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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 1997-1998

Summary of Research Grants

In Vitro Evaluation of Pharmacological Interventions Aimed to Prevent Atherosclerosis
Rita B. Alevriadou, PhD
Johns Hopkins University, Baltimore, Maryland
Studies of animals with artificially-induced hypercholesterolaemia have shown arteries that contain retracted endothelial cells (ECs), which provide the sites for underlying macrophages and for the formation of mural platelet thrombi. The cellular events that occur in vessels of hypercholesterolaemic animals are exactly mirrored by those observed in human atherosclerotic coronary arteries in hearts removed in transplant operations. Early events may be summarized as follows:

  1. High concentrations of native low-density-lipoprotein (LDL) promote the adhesion of blood monocytes to the ECs, suggesting that the latter undergo activation upon such exposure. Oxygen radicals from activated ECs oxidize LDL (oxLDL). Oxidized LDL (oxLDL):
    • Greatly increases adherence and migration of monocytes and lymphocytes into the subendothelial space, by inducing formation of adhesive EC-surface glycoproteins (eg. vascular cell adhesion molecule, VCAM-1), secretion of chemoattractants (eg. monocyte chemotactic protein-1, MCP-1) and inhibition of nitric oxide (NO) release.
    • Initiates platelet interactions with ECs and with subendothelial proteins exposed in the gaps between injured ECs, by inducing a procoagulant EC activity, via expression of surface proteins (eg. tissue factor, TF) leading to thrombin generation, and via inhibition of NO release.

Using intravital microscopy in animals, different groups have demonstrated that antioxidants and NO donors separately reduced the oxLDL-induced leukocyte adherence. in vitro studies of atherogenesis (initiation of atherosclerosis) have been limited to exposing cultured ECs to minimally-modified LDL (MM-LDL), assaying for the EC proadhesive/procoagulant activities, and microscopically observing and measuring the number of monocytes suspended in culture media that adhere to LDL-treated EC monolayers. Recently, an in vitro study duplicated the in vivo finding of the beneficial effect of antioxidants on monocyte adhesion to simulated ECs. We propose to systematically evaluate the merits of pharmacological interventions that target key contributors of the early atherosclerotic process, using an in vitro model that allows us to assess the effect of MM-LDL on both the leukocyte and platelet adherence to endothelium.

Specifically, we will:

  • Develop an in vitro model of MM-LDL toxicity to human aortic ECs (HAECs). We will verify the proadhesive and procoagulant status of cultured HAECs, before and after MM-LDL exposure, by measuring reactive oxygen species generation (spectrophotometrically), NO and MCP-1 release (nitrate/nitrate assay and Western blot respectively) and formation of surface of TF (protein expression and activity assay). We will then perfuse heparinized human whole blood with both fluorescently-labeled leukocytes and platelets over HAEC monolayers, treated or not with MM-LDL, and employ epifluorescence video microscopy and digital image processing to quantify the extent of leukocyte and platelet deposition on and between ECs.
  • Use the model to compare the following agents that potentially protect ECs from atherogenesis:
    • Antioxidants HAECs will be pretreated withh either alpha-tocopherl or probucol before stimulation with MM-LDL, and the effect of antioxidants on leukocyte and platelet interactions from flowing whole blood with cultured and HAECs will be quantified.
    • NO donors Inhibition of NO is shown to increase MCP-1 expression and monocyte chemotactic activity. NO is also an inhibitor of platelet aggregation. Hence, incubation with NO donors, sodium nitroprusside or SIN-1, should restore the antiatherogenic EC properties.
    • Thrombin inhibitors Administration of TF pathway inhibitor (rTFPI) that inhibits thrombin generation will be tested.

This study aims to develop an in vitro assay for the better assessment of lipoprotein toxicity to vascular endothelium, since it will closely mimic the in vivo interactions between flowing blood cells and ECs. The assay has the potential to reduce the testing of antiatherogenic drugs in cholesterol-fed animals by pointing to the most promising treatment.
Experiments Using a Computer Model of the Immune System
Franco Celada, MD, PhD
Hospital for Joint Diseases-Rheumatology, New York City, New York
A strategic move towards limiting the use of animal experimentation to the strictly indispensable is to set up experimentation in the computer, at a scale that permits a demonstration of both the capabilities and the limitations of the new tool. One example in point is the model of the humoral immune system that we have constructed and refined over half a decade.

