Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production in the cell culture. Violacein, a purple-colored pigment produced by microorganisms, has diverse bioactive properties and has been proposed for various industrial applications. In this study, we evaluated the potency of violacein for cell cycle control and improvement of recombinant immunoglobulin G (IgG) production in CHO cells. Compared with the control, 0.9 µM violacein in a 14-day fed-batch culture increased the maximum IgG concentration by 37.6% via increasing the specific production rate and cell longevity. Cell cycle analysis showed that violacein induced G1 and G2/M phase arrest. However, the G1 arrest was observed only on day 1, while G2/M arrest lasted more than 3 days, suggesting that G2/M arrest mediated the violacein-induced enhanced IgG production. Moreover, in line with the increased protein expression, the expression levels of IgG mRNA and nutrient metabolic rates were also increased. N-Linked glycosylation and charge variant profiles were barely affected by violacein treatment. Our results indicate that violacein affects the cell cycle of CHO cells and increases IgG production without changing product quality, showing promise as a mAb production enhancer in CHO cells. The study provides insight into violacein utilization in industrial mAb manufacturing and can help develop advanced, effective mAb production technologies using CHO cell cultures.

Biological Activity of High-Purity ß-1,3-1,6-Glucan Derived from the Black Yeast Aureobasidium pullulans: A Literature Review.

The black yeast Aureobasidium pullulans produces abundant soluble ß-1,3-1,6-glucan-a functional food ingredient with known health benefits. For use as a food material, soluble ß-1,3-1,6-glucan is produced via fermentation using sucrose as the carbon source. Various functionalities of ß-1,3-1,6-glucan have been reported, including its immunomodulatory effect, particularly in the intestine. It also exhibits antitumor and antimetastatic effects, alleviates influenza and food allergies, and relieves stress. Moreover, it reduces the risk of lifestyle-related diseases by protecting the intestinal mucosa, reducing fat, lowering postprandial blood glucose, promoting bone health, and healing gastric ulcers. Furthermore, it induces heat shock protein 70. Clinical studies have reported the antiallergic and triglyceride-reducing effects of ß-1,3-1,6-glucan, which are indicators of improvement in lifestyle-related diseases. The primary and higher-order structures of ß-1,3-1,6-glucan have been elucidated. Specifically, it comprises a single highly-branched glucose residue with the ß-1,6 bond (70% or more) on a backbone of glucose with 1,3-ß bonds. ß-Glucan shows a triple helical structure, and studies on its use as a drug delivery system have been actively conducted. ß-Glucan in combination with anti-inflammatory substances or fullerenes can be used to target macrophages. Based on its health functionality, ß-1,3-1,6-glucan is an interesting material as both food and medicine.

Low-concentration staurosporine improves recombinant antibody productivity in Chinese hamster ovary cells without inducing cell death.

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.

Screening of new cell cycle suppressive compounds from marine-derived microorganisms in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.

A randomized, double-blinded, placebo-controlled, multicenter trial, healing effect of rebamipide in patients with low-dose aspirin and/or non-steroidal anti-inflammatory drug induced small bowel injury.

BACKGROUND: It is not clear what kind of drug is appropriate to heal NSAID-induced enteropathy. Several reports showed the preventive effect of prostaglandin analogue or inducer using healthy subjects who took NSAIDs. However there was no report for healing effect and for patients. The aim of this study was to evaluate the healing effect of rebamipide in patients with NSAIDs-induced enteropathy. In addition, we evaluated for nutritional parameter. METHODS: This study was conducted as a randomized, double-blinded, placebo-controlled, multicenter trial. Study protocol was approved by each hospital's ethical committees. Patients with LDA and/or NSAID more than 3 months were enrolled. Patients with enteropathy were divided into the placebo and the rebamipide groups. Rebamipide 100 mg three times daily was administered during 4 weeks. Capsule endoscopies were performed at 0 and 4 week. The number of small intestinal ulcer and erosion were evaluated. Total protein was analyzed as nutritional parameter. RESULTS: Sixty one participants were completed this study. Change in number of small intestinal erosion in the rebamipide group was -2.5 ± 3.4, and 2.1 ± 3.9 in the placebo group (P < 0.0001). Change in number of small intestinal ulcer in the rebamipide group was -0.5 ± 1.6, and 0.1 ± 0.7 in the placebo group (P = 0.024). Change in serum total protein levels in the rebamipide group was 0.06 ± 0.36, and -0.27 ± 0.34 in the placebo group (P = 0.0005). CONCLUSIONS: Rebamipide has not only the healing effect for NSAIDs-induced enteropathy compared with placebo, but the improvement of nutritional condition. These results showed a tentative therapeutical strategy for chronic NSAIDs users.

