High NK Research Articles

Psychosocial factors regulating natural-killer cell activity in recurrent spontaneous abortions.

The preconceptional natural-killer cell (NK) activity predicts subsequent miscarriage among women with unexplained recurrent spontaneous abortion (RSA). Psycho-neuro-immuno-endocrine network has recently been proposed as a mechanism for abortions. We therefore examined which psychosocial factors influenced the NK activity among women with RSA. We measured the preconceptional NK activity of 61 women with a history two consecutive unexplained first-trimester miscarriages and no live births. We also administered semi-structured interviews and a battery of self-report questionnaires to assess their social support, personality, self-esteem and psychiatric symptoms. The preconceptional NK activity was negatively correlated with the women's neuroticism personality trait (r= -0.32, P = 0.01) and current depressive symptoms (r = -0.26, P= 0.05), and positively correlated with their self-esteem (r = 0.34, P = 0.01). In addition to several substances such as transforming-growth-factor beta and granulocyte-macrophase colony-stimulating factor, we found that low neuroticism, low depression scale score and high self-esteem contributed to high NK activity among women with RSA.

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation.

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

Constitutive IL-10 Production Accounts for the High NK Sensitivity, Low MHC Class I Expression, and Poor Transporter Associated with Antigen Processing (TAP)-1/2 Function in the Prototype NK Target YAC-1

Tumor cells that are treated with rIL-10 or transfected with the IL-10 gene show phenotypic changes. These include low but peptide-inducible expression of MHC class I, low sensitivity to specific CTL-mediated lysis, and increased NK sensitivity. In vitro-established mouse tumor lines were screened for IL-10 expression and production, and a large proportion of plasmocytomas or T cell lymphomas were found to produce IL-10. Since one of these lines was the prototype NK target cell YAC-1, we investigated whether the high IL-10 production of this cell line was related to its high NK sensitivity and its defects in MHC class I expression. The decrease in H-2 expression following the in vitro culture of in vivo-passaged YAC-1 cells was accompanied by a gradual increase in IL-10 production, whereas the reverse was found when passing in vitro-grown YAC-1 in vivo as an ascites tumor in syngenic mice. In addition, differences in YAC-1 MHC class I expression correlated with alterations in the functional activity of TAP-1/2 proteins. YAC-1 cells that were transduced with a retroviral IL-10 antisense construct (Y-IL-10 AS) only produced about half of the IL-10 that was produced by YAC-1 transduced with the control construct (Y-IL-10 Mock). Relative to Y-IL-10 Mock cells, the expression of H-2 on Y-IL-10 AS cells was markedly increased, and NK sensitivity was decreased. These data argue for a mechanism wherein IL-10 production is causally related to the low H-2 expression, decreased TAP function, and high NK sensitivity of YAC-1 cells.

The use of heterocycles for the conformational restriction of biologically active peptoids

A series of piperazinone ring systems have been synthesised as a means of evaluating the effect of conformational restriction on high affinity non-peptide NK 1, NK 3 and CCK-B receptor ligands. The synthesis of the targeted heterocycles is described along with a discussion of their affinities for their respective receptor types.

IL-15 Mediates Anti-tumor Effects after Cyclophosphamide Injection of Tumor-Bearing Mice and Enhances Adoptive Immunotherapy: The Potential Role of NK Cell Subpopulations

