Insufficient ex vivo expansion of Valpha24(+) natural killer T cells in malignant lymphoma patients related to the suppressed expression of CD1d molecules on CD14(+) cells.

BACKGROUND: Valpha24(+) natural killer T (NKT) cell is a human counterpart of mice Valpha14(+) NKT cell that has a regulatory role for innate and acquired potential antitumor activity. The efficient expansion of NKT cells is an obstacle to the clinical application of Valpha24(+) NKT cells for immunotherapy. METHODS: We used mononuclear cells (MNC) obtained from the peripheral blood (PB) of normal healthy donor (HD) and malignant lymphoma (ML) patients before and after granulocyte colony-stimulating factor (G-CSF) treatment. MNC were cultured for 12 days with alpha-galactosylceramide (100 ng/mL) and interleukin-2 (IL-2; 100 U/mL). RESULTS: The fold expansion of Valpha24(+) NKT cells was higher in HD than in ML patients (208 versus 0.00), despite comparable numbers of Valpha24(+) NKT cells before culture. G-CSF administration enhanced the predominance of Valpha24(+) NKT cell fold expansion in HD compared with ML patients (1935 versus 1.95). After treatment with G-CSF, the expression of CD1d molecules was up-regulated in CD14(+) cells from HD but not ML patients. The fold expansion of Valpha24(+) NKT cells and CD1d expression on CD14(+) cells was strongly correlated in both HD and ML patients (r(2)=0.84). However, replacement of a patient's CD14(+) cells with HD cells did not increase the efficacy of Valpha24(+) NKT cell expansion. DISCUSSION: G-CSF-mobilized PB from ML patients has inhibitory characteristics for Valpha24(+) NKT cell expansion as a result of both monocytes and Valpha24(+) NKT cells. Multiple procedures would be needed for the expansion of patients' Valpha24(+) NKT cells.

Immune reconstitution following reduced-intensity transplantation with cladribine, busulfan, and antithymocyte globulin: serial comparison with conventional myeloablative transplantation.

The primary object of the conditioning regimen for allogeneic reduced-intensity stem cell transplantation (RIST) is immunosuppression to achieve stable engraftment of donor cells, rather than bone marrow ablation. Therefore, immune reconstitution after RIST might be different from that after conventional stem cell transplantation (CST). In this study, 22 patients underwent RIST and 28 underwent CST. The RIST regimen consisted of cladribine (2-CdA; 0.11 mg/kg/day for 6 days), BU (4 mg/kg/day for 2 days), and rabbit anti-thymocyte globulin (ATG; 2.5 mg/kg/day for 2-4 days). The CST group received either the BU (4 mg/kg/day x 4 days)/CY (60 mg/kg/day x 2 days) (n=13) or CY (60 mg/kg/day x 2 days)/TBI (4 Gy/day x 3 days) regimen (n=15). All patients underwent transplantation with G-CSF-mobilized blood stem cells. Engraftment speed after RIST was fast and seven of 22 patients did not require platelet transfusion. We noted that the numbers of CD4+, CD4+CD45RA+, and CD4+CD45RO+ T cells after transplant in the RIST group were significantly lower than those in the CST group (P=0.0001 for both the comparisons). However, the reconstitution of CD20+ B cells was faster in the RIST group (P=0.0001). The response of T cells to PHA stimulation was lower in the RIST group (P=0.0001 on day 30 and P=0.02 on day 90). Nevertheless, there were no significant differences in the incidence of bacterial, fungal, or viral infections between the two groups. We concluded that our RIST regimen might delay laboratory-evaluated T-cell immune reconstitution compared to CST; however, the observed setbacks did not directly translate into clinically significant increases in infectious episodes.

Induction of vasculogenesis in breast cancer models.

