Envelope protein and p18(IIIB) peptide recognized by cytotoxic T lymphocytes from humans immunized with human immunodeficiency virus envelope.

Cytotoxic T cells are the main antigen-specific effector cells of the cellular immune system and MHC class I restricted cytotoxic T-lymphocyte (CTL) responses in mice, acting against the HIV-1 envelope protein, are known to be predominantly directed against an amino acid sequence in the third hypervariable domain. We have investigated the epitope specificity of anti-HIV-1 CTL in healthy human volunteers inoculated with a recombinant vaccinia expressing the HIV-1 gp160 envelope gene. Their isolated lymphocytes were stimulated in vitro with autologous HIV-1 infected cells. Our results show that immunization with recombinant virus is able to generate virus-specific CTLs to the HIV-1 gp160 envelope protein and to a 15-residue synthetic peptide corresponding to a highly variable region of the envelope p18(IIIB). The CTL response was restricted by class I MHC molecules HLA-A2 and A3 that commonly occur in the human population.

Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat LTR (HIV-1) responsive to steroid hormone stimulation.

Within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1) provirus there exists a steroid hormone-responsive element corresponding to the TGTTCT sequence identified as the glucocorticoid receptor binding element within the LTR of mouse mammary tumor virus. We have used an LTR(HIV-1)-chloramphenicol acetyl transferase (CAT) plasmid construct to transfect infected H9V3 and noninfected H9 cells. Four hours before harvest the cells were divided into two parts and half was treated with hydrocortisone (10(-7) M). The cells were harvested and washed, and the CAT activity was measured. In eight repeat experiments an increased expression of the CAT gene has consistently been observed in H9V3 cells in response to the glucocorticoid but no significant effect of the steroid was observed in noninfected cells. Double transfection of LTR(HIV-1)-TAT and LTR(HIV-1)-CAT into noninfected H9 cells results in a cell population in which the CAT gene was responsive to glucocorticoid stimulation. A time course and dose response for the steroid effect have been determined and the binding of steroid receptor fo the LTR-DNA characterized by gel retardation experiments.

Variable stringency hybridization of polymerase chain reaction amplified HIV-1 DNA fragments.

DNA isolated from the peripheral blood mononuclear cells of HIV-1 seropositive individuals was used for polymerase chain reaction (PCR) amplification of gag and envelope regions. Eight aliquots of the amplified DNA fragments have been subjected to Southern/dot blot analysis, hybridizing with 32P-labelled-BH10 (HIV-1 strain IIIB) at low stringency. After the filters had been autoradiographed, they were cut so that each hybridized band/dot could be subject to variable stringency washing using various ionic concentrations at a fixed temperature. The filter was reconstructed so that the effect of the variable stringency wash might be visualized following a second exposure to Kodak film. The level of activity for each band/dot was measured by counting the 32P or by densitometry analysis of the photographic record. The results allow a plot to be made of the decrease in bound radioactivity against ionic strength. By comparison with a standard curve obtained for HIV-1 strain IIIB amplified fragments subject to similar hybridization and analysis, an estimation of the degree of nucleotide mismatch relative to the BH10 DNA probe can be obtained. The technique provides a rapid means of characterizing PCR amplified fragments.

Characteristics of the calf uterine androgen receptor.

The highest molecular weight form of the calf uterine androgen receptor separates as an 11S form in glycerol gradients. This "cytosolic" receptor, prepared in the presence of molybdate, polyethyleneimide and low ionic strength, dissociates into 9S and 7.2S forms with increasing KCl concentration. A 4.5S androgen binding component appears as the predominant form of the receptor in the absence of polyethyleneimide and this unit quantitatively converts to a stable 3.5S form in the absence of molybdate. Renaturation of partially purified protein, separated by SDS-PAGE electrophoresis, demonstrates the presence of an androgen binding component in the 110 kDa region of the gel. This renatured protein separates as a 4.5S component in glycerol gradients and has a Stokes radius of 6 nm. Photoaffinity labelling of partially purified receptor preparations, followed by SDS-PAGE electrophoresis, reveals the presence of an androgen binding component having a molecular weight of 115 kDa. The binding characteristics and specificity of the receptor binding to R1881 have been studied and a DHT-affinity chromatography resin used to purify the receptor.

