Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

Introduction

Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment.

Methods

An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling.

Results

Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment.

Conclusions

These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity.     

Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

Objective

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells.

Methods

High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays.

Results

Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting.

Conclusion

Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).     

Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

Background

Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control.

Methodology/Principal Findings

In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3–1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10^6–7 CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10⁴ CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression.

Conclusions/Significance

The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens.     

Novel hybrid method for the evaluation of parameters contributing in determination of protein structural classes

Due to the increasing gap between structure-determined and sequenced proteins, prediction of protein structural classes has been an important problem. It is very important to use efficient sequential parameters for developing class predictors because of the close sequence-structure relationship. The multinomial logistic regression model was used for the first time to evaluate the contribution of sequence parameters in determining the protein structural class. An in-house program generated parameters including single amino acid and all dipeptide composition frequencies. Then, the most effective parameters were selected by a multinomial logistic regression. Selected variables in the multinomial logistic model were Valine among single amino acid composition frequencies and Ala-Gly, Cys-Arg, Asp-Cys, Glu-Tyr, Gly-Glu, His-Tyr, Lys-Lys, Leu-Asp, Leu-Arg, Pro-Cys, Gln-Met, Gln-Thr, Ser-Trp, Val-Asn and Trp-Asn among dipeptide composition frequencies. Also a neural network model was constructed and fed by the parameters selected by multinomial logistic regression to build a hybrid predictor. In this study, self-consistency and jackknife tests on a database constructed by Zhou [1998. An intriguing controversy over protein structural class prediction. J. Protein Chem. 17(8), 729-738] containing 498 proteins are used to verify the performance of this hybrid method, and are compared with some of prior works. The results showed that our two-stage hybrid model approach is very promising and may play a complementary role to the existing powerful approaches.     

Status of Haemaphysalis tick infestation in domestic ruminants in Iran

The geographical distribution and ecological preferences of Haemaphysalis in domestic animals in Iran were studied 4 times a year from April 2003 to March 2005. A total of 1,622 ixodid tick specimens were collected from 3 different zones. Among them, 108 (6.7%) Haemaphysalis ticks, consisting of 6 species, were identified; H. punctata (3.4%), H. parva (0.5%), H. sulcata (0.6%), H. choldokovskyi (1.7%), H. concinna (0.06%) and Haemaphysalis sp. (0.6%). H. punctata was the most abundant species, whereas H. concinna was the rarest species collected in humid and sub-humid zones on cattle, sheep and goats. H. choldokovskyi was principally collected from sheep and goats grazed in cold mountainous areas. The infested areas consisted of Caspian Sea (Guilan, Mazandaran, Golestan, and central provinces), mountainous (Azarbaiejan, Ardebil, Kohgilouyeh, and Kordestan) and semi-dessert (Khorasan, Semnan, Kerman, Sistan, and Baluchestan) zones. The Caspian Sea zone (23.6%) was the most highly infested region. The results show that various species of Haemaphysalis ticks infest domestic ruminants in Iran and each tick species show characteristic geographical distributions.     

Prevalent mutations of human prion protein: a molecular modeling and molecular dynamics study

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144-152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

Stabilized β-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis

The precise mechanisms by which β-catenin controls morphogenesis and cell differentiation remain largely unknown. Using embryonic lung development as a model, we deleted exon 3 of β-catenin via Nkx2.1-cre in the Catnb[+/lox(ex3)] mice and studied its impact on epithelial morphogenesis. Robust selective accumulation of truncated, stabilized β-catenin was found in Nkx2.1-cre;Catnb[+/lox(ex3)] lungs that were associated with the formation of polyp-like structures in the trachea and main-stem bronchi. Characterization of polyps suggests that accumulated β-catenin impacts epithelial morphogenesis in at least two ways. “Intracellular” accumulation of β-catenin blocked differentiation of spatially-appropriate airway epithelial cell types, Clara cells, ciliated cells and basal cells, and activated UCHL1, a marker for pulmonary neuroendocrine cells. There was also evidence for a “paracrine” impact of β-catenin accumulation, potentially mediated via activation of Bmp4 that inhibited Clara and ciliated, but not basal cell differentiation. Thus, excess β-catenin can alter cell fate determination by both direct and paracrine mechanisms.     

