A novel strategy inducing autophagic cell death in Burkitt's lymphoma cells with anti-CD19-targeted liposomal rapamycin.

Relapsed or refractory Burkitt's lymphoma often has a poor prognosis in spite of intensive chemotherapy that induces apoptotic and/or necrotic death of lymphoma cells. Rapamycin (Rap) brings about autophagy, and could be another treatment. Further, anti-CD19-targeted liposomal delivery may enable Rap to kill lymphoma cells specifically. Rap was encapsulated by anionic liposome and conjugated with anti-CD19 antibody (CD19-GL-Rap) or anti-CD2 antibody (CD2-GL-Rap) as a control. A fluorescent probe Cy5.5 was also liposomized in the same way (CD19 or CD2-GL-Cy5.5) to examine the efficacy of anti-CD19-targeted liposomal delivery into CD19-positive Burkitt's lymphoma cell line, SKW6.4. CD19-GL-Cy5.5 was more effectively uptaken into SKW6.4 cells than CD2-GL-Cy5.5 in vitro. When the cells were inoculated subcutaneously into nonobese diabetic/severe combined immunodeficiency mice, intravenously administered CD19-GL-Cy5.5 made the subcutaneous tumor fluorescent, while CD2-GL-Cy5.5 did not. Further, CD19-GL-Rap had a greater cytocidal effect on not only SKW6.4 cells but also Burkitt's lymphoma cells derived from patients than CD2-GL-Rap in vitro. The specific toxicity of CD19-GL-Rap was cancelled by neutralizing anti-CD19 antibody. The survival period of mice treated with intravenous CD19-GL-Rap was significantly longer than that of mice treated with CD2-GL-Rap after intraperitoneal inoculation of SKW6.4 cells. Anti-CD19-targeted liposomal Rap could be a promising lymphoma cell-specific treatment inducing autophagic cell death.

Fucosylated TGF-ß receptors transduces a signal for epithelial-mesenchymal transition in colorectal cancer cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a major inducer of epithelial-mesenchymal transition (EMT) in different cell types. TGF-ß-mediated EMT is thought to contribute to tumour cell spread and metastasis. Sialyl Lewis antigens synthesised by fucosyltransferase (FUT) 3 and FUT6 are highly expressed in patients with metastatic colorectal cancer (CRC) and are utilised as tumour markers for cancer detection and evaluation of treatment efficacy. However, the role of FUT3 and FUT6 in augmenting the malignant potential of CRC induced by TGF-ß is unclear. METHODS: Colorectal cancer cell lines were transfected with siRNAs for FUT3/6 and were examined by cell proliferation, invasion and migration assays. The expression and phosphorylation status of TGF-ß downstream molecules were analysed by western blot. Fucosylation of TGF-ß receptor (TßR) was examined by lectin blot analysis. RESULTS: Inhibition of FUT3/6 expression by siRNAs suppressed the fucosylation of type I TßR and phosphorylation of the downstream molecules, thereby inhibiting the invasion and migration of CRC cells by EMT. CONCLUSION: Fucosyltransferase 3/6 has an essential role in cancer cell adhesion to endothelial cells by upregulation of sialyl Lewis antigens and also by enhancement of cancer cell migration through TGF-ß-mediated EMT.

Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms.

Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34(+) cells, CD34(+) blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34(+) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO(+) leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO(+) leukemic cell proliferation. The demethylating agent, 5-aza-2'-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells.

Successful treatment of advanced peripheral T-cell lymphoma with an angiocentric growth pattern complicated with hemophagocytic syndrome by high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Peripheral T-cell lymphomas (PTCL) account for about 10% of all lymphomas in Western countries, respond poorly to therapy, and have short survival with no sustained remission. Furthermore, the complication of hemophagocytic syndrome (HPS) sometimes makes the prognosis of this disease extremely worse. We report here a case of PTCL with an angiocentric growth pattern complicated with HPS successfully treated by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Our case suggests this approach is an excellent candidate for the treatment of this disease.

A case of true malignant histiocytosis: identification of histiocytic origin with use of immunohistochemical and immunocytogenetic methods.

We report here an autopsy case of true malignant histiocytosis. The patient was a 67-year-old woman who exhibited fever, wasting, hepatosplenomegaly, and progressive pancytopenia. The bone marrow aspiration disclosed hemophagocytosing cells, which resembled histiocytes. The molecular analysis did not show the clonal gene rearrangement of T-cell receptor or immunoglobulin heavy chain. Although the patient had been started on methylprednisolone pulse therapy and chemotherapy with etoposide, she died from cerebral hemorrhage. The autopsy specimens of spleen and liver showed extensive infiltration of atypical cells, for which histiocytic origin was identified with an immunohistochemical method using monoclonal antibodies against CD11c, CD68, macrophage colony-stimulating factor (M-CSF), M-CSF receptor, lysozyme, antitrypsin and alpha1-antichymotrypsin. Recent investigations have disclosed that in most cases diagnosed as malignant histiocytosis, hemophagocytosis was reactive and not evoked by histiocytic malignancy. True malignant histiocytosis, for which histiocytic origin is confirmed, is extremely rare.

