Secretion of immunoreactive ANF from FRTL-5 rat thyroid cells: regulation by calcium ionophore A23187.

Atrial natriuretic factor-like immunoreactivity (ir-ANF) was characterized in a continuous line of rat thyroid follicular cells (FRTL-5) and the influence of the calcium ionophore A23187 on ir-ANF secretion was examined. Ir-ANF was identified by immunohistochemical staining as primarily reticular and juxtanuclear in short-term cultures, and more peripheral and granular in longer-term cultures, suggesting a process of ir-ANF packaging into secretory granules. The accumulation of ir-ANF granules was dependent upon the presence of thyrotropin (TSH) in the medium. Secreted ir-ANF was characterized using reversed-phase, high-performance liquid chromatography (RP-HPLC) and radioimmunoassay as a single peak eluting one fraction earlier than 125I-labeled rat ANF (99-126) (i.e., circulating atrial ANF) included as an internal standard. A23187 treatment of cells exhibiting primarily reticular ir-ANF caused a change to a pattern of more distinct, peripherally localized granules. This change occurred within 1 h after A23187 treatment and was dependent on the presence of Ca2+ in the medium. In cultures containing primarily ir-ANF granules, A23187 (0.5 micrograms/ml) induced a peripheral translocation of the granules at 30 min and a complete degranulation by 7 h. Enzyme-linked immunoadsorbent assay (EIA) confirmed a dose-dependent effect of A23187 on ir-ANF release into the medium. These results suggest that some of the effects of Ca2+ in the thyroid could be ascribed to its mobilization and release of ir-ANF, which in turn may have autocrine effects on thyroid follicular cells.

Transfer factor for the prevention of varicella-zoster infection in childhood leukemia.

Sixty-one patients with leukemia and no immunity to chickenpox were given dialyzable transfer factor or placebo and followed for 12 to 30 months in a double-blind trial designed to examine the clinical efficacy of transfer factor. Sixteen patients in the transfer-factor group and 15 in the placebo group were exposed to varicella zoster, and most of them had a rise in antibody titer. Chickenpox developed in 13 of 15 exposed patients in the placebo group but in only one of 16 in the transfer-factor group (P = 1.3 x 10(-5)). In the patients treated with transfer factor and exposed to varicella without acquiring chickenpox the titer of antibody to varicella zoster was equal to that in the patients given placebo who became infected with chickenpox. Transfer factor converted negative results on skin tests for varicella zoster to positive in approximately half the recipients. Passive immunization with dialyzable transfer factor appears useful in nonimmune persons.

Specific impairment of cell-mediated immunity in mothers of infants with congenital infection due to cytomegalovirus.

Specific lymphocyte-mediated cytotoxicity to cytomegalovirus (CMV) in eight infants (six to 27 months old) with congenital CMV infection and in the mothers of six of these infants was evaluated with use of a 51chromium (51Cr)-release microassay. The control population consisted of 25 normal newborns, children, and adults. The titers of indirect hemagglutinating (IHA) antibody to CMV in the infected infants ranged from 1:16 to 1:1,024. All of these infants had detectable specific immune release of 51Cr that ranged from 3.3% to 48.9% (mean +/-SE, 21.0%+/-5.6%). The mothers of these infants demonstrated significantly elevated titers of IHA antibody to CMV (geometric mean titer, 1:410) as compared with a mean titer of 1:22 in controls (t = 5.71; P less than 0.001) but showed significantly depressed specific immune release (9.2% +/- 3.2%) compared with that of normal seropositive controls (24.8% +/- 2.8%; t = 3.31; P less than 0.001). In addition, two adult nulliparous women with persistent CMV viruria were also found to have depressed specific immune release to CMV (10.8% and 16.2%). These data suggest that a specific impairment in cell-mediated immunity to CMV occurs in mothers of infants with congenital CMV infection and in some persons who persistently excrete CMV.

51Chromium-release microassay technique for cell-mediated immunity to mumps virus: correlation with humoral and delayed-type skin hypersensitivity responses.

Lymphocyte-mediated immune responsiveness to mumps virus was studied with use of a 51chromium (51Cr)-release microassay of lymphocytotoxicity to target cells persistently infected with mumps virus. The release of 51Cr from immune cells was found to be virus-specific and reproducible and correlated well with the presence of antibody but not with the magnitude of antibody levels. Delayed-type skin hypersensitivity and specific immune release of 51Cr did not correlate well, which suggests that the two events may be mediated by different populations of cells.

