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.

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.

In vitro potentiation of human natural killer cell activity by a streptococcal preparation, OK-432: interferon and interleukin-2 participation in the stimulation with OK-432.

Inasmuch as human natural killer (NK) cell activity was markedly augmented by a streptococcal immunopotentiator, OK-432, both in vivo and in vitro, the mechanism in which OK-432 augmented human NK cell activity was analyzed. Culture supernatants of nonadherent lymphocytes stimulated with OK-432 significantly augmented NK cell activity. Significant activity of both interferon (IFN) (both alpha- and gamma-types) and interleukin-2 (IL-2) was detected in the culture supernatants from nonadherent lymphocytes. Concomitant treatment of supernatants with anti-IFN-alpha antiserum and pH-2 glycine-HCI buffer or the absorption of supernatants with an IL-2-dependent cell line completely abrogated the NK-augmenting activity, whereas the treatment with either one of these resulted in only partial elimination of the activity. These results indicate that OK-432 stimulates human nonadherent lymphocytes to produce IFN and IL-2 and that both factors are primarily responsible for the NK augmentation by OK-432.

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.

Induction of thioredoxin in human lymphocytes with low-dose ionizing radiation.

Induction of the expression of the thioredoxin (TRX) gene, producing a key protein in regulating cellular functions through redox reaction as well as being a radioprotector, was followed after ionizing irradiation of lymphocytes from human donors. The TRX mRNA level increased to a peak, 5.7-fold higher than the control at maximum, 6 h after irradiation, and then decreased. The optimum radiation dose for enhancement of induction of the TRX mRNA was 0.25 Gy. The TRX protein also increased to a peak, a 3-fold increase at maximum, with the same timing as that for TRX mRNA.

Induction of mRNAs for glutathione synthesis-related proteins in mouse liver by low doses of gamma-rays.

We examined the elevation of the reduced form of glutathione (GSH)level and the induction of MRNAs for proteins involved in the synthesis and regeneration of GSH in the liver of mice after low-dose gamma-ray irradiation. The liver GSH level increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at around 12 h post-treatment. The mRNA of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for de novo synthesis for GSH, showed a small increase that peaked at 6 h after gamma-ray irradiation at a dose of 50 cGy. Only a small increase in gamma-GCS activity was observed throughout the 24-h post-irradiation period. In the case of glutathione reductase (GR), which is involved in the regeneration of GSH from the oxidized form (GSSG), the mRNA level peaked strongly at 1 h, while the activity peaked at twice the control level 12 h after irradiation. The level of mRNA for thioredoxin (TRX), which contributes to GSH biosynthesis by supplying cysteine to the de novo pathway, peaked at 1 h and declined thereafter, while the activity peaked at 3 h and then declined sharply. These results indicate that the increase in endogenous GSH immediately following low-dose gamma-ray irradiation is predominantly due to operation of the regeneration cycle and not de novo synthesis. We also examined the dependence of mRNA induction on the gamma-ray dose.

Phase I and pharmacokinetic study of a new taxoid, RPR 109881A, given as a 1-hour intravenous infusion in patients with advanced solid tumors.

PURPOSE: RPR 109881A is a new semisynthetic taxoid compound that has a similar mechanism of action to docetaxel. The purpose of this phase I study was to characterize the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetic profile, and antitumor effects of this agent. PATIENTS AND METHODS: Nineteen eligible patients with advanced solid tumors were enrolled. RPR 109881A was administered as a 1-hour intravenous infusion every 3 weeks at doses ranging from 15 to 75 mg/m(2). Pharmacokinetic evaluation was performed at the first cycle. RESULTS: Neutropenia (febrile neutropenia) and fatigue were dose-limiting toxicities at doses of 60 and 75 mg/m(2) and seemed to be dose-related. Both thrombocytopenia and anemia were infrequent. Nonhematologic toxicities were generally mild. Pharmacokinetic studies indicated that RPR 109881A plasma disposition was bi- or triphasic, with a high total plasma clearance, a large volume of distribution, and a long terminal half-life. The area under the concentration-time curve (AUC) and the peak concentration of RPR 109881A seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics. Urinary excretion over 48 hours was low, with a mean of 0.8 +/- 0.36% of the administered dose. A significant relationship existed between the percentage decrease of neutrophil counts and the AUC of RPR 109881A. Among 18 assessable patients, two partial and two minor responses were documented. CONCLUSION: RPR 109881A was found to be a well-tolerated and promising taxoid agent. The MTD was 75 mg/m(2), and the recommended dose for phase II study was 60 mg/m(2) as a 1-hour infusion every 3 weeks.

