Transcutaneous immunotherapy via laser-generated micropores efficiently alleviates allergic asthma in Phl p 5-sensitized mice.

BACKGROUND: Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser-generated micropores to subcutaneous injection. METHODS: BALB/c mice were sensitized by intraperitoneal injection of recombinant grass pollen allergen Phl p 5 together with alum. Subsequently, lung inflammation was induced by repeated intranasal challenge. During the treatment phase, adjuvant-free Phl p 5 was applied in solution to microporated skin or was subcutaneously injected. Lung function and cellular infiltration; Phl p 5-specific serum levels of IgG1, IgG2a, and IgE; and cytokine levels in bronchoalveolar lavage fluids as well as in supernatants of splenocyte cultures were assessed. RESULTS: Both therapeutic approaches reduced airway hyperresponsiveness and leukocyte infiltration into the lungs. Whereas subcutaneous immunotherapy induced a systemic increase in Th2-associated cytokine secretion, transcutaneous application revealed a general downregulation of Th1/Th2/Th17 responses. Successful therapy was associated with induction of IgG2a and an increase in FOXP3+ CD4+ T cells. CONCLUSIONS: Transcutaneous immunotherapy via laser microporation is equally efficient compared with conventional subcutaneous treatment but avoids therapy-associated boosting of systemic Th2 immunity. Immunotherapy via laser-microporated skin combines a painless application route with the high efficacy known from subcutaneous injections and therefore represents a promising alternative to established forms of immunotherapy.

Extracellular neutrophil traps in periodontitis.

BACKGROUND AND OBJECTIVE: Chronic periodontitis, the chronic inflammatory disease of the periodontium, is caused by bacteria and is characterized by an influx of neutrophils into the gingival crevice. Recently, a 'new' extracellular neutrophil defense mechanism - neutrophil extracellular traps - has been described. However, their role in periodontitis has not yet been investigated. MATERIAL AND METHODS: Clinical examinations, transmission and scanning electron microscopy, as well as cytology and confocal laser-scanning microscopy, were employed to analyze gingiva biopsies and crevicular exudate from patients with chronic periodontitis. RESULTS: An abundance of neutrophil extracellular traps and some phagocytic neutrophils was found on the gingival pocket surface and in the purulent crevicular exudate. Finding neutrophil extracellular traps in the spontaneously effused purulent crevicular exudate clearly indicated that they are flushed from the pocket by the crevicular exudate. In cases of dispersal of subgingival plaque bacteria, their trapping by neutrophil extracellular traps in purulent crevicular exudate and on the gingival surface was demonstrated. CONCLUSION: Trapping the crevicular bacteria prevents their adhesion to and invasion of the gingiva. The combination of neutrophil extracellular traps and crevicular exudate outflow appears to be a 'novel' defense mechanism for the clearance of crevicular bacteria in chronic periodontitis.

Ex vivo gingival-biofilm consortia.

AIMS: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. METHODS AND RESULTS: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. CONCLUSIONS: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.

Chlorhexidine-induced ultrastructural alterations in oral biofilm.

Chlorhexidine, the most used biocide in periodontology, alters the permeability of the bacterial cell membrane. However, the chlorhexidine-induced morphological alterations in the oral biofilm have not been studied. To examine the effects of chlorhexidine on oral biofilm on an electron microscopic level, gingival epithelial cells with attached biofilm were collected from 10 volunteers, subjected to 0.1% chlorhexidine for 1 or 5 min, stained with ruthenium red-tetroxide, and analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM visualized the bacterial glycocalyces and the biofilm matrix on the biofilm surface; however, no chlorhexidine-induced alterations were observed. TEM revealed loss of bacterial membrane integrity and fimbrial disintegration in a few bacteria. In the proximity of these alterations, a restricted matrix disintegration was also observed. However, the chlorhexidine-induced alterations only effected a minor part of the oral biofilm and did not cause its disintegration. These findings suggest the insufficient efficiency of chlorhexidine against oral biofilm.

Bacterial internalization in periodontitis.

BACKGROUND: Bacterial invasion of host epithelial cells plays an important role in the pathogenesis of periodontitis; however, the exact mechanism of the invasion has not been investigated. METHODS: Pocket epithelium biopsies in periodontitis were analysed via scanning and transmission electron microscopy using ultra-histochemical staining with ruthenium red for glycocalyx visualization. RESULTS: We demonstrated that oral bacteria adhered via fimbriae-mediated adhesion only. The bacterial internalization in periodontitis was marked by the hallmark of the fimbriae-induced zipper mechanism--the phagocytic cup formation--but we found no sign of the trigger mechanism of internalization. In addition, we frequently observed apoptosis in the phagocytizing epithelial cells. CONCLUSION: Fimbriae-mediated adhesion is a prerequisite for bacterial invasion in periodontitis. This occurs by the fimbriae-induced zipper mechanism of internalization. As internalization induces apoptosis, the subsequent exfoliation might play a significant role in the clearance of periodontal pathogens.

Analysis of microvascular trees by means of scanning electron microscopy of vascular casts and 3D-morphometry.

