Persistent T-cell mixed chimerism in a case of malignant lymphoma after nonmyeloablative allogeneic peripheral blood stem cell transplantation

We describe a 51-year-old woman with recurrent follicular lymphoma from the age of 47 despite chemo-radio therapy, who subsequently underwent nonmyeloablative stem cell transplantation with conditioning consisting of fludarabine and low-dose total body irradiation (2 Gy). Myelosuppression was very mild, so the patient required no transfusions. Chimerism analysis from peripheral blood showed that T-cell mixed chimerism continued over 12 months after stem cell transplantation (the percentage of recipient T-cells was approximately 20%). Despite this, the lymphadenopathy disappeared, and the patient developed grade II acute GVHD (graft versus host disease). It has been considered that the establishment of full donor chimerism is required to induce GVHD and GVM (graft versus malignancy) effects. In this case, however, an allo-response was observed despite the persistence of T-cell mixed chimerism.     

Successful open stent grafting of a right aortic arch and a descending aortic aneurysm originating from a Kommerell's diverticulum: report of a case

The case of a 43-year-old man found to have an aneurysm developing from a Kommerell's diverticulum at the origin of an aberrant retroesophageal left subclavian artery is reported herein. The aneurysm was treated by the open stent grafting technique and complete revascularization was achieved. Received: February 8, 2001 / Accepted: September 11, 2001     

Mitogen-activated protein kinase inhibitor,PD98059, inhibits rat retinal pigment epithelial cell replication by cell cycle arrest

PURPOSE: To investigate the effect of PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, on the replication of rat cultured retinal pigment epithelial (RPE) cells. METHODS: Growth-phase rat RPE cells were exposed to various concentrations of PD98059 in serum-free F12 medium containing 0.1% dimethyl sulfoxide. Cell proliferation was assessed by cell counts using a hemocytometer. Cell viability was tested by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay at 24 hours after PD98059 application. Hoechst 33552 and propidium iodide staining were used to assess nuclear morphology. Immunostaining with Ki67 antibody was used for cell cycle analysis because the staining patterns produced on cells are characteristic depending on their position within the cell cycle. RESULTS: PD98059 inhibited cellular proliferation of cultured rat RPE cells in a dose-dependent manner but did not induce cell death. Twenty-four hours after the application of PD98059, cultured RPE cells were not immunopositive for Ki67, indicating that their cell cycle was arrested in the G0/G1 phase. CONCLUSION: These results demonstrated that MAPK inhibition arrested cell cycle progression of rat cultured RPE cells at the G0/G1 phase. The pharmacological induction of cell cycle arrest could be a new approach to inhibit cellular proliferation in such conditions as proliferative vitreoretinopathy.     

A novel protein with alkaline phosphatase and protease inhibitor activities in Streptomyces hiroshimensis

A novel alkaline phosphatase (S-ALP) was found in the culture filtrate of Streptomyces hiroshimensis IFO 12785. Purification was achieved on Sephadex G-75 column, palmitoylated gauze column, and Superdex 75 HR column chromatographies. The molecular weight of S-ALP was estimated to be 14200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point is 9.2. S-ALP had maximum enzyme activity at pH 9.5. S-ALP efficiently catalyzed both p-nitrophenyl phosphate and p-nitrophenyl phosphorylcholine substrates, particularly the latter. The N-terminal amino acid sequence (25 residues) of S-ALP was 60 to 72% identical to that of Streptomyces subtilisin inhibitor-like proteins. S-ALP exhibited trypsin inhibition in addition to a strong inhibition of subtilisin.     

Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells

Non-viral gene transfer into a wide range of human cells was examined in order to clarify the factors that affect the efficiency and safety of non-viral vectors and to optimize the conditions so that high efficiency and low toxicity could be achieved. Six non-viral vectors (Lipofectin, LipofectAMINE PLUS, SuperFect, Effectene, DMRIE-C and DOTAP) were used to transfect a mammalian expression plasmid pCMVbeta into 16 types of human primary cells and cultured cell lines. Transfection efficiency was quantified using a galactosidase assay. Cytotoxic effects were measured by lactate dehydrogenase (LDH) assay and WST-8 assay. In serum-free conditions, LipofectAMINE PLUS, Effectene and SuperFect, on average, transfected DNA more successfully than Lipofectin, DMRIE-C, and DOTAP, although the levels of gene expression with these vectors varied remarkably in different cells. The most effective vector also differed depending on the cell type. Serum was found to inhibit gene transfer and reduce the cytotoxicity of all of these vectors except Effectene. The efficiency and toxicity of the non-viral vectors used depended on the type of vector, the DNA/vector ratio, the type of cell, and the presence of serum. These results provided useful information for the optimization of transfer conditions of these non-viral vectors.     

Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption

The plasminogen/plasmin system in epidermis is thought to be the major protease involved in the delay of barrier recovery. However, little is known about the mechanism through which this system is activated. In order to clarify this mechanism, we first determined the distribution of proteolytic activity by using in situ zymography. As a result, plasminogen-activator activity was found to be present in the stratum corneum (SC) after barrier disruption. Next, SC subjected to repeated barrier disruption was collected to identify the protease. The protease was identified as urokinase-type plasminogen activator, because flybrinolytic activity of the collected SC was abolished by addition of anti-urokinase antibody. Urokinase activation in SC was confirmed by means of an in vitro assay, in which the precursor of urokinase (pro-uPA) became active after incubation with the insoluble component of SC homogenate. These findings indicated that urokinase-type plasminogen activator is activated in SC after barrier disruption and this activation might trigger the plasminogen/plasmin system in the epidermis.     

Does indocyanine green accurately measure plasma volume early after cardiac surgery?

Potential overestimation of plasma volume (PV) determination by the conventional indocyanine green (ICG) dilution method (PV-ICG) can occur when generalized capillary protein leakage is present, because ICG binds to proteins. We recently reported that this overestimation can be recognized by simultaneous measurement of the initial distribution volume of glucose (IDVG). We examined whether overestimation of PV-ICG and further ICG-pulse dye densitometry-derived plasma volume (PV-PDD) can occur early after cardiac surgery by using the PV-ICG/IDVG ratio as an indicator. Possible overestimation was defined as a ratio higher than 0.45. Twenty-four consecutive postcardiac surgical patients were enrolled. PV-ICG, PV-PDD, and IDVG were calculated simultaneously after admission to the intensive care unit and on the first postoperative day. The mean +/- SD PV-ICG/IDVG ratio for 47 recordings was 0.38 +/- 0.05. Four had a PV-ICG/IDVG ratio higher than 0.45, and the highest was 0.48. The mean PV-PDD/IDVG ratio for a total of 47 recordings was 0.39 +/- 0.10. There were extremely high or low ratios observed in PV-PDD determinations, but they were not observed in PV-ICG determinations. Results suggest that most of the PV-ICG measurements are accurate, but inaccuracy of PV-PDD can occur early after cardiac surgery. IMPLICATIONS: Overestimation of indocyanine green-derived plasma volume can occur in the presence of generalized capillary protein leakage. This overestimation was examined early after cardiac surgery by using the simultaneous measurement of the initial distribution volume of glucose. We suggest that overestimation of the traditional dye dilution method is negligible, but apparent over- or underestimation of pulse dye densitometry-derived plasma volume cannot be negligible.     

Alpha-tocopherol protects cultured human cells from the acute lethal cytotoxicity of dioxin

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30-40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0-31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78-97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.     

Effects of neonatal hypoxic-ischemic brain injury on skilled motor tasks and brainstem function in adult rats

In an attempt to establish more sensitive long-term neurofunctional measurements for neonatal hypoxic-ischemic brain injury, we examined skilled motor task and brainstem functions in adult rats after neonatal cerebral hypoxia-ischemia (H-I), using a staircase test and auditory brainstem response (ABR), respectively. Seven-day-old rats underwent a combination of left common carotid artery ligation and exposure to 8% O(2) for 1 h (n=16). The control animals only received sham operation (n=16). At 3 months of age, the staircase test and ABR were performed. In the staircase test, H-I animals showed marked impairment of skilled forelimb use in the side contralateral to the occluded artery, and the degree of brain damage correlated significantly to skilled forelimb use. In the ABR, H-I animals showed brainstem dysfunction assessed by measuring interpeak latencies for waves III-V and I-V. We also examined the brainstem with antibodies specific for activated caspase-3, a protein involved in initiation of apoptosis, and observed that caspase-3 was activated in the ipsilateral inferior colliculus at 24 h after H-I. The present study shows that both the staircase test and ABR are sensitive and objective long-term neurofunctional measurements that can be used in future studies to assess therapeutic intervention in this neonatal cerebral H-I model.