Our model is a cellular automaton simulation of the immune system, incorporating the core functions of its components (effector cells, helper cells and antigen presenters). The meeting of cells and molecules is probabilistic, governed by affinity and specificity (a repertoire of binary receptors will match a population of binary determinants), and each cell obeys a realistic set of rules; from the combination of these single actions, systematic phenomena emerge.

For example, immunization by antigen results in processing, presentation to T cells, T-B cell cooperation, clonal growth, antibody formation, affinity maturation by mutation and selection, and memory. Hypotheses are tested by modifying parameters or changing structural or behavioral rules. The verification of the results will still require biological experimentation, but now limited to promising directions, with substantial economy of animal lives.

With the aim of demonstrating and expanding the capabilities of the model, we propose to focus on one central activity of immunologists, the design of vaccines. The perfect vaccine should have the identical immunogenicity of the foreign invader but none of its aggressiveness (toxicity, invasiveness). This goal is never attained because e.g. any treatment of toxins that renders them harmless invariably lowers their degree of crossreaction with the wild type. This can happen by conformational changes or chemical actions on peptides or determinants.

We are going to simulate a number of conditions that can cause this immunogenicity gap, inject model vaccines with a fine degree of difference, study the effect on the titer and affinity of the antibody response and predict counteracting measures, in terms of dose and frequency of vaccine administration. We will also set up experiments to determine the conditions that can favor the outbreak of autoimmune responses by intermolecular help unexpectedly furnished by the antigen. Finally, we will study the critical parameters that may help mutable invaders to escape even a rapidly adapting response by the immune system.
Cellular-based Assays to Detect Toxic Agents and Elucidate the Underlying Genetic and Biochemical Mechanisms of Toxicity
Michael Scott Dubow, PhD
McGill University, Montreal, Quebec
The reduction and/or replacement of whole animals in the assessment of potential dangers is an increasingly coveted goal by the public, regulatory agencies and scientists. One of the major difficulties in the achievement of this goal resides in the fact that the underlying mechanisms of toxicity (which ultimately result in observable effects) in whole animals have been difficult to dissect, and thus reproduce in vitro. The major goal of our research is to identify and characterize the mechanisms of toxicity of (selected classes of) toxic compounds at the molecular level in such a manner that we obtain cellular and molecular-based assays for toxicity measurements, as well as important information for the prediction, treatment and ultimate elimination of the effects engendered by these chemicals.

The underlying tenet of our research is that cells will augment or repress the expression of specific genes, upon exposure to bioavailable doses of a toxic agent, in order to reorient cell physiology to cope with the stress. My laboratory has taken a two-pronged approach, using both bacterial (Escherichia coli) and human (HeLa) cells in culture to identify and characterize these genetically-programmed responses to chemical exposure. In bacteria, we prepared a collection ("library") of luciferase gene fusions (using the luciferase-encoding luxA,B genes from the marine bacterium Vibrio harveyi) and have identified a number of clones whose luminescence (and thus luciferase expression) is increased upon cellular exposure to important toxic compounds.

These clones are continuing to be validated for use as luminescent "biosensors" to detect particular toxic agents and, due to the luciferase "gene tagging" used in our approach, the toxin-inducible gene(s) and encoded proteins and regulatory circuits are being identified and characterized to elucidate the fundamental mechanisms of toxicity. We are also expanding our research using human cells in culture to extrapolate our knowledge in bacterial cell responses. Our approach is to isolate and identify poly(A)+mRNAs which are increased upon cellular exposure to aluminum, tributlytin and atrazine using the creation and cloning of magnetic bead-based "subtraction libraries" and the new technology of "differential display."

We have chosen to use HeLa cells as they are a fibroblast cell line, and this cell type represents the type of cell that would be first exposed to a particular toxic agent in a whole animal. In both approaches, the toxin-affected cDNAs are being cloned, identified by DNA sequence, and will ultimately be used to develop molecular probes for the elaboration of non-whole animal assays for toxicity assessment. In addition, we are continuing to screen a Lambda gt11 cDNA expression library (prepared from HeLa cells) for clones which produce human polypeptides that bind to (radiolabelled) phenol or atrazine.

