Effect of nicotine on the proliferation and differentiation of mouse induced pluripotent stem cells.

The molecular mechanisms that regulate the proliferation and differentiation of induced pluripotent stem (iPS) cells are of great interest. However, whether stimulation with nicotine enhances the proliferation and differentiation of iPS cells has not been investigated. In the present study, western blot analysis revealed that the α4-nAchR and α7-nAchR are expressed in mouse iPS cells. Mouse iPS cells were treated with nicotine for 24 h under feeder-free conditions. Mouse iPS cells were guided to differentiate into mesodermal progenitor cells on type IV collagen (Col IV)-coated dishes in differentiation medium. Mouse iPS cells were guided to differentiate into neural progenitor cells by embryoid body (EB) formation on ultra-low-attachment dishes. After 4 days of growth, all-trans retinoic acid (ATRA; 1 µM) or nicotine (300 nM) was added to the EB cultures and maintained for additional 4 days and plated onto fibronectincoated plates. A BrdU incorporation assay showed that treatment with 300 nM nicotine significantly increased the DNA synthesis of mouse iPS cells or mouse iPS cell-derived mesodermal progenitor cells. This effect was significantly inhibited by pretreatment with an α4-nAchR antagonist, an α7-nAchR antagonist, or a CaMKII inhibitor. The differentiation potential of mouse iPS cells into mesodermal progenitor cells or neural progenitor cells was not affected by the nicotine treatment. The present study indicates that stimulation of the α4-nAchR and α7-nAchR may lead to a significant increase in the proliferation of mouse iPS cells or mouse iPS cell-derived mesodermal progenitor cells through the CaMKII signaling pathway.

Effect of dietary anti-Helicobacter pylori-urease immunoglobulin Y on Helicobacter pylori infection.

Recently, chicken egg yolk was recognized as an inexpensive antibody source, and the therapeutic usefulness of egg yolk immunoglobulin Y (IgY) in oral passive immunization has been investigated. Although multiple antibiotic treatments eradicate most Helicobacter pylori (H. pylori) infections, therapy fails in 10-15% of cases due to the development of drug resistance. Consequently, it is important that new, more broadly based therapies for the treatment of H. pylori infection should be identified. The present study evaluated the effect, on H. pylori infection, of IgY prepared from egg yolk of hens immunized with H. pylori urease (anti-HpU IgY). Seventeen asymptomatic volunteers diagnosed as H. pylori-positive by the 13C-urea breath test (UBT) were orally administered anti-HpU IgY for 4 weeks. Four weeks later, UBT values were significantly decreased although no case showed H. pylori eradication. An H. pylori-positive 53-year-old female gastritis patient administered anti-HpU IgY plus lansoprazole for 8 weeks showed a decrease in serum pepsinogen (PG) I and UBT values as well as an increase in the PG I/II ratio. In conclusion, anti-HpU IgY may mitigate H. pylori-associated gastritis and partially attenuate gastric urease activity. Furthermore, anti-HpU IgY combined with antacids appears to ameliorate gastric inflammation. These encouraging results may represent a novel approach to the management of H. pylori-associated gastroduodenal disease.

Adherence protects the binding sites of Helicobacter pylori urease from acid-induced damage.

Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.

Enhanced reduction of Helicobacter pylori load in precolonized mice treated with combined famotidine and urease-binding polysaccharides.

The present study investigated the effect of a model urease-binding polysaccharide in combination with a histamine H(2) receptor antagonist on Helicobacter pylori colonization in vivo. Euthymic hairless mice were treated daily with dextran sulfate via drinking water and/or famotidine via intragastric gavage starting at 1 week postchallenge with a CagA(+) VacA(+) (type 1) strain of H. pylori. Treatment of precolonized mice for 2 weeks with dextran sulfate combined with famotidine yielded a group mean bacterial load (per 100 mg of gastric tissue) of log(10) 1.04 CFU, which was significantly lower than those of the famotidine (log(10) 3.35 CFU, P < 0.01) and dextran sulfate (log(10) 2.45 CFU, P < 0.05) monotherapy groups and the infected nontreated group (log(10) 3.64 CFU, P < 0.01). Eradication was achieved after 2 weeks of treatment in 50% or more of the test mice using drug combinations (1 or 2 weeks of famotidine plus 2 weeks of dextran sulfate) versus none in the monotherapy and positive control groups. The enhanced activity of the drug combination may be related to the daily pattern of transient acid suppression by famotidine inducing periodic bacterial convergence to superficial mucus sites penetrated by dextran sulfate from the lumen. Increased urease-dextran sulfate avidity was observed in vitro in the presence of famotidine and may partly account for the enhanced activity. With potential utility in abbreviating treatment time and eradication of antibiotic-resistant strains, the use of urease-targeted polysaccharides concurrently with a gastric acid inhibitor warrants consideration as an additional component of the standard multidrug chemotherapy of H. pylori infection.

Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides.

BACKGROUND & AIMS: The significance of acid-primed recognition of ligands by Helicobacter pylori urease is unknown. This study aimed to further characterize the specificity of urease adherence in vitro and verify whether specific inhibition will translate into in vivo suppression of colonization. METHODS: A highly sensitive competitive enzyme-linked ligand capture assay was used to quantify the capacity of each test inhibitor to compete with labeled mucin for binding sites on immobilized native urease. A model polymer that strongly bound urease was used in an in vivo trial using euthymic hairless mice as an infection model. RESULTS: The blockage of urease-gastric mucin interaction by certain inhibitors revealed an acid-functional lectin-like activity by urease, specifically recognizing bacterial lipopolysaccharides and certain species of polysaccharides, nonbacterial glycolipids, and glycoproteins. Dextran sulfate significantly (P < 0.01) suppressed colonization of mice by H. pylori when given before and/or after challenge. CONCLUSIONS: The acid-driven high-affinity adherence of H. pylori urease to mucin and lipopolysaccharides contributes to gastric mucosal colonization by the bacterium based on in vivo targeting experiments using specific polysaccharides in a mouse model with acute infection. Acid-functional urease-homing polysaccharides that can interfere with urease-mucin or H. pylori whole cell-mucin interaction in vitro can significantly interfere with colonization by the bacterium in vivo.

Hepatocarcinogenic activity of N-butyl-N-(4-hydroxybutyl)nitrosamine in rats is not modified by sodium L-ascorbate.

Male F344 and Wistar Shionogi (WS) rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks and then killed at week 36 (experiment 1). Although reduction of body weight increase was found, no effects on liver weights were noted. Formalin-fixed and paraffin-embedded liver tissues from rats killed terminally were cut and stained for glutathione S-transferase placental form (GST-P) immunohistochemically. Marked elevation of quantitative values of small GST-P positive (GST-P+) foci were apparent in both strains of rat administered BBN. In experiment 2, both sexes of F344 rats were given 0.05% BBN in the drinking water for 4 weeks and then fed diet containing 0 or 5.0% sodium L-ascorbate (SA) for 32 weeks. No body and liver weight changes were evident in any group. Quantitative values for small GST-P+ foci were increased in both sexes of rats exposed to BBN but were not modified by additional SA treatment. Thus, it was confirmed that the selective bladder carcinogen BBN also acts as a liver carcinogen. These results, from the quantitative analysis of small GST-P+ foci as end point marker lesions, indicate that the liver tumor modifying potential of test chemicals can be evaluated in rats by using an initiation/promotion protocol for urinary bladder carcinogenesis.

Primary non-Hodgkin malignant lymphoma of the male breast.

A case of primary non-Hodgkin lymphoma of the male breast is reported. The patient was a 76-year-old Japanese with a history of bilateral gynecomastia. After the patient had received sex hormone treatment for the gynecomastia, rapid growth of a tumor in the right breast was noted, with regression of a contralateral breast lesion. Clinically, inflammatory breast cancer was suspected, and right mastectomy with ipsilateral axillary lymph node dissection was performed after intraarterial infusion chemotherapy using a cis-platinum derivative. The histology of the surgical specimen was non-Hodgkin malignant lymphoma of the diffuse large cell type, with focal tumor necrosis. Immunohistochemically, the tumor cells showed a B-cell nature. The patient is currently well without disease 39 months after surgery.

[A case of unresectable liver metastasis from leiomyosarcoma of the stomach successfully treated by intra hepato-arterial chemotherapy with Infuse-A-Port].

This report describes a 55-year-old man who had a curative operation for leiomyosarcoma of the stomach 6 years ago. Unresectable liver metastases was discovered during the second laparotomy and successfully treated by intra hepato-arterial chemotherapy, which included MMC, ADM and Therarubicin with Infuse-A-Port. He remains well presently with a decrease in the size of the liver metastases and no metastasis to any other organs upon investigation by an abdominal CT 2 years and 2 months after the second laparotomy.

[A retroperitoneal leiomyosarcoma--a case of non-concurrent double cancers with kidney cancer].

