Titanium and zirconia particle-induced pro-inflammatory gene expression in cultured macrophages and osteolysis, inflammatory hyperalgesia and edema in vivo.

AIMS: The biological reaction to wear debris is critical to the osteolysis underlying aseptic loosening of joint prosthetic implants. In an attempt to reduce aseptic loosening, ceramics have been introduced. This study was designed to evaluate, compare and correlate the expression of Toll-like receptors (TLRs), their intracellular adaptors and proinflammatory cytokines in cultured macrophages challenged with titanium or zirconia particles, as well as particle-induced osteolysis in calvaria and hyperalgesia and edema in hind paw. MAIN METHODS: TLRs and their adaptors were evaluated at the mRNA level by RT-PCR, and cytokine expression was evaluated at the mRNA and protein levels. Osteolysis and hyperalgesia and edema were evaluated in vivo, in calvaria and hind paw, respectively. KEY FINDINGS: Cultured macrophages challenged with zirconia or titanium particles expressed increased mRNA for TLRs 2, 3, 4 and 9, and their adaptors MyD88, TRIF and NF-κB and cytokines TNF-α, IL-1ß and IL-6, which were also increased at protein level. Quantitative differences are evident and, in general, zirconia particle-induced pro-inflammatory gene expression was lower than that induced by titanium particles. In in vivo experiments, exposition to titanium or zirconia particles induced osteolysis in calvaria and hyperalgesia and edema in hind paw; however those induced by zirconia particles were significantly lower. There is a strong and positive correlation between the expressions of mRNA for TLR4, NF-κB, TNF-α, IL-1ß and IL-6. SIGNIFICANCE: Collectively, our data suggest that zirconia ceramic particles are less bioactive than titanium particles.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Inflammatory and bone regulators expression in murine macrophages under exposure of commercial and experimental mineral trioxide aggregate.

BACKGROUND: Mineral trioxide aggregate (MTA) has been used in a variety of surgical and non-surgical endodontic applications. The aim of this study was to evaluate the gene expression and protein production of TNF-α, IL-1ß and IL-6, as well as the gene expression of RANKL and OPG using both commercial and experimental MTA in macrophage cell cultures. METHODS: Peritoneal macrophage cell culture was performed. Viability, gene expression of cytokines, RANKL and OPG, and protein levels in experimental- and commercial-grey MTA co-cultured with peritoneal macrophages was determined by tryptan blue, real time PCR and ELISA. RESULTS: The expression of TNF-α for both commercial and experimental MTA was higher, while the expression of IL-1ß and IL-6 was similar when compared to the negative control. At protein expression level, no differences were observed between the negative control and cements. RANKL did not show a significant improvement in gene expression when compared with the negative control, but OPG expression in cement samples was higher when compared to the negative control. CONCLUSIONS: This study suggests that commercial and experimental MTA promotes anti-inflammatory processes, as well as bone healing capacity.

Expression of Xylella fastidiosa fimbrial and afimbrial proteins during biofilm formation.

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.

Surface plasmon resonance immunosensor for early diagnosis of Asian rust on soybean leaves.

Soybean rust (Asian rust) is a disease that occurs in soy cultures, negatively affecting pod formation and final grain weight and reducing value and product quality. Early identification of fungus in the plants prevents severe farming losses and spread to neighboring cultures. In this paper, a fast response sensor was developed based on surface plasmon resonance to detect Asian rust in soybean leaf extract at early stages of the disease. The antibody anti-Phakopsora pachyrhizi (pathogen) was covalently immobilized on a gold substrate via a self-assembled monolayer (SAM) of thiols using cysteamine-coupling chemistry. This immunosensor presented a linear response range for the antigen from 3.5 to 28.0 microg mL(-1) (r(2)=0.996). The effects of the antibody amount and the surface blocking to minimize non-specific adsorption on immunosensor response were evaluated. These studies provide new perspectives on using SPR technology for the development of a highly sensitive sensor for agricultural applications.

A new paradigm in the periodontal disease progression: gingival connective tissue remodeling with simultaneous collagen degradation and fibers thickening.

Bacterial dental plaque is considered to be the main cause of periodontal diseases, but progression of the disease is also related to the host inflammatory response. The earliest affected tissue is the gingiva, but the specific mechanisms involved in the onset of this condition remain unclear. Frequently, collagen degradation is pointed as the main marker of periodontal disease progression, but the organization of the fibers in the gingival tissue is still unknown. The aim of the present study was to investigate the gingival extracellular matrix in a model of ligature-induced periodontal disease. Analysis of the microbiota indicated a progressive increase in the ratio of Gram-negative/Gram-positive microorganisms. There was no difference in the organization of reticulin fibers next to the epithelial basement membrane, whereas the arrangement of collagen fibers in the gingival connective tissue was significantly affected. Animals with inflammation presented a reduction of 35% in the total area occupied by collagen fibers. However, these fibers were thicker and more densely packed. These alterations involve type I, type III and type VI collagens as determined by immunohistochemistry. The results demonstrated the occurrence of marked reorganization of the gingival extracellular matrix in response to the inflammatory process, indicating a new paradigm in the periodontal disease progression: collagen degradation and fibers thickening, simultaneously.

