Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.

Effect of interleukin (IL)-12 and IL-15 on activated natural killer (ANK) and antibody-dependent cellular cytotoxicity (ADCC) in HIV infection.

The ability of IL-12 and IL-15 to enhance natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of mononuclear cells (MNCs) from HIV+ children and their mothers was investigated. MNCs from HIV+ patients were deficient in NK and ADCC activity compared to control MNCs against several target cells. Overnight incubation with IL-15 or IL-12 augmented NK activity of MNCs from both patients and controls, and the combination of IL-12 and IL-15 resulted in the greatest enhancement. ADCC in HIV+ patients against gp120-coated CEM.NKR cells or chicken erythrocytes could also be enhanced by IL-2 or IL-15 in overnight cultures. Culturing MNCs with either IL-2 or IL-15 for 1 week increased the NK activity in patients to levels of controls treated with these cytokines. However, the response to the combination of IL-12 and IL-15 was less than that to IL-15 alone in 1-week cultures. Culturing MNCs with IL-2 and IL-15 for 1 week also increased the percentage of CD16+/CD56+ cells in both patients and controls. Thus, IL-15 can restore the deficient NK activity in patients and may be a candidate for immunomodulative therapy in HIV+ patients.

Interleukin (IL)-15 enhances antibody-dependent cellular cytotoxicity and natural killer activity in neonatal cells.

Interleukin (IL)-15 is a novel cytokine that is very similar to IL-2 in receptor specificity and biological activities. We compared the ability of IL-15 and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNC) in both natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) assays. Incubation with IL-15 (10 ng/ml) or IL-12 (1 ng/ml) for 18 h enhanced the NK activity (using K562 target cells) of both cord and adult MNC, increasing cord cell cytotoxicity threefold. Similar enhancement was seen in ADCC assays using erythrocyte targets and NK-resistant CEM cells coated with HIV gp-120 antigen. Incubation of cord cells with IL-15 or IL-12 for 1 week increased both NK and ADCC, although the combination produced less of an effect than either cytokine alone. IL-15 also increased the percentage of CD16+/CD56+ cells after 1 week incubation. This enhancement of NK and ADCC activities and the number of NK cells by IL-15 suggests it may be clinically useful in treating immunodeficient patients.

Enhancement of antibody-dependent cellular cytotoxicity of neonatal cells by interleukin-2 (IL-2) and IL-12.

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.

Antibody-directed natural cytotoxicity results in enhanced killing of HIV gp120-coated CEMNKR cells.

Cellular cytotoxicity may be an important defense in the control of HIV progression. In the present study antibodies were attached to peripheral blood mononuclear cells (PBMC) by exposing them to polyethylene glycol and phthalate oil in the presence of HIV human hyperimmune IVIG (HIVIG). The attachment procedure is known as "franking" and the resultant cytotoxicity is termed "antibody-directed." The majority of the cells that are franked with attached HIVIG are CD16+ (Fc gamma RIII), placing them in the natural killer cell population. Franking increased the cytotoxicity of PBMC from both healthy controls and HIV-seropositive patients approximately fourfold compared to conventional antibody-dependent cellular cytotoxicity using CEM cells coated with HIV gp120 antigen as targets. Use of anti-HIV monoclonal antibodies for franking was less efficient than polyclonal HIVIG. The HIVIG-franked PBMC suppressed p24 production of in vitro HIV IIIb-infected human PBMC. The ability of HIVIG to enhance and direct cytotoxicity to HIV targets may suggest a new therapeutic approach to HIV control.

Human immunodeficiency virus (HIV) type-1 GP120-specific cell-mediated cytotoxicity (CMC) and natural killer (NK) activity in HIV-infected (HIV+) subjects: enhancement with interleukin-2(IL-2), IL-12, and IL-15.

