Recurrent Spindle Cell Carcinoma Shows Features of Mesenchymal Stem Cells.

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient's quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor's cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

Hand metastasis from a sacral chordoma.

Chordomas are rare, low grade, malignant tumours derived from the ectopic remnants of the notochord that line the axial skeleton. They are characterised by their slow growth, long disease course and propensity for local relapse. Furthermore, up to 40% of non-cranial chordomas metastasise. We describe the first reported case of a hand metastasis arising from a conventional sacral chordoma after carbon ion radiotherapy. The common occurrence of distant metastasis with chordomas makes it important to perform a systemic examination, in part because their resection might improve patient prognosis.

Frequency of CD4(+)FOXP3(+) regulatory T-cells at early stages after HLA-mismatched allogeneic hematopoietic SCT predicts the incidence of acute GVHD.

Acute GVHD (aGVHD) is a major obstacle to allogeneic hematopoietic SCT (alloHSCT). Although it is thought that aGVHD is initiated in secondary lymphoid organs at a very early stage of alloHSCT, whether CD4(+)FOXP3(+) regulatory T-cells (Tregs) have an impact on aGVHD development during this period remains unclear. Here, we measured Tregs in peripheral blood as early as possible after HLA-mismatched alloHSCT, and assessed the incidence of aGVHD. Flow cytometric analyses revealed that at the second week after HSCT, patients with aGVHD had significantly (P=0.018) lower Treg:CD4(+)T-cell ratios than those without aGVHD. As these differences were seen before the development of aGVHD, these ratios can predict the incidence of aGVHD. The cumulative incidence of aGVHD in patients with ratios of <9% was significantly higher than that in patients with ratios of 9% (P=0.0082, log-rank test). Additionally, the specific ratio of Tregs:CD4(+)T-cells was the most significant value among all other possible lymphocyte-associated ratios and absolute cell counts. These findings suggest that the ratio of Tregs:CD4(+)T-cells at the second week post HLA-mismatched alloHSCT might be a potent predictor of aGVHD in these patients. The practical efficacy of this finding should be verified in further interventional studies.

"Obama system:" a new easy, safe, and economical technique for laparoscopic single-port surgery.

INTRODUCTION: Various approaches to laparoscopic single-site surgery (LESS) have been developed to reduce pain and other complications, promote recovery, and improve cosmetic outcomes, particularly relative to conventional open or laparoscopic surgery. Three-port procedures for LESS have been reported to be superior to single-port access, but they usually require expensive, technically sophisticated instruments. To avoid these problems, we have developed a modified procedure for performing LESS with a single port, referred to as the "Obama system." METHODS: From January 2009 through December 2010, we performed laparoscopic cholecystectomy (LC) in 61 patients. Conventional LC with three ports was performed in 39 patients, LESS with a SILS Port was performed in 4 patients, and modified LESS was performed using the Obama system in 18 patients. The operative results were compared. RESULTS: LC was successfully completed in all 61 patients, with no postoperative complications. The mean operating time was 102.3 min (C-reactive protein [CRP] ≤ 2) and 160.1 min (CRP > 2) in the 39 patients who underwent conventional LC, 108.3 min (CRP ≤ 2) in the 4 patients who underwent LESS with a SILS Port, and 116.5 min (CRP ≤ 2) and 186.5 min (CRP > 2) in the 18 patients who underwent LESS using the Obama system. No morbidity or mortality was associated with any technique. CONCLUSION: The Obama system is easier to use and more efficient and reliable than any other technique currently available for LESS. This system is expected to greatly contribute to the further development and wider acceptance of LESS.

Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors.

Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

Salvage haploidentical transplantation for graft failure using reduced-intensity conditioning.

Graft failure is a major concern after cord blood transplantation (CBT) or HLA-haploidentical transplantation (haplo-SCT). As patients who undergo CBT or haplo-SCT almost always lack both matched-related and -unrelated donors, salvage transplantation would also be limited to either CBT or haplo-SCT. In this study, we assessed eight patients who received haplo-SCT as salvage therapy for graft failure. Five and three patients had received haplo-SCT and CBT, respectively, which resulted in graft failure. The median interval from the failed transplantation to salvage transplantation in six patients with primary graft failure was 33.5 days. The reduced-intensity conditioning regimen consisted of fludarabine, thiotepa, rabbit antithymocyte globulin and low-dose TBI. All eight patients achieved neutrophil engraftment, and seven patients achieved platelet recovery. The median times to neutrophil recovery and platelet recovery were 10 and 20 days, respectively. Three patients died from treatment-related causes: two from GVHD and one from rupture of carotid artery aneurysm. Five patients are alive, at a median follow-up of 946 days. The probability of overall survival at 5 years was 75%. These findings may serve as a rationale for giving precedence to haplo-SCT over CBT in salvage SCT after graft failure.

Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.

A role of donor-specific HLA antibodies (DSA) in graft failure after SCT has been suggested, but the relevance of DSA in unmanipulated haploidentical SCT (haplo-SCT) remains unknown. We prospectively examined HLA antibodies using the Luminex-based single Ag assay for 79 adult patients undergoing unmanipulated haplo-SCT. Among them, 16 (20.2%) were HLA Ab-positive, including five patients with antibodies not corresponding to donor HLA Ags and 11 DSA-positive patients. Of the 11 DSA-positive patients, five received treatments to decrease DSA levels, including two, who received plasma exchange and rituximab, two who received platelet transfusions from healthy-related donors having DSA-corresponding HLA Ags and one who received bortezomib. Platelet transfusion was the most simple and effective treatment option for class I DSA. The cumulative incidence of neutrophil recovery was significantly lower in pretransplant (post-treatment) DSA-positive patients than in DSA-negative patients (61.9 vs 94.4%, P=0.026). Notably, three of five patients with high levels of DSA had graft failure. Donors should be selected on the basis of an evaluation of HLA antibodies. If haplo-SCT from donors with HLA Ags that correspond to high levels of DSA must be performed, then recipients should be treated for DSA to improve the chances of successful donor engraftment.

Incidence of extramedullary relapse after haploidentical SCT for advanced AML/myelodysplastic syndrome.

Extramedullary (EM) relapse of leukemia after allo-SCT in patients with AML/myelodysplastic syndrome has been increasingly reported. The reduced effectiveness of the GVL effect in EM sites, as compared with BM, has been suggested to underlie this problem. We retrospectively analyzed the pattern of relapse after haploidentical SCT (haplo-SCT), performed as the first or second SCT. Among 38 patients who received haplo-SCT as their first SCT, the cumulative incidences of BM and EM relapse at 3 years were 40.5 and 10.9%, respectively. Among 19 patients who received haplo-SCT as their second SCT, the cumulative incidences of BM and EM relapse were 30.9 and 31.9%, respectively. Moreover, most of the patients who underwent repeat haplo-SCT for the treatment of EM relapse had further EM relapse at other sites. Post-relapse survival did not differ significantly with different patterns of relapse. The frequent occurrence of EM relapse after haplo-SCT, particularly when performed as a second SCT, suggests that the potent GVL effect elicited by an HLA disparity also occurs preferentially in BM. Our findings emphasize the need for a treatment strategy for EM relapse that recognizes the reduced susceptibility of EM relapse to the GVL effect.

An easy and reproducible model of kidney transplantation in rats.

OBJECTIVE: Kidney transplantation in rats is an important research model. Various methods have been reported, but there is no "standard operation." We investigated a 1-stage versus a 2-stage native nephrectomy and the type of ureteral anastomosis seeking to establish a standard, reproducible and successful method. MATERIALS AND METHODS: We used PVG (RT1c-RT1Ac: B/Dc) male rats, weighing approximately 200 to 250 g, that underwent transplantation after right recipient nephrectomy. Left recipient nephrectomy was performed either 10 days later or simultaneously. The ureteric anastomosis was fashioned 2 ways: using a ureteral stent or by bladder insertion. RESULTS: Urinary complications (obstruction or reflux) were observed in 77.8% when a ureteral stent was used for the ureteric anastomosis versus 28.6% when using the bladder insertion technique (P = .0211). Transplanted rats with nephrectomy of both native kidneys at the time of grafting showed a perioperative mortality of 70%, whereas those hosts with a 2-stage nephrectomy displayed a mortality rate of 22% (P = .0025). CONCLUSIONS: The bladder insertion technique reduced the incidence of urological complications in rats. In addition, unilateral native nephrectomy at the time of operation with delayed contralateral nephrectomy was better tolerated than simultaneous bilateral nephrectomy. These 2 surgical variants allowed us to perform kidney transplantation with a high degree of success.

Decreased circulating CD34+ cells are associated with progression of diabetic nephropathy.