Use of cryopreserved virus-infected target cells in a lymphocytotoxicity 51Cr release microassay for cell-mediated immunity to cytomegalovirus.

A new 51Cr-release microassay is described for the measurement of cell-mediated immunity to cytomegalovirus, using cryopreserved target cells acutely infected with cytomegalovirus.

Specific inhibitory factors of cellular immunity in children with subacute sclerosing panencephalitis.

Employing a 51Cr release cytotoxicity microassay, and using both measles-and SSPE-infected target cells, four patients with documented SSPE were evaluated for specific cellular and humoral immunity. Mononuclear leukocytes from SSPE patients and control subjects exhibited comparable cytotoxicity. Serum and CSF from these SSPE patients inhibited the cellular response to SSPE-infected cells but not to measles-infected cells. Moreover, fresh whole serum alone from control donors produced significant 51Cr release from both cell lines, whereas SSPE whole serum was effective only against measles-infected cells. CSF from an additional ten patients with SSPE was examined for inhibitory activity: seven of these completely blocked and one partially blocked cell-mediated cytotoxicity to SSPE-infected cells. Preliminary characterization of the serum inhibitory factor suggested that it is IgM or antigen-antibody complexes. These data also suggest antigenic differences between the SSPE and measles viruses.

Depressed specific cell-mediated immunity to Herpes simplex virus type 1 in patients with recurrent herpes labialis.

Cell-mediated immunity to herpes simplex virus type 1 (HSV-1) was assessed by a lymphocytotoxicity 51-Cr-release microassay procedure, using the MA-160 human prostatic adenoma cell line chronically infected with HSV-1 and its parent cell line as control. The specific immune release mean plus or minus standard deviation for nine asymptomatic patients with recurrent HSV-1 infections was 13.7 plus or minus 8.1%, compared to 28.9 plus or minus 8.4% in seven normal seropositive controls (P is less than 0.01). In four additional patients studied serially, the cell-mediated immunity was significantly increased during the recrudescence of herpetic infection, with a mean specific immune release value of 51.7 plus or minus 27.8%, compared to 8.7 plus or minus 1.5% during the convalescent period 2 to 10 weeks later (P is less than 0.05). These findings suggest that patients with recurrent HSV-1 infections have vigorous cellular immune responses during the acute phase but a specific impairment of cell-mediated immunity during the quiescent period, which may in part account for their susceptibility to recurrent herpetic infections.

Identification of the cell population involved in viral-specific cell-mediated cytotoxicity in man: evidence for T cell specificity.

The nature of the cell population involved in lymphocyte mediated cytotoxicity to baby-hamster-kidney (BHK-21) target cells persistently infected with rubella virus was investigated by a 51Cr-release microassay. After depletion of the T cell population with an antiserum to human0thymus-lymphoid tissue antigen (HTLA), the purified B cell population showed a decrease in E-rosette formation (9.0 +/- 2.2% compared to 69.6 +/- 9.1% before treatment) and an insignificant degree of cytotoxic activity against rubella-infected target cells (specific immune release of 51Cr was 0.9 +/- 2.6% compared to 24.2 +/- 3.8 before treatment). A purified T cell population, prepared by depletion of B cells with an anti-human immunoglobulin serum and complement, was found to show no alteration in E-rosette formation (85.2 +/- 6.2%) or cytotoxicity (30.3 +/- 4.4% SIR) but showed decreased EA- and EAC-rosette formation (2.7 +/- 1.5% and 10.5 +/- 3.2%, respectively, compared to 19.4 +/- 2.9% and 28.0 +/- 4.1% before treatment). A monocyte-depleted population prepared by removal of the plastic adherent mononuclear cells showed no significant alteration of rosette formation or cytotoxicity. These experiments suggest that the predominant lymphoid population responsible for direct cell-mediated cytotoxicity to virus infected target cells in the 51Cr-release microassay appears to be effected by a thymus-dependent lymphocyte population.

Cellular immune responses to Herpes simplex virus type 1 in recurrent herpes labialis: in vitro blastogenesis and cytotoxicity to infected cell line.

