Cytomegalovirus vaccine. Specific humoral and cellular immune responses in human volunteers.

Four seronegative adult male volunteers were immunized with Towne strain cytomegalovirus (CMV) vaccine. The only reaction was transient pain and swelling at the inoculation site. Viral cultures were performed during the first 12 weeks after immunization, and CMV was not recovered from throat, urine, or peripheral blood mononuclear cells. Both CMV-specific humoral and cellular immunity developed within three weeks of vaccination. Whereas humoral antibody titers declined steadily with time, the cellular immune responses seemed biphasic. An early peak in lymphocyte proliferation to CMV antigen occurred three to six weeks after immunization. Responses then diminished but increased again six to ten months after immunization. This study in a small group of normal male volunteers indicated that CMV vaccine was safe and immunogenic. That CMV vaccine elicited CMV-specific humoral and cell-mediated immunity is important, because there is evidence that both are necessary for protection from CMV infections.

Murine splenocytes express a naloxone-insensitive binding site for beta-endorphin.

Naloxone-resistant binding sites for beta-endorphin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of beta-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I]beta-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. However, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd = 4.1 X 10(-9) M). Competition studies showed that N-acetyl-beta-endorphin (N-Ac-beta-endorphin)-(1-31) was equipotent to beta-endorphin-(1-31). beta-Endorphin-(6-31) and beta-endorphin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas beta-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I]beta-endorphin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of beta-endorphin and N-Ac-beta-endorphin. beta-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for beta-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-beta-endorphin, presumed to be an inactivation product of beta-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.

Induction of the messenger ribonucleic acid for proenkephalin A in cultured murine CD4-positive thymocytes.

Although proenkephalin A (PEA) messenger RNA (mRNA) has been detected in many types of immune cells, little understanding exists about its role or the role of enkephalin peptides in immune responses. We have studied the expression of PEA mRNA during thymocyte maturation by identifying the subpopulation of thymocytes that expresses PEA mRNA. PEA mRNA was induced in unfractionated murine thymocytes after in vitro activation of these cells with the T cell mitogen, concanavalin A (Con-A). A slight induction of PEA mRNA was seen after 48 h of Con-A stimulation; however, the maximal response occurred after 72 h of culture with Con-A. Two PEA mRNA bands were present in unfractionated thymocytes which had been cultured with Con-A for 48 and 72 h. The predominant band was 1.4 kilobases (kb), and a second band was approximately 1.7 kb. Fractionation of thymocytes into CD4, CD8, and double negative subpopulations showed that only the 1.4 kb PEA mRNA was inducible in the mature CD4 subpopulation. Induction required the presence of antigen-presenting cells in addition to CD4 thymocytes. Neither the 1.4 kb nor the 1.7 kb PEA mRNA was induced in the CD8 or double negative subpopulations. In contrast to the action of Con-A on murine thymocytes, PEA mRNA was not induced by this mitogen in murine splenic mononuclear cells at 24, 48, or 72 h. The regulated expression of PEA mRNA in murine thymocytes, but not in peripheral T lymphocytes, suggests a role for PEA mRNA and its peptides in thymocyte maturation.

Expression and function of proenkephalin A messenger ribonucleic acid in murine fetal thymocytes.

In recent years we examined the function of the endogenous enkephalins encoded by PEA messenger RNA (mRNA) expressed in murine thymocytes, the precursor cells to T lymphocytes, which are the primary effector cells in cell- mediated immune responses. In the present study, we examined the expression and function of PEA mRNA and enkephalins in fetal thymocytes. By Northern gel and in situ hybridization techniques, we show that PEA mRNA is constitutively expressed in fetal thymocytes early in gestation, with maximal expression occurring on day 15. By birth, PEA mRNA is no longer constitutively expressed, but can be induced by culturing newborn thymocytes with the T cell-specific mitogen, Concanavalin-A. Both a delta-opioid receptor antagonist, naltrindole, and a PEA mRNA-specific antisense complementary DNA enhance the spontaneous proliferation of day 15, but not day 14, fetal thymocytes, consistent with the observation that PEA mRNA is expressed in thymocytes on day 15, but not day 14, of gestation. The enhanced proliferation of day 15 fetal thymocytes is reversed by a delta-opioid receptor agonist, deltorphin. The data suggest that endogenous enkephalins encoded by PEA mRNA expressed in day 15 fetal thymocytes act to inhibit the spontaneous proliferation of these cells, perhaps so that their differentiation into mature T lymphocytes can occur.

Dw subtypes of serologically defined DR-DQ specificities restrict recognition of cytomegalovirus.

