Tamotsu Sagawa

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Primary gastrointestinal follicular lymphoma involving the duodenal second portion is a distinct entity: A multicenter, retrospective analysis in Japan

We conducted a multicenter, retrospective study to determine the anatomical distribution and prognostic factors of gastrointestinal (GI) follicular lymphoma (FL). This study included 125 patients with stage I and II1 GI–FL. Of the 125 patients, the small intestine was examined in 70 patients, with double‐balloon endoscopy and/or capsule endoscopy. The most frequently involved GI–FL site was the duodenal second portion (DSP) (81%), followed by the jejunum (40%); 85% of patients with involvement of the DSP also had jejunal or ileal lesions. The absence of abdominal symptoms and macroscopic appearance of multiple nodules were significantly present in the DSP‐positive group. During a median follow up of 40 months, six patients showed disease progression. Patients with involvement of the DSP had better progression‐free survival (PFS) than those without such involvement (P = 0.001). A multivariate analysis revealed that male sex, the presence of abdominal symptoms, and negative involvement of the DSP were independently associated with poor PFS. In conclusion, most patients with GI–FL have duodenal lesions associated with multiple jejunal or ileal lesions. Gastrointestinal follicular lymphomas involving the DSP might be a distinct entity showing a favorable clinical course. (Cancer Sci 2011; 102: 1532–1536)

Argon plasma coagulation for successful treatment of early gastric cancer with intramucosal invasion.

Background: In recent years, there has been an increasing number of cases of early gastric cancer (T1, NX) with intramucosal invasion, which are untreatable by surgical or endoscopic mucosal resection (EMR) because of their high risk. Currently, no adequate treatment is available for such patients. Aim: The main objective of this study was to evaluate whether argon plasma coagulation (APC) is an effective and safe modality for treating early gastric cancer untreatable by surgical resection or EMR. Methods: The study group comprised 20 men and seven women diagnosed with gastric cancer with intramucosal invasion who were considered poor candidates for surgical resection or EMR due to risk factors such as severe cardiac failure or thrombocytopenia. Irradiation conditions for APC treatment were determined using swine gastric mucosa. We used an argon gas flow of 2 l/min at a power setting of 60 W and a maximum irradiation time of 15 s/cm2. The follow up period of the 27 patients ranged from 18 to 49 months (median 30 months). Results: All lesions were irradiated easily, including areas anatomically difficult for EMR such as the gastric cardia or the posterior wall of the upper gastric body. In 26 of 27 patients (96%) there was no evidence of recurrence during the follow up period (median 30 months). One patient showed recurrence six months after the treatment but was successfully retreated. No serious complications were found in any of the 27 patients but three patients (11%) experienced a feeling of abdominal fullness. Interpretation: APC is a safe and effective modality for treatment of early gastric cancer with intramucosal invasion untreatable by surgical resection or EMR. However, further observations are necessary to determine the long term prognosis of patients undergoing this treatment.

Phase I study of S-1, docetaxel and cisplatin combination chemotherapy in patients with unresectable metastatic gastric cancer

The aim of this dose escalation study was to determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs) and preliminary efficacy of docetaxel, S-1 and cisplatin combination chemotherapy in patients with unresectable metastatic gastric cancer. Seventeen patients received oral S-1 (40 mg m−2 bid) on days 1–14, intravenous cisplatin (60 mg m−2) and docetaxel (60, 70 or 80 mg m−2 depending on DLT) on day 8 every 3 weeks. The MTD of this combination was presumed to be docetaxel 70 mg m−2. At this dose level, 40% of the patients (two of five) developed grade 4 neutropenia and 20% (one of five) exhibited grade 3 nausea during the first course. Therefore, the recommended dose of docetaxel was defined as 60 mg m−2. The DLT was neutropenia. The response rate (RR) was 88.2% (15 of 17), consisting of one complete response and 14 partial responses. There were two stable diseases but no progressive disease. Of these 15 responders, four (23.5%) with high VEGF expression showed rapid tumour regression and achieved downstaging, leading to subsequent curative gastrectomy. Three of these have been disease free for about 3 years, suggesting a complete cure. In conclusion, this regimen was tolerable and showed a quite high RR, with an appreciable downstaging rate in metastatic gastric cancer.

