Positive correlation between degree of parasitemia, interferon titers, and natural killer cell activity in Plasmodium falciparum-infected children

Natural killer (NK) cell activity and interferon levels have been measured in the peripheral blood of children acutely ill with Plasmodium falciparum infection. The NK cell levels were found to be raised in the malaria-infected children, with a positive correlation between the degree of parasitemia and lytic activity. Comparatively high titers of antiviral activity was discovered in sera from the majority of P. falciparum-infected children, again positively correlating with the degree of parasitemia and NK levels. The characteristics of the antiviral factor indicated alpha-type interferon to be the dominating agent involved. Addition of exogenous interferon in vitro potentiated the NK levels of PBL from normal children while having no significant impact on cells from malaria-infected children.     

Pulmonary compartmentalization of interferon and natural killer cell activity

Studies were performed to determine if natural killer (NK) activity in the mononuclear cells harvested from infected lungs was dependent on local or systemic factors. Mice were inoculated by intratracheal (it), intraperitoneal (ip), or intravenous (iv) routes with (a LD50 dose of) influenza virus A PR/8/34. At various days postinoculation cells from lungs, spleens, and peripheral blood were assayed for NK activity, and lung wash, lung homogenates, and serum were assayed for interferon. After it inoculation there was three- to fourfold increase of NK activity in the lung with little or no increase in NK activity in spleens or peripheral blood. The local augmentation of NK activity in the lung correlated with an increase in interferon (IFN) titer in the lung wash and lung homogenate of PR8 inoculated mice. The virus failed to induce IFN or augment NK activity when it was inoculated systemically. The observed local augmentation of NK activity and local induction of interferon production following it inoculation suggests that the NK population in the lung is capable of responding to locally derived regulatory factors.     

Differential effects of recombinant human interferon-gamma and interleukin 2 on natural killer cell activity of peripheral blood in early human development

Ontogenic development and the lymphokine responsiveness of human NK cell activity against K562 target cells in peripheral blood lymphocytes were evaluated in fetuses, premature infants, and term neonates by using a 4-hr 51Cr-release assay. Basal NK activity and NK boosting by lymphokines were comparatively assayed after an 18-hr incubation with medium alone, recombinant human IFN-gamma (1000 U/ml), and recombinant human IL 2 (25 U/ml), respectively. Lymphocytes from 20-wk-old fetuses lacked NK cell activity even after the pretreatment with IFN-gamma. Low, but significant levels of NK activity and NK boosting by IFN-gamma were observed in premature infants after 27 wk of gestation, with a progressive intrauterine maturation of these activities. Both basal NK activity and NK boosting by IFN-gamma in term neonates were still lower than those of adult controls. The grade of NK boosting by IFN-gamma appeared to depend on the development of basal NK activity. Contrary to IFN-gamma, IL 2 could induce marked NK activity even in 20-wk-old fetuses who lacked both basal and IFN-gamma inducible NK activities. NK boosting by IL 2 was much more efficient than that by IFN-gamma at any period of human life. The facts that IL 2-induced NK boosting could occur without any appreciable production of IFN-gamma in neonatal lymphocytes, and that ample neutralizing doses of anti-IFN-gamma antibody hardly suppressed IL 2-mediated NK boosting even in adult lymphocytes, indicated that the effect of IL 2 on NK boosting might be independent of IFN-gamma production. On the basis of the ontogenic differences in the development of the lymphokine responsiveness of NK cell activity and on the different NK boosting mechanisms of these lymphokines it was suggested that so-called human "pre-NK cells" might be divided into IFN-gamma sensitive and IL 2-sensitive cells. Whether these cell populations belong to different cell lineages or different maturation stages of the same cell line, however, remains unsettled.     

Respiratory Syncytial Virus Fusion Protein Mediates Inhibition of Mitogen-Induced T-Cell Proliferation by Contact

Human respiratory syncytial virus (HRSV) and bovine respiratory syncytial virus (BRSV) are major pathogens in infants and calves, respectively. Experimental BRSV infection of calves and lambs is associated with lymphopenia and a reduction in responsiveness of peripheral blood lymphocytes (PBLs) to mitogens ex vivo. In this report, we show that in vitro mitogen-induced proliferation of PBLs is inhibited after contact with RSV-infected and UV-inactivated cells or with cells expressing RSV envelope proteins on the cell surface. The protein responsible was identified as the RSV fusion protein (F), as cells infected with a recombinant RSV expressing F as the single envelope protein or cells transfected with a plasmid encoding F were able to induce this effect. Thus, direct contact with RSV F is necessary and sufficient to inhibit proliferation of PBLs. Interestingly, F derived from HRSV was more efficient in inhibiting human PBL proliferation, while F from BRSV was more efficient in inhibiting bovine PBLs. Since various T-cell activation markers were upregulated after presenter cell contact, T lymphocytes are viable and may still be activated by mitogen. However, a significant fraction of PBLs were delayed or defective in G0/G1 to S-phase transit.     