Cellular immunity to herpes simplex virus type 1 (HSV-1) in 12 volunteers with recurrent herpes labialis was evaluated by means of two microassays. In the blastogenesis assay, lymphocytes were incubated with tissue culture cells persistently infected with HSV-1. Uninfected cells were used as controls, and a blastogenic index was calculated. The mean blastogenic index (plus or minus SD) for subjects with recurrent herpes labialis was 26.9 (plus or minus 8.3); the mean blastogenic index (plus or minus SD) in control donors with antibody to HSV-1 was 13.4 (plus or minus 7.2). The difference between these values was statistically significant (t equals 4.154; P smaller than 0.001). In the cytotoxicity assay, cells of the same persistently infected line were used as target cells, and release of 51-Cr from these cells or from control cells served as the index of lymphocyte reactivity. Specific immune release attributable to HSV-1 averages 3.7% (plus or minus 1.8%) in subjects with recurrent herpes labialis, compared with 23.1% (plus or minus 9.8%) in controls (t equals 6.135; P smaller than 0.001). These data suggest a dissociation between mechanisms of cellular immunity, with enhanced lymphocyte blastogenesis but decreased cytotoxicity. Recurrent herpes labialis may thus result from subtle cellular immune deficiency involving at least one of the efferent mechanisms.

Inhibitory effects of bilirubin on cellular immune responses in man.

A significant depression in cell-mediated immunity as measured by lymphoproliferative responses to phytohemagglutinin and responsiveness to mixed lymphocyte culture was observed when adult lymphocytes or cord blood lymphocytes were incubated with increasing concentrations of bilirubin. The inhibitory effect of bilirubin could only be demonstrated with suboptimal concentrations of PHA (0.01 and 0.005%) and was more marked in premature infants than in term neonates or adults. This effect was partially reversible after short preincubation with bilirubin, but was more protracted with preincubations of 24 hours or more. Inhibition of MLC responsiveness of 80.1 plus or minus 5.1% was also demonstrated at a bilirubin concentration of 20 mg/dl. Specific cytotoxicity to rubella virus-infected cells, measured by a 51Cr-release microassay, was not found to be depressed. Bilirubin thus appears to have an inhibitory effect on immune responsiveness which is greater on the afferent limb than on the effrent limb of immunity.

Effects of adenine arabinoside on cellular immune mechanisms in humans.

In vitro lymphocyte blastogenic responses to the commonly employed mitogens, phytohemagglutinin, pokeweed, and concanavalin A, were evaluated when adenine arabinoside (ara-A) in a concentration of 3 mug/ml was added to the culture materials. Similarly, blastogenic and cytotoxic responses to cell cultures persistently infected with herpes simplex virus 1, herpes simplex virus 2, and varicella-zoster virus were determined in the presence of ara-A. No depression of these cellular immune responses by ara-A was demonstrated. This was in contrast to the effect of cytosine arabinoside, which at a concentration of 3 mug/ml severely inhibited these immune responses. Further studies examined lymphocyte blastogenic responses to the mitogens and blastogenic and cytotoxic responses specific for the herpes group virus infecting patients who were subsequently treated with ara-A; determinations were made before, during, and after treatment. In vitro responses during and after treatment with ara-A were unchanged or often enhanced as compared to pretreatment values. Therefore, the antiviral chemotherapeutic agent, ara-A, does not appear to depress the host's cellular immune responses, which are vital to successful elimination of invading herpes group viruses.

Impaired cellular immunity to rubella virus in congenital rubella.

Specific cell-mediated immunity (CMI) responses to rubella virus were studied in 12 children with documented congenital rubella syndrome employing a (51)Cr lymphocytotoxicity microassay. Hemagglutination inhibition antibody was detected in 11 of the 12 children, with titers ranging from 1:4 to 1:128. CMI to rubella virus was demonstrated in only 3 of the 11 antibody-positive children. The 12th child was negative for both hemagglutination inhibition and CMI. Of the three children with a positive CMI response, two had histories of reinfection with rubella virus. These data suggest that congenital rubella infection produces an impaired CMI response which subsequently may be altered by reinfection with rubella virus. The lack of CMI in the presence of antibody and concurrent excretion of live virus in the child with documented congenital rubella infection suggest a factor to be explored in the pathogenesis of this disease.