Characterization and analysis of migration patterns of dentospheres derived from periodontal tissue and the palate.

BACKGROUND AND OBJECTIVE: Stem cells derived from periodontal and palatal tissues may be useful for regenerative therapies of periodontal tissues. In addition to the use of single periodontium-derived stem cells (pdSCs) and palatal-derived stem cells (paldSCs), the application of pdSC and paldSC dentospheres, providing a pool of vital stem cells, may be a useful approach. As cell migration is a prerequisite for stem cells to regenerate a three-dimensional tissue environment, we characterized pdSCs and paldSCs and investigated the migratory activity of dentospheres within a three-dimensional environment. We also investigated the capacity of the dentospheres to grow on zirconium dioxide surfaces. MATERIAL AND METHODS: The capacity of pdSCs and paldSCs to differentiate into the neuronal and osteogenic lineages was proved by RT-PCR and immunohistochemistry through the detection of specific lineage markers, such as alkaline phosphatase, glutamate decarboxylase 1 (also known as GAD67, the 67-kDa isoform of glutamate decarboxylase), neurofilament-M and ß-III-tubulin. The expression profile of surface molecules on pdSCs and paldSCs was analyzed by flow cytometry. Adhesion and growth of pdSC/paldSC dentospheres on zirconium dioxide surfaces were determined using confocal laser-scanning microscopy. The migratory behavior of the cells was analyzed using a three-dimensional collagen matrix migration assay. RESULTS: Both pdSCs and paldSCs were positive for epidermal growth factor receptor, CC chemokine receptor 2 and CXC chemokine receptor 4 expression and were able to grow on zirconium dioxide surfaces. Cell-migration experiments revealed that both stem-cell populations responded similarly to epidermal growth factor (EGF), monocyte chemotactic protein 1 (MCP-1) and stromal cell-derived factor 1alpha (SDF-1α). Stimulation with EGF resulted in an increased migratory activity of both stem-cell types, whereas the locomotory behavior of the cells was impaired by both MCP-1 and SDF-1α. CONCLUSION: Dentospheres represent a pool of vital pdSCs/paldSCs. As a result of the migratory activity demonstrated, along with the capacity to grow on zirconium dioxide surfaces, dentospheres may be useful for regenerative purposes in periodontal tissues.

Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease.

A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.

Intrathecal antibody synthesis in Lyme neuroborreliosis: use of recombinant p41 and a 14-kDa flagellin fragment in ELISA.

The intrathecal synthesis of IgM and IgG antibodies to Borrelia burgdorferi sonicate, to recombinant flagellin (41 kDa) and to a tryptic peptide of the flagellin (14-kDa fragment) was determined by ELISA in paired cerebrospinal fluid (CSF) and serum samples from 35 patients with Lyme neuroborreliosis (LNB) and in 10 patients with neurosyphilis. The antibody index (AI = QBb/QIg) was calculated from the ratio between CSF/serum quotients for specific antibodies (QBb) and total immunoglobulins (QIg). For the examination of IgG antibodies, the sonicate ELISA was performed with and without pre-absorption with Treponema phagedenis. Of 35 patients with LNB, 31 had intrathecal IgG response to B. burgdorferi demonstrated by sonicate ELISA (24 after absorption of cross-reactive antibodies), 29 had a response demonstrated by flagellin ELISA and 21 of 35 by 14-kDa ELISA. In patients with neurosyphilis the AI (IgG) was elevated in the sonicate ELISA in 7 of 10 samples (none of 10 after absorption of cross-reactive antibodies), in the flagellin ELISA in 5 of 10 samples and in the 14-kDa ELISA in none of 10 samples. Intrathecal synthesis of IgM antibodies to B. burgdorferi was demonstrated in patients with neuroborreliosis by sonicate ELISA in 20 of 35 samples, by flagellin ELISA in 16 of 35 samples and by 14-kDa ELISA in 9 of 35 samples. No intrathecal synthesis of B. burgdorferi-specific IgM could be detected by any assay in patients with neurosyphilis.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources.

A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level.

Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis.

N-terminal amino acid sequence of the Borrelia burgdorferi flagellin.

The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44-55%), in contrast to the high degree (67-95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.

Sexual reproduction and the role of sperm attractants in monoecious species of the brown algae order fucales (fucus, hesperophycus, pelvetia, and pelvetiopsis).

Details of sexual reproduction in Fucus spiralis are reported. Slimy oogonial packets form on the outside of the receptacles held in position by hairs extending from the ostiole. Spermatozoids enter the oogonium, and fertilization takes place before zygotes are disseminated by disintegration of oogonium walls. During these events the sperm attractant fucoserratene can be detected. Fertilization in the monoecious Fucus spiralis is therefore based on the same mechanism as in dioecious members of the genus. Egg specific substances have also been detected in Californian members of the genera Fucus, Hesperophycus, Pelvetia, and Pelvetiopsis, although the biological significance of these potential sperm-attractants could not be established. The tendency towards self-fertilization in monoecious Fucales is discussed.

The Sperm Attractant of the Marine Brown Alga Ascophyllum nodosum (Phaeophyceae).

Spermatozoids of the intertidal seaweed Ascophyllum nodosum (Fucales, Phaeophyceae) are attracted to eggs prior to fertilization. The attractant has been isolated and its structure identified as 1(3E, 5Z, 8Z)-undecatetraene (finavarrene). The relation of finavarrene to sex hormones in other brown algae is discussed.

Dictyota dichotoma (Phaeophyceae): Identification of the Sperm Attractant.

Freshly released eggs of the marine brown alga Dictyota dichotoma secrete a substance that attracts spermatozoids. This compound has been identified as n-butyl-cyclohepta-2,5-diene. It is closely related to attractants in several other brown algae and confirms that a relation exists between phylogeny and attractant compounds.

Isolation of a spermatozoid-releasing and -attracting substance from female gametophytes of Laminaria digitata.

SEVERAL dioecious marine brown algae have been valuable in studying chemical communication during sexual reproduction. For example, female gametes of Ectocarpus siliculosus, Cutleria multifida and Fucus serratus secrete olefinic hydrocarbons into the seawater to attract male gametes(1-3). A more complex system is found in the order Laminariales, which includes the large kelps. Mature female gametophytes in members of this group secrete highly volatile material with spermatozoid-attracting activity, which also induces explosive discharge of antheridia(4). We report here a study of this volatile material. It is now possible to attribute spermatozoid-releasing and -attracting activity to one specific compound.