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Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, are the ideal cell sources for disease modeling, drug discovery, and regenerative medicine. In particular, regenerative therapy with hPSC-derived cardiomyocytes (CMs) is an unmet medical need for the treatment of severe heart failure. Cardiac differentiation protocols from hPSCs are made on the basis of cardiac development in vivo. However, current protocols have yet to yield 100% pure CMs, and their maturity is low. Cardiac development is regulated by the cardiac gene network, including transcription factors (TFs). According to our current understanding of cardiac development, cardiac TFs are sequentially expressed during cardiac commitment in hPSCs. Expression levels of each gene are strictly regulated by epigenetic modifications. DNA methylation, histone modification, and noncoding RNAs significantly influence cardiac differentiation. These complex circuits of genetic and epigenetic factors dynamically affect protein expression and metabolic changes in cardiac differentiation and maturation. Here, we review cardiac differentiation protocols and their molecular machinery, closing with a discussion of the future challenges for producing hPSC-derived CMs. Stem Cells 2019;37:992-1002., (© AlphaMed Press 2019.)

Background: Several studies have reported outbreaks due to human metapneumovirus (hMPV) in long-term care facilities (LTCF) for the elderly. However, most of these reports are epidemiological studies and do not investigate the clinical features of hMPV pneumonia., Methods: Three independent outbreaks of hMPV occurred at separate LTCF for intellectually challenged and elderly residents. A retrospective evaluation of hMPV pneumonia and its clinical and radiological features was conducted using available medical records and data., Results: In 105 hMPV infections, 49% of patients developed pneumonia. The median age of pneumonia cases was significantly higher than non-pneumonia cases (P < .001). Clinical manifestations of hMPV pneumonia included high fever, wheezing in 43%, and respiratory failure in 31% of patients. An elevated number of white blood cells as well as increased levels of C-reactive protein, creatine phosphokinase, and both aspartate and alanine transaminases was also observed among pneumonia cases. Evaluation of chest imaging revealed proximal bronchial wall thickenings radiating outward from the hilum in most patients., Conclusions: The aforementioned characteristics should be considered as representative of hMPV pneumonia. Patients presenting with these features should have laboratory testing performed for prompt diagnosis.

Background: Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and often results from refractory inflammatory bowel disease (IBD). Stress response proteins Cirp and HSPA4 are involved in the refractory clinical course and development of CAC. RNA-binding motif protein 3 (RBM3) is induced in response to various stresses and is upregulated in several cancers. However, the role of RBM3 in CAC is unclear., Methods: We assessed RBM3 expression and function in 263 human intestinal mucosa samples from patients with IBD and in Rbm3-deficient (Rbm3) mice., Results: Expression of RBM3 was correlated with the expression of stress response proteins Cirp, HSPA4, and HSP27 in the colonic mucosa of patients with IBD. Significant correlation was observed between the expression of RBM3 and that of Bcl-xL or stem cell markers. RBM3 expression increased and significantly correlated with R-spondin expression in the colonic mucosa of patients with refractory IBD, a condition associated with increased cancer risk, and RBM3 was overexpressed in human CACs. In the murine CAC model, Rbm3 deficiency decreased R-spondin and Bcl-xL expression and increased apoptotic cell number in the colonic mucosa, leading to reduced tumor multiplicity. Transplantation of wild-type and Rbm3 bone marrow did not alter tumor burden, indicating the importance of RBM3 in epithelial cells., Conclusions: Our findings indicated that RBM3 was required for efficient inflammatory carcinogenesis in the murine CAC model and suggested that RBM3 could be a predictive biomarker of CAC risk and a new therapeutic target for cancer prevention in patients with IBD.

Background: Expression of heat shock protein A4 (HSPA4, also called Apg-2), a member of the HSP110 family, is induced by several forms of stress. The physiological and pathological functions of HSPA4 in the intestine remain to be elucidated., Methods: We assessed HSPA4 expression and function by generating HSPA4-deficient mice and using 214 human intestinal mucosa samples from patients with inflammatory bowel disease (IBD)., Results: In the colonic mucosa of patients with IBD, a significant correlation was observed between the expression of HSPA4 and antiapoptotic protein Bcl-2, a T-cell-derived cytokine IL-17 or stem cell markers, such as Sox2. In refractory ulcerative colitis, a condition associated with increased cancer risk, expression of HSPA4 and Bcl-2 was increased in inflammatory cells of colonic mucosae. HSPA4 was overexpressed both in cancer cells and immune cells of human colorectal cancers. Patients with high expression of HSPA4 or Bmi1 showed significantly lower response rates upon subsequent steroid therapy as compared with patients with low expression of each gene. HSPA4-deficient mice exhibit more apoptosis and less expression of IL-17/IL-23 in inflammatory cells and less number of Sox2 cells after administration of dextran sodium sulfate than control mice. Transduction of HspaA4 bone marrow into wild-type mice reduced the immune response., Conclusions: Upregulation of Bcl-2 and IL-17 by HSPA4 would control apoptosis of inflammatory cells and immune response in the gut, which might develop treatment resistance in IBD. HSPA4 and Bmi1 would be a useful biomarker for refractory clinical course and a promising approach for a therapeutic strategy in patients with IBD.

Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs., (©AlphaMed Press.)

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohn's disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1β levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

Background: Perioperative enteral immunonutrition is thought to reduce postoperative morbidity in patients undergoing major gastrointestinal surgery. This study assessed the clinical effects of preoperative enteral immunonutrition in well nourished patients with gastric cancer undergoing total gastrectomy., Methods: Well nourished patients with primary gastric cancer, fit for total gastrectomy, were randomized to either a control group with regular diet, or an immunonutrition group that received regular diet supplemented with 1000 ml/day of immunonutrients for 5 consecutive days before surgery. The primary endpoint was the incidence of surgical-site infection (SSI). Secondary endpoints were rates of infectious complications, overall postoperative morbidity and C-reactive protein (CRP) levels on 3-4 days after surgery., Results: Of 244 randomized patients, 117 were allocated to the control group and 127 received immunonutrition. SSIs occurred in 27 patients in the immunonutrition group and 23 patients in the control group (risk ratio (RR) 1.09, 95 per cent confidence interval 0.66 to 1.78). Infectious complications were observed in 30 patients in the immunonutrition group and 27 in the control group (RR 1.11, 0.59 to 2.08). The overall postoperative morbidity rate was 30.8 and 26.1 per cent respectively (RR 1.18, 0.78 to 1.78). The median CRP value was 11.8 mg/dl in the immunonutrition group and 9.2 mg/dl in the control group (P = 0.113)., Conclusion: Five-day preoperative enteral immunonutrition failed to demonstrate any clear advantage in terms of early clinical outcomes or modification of the systemic acute-phase response in well nourished patients with gastric cancer undergoing elective total gastrectomy., Registration Number: ID 000000648 (University Hospital Medical Information Network (UMIN) database)., (Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

The administration of granulocyte colony-stimulating factor (G-CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)-derived c-kit(+) cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM-derived hematopoietic cells mobilized by G-CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)-transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G-CSF for 10 days. In the acute phase, a number of GFP(+) cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and alpha-smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G-CSF administration, and the enhanced myofibroblast-rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G-CSF-mobilized monocytes were harvested from the peripheral blood of GFP-transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP(+) myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G-CSF promotes this recruitment and enhances cardiac protection.

Objectives: Fluoroquinolone-resistant Streptococcus pneumoniae are increasing worldwide rapidly. In vitro activities of sitafloxacin were evaluated against clinical isolates of S. pneumoniae resistant to levofloxacin (MIC of levofloxacin > or = 4 mg/L), which were characterized genetically., Methods: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analysed by PCR-based sequencing. MICs of sitafloxacin and other quinolones were determined by a microdilution broth method., Results: All 18 strains had at least one amino acid substitution in the QRDRs of GyrA and ParC, which included Ser-81-->Tyr/Phe and Glu-85-->Lys in GyrA and Ser-79-->Phe/Ile/Tyr, Asp-83-->Tyr, Asn-91-->Asp, Ser-107-->Phe, Lys-137-->Asn and Ala-142-->Ser in ParC. Most isolates had Asp-435-->Asn/Ile-460-->Val/Ala-596-->Thr substitutions in ParE, while no amino acid substitution in GyrB was noted in all isolates. Ten isolates for which levofloxacin MICs were 16 or 32 mg/L had multiple mutations in both GyrA and ParC. The MIC80 value of sitafloxacin for levofloxacin-resistant isolates was 0.25 mg/L. The range of MICs of sitafloxacin for isolates resistant to levofloxacin (MIC 4-32 mg/L) was 0.016-0.5 mg/L., Conclusions: These findings warrant further studies to evaluate the usefulness of sitafloxacin in the treatment of levofloxacin-resistant S. pneumoniae infection.

Objectives: The activities of pazufloxacin and tosufloxacin against Legionella spp. were evaluated in vitro and compared with those of other quinolones, macrolides and azithromycin., Methods: The conventional MICs were determined by the microbroth dilution method. Intracellular activities of drugs were evaluated by a cfu count. The minimal extracellular concentration inhibiting intracellular growth of bacteria (MIEC) was determined by a colorimetric cytopathic assay., Results: MICs of pazuloxacin and tosufloxacin at which 90% (MIC90) of isolates are inhibited in 76 different Legionella spp. strains (38 ATCC strains and 38 clinical isolates) were 0.032 and 0.016 mg/L, whereas the MIC90s of levofloxacin, ciprofloxacin, garenoxacin, erythromycin, clarithromycin and azithromycin were 0.032, 0.032, 0.032, 2.0, 0.125 and 2.0 mg/L, respectively. Pazufloxacin and tosufloxacin at 4x MIC inhibited intracellular growth of Legionella pneumophila SG1 (80-045 strain), as did other quinolones, clarithromycin and azithromycin, whereas erythromycin at 4x MIC did not. MIECs of pazufloxacin, tosufloxacin, levofloxacin, ciprofloxacin and garenoxacin for the strain were 0.063, 0.004, 0.016, 0.032 and 0.008 mg/L respectively, which were superior to those of macrolides and azithromycin. Pazufloxacin showed potent activity against three additional clinical isolates of L. pneumophila SG1, one clinical isolate each of L. pneumophila SG3 and SG5, as well as Legionella micdadei, Legionella dumoffii and Legionella longbeachae SG1., Conclusions: Pazufloxacin and tosufloxacin, as well as other quinolones, were more potent than macrolides and an azalide. Present data warrant further study on the efficacy of these drugs in the treatment of Legionella infections.