Hyperthyroxinemic mice have reduced natural killer cell activity. Evidence for a defective trigger mechanism

Natural killer (NK) activity of peripheral blood lymphocytes from hyperthyroxinemic patients (Graves' disease or thyroxine (T4)-treated) is severely depressed. In order to study the relationship of thyroid hormone to NK activity, a model for hyperthyroxinemia was induced in mice by addition of T4 to the drinking water. Control mice were hypothyroid (fed propylthiouracil) or normal. Serum T4 levels were elevated (within 2 wk) in mice fed thyroid hormone. Six weeks after initiation of the diets, in vitro NK activity was undetectable in the peripheral blood, spleen, or lung mononuclear cell populations harvested from hyperthyroxinemic mice. Control mice had NK activity within the normal range. Spleen cells from mice fed thyroid hormone and control mice were tested for their ability to release lytic factors (natural killer cytotoxic factors). Lymphoid cells were incubated for 20 hr with unlabeled Yac-1 cells. Supernatants were tested for their capacity to lyse 51Cr-labeled Yac-1 cells in a 20-hr chromium release assay. Unlike controls, supernatants from hyperthyroxinemic spleen cells incubated with Yac-1 targets were unable to lyse 51Cr-Yac-1 cells. The NK cells from the mice fed T4 synthesized lytic factors because nonspecific stimuli, such as 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore A23187, induced release of lytic factors capable of lysing Yac-1 targets into the media. These data support the hypothesis that excess thyroid hormone interferes with the triggering mechanism used by NK targets to cause release of lytic molecules from NK cells.     

Pepsins in protein-energy malnutrition

This study reports on basal gastric pepsins in 40 normal, kwashiorkor and marasmic children admitted to the paediatric wards of the University of Benin Teaching Hospital, Nigeria. The activity of total pepsin was significantly depressed in the diseased states. The various fractions of pepsin separated on ion-exchange chromatography also showed dramatic reductions for both kwashiorkor and marasmus. This adaptive reduction of pepsin and its fractions was more drastic in marasmus than kwashiorkor. On disc-gel electrophoresis, four of the pepsin bands found in normal gastric aspirate were missing in children with the syndromes. Gastric electrolytes and acidity recorded for the diseased states were not conducive for maximal peptic activity. It is suggested that due to the reduced proteolytic capability of the malnourished infants, rehabilitation with intact protein should be cautious and gradual.     

Involvement of the 2'-5' A pathway in the augmentation of natural killer activity

Pretreatment of human large granular lymphocytes (LGL) or unseparated peripheral blood mononuclear cells with interferon (IFN) resulted in a significant augmentation of natural killer (NK) activity. This increase was paralleled by an increase in the 2'-5'A synthetase activity. In order to investigate the possibility that IFN might be inducing augmentation of NK cells via the 2'-5'A pathway, we tested the effects of nonphosphorylated core material [(A2'p)2A] and of the triphosphorylated form of the 2'-5'A [ppp(A2'p)2A]. The core material had no detectable effect on NK activity. In contrast, when experiments were performed with the triphosphorylated form of 2'-5'A, NK activity was stimulated. In order to achieve activation, permeabilization of LGL with calcium chloride was necessary and, under these conditions, a dose-dependent augmentation of NK activity was seen. However, the calcium treatment had considerable toxic effects on basal levels of NK activity. Collectively, these results suggest that IFN may be inducing augmentation of NK activity via the 2'-5'A pathway. Further studies will be necessary to determine the effects of IFN and/or 2'-5'A on subsequent activation steps in the process leading to cytotoxicity by NK cells.     