The daily administration of IL-15 to cyclophosphamide (CY)-injected mice bearing the 76-9 rhabdomyosarcoma was shown to prolong the period of remission induced by CY. In addition, IL-15 was shown to enhance the efficacy of adoptive immunotherapy. Cytotoxicity assays using spleens from normal and tumor-bearing mice indicated that IL-15 enhanced NK cell activity but there was no evidence for class I-restricted cytolytic T cell activity. To determine whether IL-15 was likely to induce different cytotoxic effectors at the tumor site compared with the spleen, tumors were removed after CY injection and cell suspensions were incubated with IL-15 in parallel with isolated spleen cells. Both populations were seen to expand to yield predominantly cells coexpressing NK1.1 and B220 antigens. However, tumor-associated NK cells were shown to differ from expanded spleen NK cells in terms of the proportions of LGL-1+cells and cells expressing early and late NK cell differentiation antigens. Both expanded populations expressed high NK cell cytotoxic activity but only the spleen cells expressed lymphocyte-activated killer cell activity. It was apparent that the expanded tumor-associated NK cells expressed low-level class I-restricted lytic activity. The potential of activated NK cells in the circulation to exert anti-tumor effects was shown by the adoptive transfer of expanded NK cells to tumor-bearing mice after CY injection when significant prolongation of life was seen in all cases. The data indicate that IL-15 may serve as a useful anti-cancer adjuvant by activating initially the NK cell arm of the immune network.

The tachykinin NK 1 receptor antagonist PD 154075 blocks cisplatin-induced delayed emesis in the ferret

The activity of a selective tachykinin NK 1 receptor antagonist, PD 154075 ([(2-benzofuran)-CH 2OCO]-( R)-α-MeTrp-( S)-NHCH(CH 3)Ph), was examined in radioligand binding studies, in a [Sar 9,Met(O 2) 11]substance P-induced foot-tapping model in the gerbil, and in cisplatin-induced acute and delayed emesis in the ferret. In radioligand binding studies, PD 154075 showed nanomolar affinity for the human, guinea-pig, gerbil, dog and ferret NK 1 receptors with an approximate 300 times lower affinity for the rodent NK 1 receptor. Using NK 2, NK 3 receptors and a range of other receptor ligands, PD 154075 was shown to exhibit a high degree of selectivity and specificity for the human type NK 1 receptor. Following subcutaneous administration PD 154075 dose dependently (1–100 mg/kg) antagonised the centrally mediated [Sar 9,Met(O 2) 11] substance P-induced foot tapping in the gerbil with a minimum effective dose (MED) of 10 mg/kg. The ability of PD 154075 to readily penetrate into the brain following oral administration was confirmed by its extraction and high performance liquid chromatography assay from the rat brain. PD 154075 was shown to achieve a relatively fast and sustained brain concentration (brain/plasma ratios ranged from 0.27 to 0.41 during the time period of 0.25–12 h). Further pharmacokinetic studies revealed that the absolute oral bioavailability of PD 154075 in the rat was (mean±S.D.) 49±15%. PD 154075 (1–30 mg/kg, i.p.) dose dependently antagonised the acute vomiting and retching in the ferret measured for 4 h following administration of cisplatin (10 mg/kg, i.p.) with a MED of 3 mg/kg. The administration of a lower dose of cisplatin (5 mg/kg, i.p.) in the ferret induces both an acute (day 1) and delayed (days 2 and 3) phase of emesis. The i.p. administration of PD 154075, 10 mg/kg three times a day for 3 days, almost completely blocked both the acute and delayed emetic responses. In the same study, the 5-HT 3 receptor antagonist ondansetron (1 mg/kg, i.p., t.i.d.) was also very effective against the acute emetic response observed during the first 4 h following cisplatin, but it was only weakly active against the delayed response. In conclusion, PD 154075 is a selective and specific high affinity NK 1 receptor antagonist with good oral bioavailability which is effective against both acute and delayed emesis induced by cisplatin in the ferret.

Elevated peripheral blood natural killer cells are effectively downregulated by immunoglobulin G infusion in women with recurrent spontaneous abortions.

