Enhanced phagocytosis of Aggregatibacter actinomycetemcomitans cells by macrophages activated by a probiotic Lactobacillus strain.

The activation of phagocytosis is one important approach to clearing pathogenic cells in a host. This study evaluated the ability of probiotic lactobacilli to induce phagocytic activity as well as the clearance of a periodontal pathogen, Aggregatibacter actinomycetemcomitans. First, the activation of phagocytosis was found by using lyophilized dead cells. Probiotic Lactobacillus strains significantly enhanced the phagocytic activity of macrophage cells, indicating that the probiotic lactobacilli have a remarkable ability to stimulate the macrophages. Essentially, 3 Lactobacillus strains tested did not have any critical toxic effect on the murine macrophage, and Lactobacillus johnsonii NBRC 13952 showed the least cytotoxic effect on the RAW264.7 macrophages. The expression of classically activated macrophage markers, IL-1ß, and cluster of differentiation 80 increased by L. johnsonii NBRC 13952; however, there was no significant difference for IL-18. The highest phagocytic activity by macrophages was found in a condition in which the macrophage activated by L. johnsonii NBRC 13952 functions to kill the cells of A. actinomycetemcomitans. Correlating with the result, a high amount of hypodiploid DNA (SubG1) was detected from the macrophage cells stimulated by L. johnsonii NBRC 13952. Taken together, the results suggest that macrophages activated by the Lactobacillus strain can facilitate the phagocytosis of A. actinomycetemcomitans cells by linking with enhanced apoptotic activities. In conclusion, L. johnsonii NBRC 13952 has a certain role in activating the RAW264.7 macrophages, thereby counteracting the infection of A. actinomycetemcomitans.

Platelet-rich plasma enhances the differentiation of dental pulp progenitor cells into odontoblasts.

AIM: To investigate the effects of PRP on odontoblastic differentiation using dental pulp progenitor cells derived from the dental papilla of rat incisors. METHODOLOGY: Monolayer cultures of odontoblastic lineage KN-3 cells were incubated with PRP for various time periods. The expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1) was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. To further clarify the role of PRP in odontogenesis, KN-3 cells were stimulated with PRP in the presence of ascorbic acid and ß-glycerophosphate. The cells were stained for alkaline phosphatase (ALP), and ALP activity was quantified in cell lysates. The formation of mineralized nodules was assessed by alizarin red staining. Statistical analysis was performed by one-way analysis of variance. RESULTS: PRP increased the mRNA and protein expressions of odontoblastic markers, such as DSPP and DMP-1. Furthermore, PRP stimulated the ALP activity and mineralized nodule formation induced by ascorbic acid and ß-glycerophosphate in a time-dependent manner. CONCLUSIONS: PRP enhances odontoblastic differentiation of KN-3 cells. These results indicate that PRP could be a potential candidate for use in the regeneration of dentine-pulp complex.

High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase.

OBJECTIVE: To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. METHODS: Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). RESULTS: HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. CONCLUSIONS: These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.

Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm.

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.

Evaluation of unexpected positive results from a commercial ELISA for antibodies to PRRSV.

Unexpected positive results from the widely used IDEXX ELISA for the detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) may confound investigations of the disease. Supplementing the ELISA with blocking agents and the use of IgG purified from serum samples had no effect on the unexpected positive results, suggesting that they were due to an antibody-antigen reaction. Simple competitive and blocking ELISAs were developed by modifying the IDEXX ELISA, and they and an indirect fluorescent antibody test (IFAT) were used to examine PRRSV antibodies in 33 antibody-negative, 88 antibody-positive and 73 unexpectedly positive sera. All the unexpectedly positive sera were negative by IFAT, and 89.0 per cent were negative by both the competitive and blocking ELISAs. The competitive ELISA (97.7 per cent) and the blocking ELISA (96.5 per cent) detected more positive sera than the IFAT (90.9 per cent). These results show that both ELISAs are capable of distinguishing positive and unexpectedly positive sera, and suggest that most of the unexpected positive signals are false-positives.

Local delivery system of cytotoxic agents to tumors by focused sonoporation.

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Neuronal intranuclear hyaline inclusion disease with rapidly progressive neurological symptoms.

