Using size-controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice.

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane-based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor-bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 µm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 µm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin ß1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 104 colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor-bearing mouse models.

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size.

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

The effects of a life goal-setting technique in a preventive care program for frail community-dwelling older people: a cluster nonrandomized controlled trial.

BACKGROUND: Frailty among older people is associated with an increased risk of needing care. There have been many reports on preventive care programs for frail older people, but few have shown positive effects on disability prevention. Physical exercise programs for frail older people affect elements such as physical fitness and balance, but are less effective for disability outcomes and are not followed up in the longer term. We developed a life goal-setting technique (LGST). Our objective was to determine the effect of a LGST plus standard preventive care program for community-dwelling frail older people. METHODS: We used a cluster nonrandomized controlled trial with seven intervention and nine matched control groups, with baseline assessment and follow-up at 3, 6, and 9 months. Participants were 176 frail older people, aged 65 years or over, living in the community in Izumi, Osaka, Japan. All participants attended regular 120 min preventive care exercise classes each week, over 3 months. They also received oral care and nutrition education. The intervention groups alone received life goal-setting support. We assessed outcomes longitudinally, comparing pre-intervention with follow-up. The primary outcome measure was health improvement according to the Japanese Ministry of Health, Labour and Welfare's "Kihon Checklist" for assessment of frailty and quality of life (QOL), analyzed with a two-way ANOVA and post-test comparison. Secondary outcomes included physical functions and assessment of life goals. RESULTS: The improvement on the Kihon Checklist for the intervention group was approximately 60 % from baseline to 9-months follow-up; the control group improved by approximately 40 %. The difference between groups was significant at 3-month (p = 0.043) and 6-month (p = 0.015) follow-ups but not at 9-month (p = 0.098) follow-up. Analysis of QOL yielded a significant time × group interaction effect (p = 0.022). The effect was significant at 3 months in the intervention group, but at no time in the control group. CONCLUSION: A 3-month exercise program helped to decrease frailty and improve QOL in frail older people, and the addition of LGST increased its effectiveness. The LGST is a feasible and promising intervention for reducing risk of needing care. TRIAL REGISTRATION: UMIN000021485 . Registered 15 March 2016.

Increased Insulin Secretion from Insulin-Secreting Cells by Construction of Mixed Multicellular Spheroids.

PURPOSE: We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids. MATERIALS AND METHODS: Multicellular spheroids of mouse MIN6 pancreatic ß cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot. RESULTS: In all cases, uniformly sized (about 300 µm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic ß cells, also increased in their spheroids. CONCLUSIONS: The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.

Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture.

During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content.

Transplantation of insulin-secreting multicellular spheroids for the treatment of type 1 diabetes in mice.

The efficacy of cell-based therapy depends on the function and survival of transplanted cells, which have been suggested to be enhanced by spheroid formation. However, few attempts at spheroid generation from insulin-secreting cells, which may be used to treat type 1 diabetes, have been reported. We therefore developed spheroids from the mouse insulinoma cell line NIT-1 by using polydimethylsiloxane (PDMS)-based microwells with a coating of poly(N-isopropylacrylamide) (PNIPAAm). The prepared NIT-1 spheroids or dissociated NIT-1 cells were transplanted into the subrenal capsule in streptozotocin-induced diabetic mice. NIT-1 spheroids prepared using the PNIPAAm-coated PDMS-based microwells had a uniformly sized spherical structure with a diameter of 200-300µm. The PNIPAAm coating increased cell survival in the spheroids and the recovery of the spheroids from the microwells. In diabetic mice, the transplanted NIT-1 spheroids reduced blood glucose levels to normal values faster than dissociated NIT-1 cells did. Additionally, survival was higher among NIT-1 cells in spheroids than among dissociated NIT-1 cells 24h after transplantation. These results indicate that insulin-secreting NIT-1 spheroids prepared using PNIPAAm-coated PDMS-based microwells are more effective for the treatment of type 1 diabetes than dissociated cells in suspension.

Poly(N-isopropylacrylamide)-coated microwell arrays for construction and recovery of multicellular spheroids.

Microwell arrays that have many micro-sized cavities on the device have been employed to form multicellular spheroids. However, methods to efficiently harvest the constructed spheroids from the microwell arrays have not been thoroughly investigated. We evaluated the effects of poly(N-isopropylacrylamide) (PNIPAAm) for constructing and harvesting spheroids from microwell arrays. Microwell arrays were coated with ethanol containing 1%, 2.5%, 5%, or 10% PNIPAAm by a solvent-casting method and then dried. Spheroids formed using the coated microwell arrays were harvested. Highly efficient and rapid recovery of NIH3T3 mouse fibroblast spheroids were achieved for the 5% and 10% coated wells (93.2% ± 1.6% and 93.6% ± 1.1% at 60 s, respectively), whereas recovery was not efficient for 0%, 1%, and 2.5% coated wells (0.2% ± 0.2%, 1.1% ± 0.6%, and 7.8% ± 4.0% at 60 s, respectively). Because PNIPAAm is a thermoresponsive polymer that exhibits a lower critical solution temperature (LCST) of 32°C, we examined the effects of temperature on the recovery rate. The recovery rates at 4°C (below LCST) were equivalent to or higher than those at 37°C (above LCST) for all four cell types examined. Functional assessment suggests that the PNIPAAm microwell arrays are not toxic to the formed spheroids. The PNIPAAm microwell array developed in the present study will be useful for constructing and harvesting spheroids.

