Characterization of mesenchymal progenitor cell populations from non-epithelial oral mucosa.

OBJECTIVES: The characteristics of cell populations extracted from oral mucosal non-epithelial tissues and their ability to differentiate were evaluated in vitro as a potential source of cells for mandibular and corneal regeneration. MATERIALS AND METHODS: Oral mucosal non-epithelial cells (OMNECs) were extracted from tissue samples and were studied by flow cytometry and RT-PCR. Cells differentiating into osteoblasts, adipocytes, chondrocytes, neurocytes, or keratocytes were characterized by RT-PCR and cell staining. RESULTS: OMNECs expressed CD44, CD90, CD105, CD166, and STRO-1 antigens, which are markers for mesenchymal stem cells. In addition, Oct3/4, c-Myc, Nanog, KLF4, and Rex, which are expressed by embryonic or pluripotent stem cells, were detected by RT-PCR. Expression of CD49d, CD56, and PDGFRα, proteins closely associated with the neural crest, was observed in OMNECs, as was expression of Twist1, Sox9, Snail1 and Snail2, which are early neural crest and neural markers. Specific differentiation markers were expressed in OMNECs after differentiation into osteoblasts, adipocytes, chondrocytes, or keratocytes. CONCLUSIONS: Populations of OMNECs may contain both mesenchymal stem cells and neural crest origin cells and are a potential cell source for autologous regeneration of mandibular or corneal stroma.

Perioperative complications after aorto-iliac stenting: associated factors and impact on follow-up cardiovascular prognosis.

OBJECTIVES: To investigate factors associated with 30-day perioperative complications (POC) after aorto-iliac (AI) stenting, and to compare follow-up cardiovascular prognosis between patients with and without POC. MATERIALS AND METHODS: This was a retrospective multicenter study. We used a multicenter database of 2012 consecutive patients who successfully underwent AI stenting for peripheral arterial disease in 18 centers in Japan from January 2005 to December 2009 to analyze independent predictors of POC and impact of POC on prognosis by logistic regression and a Cox proportional hazard regression model, respectively. RESULTS: Mean age was 71 ± 9 years (median: 72 years; range: 37-98 years), and 1,636 patients (81%) were men. POC occurred in 126 patients (6.3%). In multivariate logistic regression analysis, old age (≥80 years), critical limb ischemia (CLI), and Trans Atlantic Inter-Societal Consensus (TASC) II class C/D were independently associated with POC with adjusted odds ratios and 95% confidence intervals (CI) of 1.9 (1.3-2.9), 2.3 (1.5-3.4), and 2.4 (1.6-3.4), respectively. Out of 2012 patients, 1995 were followed up for more than 30 days (mean: 2.6 ± 1.5 years; range: 2-2,393 days). In a Cox hazard regression model adjusted for baseline clinical characteristics, POC was positively and independently associated with follow-up major adverse cardiac events (adjusted hazard ratio [HR]: 1.9; 95% CI: 1.3-2.8; p = .002), but not with major adverse limb events and target lesion revascularization (adjusted HR: 1.4; 95% CI: 0.7-2.7; p = .25; and adjusted HR: 1.2; 95% CI 0.6-2.6; p = .568), respectively. CONCLUSIONS: Age >80 years, CLI, and TASC C/D lesion were positively associated with POC after AI stenting. Occurrence of POC appears to adversely affect follow-up cardiovascular, but not limb and vessel prognosis.

Transplantation of corneal endothelium with Descemet's membrane using a hyroxyethyl methacrylate polymer as a carrier.

AIMS: To evaluate the histology and function of Descemet's membrane transplanted with intact endothelium. METHODS: Japanese white rabbits and human eyebank eyes were used as donors and recipients of Descemet's membrane transplantation. Donor endothelium was hydrodissected by injecting indocyanine green from a limbal incision, and then processed as a corneal scleral button. A 6 mm diameter donor sheet was trephined, and folded in half using a 6 mm diameter polymer as a carrier. Recipient endothelium was also hydrodissected from the limbus using trypan blue to stain the Descemet's membrane. Continuous curvilinear descemetorhexis (CCD) was performed to remove a circular section of the Descemet's membrane using a 27 gauge cystotome. Donor tissue was inserted into the anterior chamber through a 5 mm limbal incision and apposed to the host stroma. Polymers were removed following transplantation. Similar surgical procedures were performed in both rabbits and eyebank eyes. Haematoxylin eosin stains were performed after 28 days in rabbits, and eyebank eyes were fixed immediately following surgery for endothelial cell counts. RESULTS: Rabbit control eyes demonstrated stromal oedema caused by loss of Descemet's membrane, whereas transplanted eyes had clear corneas. The mean (standard deviation) pachymetry of operated eyes was 376.6 (SD 32.5) mum compared with 389.6 (SD 25.1) mum in the unoperated eye. Mean endothelial density immediately following surgery in eyebank eyes was 2749 (SD 288) cells/mm(2). CONCLUSIONS: Transplantation of Descemet's membrane by CCD produces a functional graft with an optically clear interface similar to control cornea.

