Steven Vroegop

T‐Cell Responses to Mls and to Bacterial Proteins that Mimic its Behavior#

The Mis locus was originally described by Festenstein (1973) as a single genetic locus with four alleles detected by the stimulation of a primary mixed lymphocyte reaction (MLR) between cells of strains identical at the major histocompatibility complex (MHC). The responses detected by Festenstein were at least as intense as those observed in MLRs between strains differing at the MHC, but differed from such responses in being unidirectional and in that no cytolytic T cells were generated. Furthermore, unlike the MHC polymorphisms that elicit primary MLR., to date it has been impossible to obtain antibodies that inhibit this response or define the product of this locus. The unique biology of this response has intrigued immunologists, leading to intensive study of many aspects of this system, as reviewed in this voltime and elsewhere (Butcher 1988, Janeway et al. 1988a, Abe & Hodes 1988). Perhaps the first question to ask about the Mis locus is: Why study it? The answer lies in the desire of immunobiologists to explain the characteristics of the T-cell response to Mis locus-disparate stimulator cells, which will be referred to simply as the T-cell response to Mis. These characteristics will be described in detail below; for the present, the most critical of these are the fmding that the T-cell response to Mis is unidirectional, stimulates about 20% of all T cells, and involves the recognition of class II MHC by the T-cell receptor (TCR);CD4 complex (see Janeway et al. 1988b), although the response does not show classical MHC restriction. A single genetic polymorphism that can stimulate such a large percent of the T-cell repertoire is certainly worth studying, as it may hold

Localization of damage induced by reactive oxygen species in cultured cells

N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single-hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process.

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus‐infected Spodoptera frugiperda (SF‐9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single‐step affinity chromatography procedure using a high‐affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF‐9 cells coded for pro‐NGF, the NGF recovered after purification was > 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3‐day primary response and a 1‐day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub‐maxillary glands.

Characterization of ultra-high affinity monoclonal antibodies with a dimeric, symmetrical antigen: Inhibition of the receptor recognition site of nerve growth factor

We generated a family of ultra-high affinity monoclonal antibodies (MAb) which inhibit competitively the binding of nerve growth factor (NGF) to its receptor. Preliminary experiments indicated that the dissociation constants (Kd) of some of the MAb:NGF complexes were substantially less than 0.1 nM. Conventional methods, such as ELISA and radioimmunoassays (RIA), were not sufficiently sensitive to measure the Kds of these MAb. Therefore, experimental conditions were developed to determine binding constants for these very high affinity MAb. The experiments establish that the Kds for our anti-NGF MAb range from 2.6 nM to 39 fM. Additionally, the inhibition of NGF binding to NGF-receptor by MAb is fully consistent with a purely competitive model but is not consistent with a model allowing the formation of a ternary complex of NGF, MAb, and NGF-receptor. One MAb, M4, immunoprecipitates NGF indicating interaction between each protomer of the NGF dimer and individual MAb molecules. We also evaluated the effects of mild denaturing conditions on the binding and biological activity of NGF and on recognition by the MAbs. Guanidine HCl or heat treatment of NGF resulted in only small, but significant, changes in binding or biological activity, in parallel with changes in recognition by the MAbs. However, binding, biological activity, and recognition by six of seven MAbs were completely eliminated by beta-mercaptoethanol reduction. Thus, our results are consistent with the MAbs interacting with the receptor recognition site on the surface of the NGF molecule. The high affinity MAbs will serve as sensitive probes of structural elements of NGF responsible for binding and biological activity.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than theKd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd=10−9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.

Inhibition of oxidative insult in cultured cells by a novel 6-chromanol-containing antioxidant

N18-RE-105 neuronal hybridoma cells were used in a cell culture system to evaluate the protective effects of a novel 6-chromanol-containing antioxidant, U78517F. First, the incorporation of the compound into the cells was evaluated, using a serum albumin carrier. Then the cells were exposed to peroxide-generating compounds, and the cell injury was estimated from the loss of alpha-aminoisobutyric acid (AIB) transport. We found that U78517F only protected the cells significantly when the degree of oxidative insult was below a certain limit; the measurable protection of cells by U78517F against either cumene hydroperoxide or H2O2 was limited to a narrow range of concentrations of the reactive oxygen species generator. Additionally, the protection provided by U78517F was largely localized to the cell membrane and did not extend to protection of mitochondrial function. The action of U78517 was fully consistent with a direct radical scavenging in the cells. The results indicate that the following factors must be taken into account for evaluation of antioxidants in cell culture: (a) the delivery of a compound to cells, especially when the compound is lipophilic; (b) the nature and extent of the oxidative insult used to evaluate protection; and (c) the location of the protective agent in the cells.

Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.