[Evaluation of liaison-clinical pathway for patients with breast cancer undergoing adjuvant therapy after curative operation--data from questionnaire survey of 56 clinics and 105 patients].

To evaluate the liaison-clinical pathway for patients with breast cancer introduced since May 2008, the data from a questionnaires survey of 56 clinics and 105 patients were reviewed. Half of the clinics specialized in internal medicine. 93% of physicians recognized the utility of the pathway while 24% made the most of the pathway. About 40% of the clinics wished to enlarge both the patient number and treatment materials. Half of the patients were employed. 55% of patients valued the pathway as helpful. And 29% of patients used the patient booklet at all times. 8% of patients replied they had complaints went to clinics. There has been no serious problem in using the pathway. Countermeasures to electronic health records in clinics, and responses to requests from each patient will be needed.

Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.

The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca(2+). One of the sites was identified as Gly(154)-Leu(155), and Leu(155) was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80 degrees C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly(154)-Leu(155) bond to the X(155)-Ile(156) one with the mutation at Leu(155). Furthermore, the Ile(164)-Asp(165) bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.

Outcomes of small nodular lesions in heavy drinkers.

BACKGROUND: Increased detection of nodular lesions in patients not yet definitively diagnosed with hepatocellular carcinoma (HCC) has occurred with the use of advanced imaging techniques. In heavy drinkers, decrease in the size of nodular lesions during on-going observation, and negation of diagnoses of HCC after surgical resection have been reported, suggesting the need for caution in diagnosis in such cases. METHODS: The subjects were eight heavy drinkers with small nodular lesions, 20 mm or less in diameter. All patients were male, with a mean age of 53 years. Five had single and three had multiple nodular lesions. Five of the eight patients were followed up for more than 4 years after an initial biopsy, while three patients were recent cases. Of the three recent cases, two were positive for antibody to hepatitis C virus (HCV) and two had hypervascular nodular lesion. Biopsies were performed percutaneously, under ultrasonography, for histological diagnosis of all cases, and the recent cases were also assessed using a variety of imaging techniques. RESULTS: On initial biopsy, no atypism (NO) was found in two patients, and borderline lesions (Border) were present in six patients. Of the five cases followed up long-term, only one of the two with NO progressed to HCC, and the three with Border showed disappearance, decrease, and no change, respectively, during the follow-up period. Of the three recent cases, no changes in size or morphology as revealed by imaging were observed following biopsy. CONCLUSIONS: In heavy drinkers, no fixed relationship was observed between initial biopsy finding and clinical course, suggesting that indication for biopsy requires reassessment, refinement, and discussion. Furthermore, tumor staining may occur in hyperplastic nodules, which are histologically similar to early HCC by needle biopsy, and care needs to be exercised for diagnosis in heavy drinkers.

A case of complete necrosis of well-differentiated early hepatocellular carcinoma by percutaneous transhepatic portal embolization.

The patient was a 49-year-old male under observation for chronic hepatitis B. In July 1997, abdominal ultrasonography showed multiple hyperechoic nodules in the right lobe, the largest with a diameter of 3.5 cm. Abdominal computed tomography did not reveal enhanced nodules, and the patient was hospitalized for suspected multiple liver tumors. All biochemical tests were normal, except for a slight decrease in platelets. The patient was positive for HBs antigen, negative for HBe antigen and positive for anti-HBe antibody. Hepatocellular carcinoma (HCC) with patent portal blood flow was strongly suspected based on the results of various imaging techniques. A tumor biopsy was conducted, and findings of multiple early hepatocellular carcinoma of a well-differentiated type were observed. Based on the HCC stage and liver function, an extensive right hepatectomy was indicated. Before the surgical resection, a percutaneous transhepatic portal embolization (PTPE) was performed using gerfoam sponzel in the right portal vein. Complete necrosis of the tumor lesions was observed in the resected liver. Early HCC thought to have developed multifocally concurrently with chronic inactive hepatitis was observed. It is highly possible that complete necrosis of these tumors occurred due to PTPE, suggesting that they are supplied by the portal blood flow.