Secreted frizzled related protein 1 (sFRP1) is a modulator of Wnt signaling. Recently, aberrations of Wnt signaling were reported to be involved in the pathology of various human neoplasms. We investigated the expression and function of sFRP1 in uterine leiomyomas. Secreted FRP1 expression was increased in leiomyomas, compared with normal myometrium using Northern and Western blot analyses. Expression was strongest in the late follicular phase (high estrogenic milieu) of the menstrual cycle. Interestingly, expression was negligible in leiomyomas treated with GnRH agonist. Expression was also prominent in cells during E2 treatment, serum deprivation, and hypoxia. Moreover, induction of apoptosis by serum deprivation in a leiomyosarcoma cell line was enhanced by antisense inhibition of sFRP1. These results suggest that sFRP1 expression was associated with uterine leiomyomas, particularly under high estrogenic conditions. Secreted FRP1 expression was not associated with cell proliferation but rather occurred during cell protection against apoptosis in vitro. Strong sFRP1 expression under high estrogenic conditions seems to contribute to the development of uterine leiomyomas through the antiapoptotic effect of sFRP1, which appear to be independent of cell proliferation.

It has been suggested that the humoral immune system plays a role in the pathogenesis of non-specific interstitial pneumonia (NSIP). Although some circulating autoantibodies to cytoskeletal protein(s) have been suggested, the antimyofibroblast antibody has not been investigated in patients with idiopathic pulmonary fibrosis (IPF) and NSIP. The purpose of this study is to evaluate the existence of antimyofibroblast antibody in the sera of patients with IPF and NSIP. The MRC5 cell line was used as a model of myofibroblast. The anti-MRC5 cell antibody was characterized in a patient with NSIP using Western blotting. Since we found that one of the anti-MRC5 antibodies was an antivimentin antibody, we established an enzyme-linked immunosorbent assay (ELISA) to measure the levels of antivimentin antibody in the sera of patients with IPF (n = 12) and NSIP (n = 23). Initially, two anti-MRC5 cell antibodies were detected in the sera of patients with NSIP, one of which was characterized as the antivimentin antibody by Western blotting. The other was characterized as an antivimentin fragment antibody. We established an ELISA to measure the antivimentin antibody and found significantly higher levels in patients with IPF and NSIP than in normal volunteers. One of the anti-MRC5 cell antibodies in the serum of a patient with NSIP was against vimentin. The serum levels of antivimentin antibody were increased in patients with IPF and NSIP compared with that of normal volunteers. These results suggest that the antivimentin antibody may be involved in the process of lung injury in IPF and NSIP.

Antibodies to cytokeratin (CK) are found in some patients with autoimmune hepatitis (AIH). We hypothesized that serum antibodies to CK8, CK18 and CK19 may be formed in patients with AIH. We established an enzyme-linked immunosorbent assay (ELISA) to quantify anti-CK8, anti-CK18 and anti-CK19 antibodies in sera of patients with AIH. In addition, we quantified circulating CK8:anti-CK8 antibody as well as CK18:anti-CK18 antibody immune complexes in patients' sera, by an enzyme-linked immunosorbent assay (ELISA). Furthermore, to evaluate the expression of CK8, CK18 and CK19 in liver tissue, immunohistochemical stainings were performed. Significantly high levels of anti-CK8, anti-CK18 and anti-CK19 antibodies were demonstrated in patients with AIH compared with normal volunteers and patients with chronic active hepatitis C (CH-C). In addition, these antibodies were significantly decreased after steroid treatment. Levels of CK8:anti-CK8 and CK18:anti-CK18 immune complexes in sera of patients with AIH were significantly high compared with those of patients with CH-C and normal volunteers. Immunohistochemically, CK8 or CK18 were absent from some hepatocytes of AIH. CK19 was aberrantly expressed in periportal hepatocytes in patients with AIH, but not CH-C. This is the first study to quantify anti-CK8, anti-CK18, anti-CK19 antibodies and immune complexes in patients with AIH. The clinical significance of anti-CK antibodies and their immune complexes of AIH is also discussed.

