Cell activation by CpG ODN leads to improved electrofusion in hybridoma production

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.     

Generation of a monoclonal antibody reactive to prefusion myocytes

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion–incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell–cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.     

Comparison of two spike areas in bileaflet mechanical valve closing sound

We previously reported our development of a wavelet analysis system which demonstrates that in vivo bileaflet mechanical valve sound splits into two spikes at higher frequency levels and, based on this system, proposed criteria for detecting malfunctioning bileaflet valves (MBVs). However, the results of that study were only tentative due to the small number of patients with MBVs enrolled in the study. Here, we discuss the possibility of new criteria based on the scalographic properties of two spikes of bileaflet valve sound. The study cohort comprised 12 patients who each received a Carbomedics valve. Based on cinefluoroscopy findings, seven valves were classified into a ??normal?? group, and the other five were classified into a ??malfunction?? group. Five consecutive valve sounds for each valve were collected for the wavelet analysis in order to re-evaluate the previously proposed criteria and to measure both anterior spike area (Aa) and posterior spike area (Pa) for calculating the spike area ratio (Aa/Pa). The proposed criteria, namely, a single spike or coefficient of variation of <0.1120 detected only two of the five malfunctioning valves, as well as one normal valve to be malfunctioning. The mean Aa/Pa of all malfunctioning valves [2.45?±?0.63; 95?% confidence interval (CI) ±1.01, 95?% confidence limits (CL) 1.44?C3.46] was significantly higher than that of all normal valves (1.17?±?0.27; 95?% CI ±0.25, 95?% CL 0.92?C1.42). Based on this result, we determined the cutoff value of Aa/Pa to be 1.4. The combination of a single spike on the scalogram and an Aa/Pa of >1.4 detected more MBVs than previously proposed criteria. This combination may represent new criteria for detecting MBVs.     

A case of oral paracoccidioidomycosis suspected to be pharyngeal cancer

Paracoccidioidomycosis is a fungal infection endemic to South American countries that affects the lungs, skin, and mucosae. Reports from Japan are limited by a long-term resident in South America. Some cases are incorrectly diagnosed because of a refractory buccal ulcer that resembles a malignant tumor. This is a disease that may not be correctly examined if we cannot suspect by a case history. We report the case of a Brazilian man who had a buccal ulcer with lung involvement, which mimicked pharyngeal cancer.     

Risk factors for bleeding after endoscopic submucosal dissection of colorectal neoplasms

AIM:To investigate the risk factors for delayed bleeding following endoscopic submucosal dissection(ESD)treatment for colorectal neoplasms.METHODS:We retrospectively reviewed the medical records of 317 consecutive patients with 325 lesions who underwent ESD for superficial colorectal neoplasms at our hospital from January 2009 to June2013.Delayed post-ESD bleeding was defined as bleeding that resulted in overt hematochezia 6 h to 30d after ESD and the observation of bleeding spots as confirmed by repeat colonoscopy or a required blood transfusion.We analyzed the relationship between risk factors for delayed bleeding following ESD and the following factors using univariate and multivariate analyses:age,gender,presence of comorbidities,use of antithrombotic drugs,use of intravenous heparin,resected specimen size,lesion size,lesion location,lesion morphology,lesion histology,the device used,procedure time,and the presence of significant bleeding during ESD.RESULTS:Delayed post-ESD bleeding was found in14 lesions from 14 patients(4.3%of all specimens,4.4%patients).Patients with episodes of delayed postESD bleeding had a mean hemoglobin decrease of2.35 g/dL.All episodes were treated successfully using endoscopic hemostatic clips.Emergency surgery was not required in any of the cases.Blood transfusion was needed in 1 patient(0.3%).Univariate analysis revealed that lesions located in the cecum(P=0.012)and the presence of significant bleeding during ESD(P=0.024)were significantly associated with delayed post-ESD bleeding.The risk of delayed bleeding was higher for larger lesion sizes,but this trend was not statistically significant.Multivariate analysis revealed that lesions located in the cecum(OR=7.26,95%CI:1.99-26.55,P=0.003)and the presence of significant bleeding during ESD(OR=16.41,95%CI:2.60-103.68,P=0.003)were independent risk factors for delayed post-ESD bleeding.CONCLUSION:Location in the cecum and significant bleeding during ESD predispose patients to delayed post-procedural bleeding.Therefore,careful and additional management is recommended for these patients.     

Diagnostic role of 18F-fluorodeoxyglucose positron emission tomography for follicular lymphoma with gastrointestinal involvement