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene, and secrete its active gene product.

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H.K. Kuramitsu, Plasmid 34 (1995) 85-95. This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb Bg/II fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3'-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3'-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.

Measurement of cytoplasmic viscosity by fluorescence polarization in phytohemagglutinin-stimulated and unstimulated human peripheral lymphocytes.

We have developed an improved technique of fluorescence polarization for measurement of the cytoplasmic viscosity of human peripheral lymphocytes. When measured using this improved method, the mean value of fluorescence polarization in lymphocytes from 20 healthy donors was 0.190 +/- 0.005. The mean value of fluorescence polarization as percent of control after stimulation of lymphocytes with phytohemagglutinin (PHA) was 83.6 +/- 3.8%. We discuss here optimal conditions for PHA stimulation of lymphocytes.

Characterization of cytokine mRNA transcripts in conjunctival cells in patients with allergic conjunctivitis.

PURPOSE: The host response to allergens appears to be regulated by specific patterns of local cytokine production. More than 20,000 conjunctival superficial cells were collected with a special brush, a smaller version of the Cytobrush used in cervical cytology, from the upper palpebral conjuntiva. METHODS: Samples were obtained by cytology brush from seven patients with allergic conjunctivitis and from seven healthy volunteers. Giemsa staining, immunocytochemistry, and flow cytometric analysis were performed. Cytokine gene expression was assayed by the reverse-transcription-polymerase chain reaction method. RESULTS: Giemsa staining of cytocentrifuged preparations from patients with allergic conjunctivitis showed conjunctival epithelial cells with lymphocytes, mast cells, and eosinophils. In an immunohistochemical study, a few CD3- and CD4-bearing cells, but not CD20- and CD14-bearing cells, were seen in patients. In 82.6 +/- 17% of the samples obtained from allergic patients, HLA-DR was present, but it was present in only 34.2 +/- 17.8% of samples from control subjects (P = 0.0001) using flow cytometric analysis. Steady state transcripts of mRNA for cytokines were analyzed with RT-PCR in conjunctival cell samples, and results showed that samples from allergic conjunctivitis expressed increased transcripts of interleukin 4 and interleukin 13 but virtually no interleukin 2 or interferon-gamma; six samples from seven healthy subjects expressed no interleukin 2, interleukin 4, interleukin 13, or interferon-gamma transcripts. CONCLUSIONS: These results suggest that the clinical features of allergic conjunctivitis in humans are associated with a specific local pattern of proinflammatory cytokine expression.

Evidence of long-term survival of donor-derived cells after limbal allograft transplantation.

PURPOSE: Severe destruction of the corneal limbus causes conjunctival invasion and subsequent visual loss. Limbal allograft transplantation (LAT) was recently proposed for the treatment of these disorders. However, whether the method functions as a stem cell transplantation of the corneal epithelium remains unclear. This study provided evidence that donor-derived corneal epithelial cells survive long after LAT. METHODS: Epithelial cells on the paracentral cornea in patients who have undergone LAT were subjected to fluorescence in situ hybridization (FISH) and polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis. X and Y chromosomes were detected using sex chromosome-specific probes in the FISH analysis, and HLA-DPBI antigens were examined in the RFLP analysis. Eyes receiving conventional penetrating keratoplasty (PKP) served as controls. RESULTS: Donor-derived epithelial cells were detected in three of five eyes (60.0%) in the FISH analysis and in seven of nine eyes (77.8%) in the RFLP analysis. Among these eyes, one and three eyes in the FISH and RFLP analysis, respectively, had both donor- and recipient-derived cells. In control PKP eyes, none of the eyes in the FISH analysis and one of eight eyes (12.5%) in the RFLP analysis had donor-derived cells. CONCLUSIONS: These results suggest that donor-derived cells survive much longer after LAT than those after PKP, and that LAT may function as stem cell transplantation of the corneal epithelium.

Automatic auditory information processing in sleep.

