Bone marrow traffic to regenerating islets induced by streptozotocin injection and partial pancreatectomy in mice.

The role of bone marrow (BM)-derived cells in the process of pancreatic islet regeneration remains unclear. The purpose of this study was to determine the role of BM cells in the repair process or regeneration of pancreatic islets in mice using chimeric green fluorescent protein (GFP) expressing BM cells. BM-infused chimeric mice were made diabetic by streptozotocin (STZ) injection or 60% partial pancreatectomy. GFP-positive cells within the islets and pancreas were studied immunohistologically. STZ treatment induced a 10-fold increase in PCNA-positive cells within the islets on day 7 posttreatment. GFP-positive cells increased in number within the islets as well as in the pancreatic parenchyma immediately after STZ injection. The partial pancreatectomy induced 2- to 3-fold increases on day 7 to 28 posttreatment. GFP-positive cells increased in number in pancreatic parenchyma but not within the islets. BM traffic to the pancreas significantly increased in the 2 models inducing islet regeneration. In both models, GFP-positive cells were not positive for antibodies against insulin, glucagon, or somatostatin, but were positive for markers of macrophages or fibroblasts, suggesting their involvement in the initiation of islet regeneration.

Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules.

A total of 83 cirrhotic nodules (pseudolobules) individually collected from 11 cirrhotic livers of hepatitis B virus carrier patient were analyzed for the frequency and mode of hepatitis B virus integration as well as histological features. Southern blot analysis disclosed discrete bands at higher molecular weight region in 26 of 83 nodules (31.3%), indicating a clonal growth of hepatocytes with viral integration. Considerable variation (0-75%) existed in the positive rates for discrete bands in nodules among livers. Molecular cloning revealed the sequence flanking an integrated viral sequence to be host DNA and thus confirmed true integration. Histological analysis, however, did not reveal any neoplastic-appearing foci of growth within nodules, despite the fact that the detection sensitivity would predict clones of more than 10(5) cells to give rise to clonal integration patterns on Southern blot analysis. The question of whether clonal expansion of hepatocytes reflects any viral integration-associated growth advantage and/or a preneoplastic condition awaits future studies.

Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation.

In order to scrutinize the possible significance of (nonatypical) mucous cell hyperplasia of the pancreas to neoplasia, we analyzed these lesions in terms of c-Ki-ras activation, which is known to be very frequent in pancreatic carcinomas. A total of 16 such mucous cell hyperplasias were collected from 10 pancreases resected for chronic pancreatitis. Tiny tissue fragments were taken from hematoxylin-stained sections by microdissection, and DNA analysis was carried out by the polymerase chain reaction amplification and oligonucleotide hybridization methods. Activating mutations of c-Ki-ras oncogene at codon 12 were detected in 10 of the 16 lesions (62.5%), a high rate as seen in carcinomas. The results indicated a clonal origin of cells comprising the mucous cell hyperplasia suggesting a neoplastic and/or precancerous nature.

Hepatocyte growth factor transforms immortalized mouse liver epithelial cells.

Transforming activity of hepatocyte growth factor (HGF) was demonstrated utilizing immortalized but not fully transformed mouse hepatocytes (MLE-10). Rat HGF cDNA, expressed under the control of a cytomegalovirus promoter, was transfected together with the neomycin resistance gene (PSV2neo) into MLE-10 cells by the calcium phosphate method, and propagated G418-resistant colonies were harvested colony by colony. After checking for integration and expression of exogenous HGF, five cell lines (MLE-10-HGF-1-5) were established. Three cell lines transfected with the vector only (MLE-10-CMV-1-3) were also established in the same manner. All MLE-10-HGF cell lines grew much faster than the MLE-10-CMV and original MLE-10 cells in culture and produced large colonies in soft agar, which colony production was blocked by the addition of anti-HGF antibody to the agar. After addition of HGF, original and MLE-10-CMV lines produced colonies in soft agar. The high-HGF-production lines (MLE-10-HGF-4 and -5) also gave rise to tumors within 2 weeks when implanted into the nude mice subcutis. In contrast, all MLE-10-CMV and original MLE-10 cells were negative in these growth assays. A rough parallelism between the level of HGF expression and the growth rate in both soft agar and nude mice subcutis was evident among MLE-10-HGF cell lines. Those with higher HGF production tended to grow in a scattered fashion in culture. High-affinity HGF receptor, HGFR/met, was expressed in MLE-10 and all the derived cell lines. Since HGF and/or HGFR/met gene expression is seen in various tumors and the serum HGF level is elevated in patients with hepatic disease, the present results indicate a possible significance of HGF and its receptor system in carcinogenesis, most probably via autocrine and/or paracrine mechanisms.

