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.

Isolation of oral epithelial progenitors using collagen IV.

OBJECTIVE: Although oral mucosal epithelial stem cells are thought to reside in the basal layer, such cells have not yet been isolated. We isolated a population of rabbit oral epithelial progenitor cells containing putative stem cells. MATERIALS AND METHODS: Epithelial cells harvested from rabbit buccal mucosa were allowed to adhere to dishes coated with collagen IV for periods ranging from 10 min to 16 h. The properties of individual cell populations were evaluated using BrdU, Ki-67, integrin beta1, integrin alpha6 and keratin 13 using colony forming efficiency (CFE). RESULTS: Cells that adhered to collagen IV-coated dishes within 10 min were enriched about sixfold in terms of BrdU incorporation, Ki-67, integrin alpha6 and integrin beta1 were strongly expressed. Interestingly, keratin 13 was faintly expressed. The CFE of rapidly adherent cells among oral epithelial cells was significant compared with other cell populations. CONCLUSIONS: These results suggested that rabbit oral epithelial cells could be isolated by depending on adhesiveness to collagen IV, especially when segregated according to progenitor cell properties. Putative progenitor cells with stem cell properties were most effectively harvested within 10 min. Our separation procedure should be a useful tool with which to isolate epithelial stem cells for regenerative medicine.

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.

[The day surgery system].

The day surgery system was established in order to satisfy the demand of healthy patients, and we have performed more than 40 types of operation under this system so far. The critical path is an indispensable tool for ensuring safety and efficiency, and the care coordinator is the key person to manage the system for that purpose. In addition, the cost management technique focusing on DRG/PPS and risk management to increase patient satisfaction are particularly important because we must manage numerous surgical cases. The day surgery system is evaluated in relation to the primary education program for surgeons. Such education not only impacts medical and surgical skills, but also is a good opportunity for training in the basic bedside manner to offer the best service to patients.

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.

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.

Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation.

BACKGROUND: Conditions that destroy the limbal area of the peripheral cornea, such as the Stevens-Johnson syndrome, ocular pemphigoid, and chemical and thermal injuries, can deplete stem cells of the corneal epithelium. The result is scarring and opacification of the normally clear cornea. Standard corneal transplantation cannot treat this form of functional blindness. METHODS: We performed and evaluated 70 transplantations of corneal epithelial stem cells from cadaveric eyes into 43 eyes of 39 patients with severe ocular-surface disorders and limbal dysfunction. Medical treatment had failed in all patients. The patients had a mean preoperative visual acuity of 0.004 (only being able to count the number of fingers presented by the examiner) in the affected eyes, which satisfies the criteria for legal blindness in most countries. In 28 eyes, we also performed standard corneal transplantation. Stem-cell transplantations were performed as many as four times on 1 eye if the initial results were not satisfactory; 19 eyes had multiple transplantations. Patients were followed for at least one year after transplantation. RESULTS: A mean of 1163 days after stem-cell transplantation, 22 of the 43 eyes (51 percent) had corneal epithelialization; of the 22 eyes, 7 eyes had corneal stromal edema and 15 eyes had clear corneas. Mean visual acuity improved from 0.004 to 0.02 (vision sufficient to distinguish the largest symbol on the visual-acuity chart from a distance of 1 m) (P<0.001). The 15 eyes in which the cornea remained clear had a final mean visual acuity of 0.11 (the ability to distinguish the largest symbol from a distance of 5 m). Complications of the first transplantation included persistent defects in the corneal epithelium in 26 eyes, ocular hypertension in 16 eyes, and rejection of the corneal graft in 13 of 28 eyes. The epithelial defects eventually healed in all but two of the eyes. CONCLUSIONS: Transplantation of corneal epithelial stem cells can restore useful vision in some patients with severe ocular-surface disorders.

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.

Trabeculectomy with the use of amniotic membrane for uncontrollable glaucoma.

