Cortical expression of endothelin receptor subtypes A and B following middle cerebral artery occlusion in rats.

This work aimed to define the spatial expression of endothelin A (ET(A)) and B (ET(B)) receptors in the cerebral cortex after permanent middle cerebral artery occlusion (MCAO) and to identify the phenotype of cells expressing ET(A) and ET(B) receptors. Cortical expression of ET(A) and ET(B) receptors was determined at the mRNA level by semi-quantitative reverse transcription-polymerase chain reaction and at the protein level by immunofluorescence staining, 12, 24 and 72 h after MCAO. Cells expressing endothelin receptors were phenotyped by double labelling with antibodies, anti-protein gene product (PGP9.5) and anti-ED1, towards neurons and activated microglia/macrophages, respectively. Both ET(A) and ET(B) receptor mRNA expressions increased significantly in the ipsilateral cortex in a time-dependent manner after MCAO. Robust expression of ET(A) receptors was noted in most neurons of the ischemic core and in several neurons in laminae 3 and 4 of the peri-infarct region 24 and 72 h after MCAO. ET(B) receptor immunoreactivity was observed in activated microglia/macrophages, beginning 24 h after MCAO. These results provide the first evidence that the action of endothelin during ischemia may be mediated by neuronal ET(A) receptors and activated microglia/macrophage ET(B) receptors. This differential localization of ET(A) and ET(B) receptors suggests that endothelin is involved in some complex neuron-glial interactions in addition to its vascular modulatory activity during ischemia.

DNA methylation patterns of the gamma delta beta-globin genes in human fetal and adult erythroid tissues.

An investigation of the correlation between the gamma----beta-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and -insensitive enzymes indicated hypomethylation in the promoter region of the gamma-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the delta-globin gene and hypomethylated at the 3' region of the beta-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.

Natural killer cell cytotoxicity to herpes simplex virus-1-infected cells is not altered by pregnancy.

There is evidence to suggest a decrease in natural killer cell cytotoxicity during pregnancy, but information regarding immune responsiveness to actual infection is limited. An in vitro study was undertaken to examine the effect of herpes simplex virus infection on natural killer cell cytotoxicity with peripheral blood mononuclear cells from pregnant (N = 8) and nonpregnant (N = 5) women. The peripheral blood mononuclear cells were separated by Ficoll-Hypaque centrifugation. Effector cells were incubated with live herpes simplex virus-1, ultraviolet-inactivated herpes simplex virus-1, or media alone for 18 hours at 37 degrees C. K562 target cells were used in a sodium chromate release assay with an effector-to-target cell ratio of 100:1. Baseline natural killer cell values (mean +/- SE) for pregnant patients (13.4% +/- 2.4%) and nonpregnant patients (19.8% +/- 3.7%) were similar. Natural killer cell cytotoxicity was significantly increased by incubation with live virus for both pregnant (37.5% +/- 6.2%) and nonpregnant subjects (49.8% +/- 7.6%). There was no difference in mean values between media and ultraviolet-inactivated herpes simplex virus-1-exposed samples for either group. Results suggest that (1) infection with live virus, but not viral antigen alone, can augment natural killer cell response in vitro and (2) natural killer cell response to herpes simplex virus-1 infection is not altered by pregnancy.

Interleukin-2 protects neonatal mice from lethal herpes simplex virus infection: a macrophage-mediated, gamma interferon-induced mechanism.

Administration of human recombinant interleukin-2 (IL-2) protected neonatal mice from a lethal herpes simplex virus (HSV) infection. Protection was not associated with viral antibody production, enhanced natural killer cell cytotoxicity, or intrinsic resistance of macrophages to viral infection. Protection was associated with increased macrophage-mediated antiviral antibody-dependent cellular cytotoxicity (ADCC). Spleen cells from IL-2-treated neonatal mice and from neonatal mice that were treated in vitro with IL-2 transferred protection to neonatal mice. These cells, by adherence, silica, and asialo GM 1 antibody treatment, were shown to be macrophages. IL-2 treatment in vitro enhanced the neonatal macrophages' ADCC function and superoxide release. Similar protection was induced by gamma interferon (IFN-gamma)-treated spleen cells. Antibody to IFN-gamma ablated both IFN-gamma- and IL-2-induced protection by adherent spleen cells. Thus, IL-2-mediated protection against murine neonatal HSV infection was affected by stimulated macrophage activity, via helper T cell-produced IFN-gamma.

