Pretreatment Of Leukocytes Research Articles

The inhibition of TNF‐α‐induced leucocyte apoptosis by melatonin involves membrane receptor MT1/MT2 interaction

The pro-apoptotic signalling cascades induced by tumour necrosis factor-alpha (TNF-α) have been intensively studied in multiple cellular systems. So far, it is known that TNF-α can simultaneously activate survival and apoptotic cell death responses. The balance between these signals determines the ultimate response of the cell to TNF-α. Moreover, emerging evidence suggests that melatonin may be involved in the protection of different cell types against apoptosis. Thus, the objective of this study was to evaluate the effect of melatonin on TNF-α-induced apoptosis in human leucocytes. Cells were treated with TNF-α alone or in the presence of cycloheximide (CHX), which promotes caspase-8 activation by eliminating the endogenous caspase-8 inhibitor, c-FLIP. Treatment with TNF-α/CHX led to apoptotic cell death, as ascertained by annexin V/propidium iodide (PI) staining. Likewise, in the presence of CHX, TNF-α stimulation produced cFLIP down-regulation and subsequent caspase-8 activation, thus directly triggering caspase-3 activation and causing Bid truncation and subsequent caspase-9 activation. Conversely, pre-incubation of cells with melatonin inhibited TNF-α-/CHX-evoked leucocyte apoptosis. Similarly, pretreatment of leucocytes with melatonin increased cFLIP protein levels, thereby preventing TNF-α-/CHX-mediated caspase processing. Blockade of melatonin membrane receptor MT1/MT2 or extracellular signal-regulated kinase (ERK) pathway with luzindole or PD98059, respectively, abolished the inhibitory effects of melatonin on leucocyte apoptosis evoked by TNF-α/CHX. In conclusion, the model proposed by these findings is that the MT1/MT2 receptors, which are under the positive control of melatonin, trigger an ERK-dependent signalling cascade that interferes with the anti-apoptotic protein cFLIP modulating the cell life/death balance of human leucocytes.

Histoplasma capsulatum Cell Wall β-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B4 Generation and Role in HIV-1 Infection

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B(4) plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B(4) and PGE(2). Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly beta-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that beta-glucan plays a signaling role in LB formation. In agreement with this hypothesis, beta-glucan-elicited LB formation was inhibited in leukocytes from 5-LO(-/-), CD18(low) and TLR2(-/-) mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after beta-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection.

Investigation of genotoxic effects of inhalative occupational exposure to vanadium: results of a multiple endpoint study

Animal experiments showed that inhalative exposure to vanadate causes lung cancer. To assess DNA damage in exposed workers (EW, n = 52) of a metal factory, we monitored DNA migration and formation of micronuclei in blood cells of EW, who are exposed to vanadium pentoxide via inhalation and in a matched control group (CG, n = 54). Median level of vanadium serum concentrations of EW (2.23 μg/l) was found to be 7-fold increased compared to CG (0.31 μg/l). In the standard single cell gel electrophoresis (SCGE) assay with leucocytes, no differences were detected between EW and CG, but increased levels of oxidised DNA bases (detected with FPG and ENDO III) were found (p < 0.05). Pretreatment of leukocytes with bleomycin resulted in a higher extent of DNA migration (27%, p < 0.001) and reduced capacity of DNA repair (by 42%, p < 0.001) in EW. Furthermore, the cytochalasin blocked micronucleus (CBMN) assay was carried out with peripheral lymphocytes (n = 24 per group). EW showed a 2.5-fold higher MN frequency as CG and a 7fold increase of nucleoplasmic bridges (which reflect formation of dicentric chromosomes) was seen. Nuclear buds (attributable to gene amplification events) were 3fold higher in EW (p < 0.001 for all events). Apoptosis rates did not differ significantly between the two groups, whereas necrosis rates doubled in EW (p < 0.001). Taken together, our results show increased genetic damage in individuals which inhale vanadium dust, possibly indicating increased risk for cancer.