STUDY OBJECTIVES: The mismatch negativity (MMN) component of the event-related potentials reflects the automatic detection of sound change. Only a few researchers have investigated the MMN elicitation during sleep in adult human and some of them reported that MMN amplitude was decreased in sleep compared to in waking. However, it is not clear that the decrease of MMN amplitude was due to increased drowsiness or long-term response decrement. Two experiments were conducted to clarify whether or not the MMN was elicited in each sleep stage. We presented auditory stimuli to subjects continuously from waking until sleep state (Experiment 1). Using the same experimental condition, we examined whether or not MMN amplitude was influenced by long-term stimulus presentation (80min.) and by vigilance level (Experiment 2). DESIGN: N/A SETTING: N/A PARTICIPANTS: N/A INTERVENTIONS: N/A MEASUREMENTS & RESULTS: Experiment 1: MMN was obtained in both drowsiness and REM sleep. MMN was significantly smaller in amplitude and shorter in latency in the both stages than in the waking state. However, MMN was not found in another sleep stage. Experiment 2: Amplitudes were no different among 0-20 min., 20-40 min., 60-80 min. But it was decreased in 40-60 min. and power value of alpha-wave was decreased in 40-60 min. CONCLUSIONS: To obtain the reliable data, by using the automatic spectral analysis, we confirmed that MMN was elicited in REM sleep. MMN was not influenced by long-term stimulation. The result suggested that auditory stimuli could be processed in the pre-attentive sensory memory even in REM sleep.

Immunohistological analysis of tumour growth factor beta 1 expression in normal and inflamed salivary glands.

AIM: To determine whether transforming growth factor-beta 1 (TGF-beta 1) has a pathogenetic role in disease of the salivary glands. METHODS: An indirect immunohistochemical technique was used to analyse TGF-beta 1 expression in six specimens of normal salivary gland and 23 surgical specimens. RESULTS: TGF-beta 1 was strongly expressed in the ductal epithelial cells of normal salivary gland tissues (six of six cases) and in inflammatory conditions (eight of 11 cases). In contrast, TGF-beta 1 was not detectable in ductal epithelial cells expressing HLA-DR around infiltrating CD4+ CD45RO+ activated T cells, in the salivary gland tissue of patients with Sjögren's syndrome. CONCLUSION: Because TGF-beta 1 has an essential role in the mucosal immunity of salivary glands, abnormal expression of this cytokine must be regarded as a candidate in the pathogenesis of Sjögren's syndrome.

Somatosensory automatic responses to deviant stimuli.

We studied event-related potentials (ERPs) produced in response to deviant stimuli in a sequence of somatosensory stimuli which were measured under stimulus-ignoring conditions. A change in the repetitive somatosensory input elicited not only a frontal negativity which was similar to the somatosensory mismatch negativity very recently reported by Kekoni et al. [J. Kekoni, H. Hämäläinen, M. Saarinen, J. Gröhn, K. Reinikainen, A. Lehtokoski, R. Näätänen, Rate effect and mismatch responses in the somatosensory system: ERP-recordings in humans, Biol. Psychol. 46 (1997) 125-142] but also a positive deflection ranging in latency from 100 to 200 ms. This somatosensory change-related positivity was reduced with prolongation of the interstimulus interval (ISI) and was different from the responses to the deviant stimuli when they were presented alone. These findings suggest that the somatosensory change-related positivity is probably generated not by activation of new afferent elements but by a detection of change in a process of comparison with sensory memory.

The effect of deviant stimulus probability on the human mismatch process.

The present study addresses the separate activities of frontal and temporal MMN generators which might be differentially affected by a change in the probability of standard stimuli. As the probability of standard stimuli was increased, the frontal MMN component significantly increased in amplitude, while the temporal one was not affected. Correspondingly, the scalp current density (SCD) maps showed that the temporal MMN generator was activated even at low probability of standard stimuli, suggesting that even the weak memory trace could start the automatic mismatch process, whereas the frontal MMN generator was activated only with increased probabilities of standard stimuli, suggesting that the stronger the memory trace is, the easier it might trigger the involuntary switching of attention to stimulus change.

Mismatch negativity (MMN) reveals sound grouping in the human brain.

To investigate a part of the structure of the memory trace, auditory event-related potentials (ERPs) were recorded from reading subjects while they were presented with two different stimulus-series simultaneously. A clear mismatch negativity (MMN) was obtained from each series, when the stimulus sequence consisted of a high-frequency series and a low-frequency series. Moreover, the MMN showed independent elicitation within each series. However, if the frequency range of one series overlapped with that of the other series, the amplitude of the MMN was prominently reduced, suggesting that the two processing functions indexed by MMN coexisted simultaneously in the preattentive acoustic system and were produced by the respective grouping of high-frequency tones and low-frequency tones.

Delivery and cytotoxicity of RS-1541 in St-4 human gastric cancer cells in vitro by the low-density-lipoprotein pathway.

