Association of mitogen-activated protein kinase pathways with gingival epithelial cell responses to Porphyromonas gingivalis infection.

Mitogen-activated protein (MAP) kinase pathways are key factors in host signaling events and can also play important roles in the internalization of pathogenic bacteria by host cells. Porphyromonas gingivalis, a periodontal pathogen, can efficiently invade human gingival epithelial cells (GECs). In this study, we examined the activation of MAP kinase pathways in GECs infected with P. gingivalis. c-Jun N-terminal kinase (JNK) was activated after 5 min of infection with P. gingivalis, whereas noninvasive Streptococcus gordonii did not have a significant effect on JNK activation. In contrast, extracellular signal-regulated kinase (ERK) 1/2 was downregulated in a dose-dependent manner by P. gingivalis, but not by S. gordonii, after a 15-min exposure. Nonmetabolically active P. gingivalis cells were unable to modulate MAP kinase activity. U0126, a specific inhibitor of MEK1/2 (ERK1/2 kinase), and toxin B, a specific inhibitor of Rho family GTPases, had no effect on P. gingivalis invasion. Genistein, a tyrosine protein kinase inhibitor, blocked uptake of P. gingivalis. The transcriptional regulator NF-kappaB was not activated by P. gingivalis. These results suggest that P. gingivalis can selectively target components of the MAP kinase pathways. ERK1/2, while not involved in P. gingivalis invasion of GECs, may be downregulated by internalized P. gingivalis. Activation of JNK is associated with the invasive process of P. gingivalis.

Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells.

We have developed a fluorescence imaging technique using a DNA-binding dye to visualize, over time, the physical interactions between Porphyromonas gingivalis and human gingival epithelial cells in vitro. The results extend previous observations of P. gingivalis invasion of gingival epithelial cells based on indirect measurements. An intracellular location for P. gingivalis was established by optical sectioning of images in the z-plane. Kinetic analysis showed that P. gingivalis invasion of epithelial cells is a rapid and efficient process, reaching completion after 12 min. Imaging of infected monolayers revealed that over 90% of a population of gingival epithelial cells contained bacteria. Furthermore, only vital bacteria were capable of invasion, and intracellular bacteria congregated in the perinuclear region of the epithelial cells. P. gingivalis remained inside the epithelial cells over a 24 h period and induced rearrangement of the actin cytoskeleton along with alteration of the size and shape of the epithelial cells. These findings provide direct evidence that entry rates of P. gingivalis into gingival epithelial cells are high and rapid, and that internalized bacteria initially localize in a specific region of the epithelial cells.

Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis.

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.

Immunolocalization of rap1 in the rat parotid gland: detection on secretory granule membranes.

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [alpha-32P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.

Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization.

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells. This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride. Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic [Ca2+] may, therefore, be involved in the invasion process, while Ca2+ influx is not. Moreover, cytosolic [Ca2+] was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P. gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli. These findings indicate that P. gingivalis invasion of epithelial cells is correlated with activation of [Ca2+]-dependent host cell signaling systems.

Mycoplasma orale infection affects K+ and Cl- currents in the HSG salivary gland cell line.

The relations between K+ channel and Cl- channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl- channels, but only the latter decrease was statistically significant. Also, Cl- currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K+ channels relatively more than Cl- channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurred in vivo.

Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.

Hypotonically activated chloride current in HSG cells.

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl- containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca(2+)-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl- current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4'-isothiocyanostilbene), and its reversal potentials corresponded to the Cl- equilibrium potentials at high and low external Cl- concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl- currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl- conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl- conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.

Characteristics and regulation of a muscarinically activated K current in HSG-PA cells.

Whole cell currents were measured in HSG-PA cells (a proposed model for salivary gland duct cells) after muscarinic receptor activation or exposure to known signaling agents. Exposure to carbachol or oxotremorine M produced large and often oscillatory increases in outward current whose reversal potentials indicated a K current. The current was sensitive to extracellular atropine, charybdotoxin, and quinine, but not apamin, and to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. The response was prolonged or increased by guanosine 5'-O-(3-thiotriphosphate) and mimicked by D-myo-inositol 1,4,5-trisphosphate (IP3) or heparin in the pipette and by extracellular Ca ionophores. Tetraethylammonium indirectly inhibited the response via the muscarinic receptor. Fura 2 in cell suspensions showed that muscarinic agonists increased cytosolic Ca ion concentration ([Ca2+]i) five- to sevenfold, and measurements with indo 1 in individual cells showed that the oscillatory changes in outward current were tightly correlated with parallel changes in [Ca2+]i. The results indicate that muscarinic receptor stimulation of HSG-PA cells activates Ca(2+)-activated K channels through a signaling pathway involving a G protein, IP3 production, and increased [Ca2+]i levels. These findings are similar to those in salivary gland acinar cells.

