The role of glucocorticoid in SIRPα and SHP-1 gene expression in AIHA patients

The aim of this work was to evaluate the regulation of SIRPα, an inhibitory phagocyte receptor, and the phosphatase SHP-1 in monocytes of patients with autoimmune hemolytic anemia, and the role of dexamethasone on SIRPα and SHP-1 gene expression and erythrophagocytosis in vitro. SIRPα and SHP-1 expression was higher in monocytes from AIHA patients compared with normal, returning to normal after glucocorticoid therapy. SIRPα and SHP-1 mRNA expression was upregulated in healthy monocytes treated with dexamethasone compared with basal; however, the erythrophagocytic ability was not altered. Our results point to a minor role of SIRPα and SHP-1 in determining AIHA.     

Fundamental role for HIF-1α in constitutive expression of human β defensin-1

Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human β defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

IL-17 expression is correlated with hepatitis B‑related liver diseases and fibrosis

IL-17-producing T cells (Th17) have been found to play important roles in several liver diseases, but few studies have evaluated the function of such cells in hepatitis?B (HBV)?related diseases, especially in hepatic fibrosis. In this study, we examined the expression of IL?17 in patients with different chronic HBV-related diseases, and assessed the association between IL-17 expression and the degree of fibrosis. The method of immunohistochemistry was used to evaluate the localization of intrahepatic IL-17. We demonstrated significantly increased expression of IL?17 in HBV-related chronic liver diseases, especially in liver fibrosis, and that the level of IL-17 is strongly correlated with the degree of fibrosis. Furthermore, we found that intrahepatic IL?17 was mainly localized in the fibrosis region. Our data reveal important roles of IL-17 and IL-17-producing cells in the progression of HBV related chronic liver diseases, especially in the formation of liver fibrosis.     

The role of Cɛ2, Cɛ3, and Cɛ4 domains in human and canine IgE and their contribution to FcɛRIα interaction

The C?2 and C?4 domains are considered as scaffolds, allowing C?3 domains to assume an appropriate orientation to interact with Fc?RI (0130 and 0050). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of C?2 and C?4 domains to bind to and induce target cell degranulation via Fc?RIα. Our results indicate that for (1) C?3 domains in IgE of canine and human origin are the only necessary region for binding to Fc?RIα. (2) The interaction of canine IgE with human sFc?RIα is significantly enhanced by contributions from both C?2 and C?4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine C?2 and C?4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the C?3 domain to facilitate stable interaction with canine Fc?RIα. Kinetic constants for the binding of this chimera to the soluble extracellular domain of the receptor indicate an approximate 120-fold decrease in the affinity for canine sFc?RIα (k_a = 5.30 × 10² M⁻¹ s⁻¹) and a 330-fold increase in the dissociation from canine sFc?RIα (K_D = 6.9 × 10⁻⁶ M⁻¹), compared to the wild type IgE kinetic constants (K_a = 6.30 × 10⁴ M⁻¹ s⁻¹; K_D = 2.1 × 10⁻⁸ M⁻¹). Although canine IgE does engage human Fc?RIα, canine C?2 and C?4 do not contribute to the high-affinity of interaction with human Fc?RIα. Upon replacement of human C?2 and C?4 domain by the canine homologues, human IgE C?3 only retains a low affinity for the human receptor, which shows that C?2 and C?4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor.     

Loss of nuclear prothymosin-α expression is associated with disease progression in human superficial bladder cancer

In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3⁺ and CD8⁺ lymphocytes (Foxp3⁺TIL and CD8⁺TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30 % were negative, 34 % showed nuclear, and 37 % showed cytoplasmic prothymosin-α expression. Foxp3⁺TILs were detected in 11 % of patients (nonnuclear vs. nuclear, p?=?0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p?=?0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p?=?0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p?=?0.006 and 0.043, respectively). The presence of Foxp3⁺TILs was significantly associated with disease progression by univariate analysis (p?=?0.022), but not by multivariate analysis (p?=?0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-β1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3⁺TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.     

