Growth and maturation of small hepatocytes

Proliferation of adult rat hepatocytes is observed in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less-differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well-developed bile canaliculus structures are often observed in the cell-cell borders. Thus, we suggest that small hepatocytes may be considered to be ‘committed progenitor cells’ that can further differentiate into mature hepatocytes.     

Establishment and characterization of human urothelial cancer xenografts in severe combined immunodeficient mice

AIM: To establish and characterize a murine xenograft model of human urothelial cancer in severe combined immunodeficient (SCID) mice for therapeutic simulation. METHODS: Pieces of 30 freshly resected urothelial tumors (24 obtained from bladder and 6 from ureter or pelvis) were implanted subcutaneously into SCID mice, and xenograft tumors were passed in tumorigenic cases. At each passage, histopathology, TP53 mutational status assessed by yeast p53 functional assay, and the Ki-67 labeling index (LI) were examined to evaluate the preservation of original features. A growth delay assay after single-dose irradiation was performed in four representative xenografts. RESULTS: Tumor growth was observed in 18 mice (60%, 18/30). Histologically, 15 of the 18 were epithelial carcinomas similar to the original tumors, whereas the other 3 were Epstein-Barr virus-associated lymphoproliferative disease, resulting in a 50% (15/30) take rate. No correlation was found between the tumor take rate and the clinicopathologic features, TP53 mutational status, or Ki-67 LI of the patients' tumors. Of these 15 xenografts, 11 xenografts were passed from 3 to 10 generations. TP53 mutational status remained stable during the passages, and the Ki-67 LI of eight xenografts was within a range of 50% of the LI of the original tumors, although the other three xenografts increased by over 50%. Specific growth delay after irradiation, independent of the original tumor growth speed and Ki-67 LI, was observed in four xenografts. CONCLUSIONS: SCID mice are useful recipients for investigations of human urothelial cancer with a wide biological range. This easy-to-handle xenograft system can help to develop a better in vivo preclinical evaluation system for therapeutic agents as well as the investigation of tumor pathophysiology.     

Geranylgeranylacetone, an anti-ulcer drug, stimulates hexosamine production in a rat gastric mucosal cell line through binding to a specific cytosolic protein

An anti-ulcer drug, geranylgeranylacetone (GGA), stimulates hexosamine production in a rat gastric mucosal cell line (RGM-1). The aim of this study was to elucidate the mechanism of this action. The role of protein kinase A, inositol phospholipid turnover and tyrosine kinase in the stimulatory action of GGA on hexosamine production in RGM-1 was determined by observing cAMP production, [³H]-inositol phosphate turnover and western blotting of tyrosine phosphorylation, respectively. Any trophic effect of GGA on RGM-1 was also checked by [³H]-thymidine incorporation. Our experiments showed that GGA has no effect on cAMP production, inositol phospholipid turnover, tyrosine phosphorylation or DNA synthesis in RGM-1. Finally, a [¹⁴C]-GGA competitive receptor binding assay was performed on RGM-1 and we found that [¹⁴C]-GGA specifically bound to RGM-1 cytosolic protein. Although retinoic acid (RA), another polyisoprenoid compound significantly stimulated hexosamine production in RGM-1, we confirmed that the [¹⁴C]-GGA binding site in RGM-1 is different from the RA binding site. In summary, GGA stimulates hexosamine production in RGM-1 and this action is probably mediated through its binding to a specific cytosolic protein in RGM-1.     

