Tohru Tamaki

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.

Clinical significance of glucocorticoid pharmacodynamics assessed by antilymphocyte action in kidney transplantation : marked difference between prednisolone and methylprednisolone

A number of studies have demonstrated the impact of glucocorticoid response of peripheral lymphocytes on kidney allograft survival, suggesting that the better the glucocorticoid selection, the better the clinical outcome. However, individual differences in pharmacodynamics of clinically important glucocorticoids have not been taken into account. Four glucocorticoids (hydrocortisone, prednisolone, methylprednisolone, and dexamethasone) were examined for their ability to suppress in vitro blastogenesis of mitogen-stimulated PBL obtained from 122 chronic renal failure (CRF) patients waiting for renal transplantation and 98 healthy volunteers. Concentrations of steroids that gave 50% inhibition of lymphocyte blastogenesis (IC50) were determined individually in order to compare steroids and subject groups. Graft outcomes in 36 kidney transplant recipients treated with prednisolone were compared retrospectively with the prednisolone pretransplant IC50 values. Lymphocyte response to each glucocorticoid showed wide deviations among the subjects. Prednisolone IC50 values of the CRF patients showed the largest deviation, ranging from 1.0 to 10,000 micrograms/L. Thus, a significantly large population of the CRF patients (26.2%), when compared with the healthy subjects (4.1%) showed a marked decrease in lymphocyte response to prednisolone (P < 0.01). The binding capacity and affinity of lymphocyte glucocorticoid receptors did not differ significantly between the responders and nonresponders, suggesting that steroid resistance is a post-receptor event. The antilymphocyte potency of prednisolone assessed by IC50 of the steroid was less than that of hydrocortisone, whereas methylprednisolone was > 12-fold superior to prednisolone. After kidney transplantation, CRF patients who showed impaired preoperative lymphocyte response to prednisolone had a significantly high incidence of acute allograft rejection under prednisolone/CsA therapy (P < 0.01). It is concluded from these results that methylprednisolone could be of benefit to prednisolone-resistant recipients, who can be identified by the preoperative lymphocyte culture.

Successful 48-hour preservation of the rat liver by continuous hypothermic perfusion with Haemaccel-isotonic citrate solution

Eleven rat livers were stored at 7°C for 24 or 48 hr using a continuous nonpulsatile perfusion method. The perfusate was based on the isotonic citrate preservation solution but contained, in addition, gelatin polypeptides (Haemaccel, Hoechst) and fluorocarbon emulsion (FC-43, Green Cross Corp.). Isologous livers were orthotopically transplanted after preservation, and long-term survival was 3/6 following 24 hr preservation and 4/5 following 48 hr preservation. All the biopsies taken immediately after re-vascularization were histologically normal. The seven surviving animals were killed at two months and histology showed biliary obstruction, but in all cases the hepatocytes appeared to be well preserved. These late histological findings are common in any use of this transplantation model and are believed to be associated with the difficulty of obtaining a satisfactory anastomosis of the bile duct. The perfusate described here is capable of providing reliable 48 hr preservation of the rat liver.

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.

Immunosuppressive Activity Of Serum From Liver-grafted Rats: Passive Enhancement Of Fully Allogeneic Heart Grafts And Induction Of Systemic Tolerance

Immunological enhancement of allogeneic heart graft survival by serum from rats tolerized by liver grafting has been studied. Serum taken from long-term-surviving PVG rats carrying orthotopic DA liver transplants (OLT serum) was able to increase the survival time of PVG.RT1a heterotopic heart grafts in PVG recipients. Administration of 1 ml of OLT serum at the time of heart grafting led to permanent survival of the grafts in all animals. The recipients became systemically tolerant of RT1a and several weeks later were able to accept permanently skin grafts from the same donor strain, while rejecting third-party grafts. Enhancement appeared to be mediated initially by IgG antibodies in the OLT serum against class II donor RT1a antigens; significant enhancement was produced by as little as 100 μg of antibody. Recipient alloantibody responses following enhancement were studied and showed selective suppression of the anti-class-I (RT1Aa) antibody levels, while the anti-class-II antibody response was apparently unaffected. The implications of these results for mechanisms of unresponsiveness following enhancement and liver transplantation are discussed.

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.

Long-term histopathology of allografts in sensitized kidney recipients

Successful desensitization therapy has brought satisfying short‐term outcomes in the recipients with anti‐donor antibody. We analyzed the long‐term pathology of the allografts in the sensitized kidney recipients. Eleven stable recipients after desensitization against positive flow cytometry T‐cell crossmatch (FTXM) were included. They were divided into two groups, based on the protocol biopsies findings at three to eight yr (group 1: subclinical glomerulitis and/or peritubular capillaritis, n = 5 and group 2: no rejection, n = 6). Estimated glomerular filtration rate (eGFR), presence of donor‐specific antibody (DSA), mean channel shift (MCS) of FTXM, urine protein levels, acute antibody‐mediated rejection (AAMR) episodes, and protocol biopsy findings were compared. Chronic transplant glomerulopathy was found in final biopsy of all group 1 cases. DSA was positive in 60% but C4d was positive in 20% case of the group 1. The history of AAMR was only found in the group 1. There was no difference in eGFR decline or proteinuria. The MCS of FTXM was higher in the group 1. The recipients with AAMR history, high MCS in FTXM, and subclinical microvascular inflammation in the early protocol biopsies have risk for developing chronic rejection in long term.

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.