Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.

In evaluating current combination drug regimens for treatment of human immunodeficiency virus (HIV) disease, it is important to determine the existence of viral reservoirs. After depletion of CD8 cells from the peripheral blood mononuclear cells (PBMCs) of both patients and normal donors, activation of patient CD4 lymphocytes with immobilized antibodies to CD3 and CD28 enabled the isolation of virus from PBMCs of six patients despite the suppression of their plasma HIV RNA to fewer than 50 copies per milliliter for up to 2 years. Partial sequencing of HIV pol revealed no new drug resistance mutations or discernible evolution, providing evidence for viral latency rather than drug failure.

PACAP and VIP stimulate Ca2+ oscillations in rat gonadotrophs through the PACAP/VIP type 1 receptor (PVR1) linked to a pertussis toxin-insensitive G-protein and the activation of phospholipase C-beta.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are hypothalamic factors that play roles in the regulation of anterior pituitary cell activity. PACAP exists in 2 forms physiologically, a 38 amino acid form (PACAP38) and a form possessing the N-terminal 27 amino acids of PACAP38 (PACAP27). We have previously shown that PACAP38 stimulates an increase in [Ca2+]i in rat gonadotrophs. In an attempt to identify the PACAP receptor type underlying this effect, we compared the potency of PACAP38, PACAP27 and VIP to stimulate Ca2+ changes in identified single rat gonadotrophs. All 3 peptides at 100 nM were capable of stimulating high amplitude Ca2+ oscillations, which were also observed in the absence of extracellular Ca2+. The order of potency of these peptides was PACAP38 > PACAP27 > VIP, and a potent antagonist of the PACAP/VIP type II binding site ([4-CI-D-Phe6, Leu17]-VIP) failed to block these responses, suggesting that these effects are mediated through a PACAP/VIP type 1 receptor (PVR1). The Ca2+ responses to PACAP38 and VIP were unaffected by overnight treatment of the cells with pertussis toxin (PTX; 250 ng/ml) indicating that these responses are mediated by a PTX-insensitive G-protein. Finally, the Ca2+ responses stimulated by PACAP38 and VIP were blocked by the phospholipase C-beta blocker U73122 (5 microM). In summary, PACAP stimulates Ca2+ oscillations in rat gonadotrophs through the activation of the PVR1 linked to a PTX-insensitive G-protein and the activation of phospholipase C-beta. VIP can stimulate the same pathway in rat gonadotrophs, although it is at least 100 fold less potent than PACAP38.

PACAP/VIP receptor subtypes, signal transducers, and effectors in pituitary cells.

Rat anterior pituitary tissue expresses mRNA for PVR1 and PVR3, as well as a low level of PVR2. The PVR1 appears to be highly expressed in gonadotroph-like cells, while somatotroph-like cells apparently express the PVR3. We have recently demonstrated the expression of mRNA for both PVR2 and PVR3 in corticotroph-like AtT20 cells (FIG.3). If normal corticotrophs express the same mRNA as AtT20 cells, this may partly explain the low levels of PVR2 seen in normal pituitary tissue. Significant levels of at least two PVR1 splice variants mRNAs (PVR1s and PVR1hop) were expressed in clonal gonadotroph-like alpha T3-1 cells and normal rat anterior pituitary tissue. However, these splice variants are reported to have almost identical pharmacological characteristics in terms of binding, and the activation of AC and PLC. Further experiments are necessary to determine the functional consequences of differential splice variant expression in such cells. Interestingly, all three pituitary-cell lines studied expressed mRNA for the PVR3 (FIG.3), whereas earlier binding studies demonstrate a predominance of PACAP-preferring binding sites on normal anterior pituitary-cell membranes. In addition, it is clear that the different PVR subtypes can couple to different intracellular messenger systems. Thus it will be important to determine the expression of the different PVR subtypes in normal anterior pituitary-cell types if we are to begin to understand the regulation of pituitary-cell regulation by PACAP. Such questions form the basis of some of the ongoing studies in our laboratory.

Stimulation of Ca2+ influx in alpha T3-1 gonadotrophs via the cAMP/PKA signaling system.

To investigate the regulation of free cytosolic calcium concentration ([Ca2+]i) by the adenosine 3',5'-cyclic monophosphate (cAMP) signaling system in clonal gonadotrophs, microfluorimetric recordings were made in single indo 1-loaded alpha T3-1 cells. Forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or a low concentration (100 pM) of the hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated Ca2+ step responses or repetitive Ca2+ transients, which were blocked by the removal of extracellular Ca2+ by the dihydropyridine (DHP) (+)PN 200-110 or by preincubation with the protein kinase A (PKA) antagonist H-89 (10 microM). Thus activation of the cAMP/PKA system in alpha T3-1 gonadotrophs stimulates Ca2+ influx through DHP-sensitive (L-type) Ca2+ channels. In contrast, high PACAP concentrations (100 nM) stimulated biphasic Ca2+ spike-plateau responses. The Ca2+ spike was independent of extracellular Ca2+, and similar responses were observed by microperfusion of individual cells with D-myo-inositol 1,4,5-trisphosphate, suggesting the involvement of the phospholipase C (PLC) signaling pathway. The Ca2+ plateau depended on Ca2+ influx, was blocked by (+)PN 200-110, but was only partially blocked by H-89 pretreatment. In conclusion, PACAP stimulates [Ca2+]i increases in alpha T3-1 gonadotrophs through both the PLC and adenylate cyclase signaling pathways. Furthermore, this is the first clear demonstration that the cAMP/PKA system can mediate changes in [Ca2+]i in gonadotroph-like cells.

The guanine-nucleotide-binding protein subunit G alpha i2 is involved in calcium activation of phospholipase A2. Effects of the dominant negative G alpha i2 mutant, [G203T]G alpha i2, on activation of phospholipase A2 in Chinese hamster ovary cells.

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.

Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1.

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcgammaR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcgammaR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.

Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy.

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1 env were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.