Shear bond strength and debonding characteristics of a new premixed self-etching with a reference total-etch adhesive.

BACKGROUND: To determine the shear bond strength and adhesive remnant index of a new premixed self-etching primer and adhesive (Tectosan, BonaDent, Germany) for orthodontic appliances in comparison to a reference total-etch system Transbond XT. METHODS: Bovine incisors were embedded in resin and randomly divided into two groups of 16 samples each. Brackets (Discovery, Dentaurum, Germany) were bonded in group 1 (total-etch-system, Transbond XT) and in group 2 (self-etch-system, Tectosan) with curing light for 40 s. Shear bonding strengths were measured after 24 h of storage in distilled water at 37 °C with a Zwicki 1120 testing machine (Zwick Roell, Germany). A force was applied on the bracket base at the wings in occluso-gingival direction. Then the adhesive remnant index (ARI) was determined. RESULTS: No statistical differences on SBS were found for both bonding agents (p = 0.63). ARI scores however differed statistically significantly (p = 0.035): in the total-etch group more adhesive remained on the teeth, whereas in the self-etch group more adhesive remained on the brackets. There were no visible enamel damages in both groups. CONCLUSIONS: No differences in the shear bond strength were found between both bonding agents. In our study the self-etch-system shifted the adhesive remnant index from more adhesive on the teeth to more adhesive on the bracket - as other already published self-etch systems did - with the new benefit of not increased enamel damages. Tectosan might therefore be a promising alternative to adhesive systems.

Effect on enamel shear bond strength of adding microsilver and nanosilver particles to the primer of an orthodontic adhesive.

BACKGROUND: The objective of this study was to determine whether the addition of microsilver or nanosilver particles to an orthodontic primer affects shear bond strength (SBS) and bracket/adhesive failure. METHODS: Bovine incisors were randomly divided into six groups with 16 specimens in each: In group 1 (control), brackets were bonded with Transbond™ XT primer. In the experimental groups, microsilver (groups 2 and 3) and nanosilver (groups 4-6) particles of different sizes were added to Transbond XT primer and light cured for 15 seconds [group 2: 0.1% (w/w) microsilver particle size 3.5-18 µm; group 3: 0.3% (w/w) microsilver particle size 3.5-18 µm; group 4: 0.11% (w/w) nanosilver particle size 12.6-18.5 nm; group 5: 0.18% (w/w) nanosilver particle size 12.6-18.5 nm; group 6: 0.33% (w/w) nanosilver particle size 12.6-18.5 nm]. Thereafter, brackets were bonded by light curing the adhesive for 20 seconds. After 24 hours of storage in distilled water at 37 °C, SBS was measured with a Zwicki 1120 testing machine. The adhesive remnant index and the prevalence of silver spots on the specimen surface were determined under 10× magnification. Statistical two-way analysis of variance was performed to compare SBS, and a chi-square test was used to compare ARI scores and the prevalence of silver spots. RESULTS: No significant differences in SBS (control: 16.59 ± 6.82 MPa; group 2: 20.6 ± 4.19 MPa; group 3: 16.98 ± 4.84 MPa; group 4: 17.15 ± 5.92 MPa; group 5: 20.09 ± 3.35 MPa; group 6: 16.44 ± 4.51 MPa; p > 0.665) and ARI scores (p = 0.901) were found between the control group and any experimental group. Only experimental groups with nanosilver particles revealed statistically more silver spots on the remaining adhesive. CONCLUSIONS: Addition of small concentrations of microsilver or nanosilver particles affects neither SBS nor ARI scores. Addition of nanosilver particles results in silver spots in the remaining primer visible under 10× magnification. Further studies are needed to investigate the anti-caries potential and clinical performance of conventional orthodontic primer with incorporated nanosilver or microsilver particles.

Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1ß, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection. METHODS: Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR. RESULTS: MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids. CONCLUSIONS: The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Leukocyte accumulation in graft blood vessels during self-limiting acute rejection of rat kidneys.

During self-limiting acute rejection preceding chronic vasculopathy, large amounts of leukocytes, predominantly monocytes, interact with the endothelium of renal allografts. We aim to characterize them and to identify targets for functional and interventional studies. Leukocytes were harvested by vascular perfusion from Fischer 344 to Lewis renal allografts or Lewis isografts, followed by flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Leukocyte accumulation peaked in allografts on day 9. The percentage of monocytes expressing MHC class II and CD161 was increased whereas CD4, CD11a, CD43, and CD71 expression remained unchanged. IFN-γ, IL-1ß, IL-2, IL-10, TNF-α, and iNOS mRNA increased in allograft leukocytes but IL-4, IL-6, IL-12, TGF-ß, and tissue factor did not. During acute rejection, 1783 genes were differentially expressed. In conclusion, graft blood leukocytes display a unique state of partial activation during self-limiting rejection. Numerous differentially expressed genes deserve further investigation as potential factors in deciding the fate of the allograft.

