Therapeutic benefits in thalassemic mice transplanted with long-term-cultured bone marrow cells.

OBJECTIVE: Autologous bone marrow (BM) cells with a faulty gene corrected by gene targeting could provide a powerful therapeutic option for patients with genetic blood diseases. Achieving this goal is hindered by the low abundance of therapeutically useful BM cells and the difficulty maintaining them in tissue culture long enough to complete gene targeting without differentiating. Our objective was to devise a simple long-term culture system, using unfractioned BM cells, that maintains and expands therapeutically useful cells for ≥4 weeks. MATERIALS AND METHODS: From 2 to 60 million BM cells from wild-type (WT) mice or from mice carrying a truncated erythropoietin receptor transgene were plated with or without irradiated fetal-liver-derived AFT024 stromal cells in 25-cm(2) culture flasks. Four-week-cultured cells were analyzed and transplanted into sublethally irradiated thalassemic mice (1 million cells/mouse). RESULTS: After 4 weeks, cultures with AFT024 cells had extensive "cobblestone" areas. Optimum expansion of Sca-1-positive cells was 5.5-fold with 20 × 10(6) WT cells/flask and 27-fold with 2 × 10(6) truncated erythropoietin receptor transgene cells. More than 85% of thalassemic mice transplanted with either type of cells had almost complete reversal of their thalassemic phenotype for at least 6 months, including blood smear dysmorphology, reticulocytosis, high ferritin plasma levels, and hepatic/renal hemosiderosis. CONCLUSIONS: When plated at high cell densities on irradiated fetal-liver-derived stromal cells, BM cells from WT mice maintain their therapeutic potential for 4 weeks in culture, which is sufficient time for correction of a faulty gene by targeting.

Evaluation of a pylorus-preserving gastrectomy for patients preoperatively diagnosed with early gastric cancer located in the middle third of the stomach.

PURPOSE: Patients diagnosed with early gastric cancer located in the middle third of the stomach have two major surgical options, namely a conventional distal gastrectomy with Billroth I anastomosis (DG) or a pylorus-preserving gastrectomy (PPG). Pyloruspreserving gastrectomy is thought to have greater functional benefits than DG, but the evaluation of its prognosis and outcome has so far been insufficient. METHODS: Between 1997 and 2007, 133 patients were diagnosed with early gastric cancer located in the middle third of the stomach. Distal gastrectomy was performed in 87 and PPG was performed in 46 of these patients. The clinicopathological characteristics were compared between the groups. RESULTS: There were fewer dissected lymph nodes in PPG (mean: 21.9) than in DG (mean: 30.4, P = 0.001). Complications were detected in 16.1% of DG patients and in 6.5% of PPG patients. The occurrence of stasis after PPG (6.5%) was similar to that observed after DG (6.9%). One patient in the DG group died from cancer recurrence, but cancer recurrence was not detected in the PPG group. Although the difference was not significant, the overall 5-year survival rate in the 46 PPG patients (95%) was better than that in the 87 DG patients (86%, P = 0.087). CONCLUSIONS: Pylorus-preserving gastrectomy patients had fewer postoperative complications than DG patients. The long-term follow-up of these patients will clarify the nutritional and prognostic benefits of PPG.

Serum interleukin-6 and -10 levels in patients with gastric cancer.

BACKGROUND: Interleukin-6 (IL-6) is known to be a multifunctional cytokine and IL-10 is an immunosuppressive factor. Both have been reported to be related to the disease prognosis in some human solid tumors. In the present study, we evaluated the clinical significance of preoperative serum IL-6 and IL-10 levels as new tumor markers in patients with gastric cancer (GC). METHODS: Preoperative serum samples from 90 patients with GC and 9 normal healthy volunteers were assayed. Levels of IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA). The clinical significance of serum IL-6 and IL-10 levels was evaluated and compared with serum carcinoembryonic antigen (CEA) levels and serum C-reactive protein (CRP) levels in these patients. RESULTS: The serum level of IL-6 was significantly higher in the GC patients than in the healthy subjects. Serum IL-6 levels were strongly correlated with CRP levels, but did not correlate with CEA or carbohydrate antigen (CA) 19-9 levels. Serum IL-10 levels did not correlate with CEA, CA19-9, or CRP. Strong positive correlations between serum IL-6 levels and tumor size and tumor stage were observed. On the other hand, IL-10 did not correlate with such clinicopathological findings of tumors. However, high serum IL-10 levels were associated with a worse prognosis in the GC patients, independently of their tumor stage. CONCLUSION: These findings indicate that serum IL-6 may suggest gastric cancer progression. On the other hand, IL-10 may play an important role in host immunity and the prognosis of GC patients.

Isolating gene-corrected stem cells without drug selection.

Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >/=10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a "purity" of >30%, enriched >2,000-fold and with a recovery of approximately 20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >/=3 days.

Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type.

Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c-/-) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving Ren1c-/- mice have no renin mRNA expression in the kidney, hydronephrosis, thickening of renal arterial walls, and fibrosis in the kidney. Plasma renin and angiotensins I and II are undetectable. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 +/- 4 versus 116 +/- 5 mmHg in +/+) and excrete large amounts of urine (5.2 +/- 0.8 ml/d, 725 +/- 34 mOsm versus 1.1 +/- 0.1 ml/d, 2460 +/- 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in -/- mice (624 +/- 19 to 656 +/- 25 mOsm), whereas in +/+, it increases severalfold (583 +/- 44 to 2630 +/- 174 mOsm). Minipump infusion of angiotensin II to Ren1c-/- mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/- mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/- mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.

Guide oligonucleotide-dependent DNA linkage that facilitates controllable polymerization of microgene blocks.

Faster and more efficient searches of a huge protein sequence space for the purpose of conducting experiments in protein evolution can be achieved through the development of a block shuffling-based evolution system. One of the key components of such a system is the accurate and efficient linkage of gene units. Here we introduce a new method that allows accurate and controllable linkage of microgene blocks. This method employs a thermostable DNA ligase that links two single-stranded microgene blocks when they hybridize a complementary guide oligonucleotide. At high temperature, the ligation of the microgene units is fully dependent on the guide oligonucleotide, which can exclude undesired polymer formation, including the incorporation of microgenes having illegitimate sizes and "head-to-head" and "tail-to-tail" ligation of blocks. We were also able to assemble three microgene units using two guide oligonucleotides. Using this method of controllable linkage should facilitate further development of a step-by-step system for the polymerization of gene blocks, leading to a versatile block shuffling-based protein evolution system.