AIMS: Circulating progenitor cells such as CD34+ cells play a key role in maintenance of vascular endothelial function and neovascularization, and a decrease in the number of CD34+ cells is associated with cardiovascular disease. However, the contribution of circulating progenitor cells to microvascular disease, such as diabetic nephropathy, is unclear. This study was therefore designed to clarify the association between diabetic nephropathy and circulating CD34+ cells. METHODS: We measured circulating CD34+ cell numbers in 85 Type 2 diabetic patients aged 40-70 years with normo- and microalbuminuria and determined the association with urinary albumin excretion rate (UAER). RESULTS: The number of circulating CD34+ cells significantly correlated with log UAER (r = -0.289, P = 0.008). Furthermore, in patients with low numbers of CD34+ cells (0.68 > cells/microl, lowest quartile of CD34+ cell number) UAER increased significantly after 12 months compared with baseline [from 34.3 +/- 7.0 to 53.6 +/- 10.8 mg/g creatinine (gCr), P < 0.05], whereas in patients with a high number of CD34+ cells (1.0 < cells/microl, highest quartile of CD34+ cell number) UAER did not change (from 16.7 +/- 4.8 to 20.1 +/- 3.0 mg/gCr). CONCLUSIONS: These results suggest that a decreased number of circulating CD34+ cells is involved in the progression of diabetic nephropathy and may be a predictor of the disease.

[Laser therapy for endobronchial malignancies].

Photodynamic therapy (PDT), neodymium yttrium aluminum garnet (Nd-YAG) laser therapy, electrocautery and microwave coagulation therapy are therapeutic options available for management of endobronchial malignancies. All of these treatment modalities have been used for both palliation of late obstructing cancers, and more recently have been used as primary treatment of early stage lung cancers. Only PDT has the curative potential for patients with early superficial squamous cell carcinoma. Nd-YAG laser therapy is used for direct thermal ablation of tissue in endobronchial malignancy. This equipment is the most widely used type of laser for bronchoscopic interventions because it has sufficient power to vaporize tissues and produces an excellent coagulation effect. But the risks of perforation and bleeding are high. Endobronchial electrocautery is the use of high-frequency electrical current that generates heat due to tissue resistance, resulting in destruction of tissue. Argon plasma coagulation (APC) is a form of noncontact electrocoagulation. The risks of perforation and igniting are much lower than with the Nd-YAG laser therapy. Microwave coagulation therapy refers to the use of all electromagnetic methods for inducing tumor destruction by using devices with frequencies of 2450 MHz. It is important to select these treatment methods appropriately according to each case.

Interferon-beta augments eosinophil adhesion-inducing activity of endothelial cells.

Viral infections induce exacerbations of asthma. One of the earliest host responses to viral infections is the production of innate cytokines including type I interferons (IFNs), such as IFN-beta, which may act to modify airway inflammation. The objective of the present study was to investigate whether IFN-beta modifies the eosinophil adhesion-inducing activity of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with IFN-beta for 24 h in the presence or absence of tumour necrosis factor (TNF)-alpha. Eosinophils were isolated from the peripheral blood of healthy volunteers. The ability of the IFN-beta-stimulated HUVEC monolayers to induce eosinophil adhesion was assessed according to the eosinophil peroxidase assay. Eosinophil adhesion to HUVECs was significantly augmented by IFN-beta in the presence of TNF-alpha but not in its absence. The augmented adhesion was inhibited by anti-alpha(4) integrin monoclonal antibody (mAb) or anti-beta(2) integrin mAb. IFN-beta significantly enhanced the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 on HUVECs in the presence of TNF-alpha. Interferon-beta can augment the adhesiveness of endothelial cells to eosinophils, mainly through the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. This action of interferon-beta may contribute to the intensification of airway inflammation in asthma that is associated with exacerbations induced by viral infections.

Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets.

Cultivated oral mucosal epithelial sheet transplantation is a new surgical strategy to treat severe ocular surface disorders such as chemical burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome. MUC16 is thought to be the most important membrane-associated mucin on the ocular surface because it forms a protective barrier on the epithelial cell surface. In this study, we studied MUC16 expression in mRNA and protein levels and compared the expression patterns between cultivated oral mucosal epithelial cell sheet and oral mucosal tissue. Specimens (5x5 mm) of oral mucosal tissue harvested from healthy volunteers were used. The oral mucosal epithelial cells were cultured on temperature-responsive culture dishes to generate stratified cell sheets. Cultivated oral mucosal epithelial cells formed three- to five-cell thick stratified sheets for 2 weeks. Scanning electron micrographs revealed that the apical surfaces of the oral mucosal tissue and the oral mucosal sheets were covered with dense microvilli/microplicae. Real-time PCR showed significantly more MUC16 transcripts in the cultivated oral mucosal sheets and corneal epithelial sheets than in the oral mucosal tissue (P=0.023 and 0.008, respectively, Mann-Whitney rank sum test). These findings were confirmed by immunohistochemical examination using an MUC16 antibody to the protein. MUC16 protein was localized to the apical cells of the oral mucosal sheets, but the human oral mucosal tissue did not express MUC16 protein in any cell layers. In this study, interestingly, the expression of membrane-associated mucin MUC16 differs between human oral mucosal epithelia and cultivated epithelial sheets. MUC16 expressed in the oral mucosal sheets may contribute to ocular surface reconstruction after oral mucosal sheet transplantation.