Antineoplastic activity of honey in an experimental bladder cancer implantation model: In vivo and in vitro studies

OBJECTIVES: The antitumor effect of bee honey against bladder cancer was examined in vitro and in vivo. Methods: Three human bladder cancer cell lines (T24, 253J and RT4) and one murine bladder cancer cell line (MBT-2) were used in these experiments. In an in vitro study, the antitumor activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, 5-Bromodeoxyuridine (BrdU) labeling index and flowcytometry (FCM). In the in vivo study, cancer cells were implanted subcutaneously in the abdomens of mice, and the effects were assessed by the tumor growth. RESULTS: In vitro studies revealed significant inhibition of the proliferation of T24 and MBT-2 cell lines by 1-25% honey and of RT4 and 253J cell lines by 6-25% honey. BrdU labeling index was significantly lower. FCM showed lower S-phase fraction, as well as absence of aneuploidy compared with control cells. In the in vivo studies, intralesional injection of 6 and 12% honey as well as oral ingestion of honey significantly inhibited tumor growth. CONCLUSION: Bee honey is an effective agent for inhibiting the growth of T24, RT4, 253J and MBT-2 bladder cancer cell lines in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. Our results are promising, and further research is needed to clarify the mechanisms of the antitumor activity of honey.     

Arrhythmias in Centenarians

Background: Many studies have shown an increase in the prevalence of arrhythmias with advancing age. However, little is known about arrhythmias in centenarians. Method and Results: Thirty-two Japanese centenarians aged 100–106 years (14 males, 18 females) were studied. All of them had 12-lead EGGs, and 22 also had 24-hour ambulatory monitoring. As controls, 89 healthy Japanese elderly from the same geographic area underwent 12-lead ECG. Their mean age was 75 ± 6 years with a range of 63–93 years, and there were 28 males and 61 females. Twenty-three of them also had Holter ECGs. On the 12-lead ECG, the heart rate was slightly, but significantly, higher in the centenarians (76.8 ± 12.7 beats/min) than that in the elderly subjects (74.9 ± 5.9 beats/min, P < 0.005). PQ and QT_C were significantly longer in the centenarians (174 ± 29 and 439 ± 33 msec, respectively) compared with the elderly subjects (158 ± 23 and 417 ± 31 msec, P < 0.005 and P < 0.001, respectively). Supraventricular premature beats (SVPBs) were observed in 31% of the centenarians and in 4% of the elderly subjects (P < 0.001). First- and second-degree AV block was recorded in 25% of the centenarians and 1% of the elderly subjects (P < 0.001). Right bundle branch block was found in 19% of the centenarians and 7% of the elderly subjects (P < 0.05). There were no differences in the frequency of ventricular premature beats (VPBs) or QRS voltage. On the Holter ECG, there were no significant differences in average heart rate, maximum heart rate, minimum heart rate, or the longest RR interval. A subgroup of centenarians had frequent SVPBs. However, none of them had > 1,000 VPBs/day as opposed to four elderly subjects (P < 0.01). Atrial fibrillation was not observed in any records of the centenarians despite the presence of frequent SVPBs. Conclusion: These data suggest that conduction disturbances of the AV nodal—His-Purkinje system and frequent SVPBs are common in centenarians, whereas very frequent VPBs and atrial fibrillation seem less common.     

ARGYROPHILIC NUCLEOLAR ORGANIZER REGION IN PROLIFERATING CELL HAS A PREDICTIVE VALUE FOR LOCAL RECURRENCE IN SUPERFICIAL BLADDER TUMOR

PURPOSE: It has been shown in many carcinomas that the proliferation rate and number of argyrophilic nucleolar organizer regions (AgNOR) are associated with tumor aggressiveness. However, in bladder tumor the significance of the correlation between the number of AgNOR and tumor behavior remains controversial. Therefore, it would be helpful if a new technique could be developed that would allow for more accurate AgNOR counting in association with tumor behavior. We established the simultaneous staining technique of AgNOR with Ki-67 labeling to reveal the significance of AgNOR count in superficial bladder tumor. MATERIALS AND METHODS: A total of 50 paraffin sections of superficial bladder tumor were stained with AgNOR and Ki-67 (MIB-1). The numbers of AgNORs in proliferating (MIB-1 positive) or resting (MIB-1 negative) cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. RESULTS: The AgNOR count in proliferating cells was significantly higher than that in resting cells (p<0.01), and the count significantly increased with tumor grade (p<0.01). Based on recurrence-free survival analyses the local recurrence rate was significantly higher in patients with high proliferating cell NOR but not for those with resting or whole cells. However, no AgNOR score helped to select patients at high risk for disease progression. CONCLUSIONS: Proliferating cell NOR had a predictive value for local recurrence in patients with superficial bladder tumor.     