This report describes a male patient who presented with symptoms suggestive of spinocerebellar degeneration and who died of respiratory failure at the age of 7 years but was diagnosed, at autopsy, as having neuronal intranuclear hyaline inclusion disease. Neuronal intranuclear hyaline inclusion disease is a progressive and degenerative disease; diagnosis is possible only by neuropathological analysis. This is a rare disorder; few cases with early childhood onset and rapidly progressive neurologic symptoms have been documented. According to previous reports, most neurons in the central nervous system exhibited intranuclear eosinophilic inclusion bodies; neuronal depletion appeared to be restricted to the cerebellar cortex and the medullary inferior olivary nuclei, consistent with the fact that clinical deficit appears to correspond to the site of neuronal depletion and not to where eosinophilic bodies are detected. Immunohistochemical analysis revealed that these inclusions were positive for ubiquitin. The case presented herein clearly indicates that neuronal intranuclear hyaline inclusion disease should be considered as a differential diagnosis of cases involving spinocerebellar degeneration with childhood onset.

Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines.

The development of immunity to vaccine antigen was examined using three prime/boost strategies and the progression of immune activities was evaluated over the course of 8 weeks. Calves were vaccinated and multiple immune parameters were evaluated using several methods to assess humoral or cellular immunity from the same samples in parallel. The three vaccination protocols used were a killed vaccine followed by a killed boost (killed/killed), MLV vaccine and boost (MLV/MLV), or a MLV vaccine and killed boost (MLV/killed). All the vaccines used included modified live IBR/PI3 viruses to make the bystander context as similar as possible. The Singer strain of BVDV was used as the source antigen in the killed vaccine, and the NADL strain of BVDV was used in the MLV vaccine. Controls received a vaccine containing only MLV IBR/PI3. The assessment panel measured SN titers, as well as lymphocyte proliferation, cytokine mRNA expression, intracellular cytokine production, and released IFN-gamma after in vitro stimulation with three strains of BVDV virus. MLV/MLV and MLV/killed groups developed significant SN titers to the type 1 BVDV virus strains, Singer and NADL, and low crossover titers were also seen to the type 2 strain, 890 over the evaluation period. These two groups showed significant proliferation in response to the NADL virus as compared to controls. Multiple immune assessments were conducted simultaneously to attempt to provide a broader, more in depth evaluation of immune response to these BVDV vaccination protocols. We observed that the correlation among most of the assays conducted were weak; the correlation between SN titers and cellular proliferation assays demonstrated a moderate correlation.

Identification and synthetic pathway of sialyl-Lewisx-containing neolacto-series gangliosides in lens tissues. 1. Characterization of gangliosides in human senile cataractous lens.

Human lens accumulates gangliosides in association with aging and senile cataract progression. In this study we purified and characterized five major gangliosides in human cataractous lenses. Structural analyses and immunological studies revealed the presence of ganglio-series gangliosides, GM3, GM2, GM1 and GD1a, and a sialyl-Lewisx-containing neolacto-series ganglioside, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide (IV3NeuAcIII3FucnLc4). Slow-moving gangliosides, although minor components, were also found to have sialyl-Lewisx-related structures, based on anti-Lewisx antiserum binding to their asialo forms. However, sialyl-paragloboside, a possible precursor of the sialyl-Lewisx ganglioside, was not identified.

Egg-jelly signal molecules for triggering the acrosome reaction in starfish spermatozoa.

It was in the early 1950s that J.C. Dan discovered the acrosome reaction in sea urchins, starfishes and several other marine invertebrates at Misaki Marine Biological Station on the Pacific coast of Japan. We now know that in many animals including mammals the acrosome reaction is an essential, and probably the most central, change in spermatozoa for fertilization. Starfish spermatozoa undergo the acrosome reaction upon encountering the jelly coat consisting of glycoproteins, steroid saponins, oligopeptides and inorganic components. To induce the acrosome reaction, three egg jelly components act in concert on the spermatozoa: a highly sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroidal saponins named Co-ARIS, and a group of glutamine-rich tetratriacontapeptides named sperm activating peptide (SAP). The action of ARIS is quite species-specific due to the specificity of ARIS-receptors in a restricted domain of the sperm surface and depends very much on sulfated saccharide chains. Co-ARIS is not much species-specific and its action depends on the sulfate group and steroid side chain. SAPs have a ring of 25 residues and increase the intracellular pH of spermatozoa. None of them can induce the acrosome reaction by itself in normal sea water, but ARIS does induce it in high Ca2+ or high pH sea water. Although a combination of ARIS and either Co-ARIS or SAP induces the acrosome reaction in normal sea water, all three are required to mimic the full activity of dissolved jelly coat.