Nonspecific killer cells in premature and mature neonates and infants

K (killer) and natural killer (NK) cells were investigated in peripheral blood of 76 children, preterm small for date babies (n = 8), preterm babies (n = 15), fullterm small for date babies (n = 6) fullterm babies (n = 7) and infants up to 12 months age (n = 40). The K and NK cell activity of human leukocytes was analysed as compared with those cells of the K 562 cell line and murine cells covered by xenologous antibodies in Graffi erythroblast leukemia by means of the 51Cr release test. K cell activities were significantly lower in preterm small for date babies to infants with 1-12 months of age. In our results it is shown that NK capacity of preterm or term newborns and infants up to 6 months age does not differ significantly from each other. Children who are 6-12 months old will have significantly higher NK cell activities. It can be concluded that K cell activities are fully developed during pregnancy and NK cell activities later when the children are between 6 and 12 months of age.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

Augmentation of mouse natural killer cell activity by LS 2616, a new immunomodulator

The quinoline-3-carboxamide LS 2616 administered to mice in drinking water increased spontaneous cytotoxicity against YAC-1 cells in a dose-dependent manner. The enhancement of spontaneous cytotoxicity was found to be mediated by NK cells, as judged by their lack of adherence to nylon wool columns, relative resistance to treatment with antibodies to Thy-1.2 and complement, and almost total abrogation after depletion of asialo-GM1+ cells. Enhancement of NK activity was evident after 2 days of treatment, was maximal after 4 days, and remained elevated during the 14-day exposure period studied. NK activity returned to control levels 4 days after cessation of treatment. NK activity was significantly increased in spleen, peripheral blood, lymph nodes, and bone marrow of LS 2616-treated mice, while activity in peritoneal exudate cells and thymus remained low. LS 2616 was able to elevate NK activity in several mouse strains studied, including mice homozygous for the beige gene. Serum interferon levels were not increased during treatment with LS 2616. Combined injection of the interferon inducer Poly I:C and LS 2616 did not increase NK activity above that of animals injected with Poly I:C alone. However, Poly I:C, in contrast to LS 2616, increased NK activity in peritoneal exudate cells. Studies at the single cell level revealed that LS 2616 increased NK activity by increasing the number of lytically active cells via recruitment of new target-binding cells and not by increasing the lytic activity of pre-existing binders.     

Natural killer cell activity in tumor-draining lymph nodes: investigations in patients with malignant melanoma and head and neck cancer

Mononuclear cells isolated from peripheral blood (PB) of patients with malignant melanoma and head and neck cancer were tested for natural killer (NK) cell activity against K562 targets. Significantly lower levels of NK activity were observed in patients with malignant melanoma with metastatic disease in comparison to stage I and control population. In head and neck cancer NK activity was normal in tumor stage 1-2 and significantly impaired in stage 3-4. Mononuclear cells (MNC) isolated from primary tumor-draining lymph nodes (LNs) had significant NK activity in 12 of 25 (48%) of melanoma and 9 of 19 (47%) of head and neck lymph nodes at E:T 40:1. Reactivity was always significantly lower than in autochthonous Preincubation (+37 degrees C, 18 h) of lymph node mononuclear cells (LNMNC) resulted in a significant increase in cytotoxicity in 36% of melanoma and 32% of head and neck LNs. Simultaneous incubation with interferon (IFN)-alpha resulted in 24% of melanoma and 44% of head and neck LNs in a significant further augmentation of NK activity. LNMNC were furthermore found to react in 2-24% (mean 7.79 +/- 2.1) with the monoclonal antibody HNK-1, which identifies a certain proportion of NK cells. No correlation was found between percentage HNK-1+ cells and the expression of NK activity. From these studies it may be concluded that tumor draining LNs contain cells which are active against K562 targets expressing low but variable levels of cytotoxicity. NK cell activity can be increased by incubation of LNMNC at 37 degrees C with or without addition of IFN. These results suggest that modulation of NK activity in tumor draining LNs might be achieved by local application of biological response modifiers, which might finally lead to an increase of local tumor defense mechanisms.     

Aflatoxins and kwashiorkor: a study in Sudanese children.

Blood and urine samples from 252 Sudanese children were investigated for their aflatoxin content by high-performance liquid chromatography. The children comprised 44 with kwashiorkor, 32 with marasmic kwashiorkor, 70 with marasmus, and 106 age-matched, normally nourished controls. Aflatoxins were detected more often and at higher concentrations in sera from children with kwashiorkor than in the other malnourished and control groups. Aflatoxicol, a metabolite of aflatoxins B1 and B2, was detected in the sera of children with kwashiorkor and marasmic kwashiorkor but not in the controls and only once in a marasmic child. The difference between children with kwashiorkor or marasmic kwashiorkor and those in the control or marasmus groups was significant. Urinary aflatoxin was most often detected in children with kwashiorkor but their mean concentration was lower than in the other groups. Aflatoxicol was not detected in urine in any group. These findings suggest either that the children with kwashiorkor have a greater exposure to aflatoxins or that their ability to transport and excrete aflatoxins is impaired by the metabolic derangements associated with kwashiorkor. The presence of aflatoxicol in the sera of children with kwashiorkor but not in the others suggests a difference in metabolism between the two groups. Further studies are needed, and measurement of aflatoxins in the food eaten by these children is already underway.     

