Impact of Human Mutant TGFß1/Fc Protein on Memory and Regulatory T Cell Homeostasis Following Lymphodepletion in Nonhuman Primates.

Transforming growth factor ß1 (TGFß1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFß1/Fc (human IgG4 Fc) fusion protein on memory CD4+ and CD8+ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)-mediated lymphodepletion in monkeys. TGFß1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti-CD3/CD28 stimulation. In combination with IL-2, the incidence of CD4+ CD25hi Foxp3hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFß1/Fc were maintained between 2 and 7 µg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFß1/Fc infusion, CD8+ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4+ CD25hi CD127lo Treg increased at 4-6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4+ CD25hi Foxp3hi Treg/CD4+ IL-17+ cell ratios were reduced, particularly after stopping TGFß1/Fc infusion. Thus, human TGFß1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low-dose) IL-2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.

Dendritic cell therapies in transplantation revisited: deletion of recipient DCs deters the effect of therapeutic DCs.

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the alloresponse and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

Titer-dependent antagonism of cocaine following active immunization in rhesus monkeys.

Immunization may be a useful pharmacokinetic antagonist therapy for cocaine users. Three rhesus monkeys were immunized with a cocaine:bovine serum albumin conjugate in alum and later with complete and incomplete Freund's adjuvants. Monkeys developed cocaine-binding antibodies (as measured by enzyme-linked immunosorbent assay) after immunization with alum; greater antibody titers developed after immunization with Freund's adjuvants. The response rate-decreasing effect of cocaine diminished in proportion to antibody titer; there was no substantial change in the rate-decreasing effect of bupropion. Plasma cocaine concentrations increased in proportion to antibody titer. Immunizations were well tolerated and had no effect on response rates. These data suggest that the antibody response to a cocaine antigen can produce a specific pharmacokinetic shift in cocaine distribution sufficient to antagonize a behavioral effect of the drug, and can do so with minimal side effects.

Methocinnamox is a potent, long-lasting, and selective antagonist of morphine-mediated antinociception in the mouse: comparison with clocinnamox, beta-funaltrexamine, and beta-chlornaltrexamine.

The irreversible mu-opioid antagonists beta-funaltrexamine (beta-FNA) and beta-chlornaltrexamine (beta-CNA) are important pharmacological tools but have a kappa-agonist activity and, in the latter case, low selectivity. This work examines whether clocinnamox (C-CAM) and the newer analog, methocinnamox (M-CAM), represent improved long-lasting antagonists for examining mu-opioid-mediated effects in vivo. beta-FNA, beta-CNA, C-CAM, and M-CAM were compared after systemic administration in mice and in vitro. beta-FNA and beta-CNA were effective agonists in the writhing assay, reversible by the kappa-antagonist norbinaltorphimine. Neither C-CAM nor M-CAM had agonist activity in vivo. M-CAM was devoid of agonist action at cloned opioid receptors. All four compounds depressed the dose-effect curve for the mu-agonist morphine in the warm-water tail-withdrawal test 1 h after administration; at 48 h, recovery was evident. In the writhing assay, the dose-effect curve for morphine was shifted in a parallel fashion in the order M-CAM >> C-CAM > beta-CNA > or = beta-FNA. In comparison with their ability to shift the dose-effect curve for bremazocine (kappa) and BW373U86 (delta), beta-CNA was the least mu-selective, followed by C-CAM < beta-FNA < M-CAM. M-CAM (1.8 mg/kg) produced a 74-fold increase in the ED(50) of morphine while showing no effect on bremazocine or BW373U86 dose-response curves. In binding assays, C-CAM and M-CAM were 8-fold selective for mu- over kappa-receptors, whereas beta-FNA and beta-CNA were mu/delta-, but not mu/kappa, selective. However, ex vivo binding assays confirmed the mu-receptor selectivity of M-CAM. M-CAM is thus a potent, long-lasting, and specific antagonist at mu-receptors in vivo that lacks confounding agonist actions.

kappa-Opioid receptor effects of butorphanol in rhesus monkeys.

