Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology

The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 µm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h) to 91 µm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.     

Effects and consequences of nerve injury on the electrical properties of sensory neurons

Nociceptive pain alerts the body to potential or actual tissue damage. By contrast, neuropathic or "noninflammatory" pain, which results from injury to the nervous system, serves no useful purpose. It typically continues for years after the original injury has healed. Sciatic nerve lesions can invoke chronic neuropathic pain that is accompanied by persistent, spontaneous activity in primary afferent fibers. This activity, which reflects changes in the properties and functional expression of Na+, K+, and Ca2+ channels, initiates a further increase in the excitability of second-order sensory neurons in the dorsal horn. This change persists for many weeks. The source of origin of the pain thus moves from the peripheral to the central nervous system. We hypothesize that this centralization of pain involves the inappropriate release of peptidergic neuromodulators from primary afferent fibers. Peptides such as substance P, neuropeptide Y (NPY), calcitonin-gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) may promote enduring changes in excitability as a consequence of neurotrophic actions on ion channel expression in the dorsal horn. Findings that form the basis of this hypothesis are reviewed. Study of the neurotrophic control of ion channel expression by spinal peptides may thus provide new insights into the etiology of neuropathic pain.     

Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.     

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.     

The saliva of the medicinal leech Hirudo medicinalis--II. Inhibition of platelet aggregation and of leukocyte activity and examination of reputed anaesthetic effects

1. Leech saliva inhibits platelet aggregation induced by collagen, ADP and epinephrine. 2. Leech saliva inhibits superoxide production by neutrophils stimulated by tetradecanoyl phorbol acetate or polyhistidine. The effect is due in part at least to eglin. 3. Reputed anaesthetic effects of leech saliva were not detected.     

Epstein-Barr virus-transformed lymphoblastoid cell lines as antigen-presenting cells and "augmenting" cells for human CMV-specific Th clones

The ability of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) to present human cytomegalovirus (HCMV) antigen to a panel of HCMV-specific T helper (Th) clones was evaluated. Among the seven Th clones studied, only one clone (SP-CN/T3-16) proliferated well to HCMV presented by both autologous mononuclear cells (MNC) and LCL, and one clone (SP-CN/T3-9) proliferated significantly better to HCMV presented by autologous LCL than by autologous MNC. The majority of the HCMV-specific Th clones tested (five out of seven) responded much better to HCMV presented by MNC than to HCMV presented by LCL. The mechanism(s) responsible for the inefficiency of LCL to present HCMV to certain clones was studied. Our results suggested that the defect of LCL is not due to insufficient interleukin 1 production, insufficient MHC class II molecule expression, nor an inhibitory mechanism or factor. In this report, we also demonstrate that by adding a minimum amount of LCL along with MNC as antigen-presenting cells (APC), one can restimulate and expand Th clones much more efficiently than by using MNC alone as APC.     

Bio-inspired synthesis of AgNPs from lichen as potential antibacterial and mosquito larvicidal activity with negligible toxicity on Gambusia affinis

Mosquitoes serve as reservoirs for viruses and other microorganisms, posing a significant health-related issue for both humans as well as livestock. Control of these deadly disease-producing mosquito vectors is of paramount importance. The chemical analysis of Parmotrema reticulatum was examined by GC–MS. Further, lichen-mediated AgNPs were confirmed through UV–vis spectrophotometry, FTIR, TEM-EDX, and XRD. After 24 h post-treatment, the lichen-synthesized AgNPs showed considerable toxicity against distinct Aedes aegypti instars with LC₅₀ values of 44.61 (I instar), 51.27 (II instars), 61.34 (III instars), 72.95 (IV instar), and 89.84 (pupae) μg/mL, respectively. Further, both P. reticulatum extract and AgNPs greatly reduced the survival and reproductive efficiency of A. aegypti adults. Eventually, in conventional laboratory circumstances, the predatory effectiveness of Gambusia affinis against Ae. aegypti II and III instar larvae were 71.35% and 53.40%, respectively. In antibacterial assays, low concentrations of the P. reticulatum synthesized AgNPs inhibited the development of Pseudomonas aeruginosa and Citrobacter freundii. Surface damage, ROS production, and protein leakage are the antibacterial mechanisms of AgNPs. Overall, the lichen-derived AgNPs can be regarded as newer and safer Ae. aegypti control instruments.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.