Induction of prostaglandin E synthase by gastroesophageal reflux contents in normal esophageal epithelial cells and esophageal cancer cells.

The synthesis of prostaglandin E2 (PGE2) requires cyclooxygenase (COX) and prostaglandin E synthase (PGES). There are two forms of PGES: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1. In this study, we investigated the effects of gastroesophageal reflux (GER) contents on PGES and COX-2 in esophageal cells. We incubated a human normal esophageal cell line, two esophageal squamous cell carcinoma (SCC) cell lines, and two esophageal adenocarcinoma (ADC) cell lines with GER contents. The production of PGE2 by these cells was assayed with an enzyme immunoassay kit. The protein expression of COX-2, cPGES, and mPGES-1 was confirmed by immunoblot analysis. The following results were obtained: GER contents induced the expression of COX-2 in all five cell lines. In normal esophageal cells, cPGES, but not mPGES-1, was detected in the cytosolic fraction. GER contents induced the expression of cPGES in the microsomal fraction. In SCC cells, cPGES was expressed in the cytosolic fraction, and mPGES-1 was expressed in the microsomal fraction. GER contents induced the expression of mPGES-1 in the microsomal fraction. In ADC cells, cPGES was expressed in both the cytosolic and microsomal fractions. GER contents induced the expression of both cPGES and mPGES-1 in the microsomal fraction. In conclusion, our results suggest that GER contents induce PGE2 production in esophageal cells. However, there are different isoforms of PGES in normal cells, SCC cells, and ADC cells.

Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells.

Interaction between proteases and protease-activated receptor (PAR) 2 has been proposed to mediate inflammatory and immune response in the gastrointestinal tract. Recently, increase in interleukin (IL)-8 in the esophageal mucosa has been associated with the pathogenesis of esophagitis induced by reflux of gastric acids, bile acids or trypsin. The aims of the present study were to determine PAR2 expression in normal human esophageal epithelial cells (HEEC) and to evaluate the mediation of IL-8 production by trypsin-PAR2 interaction in HEEC. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis revealed that PAR2 mRNA and protein were constitutively expressed in HEEC without upregulation by the stimulation with tumor necrosis factor alpha or trypsin. IL-8 was produced in a dose-dependent fashion when cells were stimulated with a PAR2 agonist such as trypsin or SLIGKV-amide. Blocking antibody to PAR2, camostat mesilate (a trypsin inhibitor), p-38 mitogen-activated protein kinase (MAPK) inhibitors or ERK1/2 inhibitors reduced IL-8 production from trypsin-stimulated HEEC. Mutation of the NFkappaB-, AP-1- and NF-IL-6-binding site on the IL-8 gene promoter abrogated the induction of luciferase activities stimulated with trypsin by 100, 80 and 50%, respectively. These results indicate that PAR2 activation in HEEC by trypsin induces NFkappaB- and AP-1-dependent IL-8 production in association with activation of p38 MAPK and ERK1/2, suggesting that esophageal inflammation may be induced by PAR2 activation via reflux of trypsin.

A novel direct competitive repopulation assay for human hematopoietic stem cells using NOD/SCID mice.

BACKGROUND: The major problem in cord blood (CB) transplantation for adult patients is shortage of stem cell number. To overcome this disadvantage, several studies on ex vivo expansion have been performed. However, such efforts are always troubled by the lack of a reliable and simple assay system for stem cells. Our aim was to establish an in vivo assay system to compare the directly repopulating ability of two populations of human hematopoietic stem cells using a xenogeneic transplant system. METHODS: Thirty CB samples from infants of each sex were pooled and enriched for CD34(+) progenitor cells. Enriched CD34(+) cells were transplanted into irradiated NOD/SCID mice at different male to female ratios, and human hematopoietic cells recovered 7 weeks after transplantation were analyzed by a quantitative DNA sex test using competitive PCR for the amelogenin gene. Using this assay system, ex vivo cultured and non-cultured CB cells were compared for repopulating ability. RESULTS: The sex ratio of human CB cells transplanted was found to be maintained for 7 weeks in matured and progenitor cells. The competitive repopulation assay of cultured and non-cultured CB cells showed a marked defect in the repopulating ability of cultured cells, although the LTCIC count was maintained during cultivation. DISCUSSION: Our assay system is a simple and reliable quantitative method that permits direct comparison of two stem cell compartments. The assay system will be useful for the assessment of the functional abilities of various human hematopoietic stem cells.