These clones are being identified by "plaque hybridization" and will furnish potential targets for the characterization of the cytotoxicity of these compounds. In this manner, we will continue to use the tools of molecular biology and biotechnology for the development of cellular and molecular-based assays to provide quantitative measurements of biologically-relevant doses of a toxic agent and a concomitant reduction in the necessity of using whole animals for safety evaluation.
A Human Model for ACD
Jurij J. Hostynek, PhD
Euroamerican Technology Resources, Inc., Lafayette, California
Proposed overall is the development of a quantitative structure-activity relationship (QSAR) model which will predict the potential and potency for delayed type contact hypersensitivity (ACD) reactions upon skin (or systemic) contact with new or untested small molecular weight nonelectrolytes in man.

Prior to large scale development, production and commercialization of new chemicals, their potential to cause hypersensitivity disease upon dermal or systemic exposure requires investigation as part of the overall assessment of their potential for adverse health effects. In order to properly assess such risks to humans, identification of potential sensitizers currently involves the use of laboratory animals, mostly the guinea pig. However, conventional animal test methods used for risk assessment purposes are time consuming and costly, requiring a relatively large number of animals, and rely on a subjective evaluation of clinical results.

The discriminant technique of two-valued multiple regression analysis of molecular descriptors is known to successfully distinguish between allergens and non-allergens. Its application to analysis of clinical human sensitization data in particular will lead to a similar algorithm predictive of human sensitization potential that, if proven successful, will make traditional animal testing superfluous, ultimately taking animal models out of the testing loop.

In Phase II the predictive value of the model will be tested by analyzing randomly selected structures for their sensitizing ability, which had been set aside from the learning set for the purpose of validation. In Phase III, the predictive value of the QSAR model will be raised from a nes/no classification acheived in Phase I and confirmed in Phase II to that of a quantitative tool for the assessment of allergenic potency of small molecular weight chemicals. Through better resolution of clinical data on record, the relative potency of known allergens in the model will be categorized in distinct levels of ranking (non, weak, moderate, strong) and analyzed by Rank Transform Regression Analysis.
Fluorescent Probing of Oxidative Stress and Antioxidant Efficacy in a Cell Culture Model
Valerian E. Kagan, PhD
University of Pittsburgh, Pittsburgh, Pennsylvania
Our broad long-term objective is to use a cell culture model that we have perfected for evaluating oxidative stress induced by commercial chemicals to now test the efficacy of antioxidants as protective (therapeutic) agents. Both cell systems and whole animal models for testing toxic agents thought to act through oxidative stress suffer from a major drawback: metabolic turnover and repair mechanisms for lipids (a major target of oxidative stress) are extremely efficient. Hence, it is difficult to determine whether oxidative stress has actually occurred.

A further drawback in both animal and cell systems is lack of sensitive measurement techniques. The combination of these two deficiencies means that, at present, extremely large quantities of cells or many animals must be used or, alternatively, extremely large, unphysiological doses of oxidant must be applied. Our perfected model eliminates both these drawbacks, thereby providing a model for testing putative oxidants and antioxidants that is superior both to current animal testing and to current cell culture techniques.

In our first year, we completed the development of the model for testing oxidants. In the second year, we wish now to use it for testing the antioxidants. Our approach and model to test the effectiveness of antioxidants will have the following unique features:

  • It allows the testing of antioxidants (exogenous natural and synthetic antioxidants as well as endogenous metabolic antioxidant mechanisms) in biochemical environments resembling in vivo models i.e. with metabolic pathways of activation and/or repair intact.
  • It is extremely sensitive, allowing assessment of antioxidants at low doses of oxidative stress in sub-toxic exposures.

A New Approach to Modeling Contact Dermatitis
Phillip S. Magee, PhD
BIOSAR Research Project, Vallejo, California
Sensitization in allergic contact dermatitis (ACD) occurs when a hapten reacts with MHC Class 2 molecules on the surface of the Langerhans cell. This event modifies the site which enables a T2 lympocyte to recognize a normally benign resident as a modified stranger. This initial event is key to the entire subsequent process altering the immune system to later invasions of the hapten. It requires over 20 animals and more than one month to evaluate by the guinea pig maximization test and may or may not be applicable to man.

The major reactive event is clearly kinetic in nature and requires the hapten to access and react with the cell surface molecules during its brief residence in the viable epidermis. Should it bind in the stratum corneum (too reactive) or pass into the dermis as reactive (code = 1) or non-reactive (code = 0) when combined with mechanistic factors describing transport and binding, classify allergens and non-allergens about as well as clinical testing.