Argon plasma coagulation treatment of hemorrhagic radiation proctopathy: the optimal settings for application and long-term outcome

BACKGROUND No standard treatment exists for hemorrhagic radiation proctopathy (HRP). Recently it was reported that argon plasma coagulation (APC) is effective for HRP. However, previous studies documented complications such as ulcers, strictures, and perforations in as many as 20% of APC-treated patients. OBJECTIVE The aim of this study was to determine the optimal parameters for APC by using swine rectum and to assess the safety and effectiveness of APC in HRP patients. DESIGN Prospective case series. SETTING University teaching hospital. PATIENTS Sixty-five patients with HRP were prospectively enrolled between 2000 and 2010. INTERVENTIONS APC for HRP. MAIN OUTCOME MEASUREMENTS Optimal APC parameters, number of treatments, success rate, complications, clinical remissions. RESULTS APC in swine rectal wall ex vivo was optimal with a 40-W current, 1.2-L/min gas flow rate, and 2-second application, which was sufficient to treat the submucosal telangiectasia but did not adversely affect the muscle layer. Sixty-five patients (46 men, 19 women; median age 72 years) with HRP occurring at a mean of 20 months after radiotherapy were studied. Proctopathy was classified as grade A (mild) in 7 patients (10.8%), grade B (moderate) in 41 (63.1%), and grade C (severe) in 17 (26.2%). The treatment success rate was 98.5% after a median of 2 (range 1-5) APC sessions. The median clinical score for rectal bleeding was significantly decreased after APC (P < .0001), and the hemoglobin level was significantly increased (P < .0001). APC was well tolerated, and no significant side effects or complications occurred. During a mean follow-up of 34.6 months (range 3.6 -121.1 months), 4 patients (6.3%) had minor recurrent rectal bleeding and 60 (93.8%) remained in remission. LIMITATIONS Nonrandomized study. CONCLUSIONS HRP treatment with optimal APC settings yields a high success rate and long-lasting clinical remission with no significant complications.

Clinical utility of capsule endoscopy with flexible spectral imaging color enhancement for diagnosis of small bowel lesions

Background and study aims: The clinical utility of computed virtual chromoendoscopy with flexible spectral imaging color enhancement (FICE) in capsule endoscopy (CE) remains controversial. To clarify the clinical utility of FICE-enhanced CE in evaluating small bowel lesions, we quantitatively assessed white light (WL), FICE, and blue mode (BM) images and examined the sensitivity of these 3 imaging modes of small-bowel lesions from patients who underwent CE. Methods: The CIELAB color difference (∆E) and visual analogue scales (VAS) were measured in 261 CE images (3 different lesion categories) using WL and FICE set 1, 2, and 3, and BM images, respectively. Three endoscopists reviewed CE videos with WL, 3 FICE mode settings, and BM, and compared the sensitivity and detectability for small intestinal diseases from 50 patients who underwent CE. Results: In the assessment of visibility in the 152 vascular lesion images, the ∆E and VAS of FICE set 1, 2, and BM images were significantly higher than that of WL images. In 88 erosion/ulceration images, the ∆E and VAS of FICE set 1 and 2 images were significantly higher than that of WL images. In 21 tumor images, there were no significant differences in ∆E among these modalities. When analyzed on a per-patient basis, FICE settings 1 and 2 had the highest sensitivity (100 %) and specificity (97.3 – 100 %) for vascular lesions. As for erosive/ulcerative lesions, FICE setting 2 had the highest sensitivity (100 %) and specificity (97.2 %). For tumors or polyps, WL had the highest sensitivity (90.9 %) and specificity (87.1 %). In per-lesion analysis, FICE settings 1 and 2 showed significantly superior detection ability over WL for vascular lesions. In the detection of erosive/ulcerative lesions, FICE setting 2 was significantly superior to WL. In tumor images, there was no significant improvement with any of the settings relative to WL images. Conclusions: FICE is most useful for improving CE image quality and detection in cases of angioectasia and erosion/ulceration of the small intestine.

