NIH Annual Intramural Research Report
Report Title
Studies of neurocysticercosis and animal cestode infections
2013 Fiscal Year
October 01, 2012 - September 30, 2013
Principal Investigator
Theodore Nash; MD
Research Organization
Gastrointestinal Parasites Section, NIAID
Collaborators from other NIAID organizations
Siddhartha Mahanty
Stephen Porcella; PhD
Collaborators from other NIH Institutes/Centers
William H Theodore; MD (NINDS)
John A Butman; MD, PhD (CC)
Masahiro Fujita (NIMH)
Dima Ali Hammoud (CC)
Robert Innis; MD (NIMH)
Extramural Collaborators
Javier A Bustos; MD (Department of Microbiology, Universidad Peruana Cayetano Heredia)
Christina Coyle; MD (College of Medicine, Albert Einstein)
Oscar Del Brutto; MD (Department of Neurological Sciences, Hospital Kennedy)
Hector H. Garcia; MD, PhD (Neurocysticercosis unit, Universidad Peruana Cayetano Heredia)
Robert H Gilman; MD (Johns Hopkins Bloomberg School of Public Health)
Isidro Gonzalez; MD (Cisticercosis Unit, Instituto Nacional de Ciencias Neurologicas)
E Houpt; MD (Div of Infectious Diseases, University of Virginia School of Medicine)
Andres G Lescano; MSc, MHS (Universidad Peruana Cayetano Heredia)
E Javier Pretell; MD (Cysticercosis Unit,, Instituto Especializado en Cie)
Herbert Saavedra; MD (Cisticercosis Unit, Instituto Nacional de Ciencias Neurologicas)
M Zimic; PhD (Laboratorio de Bioinformatica y Biologia Molecular, , Universidad Peruana Cayetano Heredia.)
Keywords
perilesional edema and seizures, Taenia solium, Cysticercosis, cestodes, seizures
Goals and Objectives
A large effort of the laboratory is devoted to various aspects concerning cysticercosis and neurocysticercosis( NCC), which is a major cause of epilepsy worldwide and also a cause of serious brain infections. Cysticercosis is caused by infection of the larval form of the tapeworm, Taenia solium, which is found in the human small intestines. Infectious ova are excreted by humans in their feces. After ingestion by pigs, the ova hatch, migrate by way of the blood vessels to the organs and develop into cysts mostly in the muscles. When cysts in poorly cooked or raw pork are ingested by humans they develop into tapeworms. Humans are accidently infected after they ingest ova that then develop into larval cysts. Almost all disease in humans is caused by brain infections. Seizures are the most common manifestation of NCC. They mostly occur when viable cysts, which can be present for long periods of time, die and incite a detrimental immune response commonly resulting in seizures. How best to control the inflammation due to treatment with anthelminthic medications or naturally degenerating cysts is a major goal. The second clinical setting occurs in the presence of calcified granulomas that are the end result of the inflammatory response to the parasite. Perilesional edema around calcified granulomas occurs in a significant proportion of patients and is frequently associated with seizures, mostly in rural endemic regions. This is a newly recognized phenomenon and is common. Questions include the nature of the immune response to the parasite, how calcified dead parasites initiate a likely immune response causing seizures and/or perilesional edema, how best to treat different forms of the parasitic infection, and how to prevent treatment related exacerbations of inflammation as a consequence of anthelminthic treatment. A third aspect of study is discovery of new drugs that can be used to treat neurocysticercosis and a major aspect of this is developing method to study efficacy of drugs in vitro so that they can be tested in animals. Understanding the pathophysiology of different aspects of the infection and disease is required to lessen the burden of disease worldwide and to effectively treat individuals who develop complicated disease. Our program is multifaceted and consists of components at NIH and in Lima, Peru. Both laboratory and clinical studies are performed at both sites. In Peru we take advantage of a large number of patients with NCC and in the laboratory we use naturally or experimentally infected pig, a resource that is limited to a few laboratories worldwide, to understand pathophysiology of disease and devise better treatments.
Summary
Seizures are the most common manifestation of neurocysticercosis (NCC) and understanding the pathophysiology is necessary to prevent and effectively treat them. The pig naturally infected model was employed to study treatment-induced inflammation that is a common cause of seizures in treated infected persons. The vital dye Evans Blue (EB) was injected to 11 pigs naturally infected with Taenia solium cysts to visualize disruption of the blood brain barrier (BBB). A total of 369 cysts were recovered from the 11 brains and classified according to the blue staining or lack of it of their capsules. The proportion of cysts with blue capsules was significantly higher in brains from pigs that had received anthelmintic treatment 48 and 120 h before the EB infusion, indicating a higher compromise of the blood brain barrier due to treatment. Measures to decrease BBB disruption and subsequent inflammation would be expected to prevent or control treatment induced seizures.

