Comparative phenotypic characterization of keratinocytes originating from hair follicles

The principal pool of epidermal stem cells is located in the bulge region of the hair follicle root sheath. In this research project, we have used a refined procedure to isolate porcine hair follicles including their root sheath and for comparison purposes also human cell material. These cells migrating from the hair follicles were then cytochemically characterized. A panel of antibodies and two labeled plant lectins were tested on cell material obtained under a range of assorted experimental conditions. Due to their role in growth regulation we also studied two endogenous lectins, specifically monitoring their expression and the presence of accessible ligands. These in vitro results were compared with findings on porcine and human hair follicles and human basal cell carcinomas in situ. The keratinocytes originating from hair follicles in the presence of feeder cells are rather undifferentiated and express galectin-1/galectin-1-binding sites but not galectin-3 in their nuclei associated with Np63 positivity. Nuclear reactivity for galectin-1 was rarely observed in the bulge of the outer root sheath of the human hair follicle and of basal cell carcinomas and absent in porcine tissue samples. Exclusion of feeder cells from our cultivation system of porcine hair follicles led to the formation of spheroid bodies from these keratinocytes. Ki67 as a marker of proliferation was not present in the nuclei of cells forming these spheroids. One part of these bodies is positive for markers of post-mitotic differentiated cells, while the other spheroids are composed of poorly differentiated cells, which are able to adhere to feeder cells and form growing colonies. In summary, the detection of galectin-1 and also nuclear binding sites for this endogenous effector points to intracellular functionality of this lectin. It can be considered a potential marker of a distinct cell population, probably at the beginning of a differentiation cascade of keratinocytes.     

Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord Ischemia Model: Nestin and Fluoro-Jade B Observations

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival. 2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L(1-3) spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4-S2 segments, points out the most injured spinal cord region by ischemia. 3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which "should have been affected" by ischemia indicates protection of this region by the IPC treatment. 4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4-S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods. 5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.     

Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.     

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an -coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca²⁺-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.     

Google Trends reflect allergic rhinitis symptoms related to birch and grass pollen seasons

Google Trends (GT) describes the variation of the relevant interest of internet searches toward medical conditions and related symptoms. Allergic rhinitis symptom levels result from the intensity of exposure to aeroallergens in combination with relevant medication use. We analyze data from Germany to examine the relationship between hay fever-related Google search terms, symptom levels, medication use, and pollen count levels. For doing so, we also employ the new definitions on pollen season and peak pollen period start and end as proposed by the European Academy of Allergy and Clinical Immunology in a recently published position paper. We extract GT data for a number of search terms related to allergic rhinitis for Germany. We use total nasal symptom and mediation scores as reported by patients via a patient hay fever diary in the Berlin and Brandenburg areas in Germany for 3 years (2014–2016), accompanied by pollen data. Then a Pearson and Spearman correlation analysis is performed between symptom data and GT data. A graphical analysis is conducted, and the identification of pollen season and peak pollen periods is done based on the EAACI criteria. The analysis reveals that GT data are highly correlated with symptom levels and follow peak pollen period start–end, concerning grass and birch pollen-induced allergic rhinitis symptoms. GT data can be used as a proxy for the identification of the onset and variation of nasal symptom and medication score for allergic rhinitis sufferers.     

Rapid Identification of Medically Important Candida Isolates Using High Resolution Melting Analysis

An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature—shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.