Cell-mediated cytotoxicity (CMC), as mediated by cytophilic antibody to human immunodeficiency virus (HIV) antigens, may be an important defense in HIV-infected (HIV+) patients in response to the virus. In this study the ability of interleukin (IL)-2, IL-12, and IL-15 to enhance natural killer (NK) and gp120-specific CMC of mononuclear cells (MNCs) from HIV+ children and adults was examined. NK activity against K562 cells was deficient in HIV+ patients compared to controls and could be enhanced by IL-2, IL-12, or IL-15, with the combinations of IL-2 + IL-12 and IL-12 + IL-15 producing more cytotoxicity than individual cytokines. Gp120-specific CMC was significantly higher in patients than in controls. It could be increased by IL-2, IL-12, and IL-15 and further by combining IL-2 and IL-12. When an exogenous source of antibody in the form of hyperimmune HIV-specific immunoglobulin (HIVIG) was present, the response of control MNCs was much higher than that of patients, although gp120-specific cytotoxicity of patients' MNCs was significantly enhanced (two- to threefold) by the addition of HIVIG. This increment in cytotoxicity due to HIVIG, however, could not be further augmented by cytokines in controls or patients. Our findings suggest multiple cytokine administration to boost NK cell function, together with passive immunotherapy, might offer a new therapeutic approach to benefit HIV+ patients.

Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight.

The effects of immediate versus delayed cell separation, storage temperature, presence of serum, and type of anticoagulation on the natural killer (NK) cytotoxicity of human mononuclear cells were assessed. The NK cytotoxicity of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMC) was tested in a 3-h chromium-51 release assay with K562 cells at various effector/target cell ratios. The NK activities of PBMC from blood anticoagulated with either heparin or EDTA and then immediately separated and assayed were not different (42.9 +/- 2.5% for heparin and 40.3 +/- 4.6% for EDTA). When these separated cells were cultured in medium with 10% fetal calf serum and stored at 4,25, or 37 degrees C for 18 h before the assay, there was a significant increase in cytotoxicity. PBMC from blood stored in heparin or EDTA for 18 h before separation had reduced NK cytotoxicity, particularly if they were kept at 37 degrees C. When separated PBMC were cultured in medium with 10% human AB serum, however, samples held at 25 and 37 degrees C decreased in cytotoxicity but samples held at 4 degrees C maintained the cytotoxicity demonstrated at the baseline level with fresh cells. We recommend that heparinized blood be used for NK assays and that the PBMC be isolated immediately and held overnight at 4 degrees C in medium with 10% AB serum if the assay must be delayed. The NK cytotoxicity under these storage conditions most closely matches the results obtained when the PBMC are isolated and tested on the same day. IF PBMC isolation must also be postponed, it is best to store the blood in heparinized tubes at 25 degrees C to prevent loss of cytotoxic function.

N-acetylcysteine enhances antibody-dependent cellular cytotoxicity in neutrophils and mononuclear cells from healthy adults and human immunodeficiency virus-infected patients.

Patients with AIDS have decreased levels of the intracellular antioxidant, glutathione, in their circulating lymphocytes and plasma. N-acetylcysteine (NAC) increases intracellular stores of glutathione and has direct antioxidant properties. In this study, the effects of glutathione and NAC on the cytotoxicity of neutrophils and mononuclear cells were tested using cells from healthy controls and human immunodeficiency virus (HIV)-infected patients. NAC (1 and 5 mM) enhanced the antibody-dependent cellular cytotoxicity (ADCC) of neutrophils from healthy adult controls and HIV-infected adults and children. The antineoplastic drug, 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), which depletes intracellular glutathione, inhibited the ADCC of neutrophils; the addition of NAC partially reversed this inhibition. Similar effects of BCNU and NAC were seen when the cytotoxicity of mononuclear cells was tested using CEM tumor cells bearing the HIV gp120 antigen as targets. Thus, NAC enhances various forms of cytotoxicity and may be beneficial to AIDS patients whose defects in leukocyte cytotoxicity may be due to glutathione depletion.

Antiviral properties of neonatal and adult human neutrophils.

The antiviral properties of neonatal and adult human neutrophils were investigated by their ability to inhibit the formation of herpes simplex virus (HSV) plaques using an extrinsic viral resistance (EVR) assay. The EVR assay was performed by incubating neutrophils or mononuclear cells (MNC) with HSV-infected Vero or CEM tumor cells for 48 h. The cells were then frozen and viable HSV was quantitated by the ability of the lysate to form viral plaques. Activation of neutrophils from normal adults with phorbol myristate acetate increased their ability to inhibit HSV plaque formation more than 10-fold. The EVR response of neutrophils from newborn infants was much lower, and no significant inhibition occurred using activated neutrophils from patients with chronic granulomatous disease. The presence of rabbit anti-HSV antiserum slightly increased the EVR response of neutrophils but produced a greater increase in the response of the MNC in both adults and newborns. However, the combination of antiserum plus cytokines (granulocyte-macrophage colony-stimulating factor, IL-2, and interferon-gamma) greatly augmented the neutrophil EVR response to the level of the MNC response. Thus, neutrophils are capable of exerting a strong antiviral response comparable to that of MNC that may be important as a second line of defense in the immunocompromised patient.

Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines.

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.

Peripheral immunoglobulin secreting cells in immunodeficiencies; effects of intravenous immune globulin.

Peripheral blood B cells secreting IgG, IgA, IgM, and IgE were quantitated in normal adults (n = 12), newborns (n = 8), patients with antibody deficiency (n = 5), and patients with elevated IgE (four patients) using a reverse enzyme-linked immunospot (RELISPOT) assay. This technique measures immunoglobulin secreted by B cells by capture on an antibody-coated plate, and identified as a plaque on a nitrocellulose-membrane plate. Hypogammaglobulinemic patients and newborns (cord blood) showed no detectable IgG, IgA, or IgE secreting cells. Several cord blood and hypogammaglobulinemic patients, however, showed normal adult numbers of IgM secreting cells. One IgA-deficient patient showed increased numbers of IgA secreting cells, but a second IgA-deficient patient showed normal numbers of IgA secreting cells. Three patients with the hyper-IgE syndrome and a patient with severe eczema had very high numbers of IgE secreting cells. The effects of intravenous immunoglobulin on this system in vivo and in vitro were also examined. High dose intravenous immunoglobulin therapy did not decrease the immunoglobulin secreting cells in two neurologic patients given high dose IVIG. In vitro exposure of normal B cells to either IVIG or cycloheximide (a protein synthesis inhibitor) decreased the number of IgA and IgM secreting B cells. Cycloheximide also decreased the number of IgG secreting B cells in vitro. IgG spots, however, were present when cycloheximide-treated cells were incubated with a high concentration of IVIG. Since IVIG may bind directly to cells, its effect on in vitro B-cell IgG synthesis could not be determined.

Decreased superoxide anion and hydrogen peroxide production by neutrophils and monocytes in human immunodeficiency virus-infected children and adults.

The higher susceptibility to serious bacterial infections of patients, particularly children, infected with the human immunodeficiency virus (HIV) may be due in part to defective function of their phagocytic cells. We examined the ability of polymorphonuclear cells and monocytes of HIV-infected children and adults to generate superoxide anion (SO) and hydrogen peroxide (HP) and compared it with that of cells from normal children and adults. SO was measured by reduction of cytochrome c and HP by horseradish peroxidase-dependent oxidation of phenol red. The cells were incubated in 96-well plates at 37 degrees C for 2 h before the assay and the nonadherent cells then removed. Readings for SO were taken at 10, 30, 60, and 120 min after stimulation with phorbol myristate acetate; HP production was assayed after 90 min. The SO and HP production by polymorphonuclear cells and monocytes from both HIV-infected children and adults was consistently found to be markedly lower than that of cells from age-matched controls. The magnitude of the difference in response between patients and control cells also increased with increasing incubation time. Thus, phagocytic cells from HIV-infected children and adults are defective in their ability to generate reactive oxygen intermediates, and this defect may make them more vulnerable to bacterial and fungal infections.

Role of oxygen intermediates in cytotoxicity: studies in chronic granulomatous disease.

The ability of human neutrophils to lyse various target cells was investigated using cells from normal individuals and from patients with chronic granulomatous disease (CGD) whose cells lack the ability to form reactive oxygen intermediates (ROI). Cytolysis was stimulated by phorbol myristate acetate (PMA), rabbit antiserum, and a heteroantibody that binds to both the FcRII receptor of neutrophils and to the target. The PMA-activated CGD neutrophils were deficient compared to controls in killing both tumor and chicken erythrocyte (CRBC) targets at all effector-target ratios in 18-h assays. When CRBC were sensitized with rabbit antiserum, the normal cells still killed slightly more. When killing of CRBC was mediated by the heteroantibody, however, cytotoxicity of CGD neutrophils exceeded that of normal cells. CGD mononuclear cells (MNC) killed tumor cell targets as well as or better than normal MNC. Thus, PMA-mediated cytolysis appears to depend primarily upon the ability of the cell to generate ROI whereas antibody-mediated cytotoxicity and MNC-mediated lysis of tumor cells do not require ROI formation.

