Involvement of tazarotene-induced gene 1 in proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells.

OBJECTIVE: Mesenchymal stem cells (MSC) have both self-renewal and multilineage differentiation potential, and bone marrow-derived MSC have been applied for tissue regeneration and repair. Although adipose tissue-derived MSC (ASC) have emerged as an alternative cell source, little information is available regarding the biologic difference between ASC derived from visceral and subcutaneous fat. Therefore, we aimed to compare the proliferation and gene expression profile of cultured human visceral ASC (VASC) and subcutaneous ASC (SASC), and to identify a novel gene involved in proliferation and differentiation of ASC. MATERIALS AND METHODS: We performed microarray analysis of cultured VASC and SASC, and investigated the role of tazarotene-induced gene 1 (TIG1), a most differentially expressed gene, in the proliferation and differentiation of ASC. RESULTS: SASC proliferated faster than VASC for over 10 passages, and TIG1 expression was consistently up-regulated in VASC of humans, rats and mice. Overexpression of the TIG1 gene in human SASC inhibited cell proliferation, whereas knockdown of TIG1 expression by siRNA promoted cell proliferation. In addition, overexpression of the TIG1 gene in SASC enhanced their differentiation into adipocytes, and promoted up-regulation of peroxisome proliferators-activated receptor gamma and CCAAT/enhancer binding protein alpha. On the other hand, TIG1 overexpression in SASC inhibited their differentiation into osteocytes and the expression of osteocalcin. CONCLUSION: TIG1 plays an important role in regulating proliferation and differentiation of ASC.

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation.

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.

A rat model of postthoracotomy pain: behavioural and spinal cord NK-1 receptor assessment.

PURPOSE: To develop a new rat model of postthoracotomy pain for investigating its mechanisms and clarifying neurochemical changes. METHODS: Male Wistar rats were randomly assigned to three groups that underwent either fourth and fifth intercostal nerve ligation, cutting of the fourth and fifth ribs, or a sham operation in which only pleura was cut. For behavioural response assessment during the following month, pinch and touch were used as mechanical stimuli, and acetone was used as a cold thermal stimulus. In addition, (125)I-substance P autoradiography was used to determine neurokinin (NK) receptor density in spinal cord laminae I and II at one to six weeks after surgery. RESULTS: In rats with nerve ligation, hypersensitivity to noxious and non-noxious stimuli continued throughout the month. The "mirror phenomenon" was observed. The lowest threshold was obtained in the dorsomedial portion of the T4 dermatome on the side of surgery. In rats with rib cutting, a lowered threshold to noxious and non-noxious stimuli was observed for two weeks. In rats with sham operations, hypersensitivity was seen only at postoperative day one. NK-1 receptor density on the side of operation increased significantly in rats with nerve ligation from day seven to 28. Receptor density was highest on day 14 (22.97 +/- 1.04 fmol x mg(-1) tissue vs. control, 16.22 +/- 0.43), representing a 50% receptor excess on the side of ligation compared to the contralateral side. CONCLUSION: Intercostal nerve damage induces long-term postthoracotomy pain and an increase of spinal NK-1 receptors in rats. This model may be useful for investigation of postthoracotomy pain.

[Detection of visceral ischemia with doplex scanning method of mesenteric arteries: in patients with acute type B aortic dissection].

Between March, 1997 and January, 1999, 11 patients with acute type B aortic dissection underwent doplex scanning evaluation of mesenteric arteries for the early detection of visceral ischemia. Peak systolic velocity (PSV) of the celiac artery (CeA) and superior mesenteric artery (SMA) was measured on their admission. Mean PSV of CeA in the non-ischemic group (8 patients) and in the ischemic group (3 patients) was 1.66 +/- 0.34 m/sec and 3.60 +/- 0.49 m/sec (p = 0.0481), respectively. Mean PSV of the SMA in the non-ischemic group and in the ischemic group was 1.93 +/- 0.52 m/sec and 3.33 +/- 0.37 m/sec (p = 0.00768), respectively. All patients with PSV of the mesenteric arteries above 3.00 m/sec presented visceral ischemia that required emergency operation. If PSV of the mesenteric arteries exceeds 3.00 m/sec, urgent surgical repair should be considered.