Treatment of hepatocellular carcinoma by AdAFPep/rep, AdAFPep/p53, and 5‐fluorouracil in mice

Although conditionally replicable adenovirus (CRAd) has been used in the clinical treatment of hepatocellular carcinoma (HCC), it suffers from the inherent drawback of having relatively low antitumor activity. Here, we have sought to overcome this drawback. First, we combined CRAd (AdAFPep/Rep) driven by α‐fetoprotein enhancer/promoter (AFPep) with a replication‐incompetent adenovirus carrying a p53 transgene that is also driven by AFPep. The synergism of this combination produced a significantly improved tumoricidal effect on the human HCC cell line Hep3B, which has a relatively short doubling time in comparison with other human HCC cell lines, through the transactivation of p53 by early region 1A transcribed by AdAFPep/Rep. This synergistic interaction was augmented by the addition of a subtumoricidal dose (0.5 μg/mL) of 5‐fluorouracil (5‐FU), which enhanced p53 expression and facilitated the release of virions from tumor cells. When relatively large (10‐mm‐diameter) Hep3B tumors grown in nude mice were injected with the two viruses in combination, they showed significantly impaired growth in comparison with those treated with each virus separately. The growth suppression effect of the virus combination was enhanced by a low dose (600 μg) of 5‐FU. Survival of the tumor‐bearing mice treated with these three agents was significantly longer than that of control mice. Moreover, the tumor completely disappeared with the repeated injection of these agents. Conclusion: This combination strategy holds promise for the treatment of relatively large and rapidly growing HCCs that may be encountered clinically. (HEPATOLOGY 2008;48:828–840.)

Amelioration of murine dextran sulfate sodium‐induced colitis by ex vivo extracellular superoxide dismutase gene transfer

Background: Although the etiology of inflammatory bowel disease has not been fully clarified, reactive oxygen species is speculated to be involved. Extracellular superoxide dismutase (EC‐SOD), an isozyme of SODs, is known to function mainly in body fluids. We investigated the efficacy of an ex vivo EC‐SOD gene transfer into dextran sulfate sodium (DSS)‐induced colitis mice. Materials and Methods: Experimental colitis was induced by providing Balb/c mice with DSS in sterile distilled water provided as desired. The syngenic fibroblasts were obtained from Balb/c mice embryos and retrovirally transduced with the hEC‐SOD gene. These engineered cells were confirmed to secrete EC‐SOD in culture medium by enzyme‐linked immunosorbent assay and were inoculated subcutaneously in the backs of DSS‐treated mice. Mucosal injury of the colon was evaluated by the disease activity index (DAI: body weight, rectal bleeding, and stool consistency), grading of histologic disease severity, and levels of cytokine (tumor necrosis factor‐&agr;, interleukin‐1&bgr;) production. 8‐Hydroxydeoxyguanosine (8‐OHdG) levels in the mucosal tissue were assessed by immunohistochemical staining. Malondialdehyde (MDA) was measured using a colorimetric assay. Results: A significant improvement was observed in DAI score and histologic severity as well as in mucosal tissue levels of inflammatory cytokines, 8‐OHdG, and MDA of mice treated with the EC‐SOD gene as compared with those without gene therapy, not only in a mild colitis model but also in a severe colitis model. Survival of treated mice in these models was significantly prolonged. Conclusions: Ex vivo transfer of the EC‐SOD gene was feasible for treatment of DSS‐induced colitis.

Erratum to: Conversion therapy for inoperable advanced gastric cancer patients by docetaxel, cisplatin, and S-1 (DCS) chemotherapy: a multi-institutional retrospective study

Background Conversion therapy is an option for unresectable metastatic gastric cancer when distant metastases are controlled by chemotherapy; however, the feasibility and efficacy remain unclear. This study aimed to assess the feasibility and efficacy of conversion therapy in patients with initially unresectable gastric cancer treated with docetaxel, cisplatin, and S-1 (DCS) chemotherapy by evaluating clinical outcomes.

ENDOSCOPIC FINDINGS OF ENTEROPATHY‐TYPE T‐CELL LYMPHOMA BY DOUBLE‐BALLOON ENTEROSCOPY AND CAPSULE ENDOSCOPY

Enteropathy‐type T‐cell lymphoma (ETL) is a rare primary intestinal disorder, particularly in Japan, and there have been few reports on the endoscopic findings of the disease. Here we report detailed endoscopic findings of ETL based on double‐balloon enteroscopy and capsule endoscopy. Double‐balloon enteroscopy and capsule endoscopy may be useful tools for diagnosing and monitoring the effects of therapy in patients with ETL.