Influence of platelet-activating factor, its cell analogs, and antagonist on the production of superoxide radicals by blood leukocytes of healthy and hypercholesterolemic individuals.

The influence of the phospholipid platelet-activating factor (PAF), its cell analogs, and lipid PAF antagonist on the production of superoxide radicals by leukocytes isolated from the blood of healthy and hypercholesterolemia IIA individuals was studied. It was found that endogenous superoxide production level in the leukocytes of hypercholesterolemic individuals more than 4-5 times higher than in the leukocytes of healthy individuals. Exogenous PAF stimulates the superoxide production in the leukocytes of healthy individuals but significantly inhibits the superoxide production in the leukocytes of hypercholesterolemic individuals. The compounds 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) and 1-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) only slightly inhibited the endogenous superoxide production in the leukocytes of hypercholesterolemia individuals. However, pretreatment of leukocytes by 1-alkenyl-PAF or PAF-antagonist (1-O-alk-1;-enyl-2-(2;-acetoxybenzoyl)-sn-glycero-3-phosphocholine) results in a 50% inhibition of the PAF-induced superoxide production by leukocytes of healthy individuals. This PAF-antagonist alone or in combination with PAF induces a substantial (65-70%) inhibition of superoxide production in the leukocytes of hypercholesterolemic individuals. It is concluded that superoxide production by leukocytes of healthy individuals and especially by leukocytes of hypercholesterolemic individuals is process that depends on PAF or PAF-like lipids.

Reduced neointima hyperplasia of vein bypass grafts in intercellular adhesion molecule-1-deficient mice.

Recently, we established a new mouse model of vein graft arteriosclerosis through the grafting of vena cava to carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease. With this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1-deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 -/- mice was reduced 30% to 50% compared with that of wild-type control animals. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wild-type, but not ICAM-1 -/-, mice. MAC-1 (CD11b/18)-positive cells that adhered to the surface of vein grafts in ICAM-1 -/- mice were significantly less as identified with en face immunofluorescence, and these positive cells were more abundant in the intimal lesions of vein grafts in wild-type mice. Furthermore, aortic SMCs cultivated from wild-type mice exhibited high ICAM-1 expression in response to tumor necrosis factor-alpha. When tumor necrosis factor-alpha-stimulated SMCs were incubated with mouse spleen leukocytes, the number of cells that adhered to ICAM-1 -/- SMCs was significantly lower than the number that adhered to ICAM-1 +/+ SMCs, which was markedly blocked through pretreatment of leukocytes with the anti-MAC-1 antibody. Taken together, our findings demonstrate that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention for vein graft disease in patients undergoing bypass surgery.

Cyclosporine enhances leukocyte adhesion to vascular endothelium under physiologic flow conditions

We investigated the effect of cyclosporine (CyA) on leukocyte adhesion to endothelium under flow conditions. Confluent human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with CyA (1, 5, and 10 μmol/L) and then exposed to a total human leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dynes/cm 2). Human umbilical vein endothelial cells stimulated with interleukin-1β (20 U/mL) were used as a positive control. Adherent cells were measured by digital image analysis. Results showed that CyA dose-dependently increased the number of leukocytes adhering to HUVECs compared with control cells. Leukocyte adhesion markedly increased on HUVECs incubated with interleukin-1β, one of the most potent inducers of endothelial cell adhesiveness. Exposure of endothelial cells to CyA did not affect the number of rolling leukocytes, which was similar to control values. To examine the role of adhesion molecules in CyA-induced leukocyte adhesion, HUVECs were incubated with monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin before adhesion assay. Functional blocking of ICAM-1, VCAM-1, and E-selectin on endothelial cells significantly inhibited CyA (10 μmol/L)-induced leukocyte adhesion. Confocal fluorescence microscopy studies showed that CyA induced an increase in the endothelial surface expression of ICAM-1, VCAM-1, and E-selectin. Pretreatment of leukocytes with the platelet activating factor receptor antagonist L659,989 significantly reduced the number of leukocytes adhering to CyA-treated HUVECs. We suggest that CyA enhances leukocyte adhesion to endothelium by upregulating adhesive proteins on endothelial surface membrane. Blocking leukocyte receptor for platelet-activating factor partially prevents adhesion, suggesting a role for endothelial cell-associated platelet-activating factor in the interaction between leukocytes and CyA-treated endothelium.