We investigated the hypothesis that elevated peripheral blood natural killer cells (NK) are decreased by immunoglobulin G infusion (IVIg) therapy in women with recurrent spontaneous abortions (RSA) and elevated NK cells. Seventy-three women with RSA and elevated NK cells received IVIg therapy (400 mg/Kg/day for 3 days ever 4 wks) and anticoagulation treatment. Peripheral blood immunophenotype assay by flow cytometry was done prospectively prior to and 7 days after first IVIg therapy, every 2 wks until 20 wks gestation and then monthly. Controls were 95 women with RSA and normal NK cells who received anticoagulation treatment. (1) 86.3% of women with elevated NK cells who received the IVIg and anticoagulation therapy had a successful pregnancy outcome; (2) Peripheral blood CD56+ NK cells and CD56+/16+ NK cells were significantly suppressed 7 days post IVIg infusion (P < 0.0005); (3) Pre-IVIg infusion levels of other lymphocyte subsets were not different as compared with those of 7 days post-IVIg therapy; (4) Women who delivered a liveborn infant with IVIg therapy demonstrated downregulation of peripheral blood NK cells (CD56+, CD56+/16+) during early pregnancy when compared to women who miscarried the index pregnancy (P < 0.05); (5) Women with normal NK cells who miscarried while on anticoagulation therapy demonstrated significantly elevated CD56+ NK cells during early pregnancy as compared with that of women who delivered a liveborn infant (P < 0.05); (6) CD19+ B cells were significantly downregulated during pregnancy in women with anticoagulation and IVIg therapy when compared to women with anticoagulation therapy (P < 0.05). Downregulation of NK cells in women with RSA is associated with a favorable pregnancy outcome. Peripheral blood NK cells (CD56+, CD56+/16+) are effectively suppressed after IVIg therapy. Women with RSA and high NK cells benefit from IVIg therapy and experience suppression of CD56+ and CD56+/16+ NK cells.

The expression of a progesterone-induced immunomodulatory protein in pregnancy lymphocytes.

The immunological effects of progesterone are mediated by a protein, named the progesterone-induced blocking factor (PIBF). The PIBF blocks NK activity in vitro and therefore prevents the abortive effect on high NK activity in mice. Increased NK activity has been suggested to play a role in pregnancy termination; thus NK inhibitory effect of the PIBF should contribute to the maintenance of normal gestation. This study was designed to investigate the relationship between n vivo PIBF-producing capacity and in vitro cytotoxic activity of pregnancy lymphocytes, as well as the clinical status or the outcome of pregnancy. Lymphocytes of 168 pregnant women (96 normal pregnancies, 16 showing clinical symptoms of threatened preterm pregnancy termination, 46 recurrent aborters, and 10 women sampled at the onset of spontaneous abortion or preterm delivery) were isolated on Ficoll-Paque gradient. The lymphocytes were tested for reactivity with a PIBF-specific antibody by immunocytochemistry, and simultaneously for cytotoxic activity to human embryonic fibroblast targets. The percentage of PIBF-positive lymphocytes in peripheral blood of healthy pregnant women was significantly higher than in that of women at risk for premature pregnancy termination. In peripheral blood of patients undergoing spontaneous pregnancy termination at the time of sampling, and in those of women showing symptoms of premature pregnancy termination we found lower than normal percentage of PIBF-positive cells. PIBF expression of the lymphocytes showed an inverse correlation with NK activity, and the rate of PIBF positive lymphocytes was related to the outcome of pregnancy. These data suggest a strong relationship between PIBF producing capacity as well as NK activity of the lymphocytes and the success of gestation.

The design of polar β-turn dipeptide mimetics

The design and synthesis of conformationally constrained, non-peptide templates (3-substituted pyrrolidines) which allow the incorporation of two adjacent amino acid side-chains in an orientation similar to that found in the i+1 and i+1 positions of a β-turn are reported. The NK 2 tachykinin receptor affinity of a Trp-Phe mimetic ( 7a) which mimics the β-turn in MEN10627 ( 8), a constrained cyclic hexapeptide, high affinity NK 2 tachykinin receptor antagonist, was determined and shown to have no significant binding affinity (NK 2, IC 50 > 10,000 nM).