[Endoventricular circular patch plasty (dor operation) for postinfarction left ventricular aneurysm and ischemic cardiomyopathy].

The determination of purse-string suture line is one of the most important point in endoventricular circular patch plasty (Dor operation) for postinfarction left ventricular aneurysm (LVAN), especially for ischemic cardiomyopathy (ICM). We suggest following three points to decide appropriate suture line. First, the purse-string suture on the basal side should be placed on the 1-2 cm level under diagonal branch. Secondly, lateral wall should not be over excluded to maintain left ventricular function. And the third, akinetic or dyskinetic lesion of apex and septal wall should be excluded as much as possible. Nine cases of five LVAN and four ICM were underwent Dor operation in our institute from Dec. 1999 to Jan. 2000. All patients were weaned from cardiopulmonary bypass easily except one patient, who was operated under IABP support, because of his preoperative severe heart failure. All patients recovered well without any serious complications and postoperative left ventricular graphies were satisfactory. Left ventricular ejection fraction and stroke volume index were increased from 34 +/- 17 to 55 +/- 16% and from 38 +/- 7 to 47 +/- 6 ml/m2, end-diastric and systric volume index decreased from 141 +/- 37 to 88 +/- 19 ml/m2 and from 96 +/- 41 to 41 +/- 23 ml/m2 respectively. The Dor procedure adopted our idea led to satisfactory result in hemodynamic and also in morphologic study.

Determination of picomolar levels of iron in seawater using catalytic cathodic stripping voltammetry.

A new procedure for the direct determination of picomolar levels of iron in seawater is presented. Cathodic stripping voltammetry (CSV) is preceded by adsorptive accumulation of the iron(III)-2,3-dihydroxynaphthalene (DHN) complex from seawater, containing 20 microM DHN at pH 8.0, onto a static mercury drop electrode, followed by reduction of the adsorbed species. The reduction current is catalytically enhanced by the presence of 20 mM bromate. Optimized conditions include a 60-s adsorption period at -0.1 V and a voltammetric scan using sampled dc modulation at 10 Hz. In these conditions, a detection limit of 13 pM iron in seawater was achieved which can be lowered further by extending the adsorption time to 300 s. The new catalytic CSV method is approximately 5 times more sensitive than existing CSV methods and was tested on samples from the Atlantic Ocean.

Twenty-seven-year follow-up of arrhythmogenic right ventricular dysplasia.

This case report describes clinical features, especially of surface ECG changes, observed for 27 years in a patient with arrhythmogenic right ventricular dysplasia (ARVD). The course of this patient was characterized by progressive deterioration of right ventricular function and progression of delayed potentials (so-called epsilon waves) following QRS complexes. However, the relation between ventricular arrhythmias and ECG changes or the degree of right ventricular abnormality was difficult to discern.

[Minimally invasive direct coronary artery bypass grafting using subxiphoid approach].

We report twelve cases of minimally invasive coronary artery bypass grafting via small laparotomy using right gastroepiploic artery (RGEA) to the right coronary artery. Seven patients were operated on without partial sternotomy (N group), and lower partial sternotomy with Octopus 2 stabilization system was used in five of the cases (P group). Postoperative Doppler echographic imaging or angiographic study showed all RGEA grafts were functioning well in P group. However, graft occlusion was observed in two of the cases in N group. Inferior partial sternotomy combined with Octopus 2 stabilization system produce a clear surgical view, which avoids of both full-sternotomy and cardiopulmonary bypass. This method might be useful especially for reoperation.

A novel human HER2-derived peptide homologous to the mouse K(d)-restricted tumor rejection antigen can induce HLA-A24-restricted cytotoxic T lymphocytes in ovarian cancer patients and healthy individuals.

