Applicability of buccal fat pad grafting for oral reconstruction.

This study evaluated the applicability of pedicled buccal fat pad grafting for the reconstruction of defects surgically created during oral surgery. A buccal fat pad graft was applied in 23 patients (5 males, 18 females; mean age 68.3 years) between 2003 and 2011. The graft was used to cover surgical defects of the palate, maxilla, upper gingiva, buccal mucosa, lower gingiva, oral floor, and temporomandibular joint region. Size of the surgical defects ranged from 15mm×12mm to 30mm×40mm; size of the buccal fat pad ranged from 15mm×12mm to 43mm×38mm. A pedicled buccal fat pad was prepared by incising the maxillary vestibule following primary surgery, and the surrounding connective tissue was preserved to supply nutrition to the pedicle during surgery. The buccal fat pad was placed on the raw surface of soft tissue or bone surface and sutured to the surrounding tissue of the defect. Complete epithelialization was observed within 4 weeks postoperatively. There were no complications or functional disorders during follow-up. Buccal fat pad grafting appears to be feasible for the reconstruction of surgically induced defects, and can be extended to the palate, mandible, mouth angle, and temporomandibular joint region.

Development of an in vitro model for radiation-induced effects on oral keratinocytes.

Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1alpha and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submucosal cellular components was a useful model.

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells.

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.

Isolation of human oral keratinocyte progenitor/stem cells.

Progenitor/stem cell populations of epithelium are known to reside in the small-sized cell population. Our objective was to physically isolate and characterize an oral keratinocyte-enriched population of small-sized progenitor/stem cells. Primary human oral mucosal keratinocytes cultured in a chemically defined serum-free culture system, devoid of animal-derived feeder cells, were sorted by relative cell size and characterized by immunolabeling for beta1 integrin, nuclear transcription factor, peroxisome proliferator-activated receptor-gamma, and cell-cycle analysis. Sorted cells were distinguished as progenitor/stem cells by functional assays and their ability to regenerate an oral mucosal graft. Small-sized cells demonstrated the lowest expression of peroxisome proliferator-activated receptor-gamma, the highest colony-forming efficiency, a longer long-term proliferative potential, an enriched quiescent cell population, and the ability to regenerate an oral mucosal graft, implying that the small-sized cultured oral keratinocytes contained an enriched population of progenitor/stem cells.

Cystic lymphangioma arising in the tip of the tongue in an adult.

Cystic lymphangioma is a benign malformation of the lymphatic channels. Most cystic lymphangiomas are present at birth and are usually diagnosed in infancy or early childhood. The head and neck region appears to be the favored site for cystic lymphangiomas. We present the first reported case of a cystic lymphangioma arising from the tip of the tongue in a 75-year-old male.

[A case of squamous cell carcinoma of the oral floor showing a complete response to oral administration of UFT].

A 73-year-old male with squamous cell carcinoma of the oral floor (T1N0M0), who had not consented to radical treatment was treated with UFT by oral administration alone. After commencement of the administration, tumor remission was observed at 2 weeks and disappeared clinically at 8 weeks. The total period of oral administration of UFT was twenty-three months, and the total dose was 186.5 g as tegafur. Major adverse effects were pigmentation of hands and liver dysfunction. He remains under observation, and there is no evidence of tumor recurrence.

Solid papillary carcinoma of the breast: report of two cases.

Two cases of solid papillary carcinoma of the breast were pathologically studied. Subjects included two female patients: a 76-year-old (Case 1) and a 43-year-old (Case 2). Both cases showed solid and papillary proliferation of spindle cells in expanded ducts, perivascular pseudorosettes, eosinophilic fine granules in an abundant cytoplasm, intracellular mucin production, and positivity for chromogranin A. Case 1 showed an aggregation form involving multiple ducts, admixture of signet-ring like cells, mucin pools, and small and large electron-dense granules and flocculent granules in electron microscopy. Case 2 showed a solitary and compact form in a dilated duct and an interlacing bundle pattern.

[Anesthetic management with veno-venous extracorporeal membrane oxygenator (VV-ECMO) in a patient with severe tracheobronchial stenosis].

We conducted an anesthetic management to perform tracheostomy and tracheolysis in a 33 year-old female with severe stenosis extending to the lower trachea and right main bronchus. The minimal diameter of the stenotic lesion of the trachea was 3 mm according to the preoperative examinations including tomography, CT scan and magnetic resonance imaging. Since there was a high risk of airway collapse during anesthetic induction that could have made ventilation impossible, we decided to apply VV-ECMO to support gas-exchange prior to anesthetic induction. Blood gas analysis showed good results, and sufficient oxygenation and stable circulation were achieved during surgical procedures. Total intravenous anesthesia with propofol and fentanyl could provide adequate depth of anesthesia during surgery and rapid recovery with good spontaneous respiration after the termination of the infusion. VV-ECMO was a useful method to support gas-exchange in a case not requiring circulatory assistance without uneven oxygenation sometimes observed in VA-ECMO.