Low frequency of ras activation in 2-acetylaminofluorene- and 3'-methyl-4-(dimethylamino)azobenzene-induced rat hepatocellular carcinomas.

Activation of ras protooncogenes in 11 2-acetylaminofluorene (AAF)-induced hepatocellular carcinomas (HCCs) or liver cell lines and 9 3'-methyl-(dimethylamino)azobenzene (3'-Me-DAB)-induced HCCs in rats was examined using the NIH3T3 cell transfection assay and oligonucleotide hybridization analysis. Only one cell line established from a AAF-treated rat liver demonstrated transforming activity with a point mutation and ATTA transversion at the second position of H-ras codon 61. The rates of ras activation were thus very low for both AAF- and 3'-Me-DAB-induced rat HCCs, the results thus extending and confirming the findings indicating that ras activation in rat HCCs induced by various type of carcinogens is infrequent.

Frequent spontaneous sister chromatid exchange in hepatocytes of transgenic mice harboring the SV40-T antigen gene.

In order to shed light on the causal mechanisms of hepatocarcinogenesis in the transgenic mouse into which the albumin-promotor-regulated SV40-T antigen gene has been introduced (T+ mouse), and especially on the frequent chromosomal aberrations seen in cultured hepatocytes and hepatocellular neoplasms derived from such animals, the frequency of sister chromatid exchange (SCE) and karyotype abnormalities were investigated in a hepatocyte primary culture system. Cells were obtained through collagenase perfusion from T+ mice at 16-18 days of age, when no morphological changes are apparent, and from nontransgenic littermates, and cultured in the presence of bromodeoxyuridine. SCE was seen in transgenic hepatocytes twice as frequently as in their normal counterparts. No karyotype abnormalities in terms of numerical change or gross aberration were detected at this phase. The results thus suggest mutagenic properties for the T antigen, which may play an important role in hepatocarcinogenesis in this transgenic mouse.

Absence of H-ras point mutation at codon 12 in N-methyl-N-nitrosourea-induced hepatocellular neoplasms in the rat.

In order to assess the possibility that activated ras-associated hepatic carcinomas might be much rarer in rats than mice because of the more frequent or rapid occurrence of powerful carcinogenic event(s) other than ras point mutations in the former animals, precancerous lesions and hepatocellular carcinomas induced by a weak hepatocarcinogen N-methyl-N-nitrosourea (MNU) in the rat liver were analyzed for the presence or absence of ras point mutations. MNU was chosen because it is well known that MNU-induced rat mammary carcinomas contain activated H-ras at very high frequency. Male Fisher rats were treated with a single dose of MNU after partial hepatectomy, and then administered dietary phenobarbital or repeated s.c. injections of carbon tetrachloride as promoting procedures. Analyses by oligonucleotide hybridization, MnlI-restriction-fragment-length polymorphism and NIH3T3 cell transfection assays revealed neither H-ras point mutations nor transforming ability of the DNA from 36 MNU-induced rat hepatic neoplasms. The results were in agreement with previous results for rat hepatocellular carcinomas induced by other potent liver carcinogens and did not support our hypothesis that the frequency of finding ras activation might be dependent on the strength of the carcinogen.

Glutamate release and neuronal injury after intrathecal injection of local anesthetics.