We have investigated the relationship between serologically defined (Ia) and T lymphocyte-defined (LD/Dw) determinants in restricted recognition of cytomegalovirus (CMV) by human T lymphocytes. T lymphocytes isolated from CMV seropositive individuals expressing DQw3/DR4/Dw4 antigens were "sensitized" to CMV in vitro; CMV-specific blasts were isolated and tested for their ability to recognize CMV presented by cells expressing different DR4-associated Dw antigens (i.e., Dw4, Dw10, Dw13, Dw14, and Dw15). Similar studies were also performed using T lymphocytes from individuals expressing DQw1/DR2/Dw2 specificities and antigen presenting cells (APC) expressing the DR2-associated Dw/LD subtypes, Dw2, Dw12, and LD-MN2. CMV-specific T cell blasts were used as responding cells in order to reduce nonspecific background alloresponses which occur with allogeneic APC. In all cases it was found that the determinants involved in restricted recognition of CMV were subtypic to the DR-associated Ia specificities. To distinguish whether Dw specificities associated with DQ or with DR molecules, or both, were involved in these responses, we used anti-DR (L243) and an anti-DQwl (S3/4) monoclonal antibodies (MoAb) to block CMV-specific responses. Both MoAb significantly blocked responses, suggesting that determinants associated with both DR and DQ molecules are involved in restricted recognition of CMV by T cells.

The delta1-opioid receptor antagonist, 7-(benzospiroindanyl)naltrexone [correction of 7-benzylspiroindanylnaltrexone], prolongs renal allograft survival in a rat model.

In this study we demonstrate allograft survival in a rat model of renal transplantation using the delta1-opioid receptor antagonist, 7-(benzospiroindanyl)naltrexone [corrected]. Treatment with 7-(benzospiroindanyl)naltrexone [corrected] caused 50% of the rats to survive longer than 100 days (untreated, 11 +/- 3 days). Naltrindole, a delta-opioid receptor antagonist without subtype selectivity, also promoted graft survival but was substantially less effective, suggesting that antagonism at delta1-opioid receptors is involved in allograft survival.

IL-1 beta modulates the concanavalin-A-induced expression of proenkephalin A mRNA in murine thymocytes.

We have previously shown that proenkephalin A (PEA) messenger RNA (mRNA) is induced in murine thymocytes by the T cell-specific mitogen concanavalin-A (Con-A). We now show that this Con-A-induced expression of PEA mRNA is modulated by the cytokine murine interleukin-1 beta (mIL-1 beta) in a biphasic, dose-dependent manner. Murine thymocytes were cultured for 72 h with Con-A and with varying concentrations of mIL-1 beta. PEA mRNA expression was analyzed by Northern gel and solution hybridization techniques. Concentrations of mIL-1 beta of 10(-14) and 10(-13) M enhanced the Con-A-induced expression of PEA mRNA in cultured murine thymocytes up to 2.5-fold, whereas higher concentrations of mIL-1 beta (10(-11) and 10(-10) M) inhibited its expression 60 and 85%, respectively. Both the enhancing and inhibiting effects of mIL-1 beta in the Con-A-induced expression of PEA mRNA were reversed by a 100-fold excess of interleukin-1 receptor antagonist protein, but not by a 10-fold excess of interleukin-1 receptor antagonist protein. The effects of mIL-1 beta on PEA mRNA expression in Con-A-activated thymocytes are different from its effects on Con-A-stimulated thymocyte proliferation. In the latter case, only enhancement of thymocyte proliferation was seen, as measured by [3H]thymidine incorporation. The present study demonstrates that PEA mRNA expression is regulated by IL-1 beta, which is thought to play a role in thymocyte maturation.

Interleukin-1 alpha and interleukin-1 beta stimulate adrenocorticotropin secretion in the rat through a similar hypothalamic receptor(s): effects of interleukin-1 receptor antagonist protein.

Numerous reports have demonstrated that interleukin-1 beta (IL-1 beta) is a potent secretagogue for adrenocorticotropin (ACTH) and that IL-1 alpha appears to be considerably less efficacious. To clarify apparent differences in the potency of IL-1 alpha vs. -beta on ACTH secretion from a functional perspective, the IL-1 receptor antagonist protein, IRAP, was utilized. Following administration to rats either intravenously (i.v.) or adjacent to the median eminence (intra-ME), IL-1 beta was approximately 8-fold more potent than IL-1 alpha. IRAP, delivered i.v. or intra-ME, inhibited ACTH secretion due to the administration of IL-1 alpha or -beta by the corresponding route. Similar amounts of IRAP were required to attenuate ACTH responses to approximately equieffective i.v. doses of IL-1 alpha (200 ng) or -beta (25 ng): IC50 for IRAP inhibition of IL-1 alpha vs. -beta was approximately 2.5 or 5.5 micrograms, respectively. At these IC50 doses, the ratios of IRAP/IL-1 were 12.5 and 220 for IL-1 alpha vs. -beta, respectively. These ratios are compatible with mediation by a type I-like IL-1 receptor. To compare these properties of the central IL-1 receptor to a peripheral type I IL-1 receptor in the same species, the IL-1-enhanced rat thymocyte comitogenesis assay was utilized. Thymocyte proliferation in response to equieffective doses of IL-1 alpha or -beta was similarly inhibited by IRAP:approximate IC50 for inhibition of IL-1 alpha vs. -beta was 12.5 or 25 ng/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Met-enkephalin-containing peptides encoded by proenkephalin A mRNA expressed in activated murine thymocytes inhibit thymocyte proliferation.