The role of TNF-α in regulating ketamine-induced hippocampal neurotoxicity

Introduction

Ketamine is commonly used in pediatric anesthesia but recent studies have shown that it could induce neurotoxicity in the developing brain. The inflammatory cytokine, tumor necrosis factor α (TNF-α) is involved in the pathogenesis of various types of neurodegenerations. In the present study, we examined whether TNF-α may regulate ketamine-induced neurotoxicity in the hippocampus of neonatal mouse.

Material and methods

The in vitro organotypic culture of hippocampal slices was used to investigate the gain-of-function and loss-of-function effect of TNF-α modulation on ketamine-induced hippocampal neurotoxicity. Also, western blotting analysis was used to examine the relative pathways associated with TNF-α modulation. In the in vivo Morris water maze test, TNF-α was genetically silenced to see if memory function was improved after anesthesia-induced memory impairment.

Results

In in vitro experiments, adding TNF-α enhanced (112.99 ±5.4%, p = 0.015), whereas knocking down TNF-α ameliorated (46.8 ±11.6%, p = 0.003) ketamine-induced apoptosis in hippocampal CA1 neurons in the organotypic culture. Western blotting showed that addition of TNF-α reduced (67.1 ±3.7%, p = 0.022), whereas downregulation of TNF-α increased (126.87 ±8.5%, p = 0.004) the phosphorylation of PKC-ERK pathway in ketamine-treated hippocampus. In in vivo experiments, genetically silencing TNF-α markedly improved the ketamine-induced memory impairment through Morris water maze test.

Conclusions

Our results clearly demonstrated a protective mechanism of down-regulating TNF in ketamine-induced hippocampal neurotoxicity. This study may present a new target for pharmacological intervention to prevent anesthesia-related neurodegeneration in brain.     

Exercise with low glycogen increases PGC-1α gene expression in human skeletal muscle

Recent studies suggest that carbohydrate restriction can improve the training-induced adaptation of muscle oxidative capacity. However, the importance of low muscle glycogen on the molecular signaling of mitochondrial biogenesis remains unclear. Here, we compare the effects of exercise with low (LG) and normal (NG) glycogen on different molecular factors involved in the regulation of mitochondrial biogenesis. Ten highly trained cyclists (VO_2max 65 ± 1 ml/kg/min, W _max 387 ± 8 W) exercised for 60 min at approximately 64 % VO_2max with either low [166 ± 21 mmol/kg dry weight (dw)] or normal (478 ± 33 mmol/kg dw) muscle glycogen levels achieved by prior exercise/diet intervention. Muscle biopsies were taken before, and 3 h after, exercise. The mRNA of peroxisome proliferator-activated receptor-γ coactivator-1 was enhanced to a greater extent when exercise was performed with low compared with normal glycogen levels (8.1-fold vs. 2.5-fold increase). Cytochrome c oxidase subunit I and pyruvate dehydrogenase kinase isozyme 4 mRNA were increased after LG (1.3- and 114-fold increase, respectively), but not after NG. Phosphorylation of AMP-activated protein kinase, p38 mitogen-activated protein kinases and acetyl-CoA carboxylase was not changed 3 h post-exercise. Mitochondrial reactive oxygen species production and glutathione oxidative status tended to be reduced 3 h post-exercise. We conclude that exercise with low glycogen levels amplifies the expression of the major genetic marker for mitochondrial biogenesis in highly trained cyclists. The results suggest that low glycogen exercise may be beneficial for improving muscle oxidative capacity.     

Transmission and clearance of human papillomavirus infection in the oral cavity and its role in oropharyngeal carcinoma – A review

The majority of sexually active individuals becomes infected with human papillomavirus (HPV) at least once in their lifetime. Pathways for HPV transmission vary across different mucosal sites per individual. They include autoinoculation within one host, direct transmission between individuals (including perinatal transmission and transmission during sexual activity), and indirect transmission through contact with hands. The authors aim to clarify the prevalence and route of transmission per anatomic site, inter- and intra-individually, using a narrative review of the literature. In conclusion, transmission of HPV to the oral cavity and oropharynx is hypothesised to occur mainly through sexual contact. Transmission of particles through saliva has not been proven and daily living activities are not a documented source of HPV infection. Oropharyngeal HPV related cancer survivors and their partners do not show increased risk of infection during sexual intercourse. Transmission of HPV to the oral cavity (autoinoculation with fingers or transmission through saliva in deep kissing) is probably of limited importance.     