Our lab has developed two successful models, one of which is published, and these have been applied through several publications to drugs, pesticides and other models to describe the intensity of ACD (1-4, non, weak, moderate, strong). This has only been partially successful and the problem appears to be our use of dichotomous substructure descriptors which have only 0 and 1 values.

Recently, I discovered that quinone haptens could be modeled relatively by simulating the protein-hapten reaction with simple nucleophiles (MeO-, MeS-). The reactants and first intermediate (adduct) were modeled by the semi-empirical program, AM1. The heat of reaction is derived from the difference between computed reactant and product heats of formation. The values are large and well-spread, allowing a simple ranking of each quinone in reactivity by the exotherm of the model reaciton. In addition, I was able to generate correlations with electronic sigma values that permit the prediction of structures beyond the study set. This work was presented last summer at the Gordon Conference on QSAR.

There are approximately 40-50 different hapten substructures (ACD Alerts) that react with protein nucleophiles to form anionic intermediates. I propose to study each case by the same technique applied to the quinones with the purpose of developing a graded reactivity scale for each hapten structure. These relative values will represent a major step in converting qualitative concepts of haptenization into descriptors suitable for the study and prediction of graded responses.

Although some models have appeared in the literature in addition to our own, all are currently incapable of a graded response and animal testing remains the standard. The success of this proposal will not eliminate the animal test, but should go far in minimizing its routine use for non-commercial products.
R3-Organotypic (Raft) Epithelial Culture System for Contact Dermatitis Testing
Craig Meyers, PhD
Pennsylvania State University, Hershey, Pennsylvania
The general dogma that skin is merely a protective covering of the body is rapidly changing. New information from in vivo and in vitro studies provides compelling evidence that the keratinocyte can initiate and actively participate in diverse inflammatory reactions. In response to environmental insult, keratinocytes independently release several types of immunoregulatory molecules. Additionally, keratinocytes express adhesion molecules immunoinflammatory associated with reactions, such as the intercellular adhesion molecule-1 (ICAM-1). keratinocytes can play a pivotal role in allergic Therefore, contact dermatitis (ACD) as well as irritant contact (ICD). We have developed a working hypothesis that an in dermatitis vitro keratinocyte culture system is assessing commercial and therapeutic products to induce ACD and ICD.

A crucial component of testing our working hypothesis is using a proper in vitro keratinocyte system. An assortment of techniques have been tried by various investigators to culture epithelial cells in culture. The organotypic (raft) culture system has been shown to most accurately mimic the in vivo physiology of the epidermis. Growing epithelial cells in the raft system has allowed for a "complete" differentiation program in contrast to what is achieved in monolayer cultures. We propose to evaluate the human epithelial raft culture system for its efficacy as a mechanistic system to test toxicity of commercial and therapeutic products. To accomplish this we will define the expression of immunoregulatory molecules in human epithelial raft culture tissues following treatment with a contact dermatitis inducing chemical. We propose that a set of indices can be defined based on the expression patterns of interleukin-1a (IL-1a), IL-aB, IL-6, tumor necrosis factor-a (TNF-a), granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factor B (TGFB) and intercellular adhesion molecule-1 (ICAM-1) that will allow us to identify contact dermatitis-inducing compounds.

In Specific Aim 1 we will continue to define the expression of immunoregulatory molecules by human epithelial raft culture tissue. This will include:

  1. Using keratinocyte lines derived from different donors.
  2. Testing SLS and NS at continuous 10-fold dilutions to determine the sensitivity of the system.
  3. Comparisons of organotypic epithelial tissues with keratinocyte monolayer cultures of cytokine expression levels both transcriptionally and translationally.
  4. Beginning investigations with a second ICD inducing compound, phenol.
  5. Begining investigations with a second SCD inducing compound, urushiol.
  6. Continuing using immunohistochemistry to detect the spatial expression of the cytokines in the context of the three-dimensional tissue architecture.
  7. Using ELISA assays to gain a better measurement of the expression levels of the cytokines.

Our second Specific Aim will be to define the expression of IL-1a, IL-1B, IL-6, TNF-a, GM-CSF, TGFB, and ICAM-1 following multiple exposures with dilutions of SLS and NS that exhibit little or no effect on the epithelial tissue when applied only once. Techniques used to analyze multiple exposed tissues will be the same as used for Specific Aim 1.