Clinical implications of the serum level of CD23 in patients with undifferentiated nasopharyngeal carcinoma.

PURPOSE: In contrast with other carcinoma cells, cells from nude mice transplanted undifferentiated carcinoma of nasopharyngeal type (UCNT) release the soluble fragment of the CD23 antigen (sCD23). We sought to study the level of sCD23 in sera of untreated UCNT patients. PATIENTS AND METHODS: Pretherapeutic sera from 65 consecutive, locally advanced, initially nonmetastatic UCNT patients were assayed for sCD23. Patients were treated with a neoadjuvant chemotherapy/full-dose radiotherapy sequence. The mean follow-up duration is 50.5 months (range, 28 to 77). The Cox proportional hazards model was used to study the association between sCD23 levels and clinical signs and disease evolution. RESULTS: sCD23 levels showed an association with disease-free survival (DFS; P = .08) and overall survival (OVS; P = .08). Patients with sCD23 levels greater than a cutoff value of 0.6 ng/mL (greater cutoffs were found to be equally significant, but less sensitive), have a relative risk (RR) of relapse of 3.3 (95% confidence interval, 1.6 to 6.9; P = .002), and an RR of death of 2.9 (95% confidence interval, 1.2 to 7.3; P = .02), when taking other prognostic factors into account. CD23 does not correlate with either the response to treatment or the development of metastases, but appears to be related to local control (cutoff, 0.6 ng/mL; RR = 5.1 [95% confidence interval, 1.2 to 21.7]; P = .02). CONCLUSION: The serum level of sCD23 appears to be an independent prognostic factor for initially nonmetastatic, locally advanced UCNT patients, treated with chemotherapy and radiotherapy. Our data indicate an association between this marker and local relapses. Thus, a simple enzyme-linked immunoadsorbent assay (ELISA) could help to identify a high-risk group among nonmetastatic UCNT patients. CD23 could be a marker for two groups of UCNT tumors, with distinct biologic characteristics and clinical behaviors.

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.

Role of ATL-derived factor (ADF) in the normal and abnormal cellular activation: involvement of dithiol related reduction.

HTLV-I transformed T cells not only express a large number of interleukin-2 receptors (IL-2R/p55(Tac], but also produce an IL-2R/Tac inducer named ATL-derived factor (ADF). We have cloned the ADF cDNA and found that ADF production in human lymphocytes can be enhanced by cellular activators such as mitogens or phorbol esters. Recombinant ADF produced by E. coli was shown to have growth-promoting activity in combination with interleukin-2 or suboptimal mitogenic stimuli on several lymphoid cells including human PBMCs, besides the originally reported IL-2R/Tac inducing activity. Homology analysis revealed an unexpected structural relationship between ADF and dithiol-reducing enzyme, thioredoxin, which had been characterized originally in prokaryotic system. Recombinant ADF also has a reducing activity, suggesting the presence of still unknown features of ADF action in vivo. The requirement of dithiol reduction in the biological activities of ADF, together with the possible involvement of ADF production in the normal and abnormal activation of human cells are discussed.

HLA class-I and class-II antigen expression by human leukemic K562 cells and by Burkitt-K562 hybrids: modulation by differentiation inducers and interferon.