Differences between human eosinophils and neutrophils in the function and expression of sialic acid-containing counterligands for E-selectin.

Both neutrophils and eosinophils have been shown to bind to the inducible endothelial cell adhesion molecule E-selectin. For neutrophils, one of the reported ligands for E-selectin is the sialylated Lewis X Ag (sLe(x)). To analyze the counterligands on eosinophils for E-selectin, adhesion assays were performed in which purified leukocytes were allowed to adhere to a soluble recombinant form of the molecule immobilized on plastic plates. Eosinophils, like neutrophils, bound to immobilized E-selectin, but significantly more neutrophils than eosinophils adhered in this assay. Consistent with the greater ability of neutrophils to bind E-selectin was the observation by flow cytometry that neutrophils expressed significant levels of sLe(x) and a sialylated dimeric form of the Le(x) Ag (sialyl-dimeric Le(x), or sialyl-stage-specific embryonic Ag-1, recognized by mAb FH6), whereas the expression of these epitopes on eosinophils was extremely low or undetectable. Expression was similar on eosinophils from allergic and nonallergic donors, and was not altered on eosinophils after induction of L-selectin shedding in vitro by treatment with platelet-activating factor. For both eosinophils and neutrophils, treatment with sialidase was associated with the complete elimination of sLe(x) and sialyl-dimeric Le(x) surface expression, and abolished leukocyte adhesion to E-selectin. Another glycosidase, endo-beta-galactosidase, which specifically cleaves the beta 1-4 galactose linkage to N-acetyl-glucosamine when it exists in an extended chain form such as that found in sialyl-dimeric Le(x), significantly inhibited eosinophil and neutrophil adhesion and expression of sialyl-dimeric Le(x). Such treatment also reduced sLe(x) expression on eosinophils, while having little effect on total neutrophil sLe(x) expression. For both eosinophils and neutrophils the sialylated ligand did not appear to be a glycoprotein because pretreatment of leukocytes with several proteases had no effect on adhesion to E-selectin or on expression of sLe(x) and sialyl-dimeric Le(x). These data suggest that eosinophils, like neutrophils, use sialylated, protease-resistant structures to bind to E-selectin, although the eosinophil expresses much lower levels of these structures on its surface. A major proportion of the sLe(x)-containing E-selectin ligand on the surface of eosinophils appears to be in the form of sialyl-dimeric Le(x), whereas this represents a minor proportion on the surface of neutrophils. Based on results using endo-beta-galactosidase, it appears that these cells may rely disproportionately upon the cell surface sialyl-dimeric Le(x) to bind to E-selectin.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased activity of circulating polymorphonuclear leukocytes in acute myocardial infarction

Acute myocardial infarction results in an increased sensitivity of circulating polymorphonuclear leukocytes to ex vivo aggregation. This increase was prevented by pretreatment of leukocytes with BW755, but not with aspirin, suggesting that the activation of blood leukocytes in infarction is due to a stimulation of cellular lipoxygenase.

A novel method for labelling of leukocytes with technetium-99m and its comparative evaluation for abscess scintigraphy

A new method, based on the pretreatment of leukocytes with glucoheptonate prior to treating with reduced 99mTc, has been developed for the preparation of 99mTc labelled leukocytes. The leukocytes labelled with a 99mTc concentration (5.59%/g tissue) similar to that of 111In-leukocytes (6.27%/g tissue), in the experimental abscess were in rat thigh. Concentration of 99mTc-leukocytes in blood at 24 h was only about 35% as compared to that of 111In-leukocytes. Biodistribution in the rat organs was similar in both cases, except in the liver where 99mTc-leukocytes exhibited about 4-fold greater concentration. Images of experimental abscess in rat by using 99mTc-leukocytes were comparable to those obtained with 111In-leukocytes.