The third Specific Aim will be to determine if organotypic epithelial tissues can recover from exposure to dermatitis inducing agents and regain normal epithelial stratified and differentiated phenotype. First, tissues will be treated with SLS or NS as described but following treatment some of the test samples will be incubated for greater time periods allowing more time for recovery. Second, SLS or NS will be applied to only half the organotypic tissue. This is to determine if the untreated epithelium can enhance the ability of the treated half to recover a normal differentiation phenotype. Techniques used to analyze multiply exposed tissues will be the same as used for Specific Aim 1.

Finally in Specific Aim 4 we will continue to study changes in the human epithelial differentiation program as a result of ICD and ACD induction. In correlation with the continued investigations of cytokine expression as proposed in Specific Aim 1, we will also continue to correlate morphological and biochemical changes in the differentiation program. This will include cataloguing disturbances in the differentiation phenotype. As the dilutions of the toxicants become greater we will determine which disturbances in the differentiation phenotype are the most sensitive to insult. We will determine if phenol and urushiol also disturb the differentiation phenotype similar to SLS and NS or are there significant differences.
Development of Brain Phantom Models for Head Injury Research
William W. Orrison, Jr., MD
New Mexico Reg. Federal Medical Center, Albuquerque, New Mexico
According to the Brain Injury Association, 2 million individuals in the United States sustain a traumatic brain injury (TBI) each year. Of those, 100,000 individuals die as a result of the trauma and approximately 80,000 suffer lifelong debilitation. There is a pressing need for improved understanding of the mechanisms of TBI and the development of tools to combat its occurence. This requires research into the basic types of head injury for which there are limited models other than animals.

Currently, TBI research is conducted on animals in the form of pre-planned brain lesion studies to determine predictive factors, and human subjects who have suffered TBI. Post-TBI studies on humans yield little information about what happens to the brain during traumatization. Through animal modeling of specific brain trauma, information can be obtained about particular consequences of experimentally identified forms of TBI. However, in addition to the inhumaneness of such forms of analysis, there are still severe limitations on the ability to visualize the brain during the act of traumatization. Due to these limitations, TBI remains extremely difficult to research.

The New Mexico Institute of Neuroimaging (NMIN) has performed extensive CT and MR evaluations of consequences of TBI. Additionally, NMIN staff are recognized leaders in the field of functional brain imaging. To begin study on what occurs during the event of human brain trauma, we have developed a prototype phantom brain model made of silicone mixtures which looks, feels and acts strikingly similar to real brain tissue.

We are requesting support to further modify the composition of the brain phantom so that it accurately mimics physical properties and MR analyses of real brains. While these phantoms would not approximate real brains in function, we feel that they could be very useful in the research of TBI through controlled analysis of the dynamics of trauma as it occurs. That data would then be compared to existing data on brain structure and that would in turn be analyzed against what we know about functional aspects of the brain. Our long range objective is for this technology to be used to alleviate the use of animals in lesion studies and to assist in the development of more functional protective devices and preventive strategies, as well as providing better diagnostic and treatment capabilities for patients suffering TBI.

Specifically, in the first year of the program, we propose to add to our existing brain models materials such as calcium and gadolinium to give the model physical properties similar to real brain tissue and to enhance the ability to obtain accurate MR and CT images. We will apply measurements of basic mechanical properties of stress and strain to the phantoms in order to determine if they possess similar physical properties of real brains, including shape memory. We will also conduct a review of the relaxation literature for the different types of brain tissue so that other properties can eventually be introduced into the phantom matrix.

Later phases of the program will include the addition of damage indicators, such as synthetic bruises in the form of microscopic vesicles of material that when disrupted, mix to form colors as with Ph sensitive dyes. Other augmentations will include the addition of more complicated structures including a brainstem and nuclei. In order to conduct these experiments we will also be developing clear skull models that will contain the brain in a closed system surrounded by synthetic cerebrospinal fluid and comprise accurate suspensions points. We anticipate that by using photolithography of real data we will be able to create accurate matches of real brain/skull systems. The models can then be used to investigate the dynamics as well as the physical effects of TBI, through evaluation by MRI and possibly high speed photographic techniques.
In Vitro Assay to Assess S. aureus Enterotoxin A Activity in Food
Linda Rassoly, PhD
Johns Hopkins University School of Public Health, Baltimore, Maryland
S. aureus enterotoxin A is one of the leading causes of food poisoning in North America with as little as 100 ng of SEA producing the symptoms. The most commonly used assay to measure SEA is an ELISA. The drawbacks of the ELISA assay are occurences of false positive results and inability to measure the activity of the toxin. The only test that measures the activity of SEA is an animal assay using kittens or monkeys. This test involves intravenous injection or reeding of monkeys or kittens with contaminated samples. The drawbacks of this animal assay are: low reliability, lack of quantitation, high cost, low sensitivity and requirement for animals and suitable animal facitilities.

that causes T cell proliferation, we are suggesting an alternative and far more sensitive method for SEA activity measurement. It does not require the use of animals and will specifically detect biologically active SEA (but not heat denatured SEA).