A mouse HER2-derived peptide, HER2p63 (A) (TYLPANASL), can induce K(d)-restricted mouse cytotoxic T lymphocytes (CTL) and also function as a tumor rejection antigen in an in vivo assay. Since the anchor motif of mouse K(d) for peptide binding has much similarity to that of human HLA-A2402, we asked if human HER2p63 (T) (TYLPTNASL) could induce HER2-specific CTL in HLA-A2402-positive individuals. Peripheral blood mononuclear cells (PBMC) of HLA-A2402-positive individuals were sensitized in vitro with HER2p63-pulsed autologous dendritic cells prepared from PBMC. CTL clone derived from these specifically lysed HER2-expressing cell lines bearing HLA-A2402. Cytotoxic activity of the CTL clone against the HER2-expressing cell line bearing HLA-A2402 was blocked by antibodies against CD3, CD8, HLA-A24 or MHC class I, and was also inhibited by the addition of excess HER2p63-pulsed C1R bearing HLA-A2402. Killer cells were generated from PBMC of seven healthy individuals and five ovarian cancer patients, all of HLA-A2402 type, by in vitro sensitization with HER2p63-pulsed autologous antigen presenting cells. These killer cells selectively lysed HER2-expressing SKOV3 transfected with HLA-A2402 cDNA, indicating high immunogenicity of HER2p63 in all 12 individuals examined.

Occupational asthma due to exposure to African cherry (Makore) wood dust.

A 35-year-old man who had been a carpenter and a cabinet worker for over 15 years, was referred to our clinic with a 4-month history of cough, chest tightness and difficulty in breathing which occurred within minutes of exposure to African cherry wood (Makore). He developed a dual asthmatic reaction on specific challenge test with an extract of African cherry wood dust. Thus, the diagnosis of occupational asthma due to exposure to African cherry wood dust was confirmed by the specific challenge test. The mechanism of asthma due to African cherry wood dust exposure is not clear.

[A recurrent case of amebic liver abscess seventeen years after the first occurrence].

A 49-year-old male who had been diagnosed as having amebic liver abscess when he was 32-year-old was admitted to our hospital with fever and watery diarrhea. Ultrasonography and CT examination demonstrated a solitary abscess in the right lobe of the liver. Cysts of Entamoeba histolytica were detected in the stool and an aspiration of the liver abscess looked like anchovy paste. Serum amebic antibody by the IFA method was positive and the case was diagnosed as amebic liver abscess. The patient was treated with metronidazole, and percutaneous transhepatic abscess drainage was performed. The liver abscess decreased remarkably in size and serum amebic antibody was negative after the treatment. Recurrence of amebic liver abscess is rare and we report this case with some literature.

Serum total bile acid level as a sensitive indicator of hepatic histological improvement in chronic hepatitis C patients responding to interferon treatment.

BACKGROUND AND METHODS: Serum total bile acid (TBA) levels are used clinically as a sensitive and reliable index of hepatobiliary diseases. In the present study, to assess the clinical usefulness of determining TBA in interferon (IFN)-treated patients, changes in liver function test values, including TBA and liver histology, were examined in 36 chronic hepatitis C patients for 3 years after a sustained response to IFN treatment. RESULTS: Alanine aminotransferase and gamma-glutamyl transpeptidase values significantly decreased during the period of IFN treatment compared to the same measures before IFN treatment. Albumin, cholinesterase, total cholesterol and platelet count values temporarily decreased during IFN treatment, then increased significantly and reached a plateau 6-12 months after the end of IFN treatment. The zinc sulphate turbidity test and TBA values began to decrease during IFN treatment and continued to decrease during the 3-year follow-up period. The histological activity index of the liver (Knodell's score) significantly decreased, whereas the staging score was unchanged 1 year after the end of IFN treatment. In patients who had a TBA value > 10 micromol/L before IFN treatment, a significant correlation was observed between the decrease of TBA and liver histology grading score. CONCLUSIONS: A decrease in serum TBA level reflected histological improvement in the liver more precisely than did changes in the other liver function test values following IFN therapy.

[Aortic valve replacement with a stentless porcine aortic root bioprosthesis].

