Laboratory research is the vital component of the biological sciences since they are empirical disciplines. Without laboratory work, a biological scientist can not succeed. My research projects have continued to make excellent progress here at Seton Hall University. I would like to state that all of my achievements could not have happened without the encouragement and collaboration of my Biology colleagues and Seton Hall’s administration. I am especially indebted to my research group, including my undergraduate and graduate students, research assistants, post-doc fellows, and collaborators because, without them, my successful research progress would not be possible. My research project has been funded by the National Institutes of Health (grant number DA 07058) since 1992, and was successfully renewed in March 1997 for five years. A renewal application of this award has been submitted for review at the current time. This grant support is the centerpiece of all my research-related activities.

A. Areas of Research

The overall goal of my research-in-progress is to delineate the effects of chronic opiate exposure on the penetration of the HIV-1 virus through the vascular endothelial cell (VEC) barrier in the immune-challenged individual. Although the VEC is effective at inhibiting penetration of most viruses, ample evidence has shown that the VEC fails to stop the invasion of HIV-1. Bacterial pathogens, which stimulate the production of the immunocytokine, interleukin-1-beta (IL-1ß), can also breach the blood-brain barrier (BBB) and invade the central nervous system (CNS) via paracellular and transcellular mechanisms. During the period since my last promotion application, we have shown that chronic morphine exposure potentiates
IL-1ß-mediated leukocyte-endothelial adhesion (LEA), thus, potentially promoting the transcellular migration of leukocytes across the VEC barrier. Morphine stimulates the hypothalamic-pituitary-adrenal (HPA) axis through activation of mu opioid receptors (MOR); whereas we have shown that chronic morphine inhibits IL-1ß-induced secretion of anti-inflammatory HPA products. Taken together, these data led us to hypothesize that opioid-dependent and IL-1ß-dependent pathways converge intracellularly; thus, morphine use, combined with inflammation, enhances the permeability of the vascular endothelial cell barrier, leading to increased penetration of HIV-1. Due to the limitations of the biological laboratory facilities at Seton Hall University, the HIV-1 work as described will be conducted via a collaborative setting with Dr. Jean Bell at the University of Edinburg in Scotland. The following specific aims and studies are designed to test our hypothesis.

Specific Aim 1:

To investigate the convergence of MOR- and IL-1 receptor-mediated pathways in vitro by determining if:

1. Morphine or IL-1ß modulates the activity of FOS in IL-1ß receptor positive Madin-Darby canine kidney (MDCK) cells stably transfected with MOR (designated MDCK-MOR1 cells).

2. Morphine or IL-1ß modulates the activity of NFkB in MDCK-MOR1 cells.

3. Treatment with morphine modulates MOR and IL-1ß receptor (IL-1R) expression in MDCK-MOR1 cells.

4. Treatment with IL-1ß modulates MOR and IL-1R expression in MDCK-MOR1 cells.

Specific Aim 2:

To characterize how opiate-dependent pathways are altered by bacterial and viral immune challenges in vivo by determining if:

1. Cytokines, such as IL-1ß, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), alter MOR expression and the levels of the endogenous opiate peptides, such as ß-endorphin and endomorphin, in the rat.

2. The bacterial endotoxin, lipopolysaccharide (LPS), alters MOR expression and endogenous opiate peptide levels in the rat.

3. There is a difference in expression of MOR and endogenous opiate peptide levels in uninfected versus HIV-1-infected post-mortem brain tissue.

Specific Aim 3:

To explore chronic morphine’s effects on bacterial-induced inflammation in vivo at the molecular, cellular, and systemic levels by determining if:

1. Morphine alters the LPS-induced plasma levels of pro-inflammatory molecules, such as IL-1ß, TNF-alpha, and IL-6, and anti-inflammatory agents, such as corticosterone, in the rat.

2. Morphine alters LPS-induced leukocyte-endothelial cell adhesion (LEA) and white blood cell flow (FLUX) in the rat.

3. Morphine promotes the progression of LPS-induced inflammation in response to endotoxin shock.

Specific Aim 4:

To delineate the effects of chronic morphine exposure and LPS-induced inflammation on the permeability of the vascular endothelial cell (VEC) barrier by determining if:

1. Morphine, LPS, or the combination of morphine and LPS enhances the permeability of the VEC barrier using an in vitro VEC barrier culture model.

2. Morphine, LPS, or combined morphine and LPS increases HIV-1 penetration of an in vitro VEC barrier model.

3. There is a difference in CD68 immunoreactivity, a marker for infiltrating macrophages, in post-mortem brain tissue from HIV-1-infected individuals with and without opiate use.

B. Grant Support

This is a $100,000 research instrumentation grant to pay, in part, for the following equipment: (1) Cytofluor 4000 Fluorescence Multi-Well Plate Reader w/ Temp. Control: (2) Fluorescent imager, and (3) ABI Prism 7000 Sequence Detection System. Not only will this funding provide the state-of-the-art tools needed so that my research project will further progress, but, as Chair of Biology, this will also allow me to continue to strengthen the research facilities and activities of the Department of Biology in order to enhance our recruitment competitiveness for both faculty and students.

As a National Institute on Drug Abuse (NIDA) RO1 award grantee, I am eligible to accept NIDA Summer Research Replacement awards for undergraduates. The above listed two grants were two administrative supplements to support Bernardo Felix and Jenine Anday, two minority students, who did research work in the summers of 1998 and 1999, respectively. Following their summer research in my laboratory, both Jenine Anday and Bernardo Felix decided to continue to pursue a Master’s degree in the Department of Biology at Seton Hall University.

As a National Institute on Drug Abuse (NIDA) RO1 award grantee, I am also eligible to apply for supplemental support for under-represented minorities for their graduate study in my laboratory. The above listed two grants are funding to support Bernardo Felix and Jenine Anday while working on their Master’s degree in my laboratory. Bernardo Felix is now a Research Associate at Merck, Inc., and Jenine Anday is a Doctoral degree student at the University of Connecticut Medical School.

C. Research Related Professional Presentations

1. Presentations of papers at meetings:

2. Invited speaker at symposia (1998 to 2001):