Acute rejection of experimental lung allografts: characterization of intravascular mononuclear leukocytes.

Leukocytes interacting with endothelia of lung allografts probably play a seminal role in acute rejection, but have not been characterized before. Transplantation was performed in the Lewis to Lewis and in the Dark Agouti to Lewis rat strain combinations. DNA replication was detected in T-cells on day 2 after pulse-labelling in vivo with 5-bromo-2'-deoxyuridine (BrdU). On day 5, leukocytes were isolated by intensive perfusion the graft, subject to flow cytometry and to quantitative RT-PCR. About 34 million leukocytes accumulated in allograft vessels, but only 10 and 6 million cells in isografts and control lungs, respectively. During rejection, IFN-gamma, IL-1beta and IL-10 mRNA expression increased, IL-12 mRNA decreased, whereas IL-2, IL-6, TNF-alpha, and TGF-beta mRNA did not change. The phenotype of graft monocytes was partially activated and intravascular T-cells proliferated. In conclusion, during rejection, monocytes with unusual properties accumulate and T-lymphocytes are activated in lung allograft blood vessels.

Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor.

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.

CREM activator and repressor isoform expression in human male germ cells.

The transcription factor cAMP-responsive element modulator (CREM) is known to play a vital role for male fertility as it has been demonstrated that male mice lacking a functional CREM gene are infertile. The CREM gene consists of 14 exons. Owing to alternative exon splicing, CREM gene expression results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Infertile men have been reported to reveal a substantial reduction of CREM activators and additional inaccurately spliced CREM transcripts. In the present study, we analysed the expression of CREM transcripts with the recently reported leader exons theta1 and theta2 and identified a new putative CREM repressor, namely theta1-F-H, in patients with impaired spermatogenesis. In addition, we applied single cell microdissection followed by RT-PCR with leader exons B, theta1 and theta2 to assign the expressed CREM activator and repressor isoforms to specific germ cell types within the seminiferous epithelium. Contrary to dogma, we demonstrated CREM activator and repressor isoforms in all germ cell types, but not in Sertoli cells. However, the percentage of germ cell samples that revealed positive RT-PCR signals for these CREM activators was higher in spermatocytes and round spermatids than in spermatogonia and elongated spermatids. It remains unknown whether these activator transcripts are physiologically active. Our data suggest a fine-tuning between CREM activator and repressor isoforms in normal germ cells that might be disturbed during impaired spermatogenesis.

Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis.

Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibiting enlarged sperm heads. The CREM-gene consists of 14 exons. Alternative exon splicing results in the production of both activator and repressor proteins. To further clarify the role of different CREM-isoforms for male infertility, the expression pattern of various CREM-isoforms during equine and human normal and impaired spermatogenesis was investigated by RT-PCR. Stallions with normal spermatogenesis expressed six activators and three repressors. In men three activators and seven different repressors were detected. In one stallion and patients with impaired spermatogenesis, only repressors were found. It is concluded that (i). stallion and man reveal a different CREM expression pattern, (ii). the expression of CREM activators is a prerequisite for normal spermatogenesis, and (iii). the lack of CREM activator expression results in male infertility.

Bradykinin increases intracellular calcium levels in rat testis peritubular cells via the B2 receptor subtype.

1. RT-PCR and Western blots were used to detect bradykinin B(2) receptors in testis and isolated peritubular cells of pre-pubertal rats. RT-PCR demonstrated expression of a single transcript, whereas Western blots showed up to three specific bands that were in accordance with the described native, glycosylated and dimeric form of B(2) receptor proteins, respectively. 2. Fura-2-loaded peritubular cells responded with an instantaneous, linear and transient rise in [Ca(2+)](i) after adding bradykinin. Stimulation of cells with bradykinin concentrations between 1 micro M and 1 pM showed a dose dependent increase of [Ca(2+)](i). The calcium response to bradykinin was diminished after stimulation of peritubular cells in calcium-free buffer. After blocking the SERCA-pumps by thapsigargin and subsequent stimulation with bradykinin, no rise of [Ca(2+)](i) was appreciated. 3. Multiple stimulation of a single peritubular cell by local perfusion with a brief addition of BK (10 nM) resulted in a fast and immediate response. However, the second and third stimuli had slower rise rates and diminished [Ca(2+)](i) peaks, showing desensitization of the kinin receptor. 4. Addition of the bradykinin B(1) receptor agonist [des-Arg(9)]-bradykinin (100 nM) to Fura-2-loaded peritubular cells did not change the [Ca(2+)](i). However, the B(2) receptor antagonist HOE 140 (100 nM) strongly inhibited the bradykinin-induced calcium response. 5. We conclude that the bradykinin-induced increase in [Ca(2+)](i), in testicular peritubular cells is mediated by the stimulation of kinin receptors of the B(2) subtype.

Expression and location of the bradykinin B2 receptor in rat testis.

To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B(2) receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B(2) receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B(2)-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B(2)-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B(2) receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.