Expression of HIV antigens at the surface of infected T4 cells: immunoelectron microscopic evidence of an immunogenic phase prior to the viral release.

HIV antigens were detected by immunoelectron microscopy at the surface of human and simian T4 lymphocytes that had been infected in vitro. HIV antigens were detected at the surface of cells exhibiting viral particles but also at the surface of cells before the release of virions. The latter cells may be considered immunogenic since they are capable of triggering specific immune responses without the cytopathic effects due to viral release.

Use of Old World monkeys for acquired immunodeficiency syndrome research.

Mangabeys, macaques, and baboons persistently infected with human immunodeficiency virus (HIV)-2 NIH-DZ demonstrated no signs of immunodeficiency disease after 6-11 months following seroconversion. Thus Old World monkeys provide an animal model to investigate the effects of passive immunization (anti-HIV-2 antibodies) on HIV infection in primates.

Persistent HIV-2 infection of rhesus macaque, baboon, and mangabeys.

Six monkeys of three different species (mangabey, macaque and baboon) were infected with human immunodeficiency type 2 (HIV-2) NIH-DZ using intraperitoneal or intravenous injections of cell-free HIV-2 or autologous HIV-2-infected cells with no prior immunostimulation. Viral expression was demonstrated by reverse transcriptase activity in cells after coculture with human peripheral blood lymphocytes or by electron microscopy. Serum was analyzed by western blot, enzyme-linked immunosorbent assay (detection of antigen and antibody), and neutralization assay carried out using immunofluorescence techniques. The 6 inoculated animals seroconverted during the 1st month after inoculation and remained persistently infected after 6-11 months. We also observed proviral DNA by genomic analysis in the six tested samples. No sign of immunodeficiency disease has been observed so far. The data suggest that HIV-2 infection of nonhuman primates provides an acceptable animal model to investigate vaccination or specific immunotherapeutic procedures.

Role of cyclic AMP in steroid action in rat intestine.

We have investigated the effect of mineralocorticoids on intestinal fluid and electrogenic glucose-linked Na+ transport across isolated sections of the rat small intestine. A rapid in vitro response is observed that contrast with the delay normally associated with steroid hormone responses. Cyclic AMP is known to affect intestinal glucose, water and Na+ transport and the effects of the steroids may be understood in terms of an inhibitory effect on intestinal cyclic AMP production. An inhibitory effect of the steroids on membrane-bound adenylate cyclase has been demonstrated and dose-response effects suggest the presence of specific membrane-bound glucocorticoid receptors.

Developmental aspects of steroid-induced ammonia release from isolated sections of rat intestine.

Release of ammonia from isolated intestinal sections of adult male rats is higher than that measured using immature animals. The increase appears to be Na+ dependent and develops during the spurt of growth at puberty. Developmental changes in Na+-dependent ammonia release from isolated sections of the intestine and growth of the small intestine in male and female rats have been compared. Intestinal growth increases far more rapidly than body weight and in the males critical developmental changes occur early during weaning and during puberty. In females the major change is at weaning and little further change occurs during puberty. Treatment of young animals with aldosterone or testosterone increases the Na+-dependent ammonia release precociously. Dose-response effects of testosterone and aldosterone in distal sections of the small intestine have been compared.

Effects of saline exposure on the response of toad bladder (Bufo marinus) to aldosterone.

1. The short-circuit current response of toad bladders to high concentrations of aldosterone (2 x 10(-7) M) has been investigated following saline exposure of toads. 2. Limited saline exposure appears to enhance the aldosterone-stimulated response. 3. Additional saline exposure results in a reduced aldosterone response. 4. Overnight pre-incubation of isolated bladders proved a better pretreatment condition for study of an aldosterone short-circuit current response. 5. Plasma aldosterone concentrations and studies of [3H]aldosterone binding in the bladders have failed to demonstrate significant effects of saline exposure on endogenous aldosterone concentrations.