Human interferons alpha and beta have more potent priming activities than interferon gamma.

The priming activities of human IFN-alpha, IFN-beta and IFN-gamma were compared on the same antiviral basis using human buffy coat leukocytes stimulated with Sendai virus or concanavalin A (Con A) to produce IFN-alpha or IFN-gamma, respectively. Pretreatment of leukocytes with any type of IFN enhanced their IFN-alpha and IFN-gamma production, but IFN-alpha and IFN-beta had more potent priming activities than IFN-gamma. IFN-alpha and IFN-gamma did not potentiate the priming activity of each other in either the IFN-alpha or the IFN-gamma producing system. Pretreatment of leukocytes with relatively high doses of IFN-alpha or IFN-beta (1000 to 3000 IU/ml) resulted in a 40- to 50-fold increase in the IFN-gamma production of Con A-stimulated leukocytes. This observation will be of use in producing IFN-gamma with a high titre.

Basophil histamine release in atopic dermatitis and its relationship to disordered cyclic nucleotide metabolism.

Maximal histamine release (HR) from leucocytes, in response to Concanavalin A (Con A) was significantly higher in a group of 16 adults with moderate to severe atopic dermatitis (AD) when compared to 13 non-atopic adults. In a further 4 adults with AD, HR was similar to that in the normals, suggesting the existence of 'high releaser' and 'low releaser' subsets within the AD group. Leucocyte cyclic AMP phosphodiesterase (PDE) activity was significantly higher in the 'high releaser' group compared to the 'low releaser' and normal groups. High and low HR responses showed strong correlations with high and low PDE. Pre-treatment of leucocytes from 'high releasers' with the experimental PDE PDE inhibitor RO-20-1724 reduced the HR to normal levels. These findings suggest that increased histamine 'releasability' in AD is related to abnormalities in cyclic nucleotide regulation. No significant HR could be demonstrated in response to a range of concentrations of methacholine in 'high releaser' atopics and normals. Methacholine also did not affect HR in response to maximal Con A stimulation in 'high releaser' atopics. Basophil percentages within the leucocyte preparation and the histamine content per basophil, were not significantly different between the atopics and normals. Con A-stimulated histamine release did not correlate significantly with serum IgE levels.

Stimulation of DNA Synthesis in Cultured Mouse Epidermal Cells by Human Peripheral Leukocytes

Primary epidermal cells derived from the skin of newborn mice can be arrested in G1(G0) of the cell cycle when kept in serum-free medium with 0.06 mM Ca++ for 4 days. These cells can be restimulated to grow by the addition of Chelex 100-treated inactivated fetal calf serum, as shown by liquid scintillation counting and autoradiography after [3H]-thymidine incorporation and by counting mitotic figures. This system may be useful in studying growth-promoting and -inhibiting substances in vitro, and it is shown here that leukocytes from peripheral human blood are able to stimulate the DNA synthesis of epidermal cells although the effect is smaller than that observed with serum. The stimulation was not inhibited by pretreatment of leukocytes with mitomycin C, excluding the possibility that the increased incorporation of radioactivity in mixed epidermal-leukocyte cultures is due to increased DNA synthesis in leukocytes stimulated by epidermal cells. Therefore, this system may also be helpful in the investigation of the role of leukocytes in the hyperproliferation of the epidermis.

Effect of Escherichia coli alpha-hemolysin on human peripheral leukocyte function in vitro.

