Natural history of lymph pumping pressure after pelvic lymphadenectomy.

Lower limb lymphedema is difficult to prevent and diagnose early because its natural history is unclear. Therefore, the aim of this study was to clarify its pathogenesis and to identify risk factors that may lead to early diagnosis. In 29 patients, aged 25 to 74 years with cervical, uterine, or ovarian cancer who underwent pelvic lymphadenectomy, indocyanine green fluorescence lymphangiography was performed with an infrared camera system, and lymph pumping pressure was measured indirectly preoperatively, and one, two, three, and six months postoperatively. Of these 29 patients, 22 (75.9%) completed the examinations. In the non-lymphedema group, the average lymph pumping pressure did not change significantly at postoperative follow-up compared with preoperative values. On the other hand, lymph pumping pressure increased at various time points in five patients who developed early lymphatic changes with dermal diffusion at the level of the proximal femur. An increase in lymph flow path resistance due to pelvic lymphadenectomy resulted in an initial increase in lymph pumping pressure, followed by a subsequent decrease, in the early lymphatic changes group. This trend in the pressure change signifies that the lymph vessels became dysfunctional as they were overwhelmed by the overload condition and this feature may be a clinically useful signal for the early diagnosis of developing lymphedema.

Effect of JNK inhibitor during islet isolation and transplantation.

Although islet transplantation has been remarkably improved by the Edmonton protocol, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases; many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hours. In this study, we show the effect of a JNK inhibitor during islet isolation and transplantation. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet cell apoptosis and improves islet graft function. These findings suggest that inhibition of JNK could prevent the impairment of islet cells and improve outcomes after pancreatic islet transplantation.

Continuous, but not occasional, oral ethanol intake reduces the success of intraportal transplanted islets of Langerhans: an experimental study.

BACKGROUND: Islet transplantation is gradually gaining acceptance for the treatment of type 1 diabetes mellitus. One of the unknown questions is alcohol intake; we have prohibited alcohol intake after islet transplantation although there is no solid evidence to support this. MATERIALS AND METHODS: In this study, we employed a mouse model to determine the effect of oral ethanol intake on transplanted islets. Either 500 or 150 islets were infused selectively into the right liver lobe of chemically induced diabetic mice. After transplantation, mice were orally administered either water (as a control) or various concentrations of ethanol for 14 consecutive days occasionally (once per day) or continuously (all intake was alcohol). Blood glucose levels were monitored and oral glucose tolerance tests (OGTT) performed. RESULTS: After 500 islets had been transplanted, all mice were cured from diabetes, but the continuous alcohol intake group showed significantly prolonged time to diabetes reversal and significantly lower glucose clearance rates by OGTT compared with the control group. After 150 islet transplantations, the diabetes cure rate in the continuous alcohol intake group was significantly lower than the control group (continuous alcohol vs control: 3/8 vs 11/12, P < .05). However, the occasional alcohol intake group showed no difference from the control group, even with as few as 150 islets transplanted per mouse. CONCLUSION: The present results demonstrated that continuous but not occasional alcohol intake reduced the success of intraportal islet transplantation.

Kyoto islet isolation method: the optimized one for non-heart-beating donors with highly efficient islet retrieval.

The availability of pancreata for clinical cadaveric islet transplantation is restricted to non-heart-beating donors (NHBDs) in Japan. This forced us to modify the current standard islet isolation protocol that was made up for brain-dead donors and make it suitable for NHBDs. The Kyoto islet isolation method is the one with induction of several steps based on the ideas both already reported literally and invented originally by ourselves. Using this islet isolation method, we isolated islets from 13 human pancreata of NHBDs and transplanted 11 preparations to six type-1 diabetic patients. The rate to meet release criteria of Edmonton protocol was 84.6%. Establishment of this method allowed us to begin a clinical islet transplantation program in Japan and to continue to perform the preparation of islets from NHBDs with high rate to meet the release criteria of the Edmonton protocol.

In situ cooling of pancreata from non-heart-beating donors prior to procurement for islet transplantation.

BACKGROUND: The specific aim of this study was to develop an effective technique for pancreas procurement for islet transplantation from a non-heart-beating donor (NHBD). METHODS: Between January 2004 and November 2004, 11 human pancreata were procured and processed for islet isolation at a cell processing center. After confirmation of brain-death status, a double-balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for kidney procurement. RESULTS: Warm ischemic time was controlled with the modified in situ regional cooling system at 6.0 +/- 0.9 minutes (mean +/- SE). The operations for procurement of the kidneys and pancreata lasted 48.1 +/- 3.6 minutes and 9.9 +/- 4.8 minutes, respectively. The islet yield per isolation was 396,767 +/- 142,842 IE (islet equivalents). Ten of the 11 cases met the criteria for pancreatic islet transplantation based on the Edmonton protocol. CONCLUSIONS: We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from an NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

Analysis of large-scale nonhuman primate islet isolations.