Background: Small cell lung cancer (SCLC) has a higher percentage of hilar masses than other histological types of lung cancer. The primary site is usually adjacent to the hilum, but we often observe primary sites in the peripheral lung field. In this study, our objectives were to elucidate whether peripheral-located small cell lung cancer (PSCLC) is an independent entity and whether it differs clinically from central-located small cell lung cancer (CSCLC)., Methods: We reviewed the clinical and pathological features of 52 patients treated at Kagawa Medical University Hospital between 1987 and 1996 with a diagnosis of SCLC. We defined CSCLC as a tumor whose primary site is located in the segmental bronchi or more proximally and PSCLC as a tumor located distal to the subsegmental bronchi. Twenty-one PSCLC patients and 31 CSCLC patients were identified. Kaplan-Meier survival curves were constructed and comparisons were made between PSCLC and CSCLC by the log-rank test. The carcinoembryonic antigen (CEA) level was also evaluated in each group., Results: Although the percentage of limited disease (LD) in the patients with PSCLC was higher than that in the patients with CSCLC, the 3-year survival rate of PSCLC tended to be worse than that of CSCLC (9% for patients with PSCLC and 29% for those with CSCLC). Survival curves of patients with PSCLC also tended to be worse than those of patients with CSCLC, not only in the limited disease group but also in the extensive disease (ED) group. In addition, the mean CEA value in patients with PSCLC was higher than that in patients with CSCLC (p < 0.001), whereas the neuron specific enolase (NSE) level was not significantly different between PSCLC and CSCLC. The median survival of patients with pretherapeutic CEA titers of > or =5 ng/ml was significantly shorter than that in patients with CEA levels <5 ng/ml., Conclusion: These findings suggest that the survival of SCLC patients with a high CEA level was significantly shorter than that of patients with a low CEA level. In addition, CEA levels in PSCLC patients were significantly higher than those in CSCLC patients. However, the survivals of LD or ED patients with PSCLC and CSCLC were not statistically different.

Two men with occupational exposure to asbestos were admitted to our hospital with minute pleural changes on their chest CT image. Conventional computed tomography (CT) scans of the chest showed slightly thickened interlobar fissures and a small amount of pleural effusion. In addition, high-resolution CT showed small nodular opacities on interlobar fissures. There were no intrapulmonary mass shadows, pleural plaques or other extrapulmonary mass shadows. These roentgenographical findings were very similar to each other. Hyarulonic acid values obtained from their pleural fluid were extremely high. Finally, we diagnosed them as having malignant mesothelioma using an immunocytochemical technique and electronmicroscopy. We conclude that HRCT is helpful in the diagnosis of malignant mesothelioma, particularly in its early manifestation such as nodular opacities of interlobar fissures.

The effect of 1alpha,25-dihydroxylumisterol3 (1alpha,25(OH)2lumisterol3) on insulin release from rat pancreatic beta-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1Alpha,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mM glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1beta,25-dihydroxyvitamin D3 (1beta,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1Alpha,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx through voltage-dependent Ca2+ channels on the plasma membrane, being also linked to metabolic signals derived from glucose in pancreatic beta-cells. However, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D through mVDR.

We have previously reported that intravenous administration of splenocytes prepared from mice in the early stages of pregnancy promoted embryo implantation in pseudopregnant mice. Since a T-lymphocyte-rich, but not a monocyte-rich preparation from splenocytes enhanced embryo implantation, similar effects of thymocytes from non-pregnant mice on implantation were examined in this study. Thymocytes were prepared from immature 21 day old ICR female mice and the supernatant of a thymocyte suspension (Th-sup) was used as the control. Thymocytes or Th-sup were injected into the caudal vein of recipient mice on pseudopregnancy day 2, and blastocysts were transferred into the endometrial lumen. The implantation rates per recipient were significantly higher in the thymocyte-treated group. ICR mice were then oophorectomized on pseudopregnancy day 3. After 3-day progesterone supplementation, blastocysts were transferred with intravenous injection of thymocytes or Th-sup. Under progesterone supplementation, successful implantations were observed in the thymocyte-treated group, but not in the Th-sup-treated group. Reverse transcriptase-polymerase chain reaction analysis revealed that mRNA expression of leukaemia inhibitory factor in the uterus was induced by thymocyte administration, but not by Th-sup. Thymocytes were divided into two populations, CD4(+/-)CD8(-) group and CD4(-)CD8(+/-) group, by separation columns. On pseudopregnancy day 2, the separated thymocytes in each group or their supernatant were injected into the endometrial stroma of the recipient mice, and blastocysts were transferred into the endometrial lumen. The administration of CD4(+/-) CD8(-) lymphocytes significantly promoted implantation rates, but no effect was observed in the CD4(-) CD8(+/-) group. These findings showed that thymocytes, especially CD4-positive lymphocytes, facilitate embryo implantation, probably by regulating endometrial differentiation.