Recently, there have been reports of postnatal vasculogenesis in cases of ischaemia models. The aim of the present study is to provide evidence of postnatal vasculogenesis in breast-cancer-bearing mice. Based on cell surface antigen expression, we isolated endothelial precursor cells from bone marrow, peripheral blood and tumour-infiltrating cells from mice that had received six human breast cancer xenografts. In all three areas (bone marrow, peripheral blood and tumour-infiltrating cells), endothelial precursor cell population was elevated in all transplanted mice. Differentiation and migration activities of endothelial precursor cells were measured by comparing levels of the endothelial precursor cell maturation markers Flk-1, Flt-1, Tie2, VE-cadherin and CD31 among these three areas. The endothelial precursor cell population was 14% or greater in the gated lymphocyte-size fraction of the inflammatory breast cancer xenograft named WIBC-9, which exhibits a hypervascular structure and de novo formation of vascular channels, namely vasculogenic mimicry (Shirakawa et al, 2001). In vitro, bone marrow-derived endothelial precursor cells from four human breast cancer xenografts proliferated and formed multiple clusters of spindle-shaped attaching cells on a vitronectin-coated dish. The attaching cells, which incorporated DiI-labelled acetylated low-density lipoprotein (DiI-acLDL) and were negative for Mac-1. The putative bone marrow derived endothelial precursor cell subset, which was double positive of CD34 and Flk-1, and comparative bone marrow derived CD34 positive with Flk-1 negative subset were cultured. The former subset incorporated DiI-acLDL and were integrated with HUVECs. Furthermore, they demonstrated significantly higher levels of murine vascular endothelial growth factor and interleukin-8 in culture supernatant on time course by enzyme-linked immunosorbent assay. These findings constitute direct evidence that breast cancer induces postnatal vasculogenesis in vivo.

Antitumor action of the PKC activator gnidimacrin through cdk2 inhibition.

Daphnane-type diterpene gnidimacrin (NSC252940), isolated from a Chinese plant, exhibited antitumor activity against murine leukemias and solid tumors. At concentrations of 10(-9) to 10(-10) M, this agent strongly inhibited the growth of human tumor cell lines. In sensitive human leukemia K562 cells, gnidimacrin is a PKC activator that arrests the cell cycle in the G(1) phase by inhibiting cdk2 activity. A 4 hr exposure of K562 cells to gnidimacrin induced the CDK inhibitor p21(WAF1/Cip1), but this effect was transient and did not correlate temporally with the onset of G(1) arrest. Expression of cdc25A, a phosphatase that activates cdk2, was reduced during 24-hr exposure to gnidimacrin. Moreover, the suppression corresponded in a concentration- and time-dependent manner to both the inhibition of cdk2 activity and the mobility shift observed when cdk2 was electrophoresed on SDS-PAGE, indicating that the phosphorylation state of cdk2 must change. Cyclin E, the other regulator of cdk2 activity, was not influenced by gnidimacrin. These results suggest that gnidimacrin exerts antitumor activity through suppression of cdc25A and inhibition of cdk2 activity.

[Effect of probucol on the concentration of cyclosporin A in patients with nephrotic syndrome].

The first choice of therapy for nephrotic syndrome is steroid, and cyclosporin A(CyA) or other immunosuppressants are selected for steroid resistant or recurrent cases. Nephrotic syndrome accompanies hyperlipidemia for which HMG-CoA reductase inhibitors are mainly used. On the other hand, probucol is used in cases showing inadequate effects or some adverse reactions under treatment with HMG-CoA reductase inhibitors. Recently, we experienced several cases whose blood levels of CyA were decreased to about half that before the combined use of probucol, and concomitant administrations were discontinued. Based on these cases, we considered that the use of probucol should be prescribed in patients with nephrotic syndrome accompanying hyperlipidemia giving preference to CyA treatment. In cases of unavoidable usage of probucol, CyA dose adjustments are required on the basis of frequent CyA blood level monitoring.

T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells.

We present evidence that T-cell-conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell-free supernatants of anti-CD3-activated T cells contained several soluble factors including CD40-ligand (sCD40L), TNF-alpha, and IFN-gamma. In contrast to moderate up-regulation of costimulatory molecules by the addition of individual cytokines or monocyte-conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose-dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF-alpha, and IFN-gamma, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM-treated DC can produce significantly higher levels of IL-12 and are highly effective stimulators in allogenenic and autologous mixed-lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen-specific immune responses against cancer and viruses.

Clinical observations of pancreatic diabetes caused by pancreatic carcinoma, and survival period.