Long-term response to zonisamide in patients with West syndrome.

The long-term effectiveness of zonisamide (ZNS) was evaluated in 11 patients with West syndrome (7 symptomatic) who had cessation of spasms with ZNS monotherapy. During the follow-up period (24 to 79 months, mean = 53 months), this response was maintained in 7 patients (3 symptomatic, relapse rate = 36%), including 2 children in whom ZNS was successfully discontinued. No serious adverse reactions were noted. ZNS may be both effective and well tolerated for the treatment of West syndrome.

Characterization of the sulfated fucose-containing trisaccharides by fast atom bombardment tandem mass spectrometry in the study of the acrosome reaction-inducing substance of the starfish, Asterias amurensis.

Fast atom bombardment mass spectrometry (FABMS) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) were applied to the investigation of the anomeric isomerism of synthetic trisaccharides consisting of xylose, galactose and sulfated fucose {Xyl1-->3Gal alpha 1-->3(4-OSO3Na)Fuc} and {Xyl1-->3Gal alpha 1-->4(3-OSO3Na)Fuc} and the linkage position of the sulfate group. It was possible to differentiate between various glycosidic linkages in several synthetic trisaccharides. The position of a sulfate group in synthetic methyl O-sulfo-alpha-L-fucopyranoside isomers was elucidated from the fragmentation patterns. Comparing the data from the synthetic sulfated trisaccharides with the spectra from the natural compound derived from glycan chains of the acrosome reaction-inducing substance (ARIS) from starfish, the anomeric structure and the position of the sulfate group in the natural sample were determined without ambiguity as Xyl beta 1-->3Gal alpha 1-->3(4-OSO3-)Fuc, in agreement with the result from an independent study based on nuclear magnetic resonance.

Interstitial deletion of 14q, 46, XY, del (14) (q24.3q32.1) associated with status nonepileptic myoclonia and delayed myelination.

A Japanese boy with interstitial deletion of the long arm of chromosome 14, including band 14q31, is described. The characteristic dysmorphic facial features, such as dolichocephaly, bushy eyebrows, horizontal narrow palpebral fissures, long philtrum, etc, and mental and motor developmental delay were observed. Other characteristic clinical manifestations were anuresis and status nonepileptic myoclonia The finding of delayed myelination of the cerebral white matter was observed on magnetic resonance examination, suggesting that an unknown factor related to myelination in the central nervous system might be localized in band 14q31.

A. actinomycetemcomitans LPS enhances foam cell formation induced by LDL.

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.

Examination of the hdrRM regulon yields insight into the competence system of Streptococcus mutans.

Previous studies have identified the hdrRM operon as a novel regulatory system induced by conditions of high cell density. Little is known about the genes under the control of this system, but a variety of important phenotypes are associated with either hdrR overexpression or mutation of hdrM. To characterize the regulatory function of the HdrRM system in Streptococcus mutans we used a microarray approach to compare the transcriptional profiles of an hdrR overexpression strain with an hdrM mutant. Both strains exhibited almost identical profiles, which included all of the known late competence genes as well as a variety of competence-induced bacteriocins. Through a combination of real-time reverse transcription-polymerase chain reaction (PCR), reporter gene analysis and random amplification of complementary DNA ends PCR, we confirmed the role of comX as a central intermediate regulator of numerous genes in the hdrRM regulon. Through these studies, we also identified novel comX-regulated genes required for natural competence. Taken together, our results suggest that the primary function of the HdrRM system is to regulate the late competence genes together with various bacteriocins. This occurs independently of the ComCDE system, even though both systems regulate nearly identical genes. This suggests that S. mutans has multiple parallel input sensory systems that control the same output response: the induction of natural competence and concurrent production of bacteriocins.