In vitro augmentation of macrophage-activating-factor release from peripheral blood cells of cancer patients by a DNA fraction from Mycobacterium bovis BCG

Effect of a DNA-rich fraction from Mycobacterium bovis BCG (MY-1) on the macrophage-activating-factor (MAF)-activity release from peripheral blood lymphocytes (PBL) of patients with benign diseases and patients with gastric cancer or colorectal cancer was studied in vitro. The MAF activity was augmented by the addition of 10 micrograms/ml of MY-1 in many cases. Augmentation of the MAF activity in benign disease, gastric cancer and colorectal cancer in stages I to III was observed in five of six cases, eight of 10 cases and five of eight cases, respectively. The activity of MY-1-induced MAF was partially abolished by the treatment with monoclonal anti-interferon-gamma (IFN-gamma) antibody. These results suggest that MY-1 augments MAF-activity release from PBL by inducing IFN-gamma and other MAF-like substances.     

In situ infiltration of natural killer-like cells induced by intradermal injection of the nucleic acid fraction from BCG

Intradermal injection of MY-1, a nucleic acid fraction extracted from Mycobacterium bovis strain BCG, induced in situ infiltration of mononuclear cells, most of which were asialo GM1 (GA1)-positive as determined by immunofluorescence microscopy. The infiltration occurred with as little as 1 microgram of MY-1 and lasted for a week. Double immunofluorescence microscopy revealed that the infiltrating GA1-positive cells were all positive for Ly-5, and partially positive for Thy-1.2, but negative for Mac-1, Ia, mu-chain, Lyt-1, Lyt-2, L3T4, and Fc receptor II. They contained neither peroxidase nor nonspecific esterase. The infiltrating cells thus markedly resembled natural killer (NK) cells in their cytochemical characteristics and surface markers. DNase and RNase destroyed the GA1-positive cell-inducing activity of MY-1. These results indicate that the nucleic acid components of MY-1 are responsible for this effect.     

Autologous enhancement by interferon of natural killer activity of human peripheral blood lymphocytes

The in vitro action of interferon (IFN)-alpha and IFN-gamma from six healthy donors and ten patients with multiple sclerosis (MS) on natural killer (INK) activity of peripheral blood lymphocytes (PBL) was studied in an autologous system. The NK activity of PBL was detected by a cytotoxic test using (3)H-uridine human erythromyeloblast K562 cells. Autologous IFN-alpha and IFN-gamma did not augment NK activity of PBL from healthy donors in vitro, whereas in samples from MS patients the IFNs strongly stimulated NK cell cytotoxic function. This stimulation suggests the existence of an inhibitor of regulatory IFN action, that is produced in healthy donors simultaneously with IFN in response to IFN induction, but which is lacking in commercial IFN preparations. The factor-containing supernatants from healthy donors reduced the stimulatory action of autologous IFNs in patients with MS almost until complete blockade. Because this inhibitor was absent in patients with MS, deficiency of an inhibitor of IFN regulatory action in MS could open the way to treatment of this compartment of the immune system.     

Interleukin 2 enhances the natural killer cell activity of acquired immunodeficiency syndrome patients through a gamma-interferon-independent mechanism

Patients with the acquired immunodeficiency syndrome (AIDS) exhibit a variety of disorders of cellular immunity, including a deficient ability to generate cytotoxic T cells and depressed levels of natural killer (NK) cell activity. Interleukin 2 (IL 2) in vitro can markedly augment these depressed immune functions. Because IL 2 can induce the release of interferon-gamma (IFN-gamma) from normal peripheral blood lymphocytes (PBL), and because IFN-gamma may play a role in the regulation of NK cell activity, this study was performed to determine if the IL 2 enhancement of the NK cell activity of patients with AIDS was an IFN-gamma-dependent effect. PBL from eight healthy heterosexual donors and from nine patients with AIDS were studied for their ability to release IFN-gamma in response to IL 2 at a concentration of 100 U/ml. After 60 hr of culture, the PBL of all eight healthy donors produced IFN-gamma with a mean titer of 113 U/ml (range 40 to 320 U/ml). In contrast, the PBL from only two of nine patients with AIDS released measurable amounts of IFN-gamma (40 U/ml each) in response to IL 2 with a mean titer of 13.5 U/ml for all nine. Although the PBL from patients with AIDS were deficient in their capacity to produce IFN-gamma in response to 100 U/ml of IL 2, significant enhancement of NK cell activity could be obtained after only 1 hr of PBL treatment with 10 U/ml of IL 2, with an optimal NK enhancing effect occurring at doses of 50 to 100 U/ml of IL 2. The use of an anti-IFN-gamma monoclonal antibody resulted in complete neutralization of the IFN released from the normal PBL cultured with IL 2, but failed to inhibit the IL 2 enhancement of NK cell activity. Exogenous IFN-gamma exhibited different kinetics of enhancement of NK cell activity when compared to IL 2, requiring substantially more than 1 hr of pretreatment of PBL. These results indicate that the PBL from patients with AIDS usually do not release IFN-gamma when cultured with IL 2, and that IL 2 enhancement of the depressed NK cell activity of these patients may be an IFN-gamma-independent event. These results may have important implications for the therapy of AIDS.     

