Akio Kawamura

Clinical study of therapeutic angiogenesis by autologous peripheral blood stem cell (PBSC) transplantation in 92 patients with critically ischemic limbs

Patients with critically ischemic limbs due to maintenance hemodialysis and diabetes are increasing in number markedly in Japan. The difficulty of treating critically ischemic limbs is well recognized. Despite active medication and surgical therapy, many critically ischemic limbs are amputated. Ninety-two patients with critically ischemic limbs were treated by transplantation of autologous peripheral blood stem cells (PBSCs). The stem cells were mobilized into the peripheral blood by administration of granulocyte colony stimulating factor (G-CSF). The mobilized mononuclear cells were separated by an apheresis technique using a centrifuge. The separated mononuclear cells contained approximately 4.0 × 107 CD34-positive cells. The collected cell suspension was divided into aliquots of 0.5–1.0 ml and transplanted into the muscle of ischemic limbs at 50–70 transplantation points. At 1.5 months after PBSC transplantation, a strong immunostaining of CD34-positive cells and factor VIII, as well as capillary formation, was observed in the muscles into which stems cells had been transplanted. In each patient tested, the serum vascular endothelial growth factor (VEGF) level increased after stem cell transplantation; the mean VEGF level increased by 176%. Of 11 diabetic patients (DM) who were not receiving hemodialysis (HD), there were no amputees regardless of their Fontaine classification. Of 19 patients in the HD(+)DM(−) category, there were no amputations in Fontaine stage I, II, and III patients, whereas three limbs and one toe were amputated in Fontaine stage IV patients. Of 13 patients in the HD(−)DM(+) category, none of the Fontaine stage I, II, or III patients underwent amputation, but six Fontaine stage IV patients underwent amputation. Of 49 patients in the HD(+)DM(+) category, 38 (78%) were classified as Fontaine stage IV, 71% (27/38) of whom had a toe or a limb amputated. In nine patients over 80 years of age, one toe and one limb were amputated. Nondiabetic, nondialyzed patients with ischemic limbs are strongly indicated for stem cell transplantation regardless of Fontaine classification. Therapeutic angiogenesis is effective for critically ischemic limbs resulting from hemodialysis and diabetes until Fontaine stage III, but is of limited effectiveness for stage IV cases.

Prevention of Limb Amputation in Patients with Limbs Ulcers by Autologous Peripheral Blood Mononuclear Cell Implantation

Abstract:  There are many cases of amputation of ischemic limbs of dialysis patients due to diabetes, despite the availability of medicine therapy and vascular by‐pass operations. As there is extensive ruin of the vascular bed due to diabetes, vascular regeneration therapy by stem cell implantation is effective. Thirty patients with ischemic limbs due to diabetes (not including type‐I) and on dialysis for chronic renal failure (19 cases), diabetes (5 cases), dialysis patients without diabetes (4 cases), and arteriosclerosis obliterans (ASO, 2 cases) were treated by autologous peripheral blood stem cell (PBSC) implantation where imminent amputation was under consideration. Granulocyte Colony Stimulate Factor (G‐CSF: 5 µg/kg/day) was administered subcutaneously for 4 days before PBSC collection, that was carried out using a centrifuge (Spectra and/or CS3000) via the vein. The collected PBSC, containing 4.2 × 107 of CD 34 positive cells, was divided into units of 0.5–1.0 mL and implanted, without any purification, to the ischemic area of the limbs in about 65 points. In 21 cases, normalization of limb temperature was observed by thermograph, and symptoms also improved. The result of this first attempt of PBSC implantation is that we were able to save 22 ischemic limbs. This is the first large report of the application of regenerative medicine to peripheral ischemic limbs.

Long-term clinical outcomes for patients with lower limb ischemia implanted with G-CSF-mobilized autologous peripheral blood mononuclear cells