K562 cells, which could be regarded as pluripotent hematopoietic progenitors, are usually considered as HLA class-I and class-II-negative cells. We show here that differentiation induction (with either sodium butyrate, 12-O-tetradecanoyl-phorbol-13-acetate, or teleocidin) or recombinant alpha- or gamma-interferon (IFN) treatment resulted in the augmentation of HLA class-I antigen expression. This augmentation of HLA class-I antigens was also observed in the Burkitt X K562 hybrid cells PUTKO and DUTKO (the latter coming from two presumably HLA-A, B-negative parents). HLA class-I genes are thus functional in K562 cells. In this system, alpha- and gamma-IFN had no clear differentiating capacity, since they were not able to modulate the expression of various hematopoietic markers, as chemical differentiation inducers did. On the other hand, neither differentiation induction nor interferon treatment could induce HLA class-II antigen expression on K562 cells. These molecules could be very faintly induced in PUTKO and DUTKO hybrids, in contrast with strong HLA class-II expression on the B parental lines. Whether these results are due to "lineage infidelity" in K562 cells or whether K562 cells represent the proliferation of HLA class-I-positive class-II-negative hematopoietic cells, with active suppression of HLA class-II antigen expression, is discussed.

Somatostatin-secreting islet cell tumor (somatostatinoma): suppression of growth hormone (GH) release induced by GH-releasing hormone.

A patient with a somatostatin (SRIH)-secreting islet cell tumor, whose only symptoms were dyspepsia and anemia, is described. The diagnosis of somatostatinoma was based on high plasma SRIH concentrations and immunocytochemical findings. The pancreatic exocrine response to secretin was decreased, whereas the insulin and/or glucagon responses to glucose and arginine were normal. Although the basal plasma GH concentration was normal, the plasma GH response to GHRH was subnormal. Gel permeation chromatography studies indicated that SRIH-14 was the predominant form of SRIH in plasma as well as in tumor tissue.

Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-1N).

Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

Hypercalcemia caused by ectopic production of parathyroid hormone in a patient with papillary adenocarcinoma of the thyroid gland.

Hypercalcemia and elevation of a serum PTH level (9800 pg/mL (normal: 160-520) were found in a 72-yr-old woman who had a lung cancer. She underwent pulmonary lobectomy for a suspected PTH-producing lung cancer. However, hypercalcemia and elevation of the serum PTH level were persistent postoperatively. Subsequent examination, using parathyroid scintiscanning, revealed a hot spot in the right lower part of the thyroid gland, suggesting hypercalcemia caused by a parathyroid tumor. She underwent bilateral exploration of the neck; however, four apparently normal parathyroid glands were seen. Therefore, hemithyroidectomy was performed for the possibility of an intrathyroidal parathyroid adenoma. Serum calcium and PTH levels declined after this operation. A nodular lesion was found in the cut sections of the resected specimen, which was consistent with the result of the scintiscanning. Histological examinations revealed a papillary adenocarcinoma of the thyroid gland, and the PTH-immunoreactivity in the tumor cells was confirmed. These findings strongly suggest that PTH could be produced ectopically by the papillary adenocarcinoma of the thyroid gland.

Plasma pancreastatin-like immunoreactivity in various diseases.

Plasma pancreastatin (PST)-like immunoreactivity in normal subjects and patients with various diseases was estimated by a RIA, using antiserum raised against a synthetic C-terminal peptide of human PST deduced from the sequence of human chromogranin-A. The mean level +/- SEM was 13.2 +/- 0.6 pmol/L in normal subjects, but was significantly higher in patients with chronic renal failure (526.7 +/- 48.5). An immunoreactive form corresponding to a human PST-like sequence [human chromogranin-A-(250-301)] and a larger form were detected by gel filtration of plasma from these patients, suggesting accumulation of the larger molecular form in these patients. A significant increase in PST-like immunoreactivity was also found in patients with liver cirrhosis (20.8 +/- 3.0 pmol/L), but not in patients with noninsulin-dependent diabetes mellitus, chronic pancreatitis, or pancreatic cancer. Elevated levels were found in 16 of the 21 patients with small cell lung carcinoma examined. High levels were also found in 3 of 11 patients with islet cell tumor.