High concentrations of local anesthetics are neurotoxic, but the mechanism for this neurotoxicity is obscure. Here, we report increased concentrations of glutamate in the cerebrospinal fluid after intrathecal injections of high concentrations of tetracaine (a local anesthetic). The peak concentrations of glutamate after administration of 1%, 2%, and 4% tetracaine were 4-fold, 6-fold, and 10-fold higher than baseline values, respectively. Animals in the 1% group were all neurologically normal one week after tetracaine injection. In the group receiving 4%, no animal was able to hop and vacuolation of the white matter and/or central chromatolysis of the motor neurons were observed. Because high concentrations of glutamate are known to be neurotoxic, our results may provide some insight into the mechanisms for neurotoxicity of intrathecal local anesthetics.

Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis.

We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure-volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure-volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis.

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone from the rat stomach in vitro.

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone (ir-TRH) from the rat stomach in vitro were studied. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The ir-TRH release from the rat stomach was enhanced significantly in a dose-related manner with the addition of histamine and inhibited with the addition of famotidine, but not with mepyramine. The stimulatory effect of histamine on ir-TRH release from the stomach was partially blocked with the addition of famotidine, but not with mepyramine. The elution profile of acid-methanol-extracted rat stomach on Sephadex G-10 was identical to that of synthetic TRH. These findings suggest that histamine stimulated ir-TRH release from the rat stomach in vitro, and that histamine's effects may be mediated via a H2-receptor.

Instability of integrated hepatitis B virus DNA with inverted repeat structure in a transgenic mouse.

We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.

Effects of the anticancer drug cocktail "UFT" on metastatic potentials of squamous cell carcinoma (O-1N) in a hamster model.

UFT, a mixture of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil, is one of the most widely used anticancer agents. The tissue localization and the efficacy of UFT in preventing lymph node metastasis were studied in hamsters transplanted with oral squamous cell carcinoma (termed "O-1N") which has high lymph node metastatic potency. After UFT administration for 14 consecutive days, the incidence of lymph node metastasis was significantly decreased (16% vs 58%). When UFT was administered orally for 3 consecutive days to hamsters bearing O-1N, and tissue concentrations of tegafur and 5-FU were measured by gas chromatography, the value of 5-FU was significantly higher in the primary transplant tumour, cervical lymph nodes with or without metastasis, and liver, than in the normal oral tissues. When 14C-UFT(14C-tegafur + uracil, a uracil/FT molar ratio of 4) was administered orally for 3 consecutive days to hamsters bearing O-1N, the 14C-tegafur level of tissue homogenates in the buccal and metastatic tumours was within the range of normal oral tissues, but the localization of silver grains was significantly higher in tumour cells than in the oral normal tissues. The results suggest that UFT would be useful for treatment and prevention of lymph node metastasis of oral carcinomas because of the high accumulation of active metabolites in metastatic and non-metastatic lymph nodes.

Improved nasal absorption of drugs using poly-L-arginine: effects of concentration and molecular weight of poly-L-arginine on the nasal absorption of fluorescein isothiocyanate-dextran in rats.

The effects of the concentration and molecular weight of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of fluorescein isothiocyanate-labeled dextran (MW, 4 kDa, FD-4) in rats were studied. When poly-L-Arg with a range of different molecular weights (MW, 8.9, 45.5 and 92.0 kDa) was applied intranasally at various concentrations, the bioavailability (F(0-9 h)) of FD-4 increased with the increasing concentration of poly-L-Arg. The enhanced absorption was also dependent on the molar concentration, in that the poly-L-Arg with a higher molecular weight increased F(0-9 h) at a lower molar concentration. In addition, for each applied concentration, the poly-L-Arg exhibited a molecular weight-dependence as far as the enhancement of FD-4 absorption was concerned. On the other hand, the maximum absorption rate (MAR) of FD-4, calculated by means of a deconvolution method, tended to reach a maximum plateau level at a lower applied concentration for the poly-L-Arg with the highest molecular weight, but this plateau level was almost the same for poly-L-Arg with molecular weights of 45.5 and 92.0 kDa. Moreover, the simulated absorption profiles of FD-4 indicate that the degree of enhancement (the level of MAR and the subsequent reduction in the absorption rate) was dependent on the molecular weight of poly-L-Arg, while the effect of poly-L-Arg was maintained for a longer period, depending on the applied concentration, although the MAR was relatively similar. These results indicate that the molecular weight of poly-L-Arg appears to affect both the enhancing efficiency (absorption rate) and the time-frame of this enhancing effect, whereas the concentrations of each poly-L-Arg system applied only have an effect on the time-frame. These effects may also be associated with the charge density of a poly-L-Arg molecule.