Mutation within the Mitf gene causes, in microphthalmic Mitf(mi)/Mitf(mi) (mi/mi) mice, multiple defects, including white coat color and functional defects in macrophages and osteoclasts. Our previous mating experiments have demonstrated that the mi mutation reduces the numbers of newborns and induces uterine inversion at delivery. The present study was designed to determine the causes of these pregnancy defects. The histology and number of F4/80-positive macrophages were not different between the ovaries of 23-day-old mi/mi and +/+ mice given eCG 48 h earlier. When ovulation was induced in these mice by hCG, the number of ovulated ova was significantly smaller in mi/mi mice than in wild-type (+/+) mice (p < 0.05). When bone marrow cells from +/+ mice were transplanted i.p. into 42 mi/mi female newborns, successful transplantation was observed in 16 of them at 20 days after birth. In one of these, the upper incisors had erupted. The mean number of tubal ova in mi/mi mice significantly increased after transplantation (p < 0.05) and was almost equal to that of +/+ mice. No uterine inversion occurred at 6 deliveries in 5 mi/mi females after bone marrow transplantation, while it occurred at 4 of 5 deliveries in mi/mi females during the same observation period (p < 0.05). These results indicate that bone marrow-derived cells, defective in mi/mi mice, are necessary for normal ovulation and delivery; the findings are consistent with the notion that macrophages play major roles in ovulation.

We have reported that i.v. administration of splenocytes prepared from early pregnant mice promoted embryo implantation in pseudopregnant CD-1 (ICR) (closed colony) mice. In this study, the similar effects of splenocytes were confirmed using an inbred strain, BALB/c mice, and the mechanism was further investigated. Splenocytes were prepared from pregnancy day 4 (P4-spl) and dioestrus mice (Di-spl), and supernatant of P4-spl suspension (P4-sup) was used as controls. On pseudopregnancy day 2, splenocytes or supernatant were injected into caudal vein or endometrial stroma of the recipient mice, and blastocysts were transferred into the endometrial lumen. In both BALB/c and ICR strains, the implantation rates per recipient with i.v. and intraendometrial injection were significantly higher in P4-spl groups. Then, ICR mice were oophorectomized on pseudopregnancy day 3. After 3 day progesterone supplementation, blastocysts were transferred with i.v. injection of P4-spl and P4-sup, or s.c. oestradiol injection. Under progesterone supplementation, successful implantations were observed in the P4-spl- and oestradiol-treated groups, but not in P4-sup-treated group. Reverse transcriptase-polymerase chain reaction analysis revealed that messenger RNA expression of leukaemia inhibitory factor in the uterus was induced by P4-spl and oestradiol, but not by P4-sup. These findings showed that splenocytes of early pregnant mice promote embryo implantation by regulating endometrial differentiation.

Serum IL-6 level increased after the injection of turpentine oil into the right gastrocnemius muscle in mice. The mRNA level of IL-6 was highest in the injected muscle at 12 h after injection, but was not identified in the opposite muscle. The activities of cathepsins B and B+L started to elevate after 12 h in the injected muscle and markedly increased after day 3. Likewise, the mRNA levels of cathepsins B and L markedly increased from day 1 to day 5 in the injected muscle. However, a very mild increase was also observed in the opposite muscle. Immunohistochemical staining of cathepsins B and L exhibited positive reactions as fine granules in myofibers at 12 h and strong positive reactions in the infiltrating macrophages at 3 days. Atrophy of myofibers type 1 and 2 was evident in a time-dependent manner in the injected muscle. Treatment with rat anti-mouse IL-6 receptor monoclonal antibody inhibited the increase in cathepsin activities in the injected muscle. We conclude that IL-6 produced in the inflamed muscle is involved in the process of muscle degeneration, especially through the activation of lysosomal cathepsins.

Differentiation of endometrial stromal cells (decidualization) is essential for embryo implantation and maintenance of pregnancy. By sequential complementary DNA subtractive hybridization, one of the messenger RNAs (mRNA) induced by progesterone in human endometrial stromal cells decidualized in vitro was identified as that of a tissue transglutaminase type II (TGase). TGase mRNA was induced within 6 h after the addition of progesterone to the culture, and the effect was dose dependent. Both the TGase inhibitor monodansylcadaverine and oligodeoxynucleotide complementary to the TGase mRNA inhibited the decidualization, as assessed by PRL production and morphological transformation. Expression of TGase mRNA in human decidua and endometria exposed to high levels of progesterone in vivo was demonstrated by Northern blotting and in situ hybridization. These data suggest that TGase is necessary for the decidualization of human endometrial stromal cells and that clarification of the mechanism of action of TGase will facilitate further insight into the diagnosis and treatment of infertility.

Natural killer (NK) cells become a prominent cell population in the rodent uterus during pregnancy. The mature, heavily granulated form of these cells is rare in virgin or postpartum uteri. Death, migration, or dedifferentiation could account for the disappearance of these cells from late gestation uteri. We asked whether uterine NK cells, also known as granulated metrial gland (GMG) cells, die in situ and if expression of Fas antigen is essential for their death. Late in gestation, fragmentation of nuclear DNA was detected histologically by OH-end labeling, as were ultrastructural changes suggesting cell death. NK cells developed in and were lost from the uteri of pregnant Fas antigen-deficient lpr/lpr mice. Postpartum samples of retained placentas contained some residual NK cells that had moved from regions of uterine musculature toward the uterine lumen and were being expelled with the placenta. Thus, both cell death and placental separation remove NK cells from the peripartum uterus.