A retroperitoneal leiomyosarcoma in a 50-year-old man is reported. A laparotomy was performed on Dec. 17, 1987 and the tumor, weighing 120 g, was completely excised. The patient was followed up and has stayed healthy (1 year after operation). Although CT ultrasonography and arteriography are helpful in the diagnosis of retroperitoneal tumors, it is felt that a histological examination is indispensable for a qualitative assessment of the lesion. Surgical excision constitutes the fundamental treatment for a retroperitoneal leiomyosarcoma. However, the prognosis is usually poor and depends on successful radical surgery. This case involved non-concurrent double cancers with a retroperitoneal leiomyosarcoma and a kidney cancer.

[Experimental study on preventive effects of lung metastases using LAK cells induced from various lymphocytes--special references to enhancement of lung metastasis after laparotomy stress].

In 5 week-old female SD rats, ether anesthesia or laparotomy for 30 minutes was done, followed by iv inoculation of 1 x 10(6) MRMT-1 cells. On day 14, number of lung metastatic nodules was examined. Preventive effect of LAK cells on lung metastases was also observed. LAK cells were induced from regional lymph node lymphocytes (LN), thymus cells (TC) and spleen cells (SC) of the rats on day 7 after tumor inoculation by incubation with 1 micrograms/ml of R-IL2 (TGP-3) for 4 days. 1) Lung metastases were noted in 100% after MRMT-1 intravenous inoculation, while number of lung metastatic nodules was reduced after iv administration of LAK cells. 2) When MRMT-1 cells were inoculated intravenously after laparotomy stress for 30 minutes, number of lung metastatic nodules was significantly increased and LAK cells reduced it significantly. 3) When R-IL2 sc administration for 4 days was accompanied, number of lung metastatic nodules was less than that of without R-IL2 substitution. 4) The antitumor effect of LAK cells was the strongest in LN-LAK, while it was the weakest in SC-LAK. In conclusion, usefulness of LAK cells for preventing lung metastases by surgical stress was indicated. This fact suggested that possibility of adoptive immunotherapy using LAK cells might be useful for prevention of lung metastases after laparotomy.

[In vivo antitumor effect of LAK cells induced from various lymphocytes--experimental study in rats].

A MRMT-1 was inoculated in the thigh of SD rats. On day 7, regional lymph node lymphocytes (TB-LN), thymus cells (TB-TC) and spleen cells (TB-SC) or non-tumor bearing spleen cells (NB-SC), were obtained. LAK were induced with lug/ml of R-IL2 (TGP-3) using above lymphocytes, and were examined as for their in vivo and in vitro cytotoxic activities. In vitro: Against MRMT-1, cytotoxic activity of LAK increased with lapse of culturing time. Thus on day 4 of culture, it was the highest (46.5%) in TB-SC-LAK, while that of TB-LN-LAK was the lowest (5.8%). Against YAC-1, they also increased except TB-LN-LAK. In vivo: When the mixture of MRMT-1 and LAK was sc inoculation, diameter of the tumor were the smallest in TB-LN-LAK and the largest in TB-SC-LAK. When it was injected intraperitoneally, in non-irradiated rats, the survival prolongation was the best in TB-LN-LAK and the worst in TB-SC-LAK. In pre-irradiated rats; the cytotoxicity was almost the same as the results of in vitro. When it was injected intravenously, number of lung metastatic nodes were the smallest in TB-LN-LAK and the largest in TB-SC-LAK. R-IL2 substitution therapy was also effective. In conclusion, the effectiveness of LAK-AIT was estimated more accurately by in vivo cytotoxic assay. TB-LN was most useful as a source of LAK cells.

[Case report of a second resection of rectal leiomyosarcoma due to recurrence after 10 years].

A leiomyosarcoma of the rectum is a rare disease, and cases of a recurrence are even more rare. Reported is the case of a patient who, in 1987, underwent a per sacral resection of a rectal tumor which was diagnosed as leiomyosarcoma. In 1987, after symptoms that showed that the patient's feces had become narrower, a recurrence of this tumor was diagnosed, so that the patient again underwent an abdominoperineal rectomy. The postoperative course has been good. Details of this disease still remain unclear. In Japan, there have been only 10 such cases of a recurrence reported. Of these, a liver metastasis and a localized recurrence are the most common. Long term follow-ups are considered necessary.

[Facilitation of tumor metastasis to the lung by operative stress in the rat--influence of adrenocortical hormones and preoperative administration of OK-432].