Preconceptional natural-killer-cell activity as a predictor of miscarriage

There is no immunological test for the prospective identification of alloimmune causes of miscarriage. We investigated whether activity of natural killer cells was predictive of subsequent abortion in women who had had unexplained recurrent abortions and had received no treatment. 24 women with high preconceptional NK activity defined as mean plus 1 SD of NK activity of 47 controls, had a significantly higher abortion rate in the next pregnancy than 44 women with normal levels of NK activity (71 vs 20%; relative risk 3·5; 95% CI 1·8–6·5).The preconceptional evaluation of NK activity in women with recurrent miscarriages may thus be predictive of the risk of pregnancy loss at the next conception.

Murine Strain Variation in the Natural Killer Cell and Proliferative Responses to the Immunostimulatory Compound 7-Allyl-8-oxoguanosine: Role of Cytokines

7-Allyl-8-oxoguanosine (loxoribine) is a di-substituted guanine ribonucleoside which has been shown previously to enhance murine NK activity, B lymphocyte proliferation, and antibody synthesis. In this study we examined the relationship among enhancement of NK activity, proliferation, and cytokine synthesis in the responses of different strains of mice to loxoribine to provide insight into the role of cytokines in these biological activities. The NK response of mice was enhanced both in vitro and in vivo in all strains tested with the exception of the NK-deficient beige (BgBg) mouse. However, there was a marked difference in the degree of NK enhancement noted in other inbred strains, with C3H and CBA mice producing the highest responses, C57BL/6, BALB/c, and DBA/2 strains giving intermediate responses, and SJL mice manifesting low responses. Striking enhancement of NK cell activity was seen in SCID mice. A somewhat different effect was seen in humans. Loxoribine treatment enhanced both the NK and LAK activity of cells from individuals with low and high spontaneous NK activity. The degree of enhancement was similar for both groups, and thus the general hierarchy of NK activity among different donors was maintained. There was less interstrain variation in the murine proliferative response to loxoribine although nude (NuNu) mice showed the highest activity and SJL mice produced substantially lower responses than other strains. All strains produced IL-6, TNFα, IFN-α/β, and IFN-γ when spleen cells were cultured for 48 hr with loxoribine. Interstrain variability of cytokine synthesis displayed no consistent pattern from one cytokine to another, and all failed to correlate with interstrain variability of NK cell activity or B lymphocyte proliferation. When anti-cytokine antibodies were tested for the ability to block the immunostimulatory effects of loxoribine, only anti-IFN-α/β and, to a lesser degree, anti-IFN-β, partially inhibited NK activation. Similarly, only anti-IFNα/β antibodies partially blocked the proliferative response to loxoribine. In both cases, reconstitution of the responses was achieved by adding back IFN-α/β to cultures containing antibodies against IFN-β/β. These data suggest that, although several cytokines are produced in response to loxoribine, only IFN-α and IFN-β are directly involved in the NK activation and proliferative responses. The pattern of strain variation appears to be reflective of variation in NK cell responsiveness to IFN-α/β.

Effects of RP 67580, a tachykinin NK1 receptor antagonist, on a primary afferent-evoked response of ventral roots in the neonatal rat spinal cord.