Regulation of Alström syndrome gene expression during adipogenesis and its relationship with fat cell insulin sensitivity

Alstr?m syndrome (ALMS) is an autosomal recessive genetic disease with characteristic phenotypical features including multi-organ fibrosis, insulin resistance, obesity and type 2 diabetes. ALMS1, a ubiquitously expressed gene mutated in ALMS patients, gives rise to a protein of unknown function localized to basal bodies of ciliated cells and centrosomes. Together with Bardet-Biedl syndrome, ALMS is a member of genetic ciliopathies, but the link between cilia/centrosome deficits and metabolic abnormalities remains to be determined. In this study for the first time we quantified Alms1 expression in a cellular model of adipogenesis during the differentiation of 3T3-L1 cells. An early decrease in Alms1 mRNA was observed during preadipocyte to adipocyte conversion. However, acute treatment of preadipocytes with the adipogenic factors did not result in significant change of Alms1 expression. In addition, to study the possible relationship between Alms1 and the degree of fat cell insulin sensitivity, as assessed with an insulin-dependent 2-[1-3H]-deoxyglucose uptake assay, we induced either a reduction or an increase in 3T3-L1 adipocytes insulin sensitivity by a chronic treatment with insulin or rosiglitazone respectively. In all these conditions Alms1 expression remained unchanged. In conclusion, our results show that Alms1 is expressed at higher level in preadipocytes suggesting a role of the gene in the early phase of adipogenesis. Moreover, changes in fat cell insulin sensitivity do not imply any effect on Alms1 expression.     

Induction of TNF-α in human macrophages by avian and human influenza viruses

The highly pathogenic avian influenza virus H5N1 is known to induce high level of tumor necrosis factor α (TNF-α) from primary macrophages. However, it is still unclear whether current H5N1 strains also induce high TNF-α production, as most of the data were derived from extinct clade 0 H5N1 strain. Here, we show that current clade 1 and 2 H5N1 strains induce variable levels of TNF-α that are not necessarily higher than those induced by seasonal influenza viruses. The result suggests that hyper-induction of TNF-α in human macrophages is not always associated with a highly pathogenic phenotype. We further tested the contribution of the NS gene segment from H5N1 isolates to TNF-α induction by using reverse genetics. While NS conferred some variation in TNF-α induction when incorporated into an H1N1 virus genetic background, it did not affect TNF-α induction in an H5N1 virus genetic background, suggesting that other viral genes are involved.     

Sex-dependent liver colonization of human melanoma in SCID mice—role of host defense mechanisms

The possibility that endocrine factors may influence the clinical course of malignant melanoma is suggested by the superior survival data of women. In preclinical models we observed a higher rate of colony formation by human melanoma cells in male compared to female SCID mice, but only in the case of the liver and not in other organs. The gender difference could be seen at an early phase of colony formation. On the other hand, in our human melanoma cell lines we failed to detect steroid receptor protein expression, and treatment with sex hormones did not considerably influence their in vitro behavior. Investigating the possible contribution of host cells to the observed gender difference, we performed in vivo blocking experiments applying pretreatment of the animals with Kupffer cell inhibitor gadolinium chloride and the NK cell inhibitor anti-asialo GM1 antibody. While Kupffer cell blockade enhanced melanoma liver colonization equally in the two sexes, a more prominent increase was observed in female than in male mice in the case of NK cell inhibition. Further supporting the importance of NK cells in the lower liver colonization efficiency of melanoma cells in females, gender difference in colony formation was lost in NSG mice lacking NK activity. Although in humans no organ selectivity of gender difference in melanoma progression has been observed according to data in the literature, our results possibly indicate a contribution of natural host defense mechanisms to gender difference in survival of patients with melanoma or other tumor types as well.