP2 and MMP9 mRNA expression in lymph nodes of the immunized rats, gelatinase activity was higher in DA rats. This activity was reduced by antiinflammatory cytokines interleukin (IL)-10 and IL-4. Interestingly, gelatinase activity was detected in the nuclei of cells within the CNS, but not of those in lymph nodes. Our results imply that posttranscriptional regulation of MMP2 and MMP9 expression and/or function determines low gelatinase activity within the CNS and in immune cells of EAE-resistant AO rats.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes.

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-gamma-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-gamma-pretreated astrocytes was abolished by antagonists of nuclear factor-kappaB (NF-kappaB) activation--a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1beta and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1beta and TNF-alpha for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes.

Highly reactive gaseous free radical nitric oxide (NO), generated by astrocytes and infiltrating macrophages is implicated in inflammatory destruction of brain tissue, including that occurring in multiple sclerosis. Therefore, the influence of immunosuppressive drug leflunomide on inducible nitric oxide synthase (iNOS)-dependent NO production in rat astrocytes and macrophages was investigated. Under the same cultivating conditions, leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-gamma+LPS-triggered expression of iNOS mRNA in astrocytes. In the presence of transcription inhibitor actinomycin D, A77 1726 failed to inhibit astrocyte NO production, suggesting transcriptional regulation of iNOS by leflunomide. This assumption was further supported by the ability of A77 1726 to inhibit IFN-gamma+LPS-induced expression of mRNA for an important iNOS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), but not genistein, an unselective protein tyrosine kinase inhibitor, completely mimicked cell type-specific inhibition of NO synthesis by A77 1726. Therefore, previously described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for A77 1726-mediated suppression of iNOS activation in astrocytes. Accordingly to results obtained with primary astrocytes, both A77 1726 and PD98059 significantly reduced IFN-gamma+LPS-induced NO synthesis in the cultures of rat astrocytoma cell line C6. The ability to suppress iNOS induction in astrocytes supports potential use of leflunomide in the treatment of multiple sclerosis and other NO-dependent inflammatory brain disorders.

Uric acid levels in sera from patients with multiple sclerosis.

The levels of uric acid (UA), a natural peroxynitrite scavenger, were measured in sera from 240 patients with multiple sclerosis (MS) and 104 sex- and age-matched control patients with other neurological diseases (OND). The mean serum UA concentration was lower in the MS than in the OND group, but the difference did not reach the level of statistical significance (P = 0.068). However, the mean serum UA level from patients with active MS (202.6 + 67.1 mumol/l) was significantly lower than that in inactive MS patients (226.5 + 78.6 mumol/l; P = 0.046) and OND controls (P = 0.007). We found a significant inverse correlation of serum UA concentration with female gender (P = 0.0001), disease activity (P = 0.012) and duration (P = 0.017), and a trend towards an inverse correlation with disability as assessed by EDSS score, which did not reach statistical significance (P = 0.067). Finally, multivariate linear regression analyses showed that UA concentration was independently correlated with gender (P = 0.0001), disease activity (P = 0.014) and duration of the disease (P = 0.043) in MS patients. These findings suggest that serum UA might serve as a possible marker of disease activity in MS. They also provide support to the potential beneficial therapeutic effect of radical-scavenging substances in MS.

Synthesis and estrogen activity screening of some new D-secoestrone derivatives.

Five new D-secoestrone derivatives were synthesized, starting from 3-methoxy-17-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile. Newly synthesized compounds showed a complete loss of estrogenic activity.

Cell-type dependent response of melanoma cells to aloe emodin.

Intrinsic characteristics of melanoma cells such as expression of inducible nitric oxide synthase (iNOS), redox status, and activity of signaling pathways involved in proliferation, differentiation and cell death define the response of the cells to the diverse treatments. In this context we compared the effectiveness of herbal antaquinone aloe emodin (AE) against mouse B16 melanoma and human A375, different in initial activity of ERK1/2, constitutive iNOS expression and basal level of reactive oxygen species (ROS). Both cell lines are sensitive to AE treatment. However, while the agent induces differentiation of B16 cells toward melanocytes, in A375 cells promoted massive apoptosis. Differentiation of B16 cells, characterized by enhanced melanin production and tyrosinase activity, was mediated by H(2)O(2) production synchronized with rapid p53 accumulation and enhanced expression of cyclins D1 and D3. Caspase mediated apoptosis triggered in A375 cells was accompanied with Bcl-2 but not iNOS down-regulation. In addition, opposite regulation of Akt-ERK1/2 axis in AE treated B16 and A375 cells correlated with different outcome of the treatment. However, AE in a dose-dependent manner rescued both B16 and A375 cells from doxorubicin- or paclitaxel-induced killing. These data indicate that caution is warranted when AE is administrated to the patients with conventional chemotherapy.