1. The pharmacological characteristics of RP 67580, a non-peptide tachykinin NK1 receptor antagonist, and its effects on a reflex response evoked by stimulation of primary afferent fibres, were examined in isolated neonatal spinal cord preparations of the rat. Potentials were recorded extracellularly from a lumbar ventral root and drugs were bath-applied in normal artificial cerebrospinal fluid (CSF) or in the presence of tetrodotoxin (TTX). 2. In normal artificial CSF, RP 67580 (0.1-0.3 microM) caused rightward shifts of the concentration-response curves for substance P (SP), neurokinin A (NKA) and substance P methyl ester (SPOMe), an NK1-selective agonist, with pA2 values of 7.25, 7.47 and 7.49, respectively. 3. In the presence of TTX (0.3 microM), RP 67580 also caused rightward shifts of the concentration-response curves for SPOMe and NKA. The pA2 value of RP 67580 against SPOMe (6.75) was significantly lower than that against NKA (7.22). RP 67580 (0.3-1 microM) did not cause a clear parallel shift of the concentration-response curves for SP, and it depressed the depolarizations induced by low concentrations of SP, but slightly potentiated those induced by high concentrations of SP. 4. RP 67580 (1 microM) did not depress the depolarizing responses to bombesin, L--glutamate, gamma-aminobutyric acid (GABA), thyrotropin-releasing hormone and muscarine. RP 67580 (1 microM), however, depressed the response to acetylcholine in the presence of atropine and the response to nicotine. RP 68651 (1 microM), the enantiomer of RP 67580 devoid of activity at tachykinin NK1 receptors, also depressed the response to acetylcholine in the presence of atropine. 5. RP 67580 (1 gAM) did not induce GABA release from the rat spinal cord.6. In the neonatal gerbil spinal cord, the antagonist effects of RP 67580 (0.3-1 JAM) against SPOMe were much less potent than in the neonatal rat spinal cord.7. In the rat spinal cord-saphenous nerve preparation, electrical stimulation of the saphenous nerve atC-fibre strength evoked a prolonged depolarization of the ipsilateral L3 ventral root (slow VRP).RP 67580 (0.1-1 JM) depressed the saphenous nerve-evoked slow VRP. In contrast, RP 68651 (0.3 JAM)had no effect on the slow VRP.8. The results of the present study indicate that RP67580 acts as a high affinity NK, receptor antagonist in the neonatal rat spinal cord, although it also possesses an antinicotinic action. This study further suggests the existence of a subpopulation of tachykinin NK, receptors that are activated by NKA and SPOMe, as well as by low concentrations of SP, and are sensitive to the antagonist action of RP 67580 in the neonatal rat spinal cord. This study also provides further evidence for the involvement of SP and NKA in the slow VRP evoked by C-fibre stimulation in the neonatal rat spinal cord.

Rational design of high affinity tachykinin NK 1 receptor antagonists

The rational design of a non-peptide tachykinin NK 1 receptor antagonist, [(2-benzofuran)-CH 2OCO]-( R)-α-MeTrp-( S)-NHCH(CH 3)Ph ( 28, PD 154075) is described. Compound 28 has a K i = 9 and 0.35 nM for the NK 1 receptor binding site in guinea-pig cerebral cortex membranes and human IM9, cells respectively (using [ 125I] Bolton-Hunter-SP as the radioligand). It is a potent antagonist in vitro where it antagonises the contractions mediated by SPOMe in the guinea-pig ileum (K B = 0 3 nM). Compound 28 is active in vivo in the guinea-pig plasma extravasation model, where it is able to block the SPOMe-induced protein plasma extravasation (monitored by Evans Blue) in the bladder with an ID 50 of 0.02 mg kg −1 iv.

Rational design of high affinity tachykinin NK 2 receptor antagonists

The rational discovery of a high affinity NK 2 receptor antagonist is described utilizing a general strategy for peptoid design. The contribution to NK 2 receptor binding affinity for each amino acid of the hexapeptide ‘inimum fragment’: Leu-Met-Gln-Trp-Phe-GlyNH 2 ( 8c), was examined by preparing derivatives where each amino acid in turn was replaced with Ala in an ‘alanine scan’. The results from this study indicated the primary importance of the Trp and Phe side-chain for binding and led to the observation that Z-Trp-PheNH 2 ( 9a) is a micromolar affinity NK 2 receptor dipeptide lead. Further exploration of structure-affinity via conformationally restricted analogues and N- and C-terminus modifications gave a selective, nanomolar affinity NK 2 receptor antagonist, (2,3di-CH 3OPh)CH 2OCO( S)Trp( S)α-MePheGlyNH 2, PD 147714 ( 19) with an K i = 1.4 nM (hamster urinary bladder membranes and using [ 125I]-iodohistidyl-NKA (0.1 nM) as the radioligand).