BACKGROUND: Pancreatic carcinoma is often associated with diabetes mellitus. The interrelationship between them is very interesting and important for clinical examination and treatment. METHODS: We examined diabetes mellitus in our patients with pancreatic carcinoma, especially those with invasive ductal pancreatic carcinoma, who were admitted to the National Kyushu Cancer Center between 1972 and 1998, in relation to secondary (pancreatic) diabetes, obstructive pancreatitis, angiopathies, treatment, and prognosis. RESULTS: Diabetes mellitus was found at a high frequency (53.1%) in patients with invasive ductal pancreatic carcinoma and was mostly thought to be secondary diabetes (45.9%), caused, in part, by obstructive pancreatitis following pancreatic tumor recognized on the first admission. Control of blood glucose with insulin was sometimes difficult, but was indispensable for the treatment of pancreatic carcinoma. Diabetic angiopathies are usually not seen in patients with pancreatic diabetes caused by pancreatic carcinoma, because the survival period of patients with pancreatic carcinoma has been limited. Furthermore, in spite of the absence of angiopathies, the survival period was significantly lower in pancreatic carcinoma patients with diabetes than in those without diabetes. CONCLUSION: Diabetes in patients with invasive ductal pancreatic carcinoma is usually secondary diabetes, occurring in part as a consequence of obstructive pancreatitis shown at the beginning of the clinical course. The duration of diabetes is too short for marked diabetic angiopathies to develop, and the survival period in patients with invasive ductal pancreatic carcinoma with diabees is also short compared with that of those patients without diabetes.

Early full donor myeloid chimerism after reduced-intensity stem cell transplantation using a combination of fludarabine and busulfan.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate lineage-specific chimerism reconstitution after reduced-intensity allogeneic stem cell transplantation (RIST) using a combination of fludarabine (30 mg/m2 for 6 days) and busulfan (4 mg/kg for 2 days). DESIGN AND METHODS: We prospectively enrolled 8 consecutive patients with hematologic malignancies who were not candidates for conventional transplantation because of either high age or organ dysfunction. Host-donor chimerism was evaluated using polymerase chain reaction-based amplification of a polymorphic short tandem repeat region. RESULTS: All of our patients achieved engraftment within a median of 11 days after transplantation. On day 30, full donor myeloid cell chimerism (>90%) was achieved in 7 patients whereas full donor T-cell chimerism was achieved in only one patient. Thus, in contrast to other reported results, full donor chimerism was achieved earlier in the myeloid lineage than the T-cell lineage. On day 60, however, T-cell chimerism caught up with myeloid chimerism. Two patients developed grade II-IV acute graft-versus-host disease (GVHD) before the detection of full donor T-cell chimerism. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the kinetics of lineage-specific chimerism depend on the agents used in the conditioning regimen, and may provide insight into the chimerism kinetics and pathogenesis of GVHD. Thus, the strategy for controlling immunosuppression after RIST should be modified according to the type of conditioning regimen applied.

Dendritic cell maturation overrules H-2D-mediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon gamma production.

Given the broad expression of H-2 class Ib molecules on hematopoietic cells, antigen presentation pathways among CD1d expressing cells might tightly regulate CD1d-restricted natural killer T (NKT) cells. Bone marrow-derived dendritic cells (BM-DCs) and not adherent splenocytes become capable of triggering NK1.1(+)/T cell receptor (TCR)(int) hepatic NKT cell activation when (a) immature BM-DCs lack H-2D(b)-/- molecules or (b) BM-DCs undergo a stress signal of activation. In such conditions, BM-DCs promote T helper type 1 predominant CD1d-restricted NKT cell stimulation. H-2 class Ia-mediated inhibition involves more the direct H-2D(b) presentation than the indirect Qa-1(b) pathway. Such inhibition can be overruled by B7/CD28 interactions and marginally by CD40/CD40L or interleukin 12. These data point to a unique regulatory role of DCs in NKT cell innate immune responses and suggest that H-2 class Ia and Ib pathways differentially control NKT cell recognition of DC antigens.

Induction of tumor specific cytotoxic T lymphocytes in prostate cancer using prostatic acid phosphatase derived HLA-A2402 binding peptide.

PURPOSE: Human prostatic acid phosphatase is a prostate specific differentiation antigen. Prostatic acid phosphatase levels increase in the serum of patients with prostate cancer and its peptide from positions 299 to 307 (PAP 299-307) is recognized by HLA-A2 restricted cytotoxic T lymphocytes. We investigated whether HLA-A2402 binding prostatic acid phosphatase derived peptides induce HLA-A2402 restricted, tumor specific cytotoxic T lymphocytes from the peripheral blood mononuclear cells of patients with prostate cancer. MATERIALS AND METHODS: Peptide binding activity was measured with RMA-S-A*A2402 cell lines and flow cytometry. Cytotoxic T-lymphocyte activity of the peripheral blood mononuclear cells of patients with prostate cancer and healthy donors was measured by interferon-gamma and (51)creatinine release assays. Prostatic acid phosphatase expression in the tumor cell lines at the messenger RNA and protein levels was investigated by reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, respectively. RESULTS: An HLA-A2402 binding, prostatic acid phosphatase derived peptide consisting of the prostatic acid phosphatase amino acid sequence from positions 213 to 221 (PAP 213-221, LYCESVHNF) showed the ability to induce HLA-A2402 restricted and tumor specific cytotoxic T lymphocytes, which are cytoxic to prostatic acid phosphatase positive tumor cells from the peripheral blood mononuclear cells of patients with prostate cancer. CONCLUSIONS: PAP 213-221 may be appropriate as a cancer vaccine for specific immunotherapy in patients with HLA-A2402 positive prostate cancer.