Opposing effects of omega-3 and omega-6 long chain polyunsaturated Fatty acids on the expression of lipogenic genes in omental and retroperitoneal adipose depots in the rat.

This study aimed to determine the effect of varying dietary intake of the major n-3 PUFA in human diets, alpha-linolenic acid (ALA; 18 : 3n-3), on expression of lipogenic genes in adipose tissue. Rats were fed diets containing from 0.095%en to 6.3%en ALA and a constant n-6 PUFA level for 3 weeks. Samples from distinct adipose depots (omental and retroperitoneal) were collected and mRNA expression of the pro-lipogenic transcription factors Sterol-Retinoid-Element-Binding-Protein1c (SREBP1c) and Peroxisome Proliferator Activated Receptor-gamma (PPARgamma), lipogenic enzymes Sterol-coenzyme Desaturase1 (SCD-1), Fatty Acid Synthase (FAS), lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (G3PDH) and adipokines leptin and adiponectin determined by qRT-PCR. Increasing dietary ALA content resulted in altered expression of SREBP1c, FAS and G3PDH mRNA in both adipose depots. SREBP1c mRNA expression was related directly to n-6 PUFA concentrations (omental, r(2) = .71; P < .001; Retroperitoneal, r(2) = .20; P < .002), and inversely to n-3 PUFA concentrations (omental, r(2) = .59; P < .001; Retroperitoneal, r(2) = .19; P < .005) independent of diet. The relationship between total n-6 PUFA and SREBP1c mRNA expression persisted when the effects of n-3 PUFA were controlled for. Altering red blood cell concentrations of n-3 PUFA is thus associated with altered expression of lipogenic genes in a depot-specific manner and this effect is modulated by prevailing n-6 PUFA concentrations.

Predictors of infarct artery patency after prehospital thrombolysis: the multicentre, prospective, observational OPTIMAL study.

OBJECTIVE: To identify predictors of early TIMI 3 flow patency of the infarct-related artery after prehospital thrombolysis in patients with ST-segment elevation myocardial infarction (STEMI) using data from a "real-world" population, and to develop a nomogram for triaging patients to emergency angiography. DESIGN: Multicentre, observational, prospective, cohort study. SETTING: 79 Hospitals in France with a prehospital mobile intensive care unit and a coronary care unit with 24 h access to coronary angiography. PATIENTS: 997 Patients with STEMI. INTERVENTIONS: All patients received prehospital thrombolysis within 6 h of symptom onset and angiography was performed within 6 h of thrombolysis. MAIN OUTCOME MEASURES: Coronary patency (TIMI flow). RESULTS: The median age of the population was 59 years and the sample comprised 18% women. After multivariable logistic regression analysis, predictors of TIMI 3 flow in the infarct-related artery were current/previous smoking (odds ratio (OR) = 1.60, 95% confidence interval 1.15 to 2.22), < or =5 leads with ST-segment elevation before thrombolysis (OR = 1.59, 1.12 to 2.25), Killip class I (OR = 1.96, 1.05 to 3.67), chest pain relief (OR = 1.62, 1.17 to 2.25) and ST-segment resolution > or =70% (OR = 1.76, 1.29 to 2.38). A nomogram was developed to assess the probability of TIMI 3 flow, according to smoking status, number of leads with ST elevation before thrombolysis, Killip class, chest pain relief and ST-segment resolution. CONCLUSIONS: This study provides quantitative data for predicting success of prehospital thrombolysis. The nomogram is a simple tool for predicting likelihood of coronary patency, based on clinical and electrocardiographic data. It may help to identify patients who require emergency angiography and rescue percutaneous coronary intervention.