The influence of type I diabetes mellitus in periodontal disease induced changes of the gingival epithelium and connective tissue.

Periodontal disease constitutes the most frequent chronic diseases in human dentition. Bacterial plaque is the main etiologic agent, although it is the host immune response that causes periodontal tissue destruction. Diabetes is considered an important risk factor, not only for the onset but also for progression of the disease. The aim of this study was to analyze structural changes in the rat gingival epithelium and connective tissue in response to the experimental periodontal disease induced by the ligature technique, under the influence of diabetes. The results showed that experimental periodontal disease is characterized by marked inflammation, affecting both the epithelial and connective tissues, causing degeneration of the dermal papilla, increase in the number of inflammatory cells, destruction of reticulin fibers, and accumulation of dense collagen fibers (fibrosis). These changes were worsened by diabetes, apparently by hampering the inflammatory response and affecting tissue repair of the affected tissues.

The influence of type I diabetes mellitus on the expression and activity of gelatinases (matrix metalloproteinases-2 and -9) in induced periodontal disease.

BACKGROUND AND OBJECTIVE: Periodontal disease corresponds to a group of lesions that affect the tooth-supporting tissues present in the dental follicle. Although bacterial plaque is important, the immune response also contributes to the destruction of periodontal tissues. Diabetes mellitus is closely associated with the development, progression and severity of periodontal disease because it not only affects extracellular matrix organization but also the tissue response to inflammation. The objective of the present investigation was to study the influence of diabetes on experimental periodontal disease by evaluating the degradation of extracellular matrix through the analysis of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity, using immunofluorescence, zymography and real-time reverse transcription-polymerase chain reaction. MATERIAL AND METHODS: Wistar rats were divided into normal and diabetic groups and evaluated 0, 15 and 30 d after the induction of periodontal disease by ligature. RESULTS: MMP-2 and -9 were detected in epithelial cells, in the blood vessel endothelium and in connective tissue cells. The same profile of enzymatic expression of MMP-2 and -9 was observed in normal and diabetic animals, with a peak in activity at day 15 of inflammation. However, in diabetic animals, MMP-2 gelatinolytic activity was reduced after the inflammatory stimulus, whereas that of MMP-9 was increased. MMP-2 gene expression decreased with inflammation in both normal groups and groups with diabetes. In contrast, MMP-9 expression increased in normal animals and decreased in diabetic animals after inflammation. CONCLUSION: The results suggest the involvement of MMP-2 and -9 in the dynamics of periodontal disease and that variation in their expression levels results in differences in tissue organization and wound healing in normal and diabetic animals.

Trypanosoma cruzi: early resistance induced by culture-derived trypomastigotes.

Previous observations in this laboratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 10(5) blood trypomastigotes (BT) corresponding to 2000 LD50%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permitting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 +/- 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor receptor expression, proliferation, and colony stimulating activity production in the urinary bladder carcinoma cell line 5637.

Addition of epidermal growth factor (EGF) to cultures of the urinary bladder carcinoma cell line 5637 regulated proliferation and production of colony stimulating activity (CSA). The optimal concentration range of EGF for stimulation of cell proliferation was 5-20 ng/ml EGF and for production of CSA 2-20 ng/ml EGF. High EGF concentrations (100-200 ng/ml) showed inhibitory effects on proliferation and to a greater extent on CSA production. Also, EGF binding sites of high affinity (kd:3.25 nM) were demonstrated on the cell surface. In the optimal concentration range for stimulation (5-20 ng/ml EGF) EGF binding sites were occupied half-maximally. The loss in EGF binding after long incubation at 37 degrees C was prevented by the lysosomal inhibitory agent, chloroquine. Nonspecific binding of EGF was very low, the amount of maximally bound EGF was 1430 fmol/mg protein (130,000 bound EGF molecules/cell). A strong band of approximately 170,000 daltons could be detected by means of an anti-erbB serum which recognizes the EGF receptor protein. The protein became phosphorylated upon addition of gamma-32P ATP. The data suggest that EGF initiates its action by binding to specific high affinity receptors and plays a role in growth regulation and differentiation of the urinary bladder carcinoma cell line 5637.

Karyotype and ultrastructure of a colony stimulating factor (CSF) producing cell line (5637) originated from a carcinoma of the human urinary bladder.

The cell line 5637 which originated from a human urinary bladder carcinoma is known to produce GM-CSF and Multi-CSF ectopically. Determination of cell surface antigens defined by monoclonal antibodies was recently reported. Here we report on the ultrastructure and karyology of this CSF secreting cell line. At the ultrastructural level the monolayer in vitro culture and the solid tumors formed in nude mice showed all characteristics consistent with a well-differentiated transitional cell carcinoma (TCC). A subclone was found to grow in suspension and did not secrete any CSF activity. High resolution chromosome analysis revealed chromosomal abnormalities which agreed only in few particulars with nonrandom chromosomal aberrations usually found in TCC. Analysis of the cytogenetic results showed that nearly all structural abnormalities present are known to be associated with acute or chronic human leukemia. The possibility that the ectopic production of CSF in this cell line may be correlated to one or more of the described chromosomal aberrations is discussed.