Antibody to cloned HSV glycoproteins B and D plus adult human leukocytes protect neonatal mice from lethal HSV infection.

Antisera produced by HSV infection or following vaccination of guinea pigs with the cloned herpes simplex virus (HSV) glycoproteins gB and gD were compared for in vitro antibody-dependent cellular cytotoxicity (ADCC) activity and for in vivo protection. Antibody from guinea pigs was able to participate in ADCC with human mononuclear cells in vitro, anti-gBgD serum being equivalent to HSV convalescent sera. In vivo, each of the guinea pig sera was able to protect neonatal mice from a fatal HSV-1 infection when given with human mononuclear cells but not when given alone. The anti-gBgD serum was the most effective in vivo, protecting 15 of 17 (88%) neonatal mice when given at a 10(-4) dilution with human mononuclear cells and was the only guinea pig serum protective at a 10(-6) dilution (5 of 7 neonatal mice).

Use of interleukin-2 and macrophages to treat herpes simplex virus infection in neonatal mice.

We have developed a model of therapy for herpes simplex virus (HSV) infection in neonatal mice. Using a lower dose of virus (10(2) plaque-forming units administered intraperitoneally), we have been able to treat infected mice with syngeneic or allogeneic adult mouse macrophages in combination with three doses of human recombinant interleukin-2. Therapy resulted in a 42%-85% survival rate. Although production of antibody to HSV was associated with successful treatment, early administration of antibody did not improve survival.

Natural killer cell cytotoxicity and antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells in human pregnancy.

Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a 51Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.

The genetic deficiency of leukocyte surface glycoprotein Mac-1, LFA-1, p150,95 in humans is associated with defective antibody-dependent cellular cytotoxicity in vitro and defective protection against herpes simplex virus infection in vivo.

The role of the Mac-1, LFA-1, p150,95 leukocyte glycoprotein family in mediating antiviral host defense was investigated by utilizing mononuclear cells (MC) obtained from eight patients with a genetic deficiency of Mac-1, LFA-1, and p150,95, and normal MC incubated with subunit-specific monoclonal antibodies (MAb) directed against these glycoproteins. As shown with an in vitro chromium-release cytotoxicity assay to herpes simplex virus (HSV)-infected Chang liver target cells, MC of these patients with the severe phenotype or normal MC preincubated with a combination of MAb against Mac-1 glycoprotein subunits were deficient in antibody-dependent cellular cytotoxicity (ADCC). When used individually, MAb directed at LFA-1-alpha or -beta also inhibited ADCC and natural killer cytotoxicity (NKC). In a single cell agarose assay, MC of Mac-1-deficient patients formed fewer effector-target cell conjugates in the presence of specific anti-HSV antibody. To investigate the in vitro contributions of these glycoproteins to cytotoxic host defense mechanisms, two in vivo adoptive transfer models were explored in which neonatal mice are protected against a lethal HSV challenge by normal human MC plus anti-HSV antibody (in vivo ADCC) or human interferon-alpha (NKC stimulated in vivo). In each model, MC from patients with "severe" or "moderate" phenotypes of Mac-1 deficiency, or normal MC incubated with a combination of anti-LFA-alpha, Mac-1-alpha, p150,95-alpha plus -beta MAb failed to protect neonatal mice against lethal HSV infection. These studies further indicate requirements for adhesion-dependent mechanisms in the mediation of MC-ADCC, and suggest that Mac-1-dependent cellular adhesive properties are necessary for normal cytotoxic functions in vivo in experimental models of human ADCC or interferon-stimulated NKC. These findings, in addition to the recognized occurrence of severe or even lethal viral infections in some Mac-1-deficient patients, suggest that glycoproteins of the Mac-1 family may be important determinants of antiviral host defense.

Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice.

Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti-T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection.

Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency.

Four children with an immunodeficiency involving the absence of leukocyte membrane glycoproteins reacting with anti-LFA-1 and OKM-1 monoclonal antibodies were unable to mediate adherence-dependent leukocyte functions. Even with normal Fc receptor function, their PMN-ADCC and MC-NKC were markedly deficient. Single cell analysis demonstrated deficient antibody-mediated PMN-target cell adherence. Monoclonal antibodies against LFA-1 and OKM-1 reproduced this immunodeficiency in leukocytes from normal adults. LFA-1/OKM-1 mediates a PMN-target cell adhesive step.

Influence of naproxen therapy on natural killer cytotoxicity and antibody-dependent cellular cytotoxicity against cells infected with herpes simplex virus.

Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) represent one of the body's primary lines of defense against viral infections, including herpes simplex (HSV). This immune defense system is negatively influenced by prostaglandins. A project was undertaken to evaluate the influence of an antiprostaglandin agent in vivo on these cytotoxic effects against cells infected with HSV. Thirteen subjects without previous histories of clinical herpes simplex infection were studied during menses with and without naproxen therapy. A statistically significant augmentation (p = 0.05) of natural killer-cell function was identified in mononuclear cells during therapy. Subjects with baseline cytotoxicity of less than 45% demonstrated consistent elevations during naproxen therapy. No other significant differences could be found for mononuclear or polymorphonuclear cells with regard to NKC and ADCC. There appears to be a subset of patients who may benefit from immunologic augmentation with antiprostaglandin agents when experiencing herpes simplex virus infection.

Analysis in human neonates of defective antibody-dependent cellular cytotoxicity and natural killer cytotoxicity to herpes simplex virus-infected cells.

Human neonatal mononuclear cells (MCs) had low antibody-dependent cellular cytotoxicity (ADCC) compared with cells from adults in a chromium-release assay against Chang liver cells infected with herpes simplex virus (HSV). Polymorphonuclear leukocyte (PMNL) ADCC of neonates was similar to that of adults. In a single-cell agarose conjugation assay IgG antibody to HSV significantly increased conjugation by adult MCs, adult PMNLs, and cord blood PMNLs but not by cord blood MCs. Expression of the high-affinity IgG Fc receptor (FcR) assayed by erythrocyte-antibody rosetting revealed significant differences between adult MC FcR and cord blood FcR. There was no difference in PMNL FcR expression. Human interferon-alpha increased neonatal MC adhesion in the presence of IgG and FcR expression, but it had no effect on MC ADCC. Defective FcR expression and target cell adhesion may partly explain low neonatal MC ADCC. In addition, cord blood cells have a lytic or recycle, as well as an adherence, defect.

Antibody-dependent cellular cytotoxicity and natural killer cytotoxicity of peritoneal cells from nude mice to herpes simplex virus-infected cells.

Nude BALB/c mice (athymic) were more susceptible to fatal herpes simplex virus (HSV) than normal BALB/c mice (P = 0.002). The peritoneal cells of nude mice mediated levels of antibody-dependent cellular cytotoxicity (ADCC) of equal or greater magnitude than cells from normal BALB/c, heterozygote nu/+, or C57BL/6 mice. Unstimulated natural killer cytotoxicity of peritoneal cells from nude mice was higher (P less than 0.05) than that mediated by cells from C57BL/6 mice. Nude mice failed to make anti-HSV ADCC antibody 6 to 14 days post HSV inoculation, at times when nu/+, BALB/c, and C57BL/6 mice produced antibody. Passive reconstitution of nude mice with high titer intraperitoneal anti-HSV immune globulin provided circulating anti-HSV ADCC antibody and significant protection against lethal HSV infection.

Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host.