[Hepatic and splenic arterial infusion chemotherapy after transcatheter peripancreatic arterial embolization for patients with inoperable adenocarcinoma of the pancreas].

Thirty-one patients with advanced pancreatic carcinoma and liver metastases were treated by hepatic and splenic arterial infusion chemotherapy after transcatheter peripancreatic arterial embolization. The response rate for these 31 patients was 61.3%, with a mean survival period of 17.8 +/- 3.2 months and a 50% survival period of 12 months. By site of the primary tumor, the response rate for pancreatic head and body carcinoma was 81%, with a mean survival period of 21.6 +/- 4.0 months and a 50% survival period of 17 months, whereas the response rate for pancreatic caudal carcinoma was 20%, with a mean survival period of 6.1 +/- 0.5 months and a 50% survival period of 6 months. We believe that the current chemotherapy is an effective treatment for advanced pancreatic cancer with liver metastases.

[Implantation of a dual chambers port for hepatic and splenic arterial infusion chemotherapy in advanced pancreatic carcinoma with multiple liver metastasis].

Dual chambers ports were implanted in 7 patients with advanced pancreatic carcinoma and metastatic liver tumors to connect a 3.3 Fr catheter as an indwelling catheter. In comparison with the implantation of a pair of Single chamber ports, implanting a Dual chambers port entails some technical difficulties, but has some benefits in terms of stabler placement, a smaller incision, reduction of medical fees, and improved QOL of patients.

Treatment of murine collagen-induced arthritis by ex vivo extracellular superoxide dismutase gene transfer.

UNLABELLED: OBJECTIVE; Superoxide dismutase (SOD) is a potent antiinflammatory enzyme that has received growing attention for its therapeutic potential. This study was undertaken to examine the efficacy of extracellular SOD (EC-SOD) gene therapy in murine collagen-induced arthritis. METHODS: Embryonic DBA/1 mouse fibroblasts were infected with a recombinant retrovirus expressing human EC-SOD. DBA/1 mice that had been treated with type II collagen were administered subcutaneous injections of 2 x 10(7) EC-SOD-expressing fibroblasts on day 29, when symptoms of arthritis were already present. The severity of arthritis in individual mice was evaluated in a double-blind manner; each paw was assigned a separate clinical score, and hind paw thickness was measured with a caliper. Mice were killed on day 50 for histologic examination of the joints. RESULTS: High serum concentrations of EC-SOD were maintained for at least 7 days. Mice treated with the transgene exhibited significant suppression of clinical symptoms such as disabling joint swelling, deformity, and hind paw thickness, compared with the untreated group (mean +/- SD maximum clinical score in the untreated and the transgene-treated groups 2.71 +/- 1.08 and 1.35 +/- 1.22, respectively; P < 0.01, and hind paw thickness 3.04 +/- 0.18 mm and 2.56 +/- 0.12 mm, respectively; P < 0.05). Histologic abnormalities, including destruction of cartilage and bone, infiltration of mononuclear cells, and proliferation of synovial cells, were also markedly improved in the EC-SOD-treated mice compared with the control group (histopathologic score 7.50 +/- 1.13 and 4.13 +/- 1.88 in the untreated and transgene-treated groups, respectively; P < 0.05). CONCLUSION: These results indicate that EC-SOD gene transfer may be an effective form of therapy for rheumatoid arthritis.

C-reactive protein is an independent predictor of the rate of increase in early carotid atherosclerosis.

BACKGROUND: An elevated plasma concentration of high-sensitivity C-reactive protein (hs-CRP) is a strong predictor of cardiovascular events. However, there have been no longitudinal studies of the relations between development of atherosclerotic lesions and hs-CRP concentrations. Furthermore, it remains unknown whether increased hs-CRP concentrations result in the development of atherosclerosis. METHODS AND RESULTS: The study included 179 outpatients 40 to 79 years of age who were treated at our institute for traditional risk factors for cardiovascular disease. The patients had no evidence of advanced carotid atherosclerosis at the time of baseline examination. Patients underwent repeated ultrasonographic evaluation of the carotid arteries for 35+/-10 months. Blood samples were collected for hs-CRP measurements. Based on focal intima-media thickening >/=1.1 mm representing plaque, plaque number (PN) and plaque score (PS; the sum of all plaque thicknesses) were calculated. The development of atherosclerosis was estimated by the formula Deltavalue/year=(last value-baseline value)/number of follow-up years. Multivariate linear regression analysis revealed that the log-transformed value for hs-CRP concentration was not related to baseline PN or PS but was related to DeltaPN/year and DeltaPS/year (beta=0.29 and 0.30; P<0.001 for both) independently of the effect of traditional risk factors. CONCLUSIONS: During the early stages of carotid atherosclerosis, the hs-CRP concentration is a marker of carotid atherosclerotic activity rather than extent of atherosclerosis.

Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT 2000 cyclosporine specific assay with the COBAS MIRA-plus analyzer.

We describe the evaluation of the EMIT 2000 cyclosporine specific assay kit, an enzyme-multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA-plus analyzer. The enzyme used for the assay was glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH-labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within-assay coefficient of variation (CV) of this method was 2.7-4.2% (n = 10) at the levels of 56.2-339.7 microg/L. Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L. The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L. The functional detection limit was 24.9 microg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81-109%. The correlation between values obtained with the EMIT 2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x - 13.053 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique.

Synergistic suppressive effect of double transfection of tumor necrosis factor-alpha and interleukin 12 genes on tumorigenicity of Meth-A cells.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer / lymphocyte-activated killer (NK / LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK / LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK / LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-alpha and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.

[Pure red cell aplasia induced by clomipramine hydrochloride in a patient with SLE].

A 48-year-old woman, who had been suffering from systemic lupus erythematosus (SLE), developed normochromic normocytic anemia after receiving clomipramine hydrochloride. Her reticulocyte count was low, and a bone marrow aspirate revealed erythroid hypoplasia without involvement of other cell lines. Thus a diagnosis of pure red cell aplasia (PRCA) was made. The anemia gradually resolved following withdrawal of the drug. Although several drugs are known to cause PRCA, this is the first time that clomipramine hydrochloride has been reported to have such an effect. The underlying SLE in this case suggested the possible immunological pathogenesis of drug-induced PRCA.

The effect of reaction temperature for nephelometric assays for rheumatoid factor.

Even using the same assay parameter, reagent and calibrator (N-latex RF kit II), the results of the assay for serum rheumatoid factors (RFs) with the Behring Nephelometer Analyzer (BNA) were higher than those with the Behring Nephelometer II Analyzer (BNII) ([BNII]=0.76 [BNA]-5.7 kIU/l, r=0.997, Sy/x=60.73, n=99). The mean bias (BNA minus BNII)+/-S.D. was 52.7+/-85.5 using the Bland and Altman plot method, and the bias was not constant. The only difference in the assay condition with the two methods was the reaction temperature with the BNA being performed at room temperature (25+/-1 degrees C) and the BNII being performed at 37 degrees C. The ratio of the results with the BNII to the BNA (BNII/BNA) ranged from 0.23 to 1.18. A significant difference was observed in the BNII/BNA ratio in patients with high levels of C-reactive protein (CRP) over 2.0 mg/l (mean BNII/BNA ratio; 0.78) in comparison to patients with normal CRP levels under 2.0 mg/l (mean BNII/BNA ratio; 0.65) (P<0.01). The RF concentrations with the BNA were reduced by addition of urea, which has been used as a mild protein-denaturing agent, and there was a significant correlation between the values calculated as (1-value treated with urea/original value without urea)x100 and the BNII/BNA ratio (r=0.652, P<0.01). These data suggested that the bias between the RF values obtained by the BNA and BNII might be caused by the variation in the reactivity of autoantibodies, which might be decreased in some inflammatory diseases.

Enzymatic assay for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD).

We described an automated enzymatic assay for conjugated bilirubin (Bc) in serum using the Iatro D-Bil kit, with bilirubin oxidase (EC 1.3.3.5 BOD) from Myrothecium species. The specificity of the enzyme in the Iatro D-Bil kit was examined by analyzing unconjugated bilirubin (Bu), delta bilirubin (Bdelta), and Bc with high-performance liquid chromatography (HPLC), before and after the enzymatic reaction using BOD. The within-assay coefficients of variation (CV) of this method were 0.58 to 5.00% (n = 20) at 1.4 to 155.8 micromol/L. Day-to-day Cvs ranged from 1.61 to 7.14% at 1.2 to 182.1 micromol/L. The analytical recovery was 96 to 101%. The presence of ascorbic acid, reduced glutathione, L-cysteine, uric acid, urea, creatinine, glucose, lipemic material, anticoagulants, hemoglobin, or human serum albumin did not affect this assay system. The correlation coefficient between values obtained with the Iatro D-Bil kit (y) and HPLC method as reference for conjugated fractions (x) was; r = 0.983, y = 0.952x + 8.851 micromol/L, Sy/x = 11.97 (n = 56). We studied serum Bc levels, not including Bu and Bdelta, in patients with hepatic diseases or autoimmune hemolytic anemia. Levels of Bc obtained by the proposed method changed more rapidly than did those of direct bilirubin (D-Bil) obtained by diazo-dye method during the course of the diseases.