Human eosinophils are more toxic than neutrophils in antibody-independent killing.

Eosinophils (EOSs) are implicated in damaging host tissues in diseases such as asthma and eosinophilic gastroenteritis. In the present study, we assessed the cytotoxicity of human EOSs from peripheral blood of patients with eosinophilia and from peritoneal fluid of patients undergoing continuous peritoneal dialysis and compared them to normal neutrophils. Cytotoxicity was measured by the release of 51chromium from cultured tumor cells and chicken erythrocytes. Both EOSs and neutrophils were separated on discontinuous Percoll gradients with greater than 95% purity. The granulocytes were activated by preincubation in an ice bath with phorbol myristate acetate and washed before incubation with the target cells. The EOSs lysed significantly more tumor cells (K562, Raji, and CEM lines) in an 18-hour assay than did neutrophils, and no significant difference was found between the peritoneal and blood EOSs. The EOSs were also much more efficient than neutrophils in lysing chicken erythrocytes when they were activated by granulocyte-macrophage colony-stimulating factor instead of phorbol myristate acetate. Cytolysis by EOSs is mediated by both oxidative and nonoxidative mechanisms, as indicated by experiments with cells from patients with chronic granulomatous disease. Thus, EOSs are much more cytotoxic than neutrophils and potentially much more damaging to patients with eosinophilia.

Effects of herpes simplex virus on induced cell-mediated cytotoxicity in neonates and adults.

Deficient cellular cytotoxic mechanisms are present in neonates, contributing to their increased susceptibility to certain viruses, notably herpes simplex virus (HSV). Significant lymphokine-activated killer (LAK) cell activity has been described in cord blood, suggesting a possible role for LAK and/or interleukin-2 (IL-2) therapy in newborns with serious viral infections. The effect of HSV (type 1) on the activation of cord versus adult LAK cells was investigated by adding virus (multiplicity of infection, MOI = 10) to cells that had been previously incubated for 4-6 days with IL-2 (50-100 U/ml). The cells were then tested 24 h after virus exposure for cytotoxic activity against 51Cr-labelled K562 and Raji target cells. HSV inhibited LAK cytotoxicity of adult cells against K562 by 44% (72 +/- 2.4%, SEM; specific lysis to 40 +/- 6.2%, n = 15) and by 62% against Raji targets (50 +/- 5.6 to 19 +/- 4.4%). A similar degree of inhibition was observed for cord cells against K562 (76 +/- 2.0 to 46 +/- 5.3%) and Raji (60 +/- 4.6 to 24 +/- 6.2%). The degree of inhibition was correlated with the dose of virus in dose-response experiments. Inhibition was also noted with irradiated (10,000 rad) but not with heat-inactivated (56 degrees C for 60 min) virus. No inhibition was found when virus was added directly to the cytotoxic assay or when virus was added at the initiation or end of culture of cells with IL-2 (i.e. day 0 or day 5-7). In contrast, HSV stimulated cytotoxic activity against both the natural killer (NK)-sensitive (K562) and NK-resistant (Raji) targets in cells not incubated with IL-2. The cytotoxicity of adult cells incubated with infectious HSV (MOI = 10) for 5-7 days increased from 5.5 +/- 1.9% in the absence of virus to 25 +/- 6.0% against K562 in the presence of virus and from 3.5 +/- 1.0 (no virus) to 16 +/- 4.3% (with virus) against Raji targets (n = 8). The cytotoxicity of cord cells was also stimulated, but to a lesser degree. Irradiated virus also stimulated cytotoxic activity but to a lesser degree in cord cells. Virus-induced nonspecific cytotoxicity may represent an important component of the host's antiviral defense that is present at birth, but somewhat diminished compared to normal adults.

Purification and properties of peritoneal eosinophils from pediatric dialysis patients.