AIMS: It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. METHODS: Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. RESULTS: Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. CONCLUSIONS: TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.

Simple evaluation of engraftment by secretory unit of islet transplant objects for living donor and cadaveric donor fresh or cultured islet transplantation.

BACKGROUND: Evaluation of engraftment is important to assess the success of islet transplantation. Recently we developed secretory unit of islet transplant objects (SUITO) index for simple evaluation of engraftment. Assuming that normal subjects aged <40 years have 100% pancreatic beta-cell function, SUITO index was calculated by the formula: 1500 x fasting C-peptide immunoreactivity [ng/dL]/(fasting blood glucose [mg/dL] - 63). In this study, we compared the efficacy of islet transplantation from cadaveric and living donors using the SUITO index. METHODS: We performed eight islet transplantations with non-heart-beating donors (NHBDs) into five patients. Two patients received fresh islets once, one patient received fresh islets twice, one patient received cultured islets once, and one patient received cultured islets twice plus fresh islets once. In addition, one patient received fresh islets from a living donor. We calculated the SUITO index from postoperative days 3 to 30 for each case. RESULTS: Mean SUITO index after one fresh islet transplant was 11.7 +/- 1.0, after two fresh islet transplants was 28.5 +/- 3.4, after one cultured islet transplant was 2.1 +/- 0.4, after two cultured islet transplant was 12.1 +/- 1.9, and after two cultured islet transplant plus one fresh islet transplant was 26.7 +/- 1.7. The mean SUITO index after single living donor islet transplant was 40.7 +/- 2.6, which was significantly higher compared with all other groups. Insulin independence was obtained when the SUITO index was >26, which might reflect that 26% beta-cell mass was required for insulin independence. CONCLUSION: SUITO index is useful to evaluate islet engraftment and to predict the possibility of insulin independence.

Insulin independence of unstable diabetic patient after single living donor islet transplantation.

BACKGROUND: Current success in islet transplantation will lead to a donor shortage. Living donor islet transplantation could be an alternative approach to expand the potential donor pool. In this study we describe the first successful living donor islet transplantation for unstable diabetes, performed at Kyoto University Hospital on January 19, 2005. METHODS: The donor was a healthy 56-year-old woman and mother of the recipient. The recipient was a 27-year-old woman with insulin-dependent diabetes since the age of 15 years. She experienced frequent hypoglycemic unawareness episodes. Her blood glucose concentration was difficult to control and C-peptide level was negative after glucagon stimulation. She needed an average 28 of units of insulin per day. The donor underwent a distal pancreatectomy and islets were isolated from the resected pancreas graft. The total islet yield was 408,114 islet equivalents and isolated islets were immediately transplanted into the recipient's liver. RESULTS: After transplant, the blood glucose level of the recipient was tightly controlled without hypoglycemic episodes. She was discharged on day 37 with a normal oral glucose tolerance test (OGTT). The recipient remained insulin-independent for >3 months, since day 22 posttransplant. The donor's postoperative clinical course was uneventful. She was discharged on postoperative day 18 and returned to her job within 1 month. CONCLUSIONS: We report the first successful living donor islet transplantation for the treatment of unstable diabetes. We believe that living donor islet transplantation may become an option in the treatment of insulin-dependent diabetes.

Rapidly functional immobilization of immortalized human hepatocytes using cell adhesive GRGDS peptide-carrying cellulose microspheres.

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay.

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.

Mutagenic activities of gentisin and isogentisin from Gentianae radix (Gentianaceae).

The mutagenic activities of 2 hydroxyxanthones, gentisin and isogentisin, obtained from the methanol extract of Gentianae radix (Gentianaceae) were investigated. The methanol extract of Gentianae radix, which showed mutagenicity in the Ames test in Salmonella typhimurium strain TA100 with S9 mix, was fractionated by column chromatography on Sephadex LH-20, and the fractions were purified by preparative TLC and column chromatography on polyamide. 2 mutagenic materials thus obtained, S1 and S2, each gave a single band on TLC. Identification of S1 and S2 was accomplished by comparing the analytical (mps, elementary analyses) and spectral (UV, IR, mass, NMR) results for S1 and S2 with literature data for gentisin and isogentisin. At doses below 10 micrograms, S1 (gentisin) and S2 (isogentisin) had similar specific mutagenic activities. At doses of over 10 to 50 micrograms, the mutagenic activities of S2 and S1 were 19.1 and 6.94 revertants per microgram respectively. This much lower activity of S1 than S2 may be a result of its poor solubility owing to the presence of the OMe group at C-3. The combined yield of S1 and S2 was about 76 mg (40 mg as S1 and 36 mg as S2), which accounted for 76% of the content of mutagenic compounds (100 mg) estimated roughly from the total mutagenic activity in the extract of the starting materials (100 g).