Arterial and capillary trees form by consecutive branching (mostly bifurcations) from a stem vessel, venous trees form by repeated merging of blood vessels. Diameters of stem (parent, mother) vessels and daughter vessels (branches), interbranching distances and branching angles between stem and daughter vessels lastly define the overall three-dimensional structure of the vascular network as well as the basic transport capacity of the system. Here we use scanning electron microscopy and 3D-morphometry to measure these variables from stereo paired images of vascular corrosion casts of the anterior cerebral artery and its main branches and from arteriolar bifurcations of the mesencephalic optic tectum in the actinopterygian fish, Acipenser ruthenus. We then calculate bifurcation indices, area ratios, asymmetry ratios and test for the optimality principles underlying the bifurcations studied. Our results show that arteriolar bifurcations in the optic tectum are in favor of the principles of minimum pumping power and minimum volume rather than the principles of minimum surface and minimum drag. We conclude that scanning electron microscopy of vascular corrosion casts in conjunction with 3D-morphometry is an excellent tool to thoroughly analyze vascular trees in healthy and diseased tissues and organs, as well as on an ontogenetic and phylogenetic scale.

Candida attachment to oral epithelium.

Inflamed oral mucosa biopsies from patients with thrush and high candidal density were observed in a transmission electron microscope (TEM) using ultra-histochemical staining with ruthenium red for glycocalyx visualization. Fimbriae comprising the glycocalyx and enabling yeast adhesion to epithelial cells were clearly visualized by ruthenium red. All internalized portions of the yeast walls were devoid of glycocalyx, indicating that the growing tips of the hyphae mechanically penetrated the host cells. The attachment of Candida occurred in two ways: by fimbria-mediated adhesion enabling colonization of the epithelial surface, and by invasion of the superficial epithelial cells via hyphae. As the interaction between adhesin receptors and adhesins stimulates the production of proinflammatory cytokines, Candida adhesion itself is assumed to induce mucosal inflammation.

Fimbria-mediated bacterial adhesion to human oral epithelium.

Oral mucosa biopsies and saliva samples from 12 individuals were processed for transmission (TEM) and scanning (SEM) electron microscopy with and without ruthenium red staining. Additionally performed microbiological estimations indicated in all bacteriological samples a facultative pathogenic flora. SEM and TEM investigation showed a diverse bacterial flora attached to the mucosal surface. Fimbriae comprising the glycocalyx and enabling bacterial attachment to the epithelial cells could be clearly visualised by ruthenium red. The only mode of bacterial attachment to the oral mucosa detected in the present investigation was fimbria-mediated adhesion and co-adhesion. The fimbria-mediated adhesion enables the bacterial persistence in the oral cavity and is the first step in the bacterial colonisation process.

Glycaemic disorders in denture stomatitis.

The prevalence of glycaemic disorders was investigated in native Upper-Austrians with Candida-associated denture stomatitis. All patients with previously unknown diabetes mellitus were subjected to an oral glucose tolerance test (OGTT) and as a result diabetes was diagnosed in 13% of the patients over 50 years of age. Thirty-five percent of all inspected patients over 50 years of age with denture stomatitis had type 2 diabetes mellitus and 36% had impaired glucose tolerance (IGT). The correlation between Candida-associated denture stomatitis and diabetes mellitus indicates a means for the early diagnosis of diabetes. Hyperglycaemia could not be a predisposition to denture stomatitis, since all patients with denture stomatitis in the age-bracket 26-50 years were without diabetes and only very few of the older patients with diabetes were obese. The correlation between Candica-associated denture stomatitis and type 2 diabetes mellitus could be traced back to a reduced resistance to Candida that preceded the diabetes.

Ontogeny of the solitary chemosensory cells in the zebrafish, Danio rerio.

Secondary epidermal solitary chemosensory cells (SCCs) are widespread among the primary aquatic vertebrates. They resemble taste bud sensory cells in fine structure and may be innervated from facial or spinal nerves. According to previous studies, SCCs may constitute a water sampling system in the contexts of predator avoidance, habitat recognition and, in some cases, finding food. By quantitative scanning (SEM) and transmission electron microscopy (TEM) in 60 specimens (57 SEM, 3 TEM) of 16 developmental stages, from pre-hatchlings to adults, we describe the ontogenetic development of SCC densities and shapes of sensory apices in the zebrafish, Danio rerio. This is put into perspective with the ontogeny of external taste buds. Just prior to hatching, 3 days after fertilization (3d AF), sensory apices of SCCs penetrate between the squamous epidermal cells, whereas taste bud pores only appear at the onset of exogenous feeding (5d AF). SCC densities increase sharply from hatching shortly after metamorphosis (25d AF) up to 6 x 10(3) per mm2 on the head and remain relatively constant in density thereafter. Conservatively estimated, there may be approximately 3.2 x 10(5) SCCs on the head and 1 x 10(6) SCCs on the entire body surfaces of a zebrafish 180d AF. SCCs are spread evenly, but are 2- to 5-fold higher in density along the head than along the body. Sensory apices are brush-like in hatchlings and early juveniles, but tend to consist of a single villus in the adults. This ontogenetic change of SCC apices parallels the evolutionary change from 'oligovillous' cells in lampreys and elasmobranchs to the 'monovillous' SCCs in the advanced actinopterygian teleosts.