Uncontrolled diabetes hinders bone formation around titanium implants in rat tibiae. A light and fluorescence microscopy, and image processing study.

This study examined the influence of diabetes mellitus on bone formation around cylindrical titanium (Ti) implants (1.0 mm in diameter and 1.5 mm in length) inserted transcortically and extending into the medullary canal of rat tibiae using light and fluorescence microscopies and image processing. Forty-eight male Wistar King A rats (age 5 weeks) were used in this experiment. Streptozotocin was injected intraperitoneally to induce diabetes and the serum glucose concentration was checked to ensure the induction of diabetes prior to implant placement and at the time of sacrifice. The animals were sacrificed 7, 28, 56, or 84 days after placement. Toluidine blue-stained undecalcified sections were prepared for histological observation and image analysis. The Ti implants in the control group became increasingly encapsulated with a bone layer. The implants in the diabetes-induced (DI) group were also surrounded with a thin bone layer. Abundant adipocytes were observed in the DI group as compared with the control group. Quantitative evaluation indicated that the control group showed a significantly higher percent of bone contact, and thickness of surrounding bone and area than the DI group. Consequently, the present study suggests that uncontrolled diabetes would hinder bone formation around Ti implants in rats.

Long-term evaluation of bone-titanium interface in rat tibiae using light microscopy, transmission electron microscopy, and image processing.

We conducted a 2-year histologic and histometric evaluation of the tibial bone-titanium (Ti) implant interface in male rats. Thirty male 6-week-old rats were used in this study. They were divided into two groups: 15 for day 28 and 15 for day 730. Microscopic observation at day 28 revealed that the newly formed bone around the implant almost surrounded the implant, but fibroblastlike cells were interposed in some histologic sections. At day 730, in contrast, such cells were rarely seen, and the bone around the implant presented a lamellar structure. Transmission electron microscopic observation at day 28 disclosed mature or poorly mineralized bone near the implant; however, an electron-dense amorphous zone about 50 nm in thickness was interposed between the bone and Ti. In places slender cells were interposed between the bone and Ti. The amorphous zone was also observed at the cell-Ti interface. At day 730, a poorly mineralized layer remained in some areas between the mature bone and the titanium, and the interposed amorphous zone was still observed. Occasionally, a 200-nm-thick layer, thought to be cell remnant, was seen. As calculated in an image-processing, system analysis, the percent bone contact and the thickness and area of the surrounding bone for the Ti implant at day 28 were 43.6%, 30.4 microns, and 0.10 mm2, respectively, and those at day 730 were 89.9%, 53.5 microns, and 0.19 mm2, respectively. In summary, although the passage of time may affect bone maturity, interfacial cells remain at the bone-Ti interface as a uniform layer together with unmineralized bone.

Histological comparison of early wound healing following dense hydroxyapatite granule grafting and barrier placement in surgically-created bone defects neighboring implants.

The purpose of this study was to examine early wound healing following grafting of dense hydroxyapatite granules (HA granules) and barrier placement in surgically-created bone defects surrounding implants. Eight healthy adult dogs with an average weight of 15 kg were used in this study. Thirty-two bone defects measuring 4 mm x 4 mm were removed with a surgical bur to form continuous bucco-lingual bone defects and 32 implants (16 titanium [Ti]) and 16 hydroxyapatite-coated [HA]) were then placed into the defects. Four implant groups were created: 1) grafting HA; 2) covering with an expanded polytetrafluoroethylene (ePTFE) membrane; 3) grafting HA and covering with ePTFE membrane; and 4) control (no treatment). Animals were sacrificed 28 days after surgery. Histological sections revealed large amounts of newly-formed bone in all bone defects surrounding the implants treated with ePTFE membranes alone. Fibrous encapsulation of HA granules was observed in the defects of the HA granules grafting group. In the group with grafting of HA granules and covering with ePTFE membranes, small amounts of bone tissue were observed among HA granules, but most HA granules were surrounded with fibrous tissue. Bone defects were completely filled with connective tissue in the control group. There were no differences in the histological findings between Ti and HA-coated implants in all cases. Histomorphometric data disclosed that the presence of HA granules in the bone defects significantly arrested bone formation. Our study suggests that the grafting of dense HA into bone defects surrounding implants will result in fibrous healing during the early healing stage.