Transforming growth factor-beta expression and natural killer cell responses during virus infection of normal, nude, and SCID mice.

The experiments presented here assess the contribution of T cells to NK cell regulation and transforming growth factor-beta (TGF-beta) production. The kinetics of NK cell activation in the absence of T cells was evaluated by comparing responses in athymic nude and SCID mice to those in normal C3H/HeN, C57BL/6, and BALB/c mice during lymphocytic choriomeningitis virus (LCMV) infection. T cell-deficient mice exhibited high NK cell activity for longer periods of time post-infection. To determine whether the reduction in NK cell activation at later times post-infection of normal mice resulted from induction of negative regulatory elements, mice were treated with the chemical IFN and NK cell inducing agent polyinosinic-polycytidylic acid (poly I:C). Although poly I:C induced plasma IFN in both uninfected and LCMV-infected normal mice, NK cell activity was elevated in the uninfected mice only. In vivo expression of TGF-beta protein in normal and T cell-deficient mice was evaluated by immunohistochemical studies on splenic sections. TGF-beta proteins were not detected in sections from uninfected mice but were induced in all strains during infection. High levels of TGF-beta proteins were localized to discrete cells, and diffuse staining was observed along marginal zones. The ability of the splenic leukocytes to release TGF-beta into conditioned media was evaluated in the Mv 1 Lu biologic assay. Transient acid treatment to release active factor from the latent form and use of specific neutralizing antibodies demonstrated that leukocytes from all strains of infected mice had released latent TGF-beta 1 into conditioned media. However, cells from infected nude or SCID mice produced substantially less active TGF-beta than those isolated from normal mice. Production of biologically active TGF-beta 1 was dependent upon the presence of T cells in normal mice, as in vivo depletion of CD4+ and CD8+ T cells by antibody treatment resulted in augmented NK cell activity and loss of active factor production. The lack of TGF-beta production was accompanied by increased NK cell numbers; at late times post-infection, T cell-deficient C57BL/6-nude mice had almost twice as many NK1.1+CD3- cells per spleen compared with T cell-containing C57BL/6 littermate control mice. Treatment with active TGF-beta 1 reduced the numbers of NK cells per spleen in infected nude mice to the levels found in infected littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Elderly high NK responders are characterized by intensive proliferative response to PHA and Con A and optimal health status

In this paper we verify a hypothesis emerging from recent immunogerontological studies assuming that an elevated level of NK activity in the elderly may be regarded as a factor compensating for a decreased reactivity of T cells. According to this assumption a high NK activity should be associated with a poor reactivity of T cells. In our study the mitogenic responses of lymphocytes of the elderly and the young high and low NK responders to phytohemagglutinin (PHA) and Concanavalin A (Con A) were measured. A high level of NK activity was not found, as would be expected from the hypothesis, to be associated with a poor reactivity to PHA and Con A. In contrast, the mitogenic response to PHA and Con A was intensive in both the young and old high NK responders while weak proliferative responses to T cell mitogens were characteristic of the young and old low NK responders. Thus, our results do not support the assumption of an association between a high NK activity and a low proliferative response to T cell mitogens in the elderly. We also found that both the elderly and the young high NK responders were characterized by an optimal health status.