Murine thymocytes activated with the mitogen Con A express proenkephalin A mRNA (PEA mRNA) and met-enkephalin and/or met-enkephalin-containing peptides ("enkephalins"). This Con A-induced expression of PEA mRNA is modulated by the delta opioid receptor agonist, deltorphin I, in a biphasic, dose-dependent manner. That is, 10(-13) M to 10(-11) M deltorphin enhanced PEA mRNA expression 3- to 3.5-fold over the level induced by Con A alone, and 10(-9) M to 10(-7) M deltorphin inhibited it 40 to 70%. delta opioid receptor antagonists recognizing the delta-2 (naltrindole (NTI) and naltriben (NTB)), but not the delta-1 (7-benzylidenenaltrexone (BNTX)), subtype of opioid receptor described in brain, reversed both the enhancing and inhibiting effects of deltorphin on Con A-induced PEA mRNA expression. In addition, the delta-2 receptor-specific antagonists, NTI and NTB, directly inhibited Con A-induced PEA mRNA expression. The function of the enkephalins expressed by thymocytes was examined by using 1) delta opioid receptor antagonists, 2) PEA mRNA-specific antisense cDNA, and 3) Ab to met-enkephalin, and measuring cell proliferation. All three reagents caused enhancement of Con A-induced proliferation, with effects ranging from two- to fourfold over the response to Con A alone. Again, the delta-2 subtype-specific antagonists, NTI and NTB, were functional and the delta-1 subtype-specific antagonist, BNTX, was not. The PEA mRNA-specific antisense cDNA blocked translation but not transcription of PEA mRNA. The data suggest that 1) endogenous enkephalins induced in thymocytes modulate their own expression through delta-2-like opioid receptors, and 2) these endogenous enkephalins function to inhibit the proliferation of activated thymocytes.

Defective membrane function in a patient with severe combined immunodeficiency disease.

We report a girl with severe combined immunodeficiency with functional impairment of both humoral and cellular immunity despite normal numbers of B and T lymphocytes. Immunologic studies revealed hypergammaglobulinaemia, absent migratory response by polymorphonuclear leucocytes to chemoattractants, and diminished lymphocyte proliferative responses to mitogens, antigens and allogeneic leucocytes. However, stimulation of the patient's mononuclear leucocytes with the calcium ionophore, A23187, resulted in a normal proliferative response. We therefore postulate a membrane defect as the basis for immunologic dysfunction in this child.

A longitudinal analysis of lymphocyte proliferative responses to mitogens and antigens during human pregnancy.

T-cell number and mitogen- and antigen-induced lymphocyte proliferative were assessed longitudinally in 18 normal human pregnancies to examine the effects of pregnancy on cellular immunity. The T-cell percentage and mitogen-induced responses did not change significantly in pregnant women as compared to nonpregnant, non-postpartum control adults. However, cell-mediated immune responses to three antigens were dramatically depressed during the third trimester and then returned to early pregnancy levels by 90 days post partum. This reduction in antigen-specific cellular immunity may be necessary to prevent rejection of the histoincompatible fetus by the mother and at the same time may render women in late gestation more susceptible to infection.

Cytomegalovirus-specific humoral and cellular immune responses in human pregnancy.

Cytomegalovirus (CMV)-specific humoral and cellular immunity was evaluated prospectively during and after 19 normal human pregnancies. Seropositive pregnant subjects had lymphocyte proliferative responses to purified CMV antigen that were markedly depressed by the end of the third trimester of pregnancy despite persistent levels of complement-fixing and immunofluorescent antibodies to CMV. These reduced lymphocyte proliferative responses returned to levels detected early in pregnancy by one year after delivery. None of the subjects excreted CMV during the study period. General parameters of cellular immunity, including thymus derived-cell counts as determined by formation of erythrocyte rosettes and mitogen-induced lymphocyte proliferation, were unaffected. Reactivation of latent CMV during pregnancy might be related to transient depression of CMV-specific cellular immunity.

Cytomegalovirus infection in infancy: virological and immunological studies.

Immunological and virological studies on 18 infants with cytomegalovirus (CMV) infection were performed. Eleven of these infants were studied on multiple occasions over a period of 1 year. The patients were divided into three clinical groups based on the probable time of infection and the resulting variation in clinical presentation. General parameters of cell-mediated immunity as determined by E-rosette formation and lymphocyte proliferative responses to mitogens and antigens were found to be normal. Quantitation of CMV excretion in urine, CMV-specific immunofluorescent (IF) and complement-fixing (CF) antibody titres and CMV-specific cell-mediated immune responses were done on all patients at approximately monthly intervals. Throughout the study period all patients continued to excrete CMV despite the presence of high antibody titres to the virus. CMV-specific lymphocyte proliferative responses were absent or diminished in 15 of the 18 patients. The immunological and virological status of all patients was similar regardless of the clinical manifestation of infection.