Our laboratory performs studies focused on the investigations of two aspects of human disease: the dementia induced by HIV, the virus responsible for AIDS; and the sexual dimorphism of autoimmunity, wherein women suffer disproportionately from the majority of autoimmune diseases. Our current experiments explore the etiology and pathogenesis of these disorders, as well as investigate basic biological mechanisms in the physiological systems affected by these conditions.

SIV model of neuroAIDS

In HIV infection, the HIV-1-associated cognitive/motor disorder, also known as the AIDS dementia complex or neuroAIDS, occurs in approximately one-third of patients. Symptoms range from a "minor" disorder, affecting 25% of individuals, to dementia, affecting 15 to 20% of those with AIDS. Although recent therapeutic advances have reduced mortality from HIV, most of these agents do not show significant penetration of the blood-brain barrier (BBB). The central nervous system (CNS) may remain a reservoir as well as suffer continued damage over the treatment-prolonged course of infection.

The etiology of the HIV-induced CNS alterations is unknown. We are studying the pathogenesis of neuroAIDS using the SIV/rhesus monkey model. Our working hypotheses are that viral infection of the brain, occurring via a "Trojan horse" mechanism of infected macrophages trafficking to the brain, initiates a sequence of events leading to neuronal dysfunction. However we believe that neurons, at least early, are not irreversibly damaged by a virus-induced pathological cascade. Thus HIV-induced CNS damage may be amenable to timely therapy to prevent the chronic consequence of CNS dysfunction. Using a multisystem approach, we are investigating the host and viral factors leading to CNS dysfunction, and investigating ways to prevent or ameliorate these untoward effects.

Viral factors

In order to obtain a reproducible infection of rhesus monkeys with SIV resulting in CNS effects, we performed a serial passage of microglia to enrich for a virus that could then infect the CNS of other monkeys by the intravenous route. Sequence analysis indicated that the serial passage resulted in an enrichment/selection of a viral quasispecies which correlated with the neuroinvasiveness of the virus and neuropathology. Furthermore, the tropism of the viral strain was altered in that it was now capable of infecting brain microvascular endothelial cells, primary components of the BBB. Potentially related to this result is our finding of sporadic disruption of the BBB throughout the course of SIV infection. The role of the BBB and cellular tropism of virus in SIV pathogenesis is the subject of current active investigation.

Host factors

In both HIV and SIV infection, numerous infiltrating macrophages can be found in the CNS. We have found an acute peak elevation of monocyte chemotactic protein 1 (MCP-1) in the CSF (and greatly increased CSF to plasma ratios of MCP-1) following SIV inoculation. A gradient of chemokine concentration is required to induce chemotaxis of immune cells; thus the increased level found in the CSF relative to plasma likely contributes to an initial influx of macrophages and possibly lymphocytes into the CNS. MCP-1 is indeed elevated in the CSF of those with AIDS dementia, and an early increase in MCP-1 may help seed the CNS with activated immune cells.

Interestingly, MCP-1 is a potent activator of cytotoxic T lymphocyte (CTL) activity. We have demonstrated that a cellular immune response, consisting of CTL specifically reactive against SIV, is present in the cerebrospinal fluid as early as one week post-viral inoculation. SIV-specific CTL could also isolated from brain parenchyma of SIV-infected macaques. Although crucial for the control of viral infection, the presence and activity of CTL in the CNS may contribute to neuronal dysfunction. The products of activated immune cells are prime candidates in mediating indirect damage to the CNS initiated by HIV infection.

Both resident cells of the CNS as well as these infiltrating immune cells can produce a variety of host defense molecules. We have found expression of a number of such molecules, including the pro-inflammatory cytokines IL-1b, TNF-a, and IFN-g, as well as the inducible form of nitric oxide synthase (iNOS) in the brains of SIV-infected monkeys. Interestingly, iNOS expression was later linked to clinical symptomatology in HIV-infected individuals with severe, rapidly progressive dementia. Again, while protective against the virus, such molecules can have deleterious effects on the CNS. In addition to viral factors, host factors likely play an important role in CNS pathogenesis. We have demonstrated that a cellular immune response, consisting of cytotoxic T lymphocytes (CTL) specifically reactive against SIV, are present in the cerebrospinal fluid as early as one week post-viral inoculation. SIV-specific CTL could also isolated from brain parenchyma of SIV-infected macaques. Although crucial for the control of viral infection, the presence and activity of CTL in the CNS may contribute to neuronal dysfunction.