Enhancement of human natural killer cell activity by subcellular components of Toxoplasma gondii

The ability of sonicates and subcellular fractions of the intracellular parasite Toxoplasma gondii to enhance in vitro human natural killer (NK) cell activity was examined. Incubation of nylon-wool-non-adherent human peripheral blood lymphocytes (PBL) with sonicates of T. gondii for 18-72 hr resulted in increased NK activity against an NK-sensitive, as well as an insensitive, target cell. Single-cell assays revealed that augmentation of NK activity was not due to an increased binding of K562 target cells to effector cells. Differential centrifugation studies indicated that NK-augmenting activity was distributed in membrane-enriched and cytoplasmic fractions. This activity was found to be resistant to treatment with ribonuclease (RNase) and deoxyribonuclease (DNase), but susceptible to proteolysis. Antibodies present in the serum of humans infected with Toxoplasma blocked the NK cell-augmenting effect of the membrane-enriched fractions. Enhancement of NK activity by PBL incubated with Toxoplasma sonicate was accompanied by a concomitant increase in interferon (IFN), but not of interleukin 2 (IL-2), levels in supernatants of the cell cultures.     

艾氏腹水癌细胞核糖核酸体外对C_(57)BL/6脾自然杀伤活性的影响

本文采用~(51)Cr释放法,研究了艾氏腹水癌细胞核糖核酸(EAC-RNA)体外对C_(57)BL/6小鼠脾自然杀伤细胞(NK)对YAC-1靶细胞杀伤活性的影响。结果表明,EAC-RNA能显著抑制NK活性。经RNase处理后,其抑制活性消失,DNase或Pronase的处理不改变其抑制活性。     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Transcriptional activity in lysates of cultured mesenchymal cells from embryonic tooth germs

Mesenchymal cells isolated from the papilla of embryonic tooth germs of the mouse were cultured in a complex medium for five to six days. Liquid nitrogen lysates, prepared from these cells, incorporated nucleoside monophosphates into a cold acid-insoluble product. The product was sensitive to RNase and no product was formed if the lysate was pretreated with DNase. The reaction was sensitive to EDTA and, in its presence, optimum activity was obtained with 2 mM MgCl2. On sucrose gradients, the reaction product was distributed between two broad peaks; one centered about 18S and the other above 28S. The RNA polymerase inhibitor alpha-amanitin inhibited approximately 50% of the activity at a concentration of 10 microgram/ml.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Orally administered streptococcal preparation,OK-432 augments the antitumor immunity of patients with gastric or colorectal cancer

A streptococcal preparation, OK-432, was orally administered at a dose of 5 KE to patients with gastric or colorectal cancer for 7–14 days before their operations, and its immunomodulatory effects on peripheral blood lymphocytes (PBL), regional node lymphocytes (RNL) and tumor infiltrating lymphocytes (TIL) were assessed. The group treated with OK-432 included 8 gastric and 6 colorectal cancer patients, and the control group included 8 gastric and 8 colorectal cancer patients. The NK cell activity of PBL was significantly augmented by the oral administration of OK-432, and the proportions of Leu 7+ and Leu 11+ cells in PBL also increased. The responses of PBL and TIL to autologous tumor extracts in the presence of interleukin-2 were enhanced after the oral administration of OK-432. The proportion of OKT8+ cells in PBL increased after treatment with oral OK-432, whereas the proportion in RNL significantly decreased. These results indicate that oral OK-432 affects NK and T cells and may augment the antitumor immunity of patients with gastrointestinal cancer.     

Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

Summary Using leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI. Abbreviations used: I-RNA, immune RNA; LAI, leukocyte adherence inhibition; KLH, Keyhole limpet hemocyanin; PBS, phosphate buffered saline; RNase, ribonuclease; DNase, deoxyribonuclease     

Analysis of natural killer activity and natural killer cell subsets in patients with bladder cancer

Summary In order to analyze the state of the natural resistance system of bladder cancer patients in vivo, we measured natural killer (NK) activity and NK cell subsets of peripheral blood lymphocytes (PBL) from 46 patients with bladder cancer and 25 age- and sex-matched healthy volunteers. The mean NK activity in patients with lowstage bladder cancer was similar to that in the controls, while NK activity in patients with high-stage bladder cancer was significantly depressed. The mean proportions of Leu7⁺ cells in patients with both low-stage and highstage bladder cancer were significantly higher than that in the controls. The mean proportion of Leu11a⁺ cells in patients with low-stage bladder cancer was similar to that in the controls, while in patients with high-stage bladder cancer it was significantly higher. This study demonstrates the abnormal immunological state of bladder cancer patients; namely, abnormalities exist not only in NK activity but also in the proportions of circulating NK cell subsets.     