Vascularization of the telencephalic choroid plexus of a ganoid fish [Acipenser ruthenus (L.)].

The vascularization of the telencephalic choroid plexus of the sterlet Acipenser ruthenus, a ganoid fish, was examined by vascular corrosion casting and by light and transmission electron microscopy. The arterial supply is from the dorsal mesencephalic artery via: 1) the ventral choroidal arteries (left and right); 2) the dorsal choroidal arteries (left and right); 3) the caudal choroidal arteries (left and right); 4) the ventral arteries of the dorsal sac; and, from the olfactory arteries, via 5) the rostral choroidal arteries. The venous drainage is mainly through a single main choroidal vein that can take various courses either directly to the anterior cardinal vein or via the middle cerebral vein to the anterior cardinal vein. To a lesser extent, the plexus is drained via the lateral telencephalic veins and the ventral vein of the dorsal sac to the middle cerebral vein. By angioarchitecture and form, the plexus can be subdivided into five distinct parts: the surface network, the median folds, the large lateral folds, the small lateral folds, and the area common to the bottom of the dorsal sac and the telencephalic plexus. Diameters of terminal vessels as measured from vascular corrosion casts and from paraplast, semithin, and ultrathin sections were never less than 10 micron. It is suggested that the different areas in one plexus may have different functions with respect to secretion and absorption of cerebrospinal fluid.

Distribution of aminergic neurons in the brain of the sterlet, Acipenser ruthenus (Chondrostei, Actinopterygii).

Aminergic nuclear areas were identified in the brain of Acipenser ruthenus using formaldehyde-induced fluorescence (FIF). One group of scattered, green fluorescent cerebrospinal-fluid (CSF)-contacting neurons is located rostral to the commissura anterior. A second very prominent field of green fluorescent neurons is situated within the recessus preopticus, the recessus preopticus organ (RPO). Caudal to the latter there is a group of green and yellow fluorescent CSF-contacting neurons, the anterior part of the paraventricular organ (PVO). Further caudally, towards the dorsorostral entrance to the recessus lateralis the latter group is continuous with the main part of the PVO, which consists of large green and yellow, fluorescent CSF-contacting neurons in a stratified arrangement. The PVO is caudally continuous with small, green fluorescent CSF-contacting neurons situated within the roof of the recessus lateralis. The nucleus recessus posterior is made up of green and yellow fluorescent CSF-contacting neurons, which cover not only the walls of the recessus posteriores, but also line the lateral, dorsal and caudal aspects of the caudal hypothalamus. Small groups of green and yellow fluorescent CSF-contacting neurons can be found also in other areas of the hypothalamic ventricular walls, especially dorsal to the PVO and within the hypothalamic floor. Three large groups of aminergic neurons are present within the brain stem: One, yellow-fluorescent raphe-system and two green fluorescent groups. The floor of the canalis centralis is lined by green fluorescent CSF-contacting neurons. There is a very low density of aminergic terminals within the brain of Acipenser. A prominent fibre tract connects the PVO and the nucleus recessus posterior.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative scanning electron microscopy of the ventricular surface in four actinopterygian fishes: Acipenser ruthenus, Salmo gairdneri, Tinca tinca and Blennius sanquinolentus (Pisces).

The ventricular surface structure of the brains of actinopterygian fishes representing four distinct evolutionary levels was investigated by scanning electron microscopy (SEM). In the chondrostean Acipenser ruthenus ventricular spaces are wide and the ependymal surface is for the greater part densely covered with cilia; apart from macrophages supraependymal cells (SE) are very scarce. In the teleosts Salmo gairdneri, Tinca tinca and Blennius sanquinolentus the ventricles are slit-like, the densely ciliated areas decrease in size. The following regions carry a variety of supraependymal (SE) cells and fibres: the rostral recessus supraopticus, the hypothalamic walls, especially the infundibulum and the dorsal walls of the rhombencephalic ventricle. There is no tight correlation between areas devoid of cilia and the circumventricular organs in teleosts. The long evolutionary history, independent of other vertebrate lines has caused a series of peculiarities in the brain of actinopterygian fishes, including a peculiar ventricular topography. Observations indicate that the rich spectrum of SE cells found in teleosts reflects a parallel evolution rather than a common heredity of teleostean fishes and higher vertebrates.

[Crown cells in the diencephalon of Acipenser ruthenus (Acipenseridae, Chondrostei)]. / Krönchenzellen im Zwischenhirn von Acipenser ruthenus (Acipenseridae, Chondrostei).

Crown cells are considered to be typical for the Saccus vasculosus of gnathostome fishes. In Acipenser ruthenus these cells could be found on the entire floor of the diencephalon by SEM. There is one accumulation of crown cells in the frontal Recessus praeopticus, one caudal to the optic chiasma and one on the floor of the Recessus lateralis. The number of apical processes of the crown cells decreases significantly according to their distance from the Saccus vasculosus. In all basal gnathostome fishes examined until now, crown cells were found on the floor of the diencephalon outside the Saccus vasculosus.