Although trabeculectomy is an established surgical technique for glaucoma, in some cases it does not achieve a good filtering effect despite the use of mitomycin-C (MMC). The authors have developed a new surgical technique for uncontrollable glaucoma that uses amniotic membrane to prevent postoperative adhesion of conjunctiva and sclera. They performed trabeculectomy with a limbal-based conjunctival flap using 0.4 mg/ml of MMC for 2 minutes. Amniotic membrane was then placed under the scleral flap and sutured using 10-0 nylon. Among 14 eyes of 13 patients who underwent this procedure, intraocular pressure was controlled to less than 20 mm Hg after surgery in 13 eyes, including 3 eyes that underwent a second surgery with the same technique and 2 eyes that underwent laser trabeculoplasty. The authors' results suggest that this technique is efficacious for the reduction of intraocular pressure in high-risk glaucoma patients.

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.

Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders.

PURPOSE: Although penetrating keratoplasty is an established surgical procedure, it often is ineffective for severe ocular surface diseases such as alkali burns or limbal deficiency. The authors have performed limbal allograft transplantation for the reconstruction of the corneal epithelium. METHODS: A total of nine patients (3 with chemical injury, 3 with limbal deficiency with unknown etiology, 2 with moderate ocular pemphigoid [OCP], and 1 with traumatic limbal deficiency) were treated by limbal allograft transplantation. Penetrating keratoplasties were performed in all patients with the exception of one with OCP. Patients received cyclosporine both systemically (10 mg/kg) and topically (0.05%) as well as high-dose intravenous dexamethasone (8 mg). RESULTS: The corneal epithelium was reconstructed in all patients, although two showed partial increased fluorescein permeability and two others required a second surgery. The other five epithelia remained clear at mean follow-up of 12.3 months, with two episodes of graft rejection which were controlled successfully by medication. CONCLUSIONS: Limbal allograft transplantation with intensive immunosuppression by cyclosporine and high-dose steroids appears to be a promising surgical intervention for the reconstruction of corneas affected by severe ocular surface disease.

Colorectal carcinoma: evaluation with ultrafast CT.

We investigated the value of ultrafast computed tomography for the preoperative assessment of colorectal carcinoma. Ultrafast CT demonstrated the primary tumor in 25 (89%) of 28 patients. Local tumor extension was detected with a sensitivity of 90%, a specificity of 78%, and an overall accuracy of 82%. Invasion into adjacent organs was detected with a sensitivity of 80%, a specificity of 91%, and an overall accuracy of 89%. Diagnoses of pericolic or perirectal lymph node metastasis were true-positive in ten, true-negative in 12, false-positive in one, and false-negative in two and those for distant lymph node metastasis were true-positive in four, true-negative in 24, and false-negative in one. Ultrafast CT may be more useful than conventional CT in evaluating local extension and pericolic or perirectal lymph node metastases because it avoids motion artifact.

Immunohistochemical and flow cytometric study of neuroendocrine carcinoma of the skin.

Immunohistochemical and flow cytometric analysis using formalin-fixed, paraffin-embedded sections was performed on 10 neuroendocrine carcinomas of the skin (NCS). Grimelius staining was positive in seven tumors. All tumors showed coexpression of CAM 5.2 and neuron-specific enolase with paranuclear dot-like or diffuse cytoplasmic reactivity. Neurofilament was positive in five cases, chromogranin in six, calcitonin and carcinoembryonic antigen in two each, and somatostatin and S-100 protein in one each. Eight primary lesions were diploid and the remaining two were aneuploid; however, two diploid NCS presented as aneuploid metastatic tumors. The follow-up periods ranged from 3 to 66 months (mean 13.6). Six patients died of metastatic diseases between 3 and 33 months (mean 9.2) after the diagnosis. There were no significant correlations among histologic features, DNA ploidy, S-phase fraction, and clinical outcome of the patients with NCS. These results indicate that a panel of antibodies may be required for immunohistochemical confirmation of neuroendocrine differentiation and that a flow cytometric analysis is not a good tool to predict the biologic behavior of NCS.