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.     

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.     

Effect of interleukin-2 on the monomeric IgG-induced inhibition of human natural killer cell activity

Monomeric IgG (mIgG) has been previously shown to inhibit human natural killer (NK) cell activity when effector cells were treated prior to the cytotoxic assay. In the present study the interaction between negative regulation by mIgG and positive regulation by interleukin-2 (IL-2) was examined. Although a dose-dependent boosting of NK activity was found upon incubation of nonadherent lymphocytes (NAL) with recombinant or natural IL-2 for 2 h at 37 degrees C, the NK effector cells remained responsive to down-regulation to mIgG. However, when NAL were treated with IL-2 under supraoptimal conditions (higher doses and longer periods of incubation than required for optimal boosting of NK activity) the subsequent addition of mIgG had a significantly reduced inhibitory effect. This partial resistance to suppression by inhibitory IgG was observed only when the second treatment was performed without washing the IL-2-pretreated effector cells. Moreover, addition of antihuman interferon gamma antibodies during the incubation of NAL with IL-2 almost abolished the loss of responsiveness of the IL-2-activated killer cells to mIgG-induced inhibition. These data provide additional evidence for the ability of interferon gamma to reverse or block the down-regulation of NK activity by mIgG.     

Cells with natural killer activity in the cerebrospinal fluid of normal mice and athymic nude mice with acute Sindbis virus encephalitis

Sindbis virus causes an acute, nonfatal inflammatory encephalitis in weanling BALB/c mice. Mononuclear inflammatory cells are present in the cerebrospinal fluid (CSF) as well as in the parenchyma of the brain. Both aspects of this inflammatory response were eliminated by treatment with cyclophosphamide. Athymic nude (nu/nu) mice developed no inflammation in the brain, but did develop a CSF pleocytosis that peaked on day 2 after infection. The time course of the appearance of cells in the CSF was earlier in nu/nu mice than their heterozygote (nu/+) littermates. The pleocytosis in nu/nu mice reached a peak on day 2, whereas in nu/+ mice the peak was on day 4, as it is in normal BALB/c mice. To determine whether some of the CSF cells in nu/nu mice may be natural killer (NK) cells, NK activity was measured in a 4-hr assay by using a YAC-1 target cell. NK cell activity in the spleen and peripheral blood was induced by infection with Sindbis virus in nu/nu mice with a similar time course to that of nu/+ mice (peak 1 day after infection). CSF from nu/nu mice had NK activity present 2 days after infection that was greater than that present in either the peripheral blood or spleen. BALB/c and nu/+ mice had insufficient cells present for assay at day 2, but BALB/c mice had NK activity present in the CSF 3 and 5 days after infection that exceeded that in the peripheral blood or spleen. Brain interferon was detectable on day 1 in nu/nu mice, but not until day 2 in nu/+ mice even though the amounts of brain virus were the same in the two groups at all time points. It is concluded that cells with NK activity contribute to the CSF pleocytosis induced by acute Sindbis virus encephalitis.     

Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.     

Nutritional regulation of serum osteocalcin: study in kwashiorkor

The serum levels of osteocalcin (bone gla protein) in two groups of Senegalese children, healthy controls and severely malnourished (kwashiorkor) children during nutritional rehabilitation, were measured. The serum osteocalcin of all kwashiorkor children was dramatically decreased on admission to hospital, but increased fourfold during rehabilitation. Serum osteocalcin was low in the control group. In both groups these low levels seemed to be independent of those of 1,25-dihydroxyvitamine D3 which were in the normal range. The results suggest that serum osteocalcin levels might be related to protein-energy status.     

Inhibition by cortisol of human natural killer (NK) cell activity

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10(-8) to 1 X 10(-5) M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-gamma). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 (PgE2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.     

Effect of interferon on the inhibition of malignant cell growth by human peripheral leukocytes. III. Effect of systemic administration

Human leukocyte interferon preparation (HuIFN-alpha LE) was given to the patients with cancer or with chronic hepatitis. Spontaneous tumor cell growth inhibition by human peripheral lymphocytes (STGI) and NK activity were enhanced by the systemic administration of HuIFN-alpha LE, although there were differences in the kinetics between the two activities after one time administration or by the repeated administration. This suggests that IFN acts indirectly on the tumor cells by the medium of normal lymphocytes or NK cells, and that tumor cell growth inhibition is different from NK activity.