AIM:To investigate the capacity for 18F-fluorodeoxyglucose(18F-FDG) positron emission tomography(PET) to evaluate patients with gastrointestinal lesions of follicular lymphoma.METHODS:This retrospective case series consisted of 41 patients with follicular lymphoma and gastrointestinal involvement who underwent 18F-FDG-PET and endoscopic evaluations at ten different institutions between November 1996 and October 2011.Data for endoscopic,radiological,and biological examinations performed were retrospectively reviewed from clinical records.A semi-quantitative analysis of 18F-FDG uptake was performed for each involved area by calculating the maximum standardized uptake value(SUVmax).Based on the positivity of 18F-FDG uptake in the gastrointestinal lesions analyzed,patients were subdivided into two groups.To identify potential predictive factors for 18F-FDG positivity,these two groups were compared with respect to gender,age at diagnosis of lymphoma,histopathological grade,pattern of follicular dendritic cells,mitotic rate,clinical stage,soluble interleukin-2 receptor levels detected by 18F-FDG-PET,lactate dehydrogenase(LDH) levels,hemoglobin levels,bone marrow involvement,detectability of gastrointestinal lesions by computed tomography(CT) scanning,and follicular lymphoma international prognostic index(FLIPI) risk.RESULTS:Involvement of follicular lymphoma in the stomach,duodenum,jejunum,ileum,cecum,colon,and rectum was identified in 1,34,6,3,2,3,and 6 patients,respectively.No patient had esophageal involvement.In total,19/41(46.3%) patients exhibited true-positive 18F-FDG uptake in the lesions present in their gastrointestinal tract.In contrast,false-negative 18F-FDG uptake was detected in 24 patients(58.5%),while false-positive 18F-FDG uptake was detected in 5 patients(12.2%).In the former case,2/19 patients had both 18F-FDG-positive lesions and 18F-FDGnegative lesions in the gastrointestinal tract.In patients with 18F-FDG avidity,the SUVmax value of the involved gastrointestinal tract ranged from 2.6 to 17.4(median:4.7).For the 18F-FDG-negative(n = 22) and-positive(n = 19) groups,there were no differences in the male to female ratios(10/12 vs 4/15,P = 0.186),patient age(63.6 ± 2.4 years vs 60.1 ± 2.6 years,P = 0.323),presence of histopathological grade 1 vs 2(20/2 and 17/2,P = 1.000),follicular dendritic cell pattern(duodenal/nodal:13/5 vs 10/3,P = 1.000),mitotic rate(low/partly high,14/1 vs 10/3,P = 0.311),clinical stage according to the Ann Arbor system(stages ⅠE and ⅡE/other,15/7 vs 15/4,P = 0.499),clinical stage according to the Lugano system(stages Ⅰ and Ⅱ-1/other,14/8 vs 14/5,P = 0.489),soluble interleukin-2 receptor levels(495 ± 78 vs 402 ± 83,P = 0.884),LDH levels(188 ± 7 vs 183 ± 8,P = 0.749),hemoglobin levels(13.5 ± 0.3 vs 12.8 ± 0.4,P = 0.197),bone marrow involvement(positive/negative,1/8 vs 1/10,P = 1.000),detectability by CT scanning(positive/negative,1/16 vs 4/13,P = 0.335),and FLIPI risk(low risk/other,16/6 vs 13/6,P = 0.763),respectively in each case.CONCLUSION:These findings indicate that it is not feasible to predict 18F-FDG-avidity.Therefore,18FFDG-PET scans represent a complementary modality for the detection of gastrointestinal involvements in follicular lymphoma patients,and surveillance of the entire gastrointestinal tract by endoscopic examinations is required.     

A useful case of ultrasound-guided axillary lymph node aspiration in a breast cancer patient with improved needle visibility

Ultrasound-guided, lymph node, fine-needle aspiration cytology is important in diagnosing axillary lymph node metastasis in breast cancer. However, poor needle visibility can render the procedure difficult. We describe a case in which state-of-the-art enhancement techniques using matrix linear probes can provide better needle visibility and improve the certainty and efficiency of the examination.     

Ethnic differences in the metabolism, toxicology and efficacy of three anticancer drugs

INTRODUCTION: Large inter-individual and inter-ethnic differences are observed in efficacies and toxicities of medical drugs. To improve the predictability of these differences, pharmacogenetic information has been applied to clinical situations. Expanding pharmacogenetic information would be a valuable tool to the medical community as well as the patient to fulfill the promise of personalized anticancer drug therapy. AREAS COVERED: This review highlights genetic polymorphisms and ethnic differences of genes, UGT1As, CYP3A4, CES1As, ABCB1, ABCC2, ABCG2, SLCO1B1, CDA and CYP2D6, involved in metabolism and disposition of three anticancer drugs: irinotecan, gemcitabine and tamoxifen. EXPERT OPINION: Recent pharmacogenetic studies have successfully identified distinct ethnic differences in genetic polymorphisms that are potentially involved in efficacies and toxicities of anticancer drugs. This achievement has led to personalized irinotecan therapy, reflecting ethnic differences in UGT1A1 genotypes, and possible benefits of genetic testing have also been suggested for gemcitabine and tamoxifen therapy, which still requires further validation. The ultimate goal for patients is a high rate or even perfect prediction of efficacies and toxicities of anticancer drugs in each ethnic population. For this challenge, more clinical studies combined with comprehensive omics approaches are necessary to further advance the field.     

Sudden onset blindness treated with pulse steroid therapy in a patient with microscopic polyangiitis

We report a 71-year-old male with microscopic polyangiitis (MPA) who developed sudden-onset, progressive, bilateral visual loss associated with a relapse of MPA symptoms. The patient was referred to our hospital, and treated with intravenous pulse steroid therapy and high-dose oral prednisolone. Although the right eye remained vision deficient, visual acuity in the left eye recovered. Ocular manifestations of MPA are quite uncommon. This case emphasizes the necessity of early detection and initiation of prompt therapy where ocular manifestations of MPA occur.     

Molecular mechanisms of liver metastasis

Colorectal cancer is the second most common cancer, and is the third leading cause of cancer-related death in Japan. The majority of these deaths is attributable to liver metastasis. Recent studies have provided increasing evidence that the chemokine-chemokine receptor system is a potential mechanism of tumor metastasis via multiple complementary actions: (a) by promoting cancer cell migration, invasion, survival and angiogenesis; and (b) by recruiting distal stromal cells (i.e., myeloid bone marrow-derived cells) to indirectly facilitate tumor invasion and metastasis. Here, we discuss recent preclinical and clinical data supporting the view that chemokine pathways are potential therapeutic targets for liver metastasis of colorectal cancer.