Experimental studies were performed to clarify the mechanism of facilitation of tumor metastasis to the lung by operative stress, using an experimental model in which 5-week old female Sprague-Dawley (SD) rats were inoculated with a low antigenic and easily metastasizable tumor, MRMT-1. In particular, relevance of adrenocortical hormones to the facilitation of tumor metastasis was examined. Furthermore preventive effects of preoperative administration of a nonspecific immunopotentiator, OK-432, were examined. Number of metastatic nodules was increased significantly by operative stress and the increase was proportionate to severity of the stress. The facilitation of metastasis by operative stress was significantly inhibited by preoperative OK-432 administration. In adrenalectomized rats, no such facilitation of metastasis by operative stress was observed. After administration of 2.5 to 20mg of hydrocortisone, number of metastatic nodules increased dose-dependently. The increase in metastatic nodules by administration of 5mg of hydrocortisone was inhibited by preoperative OK-432 administration. Thus it was concluded that facilitation of tumor metastasis by operative stress was proportionate to severity of the stress, and one of its essential causative factors was the stress-induced adrenocortical hypersecretion, which suppressed immunity of the host. Administration of OK-432 was effective for counteracting the stress-induced facilitation of metastasis.

[Effects of serum immunosuppressive factors on the cytotoxicity of lymphokine-activated killer (LAK) cells induced by recombinant interleukin 2(R-IL2)].

Fundamental studies were performed on adoptive immunotherapy, especially on effects on lymphocytic cytotoxic activity, of nonspecific immunosuppressive factors (ferritin, IAP, AFP) and of serum factors obtained from gastric cancer patients, and possible intervention of suppressor T cells in serum immunosuppressive activity on the cytotoxicity was also examined. The following results were obtained. 1) Cytotoxicity of LAK cells induced by culturing normal peripheral blood lymphocytes (PBL) with R-IL2 in the medium containing normal AB-type sera, was higher than that of PBL. 2) Effect of nonspecific immunosuppressive factors on cytotoxicity of LAK cells was lower than that on cytotoxicity of PBL. 3) Cytotoxicity of PBL was inhibited in a relatively specific fashion by sera from patients of the cancers which were of identical histological types with the target tumor cells, while that of LAK cells was hardly inhibited by patients' sera. 4) Cytotoxicity of Leu 15-PBL was inhibited by nonspecific immunosuppressive factors and also by cancer patients' sera in a relatively specific fashion in relation to histology. 5) Cytotoxicity of Leu 15-LAK cells was hardly inhibited by serum nonspecific and specific immunosuppressive factors. The above results showed that serum immunosuppressive factors might act on PBL cytotoxicity without intervention of suppressor T cells, and that LAK cells were hardly inhibited by such immunosuppressive factors. All these results suggested usefulness of adoptive immunotherapy with LAK.

[Experimental studies on induction of various lymphokine-activated killer cells using recombinant interleukin 2].

Fundamental studies were performed on adoptive immunotherapy, especially on promotion of cell proliferation and on augmentation of cytotoxicity of various lymphokine-activated killer cells, induced by recombinant interleukin 2(R-IL2) with (P-LAK) or without (LAK) PHA, from peripheral blood lymphocytes (PBL) of healthy volunteers. The following results were obtained. 1) In LAK induced by culturing normal PBL with R-IL2, a cell proliferation was observed in 14 days. Their cytotoxic activity against all the strain cells examined, was higher than PBL already on the third day of culture. 2) Culturing normal PBL with PHA (with or without R-IL2) for 3 days, followed by culturing with R-IL2 for 11 days, caused a marked increase in cell number by about 65 times in 14 days. Cytotoxicity of these cells against MKN-28, MKN-45 and KATO III was found to increase with lapse of culture time. 3) On the 14th day of culture, cytotoxicity of LAK was higher than that of P-LAK. 4) Surface phenotype analysis of LAK revealed that OKT3+ (cell ratio) tended to increase, OKT8+ increased significantly, OKT4+ and Leu7+ tended to decrease, and OKT4+/OKT8+ ratio decreased significantly. Analysis of P-LAK revealed that OKT3+ and OKT8+ increased significantly and OKT4+, Leu7+, and OKT4+/OKT8+ ratio decreased significantly. 5) The rate of total increase in cytotoxicity, calculated in multiplying the rate of cell number increase by the rate of increase in cytotoxicity, was higher in P-LAK than in LAK. The above results showed that P-LAK induced by addition of PHA for the first few days, could cause marked increase both in cell number and in total cytotoxicity.