Tympanograms in ears with small perforations of the tympanic membranes.

It has been believed that in an ear with a perforation, the tympanogram becomes a straight line due to the lack of compliance change in response to the pressure change in the external auditory meatus. Recently, however, we found two types of tympanogram with distinctive characteristics in ears with small perforations. In the first tympanogram type, when the external auditory meatus pressure was changed in the decreasing direction, the compliance peak was formed in the positive-pressure area, whereas it was formed in the negative-pressure area when it was changed in the increasing direction. The difference of the pressure at which compliance peak was obtained was great. The second tympanogram type was unique, with multiple notches similar to those of electronystagmographic recordings of the jerky nystagmus. Knowledge of these tympanograms helps in recognizing small tympanic-membrane perforations.

Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.

Partial hepatectomy and subsequent radiation facilitates engraftment of mouse embryonic stem cells in the liver.

For liver-targeted regenerative medicine, embryonic stem (ES) cell-derived hepatocyte-like cells proffer great expectation. In vitro exposure to a combination of various growth factors, such as hepatocyte growth factor and fibroblast growth factor-4, as well as cytokines, leads to differentiation of ES cells into hepatocyte-like cells. We sought to determine the in vivo environment that allowed engraftment of ES cells transplanted to the liver. Thus, we examined the effect of partial hepatectomy (50%) (PHT) and subsequent radiation (RT) of the male Balb/c mouse host liver on ES cell engraftment. ES cells (5 x 10(6)) derived from 129Sv mice were transplanted into the residual liver. The controls were ES cells transplanted into a normal liver. Bromo-deoxy-residine (BrdU)-uptake was performed to evaluate the effect of hepatectomy and RT on hepatocyte regeneration. Mouse ES cells engrafted, forming teratomas in the normal liver without showing any mononuclear infiltration. A liver modified by PHT and RT facilitated engraftment of mouse ES cells compared with a normal liver. Hepatic RT significantly suppressed hepatocytic uptake of BrdU.

Transduction of human islets with the lentiviral vector.

Establishment of an efficient gene delivery system for human pancreatic beta cells is important for the development of diabetes-targeted cell therapies. The human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector is well documented to be an effective gene transfer tool for various types of cells. Thus, we examined the efficiency of lentivirus-mediated gene delivery for human islets. Human islets were isolated using defined protocols for enzymatic dissociation and purification using discontinuous Ficoll gradients with a refrigerated Cobe 2991 machine. Isolated islets were shipped to Japan, cryopreserved for 3 months, and then subjected to transduction. A vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector LtV-NLS/LacZ was produced in 293T cells under the Fugene6 method. 804G extracellular matrices were applied for monolayer formation of islets. Detection of NLS/LacZ expression was performed using X-gal staining. Lentiviral transduction was effective in these monolayer islets.

Additional O antigens of Vibrio fluvialis and Vibrio furnissii.

A total of 297 strains of Vibrio fluvialis and Vibrio furrnissii, which were collected from various countries for the past 15-year period of 1984-1998, were serogrouped. Of those examined, 239 strains of V. fluvialis and V. furnissii were classified into 29 known O serogroups; 9 strains were found to belong to R-form cultures, and the rest of the 49 strains could not be serogrouped. Of those serologically untypable strains, 26 novel O serogroups (O36 to O61) were established and added to our reference of the V. fluvialis and V. furnissii antigenic scheme. As all antisera against the O reference strains of the organisms contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera were absorbed with the reference rough strain, V. fluvialis GF25.

Middle ear inflation as a treatment for secretory otitis media in children.