To gain further evidence for the role of the Escherichia coli alpha-hemolysin in pathogenesis, its in vitro effects on human peripheral leukocyte function were studied. Leukocytes exposed to low doses of alpha-hemolysin responded with a marked chemiluminescence response, indicating activation of oxidative metabolism. This response was time and dose dependent. Pretreatment of leukocytes with doses of alpha-hemolysin at which nearly 80% of the cells survived decreased the ability of the cells to phagocytize bacteria and particles and to undergo chemotaxis. Premature activation of leukocytes and inhibition of phagocytosis and chemotaxis by alpha-hemolysin, if they occur in vivo, would greatly enhance the survival of an invading E. coli strain.

Involvement of human membrane-associated complement components in the rosette formation between Marmoset red blood cells and human leukocytes

The majority of human monocytes, a subpopulation of B lymphocytes, and all B lymphoblastoid cell lines (B-LCL) tested but one form rosettes with Marmoset red blood cells (MaRBC). None of the human peripheral T cells, T-LCL, and B chronic lymphoid leukemia cells (B-CLL) used bind to MaRBC. The binding could not be correlated with any membrane markers or antigens present on cultured cells or peripheral blood leukocytes (PBL). Blocking of the rosette formation by preincubation of MaRBC with purified human complement (C) components and cobra venom or by pretreatment of leukocytes and cultured cells with antisera to human C components suggested that membrane-associated C components present on PBL or B-LCL are involved in the binding to MaRBC.

Mannose-sensitive interaction of Escherichia coli with human peripheral leukocytes in vitro.

The ability of Escherichia coli which possess or lack mannose-sensitive adherence factors (adhesins) to associate with human peripheral leukocytes in vitro in the absence of serum was studied. E. coli 19+, which have mannose-sensitive adhesins, were derived from E. coli strain 19 by culturing in static Trypticase soy broth at 37 degrees C. E. coli 19-, which lack mannose-sensitive adhesins, were derived from E. coli 19 by culturing in agitated Trypticase soy broth at 30 degrees C. E. coli 19+ attached to leukocytes and stimulated the release of lysozyme but not beta-glucuronidase or lactate dehydrogenase. In contrast, E. coli 19- showed poor attachment to the leukocytes and failed to stimulate lysosomal enzyme release. During a 60-min incubation with the leukocytes, the number of viable 19+ organisms decreased, whereas the number of viable 19- remained constant. Purified type 1 pili from E. coli 19+ agglutinated the leukocytes but did not stimulate lysosomal enzyme release. Pretreatment of leukocytes with type 1 pili failed to prevent the adherence of E. coli 19+. The association of 19+ with leukocytes and subsequent release of lysozyme could be blocked by alpha-methyl-D-mannoside but not by equivalent concentrations of dextrose and sucrose. These results show that mannose-sensitive adhesins on E. coli mediate association of the organisms with leukocytes in the absence of serum components. The identity of the adhesins involved in leukocyte association has yet to be determined.

The detection of membrane-associated complement components (C 3 and C 4) on circulating human normal and leukemic leukocytes and on cultured cells with monkey erythrocytes.

Monkey erythrocyte (Mk) rosette formation is described as an exquisitely sensitive method for the detection of complement (C) components on the membrane of human leukocytes. Blocking of the immune adherence receptor on Mk blocked subsequent rosette formation as did pretreatment of leukocytes with antiserum to the C components C 3 and C 4. In vitro C deposition by immune complex formation with normal human lymphocytes enhanced Mk rosette formation, and this could be inhibited with antiserum to C 3. The use of Mk rosette formation revealed that cells from a wide variety of human lymphoid and myeloid leukemias carry membrane-bound C. It was also shown that several lymphoblastoid cell lines, including a T cell line, probably synthesize both C 3 and C 4. Mk rosette formation is not dependent on metabolic activity of the rosetting leukocyte, and it is suggested that this technique will be of value in detecting C deposition in a variety of situations.

Relationship of pregnancy-associated alpha2-glycoprotein (alpha2-PAG) to peripheral blood leucocytes.