Peritoneal eosinophilia frequently occurs in patients undergoing peritoneal dialysis. We have devised a method for isolating large numbers of these peritoneal eosinophils from pediatric patients on continuous peritoneal dialysis. Patients were selected on the basis of previous high peritoneal eosinophil counts and had an age range of 1.5-11 years. The unfractionated peritoneal fluid contained 7.9 +/- 3.7% neutrophils, 3.8 +/- 1.0% lymphocytes, 11.0 +/- 3.7% monocytes/macrophages, and 77.3 +/- 6.3% eosinophils (based on Wright stain) and up to 2 x 10(9) cells could be recovered from 1 liter of peritoneal dialysate. The cells were concentrated by centrifugation and the cell suspension then layered over a discontinuous Percoll gradient consisting of layers of 45%, 55%, 65%, and 75% Percoll. The gradients were centrifuged resulting in the formation of bands of cells at the interfaces of the layers. The densest band of cells (above 75% Percoll) contained 94.7 +/- 1.8% eosinophils (mean with median of 98%) and 4.3 +/- 16% neutrophils. The eosinophil counts were 72.2 +/- 7.1% above the 65% layer, 57.1 +/- 8.7 above 55%, and 40.9 +/- 10.9% above 45%. The monocyte/macrophage count increased from 0.1% above the 75% layer to 42.9% above the 45%. The denser eosinophils (above 75% and 65%) had the appearance of normal blood eosinophils and comparable function to blood eosinophils in cytotoxic and oxidative assays. This method provides a means of obtaining large numbers of very pure eosinophils for study of eosinophil function, eosinophil subpopulations, or eosinophil granule constituents.

Lymphokine-activated killer cells in primary immunodeficiencies and acquired immunodeficiency syndrome.

Cytotoxic mechanisms (e.g., natural killer (NK) lysis, antibody-dependent cellular cytotoxicity, and cytotoxic T lymphocyte lysis) play an important role in host defense against various infections and neoplasms. Lymphokine-activated killer (LAK) cytotoxicity, induced in vitro by incubating mononuclear cells with interleukin 2 (IL-2) for 2-5 days, may also represent an important component of the body's cytotoxic repertoire. In 10 patients with congenital cellular immunodeficiencies, including 5 with severe combined immunodeficiency, the mean LAK activity in a 3-hr chromium release assay against Raji target cells was 44 +/- 8.1%, which is equivalent to that observed in normal adults and neonates. In only one case, a patient with reticular dysgenesis, was there absent LAK cell generation. Haploidentical T cell-depleted bone marrow transplantation (BMT) restored LAK activity in this patient. LAK activity was first observed in this patient and two others 3-6 weeks following BMT, prior to other evidence of immunologic engraftment such as lymphocyte proliferation to mitogens, NK activity, or interferon-gamma production. One patient with adenosine deaminase deficiency showed normal levels of LAK activity despite absent NK activity. Three patients with chronic granulomatous disease also had normal LAK activity (57 +/- 14% specific lysis). In 9 patients with acquired immunodeficiency syndrome (AIDS), IL-2 activation resulted in a mean cytotoxic activity of 56 +/- 8.7% toward Raji targets. In addition, 9 patients with pre-AIDS complex also showed normal levels of cytotoxicity (37 +/- 3.3% toward Raji targets), equivalent to that of 8 normal controls, including two healthy homosexual males (mean lysis 38 +/- 3.9%). These results indicate that LAK cells appear early in immunologic ontogeny. Further, the mechanism of lysis is not oxygen dependent since LAK activity was present in the 3 patients with chronic granulomatous disease. The ability to generate LAK in a wide spectrum of immunodeficiencies may indicate that IL-2 could be used in therapy of such disorders.

Enhancement of normal neutrophil chemiluminescence by chronic granulomatous disease neutrophils.