From April, 1999 to November, 1999, 8 patients underwent the aortic valve replacement using Medtronic Freestyle aortic root bioprosthesis. Mean age was 67.8 years old. Aortic valve etiology was as follows, AS: 1 case, AR: 2 cases, ASR: 3 cases, IE: 1 case, mechanical valve malfunction due to subvalvular pannus: 1 case. The implanted valve size was 25: 2 cases, 23: 3 cases, 21: 1 cases, 19: 2 cases. Surgical technique was subcoronary: 7 cases, full-root: 1 case. Post-op 2D-echo revealed tribial aortic valve incompetence in two cases because of size discrepancy between the aortic annulus and the ascending aorta. Pacemaker implantation was needed in 1 case because of the complete A-V block. In comparison with 5 cases of aortic valve replacement with Hancock II performed in same period, there were no significant differences about the implanted valve size and mean systolic pressure gradient, but the effective orifice area in Freestyle cases was significantly larger than Hancock II cases.

Analysis of liquid accumulated in the distended capsular bag in early postoperative capsular block syndrome.

PURPOSE: To analyze the transparent liquid between the posterior lens capsule and the posterior chamber intraocular lens (PC IOL) in early postoperative capsular block syndrome and discuss the mechanism of posterior capsule distention. SETTING: Department of Ophthalmology, Tokyo University School of Medicine, and Tokyo Research Institute, Seikagaku Corporation, Tokyo, Japan. METHODS: This study evaluated 3 cases of capsular block syndrome presenting with transparent liquid in the distended capsular bag 1 day after cataract surgery. The transparent liquid material between the posterior capsule and PC IOL was aspirated and analyzed using high-performance liquid chromatography (HPLC). Also, sodium hyaluronate was diluted using a dialyzer to determine whether the aqueous humor was drawn into the capsular bag by an osmotic gradient across the capsule. RESULTS: The elution time of the samples was almost the same as that of sodium hyaluronate 1.0% (Healon) The concentration of the samples ranged from 3.29 to 9. 01 mg/mL by HPLC analysis. The sodium hyaluronate absorbed the physiological salt solutions through the dialyzer and expanded to 1. 9 times its original volume. CONCLUSIONS: These results indicate that the main ingredient of the transparent liquid in capsular bags is sodium hyaluronate and that the distention is caused by aqueous humor being drawn into the capsular bag by an osmotic gradient across the capsule when the capsulorhexis diameter is smaller than that of the PC IOL and by viscoelastic material retained and trapped in the bag intraoperatively.

Effect of anti-inflammatory agents on corneal wound-healing process after surface excimer laser keratectomy.

PURPOSE: To investigate the effect of anti-inflammatory agents on conjunctival inflammation and corneal haze formation after excimer laser keratectomy. SETTING: Department of Ophthalmology, University of Tokyo Faculty of Medicine, Tokyo, Japan. METHODS: After excimer laser keratectomy was performed in 21 rabbits (42 eyes), saline, betamethasone 0.1%, or diclofenac 0.1% was topically applied 6 times a day for 4 weeks and then 3 times a day for 8 weeks. The degree of conjunctival inflammation was determined 1, 2, 3, and 7 days after the keratectomy. The degree of corneal haze was quantitatively measured using a digital analyzer before and once a week after the keratectomy. The expression of type IV collagen in the corneas at baseline and 4 and 12 weeks after the keratectomy was examined immunohistochemically. RESULTS: Compared with saline, betamethasone and diclofenac significantly decreased early-phase conjunctival inflammation. Betamethasone significantly inhibited corneal haze formation compared with saline at 3 to 5 and 8 to 12 weeks. Diclofenac did not inhibit corneal haze formation significantly. Although betamethasone tended to be more effective in inhibiting corneal haze formation and deposition of type IV collagen than diclofenac, there was no statistical difference between the 2 anti-inflammatory agents. CONCLUSION: Topically applied betamethasone effectively suppressed corneal haze formation after excimer laser keratectomy. Diclofenac was not statistically effective in inhibiting corneal haze formation.

Epidural block with mepivacaine before surgery reduces long-term post-thoracotomy pain.