The high molecular weight plasma glycoprotein pregnancy-associated alpha2-glycoprotein (alpha2-PAG) can be demonstrated on the surface of blood leucocytes and is synthesized by oestrogen-stimulated cells in culture. By the use of an indirect immunofluorescence technique, pretreatment of leucocytes with anti-alpha2-PAG IgG revealed alpha2-PAG on C3 rosette-forming cells and monocytes but not on E rosettes. Lymphocytes treated with anti-alpha2-PAG showed markedly diminished ability to form Fc rosettes. A percentage ranging from 15% to 20% of leucocytes from normal adults of both sexes, pregnant and oestrogen-treated females were positive for alpha2-PAG but no obvious correlatin was observed between the incidence of alpha2-PAG bearing cells and plasma levels of the protein.

Suppression of graft versus host reactions in chickens by pretreatment of leucocytes with anti-light chain sera

Graft versus host reactions were studied in chickens by the use of the chorioallantoic (CAM) pock forming method. Adult leucocytes inducing the lesion, were first pretreated in vitro with either anti-thymocyte, anti-bursal, or anti-immunoglobulin sera. Specific anti-thymocyte sera inhibited the reaction confirming this to be a T cell mediated reaction. One anti-globulin serum specific for avian light chains also markedly suppressed the reaction. That this was due to the specific anti-light chain antibody was shown by the removal of inhibitory activity with purified light chains, but not with washed thymocytes. These studies further demonstrate the presence of a type of immunoglobulin molecule on T cells.

Cytotoxic effects of leukocyres triggered by complement bound to target cells.

Chromium-51-labeled chicken erythrocytes (E), treated with rabbit anti-Forssman antibody (A) and the first four (C1-4) or the first seven (C1-7) components of human complement (C), released isotope upon exposure to human leukocytes. Isotope release from EACJ-7 cells proceeded more rapidly and was more extensive than that from EACI-3 cells. Lysis of these cells was suppressed by pretreatment of leukocytes with antimycinA. Monocyte-enriched leukocyte preparations affected both types of target cell-complement intermediates, whereas purified lymphocytes lysed EACI-7 cells but not EACI-3 cells.

Interférons humains induits in vitro par des polynucléotides synthétiques

Human interferons induced in vitro by synthetic polynucleotides Optimal conditions of in vitro production of human interferon were studied, employing polyinosinic acid and polycytidylic acid complexes as inducers. Two cellular sources were utilized: leukocytes and the amniotic membrane. Greater amounts of interferon were obtained from leukocytes. In this system as little as 0.01 μg/ml of poly I + poly C was sufficient for the induction of detectable amounts of interferon in the extracellular medium. The production of interferon increased more than 100-fold when cells were pretreated with DEAE-dextran at a concentration of 10 μg/ml. In the presence of DEAE-dextran (10 μg/ml of the complex in leukocyte suspensions 10 7 cells/ml) titres of interferon between 1024 and 2048 were obtained. Under the same conditions, interferon titering 64 was recovered from the amniotic membrane. Physicochemical and biological properties of the polynucleotide-induced interferons were determined and found to be the same as those for viral-induced interferons. The molecular weight of polynucleotide-induced leukocyte interferon was 25000 (± 10 %). The same values were found using para influenza 1 (Sendaï) virus as inducer. Pre-treatment of leukocytes with actinomycin D (1 μg/ml, 1 h) reduced the production of interferon by 75 %. Biological activity of interferon in amniotic cells was inhibited 90 % by pretreatment with actinomycin D (0.1 μg/ml). The mixing curve of poly I + poly C was studied correlating the hypochromic effect and interferon production. The largest amounts of interferon and maximal hypocromic effect were found when poly I and poly C were mixed in equimolecular proportions. These results agree with the hypothesis that the double-stranded configuration of the RNA molecule is essential for the induction of interferon.