Endometrial stromal differentiation (decidualization) is essential for implantation of the developing blastocyst. To investigate the process of progesterone (P)-induced decidualization of human endometrial stromal cells (ESC), a complementary DNA library enriched with P-induced genes was constructed from cultured human ESC by subtractive hybridization and the polymerase chain reaction. One of the isolated clones was the complementary DNA for the tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently identified member of the human TIMP family. When human ESC were cultured in the presence of P for 6 days, the induction of TIMP-3 messenger RNA (mRNA) expression was observed by Northern blotting. In contrast, the marked induction of PRL mRNA expression and morphological changes were observed after 9 days of culture. P-induced TIMP-3 mRNA expression was dose dependent, and this induction was inhibited by the antiprogestin RU486. Estrogen did not induce TIMP-3 mRNA expression under similar conditions. In situ hybridization analysis of endometria from nonpregnant women revealed that the TIMP-3 mRNA expression was restricted to predecidualized stromal cells. At the feto-maternal interface, TIMP-3 expression was observed in fetal extravillous trophoblasts that had invaded the maternal decidual tissues as well as in the maternal decidual cells. These findings suggest that TIMP-3 is a sensitive indicator of ESC decidualization, and that the induction of TIMP-3 expression in decidual cells and trophoblasts may be important in the regulation of trophoblast invasion.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.

We devised an index to estimate the degree cross-resistance between piperacillin, ceftazidime, sulbactam/cefoperazone, amikacin, tobramycin, carumonam and imipenem against 139 separate clinical isolates of Pseudomonas aeruginosa. A negative value of the index indicated the cross-resistance whereas a positive value suggested the converse making the device an index for the absence of cross resistance (ACR). Using the ACR index, we identified pairs of antibiotics exhibiting the least degree of cross-resistance and therefore the highest potential for treating infections due to P. aeruginosa.

Progesterone (P) is known to regulate sex steroid receptors in uterine cells. However, its precise regulation at the messenger ribonucleic acid (mRNA) level is unclear. In this study we examined the effects of P and testosterone (T) on the regulation of sex steroid receptors in cultured human endometrial stromal cells (ESC), using the quantitative reverse transcriptase polymerase chain reaction method. We isolated ESC from human endometrial tissues and cultured them with or without P (10(-6) mol/L) or T (10(-8) mol/L) for 9 days. Incubation with P decreased progesterone receptor (PR), estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.56 +/- 0.04-, 0.53 +/- 0.08-, and 0.84 +/- 0.04-fold (mean +/- SE), respectively. T also decreased PR, estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.48 +/- 0.06-, 0.52 +/- 0.05-, and 0.82 +/- 0.04-fold (mean +/- SE), respectively. These decreases by P and T occurred in a dose-dependent manner. We also examined the sex steroid receptor levels in human ESC cultured for 0, 3, 6, and 9 days. The PR mRNA level in ESC without P was increased in a time-dependent manner. This increase was also inhibited by P, and the mRNA level in the presence of P was almost constant throughout the culture period. Our results demonstrated that P or T is a regulator of sex steroid receptors in ESC and that this regulation may influence the responsiveness to P of decidual change in ESC.

Increasing evidence suggests that macrophage colony-stimulating factor (M-CSF) is produced in the uterine endometrium and that it plays an important role in the reproductive process. In the present study, using an in vitro decidualization model and human endometrium, we investigated M-CSF messenger RNA (mRNA) expression in human endometrial stromal cells (ESC) by Northern blotting and in situ hybridization. The secreted M-CSF in the culture medium of ESC was measured by enzyme-linked immunosorbent assay. ESC were cultured in the presence of progesterone (P) or estrogen. After a 9-day culture with P, when in vitro decidualization was confirmed by the production of PRL, M-CSF mRNA and protein levels were 3.1 +/- 0.5- and 3.2 +/- 0.8-fold (mean +/- SEM) higher, respectively, than those in cultures without P (P < 0.01). The P-induced increase was dose dependent. On the other hand, estrogen did not increase M-CSF mRNA expression. M-CSF mRNA expression in the first trimester deciduae that expressed PRL mRNA was higher than that in the endometria. By in situ hybridization, ESC as well as epithelial cells were shown to express M-CSF both in vitro and in vivo. These findings indicate that human ESC (decidua cells) express M-CSF mRNA and suggest that they secrete M-CSF in a P-dependent manner during the process of decidualization.

Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine M1 myeloid leukemia cell line, is essential for blastocyst implantation in mice. However, its expression and the role it plays in the human uterus are unknown. To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Analysis of LIF mRNA showed two hybridization bands, with estimated mRNA sizes of about 4.0-kb pairs and 1.8-kb pairs. LIF mRNA was detected at high levels in endometrial tissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta. In the secretory phase, the endometrial tissue showed higher LIF expression than in the proliferative phase (9.5-fold; p < 0.01). The endometrial tissues were separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in each fraction were examined by quantitative RT-PCR. These levels were higher in the EF than in the SF (3.3-fold; p < 0.05). These findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.