Aldosterone effects on renal metabolism.

1. Manometric studies of the sodium dependent oxygen consumption in rat kidney slices have failed to reveal any significant effect of aldosterone treatment, adrenalectomy or sodium diet on sodium diet on sodium metabolism. 2. However, ammonia release from kidney slices was significantly reduced following adrenalectomy and this decrease was influenced by aldosterone treatment. 3. The dose-response characteristic obtained for this aldosterone-stimulated ammonia release has been determined. 4. The effect of a high Na+ diet on the ammonia release has been studied. An initial decrease after 2 days may be associated with decreased endogenous aldosterone secretion. However, aldosterone (2.5 microgram/100 g body weight)injections into these high Na+ treated animals fails to restore the normal ammonia release. 5. The effects of aldosterone (2.5 microgram/100 g body weight), dexamethasone (2.5 microgram/100 g body weight) and corticosterone (2.5 microgram/100 g body weight) injections, in adrenalectomized rats, on ammonia release and tissue tyrosine aminotransferase activities have been compared.

Role of mitochondrial Ca2+ in antidiuretic hormone action.

The calcium ion concentration measured in rat kidney mitochondria, isolated from vasopressin treated tissue, has a dose response characteristic in which the calcium concentration reached a minimum at low doses of vasopressin (2 mU/ml), at higher doses of hormone the mitochondrial calcium ion concentration increases reaching a value close to that of the controls with vasopressin (100 mU/ml). This efflux and subsequent uptake of mitochondrial calcium has been shown to be a direct effect of the varying cyclic AMP concentrations. Sodium and water permeability effects of vasopressin have been shown in toad bladder to have different dose response characteristics. Maximum sodium transport occurs at a lower dose of vasopressin (2 mU/ml) and is believed to be associated with direct permeability effects of the hormone. Maximum water transport occurs at a higher dose of vasopressin (100 mU/ml) over a concentration range associated with hormone-stimulated adenylate cyclase activity. The water transport response to low doses of vasopressin may be potentiated by aldosterone treatment, an effect that can be related to the inhibition of tissue phosphodiesterase activity and subsequent increased cyclic AMP concentrations. In steroid depleted conditions the cyclic AMP medicate efflux of mitochondrial calcium ions, that occurs at low doses of vasopressin, may prevent the release of membrane bound calcium ions and thus inhibit the water permeability effect of the hormone. Higher levels of cyclic AMP reverse this inhibitory effect and give rise to an increased water flow. It is concluded that cyclic AMP and intracellular concentrations of calcium ion act as inter-related mediators of antidiuretic hormone action.

Effect of steroid depletion on the response of toad bladder to vasopressin.

1. We have investigated the water transport and short-circuit current (s.c.c.) response to vasopressin (1 mu./ml. and 100 mu./ml.) in isolated toad urinary bladders (Bufo marinus) following overnight incubation in the presence or absence of steroid-containing Ringer solution. 2. The water transport response to the lower dose of vasopressin (1 mu./ml.) was considerably reduced in 'steroid depleted' conditions, wheras the response to the higher dose of vasopressin (100 mu./ml.) was not similarly affected. 3. Aldosterone 3. Aldosterone 3. Aldosterone (10(-7)M) potentiated the water transport response to the lower dose of vasopressin (1 mu./ml.) but had no effect on the response to the higher dose (100 mu./ml.). 4. There was no effect of 'steroid depletion' or aldosterone treatment on the vasopressin s.c.c. response when measured as a percentage increase above basal levels. 5. In 'steroid depleted' conditions vasopressin (1 mu./ml.) maximally stimulated Na+ transport (s.c.c.) but a higher dose of vasopressin (100 mu./ml.) was required for maximum water transport. 6. We have failed to obtain any potentiation effect of corticosterone (10(-7)M) on the water transport or s.c.c. response to vasopressin (1 mu./ml.).