PURPOSE: To examine the effect of continuous epidural block initiated before thoracic surgery upon early and long-term postoperative pain. METHODS: In a double-blind study, 70 patients scheduled for thoracic surgery under general anesthesia were assigned randomly to receive continuous epidural block with mepivacaine 1.5% initiated either 20 min before surgical incision (Pre group) or at completion of surgery (Post group). In both groups the initial dose was 4 ml, followed by a continuous infusion at 4 ml x hr(-1) until 72 hr after operation. Indomethacin suppositories, 50 mg, were administered on request as supplementary analgesics. Visual analogue scale at rest was assessed four hours after operation, and then every 24 hr after operation on postoperative days 1 through 7, and also days 14 and 30. At three and six months after operation, all patients were interviewed by telephone with respect to postoperative pain. The most severe pain was assessed using modified numerical rating scale. RESULTS: By a visual analogue scale, postoperative pain was less in the Pre group than in the Post group at four hours, two and three days after operation (P < 0.05). By a numerical rating scale six months after operation, pain was less in the Pre group than in the Post group (P = 0.015). The percentage of pain-free patients was higher in the Pre group than in the Post group at three (P = 0.035) and six (P = 0.0086) months after operation. CONCLUSION: Continuous epidural block initiated prior to surgery may reduce long-term post-thoracotomy pain.

NF-kappaB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications.

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.

[Labyrinthine fistulas in cholesteatoma].

The purpose of the present study was to investigate the clinical features of cases of cholesteatoma with labyrinthine fistulas, and in particular the pre and post-operative bone-conduction (BC). Cholestatoma patients with bone erosion or a defect found in the first stage operation were analyzed. The operations were conducted between 1992 and 1996. The patients were classified into four types, I, IIa, IIb, and III, according to Dornhoffer and Milewski's classification, which is based on different stages in the bone defect. A type I fistula is an erosion of the bony labyrinth with an intact endosteum. Type IIa is accompanied by an opened perilymphatic space with undisturbed perilymph while type IIb has a disturbed perilymph. A Type III fistula is an opened perilymphatic space with a disturbance of the underlying membranous labyrinth. Only 24 patients with type II and type III fistulas were included in this study. The location of the fistulas was the semicircular canals (SCCs) or/and the vestibula in 21 patients and in the cochlea in 3 patients. We examined the fistula by high-resolution computed tomography scan (CT scan) with 1 mm slice and 1 mm width axial-sections in 14 patients. A bone defect in the labyrinth was detected in 10 cases (71.5%) pre-operatively. Pre-operative BC was worse in the patients with cochlear fistulas than in those with fistulas located in SCCs or the vestibula. Within this latter group there were 13 type IIa (group IIa), patients and 8 type IIb or III (group IIb or III) patients. However, there was no difference in the pre-operative BC between these two sub-groups Tympanoplasty was conducted in all 24 patients. The postoperative BC of group IIa and group IIb or III were compared. Two of the 13 patients in group II a (15.4%) and 3 of 8 in group IIb or III (37.5%) had a deteriorated postoperative BC. Statistical analysis revealed that the postoperative BC was more inclined to become worse in patients with advanced stage IIb or III fistulas.

Creating cataract in a pig eye.

The purpose of this study was to create a uniform, reproducible hardened lens nucleus and an anterior capsule in a pig eye that approximate those in the human eye for use in cataract surgery training. The lens nuclei was hardened and anterior capsule elasticity was reduced in pig eyes by injecting the lens with mixtures of formalin and alcohol (ethanol, 2-propanol, or both). A 30 gauge needle was used to pierce the lens 3.0 mm from the limbus. Then, 0.2 mL of the formalin and alcohol mixture was injected. The most effective mixture was formalin, ethanol, and 2-propanol in the ratio 4:3:3. Fifteen minutes after this mixture was injected, the pig eye had nuclear cataract with grade III hardness and an anterior capsule that approximated the capsule in a human cataractous eye. The cataract created by this method will be useful in teaching continuous curvilinear capsulorhexis, divide and conquer and phaco chop phacoemulsification, and nuclear hydrodelineation and rotation.

Three-dimensional organization of collagen fibrils during corneal stromal wound healing after excimer laser keratectomy.

PURPOSE: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze. SETTING: University of Tokyo Hospital, Tokyo, Japan. METHODS: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microscopy after re-embedding. RESULTS: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased. The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy. CONCLUSION: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.