Bacterial neuraminidase rescues influenza virus replication from inhibition by a neuraminidase inhibitor.

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).

[Potent primary leptomeningeal lymphoma masquerading as tuberculous meningitis - a case report].

A 45-year-old man presented with fever, progressive mutism and memory loss, was admitted to our hospital. MR imaging and angiography suggested multiple foci of infarctions and vasculitis without Gadrinium-enhancement. CSF examination revealed pleocytosis with mononuclear cell dominance and elevated protein content. Adenosine deaminase activity was accelerated, and no malignant cell was found. Whole body CT imaging and Garium-scintigraphy were normal. Under the clinical diagnosis of tuberculous meningitis, anti-tubercular drugs with steroid were administered, resulting in marked attenuation of his neurological impairments. Four months later, his symptoms aggravated and restudy of Garium-scintigraphy revealed enhanced accumulation in the submandibular and abdominal lymphnodes. A lymph node biopsy revealed diffuse large B-cell lymphoma cells. In such a case of this clinical statue, careful and repeated observations should be required to establish the correct diagnosis of occult lymphoma.

[Living situation of the in-home patients suffering from amyotrophic lateral sclerosis in Aichi prefecture, Japan].

It is essential that we know the real situation of at-home patients with amyotrophic lateral sclerosis (ALS) in order to improve their medical support system. We indirectly investigated the daily living status of ALS patients and their families at home by conducting on individual questionnaires survey for nurses working at public health centers in Aichi prefecture, Japan. Detailed information about 136 cases was obtained, and we could clarify the need for variety of communication methods, plasticity of medical interrelations and care between neurologists and home doctors, incomplete utilization of social resources including various official support, overwork among single caregivers, and underdeveloped immature individual medical care support programs for them. Thus it might be important that we should promote the sure utilization of social resources and programming the individual medical care support in their earlier stages. And moreover, we should also consider constructing a general support system for at-home patients with ALS, in which each professional would owe the dividing responsibility, without role duplications. These strategies would lead to overall the better quality of life among ALS patients, and their families.

Prenatal management of the fetus with lethal malformation: from a study of oligohydramnios sequence.

47 cases of oligohydramnios sequence (OS) diagnosed at Kanagawa Children's Medical Center from 1992 to 2008 were studied retrospectively. Early termination of pregnancy was chosen in 9 cases, observed natural labor was chosen in 23 cases, and 18 cases resulted in natural deliveries. Fetal demise occurred during labor in 44.4% of terminated cases, while it occurred in 5.6% of observed cases. Preterm labors and breech presentations occurred most frequently. Most infants died within 18 hours after their births. There were 3 familial cases. These results provide important information for planning the perinatal care when fetuses are diagnosed with OS.

Effect of Obstructive Jaundice and Nitric Oxide on the Profiles of Intestinal Bacterial Flora in Wild and iNOS Mice.

We previously reported that the plasma level of endotoxin and colonic expression of IgA in the mouse increased with obstructive jaundice induced by bile duct ligation (BDL). To elucidate the mechanism of the BDL-induced increase, we analyzed the effect of BDL on intestinal flora in wild type and inducible nitric oxide synthase (iNOS)-deficient mice (iNOS(-/-)) using the terminal restriction fragment length polymorphism analysis (T-RFLP) and 16S rDNA clone libraries. The amounts of bacterial DNA detected in fecal samples from both animal groups pretreated with antibiotics were extremely low as compared with untreated groups. We found that the profiles of enteric bacteria changed markedly after BDL. The bacterial composition is significantly different between not only wild type and iNOS(-/-) mice but also those before and after BDL, respectively. Among enteric bacteria examined, Lactobacillus murinus was found to increase markedly after BDL in rectum of both animal groups. However, Escherichia coli markedly increased after BDL in the iNOS(-/-) mice. These findings suggest that profiles of enteric flora change markedly in animals during obstructive jaundice by some mechanism that is affected by bile constituents and iNOS-derived NO.

Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis.

Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O(2) (-)). To elucidate the role of O(2) (-) and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O(2) (-) and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O(2) (-) and NO may occur in and around phagocytized macrophages.

Dynamic aspect of reactive oxygen and nitric oxide in oral cavity.

Oral mucosa is a critical protective interface between external and internal environments. Therefore, it must serve as a barrier to a huge number of microbial species present in the environment. Saliva is an important factor that provides for the environment in the oral cavity, and it is indispensable to the host defense reaction in this manner. Oral neutrophils are also important contributors to maintaining the balance between health and disease in this complex environment. These produce reactive oxygen species, nitric oxide, and several antimicrobial peptides, and enzymes. Neutrophils and saliva all contribute to the maintaining the health of the oral cavity in overlapping but independent ways. In addition to production by neutrophils and macrophage, some bacteria can also generate superoxide, hydrogen peroxide, and nitric oxide. Dietary intake of nitrate-enriched vegetables might play important roles in the protection of the oral and stomach against hazardous pathogens via the gastro-intestinal-salivary cycle of nitric oxide (NO) and related metabolites. This review will focus on defense system of the human oral cavity and metabolism of reactive oxygen and NO.