The c-kit proto-oncogene encodes a tyrosine kinase receptor and is allelic with the dominant white-spotting (W) locus of the mouse. In this study we investigated the expression of human c-kit protein in various adult and fetal human tissues immunohistochemically using anti-human c-kit monoclonal antibody. To discriminate c-kit+ cells from mast cells expressing c-kit, mast cells were identified by staining with Toluidine blue. In oogonia, spermatogonia and skin melanocytes of the fetus and in oocytes of adult ovary, c-kit expression was detected. In adult uterus, c-kit+ cells were widely distributed in the basal layer of the endometrium, myometrium and cervix, the number and distribution being almost identical to those of mast cells. In fetal uterus, c-kit+ non-mast cells clustered beneath the epithelium and a few mast cells were observed in the myometrium and subserosal layer. In both adult and fetus, c-kit+ non-mast cells were detected within smooth muscle layers of the intestine, colon and oesophagus, while mast cells were observed in the mucosal and submucosal layers of these organs. In contrast to mice, no expression of c-kit protein was detected in the human placenta and decidua. Thus, the distribution of c-kit+ cells in various tissues is similar but not identical between adult and fetus and between human and mouse.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and is shown to be allelic with the white-spotting locus (W) of the mouse. In order to elucidate the role of c-kit protein during placental development, we have examined the expression of c-kit protein in the uterus and placenta of mice at pre- and post-implantation stages by the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody. At Days 3 and 5 of pregnancy and pseudo-pregnancy, c-kit protein was detected in the glandular epithelium, but little expression was observed in the luminal epithelium. At Day 7 of pregnancy, expression was detected in the stromal cells around the uterine crypts of the mesometrial portion, but not in the vigorously proliferating decidual cells around the developing embryo. At Days 9 and 10 of pregnancy, the decidua basalis facing invading trophoblasts gradually expressed c-kit protein. In the mature placenta, c-kit protein was detected in the labyrinthine and decidual layers, but in neither the giant trophoblastic nor the spongiotrophoblastic layer. By Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), c-kit mRNA was detected at the stages of periimplantation and placental development. These results suggested that the c-kit protein might be involved in the proliferation and differentiation of placenta.

The activity of 12 antibiotics, piperacillin, cefazolin, cefotiam, ceftizoxime, latamoxef, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline, against 120 isolates of Pseudomonas aeruginosa was examined. In addition, the efficacy of antibiotics against single-, double-, or triple-drug-resistant isolates of P. aeruginosa were also examined to determine the cross-resistance to each drug. There was cross-resistance between piperacillin, ceftazidime and aztreonam, but amikacin and imipenem remained effective antibiotics, especially as salvage therapy, against isolates resistant to one agent. Results also suggested that piperacillin, ceftazidime or imipenem in combination with amikacin are effective combination regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenem were also suitable antibiotics, especially as salvage therapy, against isolates of P. aeruginosa resistant to two agents. In conclusion, the results provide useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant P. aeruginosa.

We have demonstrated interleukin-6 (IL-6) production by human renal carcinoma cells. The IL-6 gene expression was detected by Northern blot analysis in 22 of 43 primary renal cell carcinoma tissues and in five of seven renal cell carcinoma cell lines. Immunohistochemical analysis confirmed the expression of IL-6 by the tumor cells. Patients with a high-level expression of IL-6 had significantly greater incidences of lymph node metastasis and a larger increase in serum C-reactive protein than those without it. We have also probed for the presence of IL-6 receptor by Northern blot analysis; we detected this receptor in 11 of the 43 primary renal cell carcinoma tissues but in none of the seven renal cell carcinoma cell lines. However, by use of the complementary DNA-polymerase chain reaction, the IL-6 receptor transcript was detected in all specimens, including the seven cell lines. No expression of the interleukin-3 (IL-3) gene was identified in any of the 43 primary renal cell tumors. These data provide evidence that IL-6 and its receptor may play a role in promoting the transformation and/or proliferation of renal cell carcinomas as well as in teh development of symptoms.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

A phase II trial of 5'-deoxy-5-fluorouridine (5'-DFUR), a new fluorinated pyrimidine analog which has been demonstrated to have potential superiority over 5-FU and tegafur for chemotherapy of murine tumors, was performed in patients with advanced non-small cell carcinoma of the lung and metastatic pulmonary tumors. 5'-DFUR at a dose of 800 mg/m2 was given per os every day for more than four weeks. None of 15 evaluable patients with non-small cell carcinoma of the lung and 15 evaluable patients with metastatic pulmonary tumors showed a complete or partial response. Toxic effects of 5'-DFUR included anorexia (29%), diarrhea (26%), nausea (23%), vomiting (10%), leukocytopenia (10%), general fatigue (10%), liver disorder (6%) and thrombocytopenia (6%).