Inhibition of cellular cytotoxicity of leukocytes for herpes simplex virus-infected cells in vitro and in vivo by intralipid.

The effect of intralipid, a lipid emulsion used in total parenteral nutrition, on cellular cytotoxicity for herpes simplex virus (HSV)-infected cells was analyzed. In vitro, intralipid inhibited antibody-dependent cellular cytotoxicity (ADCC) of lymphocytes, monocytes-macrophages, and polymorphonuclear leukocytes and natural killer cytotoxicity of lymphocytes for radiolabeled HSV-infected liver cells. This was due to an effect on the leukocytes, rather than on the target cells. Intralipid did not affect leukocyte viability but inhibited the expression of leukocyte Fc receptors necessary for cytotoxicity. In vivo, intralipid inhibited murine ADCC and completely nullified the protection against lethal infection with HSV in neonatal mice afforded by the administration of human leukocytes and antibody. These data suggest that high levels of circulating intralipid may interfere with antiviral immunity in humans and predispose hosts who are already compromised to severe viral infections.

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity.

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.

Virus-infected colostral cell cytokine stimulation of human leukocyte natural killer cytotoxicity.

Natural killer cytotoxicity is an important antiviral defense mechanism. Human peripheral blood mononuclear cells cultured with herpes simplex virus (HSV)-infected cells produced a cytokine. This substance stimulated adult natural killer cytotoxicity from 53.0 +/- 10.5% to 79.8% (P less than 0.01) against HSV-infected target cells. These data resulted in a calculated cytokine-dependent cellular cytotoxicity (CDCC) value of 65.8%. Cytokine production was not stimulated by uninfected cells and was independent of the presence or absence of antibodies to HSV in sera of donors and mononuclear cells. Cells from human colostrum also produced an HSV-stimulated cytokine which mediated CDCC by using both adult (19.8 +/- 3.9%) and neonatal (18.6 +/- 3.4%) mononuclear effectors cells. Colostral cell cytokine production was also independent of donor HSV serology. Not all colostral cultures produced the cytokine, and in general colostrum-stimulated CDCC was lower than peripheral blood leukocyte-stimulated CDCC. Colostral cell cytokine stimulation of neonatal natural killer cytotoxicity may account in part for the increased nonspecific resistance of breast-fed infants to viral infection.

Ontogeny of murine cellular cytotoxicity to herpes simplex virus-infected cells.

Mice infected with herpes simplex virus either orally or intraperitoneally had a markedly age-related mortality. All animals under 3 weeks of age died, whereas all those over 3 weeks of age survived. The ability of murine peritoneal cells to kill herpes simplex virus-infected target cells in the absence (natural killer cytotoxicity) or presence (antibody-dependent cellular cytotoxicity) of antiviral antibody was similarly correlated with age and survival. This correlation is further support for the relevance of these antiviral defense mechanisms, and it may help explain the profound susceptibility of neonatal mice to herpes simplex virus infection.

Human colostral cytotoxicity. II. Relative defects in colostral leukocyte cytotoxicity and inhibition of peripheral blood leukocyte cytotoxicity by colostrum.

Colostral cells have been previously shown to mediate antibody-dependent cellular cytotoxicity (ADCC) to herpes to simplex virus-infected cells. A comparison of colostral cells to matched peripheral blood mononuclear cells has revealed significantly lower ADCC activity, higher antibody requirements, and slower kinetics of colostral cell ADCC against infected cells. Colostral cells failed to mediate natural killer cytotoxicity. Lymphocytes, monocyte macrophages, and polymorphonuclear leukocytes isolated from peripheral blood and incubated with colostrum from virus-immune or nonimmune women markedly inhibited ADCC. Inhibitory activity was found in lipid and aqueous fractions and was due to an effect on leukocytes, not target cells. A partial explanation was inhibition of expression of leukocyte Fc receptors, a prerequisite for ADCC, by acellular colostrum. These results raise questions concerning the potential antiviral activity of colostral cells in vitro.