Butorphanol and nalbuphine have substantial affinity for mu and kappa-opioid receptor sites, yet their behavioral effects in monkeys are largely consistent with a mu receptor mechanism of action. Using ethylketocyclazocine (EKC) discrimination and diuresis assays in rhesus monkeys (Macaca mulatta), the purpose of the current investigation was to characterize the in vivo kappa-opioid activity of these compounds through the use of an insurmountable mu-opioid receptor antagonist, clocinnamox. Alone, butorphanol (0.001-0.032 mg/kg i.m.) failed to generalize to EKC, and pretreatment with the competitive opioid receptor antagonist quadazocine (0.1 or 0.32 mg/kg i.m.) did not alter this generalization. At 24 h after clocinnamox (0.1 mg/kg i.m.) administration, butorphanol fully generalized to EKC, and this generalization was maintained in two of three monkeys at 72 h. Parallel results were observed in diuresis: butorphanol alone and in the presence of quadazocine (1 mg/kg i.m.) did not alter urine output, and a marked diuretic effect was demonstrated 24 h to 2 weeks after clocinnamox administration. Clocinnamox did not alter the discriminative stimulus or diuretic effects of nalbuphine or of the kappa-opioid receptor agonists EKC or U69593. These results are consistent with an in vivo agonist activity of butorphanol at kappa-opioid receptors that can only be demonstrated when an insurmountable antagonist has substantially eliminated the dominant receptor population through which it exerts its action.

Further characterization of the quinine taste discrimination stimulus in rhesus monkeys: effects of inter-trial interval, number and distribution of test stimuli.

The purpose of the current investigation was to characterize further the taste discriminative stimulus properties of quinine. Using a food-maintained two-choice (quinine and water) discrimination paradigm, the effects of varying inter-trial intervals, and the number and distribution of test stimuli were examined in rhesus monkeys (Macaca mulatta). Monkeys were trained to respond under a chain schedule of reinforcement (FR5B-fluid, FR30B-food) with a 60-s inter-trial interval. Interestingly, the quinine discrimination remained invariant across all the conditions tested: alterations in inter-trial interval (0, 15, 30, 45, 60 s), alterations in the number of test stimuli (six at quarter-log unit increments [0.1-1 mg/ml] versus 10 at quarter-log unit increments [0.01-1 mg/ml]), and alterations in the distribution of the test stimuli (quarter-log unit increments [0.1-0.1 mg/ml] versus half-log unit increments [0.01-1 mg/ml]) failed to alter the quinine discriminative stimulus (ED50s ranged from 0.18-0.29 mg/ml). Furthermore, incorrect lever selections were very rare (less than 12% of all test sessions), independent of test stimuli order (i.e. quinine followed by water trials, water followed by quinine trials), and independent of the progress through the test session (i.e. incorrect lever selections were consistent--approximately one error for every 10 trials--within early, middle and late portions of the session). These results demonstrate the reliability and robustness of the quinine taste discriminative stimulus in rhesus monkeys and provide further validation for the experimental paradigm.

Functional characterization of the protease of human endogenous retrovirus, K10: can it complement HIV-1 protease?

To investigate the biochemical properties of the protease encoded by the human endogenous retrovirus, K10 (HERV-K), 213 amino acids of the 3'-end of the HERV-K protease (PR) open reading frame were expressed in Escherichia coli. Autocatalytic cleavage of the expressed polypeptide resulted in an 18.2 kDa protein which was shown to be proteolytically active against a fluorogenic peptide used as a substrate for HIV-1 protease. On the basis of sequence homology and molecular modeling, the 106 N-terminal amino acids of HERV-K PR were predicted to comprise a retroviral protease core domain. An 11.6 kDa protein corresponding to this region was expressed and shown to be a fully functional enzyme. The 11.6 kDa domain of HERV-K PR is unusually stable over a wide pH range, exhibits optimal catalytic activity between pH 4.0 and 5.0, and exists as a dimer at pH 7.0 with a Kd of 50 microM. Like HIV-1 PR, the HERV-K PR core domain is activated by high salt concentrations and processes HIV-1 matrix-capsid polyprotein at the authentic HIV-1 PR recognition site. However, both the 18.2 and 11.6 kDa forms of HERV-K PR were highly resistant to a number of clinically useful HIV-1 PR inhibitors, including ritonavir, indinavir, and saquinavir. This raises the possibility that HERV-K PR may complement HIV-1 PR during infection, and could have implications for protease inhibitor therapy and drug resistance.

Primary structure of bovine adenosine deaminase.

Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.