Clinical evaluations of middle ear inflation for secretory otitis media (SOM) were performed with special emphasis on the influence of seasonal and aging factors. One hundred and forty-nine children between the ages of 3 and 9 years (227 ears) were all diagnosed as SOM by pneumatic-otoscopic findings, and type B tympanogram (TG) at 3 weeks or more after the onset of acute SOM or the initial observation of SOM. Middle ears were inflated by Politzer's method or by our modified method once or twice each week for 2 months. After inflation, TG displayed two different time sequences: one group changed to the A or C type immediately after inflating the ear one or more times, but usually returned gradually to the B type (TG-improved group): and the other group remained without any changes (TG-unchanged group) for the duration of this study. The healing rate in the TG-improved group was significantly higher than in the TG-unchanged group at the 2-month endpoint. The cure rate of SOM was significantly higher in spring than in autumn in the TG-unchanged group but not in the TG-improved group. There were almost no differences between the healing rates in the 3-5 and 6-9 year-old children. When a TG-unchanged ear is found in autumn during the 2-month inflation treatment, more careful and forcible treatments should be introduced later, especially to children between the ages of 3 and 9 years.

Forward-backward tracing tympanometry in the diagnosis of middle ear pressure.

Two tympanograms (TG) were routinely recorded on each ear by changing the pressure in the external auditory meatus (EAM), one in the decreasing (forward tracing: TG-F) followed by the other in the increasing direction (backward tracing: TG-B). Both TG were drawn on the same chart and the peak locations were compared. In a normal ear the TG-F peak tended to be formed in the negative pressure area and that of TG-B in the positive area. In a model whose middle ear pressure (MEP) was adjusted to the atmospheric pressure, the TG-F peak always indicated a negative pressure and that of TG-B a positive pressure value. As long as the same model was used, the magnitude of peak shift was identical irrespective of the middle ear pressure. The magnitude of the peak shift was influenced by the speed of EAM pressure change and a linear increase was observed up to the speed of 70 mmH2O/sec, both in a normal ear and a model. These findings seem to suggest that the peak location of a unidirectionally drawn TG can not be regarded as indicating the precise MEP. A valid MEP can better be estimated by averaging the peak pressure of TG-F and TG-B of forward-backward tracing tympanogram. In ears with pathology considerable variation was noted in the magnitude of the peak shift, so that relying solely on unidirectionally drawn TG could have lead to misdiagnosis in some cases. This was especially true with ears having small perforations covered with granulation or fluid.

Tympanograms of otitis media with effusion: an experimental study.

A sufficient explanation concerning tympanogram (TG) of otitis media with effusion (OME) has not been reported. The purpose of the present study was to investigate the relationship between the types of TG and actual pathological status of the middle ear system by means of experimentally modified middle ear clefts. In the present experiments, human temporal bones, guinea pigs' bullae and plastic models were used. The results were as follows: (1) Water was gradually poured into the middle ear cleft through the Eustachian tube. When the water level rose above the umbo, the height of TG slightly exceeded that of the original TG. Further addition of water produced M-shaped TG, however, flat TG (type B) was not detected until the water-line nearly reached the aditus. A similar phenomenon was observed in the plastic models when the water level reached the aditus-equivalent-site. (2) Tympanometrically measured middle ear pressure (MEP) was almost equivalent to the actual MEP recorded by a manometer when the tympanic membrane was normal. However, this result could not be duplicated in flaccid or adhesive tympanic membrane cases. (3) A clear reduction in the height of TG in guinea pigs' bullae was observed with decreased air volume. In addition, shallow TG was produced by an occlusion of the aditus of the human temporal bones and of the aditus-equivalent-site of the plastic models. It was demonstrated that the TG pattern depends on the fluid volume and location, air volume of mastoid cavity, intratympanic pressure and eardrum condition.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation and incidence of Vibrio cholerae from river water].

The prevalence of Vibrio cholerae contamination in river water derived from 20 sites of 18 rivers in Kanagawa, Japan, was investigated during a period from July to September, 1987, and from one of the 20 sites in August, 1988 and in February, 1989, V. cholerae non-O1 was found in all samples at concentrations of 0.9-->1,400 MPN/100 ml. Higher amounts of the organism were observed in the samples from estuaries. V. cholerae O1 was detected in samples collected in August, 1988 and in February, 1989 at concentrations of 150 MPN/100 ml and 1.5 MPN/100 ml, respectively. From 1989 to 1995, water samples were collected monthly from 10 sites of 10 rivers to detect V. cholerae. V. cholerae O1 and non-O1 were detected in 3.6% (30 of 840) and in 61.1% (513 of 840) in the water samples examined, respectively. Overall, V. cholerae was found in 62.9% (528 of 840). Both types, O1 and non-O1, of organisms were detected in 15 samples. These results indicated that river water was contaminated frequently with V. cholerae non-O1 and sporadically with V. cholerae O1 throughout the year. Only one strain of V. cholerae O1 out of 543 V. cholerae strains was found to be a producer of cholera toxin. During these studies, the selectivity of 3 media for V. cholerae O1 was evaluated, and PMT agar was found to be the best.