Cytokines, MMP-2, and MMP-9 levels in patients with a solitary cysticercus granuloma
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.156279
Source of Support: None, Conflict of Interest: None
Objectives: In patients with neurocysticercosis, perilesional inflammatory activity is thought to be responsible for seizures. This study was designed to evaluate the serum and cerebrospinal fluid (CSF) concentrations of cytokines as well as matrix metalloproteinase (MMP)-2 and MMP-9 in patients with a solitary cysticercus granuloma.
Keywords: Cysticercosis; epilepsy; matrix metalloproteinase; neurocysticercosis; seizures; Taenia solium
A solitary cysticercus granuloma is considered as the most frequent variety of neurocysticercosis in India and is also the most frequent neuroimaging abnormality seen in patients with new-onset seizures. A solitary cysticercus granuloma is seen on a contrast enhanced computed tomographic scan as an enhancing, ring-shaped lesion. These enhancing lesions are usually less than 20 mm in diameter and may be surrounded by varying amounts of perifocal edema. In its natural course, a solitary cysticercus granuloma frequently disappears spontaneously; recurrences of the solitary form after resolution of the initial granuloma have, however, been reported. ,,,,,,,,,,
In the brain parenchyma, the cysticercus larva passes through four distinct stages. In the vesicular stage, the larva is in the form of a cyst filled with clear fluid. There is an eccentric scolex as well. The vesicular cysts are viable and are not associated with any inflammatory changes in the cyst wall or the surrounding brain tissue. In the colloidal stage, inflammatory changes develop in the cyst wall and in brain tissue. The cystic fluid gets converted into a jellylike material while the cyst wall becomes thick and fibrous. Later, mineralization of the cystic fluid and degeneration of the scolex leads to evolution of the granular-nodular stage. Ultimately the cyst gets mineralized in its entirety, resulting in the calcific stage. A solitary cysticercus granuloma usually represents either the colloidal stage or the "granular-nodular" stage. ,,,,,,,,, In patients with neurocysticercosis, perilesional inflammatory activity is thought to be responsible for seizures.  Similar to what has been suggested with focal cerebral calcifications (presumably related to neurocysticercosis), the presence of gliosis consequent to resolution of the granuloma may lead to recurrence of seizures in patients with a solitary cysticercus granuloma.  The resolution of the granuloma may also be affected by the use of albendazole but its exact role and the specific duration of usage still remains controversial. 
The T-helper type 1 (Th-1) cytokines (like interleukin (IL)-1β, IL-6 and tissue necrosis factor (TNF)-α) response is associated with a robust immune reaction against the neurocysticercal lesions. It is, however, also responsible for host tissue damage. In contrast to this, the viable parasites induce T-helper type 2 (Th-2) regulatory cytokines like IL-10 that help in avoiding the host inflammatory response and therefore, remain asymptomatic.  The murine model studies have shown the role of IL-1β in increasing seizure susceptibility after a central nervous system insult.  Seizures per se can cause elevation of pro- as well as anti-inflammatory cytokines.  Matrix metalloproteinase (MMP) are the zinc dependent endopeptidases responsible for degradation of the matrix as well as the non-matrix proteins. MMP-2 and MMP-9 belong to the family of gelatinase and are known as gelatinase A and gelatinase B, respectively.  Studies have shown that MMP-2 and MMP-9 play a crucial role in the breakdown of blood brain barrier and in the migration of leucocytes in the central nervous system diseases such as multiple sclerosis and tuberculous meningitis. , In central nervous system tuberculosis, pro-inflammatory cytokines IL-1β and interferon-γ have been demonstrated to up regulate the MMP-9 level. The latter has been postulated to play a significant role in causing brain tissue damage.  Recent studies have revealed the role of MMP-2 and MMP-9 in parasitic infections like Toxoplasma gondii in generating an astroglial reaction and in the recruitment of leucocytes.  There is emerging evidence that MMP-2 and MMP-9 may play a significant role in the symptomatology of patients with multiple neurocysticercosis. 
In this study, we estimated the serum and CSF cytokines and MMP-2 and 9 levels in patients with a solitary cysticercus granuloma. We also assessed the possible role of baseline cytokines and MMPs on the resolution, persistence or calcification of these lesions.
This case-control study was carried out in a tertiary care hospital from 2009 to 2013. The ethical approval was obtained from the Institutional Ethics Committee. The written informed consent was taken from all the cases and controls (or their guardians), enrolled in the study.
Consecutive patients with new onset seizures (presenting within 7 days of the first seizure) were screened using contrast-enhanced computed tomography of the brain. A solitary cysticercus granuloma was diagnosed on the basis of criteria given by Rajshekhar and co-workers. , All our patients had a solitary ring-enhancing lesion measuring less than 20 mm with or without the presence of perilesional edema. , [Figure 1].