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.

[Two cases of failure of a programmable pressure valve].

Two cases of a programmable pressure valve trouble are reported. Case #1 was that of a 49-year-old male who had received a ventriculoperitoneal shunt (VPS) using a Sophy programmable pressure valve (Model S3) after the removal of the fourth ventricle ependymoma. After the VPS, the patient went through several episodes of complaining of headache due to the spontaneous movement of the rotor from the high pressure position to the medium or low pressure position. The symptom had disappeared after exchanging the valve for a new one and by setting the valve pressure at high. Examination of the removed valve revealed that the bending-degree of the spring tip was insufficient, and that the opening pressures at the medium and high pressure positions were both lower than the desired range. Case #2 was that of a 62-year-old female who had received a VPS for normal pressure hydrocephalus using a Sophy programmable pressure valve (Model S3) with the valve pressure set a high. However, her symptoms did not improve, so an attempt was made to change the valve pressure from the high pressure position to the medium pressure position with a ring magnet. But the rotor could not be moved. The valve was exchanged and the valve pressure was set at medium, and the symptoms of the patient improved postoperatively. Examination of the removed valve revealed that the movement of the rotor with a ring magnet was not able to be carried out smoothly.(ABSTRACT TRUNCATED AT 250 WORDS)

Atypical adenoma of the thyroid. A clinicopathologic and flow cytometric DNA study in comparison with other follicular neoplasms.

A clinicopathologic and DNA flow cytometric study was performed on seven patients (three males, four females) with atypical adenoma of the thyroid gland, using formalin-fixed paraffin-embedded tissues. The results were compared with those of 30 follicular adenomas and 13 follicular carcinomas. The patients ranged in age from 32 to 74 years (mean; 55.8 years), and the mean follow-up period was 11.0 years. All patients except two who died of other diseases were free of thyroid disease after initial surgery. It showed that there was no evidence of clinical cancer in this follow-up study of patients operated on for atypical adenomas. Four of the atypical adenomas were diploid, two were aneuploid, and one was tetraploid. Twenty-seven of the 30 follicular adenomas were diploid. Three patients with aneuploid follicular adenoma were free of disease. Of the 13 follicular carcinomas with a mean follow-up period of 6.9 years, five were diploid, seven were aneuploid, and one was tetraploid. Two patients with aneuploid follicular carcinoma and one with diploid carcinoma developed lung metastases, and one patient each with diploid and aneuploid follicular carcinoma died of disease. There was no significant correlation between histologic features, ploidy status and prognosis among follicular carcinomas. The results of this study suggest that DNA flow cytometric analysis is not a useful tool for predicting the clinical behavior of follicular neoplasms.

[Five cases of intracranial lipoma; CT and magnetic resonance images].

We encountered five cases of intracranial lipoma after introduction of MRI. They were located in the quadrigeminal plate, interpeduncular fossa, pineal region and two of them were found in the cerebellopontine angle, (although intracranial lipoma in this location has been reported to be extremely rare). MRI can precisely locate a small lesion that would be overlooked by CT scans. Operative treatment was performed in two symptomatic cases (CP angle and pineal lesions) and the tumors were subtotally resected. The symptoms of the patients disappeared postoperatively. This indicated that even subtotal removal can alleviate the symptoms of intracranial lipomas and that favorable results can be obtained.

[A case of malignant melanoma, axillary lymph node metastasis proved by fine needle aspiration biopsy cytology].

We report the case of a 57-year-old man with a malignant melanoma in the subunguinal region of his right thumb associated with the palpable axillary lymph nodes. Its metastasis was confirmed by fine needle aspiration biopsy cytology. Prognosis if a malignant melanoma is protean by its stage. Thus information regarding the axillary metastasis is mandatory for deciding the therapeutic plan. Fine needle aspiration biopsy cytology is a useful technique for diagnosing lymph node metastasis.