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. RS-1541 has been shown to bind preferentially to plasma lipoproteins and to exhibit selective and sustained uptake by tumors in mice. To elucidate a mechanism of RS-1541 cytotoxicity, the cellular uptake and the cytotoxicity of a complex of RS-1541 with human low-density lipoprotein (RS-1541/LDL complex) were investigated in cultured St-4 human gastric cancer cells. Both the cellular uptake and the cytotoxicity of the RS-1541/LDL complex were greater in cells with higher LDL-receptor activities than in control cells. Excess amounts of LDL or 1 microM of monensin, a proton ionophore, significantly inhibited both the uptake and the cytotoxicity of the complex. Chloroquine, an inhibitor of lysosomal enzymes, decreased the intracellular level of rhizoxin liberated from RS-1541 and suppressed the cytotoxicity of the RS-1541/LDL complex. However, a detergent-aided solution of RS-1541 showed very low cellular uptake and cytotoxicity, irrespective of the LDL-receptor activities of these cells. These results demonstrate that the RS-1541/LDL complex is incorporated into the cells via the LDL receptor and that it manifests its cytotoxic activity after forming rhizoxin, the original antitumor agent, in the lysosomes.

Production, characterization, and application of monoclonal antibodies which distinguish four glucosyltransferases from Streptococcus sobrinus.

A 1,3-alpha-glucan synthase (GTF-I), a highly branched 1, 6-alpha-glucan synthase (GTF-U) and a 1,6-alpha-glucan synthase (GTF-T) were purified to near homogeneity from the culture fluid of Streptococcus sobrinus strain B13N (serotype d) and characterized. In addition, a crude preparation of a recombinant oligo-isomaltosaccharide synthase (rGTF-S) was prepared from a cell-free extract of Escherichia coli MD124 transformant. Using four homogeneous GTF preparations including previously purified rGTF-S as antigens for immunization, 11 murine hybridomas producing a monoclonal antibody (MAb) were established through the fusion of myeloma cells (P3X63-Ag8-U1) and spleen cells of immunized BALB/c mice. When the immunoreactivities of the resultant MAbs were tested, all five MAbs raised against GTF-I, all three MAbs raised against GTF-T, and two of three MAbs raised against GTF-U reacted specifically with the homologous enzyme alone, while one MAb (B86) raised against GTF-U cross-reacted strongly with all GTFs. Although no MAb monospecific for rGTF-S was obtained, precise recognition of GTF-S was possible using the nonspecific B86 antibody together with the MAbs monospecific for the three glucan synthases. Thus, a set of four typical MAbs (B17, B76, B19 and B86) were successfully used for the identification of gene products expressed in 24 previously constructed E. coli phage clones, and the findings suggested that six phage clones might express a gtfU gene encoding GTF-U which has not been hitherto isolated.

Real-time telemedicine in the clinical assessment of the ocular surface.

PURPOSE: To report the feasibility of real-time video and audio transmission of slit-lamp images of the eye using conventional telephone systems. METHOD: We analyzed the feasibility and benefits of telemedicine in the diagnosis and follow-up of ocular surface disorders in referral patients using a real-time audio-video system that functions through integrated services digital network lines at a transmission speed of 384 kilobytes per second. RESULTS: Real-time slit-lamp images obtained through integrated services digital network lines offered satisfactory visual resolution of the ocular surface for the diagnosis and follow-up of ocular surface and corneal disease, while simultaneous audio transmission allowed for direct communication with the patient and attending physician when necessary. CONCLUSION: Telemedicine using conventional telecommunication infrastructures offers sufficient information for the diagnosis and follow-up of ocular surface disorders in referral patients.

Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome.

PURPOSE: Ocular cicatricial pemphigoid and Stevens-Johnson syndrome often cause ocular damage and blindness not amenable to surgical correction. We present a new surgical technique for reconstructing affected eyes. METHODS: Fourteen eyes of 11 patients with cicatricial keratoconjunctivitis (seven patients with cicatricial pemphigoid and four with Stevens-Johnson syndrome; average age +/- S.D., 55.5 +/- 25.4 years) were treated with a combination of allograft limbal transplantation, amniotic membrane transplantation, and tarsorrhaphy, followed every 15 minutes by artificial tears derived from the patient's blood serum. Eight eyes required concomitant penetrating or lamellar keratoplasty because of corneal opacity. RESULTS: With a mean follow-up of 143 days (range, 10 to 608 days), we achieved successful ocular surface reconstruction in 12 eyes, with minimal recurrence of symblepharon. Failure occurred in two eyes (one each in 9- and 10-year-old boys) that developed corneal infiltration and vascularization. CONCLUSIONS: A combination of allograft limbal transplantation, amniotic membrane transplantation, and tarsorrhaphy, followed by the use of serum-derived tears, can reconstruct the ocular surface in most cases. Although in this study the follow-up period was short and relatively few patients were studied, this approach appears to offer an alternative to keratoprosthesis for treating severe cicatricial keratoconjunctivitis with dry eye.