Neutrophils and other phagocytic cells from patients with chronic granulomatous disease (CGD) lack the ability to generate reactive oxygen intermediates (ROI), although other phagocytic functions appear to be intact. The effects of CGD neutrophils on the ability of normal neutrophils to produce ROI as measured by luminol-enhanced chemiluminescence (CL) were examined. Normal neutrophils (2 x 10(5)) had a peak CL response to phorbol myristate acetate (PMA; 20 ng/ml) of 6.5 +/- 0.9 mV, while the CL response to CGD neutrophils was completely absent. However, the addition of CGD neutrophils (8 x 10(5)) to normal neutrophils (2 x 10(5)) markedly increased the peak CL response to PMA to 11.0 +/- 1.1 mV (P less than 0.001). The peak response of normal neutrophils (2 x 10(5)) alone to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-6) M) was 9.0 +/- 1.1 mV, and this increased to 22.2 +/- 3.5 mV (P less than 0.001) when 8 x 10(5) CGD neutrophils were added and to 18.9 +/- 3.6 mV (P less than 0.005) when 4 x 10(5) CGD neutrophils were added. Thus, CGD neutrophils increase the release of ROI from normal cells, suggesting nonoxidative regulatory factors in ROI production.

Replication of herpes simplex virus in blood monocytes and placental macrophages from human neonates.

Increased permissiveness of macrophages for herpes simplex virus (HSV) replication may be a mechanism for the dissemination and severity of neonatal herpetic infection. We have assessed the replication of HSV in neonatal blood monocytes and placental macrophages using several criteria for viral permissiveness. Assay of production of infectious progeny virus indicated that cord blood monocytes, like adult monocytes, were nonpermissive for HSV (about 1% of cells producing virus). In vitro culture of cord blood monocytes resulted in increased replication of HSV, but no greater extent than virus production in cultured adult cells. HSV infection of fetal placental macrophages was weak but present (4.4% of cells). Assay of production of viral antigens and electron microscopic analysis of structural elements indicated that a larger number of cord blood monocytes and placental macrophages were abortively infected than were productively infected. These results indicate that monocytes and macrophages from human neonates do not show the enhanced permissiveness for HSV demonstrated in newborn mice and suggest that dissemination of herpetic infection in human newborns cannot be explained by increased neonatal monocyte permissiveness for HSV.

Enhanced interferon production and lymphokine-activated cytotoxicity of human placental cells.

The immunologic competence of human placental mononuclear cells was compared to that of adult and cord blood mononuclear cells. Mononuclear cells were isolated from fresh placentas by digestion with collagenase and DNase, followed by Ficoll-Hypaque and discontinuous Percoll separation. Placental cells incubated with phytohemagglutinin (PHA) synthesized significantly more interferon-gamma (IFN-gamma) at 2 days (29 +/- 5.5 IU/ml) and 5 days (46 +/- 8.5 IU/ml) than PHA-activated cord cells (3.6 +/- 0.6 IU/ml at 2 days and 2.7 +/- 0.7 IU/ml at 5 days) but less than PHA-activated adult cells (81 +/- 20 IU/ml at 2 days and 270 +/- 161 IU/ml at 5 days). Placental and adult cells, but not cord cells, also synthesized significant quantities of IFN-gamma following incubation with interleukin-2 (IL-2). There was synergism between IL-2 and PHA activation for IFN-gamma production for some cord samples. After a 5- to 7-day incubation with IL-2, the lymphocyte-activated killer (LAK cell) cytotoxicity of placental cells (measured in a 3-hr chromium-release assay at an E:T ratio of 40:1) was enhanced 13-fold against K562 target cells (6 +/- 2% to 77 +/- 4%) compared to a 4-fold increase in cord cells (16 +/- 4% to 68 +/- 3%) and a 2-fold increase in normal adult cells (35 +/- 4% to 65 +/- 3%. Against the natural killer (NK)-resistant Raji target, placental cells increased their LAK cytotoxic activity (3 +/- 1% to 59 +/- 7%) compared to a 7-fold increase with cord cells (6 +/- 1% to 43 +/- 3%) and a 3-fold increase with adult cells (11 +/- 2% to 38 +/- 4%). A notable degree of cytotoxic activity in the absence of IL-2 against Molt targets was noted in 11 of 14 (79%) placental cell samples at 5 days. Only 10 of 24 (42%) adult and 17 of 37 (40%) cord samples showed spontaneous cytotoxic activity equal to or greater than 10%. Some placental samples actually showed an increase in cytotoxic activity when incubated without IL-2. The ability of placental cells to produce significant levels of IFN-gamma, to develop considerable LAK activity, and to maintain or develop cytotoxic activity in the absence of IL-2 suggests a vigorous, active immune system of the placenta compared to the relatively dormant immune system of the neonate. These observations suggest that placental cells may have a primary role in fetal defense.