Functional measures of the CNS

Many of the neuropsychological and neurophysiological tests that are performed on humans can be adapted to studying non-human primates. With our collaborators at Scripps we are making use of a variety of cognitive, motor, and neurophysiological methods to test CNS function in rhesus macaques. In order to probe CNS dysfunction resulting from SIV infection, we have utilized a variety of cognitive, motor, and neurophysiological tests. We have found that electrophysiological measurement of brainstem and cortical evoked potentials (EPs) yields a sensitive measure of CNS changes relatively early after infection. Within two months after viral inoculation, 75% of animals exhibit delayed latencies in the brainstem auditory evoked potentials (BSAEP), and all infected animals develop and maintain abnormalities in the BSAEP, as well as in the cortical AEP and VEP, over the course of infection. These data indicate that SIV infection induces reproducible alterations in neuronal circuitry.

Radiotelemetric methods were also adapted for their use in measuring physiological parameters (temperature and movement) in the SIV model of neuroAIDS. We found that, following the acute fever induced by infection, chronic SIV infection resulted in a distinct hyperthermia due to a consistent increase in the nighttime nadir of temperature. Infection also resulted in an acute decline in general motor activity, which then recovered to baseline, only to decline again by three months post-infection.

To further assess CNS function, a behavioral test battery was utilized in longitudinal studies. These studies have revealed that infection with the microglia-passaged SIV stock results in periods of poor performance over the course of infection as well as sustained abnormalities in the later stages of disease. In aggregate, our studies indicate that SIV infection affects an ensemble of functions controlled by the CNS.

Our findings of early detectable abnormalities (in EPs, movement, and temperature) following infection makes this model ideal for testing new therapies aimed at preventing or arresting cognitive decline induced by HIV. We have recently used treatment of monkeys with an anti-viral agent (PMPA) to determine if lowering systemic viral load in the monkeys can prevent, reverse, or ameliorate the CNS abnormalities that develop due to SIV infection. Indeed, the electrophysiological abnormalities were reversed by lowering viral load. In contrast, the movement decrease and body temperature increase were unaffected by this treatment. The drug used for treatment probably affected the virus directly only in the periphery, not within the CNS, because of its inability to effectively cross the BBB. Still, such treatment can be effective in preventing certain untoward effects of infection on the CNS since virally infected cells from the periphery can enter the CNS, and peripherally-produced host defense molecules can affect the CNS. Once viral load is lowered in the periphery, a normal turnover of virally infected cells in the CNS will lead to decreased virus in the CNS.

It is possible that the early seeding of the CNS by virus and/or the early influx of activated macrophages and other immune cells into the CNS result in changes that are unaffected by future drug treatment. Alternatively, the consequences of the continued viral-host interaction may inevitably lead to the movement and temperature alterations, regardless of the level of peripheral virus. Our findings support the assumptions that HIV infection can initiate damage to the CNS, and that the CNS disorders result from the indirect effects of viral infection.

Tight Junctions and the BBB

Our studies on SIV and the BBB led us to additional investigations on the BBB. Since we found gaps in our current knowledge on the molecular makeup of the BBB, we have initiated an in-depth molecular study of the BBB. Novel molecules have been identified which are expressed by the specialized BBB endothelial cells. One of these, named MBEC1, encodes a developmentally regulated membrane protein that is homologous to the Clostridium perfringens enterotoxin receptors. It is now clear that MBEC1 is a member of the claudin multigene family, and has been renamed claudin-5.

The claudins are four transmembrane spanning domain proteins that constitute tight junction (TJ) strands. TJs are key to the barrier function of the BBB, and lead to compartmentalization by creating a barrier for solute diffusion through the paracellular pathway. Endothelial cells vary in their organization of TJs. Although attempts have been made to correlate number and structure of tight junctions with tightness of the barrier, this relationship is not yet clear. We are exploring the effect of MBEC1/Cldn5 expression on TJ function. Mutational studies, investigating the role of different domains and specific amino acid residues of MBEC1/Cldn5 in TJ function, are underway. We have also produced transgenic mice expressing MBEC1/Cldn5 to probe its ability to induce TJs in vivo. The further characterization of MBEC1/Cldn5 and other novel molecules identified will lead to a better understanding of the role of the BBB in normal physiology and in disease states, including neuroAIDS.