Ras-oncogene activation was investigated in the bladder tumors of F344 male rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. DNA from one of the nine transitional cell carcinomas contained an H-ras oncogene detectable by the NIH/3T3 transfection assay. Analysis of p21 ras proteins suggested that the activating mutation resided within codon 61 of the H-ras gene and that such activating mutations were not present in other tumors. In contrast to mutational activation of ras genes, enhanced expression of p21 was observed in all tumors examined by immunohistochemical techniques with the use of Formalin-fixed paraffin-embedded tissue sections and an anti-ras p21 antibody, RAP-5. Further histochemical analysis of bladder tissues at various stages of the BBN-induced carcinogenic process indicated that the enhanced expression of p21 appeared early; the reactivity with RAP-5 was observed in diffuse hyperplastic epithelia after 5 weeks of exposure to BBN. The frequency of ras oncogenes, activated either by point mutations or overexpression of p21, in BBN-induced rat bladder carcinomas has thus been shown to be similar to that observed in human bladder carcinomas.

Sister chromatid exchanges (SCE) frequency in cultured lymphocytes from seven patients with lung cancer receiving cis-Diamminedichloroplatinum(II) (Pt(II] for chemotherapy was studied. Significantly increased SCE frequency was observed in all the patients, six of whom received intravenous infusion of Pt(II) and one of whom received the agent as an intrapleural infusion. In addition, a dose-dependent increase in the frequency of SCE was observed in lymphocytes from healthy subjects upon exposure to Pt(II) in vitro. The results of the present study revealed that Pt(II) is a potent inducer of SCE in human lymphocytes in vivo and in vitro. It thus appears that follow-up study of the long-term survivors who have been treated with this agent for cancer is necessary because of the possible occurrence of secondary neoplasms.

A phase II study of adriamycin (ADM) (60 mg/m2) was performed in 22 patients with non-small cell lung carcinoma (NSCLC). There were no responders in the 19 evaluable patients (16 with adenocarcinoma, two with squamous cell carcinoma and one with large cell carcinoma). The major side effects were alopecia (89%), leukocytopenia (73%), thrombocytopenia (58%) and upper gastrointestinal symptoms. Although ADM at 60 mg/m2 did not appear to have sufficient antitumor activity against NSCLC in this study, it is necessary to evaluate further the efficacy of ADM against NSCLC with another treatment schedule.

Natural killer (NK) and lymphokine activated killer (LAK) cell activities in 133 healthy volunteers were analyzed with regard to the volunteer's sex, age, smoking history and the familial incidence of cancer. None of these factors had any influence on NK and LAK cell activities. It was concluded that identifying individuals with increased risk of cancer development by examining NK and LAK activities would be difficult.

Injection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genetically deficient in mast cells resulted in a 10-fold increase in the histidine decarboxylase [HDC, L-histidine carboxylase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Transplantation of cells from other organs of the normal mice, such as the thymus, mesenteric lymph node and spleen, did not restore the HDC increase significantly. Transplantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treatment with anti-Thy-1,2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.

About half of the chimeras produced by aggregation of two mouse embryos are sex chimeras composed of both XX and XY cells. We developed a fast and easy method to identify sex chimeras by using electrophoretic bimorphism of an X-linked enzyme, phosphoglycerate kinase-1 (PGK-1), as a marker. When embryos resulting from the crossing of a Pgk-1b/Pgk-1b female and a Pgk-1a/Y male are aggregated, the genotype of sex chimeras is Pgk-1b/Pgk-1a----Pgk-1b/Y. Most of these were identifiable from the PGK-1 electrophoretic pattern of blood cells (i.e., AB type) and the appearance of genitalia (male type or apparently abnormal). Genotypes of functional sperm in the testes of the male-type sex chimeras were also identifiable from the PGK-1 electrophoretic pattern of progenies. Examination of gonads of the sex chimeras revealed that a considerable proportion was hermaphorditic. With this method, reasonable numbers of male-type sex chimeras and hermaphrodites may be selected and used as material for investigating sexual differentiation.

Twenty-six patients with non-small cell carcinoma of the lung and 25 with metastatic pulmonary tumors were treated by intravenous injection of 7-N-(p-hydroxyphenyl)-mitomycin C (KW2083), a derivative of mitomycin C, either at a single 70-mg/m2 dose or at a dose of 20-30 mg/m2 once a week for 3 weeks. Two patients with adenocarcinoma among 21 evaluable patients with non-small cell carcinoma of the lung, and one with embryonal cell carcinoma among 21 patients with metastatic pulmonary tumors achieved partial response lasting 5 to 7 weeks. In these three patients, KW2083 was administered by a single 70-mg/m2 dose, and no patients who received a dose of 20-30 mg/m2 weekly achieved objective response. Myelosuppression, primarily thrombocytopenia, was pronounced with either treatment regimen and it was the major dose-limiting toxicity.