Bilateral epididymal sarcoidosis presenting without radiographic evidence of intrathoracic lesion: Review of sarcoidosis involving the male reproductive tract

Sarcoidosis is a multisystem disorder that rarely involves the genitourinary tract. To date, only 59 cases of histologically proven sarcoidosis involving the male reproductive tract have been reported in the literature. We present here a case of bilateral epididymal sarcoidosis without radiographic evidence of intrathoracic lesion. A 46-year-old man presented with a one-week history of painless bilateral scrotal swellings. Physical examination detected multiple elastic firm nodules on both sides of the scrotum which showed no tenderness. The nodules seemed to involve the entire bilateral epididymides. Some irregularly shaped hypoechoic masses in the bilateral epididymides were identified on gray scale ultrasonography. On magnetic resonance images, the bilateral epididymides were seen to be enlarged, heterogeneous and nodular without any signs of testicular involvement. The lesion showed a slightly high signal intensity on the T2-weighted image. Pathological evaluation following bilateral epididymectomy found non-caseating epithelioid cell granulomas with giant cells in epididymal tissue, thus confirming a diagnosis of sarcoidosis. Gallium-67 scanning showed additional small hot spots in the anterior chest wall and extremities. Open biopsy of a superficial papular lesion in the dermis of the right upper arm was performed and pathological findings indicated sarcoid granulomas. This report also includes a review of the literature pertaining to sarcoidosis of the male reproductive tract.     

Perinatal medical support in the area surrounding the Hanshin-Awaji earthquake

After natural disaster, perinatal medical care must usually be provided from outside the disaster area, because most of the medical efforts inside the area focus on patients without special needs. This study reviews the emergency perinatal medical response after the great Hanshin-Awaji earthquake. In the present study, we summarize records of telephone calls and reports from the Neonatal Mutual Cooperative System (NMCS). The day of the earthquake, very little information was available to or from the disaster area. The day after the earthquake, Osaka City General Hospital (OCGH) and Osaka Medical Center and Research Institute for Maternal and Child Health were established as key facilities, and OCGH served as a center through which information passed to and from the disaster area. Most telephone calls to OCGH were placed on the second day after the earthquake by pregnant women concerned about their deliveries. Many high-risk pregnancies and newborn infants were transferred out of the disaster area over the next month. This analysis shows that although the emergency response was very rapid in this instance, the operation may be significantly improved in disasters of this magnitude: (i) if a communication mechanism able to serve a large volume is established; (ii) if helicopter transport was easily available; and (iii) if key facilities to handle communications were previously established so as to be available immediately.     

Blocking Effects of Maternal Serum-IgG and Placental Eluate-IgG on Materno-Fetal Mixed Lymphocyte Reaction and Their Individual Specificity*

ABSTRACT: The purpose of this study is to clarify the problem regarding individual specificity of maternal serum immunoglobulin G (IgG) and placental eluate IgG that block the unidirectional mixed lymphocyte reaction (MLR) in which maternal and cord blood lymphocytes (lys) were used as responder and stimulator, respectively. Both maternal serum IgG and placental eluate IgG were found to include blocking antibodies (BA) which showed similar blocking effect on MLR. But, in the case of serum IgG, individual specificity of BA on MLR was observed to be less great in MLR done using whole lys or T-enriched lys of the cord blood as stimulator, whereas it became greatly significant when MLR was done using B-enriched lys of the cord blood as stimulator. This was the case even in MLR done by using unrelated maternal lys as responder. On the other hand, in the case of placental eluate, it was found to be greater when compared to serum IgG even where MLR was done using not only B lys but also T lys as stimulator. It is thus strongly suggested that BA include individual specific antibodies against fetal B lys having HLA-D/DR locus determinants in addition to individually nonspecific antibodies common to pregnancy which may be produced to a “yet unknown fetal antigen.”