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats.

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.

Screening of cationic compounds as an absorption enhancer for nasal drug delivery.

Several cationic compounds were screened as potential nasal absorption enhancers to increase intranasal absorption of a model drug, fluorescein isothiocyanate labeled dextran (MW 4.4 kDa, FD-4), without nasal membrane damage in rats. Their effects were compared with those of classical enhancers. Various cationic compounds (poly-L-arginines with different molecular weights (MW 8.9, 45.5 and 92.0 kDa, poly-L-Arg (10), (50) and (100), respectively), L-arginine (L-Arg), L-lysine (L-Lys), and cetylpyridinium chloride (CPCL) were evaluated. Of the cationic compounds, poly-L-Arg and CPCL greatly enhanced the intranasal absorption of FD-4, as did chitosan, a cationic polysaccharide which has been reported to show a great effect on the transnasal delivery of peptide and protein drugs. The enhancing intensity by poly-L-Arg was dependent on its molecular weight. Rank order of the enhancing ratio, calculated from the AUC ratio for the enhancer treatment against the untreatment, was 0.5% poly-L-Arg (100) congruent with0.5% sodium dodecylsulfate congruent with0.5% CPCL?0.5% poly-L-Arg (50)?0.5% sodium deoxycholate congruent with0.5% sodium taurodihydrofusidate?0.5% polyoxyethylene-9-lauryl ether congruent with0.5% lysophosphatidylcholine?0.5% chitosan congruent with0.5% poly-L-Arg (10)>/=10% L-Arg congruent with10% L-Lys?0.5% sodium glycocholate congruent with0.5% sodium taurocholate congruent with0.5% EDTA. Only the poly-L-Args represented almost the same degree of hemolysis of cationic compounds compared with pH 7.0 phosphate buffered saline in the rat erythrocyte lysis experiment. The enhancing ratio by classical enhancers correlated with leaching of protein, phospholipids and LDH from isolated rabbit nasal mucosa. CPCL also fell on the regression lines between the enhancing ratio and their degree of leaching from classical enhancers. In contrast, the enhancing intensities by poly-L-Arg (10), (50) and (100) were greatly shifted from the regression line: the amount of leaching was markedly low in spite of a great enhancement of FD-4 absorption. These findings suggest that of the assessed enhancers only the poly-L-Args enhance the transnasal delivery of high molecular substances without severe damage to the nasal mucosal membrane. Poly-L-Arg is therefore a promising candidate having a good balance between enhancing activity and safety for nasal peptide and protein delivery.

The role of radiotherapy in the management of salivary gland carcinomas.

A retrospective study was performed on 44 patients with carcinoma originating in the major and minor salivary glands to examine the effects of postoperative radiotherapy on locoregional and distant control and survival. 22 patients were treated by surgery alone and the 22 other patients were treated by a combination of surgery and postoperative irradiation. In the surgery group, local recurrence developed in all 8 patients with evidence of residual disease at the surgical margins, whereas local control was achieved in 7 of 15 patients with positive surgical margins in the combination group and the control rate was related to the amount of residual disease. Neck metastasis, which developed in 13 patients (30%), was not affected by the status of surgical margins or by the treatment modality. On the other hand, the incidence of distant metastasis seen in 19 patients (43%) was much higher in patients with positive surgical margins and the development of distant metastasis in these patients was not prevented by postoperative irradiation. The survival rates at 5, 10 and 15 years were 54, 48 and 41%, respectively, for the irradiated patients, whereas the values for the patients treated by surgery alone were 75, 70 and 70%, respectively. The results indicate that postoperative irradiation is effective in controlling local recurrence but not neck and distant metastases. Wide excision with sufficient surgical margins followed by postoperative radiotherapy and systemic chemotherapy are essential to obtain a better prognosis.