Perilesional edema around calcifications is a recently described cause of seizures. Studies performed by us earlier indicated that about 50% of seizures experienced by persons with NCC and only calcifications will show perilesional edema at the time of seizure activity. Whether edema is consequence of the seizure, or a result of host inflammation directed against parasite antigens or other processes is unknown. We imaged a marker of neuroinflammation, translocater protein (TSPO), using positron emission tomography (PET) and the selective ligand 11C-PBR28. In nine patients with perilesional edema, degenerating cyst or both, PET findings were compared to the corresponding magnetic resonance images. Degenerating cysts were also studied because unlike perilesional edema, degenerating cysts are known to have inflammation. In three of the nine patients, changes in 11C-PBR28 binding were also studied over time. 11C-PBR28 binding was compared to the contralateral un-affected region. Results 11C-PBR28 binding increased by a mean of 13% in perilesional edema or degenerating cysts (P =0*0005, n = 13 in nine patients). Among these 13 lesions, perilesional edema (n=10) showed a slightly smaller increase of 10% compared to the contralateral side (P = 0*005) than the three degenerating cysts. In five lesions with perilesional edema in which repeated measurements of 11C-PBR28 binding were done, increased binding lasted for 2 - 9 months. These studies strongly support an inflammatory etiology of perilesional edema. . The long duration of increased TSPO binding after resolution of the original perilesional edema and the pattern of periodic episodes is consistent with intermittent exacerbation from a continued baseline presence of low level
inflammation. Novel anti-inflammatory measures may be useful in the prevention or treatment of seizures in this population.

Another study suggested that one mechanism of activation of perilesional edema is acute corticosteroid withdrawal. We report 6 cases where acute withdrawal of corticosteroids precipitated perilesional edema around calcifications. Some of the calcifications showed neither edema nor enhancement before corticosteroids indicating that there is potential for inactive calcifications to show edema. Furthermore this suggests that perilesional edema is an immune mediated phenomenon. Whether perilesional edema results from new release or recognition of sequestered parasite antigen or inhibition of suppression is unclear.

Discovery of new anthelmintics with activity against T. solium has been limited by the lack of suitable sensitive assays that allow high throughput screening. Using an in vitro culture system with T. crassiceps metacestodes we demonstrate that changes in secretion of parasite-associated alkaline phosphatase (AP) and phophoglucose isomerase (PGI) can be used to detect and quantify anthelmintic effects of praziquantel (PZQ), a drug with activity against T. solium. We applied two enzyme release assays to screen for anti-T. crassiceps activity in non-conventional anti-parasitic drugs and demonstrate that nitazoxanide and artesunate induced release of both AP and PGI in a differing time and dose related patterns. Furthermore, imatinib, a tyrosine kinase inhibitor previously reported to have cidal activity against Schistosoma mansoni, also induced release of both AP and PGI in a dose dependent manner, similar in pattern to that observed with the other anthelmintics. We also evaluated release of adenine triphosphate (ATP) into cyst supernatants as an indicator of drug effects, but did not see any differences between treated and untreated cysts. These data provide the basis for rapid and quantitative screening assays for testing for anthelmintic activity in candidate anti-cestode agents
Publications Generated during the 2013 Reporting Period
See Project Bibliography

Ordered by author name.

1.
Guerra-Giraldez C, Marzal M, Cangalaya C, Balboa D, Orrego MA, Paredes A, Gonzales-Gustavson E, Arroyo G, García HH, González AE, Mahanty S, Nash TE, The cysticercosis working group in Peru (2013) Disruption of the blood-brain barrier in pigs naturally infected with Taenia solium, untreated and after anthelmintic treatment. Exp Parasitol, in press (e-pub ahead of print)
2.
Mahanty S, Madrid EM, Nash TE (2013) Quantitative screening for anticestode drugs based on changes in baseline enzyme secretion by Taenia crassiceps. Antimicrob Agents Chemother 57:990-5
3.
Nash TE, Mahanty S, Garcia HH (2013) Neurocysticercosis-more than a neglected disease. PLoS Negl Trop Dis 7:e1964
4.
Paredes A, de Campos Lourenço T, Marzal M, Rivera A, Dorny P, Mahanty S, Guerra-Giraldez C, García HH, Nash TE, Cass QB, Cysticercosis Working Group in Peru (2013) In vitro analysis of albendazole sulfoxide enantiomers shows that (+)-(R)-albendazole sulfoxide is the active enantiomer against Taenia solium. Antimicrob Agents Chemother 57:944-9