The purpose of this research proposal is to replace current animal assay for detection of S. aureus enterotoxin A (SEA) activity with a more sensitive non-radioactive in vitro cell proliferation assay. The specific goals are the isolation of SEA-reactive T cell clones; developing an in vitro proliferation assay using the T cell clones to measure SEA levels; testing suitability of a non-radioactive assay to replace the radioactive 3H-based proliferation assay in quantitation of SEA activity in food samples; and adjusting the assay to measure SEA activity in foods.
In vitro Assay for Hapten-Specific Priming of Human T Lymphocytes
Wayne J. Streilein, MD
Schepens Eye Research Institute, Boston, Massachusetts
Our initial goal has focused on generation and characterization of hapten-specific sensitized T cells with human PBMC. We have recruited 5 DNCB-sensitized donors and 10 naive volunteers for the present study. Sensitized donors had been previously immunized in vivo with DNCB; naive donors were never exposed to this hapten. 50 ml peripheral blood was collected from these donors. Each blood sample was diluted and overlayered on lymph-opaque, then centrifuged (at 1600 rpm for 30 min). Low density mononuclear cells at interface were harvested and divided in two samples for preparation of purified T cells and adherent PBMC respectively. One sample was subjected to human T cell-enrichment column. The other sample was either used directly as fresh PBMC or cultured overnight in RPMI 1640 with human recombinant GM-CSF (10 ng/ml) to generate cultured nonadherent PBMC. The fresh and cultured PBMC were then derivatized with 5mM DNCB in vitro. For hapten-specificity control, some fresh PBMC were derivatized with 0.1% oxazolone; negative control cells were treated with acetone alone.

Autologous mixed lymphocyte reactions (AMLR) were then set up using purified T cells as responder cells (10,000 cells/well) and fresh or cultured PBMC that had been derivatized or not, and irradiated (2000 rads) were used as stimulator cells. The AMLRs were maintained for 5 days. 48-hr cultured supernatants were collected and assayed for production of IFN-y and IL-4. During the terminal 18-hr of the 5 day culture period, 3H-thymidine was added to measure incorporation as indication of T cells proliferation. To prove that in vitro primed T cells are hapten-specific, we set up secondary cultures using in vitro activated T cells and freshly prepared hapten-derivatized PBMC. T cell proliferation and cytokine production were also detected in same way.

The results from representative experiments are presented at the end of this section of application. From Figure 1, we have defined that blood T cells from sensitized donors specifically respond to stimulation of fresh PBMC derivatized with the same hapten and secrete large amounts of IFN-y and little IL-4; From Figure 2, we have determined that after cultured with GM-CSF, DNCB-derivatized PBMC acquire capacity of activating hapten-specific autologous naive T cells; From Figure 1 and Figure 3, we have proved that in vitro primed T cells display functional properties similar to T cells from peripheral blood of hapten-sensitized donors. Based on these results, we can come to the following conclusions:

  1. T cells prepared from peripheral blood of DNCB-sensitized donors display proliferative responses in hapten-specific fashion upon restimulation with heptenated fresh PBMC. In addition, the cells' specific cytokine profile can be characterized: large amounts of of IFN-y are produced, by contrast, little if any IL-4 is found. However, fresh PBMC, whether DNCB-derivatized or not, fail to activate T cells harvested from naive donors.
  2. After in vitro culture with human recombinant GM-CSF, PBMC acquire potent accessory function. Whether derivatized or not, they can significantly activate autologous naive T cells. However, DNCB-conjugated PBMC induce significantly greater T cell proliferation than their non-derivatized counterparts. Similarly, significant amounts of IFN-y are detected in cultures stimulated by cultured PBMC which have been derivatized or not, although T cells activated by haptenated PBMC produced significantly more IFN-y.