Hepatic Arterial Infusion of Interleukin-2 in Advanced Hepatocellular Carcinoma

From August 1988 to February 1991, we performed hepatic arterial cannulation after tumor mass reduction in 17 patients with advanced hepatocellular carcinoma. By a subcutaneous infusion pump the patient received a continuous infusion of interleukin-2 (IL-2) (0.35 x 10(6) Japanese Reference Unit/day) and intermittent chemoembolization with 10 mg doxorubicin emulsion. The longest period of IL-2 infusion was 32 months. In 3 patients, the IL-2 dose could not be increased to the planned level due to fever or jaundice. Eight patients received the infusion for more than 10 months. NK and LAK activity in the peripheral blood, which had been reduced after partial liver resection, arterial embolization, or ethanol injection, was enhanced significantly by the IL-2 infusion, and the levels remained high for about 8 to 10 months. During periods of high NK and LAK activity, direct effects on the liver tumors were observed. In 11 patients who received IL-2 infusion for more than 6 months there was CR in 4, PR in 2, NC in 3 and PD in 2. Thereafter, however, the levels of NK and LAK activity decreased despite increase in the IL-2 dose, and sudden appearance of metastatic tumors caused deterioration of the patients. The 1-year and 2-year cumulative survival rates were 41% and 16% respectively. Further studies are required to explore ways for longer-lasting enhancement of NK and LAK activity.

Immunosenescence of human NK cells: effects on tumor target recognition, lethal hit and interferon sensitivity.

Previous results from this laboratory have shown the preservation of non-MHC-restricted, constitutive oncolytic activity of human peripheral blood NK cells in the elderly as assessed by the chromium release assay which quantitates the lytic endpoint at the cell population level. We have now addressed this senescence-related change at single-cell level using 101 blood samples. Both the efficiency of the initial tumor target binding step i.e., recognition of K562, the NK-sensitive erythroleukemia cell line, as well as the ability of NK cells to deliver lethal hit are highly conserved during healthy aging. In fact, the elderly exhibit a statistically significant, moderately higher frequency of active killers among circulating lymphocytes. Analyzed in another way, a majority of "high NK responders" were found to be older donors, while none in the "low NK responders" group were > 70 years old. Gamma interferon, a gene product as well as an autocrine activator of NK cells, is effective in converting non-lytic "pre-NK" cells to active killers at single-cell level. This in vitro cytokine sensitivity of NK cells is unaltered during immune senescence. The intactness of the NK cell's capacity to be modulated may be vital in both tumor resistance and host viral defenses of aged humans.

Stable expression of high affinity NK 1 (substance P) and NK 2(neurokinin A) receptors but low affinity NK 3 (neurokinin B) receptors in transfected CHO cells

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabeled substance P and neurokinin A (substance K) to CHO clones expressing the NK 1 and NK 2 receptors, respectively, were saturatable and of high affinity ( K d1=0.17 nM (NK 1); 3.4 nM (NK 2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK 1 and NK 2 receptors. In contrast, the binding of eledoisin to the NK 3 receptor expressed in the transfected CHO cells was of low affinity (IC 50=240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC 50=8 nM). However, in both cases the receptor exhibited the specificity of a classical NK 3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.

Ｍａｌｉｇｎａｎｔ ｆｉｂｒｏｕｓ ｈｉｓｔｉｏｃｙｔｏｍａ樹立細胞の制癌剤および免疫担当細胞に対する感受性

A cell line was established from a patient with malignant fibrous histiocytoma, which had originated in the maxillary sinus. Using this cell line, sensitivity to chemotherapeutic agents and cytotoxicity against various immunoeffecter cells were tested. This MFH cell line was sensitive in some degree to adriamycin, 5-fluorouracil, cisplatin, peplomycin, and methotrexate at high doses, but insensitive to mitomycin-C, vincristine and cyclophosphamide. Furthermore, this cell line showed no sensitivity to cytolytic cytokines, such as tumor necrosis factor-alpha and interferon-gamma. Lymphokine activated killer (LAK) cells and lymphokine activated tumor infiltrating lymphocytes (LA-TIL), induced by culture of peripheral blood lymphocytes and TIL respectively with rIL-2 showed high NK and LK activities and remarkable anti-autologous tumor ability.