Increased incidence of bone metastases in hepatocellular carcinoma.

OBJECTIVES: Recently, we often encounter hepatocellular carcinoma patients with bone metastases. We therefore examined the changes in the incidence of bone metastases in hepatocellular carcinoma from 1978 to 1997 and tried to identify the characteristic clinical features. We also discuss the reasons for the increased incidence of bone metastasis in hepatocellular carcinoma. METHODS: A total of 673 patients with hepatocellular carcinoma during the period 1978-1997 were studied. Bone metastasis was screened by bone scintigraphy, and bone lesions were confirmed by plain radiography, computed tomography and/or magnetic resonance imaging. The serum levels of the C-terminal telopeptide of type 1 collagen, which represent osteoclastic bone resorption, were also measured. RESULTS: The incidence of bone metastasis during the decade 1988-1997 was significantly higher than that during the period 1978-1987. The median survival time of patients with hepatocellular carcinoma during 1988-1997 was also significantly longer than that during 1978-1987. Portal thrombus was found in about half of the patients with bone metastases. The most common site of bone metastases was the vertebra followed by the pelvis, rib and skull in that order. All bone lesions depicted by plain radiograph, computed tomography and/or magnetic resonance imaging were of the osteolytic type, and the serum levels of C-terminal telopeptide of type 1 collagen were significantly elevated in the patients with bone metastases. CONCLUSIONS: The increased incidence of bone metastasis in hepatocellular carcinoma in the decade 1988-1997 is first attributed to the prolonged survival rate of hepatocellular carcinoma patients due to recent progress in both the diagnosis and treatment of the disease. Dissemination of hepatocellular carcinoma cells to the vertebra through the portal vein-vertebral vein plexuses due to the presence of portal thrombus and/or portal hypertension may be related to a higher incidence of bone metastasis in hepatocellular carcinoma. Both an early diagnosis and timely treatment of bone metastases are thus called for in the follow-up of hepatocellular carcinoma patients.

Angiotensin-converting enzyme inhibitors and probucol suppress the time-dependent increase in urinary Type IV collagen excretion of Type II diabetes mellitus patients with early diabetic nephropathy.

BACKGROUND: A multicenter prospective clinical trial was carried out in 9 National Hospitals in Japan to elucidate the time-dependent change in urinary Type IV collagen excretion rate of Type II diabetes mellitus (DM) patients, and to investigate whether an angiotensin-converting enzyme inhibitor (ACE-I) or probucol is effective in preventing progression of renal involvement of diabetics by evaluating urinary Type IV collagen excretion. METHODS: Normo- and microalbuminuric patients with Type II DM were recruited. Patients were assigned to either the control (n = 88), ACE-I (n = 43) or probucol (n = 37) group and treated for 24 months. Besides albumin excretion rate (AER), urinary Type IV collagen excretion rate was also measured. RESULTS: Although, AER, urinary N-acetyl-beta-D-glucosaminidase and beta2-microglobulin excretion rates in the control group did not vary over 24 months, urinary Type IV collagen excretion rate in the control group increased time-dependently (p < 0.01 vs baseline at 18 months and p < 0.005 vs baseline at 24 months). In the ACE-I and probucol groups, time-dependent increases in urinary Type IV collagen excretion rates were not observed. In the ACE-I group, the urinary Type IV collagen excretion rate was significantly lower than that in the control group at 24 months (p < 0.05). In the probucol group, the urinary Type IV collagen excretion rate was significantly lower than that in the control group at 6 months (p < 0.05). In the ACE-I group, AER decreased significantly compared with baseline at 18 months (p < 0.05) and at 24 months (p < 0.005). CONCLUSIONS: ACE-I has a beneficial effect and probucol may have a beneficial effect in preventing the progression of early diabetic nephropathy. Measurement of the urinary Type IV collagen excretion rate in combination with AER would be useful for the management of early renal involvement in Type II DM.