BACKGROUND Many studies have described the clinical effects of treating critical limb ischemia with granulocyte colony-stimulating factor-mobilized autologous peripheral blood mononuclear cells (M-PBMNC); however, there are no long-term data available on survival, limb salvage, or prognostic factors. METHODS To investigate the long-term clinical outcomes of M-PBMNC implantation, we reviewed data for 162 consecutive patients with limb ischemia who were treated with M-PBMNC implantation at 6 hospitals between 2001 and 2006. A subset of 123 patients with homogenous clinical profiles was selected for prognostic factor analysis. RESULTS Of the 162 patients, 50 died during the follow-up period. The median follow-up time for surviving patients was 26.4 months. The 2-year survival rate was 65% for the 140 patients with arteriosclerosis obliterans (ASO), and 100% for the 11, 4 and 7 patients with thromboangiitis obliterans (TAO), diabetic gangrene (DG) and connective tissue disease (CTD), respectively. The 1-year amputation-free rates for ASO, TAO, DG and CTD were 70%, 79%, 75% and 83%, respectively. Common serious adverse events included heart failure (15 cases), myocardial infarction (15 cases), serious infection (13 cases), stroke (10 cases), and malignant tumor (9 cases). Significant negative prognostic factors associated with overall survival were ischemic heart disease and collection of a small number of CD34-positive cells. Factors associated with time-to-amputation and amputation-free survival were a combination of Fontaine classification and lower limb gangrene, and history of dialysis. CONCLUSIONS Collection of a small number of CD34-positive cells and ischemic heart disease were associated with a reduction in overall survival.

Adenocarcinoma arising in gastric heterotopic pancreas: Clinicopathological and immunohistochemical study with genetic analysis of a case

Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57‐year‐old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic‐cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19‐9, mucin (MUC) 1, and insulin, and the mutant allele‐specific amplification method revealed a point mutation at K‐ras codon 12 (GGT [Gly]→GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.

Prior induction of heat shock proteins by a nitric oxide donor attenuates cardiac ischemia/reperfusion injury in the rat.

BACKGROUND Recent studies have demonstrated that nitric oxide (NO) releasers considerably increase heat shock proteins (HSPs) in the in vitro cell system, providing resistance to oxidant damage. This study was designed to examine the cellular responses of HSPs induced by prior administration of an NO releaser, FK409 (FK), in an in vivo transplantation model. METHODS Lewis rats received either saline or FK solution intravenously administered at different time points before graft harvesting (10 micromol/kg) or for 15 min during reperfusion (0.66 micromol/kg/min). Tissue specimens were taken to determine HSP70 and heme oxygenase-1/HSP32 (HO-1) expression, and glutathione content. After 24-hr preservation with University of Wisconsin solution, heterotopic cardiac transplantations were performed, and graft survival was determined at 14 days. Tissue samples for end labeling of nuclear DNA fragments (TdT-mediated d-uridine triphosphate biotin nick end labeling; TUNEL) and propidium iodide staining were taken 15 min after reperfusion. RESULTS The gene and protein expression of HSP70 after FK administration peaked at 12 min and 60-90 min, whereas those of HO-1 peaked at 6 min and 90 min, respectively. Then, representative cardiac grafts taken 60 min after FK treatment were examined for further assay. Localization of induced HSP70 and HO-1 molecules were observed in the myocardium and vascular endothelium, respectively. Prior treatment of FK was effective in preventing the reduction of tissue glutathione contents compared with control (P<0.05). Fewer TUNEL and propidium iodide-positive cells were also observed in the FK group (P<0.0005, vs. control). The graft survival rate was higher in the FK group (9/10 vs. 1/10 of control; P<0.001), whereas the groups either harvested 10 min after FK pretreatment or continuously infused for 15 min during reperfusion were inferior, similar to that of control. CONCLUSION Prior induction of HSP70 and HO-1 with a relatively low dose of FK administration attenuates ischemia and reperfusion injury, which was due to antioxidant and antiapoptotic activities augmented by such stress proteins. Thus, NO releasers as a pharmacological maneuver may provide an innovative approach for the prevention of ischemia and reperfusion injury.

Artificial liver support at present and in the future.

Liver failure is a fatal disease. Liver transplantation is the only established treatment for liver failure; however, donor shortages remain problematic. In the United States and Europe, artificial livers as a bridge to liver transplantation are being considered. In Japan, we have taken a different approach to the treatment of end-stage liver diseases because of the characteristics of the health-care insurance system, regulated by the government. Furthermore, cadaveric liver transplantations are unsuited to the social mores of Japanese culture. Practically speaking, we believe that plasma exchange (PE) and continuous hemodiafiltration (CHDF) are the most effective therapies for the treatment of liver failure, although randomized controlled studies are needed to determine their effects. Overall, we believe that the first line of treatment for liver failure should be PE and CHDF, and the second line should be bioartificial liver support. In the near future, we hope that both gene therapy and regenerative medicine will contribute to the development of a functional artificial liver.