Influence in vitro on NK and K cell activities by cimetidine and indomethacin with and without simultaneous exposure to interferon

Summary The histamine-2 receptor antagonist cimetidine (10^–5 M) and the prostaglandin synthesis inhibitor indomethacin (10^–8 M) augmented natural killer cell activity in the majority of healthy controls and patients with advanced melanoma and in a lower frequency of patients with colorectal carcinoma. Antibody-dependent cellular cytotoxicity was increased in most melanoma patients but in a lower proportion of patients with colorectal cancer. Compared with the effect of interferon the augmentation of NK- and K-cell activities was small in most patients. Cimetidine was also demonstrated to bring about a further increase in the interferon-induced NK activation of peripheral blood mononuclear cells from a majority of healthy donors and patients with melanoma. Furthermore, cimetidine augmented the interferon-induced K-cell activation of peripheral blood mononuclear cells from most patients with melanoma and colorectal cancer.     

DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia

It is believed that damage to the membranes of brain cells of schizophrenia (SCZ) patients induces the formation of autoantigens and autoantibodies. Nevertheless, the importance of immunological changes leading to the loss of tolerance to self-antigens in the genesis of SCZ has not been established. The MALDI mass spectra of the IgG light chains of 20 healthy donors were relatively homogeneous and characterized by one peak with only one maximum. In contrast to the healthy donors, the MALDI mass spectra of IgG light chains corresponding to 20 SCZ patients demonstrated, similarly to 20 autoimmune systemic lupus erythematosus (SLE) patients, two maxima of a comparable intensity. In addition, the MALDI spectra of the IgG light chains of five SLE and four SCZ patients contained a small additional brightly pronounced peak with remarkably lower molecular mass compared with the main one. DNase autoantibodies (abzymes) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases without a significant disturbance of the immune status does not contain DNase abzymes. Here, we present the first analysis of anti-DNA antibodies and DNase abzymes in the sera of SCZ patients. Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of SCZ patients. The sera of approximately 30% of SCZ patients displayed a higher content of antibodies (compared with 37% of SLE) interacting with single- and double-stranded DNA compared with healthy donors. Antibodies with DNase activity were revealed in 80% of the patients. These data indicate that some SCZ patients may show signs of typical autoimmune processes to a certain extent.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

Synthesis of chloroplast ribonucleic acid in Chlamydomonas reinhardtii toluene-treated cells.

Chlamydomonas reinhardtii cells treated with toluene at 0 degrees C and 25 degrees C incorporate ribonucleoside triphosphates (NTPs) into chloroplast RNA at 25 degrees C and also at 35 degrees C. The incorporation requires all four NTPs and Mg2+, and is completely inhibited by DNase, RNase, actinomycin D (40 microgram/ml) and rifampicin (350 microgram/ml). However, the incorporation is almost totally insensitive to both alpha-amanitin and streptolydigin at 200 microgram/ml.     

Evidence for a nonoxidative mechanism of human natural killer (NK) cell cytotoxicity by using mononuclear effector cells from healthy donors and from patients with chronic granulomatous disease

In vitro natural killer (NK) activity expressed by blood mononuclear cells from patients with chronic granulomatous disease of childhood (CGD) was equivalent to that expressed by cells from normal, healthy volunteers. Because neutrophils and monocytes from these same donors exhibited extremely depressed oxidative functions, our data could be interpreted to show that a) NK cells derived from a unique and separate cellular lineage unaffected by the disease-related oxidative defect, or b) the in vitro cytolytic mechanism(s) of NK cells were not dependent on oxygen metabolites. These hypotheses were examined by using as NK effector cells large granular lymphocytes (LGL) from healthy donors whose monocytes and neutrophils had normal oxidative functions. Such functions were measured in the nitroblue tetrazolium dye reduction assay, which is a qualitative measurement of superoxide anion production; by reduction of ferric cytochrome c, a more specific and quantitative measurement of superoxide anion production; and in the luminol-enhanced chemiluminescence assay, an extremely sensitive measure of several reactive oxygen radicals, including superoxide anion, hydroxyl radical, and singlet oxygen. Whereas monocytes and neutrophils from healthy donors were readily stimulated with zymosan or phorbol myristate acetate (PMA) in each of these assays. LGL produced no detectable amounts of oxygen metabolites when co-incubated either with K562 erythroleukemia cells, PMA, E. coli endotoxin, or the calcium ionophore A23187. Thus, because NK cell activity is normal in CGD patients with major oxidative defects, and because no reactive oxygen metabolites could be detected in LGL that simultaneously exhibited potent NK activity, we conclude that in vitro NK activity by human mononuclear cells involves a lytic mechanism(s) independent of oxygen metabolites.