Molecular interaction of human serum albumin with paracetamol: Spectroscopic and molecular modeling studies

The interaction between paracetamol and human serum albumin (HSA) under physiological conditions has been investigated by fluorescence, circular dichroism (CD) and docking. Fluorescence data revealed that the fluorescence quenching of HSA by paracetamol was the result of the formed complex of HSA–paracetamol, and the binding constant (K_a) and binding number obtained is 1.3 × 10⁴ at 298 K and 2, respectively for the primary binding site. Circular dichorism spectra showed the induced conformational changes in HSA by the binding of paracetamol. Moreover, protein–ligand docking study indicated that paracetamols (two paracetamols bind to HSA) bind to residues located in the subdomain IIIA.     

Bcr and Abr Cooperate in Negatively Regulating Acute Inflammatory Responses

Bcr and Abr are GTPase-activating proteins for the small GTPase Rac. Both proteins are expressed in cells of the innate immune system, including neutrophils and macrophages. The function of Bcr has been linked to the negative regulation of neutrophil reactive oxygen species (ROS) production, but the function of Abr in the innate immune system was unknown. Here, we report that mice lacking both proteins are severely affected in two models of experimental endotoxemia, including exposure to Escherichia coli lipopolysaccharide and polymicrobial sepsis, with extensive microvascular leakage, resulting in severe pulmonary edema and hemorrhage. Additionally, in vivo-activated neutrophils of abr and bcr null mutant mice produced excessive tissue-damaging myeloperoxidase (MPO), elastase, and ROS. Moreover, the secretion of the tissue metalloproteinase MMP9 by monocytes and ROS by elicited macrophages was abnormally high. In comparison, ROS production from bone marrow monocytes was not significantly different from that of controls, and the exocytosis of neutrophil secondary and tertiary granule products, including lactoferrin, was normal. These data show that Abr and Bcr normally curb very specific functions of mature tissue innate immune cells, and that each protein has distinct as well as partly overlapping functions in the downregulation of inflammatory processes.BCR originally was discovered as a human gene on chromosome 22 that, in chronic myeloid leukemia, becomes fused to the c-ABL tyrosine kinase gene originating from chromosome 9 (18). The normal gene encodes a 160-kDa protein that contains a domain with GTPase-activating (GAP) activity toward Rho family GTPases (7, 11, 12, 32, 36). There is only one other gene in mouse and human, called ABR, that is closely homologous to BCR (17). Abr shares several domains with Bcr, which includes a Dbl homology (DH) domain and a GAP domain. Bcr has an additional N-terminal part consisting of a coiled-coil and a serine/threonine kinase domain that is not present in Abr, suggesting that each GAP has a distinct cellular function.Rho GTPases, including Rho, Rac, and Cdc42, play important roles in many functions of cells of the innate immune system (16). They cycle between active GTP and inactive GDP-bound conformations. GAP proteins catalyze the conversion of bound GTP to GDP on Rho GTPases and thus act as negative, inactivating regulators.In previous studies, we showed that both Abr and Bcr specifically act as GAPs for Rac and not for the related Cdc42 (6). To investigate the normal cellular function of these two related GAPs, we generated mice defective in the production of Abr or Bcr through gene targeting. Mice that lack both proteins have defects in the architecture of the inner ear, with the partial absence of otoconia and hair cells. Additionally, postnatal cerebellar development is abnormal, with a persistence of ectopic granule cells at the cerebellar surface. These combined abnormalities cause persistent circling and balance problems (20, 21).As reported previously, neutrophils from mice lacking Bcr produce increasing amounts of reactive oxygen species (ROS), and bcr^−/− mice injected with Escherichia coli lipopolysaccharide (LPS) are much more severely affected than are wild-type mice (39). We further explored the role of Bcr and Abr in the innate immune system with a detailed study of bone marrow-derived macrophages (BMM). Interestingly, macrophages isolated from double-knockout (abr × bcr^−/−) mice exhibited multiple defects. These include aberrant actin cytoskeletal organization and the increased colony-stimulating factor 1-stimulated chemotaxis and phagocytosis of opsonized zymosan or E. coli (6).In the current study, we examined whether the defects observed in vitro result in an observable phenotype in vivo, under inflammatory conditions. Here, we report that Abr plays a distinct role in negatively regulating the innate immune system in vivo, as well as exhibiting overlap with the function of Bcr. Mice lacking both Abr and Bcr have a severely impaired ability to resolve septic shock, showing that the activity of both proteins is required for the appropriate negative control of innate immune responses.