Transient increase in telomerase activity of proliferating fibroblasts and endothelial cells in granulation tissue of the human skin

Although granulation tissue formation is an important step for second‐intention wound healing, the molecular events underlying this process are still poorly understood. To investigate the role of telomerase in the formation of granulation tissue, we measured the activity of this enzyme and determined the expression and localization of human telomerase reverse transcriptase mRNA using human skin samples. Telomerase activity in the tip of the granulation tissue where fibroblasts actively proliferate was detected at a level 5.6 ± 1.5 times higher than that at the edge of the tissue when using a polymerase chain reaction‐based telomeric repeat amplification protocol assay coupled with enzyme‐linked immunosorbent assay. This, together with the findings from semiquantitative reverse transcriptase‐polymerase chain reaction and in situ hybridization of human telomerase reverse transcriptase, revealed that proliferating cell nuclear antigen‐positive fibroblasts and endothelial cells in the progressing granulation tissue showed de novo activation of telomerase with high human telomerase reverse transcriptase mRNA expression. This condition may be a prolongation of cellular replicative capacity taking advantage of the positive regulatory dynamics of cell growth. We conclude that the regulation of telomerase activity may play an important role in granulation tissue formation in wound healing.

Application of peripheral blood stem cells (PBSC) mobilized by recombinant human granulocyte colony stimulating factor for allogeneic PBSC transplantation and the comparison of allogeneic PBSC transplantation and bone marrow transplantation

Recombinant human granulocyte colony stimulating factor (rhG-CSF)-mobilized peripheral blood stem cells (PBSC) are now widely used for allogeneic PBSC transplantation (alloPBSCT). Large numbers of hematopoietic progenitor cells mobilized by rhG-CSF would be considered equivalent or better than bone marrow (BM) cells and would be used as an alternative to BM for allogeneic hematopoietic stem cell transplantation. The complications associated with the administration of rhG-CSF and apheresis in PBSC collection in formal donors are well tolerated and usually acceptable in the short term but some hazardous adverse events such as splenic rupture and cardiac arrest are reported although the incidence is very low. Protective means and stopping rules for safe donation in the collection of PBSC are established. The characteristics of PBSC were clarified; the expression of some adhesion molecules such as CD49d on CD34 positive cells of PBSC have been shown to be low compared to BM stem cells. In alloPBSCT compared with allogeneic BM transplantation (alloBMT), the incidence and frequency of graft versus host disease (GVHD) is of concern because high number of T lymphocytes are infused in alloPBSCT. The incidence and severity of acute GVHD are not increased but chronic GVHD is higher in alloPBSCT compared with alloBMT. The outcome of alloPBSCT and BMT are almost equivalent and conclusive results regarding survival are not yet available.

Successful treatment of ulcerative colitis with leukocytapheresis using non-woven polyester filter

Abstract:  Ulcerative colitis is a chronic inflammatory disease of the rectum and colon. Although the pathogenesis of ulcerative colitis is not fully elucidated, cell‐mediated immunity plays an important role in disease pathogenesis. Leukocytapheresis is a newly emerging therapy to eliminate activated leukocyte from systemic circulation. We have studied the effects of leukocytapheresis on patients with ulcerative colitis who had failed to respond to conventional therapy. A total of 51 patients with ulcerative colitis were treated with apheresis using a non‐woven polyester fiber filter (Finecell, Asahi Medical Co., Tokyo, Japan) originally developed as a microcoagulation elimination filter for massive transfusion. Of the 51 patients, 33 (64.7%) achieved clinical remission manifested by clinical activity and colonoscopic findings without any adverse effects. This result suggested that leukocytapheresis using Finecell might serve as an alternative therapy for ulcerative colitis as other leukocytapheresis using centrifugation or column.

Preoperative autologous blood donation in hip surgeries

We evaluated the effectiveness of preoperative autologous blood donation in reduction of the need for transfusion of homologous blood in hip surgeries at our hospital. The cases of 55 patients who had 67 hip surgeries, including 17 total hip arthroplasties (THA) and 41 rotational acetabular osteotomies (RAO), were studied. The patients predeposited an average of 995 ml of blood for each procedure. The calculated blood loss was an average of 961 ml. Ninety-seven percent of the procedures for which autologous blood had been predeposited were performed without transfusion of homologous blood. In the group for THA, an average of 981 ml of autologous blood was transfused for a blood loss of 1417 ml; hemoglobin levels after operation averaged 103 g/l. From this data, a 1000 ml donation seemed to be an optimal blood deposit for THA and a 800 ml blood deposit seemed sufficient for RAO, where the patients are younger.