Pelvic malignant mixed mesodermal tumor of uncertain origin: a case report.

Extra-uterine, and especially extragenital, malignant mixed mesodermal tumors (MMMT) are very rare. A large intrapelvic tumor resected from a 56-year-old woman was investigated with morphological and immunohistochemical methods. A large, soft and fragile tumor was located in the pelvic space. The tumor showed high cellularity and was biphasic; it consisted of an admixture of adenocarcinoma and various kinds of sarcomas. The latter were comprised of high-grade endometrial stromal sarcoma, pleomorphic sarcoma, and chondrosarcoma. The pleomorphic sarcoma showed a storiform pattern. The periodic acid-Schiff-positive eosinophilic hyaline droplets and globules in multinucleated giant cells revealed a typical ring-like or peripheral staining for alpha-1-antitrypsin and alpha-1 antichymotrypsin. We considered this case to be pelvic MMMT of uncertain origin, heterologous type.

[Anesthetic management for mitral valve replacement in a patient with mitral stenosis and dilated cardiomyopathy].

A 42-year-old man with dilated cardiomyopathy and rheumatic mitral stenosis underwent mitral valve replacement. Prior intravascular fluid administration and infusion of dopamine and dobutamine stabilized hemodynamics during the induction of anesthesia. High-dose fentanyl at pre-cardiopulmonary bypass period and circulatory assist with milrinone and intraaortic balloon pumping after the bypass enabled us to obtain stable hemodynamics. Preoperative dobutamine stress test provided us the effective information for circulatory management in this patient.

Simple and reliably sensitive diagnosis and monitoring of Philadelphia chromosome-positive cells in chronic myeloid leukemia by interphase fluorescence in situ hybridization of peripheral blood cells.

Philadelphia (Ph) chromosome or the bcr/abl fusion gene is the hallmark of chronic myeloid leukemia (CML) and serves as a prognostic marker during its treatment. Its detection has been primarily done by karyotype analysis of bone marrow cells. The major limitation of the karyotypic technique is an absolute need for metaphases, often difficult to obtain in an appropriate number in patients under therapy. Fluorescence in situ hybridization (FISH) is a sensitive and quantitative method to detect the bcr/abl fusion gene in cells in both metaphase and interphase. Using M-bcr and abl probes, we performed the interphase FISH in the peripheral blood of 30 healthy volunteers and in 20 hematologically normal bone marrow samples. False-positive cells were detected in 2.7 +/- 0.7% (mean +/- standard deviation) and 2.3 +/- 0.7% among 500 cells, respectively. Then we tested 31 patients with CML at various stages of disease on 50 occasions. Although there was a good correlation between the percentage of FISH-positive cells in the peripheral blood and that in the bone marrow (r = 0.977), between the percentage of FISH-positive cells in the peripheral blood and that of Ph chromosome in the bone marrow (r = 0.841), and between the percentage of FISH-positive cells and that of Ph chromosome in the bone marrow (r = 0.933), the limits of agreement in each group were not small, and thus the peripheral blood FISH test can not be interpreted as the same method with conventional karyotyping. Additionally, we could easily rule out CML in 15 individuals with leukocytosis without performing bone marrow aspiration. The present study indicates that FISH analysis in the peripheral blood is a simple and reliably sensitive test for the detection and quantitative monitoring of the M-bcr/abl fusion gene in CML in routine clinical practice, although this can not entirely replace karyotype analysis of bone marrow cells.

[Anesthetic management for pericardial fenestration in a hypertrophic cardiomyopathy (HCM) patient with massive pericardial effusion].

A 65 year-old male with HCM had progressively increased pericardial effusion. He also had atrial fibrillation (af), cardiac systolic dysfunction and chronic renal failure needing hemofiltration. Pericardial fenestration was carried out to improve diastolic function. Anesthetic management with fentanyl plus low-dose propofol infusion and postoperative analgesia with epidural morphine were effective for hemodynamic stability to prevent myocardial depression and to control ventricular response to atrial fibrillation. Intraoperative trans-esophageal echocardiography (TEE) monitoring was very useful for fluid therapy, inotropic support and estimation of systolic and diastolic function.

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia.

We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

Modulation of homeobox B6 and B9 genes expression in human leukemia cell lines during myelomonocytic differentiation.

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.

Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid.