Lack of effectiveness of radiotherapy combined with cisplatin in patients with locally advanced pancreatic carcinoma.

BACKGROUND: Cisplatin has been reported to enhance the cell-killing effect of radiation. The current study was conducted to evaluate the efficacy and toxicity of radiotherapy combined with cisplatin in patients with locally advanced pancreatic carcinoma. METHODS: Forty-one patients with pancreatic carcinoma that was unresectable but confined to the pancreatic region were treated with external beam radiation (50.4 grays [Gy] in 28 fractions over 5.5 weeks) and daily cisplatin (5 mg/m(2)/day as a 30-minute infusion just before each radiation fraction). Maintenance 5-fluorouracil (5-FU) (500 mg/m(2)) given once weekly was initiated 1 week after the completion of the chemoradiotherapy and continued until disease progression or unacceptable toxicity. RESULTS: Of the 41 patients, 31 (76%) completed the scheduled course of chemoradiotherapy. The median survival time was 7.7 months, and the 1-year survival rate was 36%. The median progression free survival time was 5.8 months. The first site of failure was distant metastases in 25 patients, locoregional recurrence in 6 patients, and both sites in 1 patient. The major toxicity was leukocytopenia and nausea/emesis. CONCLUSIONS: Radiotherapy with daily cisplatin appears to be inferior to conventional chemoradiotherapy using 5-FU in patients with locally advanced pancreatic carcinoma.

Efficient gene transduction by RGD-fiber modified recombinant adenovirus into dendritic cells.

Dendritic cells (DC) are important antigen-presenting cells in the development of an anti-tumor T cell response. To extend the range of current immuno / gene therapies, we tested luciferase-expressing RGD-adenovirus (Ad) (Ad5lucRGD)-mediated transduction into DC. Phenotypically characterized DC were generated from peripheral blood CD14(+) cells by incubation with granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor alpha. On the 7th day of culture, the cells became mature DC with a CD1a(+), CD11c(+), CD80(+), CD83(+), CD86(+), human leukocyte antigen (HLA)-DR(+), CD14- phenotype. The expression of alpha( v)beta(3) integrin was enhanced on day 3 and returned to the basal level on day 7. We then compared the transduction efficiency of an Ad5lucRGD system to that using conventional Ad, in cells harvested on days 1, 3 and 7 of culture. Luciferase activity was negligible in AdCMVLuc, but remarkable in cells processed with Ad5lucRGD. Activity was maximal in cells that had been cultured for 3 days. Recombinant Ad5 fiber knob protein blocked AdCMVLuc- and Ad5lucRGD-mediated gene transduction by 90% and 20%, respectively. Surface markers and cytokine production were not affected by Ad5lucRGD-mediated transduction.

Absence of endothelial cells, central necrosis, and fibrosis are associated with aggressive inflammatory breast cancer.

We recently established a new human inflammatory breast cancer (IBC) xenograft (WIBC-9) originating from a patient with IBC. The graft was transplantable in BALB/c nude and severe combined immunodeficient (SCID) mice. WIBC-9 was frequently accompanied by lung metastasis and exhibited erythema of the overlying skin, reflecting its human counterpart. Histological study of the original tumor and WIBC-9 revealed invasive ductal carcinoma with a hypervascular structure of solid nests and marked lymphatic permeation in the overlying dermis. In the central part of the solid nests, absence of endothelial cells, central necrosis, and fibrosis were observed. In vitro, WIBC-9 formed tube-like structures and loops, reflecting its in vivo feature and its human counterpart. WIBC-9 exhibited aneuploidy, ErbB-2 gene amplification, and an absence of estrogen receptor and progesterone receptor, which is consistent with IBC. Comparative studies of WIBC-9, three established non-IBC xenografts, and a human breast cancer cell line (SK-BR3) by reverse transcription-PCR, ELISA, and immunohistochemistry indicated that certain human genes (interleukin 8, vascular epidermal growth factor, basic fibroblast growth factor, angiopoietin 13, Flt-1, Tie-2, and Tie-1) and certain murine genes (integrin alpha(v)beta3, flt-1, tie-2, vascular epidermal growth factor, and CD31) were overexpressed in exposure to tumor cells. The molecular basis and these unique histological features may be associated with aggressive IBC on angiogenic and nonangiogenic pathways.