Collins, Thomas R.
ARTICLE IN BRIEF
Investigators created a novel model for cryptococcal meningitis, which offered new information about how different viral strains affect morbidity and mortality.
NEW ORLEANS — A new mouse model of cryptococcal meningitis is producing observations — though early — about the way that different strains of the fungus affect intracranial pressure after being injected, researchers from Emory University said here at the annual meeting of the American Neurological Association in October.
The model is a novel foray into the understudied disease, which can kill HIV patients who are not protected with antiretroviral medications, as well as others who are immunocompromised. The disease is a large problem in places with poor access to HIV treatments such as Africa, though it can be a problem for those with weakened immune systems in the United States, too.
“It's a very common opportunistic infection and not that many neurologists or neuroscientists are studying it,” said Jeffrey Rumbaugh, MD, PhD, assistant professor of neurology at Emory, who has made the study of neurological infections in people with AIDS a primary focus of his work. “People who study it are mainly infectious disease doctors so I thought I could bring a different perspective from the neuroscience side of things.”
Patients with autoimmune diseases and transplantation patients could also be at risk, with rising use of immunosuppressive drugs and monoclonal antibodies.
“There's an increasing number of non-HIV patients who are getting, or are at risk of getting, cryptococcal meningitis,” Dr. Rumbaugh said.
Previously developed models of the disease generally have been made to study the pulmonary effects of the infection or focus on the neurological effects by direct injection of the fungus into the brain. But in the Emory model, the mice were injected in the tail vein.
“I thought that would be more physiologically relevant,” he said. And by skipping the lung and going straight to the bloodstream, researchers are able to bypass the pulmonary disease and systemic effects that might complicate assessing the neurological impact.
A prevailing hypothesis is that, after an immunocompromised person breathes it in, the fungus causes intracranial pressure to build, causing debilitation. This happens, the belief goes, because the fungus' large capsules — “a gelatinous goo,” as Dr. Rumbaugh put it — clogs the arachnoid granulations that are supposed to reabsorb cerebrospinal fluid (CSF) and get it back into the blood. When clogged, the body keeps producing CSF but it has nowhere to drain, so it builds up, with sometimes catastrophic effects.
HOW THE MODEL WAS DEVELOPED
The researchers injected three different strains of Cryptococcus neoformans, each producing different amounts of gelatinous capsules: the parent strain, H99; a strain that produces more capsules, called PKR1–33; and a strain that produces fewer, called CAP59. The non-parent strains differ from the parent strains by just one gene each — a gene that accounts for the different amount of capsule produced. The gene is not thought to have any role other than the degree of capsule product, Dr. Rumbaugh said.
“We wanted to see basically if we injected these three strains into the mice, does that affect their hydrocephalus, their morbidity, their mortality, etc.?” he said.
“We would hypothesize that if what everyone believes is correct — that capsule leads to obstruction of granulations — we would think that the organism that makes more capsule would obstruct granulations faster and those animals would get sick and or die faster.”
RESULTS FROM TESTING
But the researchers were surprised by the results. PKR1, the strain producing the most capsule, actually proved to be the least harmful of the strains.
Researchers rated the health of the mice each day after injection — with a rating of 5 for the healthiest, and 1 the sickest. By day six, all of the mice in the H99 group had descended to a rating of 2 or 3; and CAP59 mice were all rated 3 on that day.
The PKR1 mice, meanwhile, were all still observed to be healthy and were rated 5. And the PKR1 mice survived a greater total number of days after their injections than either of the other groups (H99 vs. CAP59, p<.05; CAP59 vs. PKR1, p<.001).
The mice injected with the PKR1 strain actually had less capsule in the brain than the other strains.
“Probably the reason PKR1, even though it makes more capsule on a per organism basis, (leads to) less capsule in the brain is because there's less organism in the brain,” Dr. Rumbaugh explained. “It's still possible that the hypothesis or belief that people have about capsule clogging the granulations is true, but it has to be measured not on a per-organism basis but rather on how much organism gets into the brain.”
It may be that the PKR1 strain has a more difficult time crossing the blood brain barrier, he suggested, and that is something he would like to study further.
But he and his team also made observations suggesting that the host response plays an important role. They found that the levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor-alpha — TH1 cells that have a protective effect against this infection — were found at the highest levels in the PKR1 mice. “This might also explain why PKR1 is less virulent, because the body's response to it is more protective than to the other organisms,” Dr. Rumbaugh said.
A greater understanding of how different strains affect the body might help yield better treatments in the long run, he said.
“If we can identify factors — either organism factors or host factors — that contribute to the elevated intracranial pressure, then perhaps we can develop a medication or treatment that would more specifically target those factors and prevent the morbidity and mortality of the disease.”
Arun Venkatesan, MD, PhD, assistant professor of neurology at Johns Hopkins University who studies infectious diseases that affect the nervous system, said that “approaches that permit dissection of genetic determinants of disease in either the host or the microbe are hugely important, as they may provide insights into pathogenesis of disease, disease susceptibility, and individualized treatment approaches.”
But he cautioned that these data are extremely preliminary, with just five mice in each group. Also, he said, the use of intravenous inoculation might limit the applicability of the model, since the fungus gains access to people when inhaled.
“Moreover, the authors do not report on whether there are differences in the levels of the three strains in the bloodstream — and if so, whether this might underlie the differences in virulence observed, rather than differential traversal of the blood brain barrier or local replication within the brain,” he said.
Correlating specific levels of intracranial pressure with the different genetic strains also would be helpful, he noted. Greater numbers of animals studied, and a more complete characterization of the disease course would help interpret these findings, Dr. Venkatesan said.
David Clifford, MD, professor of clinical neuropharmacology in neurology at the Washington University in St. Louis and principal investigator of the Neurologic AIDS Research Consortium, said cryptococcal meningitis is an understudied area that deserves more effort toward understanding “the specifics of why the brain is targeted by just a subset of organisms in certain circumstances.”
“Virulence studies are really important — it's remarkable the limited number of organisms that attack the brain and cause meningitis in general given the frequency of potential targets and the infrequency with which meningitis develops,” he said. “We gloss over it (the disease) in the US, but worldwide it's a huge problem.”