Localization of the G-protein G(o) in exocrine glands.

The GTP-binding protein G(o) was localized immunohistochemically in the rat parotid gland and in other exocrine glands with specific G(o) antibodies. Immunohistochemical studies revealed that affinity-purified G(o alpha) polyclonal antibody (GO/85) immunoreacted primarily with duct cells of the rat parotid gland; immunoreactivity was also noted in duct cells of the rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. G(o alpha) antiserum (9072) differing in specificity for epitopes within G(o alpha) produced similar results. This antiserum also immunoreacted with rat submandibular duct cell secretory granule membranes. In contrast, in rat and mouse pancreas G(o alpha) antibodies immunoreacted primarily with islet cells. Duct cells were negative but there was light labeling of rat pancreatic acinar cells. The apparent duct specificity of G(o alpha) staining was further verified by demonstrating that G(o alpha) antibodies immunoreacted with HSG-PA cells, a human transformed salivary duct cell line. Specificity in immunohistochemical labeling of HSG-PA cells was confirmed by Western blot analysis. The results demonstrate that G(o) appears to be selectively expressed in the duct cells of rat parotid gland and other salivary glands. The selective enrichment of G(o) in duct cells suggests that this G-protein plays an important role in duct cell physiology.

Oral hairy leukoplakia: ultrastructural features.

Ten instances of a white plaque of the lateral tongue unique to homosexual males and referred to as oral hairy leukoplakia were analysed ultrastructurally. The surface epithelial layer exhibited extracellular, intracellular and intranuclear penetration by hyphae of Candida albicans, sometimes accompanied by coccobacilli in the extracellular space. The subcorneal epithelial layer included koilocytoid ballooned cells which had a paucity of cytoplasmic organelles and displayed condensation and emargination of the chromatin. Cells that exhibited these nuclear changes were found to be infected by a herpes-type virus which was visualized by electron microscopy in all ten cases. Clusters of nucleocapsids (86-110 nm in diameter) occurred in the nuclei and enveloped virions (111-175 nm in diameter) occurred in the cytoplasm and extracellular spaces. Virions showed budding from the nuclear envelope. Bundles of tubular structures (20 nm diameter) arranged in parallel occurred in the cytoplasm of some koilocytoid cells. There was no evidence by electron microscopy of the presence of papilloma virus within koilocytotic nuclei.

Clear cell odontogenic tumor: histochemical and ultrastructural features.

Clear cell odontogenic tumor, a rare epithelial jaw lesion of putative odontogenic origin, histologically resembles clear cell adenocarcinomas. Ultrastructural and histochemical features are described and support a non-glandular derivation. The intraosseous neoplasm is characterized by ovoid nests of clear or finely stippled cells with a mature collagenous stroma. These cells are PAS-positive, diastase labile and fail to bind alcian blue. Enzyme histochemical reactions disclose dehydrogenase, non-specific esterase, and acid phosphatase positivity. Fine structural characteristics include plasma membrane microvilli, desmosomes, endoplasmic reticulum, free ribosomes, glycogen rosettes and lysosomes. Many cells exhibit a paucity of cytoplasmic organelles with prominent vacuolization. Centrioles and annulate lamellae are also encountered. Summarily, clinical, radiographic, histochemical and ultrastructural features indicate that this neoplasm is probably of epithelial odontogenic origin with cytodifferentiation emulating glycogen-rich presecretory ameloblasts.

Freeze-fracture study of the tegument of larval Taenia crassiceps.

The tegument of Taenia crassiceps cysticerus is composed of 2 layers, an outer microthrix border and an underlying vesicular syncytium. Each microthrix is a long slender structure that has 2 portions, a thick proximal portion and a thinner distal portion. The inner core of each microthrix contains a cylinder of microfilaments enclosing a central bundle of microfilaments. There are numerous particles on both the protoplasmic and extracellular fracture faces of the proximal portion of the microthrix membrane while there are few particles on the fracture faces of the distal portion. The vesicles that tightly pack the distal tegument are membrane-bound biconcave discs arranged in stacks.