Vestibular nerve injury as a complication of microvascular decompression.

We report a case of hemifacial spasm in which hearing was well preserved after microvascular decompression, but the vestibular nerve was injured selectively. We review the pathophysiological mechanisms relevant to this complication in the light of results of animal experimental studies we have conducted. In addition, we discuss the clinical significance of this particular type of cranial nerve injury.

Transplantation of amniotic membrane and limbal autograft for patients with recurrent pterygium associated with symblepharon.

AIM: Treatment of recurrent pterygium associated with symblepharon requires both suppression of fibrosis and reconstruction of limbal barrier. To achieve this, human amniotic membrane was transplanted and limbal autografts performed. METHODS: Four patients with severe symblepharon resulting from multiple surgeries for pterygium were treated. Human amniotic membrane was obtained at caesarean section and preserved until surgery. After excision of the fibrous tissues, the amniotic membrane was placed on the sclera, and a limbal autograft transplantation was performed using limbal tissues taken from the affected eye. RESULTS: Recurrence of symblepharon was not observed in any of the patients and significant suppression of the subconjunctival fibrosis was achieved. Ocular movement improved in all cases. Complete remission of pterygium regrowth occurred in three cases, and a slight (about 1 mm) recurrence occurred in one case. The limbal donor site showed the presence of mild depressions without the formation of pseudopterygium. CONCLUSION: Transplantation of human amniotic membrane with a limbal autograft appears to be a promising surgical treatment for reconstructing the ocular surface in patients with recurrent pterygium associated with symblepharon.

Hydroxypropyl methylcellulose for the treatment of severe dry eye associated with Sjögren's syndrome.

Our purpose was to evaluate the efficacy of a new formulation of methylcellulose, preservative-free 0.5% hydroxypropyl methylcellulose (HPMC), for the treatment of dry eye. In the clinical part of our study, two groups of dry-eye patients, those with Sjögren's syndrome (SS) and those without (non-SS), were treated topically with 0.5% HPMC and evaluated for symptoms, ocular surface vital staining, tear breakup time (BUT), and tear evaporation rate from the ocular surface at 40% ambient humidity (TEROS40). In the in vivo part of the study, rose bengal uptake was measured in human conjunctival epithelial cells, which were cultured and incubated with or without 0.5% HPMC. Although symptoms improved in both groups, rose bengal and fluorescein staining and BUT improved significantly only in the SS group. TEROS40 increased for 30 min after instillation of 0.5% HPMC, but not after use of 0.1% sodium hyaluronate or saline-based artificial tears. Rose bengal uptake by cultured conjunctival epithelial cells was blocked by 0.5% HPMC. These findings suggest that 0.5% HPMC provides long coverage of and protection for the ocular surface. Patients with severe dry eye, such as in SS, are good candidates for this treatment.

Nucleation of Astral-shaped Microtubules from Latex Beads Conjugated with MTOG Proteins.

A model system for the formation of astral-shaped microtubules (Mts) consisting of Latex beads (diameter of 0.2 mum), a protein fraction (p51) comprised of MTOGs (microtubule-organizing granules) and tubulin was established. The Latex beads were first incubated with p51 in the presence of GTP at 0 degrees C, then the purified tubulin dimer fraction was added, resulting in the formation of an aster-like structure observed by dark-field microscopy. On preincubation of the Latex beads with GDP instead of GTP, the asters did not form. Unhydrolyzable GTP analogues such as GTP-gammaS and GMP-PNP promoted aster formation as did GTP as observed by dark-field microscopy. Polylysine, as representative of basic polymers capable of binding to the surface of the Latex beads, promoted spontaneous Mt assembly, and eventually an aster-like structure without Latex beads in the center formed. Further analyses made by measuring the optical density of the aster-forming system produced the following results. 1) preincubation of the Latex beads with GTP or GMP-PNP supported Mt assembly from the beads showing profiles typical for a sitedirected assembly without the lag phase. 2) GTP-gammaS and GDP inhibited the turbidity increase of the system, causing a decrease in both the initial velocity and the level of steady state of Mt assembly. 3) Anti-p51 monoclonal antibody (HP1) substantially inhibited the aster formation, while anti-gamma-tubulin antibody only slightly inhibited assembly.