Differentiation therapy with all-trans retinoic acid (ATRA) has marked a major advance and become the first choice drug in the treatment of acute promyelocytic leukemia (APL). However, patients who relapse from ATRA-induced complete remission (CR) have difficulty in obtaining a second CR with a second course of ATRA therapy alone. We tested the efficacy of a new synthetic retinoid, Am80, in APL that had relapsed from CR induced by ATRA in a prospective multicenter study. Am80 is approximately 10 times more potent than ATRA as an in vitro differentiation inducer, is more stable to light, heat, and oxidation than ATRA, has a low affinity for cellular retinoic acid binding protein, and does not bind to retinoic acid receptor-gamma. Patients received Am80, 6 mg/m2, orally alone daily until CR. Of 24 evaluable patients, 14 (58%) achieved CR. The interval from the last ATRA therapy was not different between CR and failure cases. The clinical response was well correlated with the in vitro response to Am80 in patients examined. Adverse events included 1 retinoic acid syndrome, 1 hyperleukocytosis, 9 xerosis, 8 cheilitis, 16 hypertriglyceridemia, and 15 hypercholesterolemia, but generally milder than those of ATRA, which all patients had received previously. Am80 is effective in APL relapsed from ATRA-induced CR and deserves further trials, especially in combination with chemotherapy.

Relapse in the external auditory canal of acute promyelocytic leukemia after treatment with all-trans retinoic acid.

A 54-year-old female was admitted to our hospital for gingival bleeding and was diagnosed as acute promyelocytic leukemia (APL). She received induction therapy according to the AML92 protocol of the Japan Adult Leukemia Study Group (JALSG) with all-trans retinoic acid (ATRA) plus chemotherapeutic agents. She achieved complete remission, but one year later had a relapse in her external auditory canal without leukemic cell in the bone marrow. Extramedullary disease is rare in APL. This case suggests the importance of careful observation for extramedullary relapse in patients who are treated with ATRA.

New retinoids and arsenic compounds for the treatment of refractory acute promyelocytic leukemia: clinical and basic studies for the next generation.

All-trans retinoic acid (ATRA) is a potent differentiation drug for acute promyelocytic leukemia (APL) and is now incorporated into first-line therapy. However, ATRA resistance has become a major clinical problem. This limitation has prompted the development of alternative agents with desirable pharmacologic properties. We describe (1) our recent clinical trial using the new synthetic retinoid Am80 to overcome acquired resistance to ATRA and (2) basic in vitro effects of arsenic trioxide, a possible alternative to ATRA, on APL cells. A total of 19 APL patients who had relapsed after ATRA-induced complete remissions (CRs) received 6 mg/m2 Am80 p.o. daily until CR; 11 (58%) patients achieved a CR between days 20 and 58 (median day 37). The in vitro sensitivity to Am80, based on PML immunostaining, correlated well with the clinical effect in all patients tested. All three patients whose blasts were sensitive to Am80 in vitro despite a poor response to ATRA achieved CRs. Thus, Am80 might be an effective compound for the treatment of refractory APL and is a promising alternative retinoid. Since arsenic compounds have reportedly induced CRs in APL patients in China, we studied the in vitro effect of arsenic and other metal ions on myeloid leukemia cell lines. The effects of arsenic were limited mainly to APL cells, and the arsenic concentration was critical for the APL cell line NB4: 1 microM As3+ induced time-dependent apoptosis, whereas 0.1 microM As3+ allowed partial NB4 cell differentiation. Arsenic trioxide was equally effective when used on ATRA-resistant NB4 cells. Among the clinical leukemia samples tested, the in vitro cytotoxic effects of As3+ were observed selectively in APL cells, regardless of their ATRA sensitivity. These data suggest that APL cells are sensitive to As3+ and that As3+ acts on APL cells via a different pathway to ATRA.

Muscle-type tropomyosin of sea urchin egg increases the actin-binding of nonmuscle-type tropomyosin.

Tropomyosin isoforms in eggs of several species of sea urchins are classified into two types, muscle-type and nonmuscle-type, based on their antigenicities. Their actin-binding abilities were investigated using muscle-type isoform (32K) and nonmuscle-type isoform (30K), which were purified by the method previously reported and separated by isoelectric focusing from eggs of sea urchin, Strongylocentrotus intermedius. Co-sedimentation assays revealed that 32K could stoichiometrically bind to actin filaments independently of the 30K, but 30K alone bound very poorly. The actin-binding of 30K was, however, considerably increased in the presence of 32K, and the molar ratio of the bound 30K and 32K was approximately 1:1. The increase in the actin-binding of 30K is probably caused by the interaction of 30K with 32K in a head-to-tail manner, as indicated by the higher specific viscosity of the mixture than that of 32K alone.