Central carcinoma of the jaw. A survey of 28 cases in the Japanese literature.

A survey of the Japanese literature revealed 28 well-documented cases of central carcinoma of the jaws. There was no sex predominance and the mandible was the site of involvement in 26 cases. The most common initial symptom was local swelling, which was followed in order of frequency by spontaneous pain, paraesthesia of the lower lip, discomfort, loosening of teeth and trismus. On clinical examination, local swelling which was often accompanied by variable symptoms was an almost constant finding. Radiographic appearance varied from unilocular to worm-eaten type radiolucencies which were often surrounded by indistinct margins on close examination. Radical surgery, combined with irradiation and/or chemotherapy was the principal treatment in most cases, but there were 4 cases in which the lesions were simply excised under a tentative diagnosis of cyst; local recurrence was noted in 5 cases. Regional lymph node metastasis and lung metastasis were observed in 8 and 2 cases, respectively. No definite conclusion was drawn with regard to the prognosis because of the short follow-up period. Histologically, epidermoid carcinoma was most frequently seen, but odontogenic cyst was confirmed to be the site of origin in 3 cases only.

Integrin VLA-5 negative primary plasma cell leukemia.

We present a case of primary plasma cell leukemia with Bence Jones proteinuria. After combination chemotherapy, leukemic cells and the urinary levels Bence Jones protein were decreased. Small lytic bone lesions were detected only in the skull. Typical plasma cells were rarely seen in peripheral blood on the hyperleukocytic phase, however they were increased in the advanced stages. The most important diagnostic sign was persistent expression of CD38 antigen on leukemic cells throughout the entire course of the illness and these leukemic cells expressed very late antigen-4 (VLA-4) but not VLA-5.

[Evaluation of the dose of endotoxin to produce endotoxin shock models in dogs].

Intravenous bolus administration of purified endotoxin (ET) is one of the most popular methods of producing experimental ET shock. In an attempt to evaluate the dose of ET in these experimental models, the kinetics of ET was assessed in various doses (1-0.002 mg.kg-1), and dose response study was performed, measuring chronological changes in hemodynamic (mean arterial pressure & cardiac output) and hematological (leucocyte & platelet counts) parameters. It was demonstrated that plasma ET concentrations were sustained extraordinarily high (greater than 5000 pg.ml-1) in doses of 0.2-1 mg.kg-1 during the observation period of several hours, while they were measurable in doses of 0.02 and 0.002 mg.kg-1, showing a biphasic decrease pattern after ET administration. In particular, plasma ET levels of low dose group (0.002 mg.kg-1) were similar to those of clinical gram negative septic patients. We conclude that the empirically used ET doses of more than 1 mg.kg-1 produce extremely severe endotoxemia and that milder degree of ET shock models produced by low dose of ET (0.002 mg.kg-1) would be more applicable for a pathophysiological study of ET shock.

[Prognostic significance of echogenic lesion within small hepatocellular carcinoma].

To evaluate prognostic significance of echogenic lesion within small hepatocellular carcinoma (SHCC, less than or equal to 2 cm in diameter), clinical and pathological findings of 32 cases with SHCC containing echogenic lesion (echogenic SHCC) were compared with those of 55 cases with non-echogenic SHCC. Compared with the non-echogenic SHCC group, the frequency of clinical stage I was significantly higher, and there were significantly more cases with solitary tumor relative to cases with multiple tumors in the echogenic SHCC group. Histologically, the incidence of the HCC composed of well-differentiated tumor cells corresponding to Edmondson's grade I was significantly higher in the echogenic SHCC group than in the non-echogenic SHCC group. Although HCCs tended to become progressively less differentiated with increasing tumor sizes in the both groups, the process of cellular change appeared to proceed more slowly in the echogenic SHCC group. Survival rate after tumor detection was 73% at three years, 56% at five years and 48% at seven years and nine years in the echogenic SHCC group, while it was 46% at three years, 42% at five years and 0% at seven years in the non-echogenic SHCC group. The present results showed that the presence of echogenic lesion within SHCC could be useful prognostic indicator.