Generation of an osteoblast-based artificial niche that supports in vitro B lymphopoiesis

B lymphocytes are produced from hematopoietic stem cells (HSCs) through the highly ordered process of B lymphopoiesis, which is regulated by a complex network of cytokines, chemokines and cell adhesion molecules derived from the hematopoietic niche. Primary osteoblasts function as an osteoblastic niche (OBN) that supports in vitro B lymphopoiesis. However, there are significant limitations to the use of primary osteoblasts, including their relative scarcity and the consistency and efficiency of the limited purification and proliferation of these cells. Thus, development of a stable osteoblast cell line that can function as a biomimetic or artificial OBN is necessary. In this study, we developed a stable osteoblastic cell line, designated OBN4, which functions as an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. We demonstrated that the production of a B220⁺ cell population from Lineage⁻ (Lin⁻) Sca-1⁺ c-Kit⁺ hematopoietic stem and progenitor cells (HSPCs) was increased ~1.7-fold by OBN4 cells relative to production by primary osteoblasts and OP9 cells in coculture experiments. Consistently, OBN4 cells exhibited the highest production of B220⁺ IgM⁺ cell populations (6.7±0.6–13.6±0.6%) in an IL-7- and stromal cell-derived factor 1-dependent manner, with higher production than primary osteoblasts (3.7±0.5–6.4±0.6%) and OP9 cells (1.8±0.6–3.9±0.5%). In addition, the production of B220⁺ IgM⁺ IgD⁺ cell populations was significantly enhanced by OBN4 cells (15.4±1.1–18.9±3.2%) relative to production by primary osteoblasts (9.5±0.6–14.6±1.6%) and OP9 cells (9.1±0.5–10.3±1.8%). We conclude that OBN4 cells support in vitro B lymphopoiesis of Lin⁻ Sca-1⁺ c-Kit⁺ HSPCs more efficiently than primary osteoblasts or OP9 stromal cells.

Protein Expression of the Bombyx mori Decapentaplegic Gene using the Baculovirus Expression Vector System

The Bombyx mori decapentaplegic gene is one of the conserved genes in vertebrate and invertebrates. The TGF-beta superfamily contains conserved polypeptide growth factors that play important roles in different cellular processes such as proliferation, apoptosis, differentiation and cell-fate determination. The B. mori dpp gene shares genetic homology with hBMPs and Drosophila dpp. Until now, only few studies have been conducted to examine the functions of B. mori dpp; and hence, its function is not yet well understood. In this study, the baculovirus expression vector system (BEVS) was used for expression of the recombinant B. mori dpp protein and in which the recombinant baculovirus is recovered in the host Sf9 cells. The selected pure recombinant baculovirus containing B. mori dpp gene (rBV-egfp-Bm dpp) was used to increase the effective protein purification by using His-tag extraction strategy. After selection of recombinant baculovirus, recombinant B. mori dpp proteins were extracted from the re-infected cells with pure rBV-egfp-Bm dpp. Herein, we summarize the efficient expression and purification of B. mori dpp proteins from the insect cells using the BEVS. This recombinant protein could be suitable for functional test and various application studies.

Effects of Sildenafil Citrate on Sodium Nitroprusside and Nitroglycerin-Induced Hypotension in Dogs / 대한마취과학회지

BACKGROUND: Nitrovasodilators are known to induce hypotension through activating nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. By inhibiting the breakdown of cGMP, sildenafil citrate may augment the nitrovasodilator-induced hypotension. The present study was aimed to investigate whether sildenafil would reduce the dose of nitrovasodilators needed to induce the hypotension. METHODS: Ten mongrel dogs were acutely instrumented with a femoral artery catheter and a pulmonary artery catheter. They were intravenously given sodium nitroprusside (SNP; 1-16ng/kg/min) or nitroglycerin (NTG; 2 - 32ng/kg/min) to induce hypotension. The study was composed of two occasions in each animal: one with sildenafil pretreatment (1 mg/kg IV followed by 0.3 mg/kg/h) and the other without to serve as control, one week apart. Hemodynamic parameters were continuously monitored. Plasma cGMP concentrations were measured by radioimmunoassay. RESULTS: Both SNP and NTG produced dose-dependent reductions in mean arterial pressure (MAP) without affecting the heart rate in both the control and sildenafil groups. Systemic vascular resistance index (SVRI) and mean pulmonary arterial pressure were also decreased. However, SNP caused a greater reduction of MAP and SVRI in the sildenafil group than in the control group; whereas, NTP caused similar reductions in both groups. Neither SNP nor NTG altered the plasma cGMP concentrations. Sildenafil increased the plasma cGMP concentration, which was further increased by SNP, but not by NTG. CONCLUSIONS: These results indicate that sildenafil may reduce the dose of SNP, but not that of NTG needed to produce hypotension in the dog. The potentiation of SNP-induced hypotension by sildenafil may be related to an augmented cGMP effect.

Retrospective Study of Oral Antifungal Agents in the Treatment of Toenail Onychomycosis / 대한의진균학회지

BACKGROUND: Clinical success rate is determined by patients compliance which depends on treatment method and period. Retrospective study to clinical success rate may give the information to promote compliance with oral medications for onychomycosis. OBJECTIVE: The purpose of this study was to evaluate clinical success rate in the different treatment group. METHOD: For the retrospective study, 618 patients diagnosed clinically and mycologically as having onychomycosis were evaluated for the cure rate and drop-out rate. RESULTS: The drop-out rate was 31.5% in itraconazole monthly pulse group, 74.3% in itraconazole daily continuous group, 77.5% in terbinafine daily continuous group and 97.8% in fluconazole weekly pulse group. Therefore the success rate was 68.5% in itraconazole monthly pulse group, 25.7% in itraconazole daily continuous group, 22.5% in terbinafine daily continuous group and 2.2% in fluconazole weekly pulse group. The drop-out rate was high in the first period of treatment schedule in most of treatment groups except itraconazole monthly pulse and daily continuous groups. CONCLUSION: We concluded that the treatment duration and method are the critical factor to treat tonail onychomycosis relating in determining patients' preference for treatment schedules.

Retrospective Study of Oral Antifungal Agents in the Treatment of Toenail Onychomycosis / 대한의진균학회지

BACKGROUND: Clinical success rate is determined by patients compliance which depends on treatment method and period. Retrospective study to clinical success rate may give the information to promote compliance with oral medications for onychomycosis. OBJECTIVE: The purpose of this study was to evaluate clinical success rate in the different treatment group. METHOD: For the retrospective study, 618 patients diagnosed clinically and mycologically as having onychomycosis were evaluated for the cure rate and drop-out rate. RESULTS: The drop-out rate was 31.5% in itraconazole monthly pulse group, 74.3% in itraconazole daily continuous group, 77.5% in terbinafine daily continuous group and 97.8% in fluconazole weekly pulse group. Therefore the success rate was 68.5% in itraconazole monthly pulse group, 25.7% in itraconazole daily continuous group, 22.5% in terbinafine daily continuous group and 2.2% in fluconazole weekly pulse group. The drop-out rate was high in the first period of treatment schedule in most of treatment groups except itraconazole monthly pulse and daily continuous groups. CONCLUSION: We concluded that the treatment duration and method are the critical factor to treat tonail onychomycosis relating in determining patients' preference for treatment schedules.