The patients with systemic diseases, malignancy, tuberculosis, HIV, pregnancy, progressive neurological deficit, raised intracranial tension and hydrocephalus were excluded from the study. Patients with a history of contrast sensitivity or a contraindication to the administration of contrast were also excluded.
Control CSF and blood samples were obtained from persons undergoing spinal anesthesia for obstructive uropathy and having no clinically active infection.
A detailed clinical history was obtained and neurological examination performed in all included subjects. The laboratory tests like complete hemogram, erythrocyte sedimentation rate, fasting blood sugar, renal function tests, liver function tests, C-reactive protein and enzyme-linked immunosorbent assay for human immunodeficiency virus were performed. The site, size and stage of the granuloma were noted along with the presence or absence of perilesional edema. An electroencephalography (EEG), using the 10-20 international system of electrode placement, was also performed.
The sera was separated by centrifugation at 1000 g for 30 minutes; and, aliquots were prepared and immediately stored at −80°C for cytokine analysis. The CSF was collected using a sterile lumbar puncture technique. It was processed immediately for cell count, proteins and sugar levels and the CSF aliquots were prepared and similarly stored at -80°C for the cytokine analysis. The parameters that were analyzed included TNF α, IL-1β, IL-5, IL-6, IL-10, MMP-2 and MMP-9.The tests were done using commercially available human ELISA kits as per the manufacturer's instructions (Bio Legend Max™, San Diego, CA, USA). The ELISA tests were performed in duplicates independently for each sample and the results were expressed as picogram/ml (pg/ml) for all cytokines and MMP-9. The results of MMP-2 were expressed in nanogram/ml (ng/ml).
The patients were treated with oxcarbazepine, 10-15mg/kg/day in two divided doses. Those patients who had a recurrence of seizures despite being on treatment were given the additional anticonvulsant, clobazam. In case of seizure recurrence, the patients were advised to report immediately to the investigator-in-charge. None of the patients were treated with albendazole or corticosteroids.
A follow-up computed tomography was done in all the patients at an interval of 6 months. The patients demonstrating partial or incomplete resolution of the lesion were considered to have a persisting lesion. The lesion was considered as "resolved" if the follow up computed tomography was reported as normal. The lesion was considered as "calcified" if the follow up imaging showed a hyperdense lesion in place of the granuloma.
Seizure recurrence was defined as occurrence of another seizure at least 1 week after the institution of the antiepileptic therapy. Status epilepticus was defined either as a continuous seizure activity or as a repetitive seizure activity without resumption of consciousness over a 30 minute period . Three or more seizure episodes were classified as multiple seizure recurrences. Serial seizures were defined as multiple seizures occurring within 24 hours.
A sample size of 47 (using a two-sided hypothesis test with a significance level of 0.05, and considering a non-response rate of 20%) was calculated. The entire clinical and the laboratory data was analysed for the normalcy of distribution using the Shapiro-Wilk test. The univariate analysis of non-parametric data was done by using the Mann-Whitney U test. Univariate analysis of the serum and CSF concentrations of TNF α, IL-1β, IL-5, IL-6, IL-10, MMP-2 and MMP-9 between the case and control groups was done using the Mann-Whitney U test. The groups based on the radiological outcome were compared in paired groups such as, persistence of the cyst versus its resolution, resolution of the cyst versus its calcification, and persistence of the cyst versus its calcification. The multivariate analysis was done using the binary logistic regression test. For parametric data, proportions were compared using the Chi-square test. The analysis was carried using SPSS version 16 and a P ≤ 0.05 was considered significant.
Of the 104 patients with new onset seizures, 57 patients were excluded. 31 patients had more than one granuloma or a conglomerate of lesions. Twenty two patients presented beyond 7 days of onset of the seizures, and 4 patients denied consent for a lumbar puncture. Therefore, 47 patients (30 males and 17 females) were enrolled in the study [Figure 2].
Baseline clinical features
The baseline demographic, clinical and laboratory characteristics of the included patients have been provided in [Table 1].
Baseline cytokines parameters
The median levels of cytokines IL-1β, IL-5, IL-6, IL-10 and TNF-α, MMP-2 and MMP-9 were significantly elevated in serum and CSF of patients with a solitary cysticercus granuloma compared with that of the controls [Table 2].
Outcome analysis after 6 months of follow up
The follow up CT revealed that in 27 (57.45%) patients, the lesions were calcified. There was a complete resolution of the lesion in 5 (10.64%) patients. In 15 (31.91%) patients, the lesions remained unchanged.
Fourteen patients had a recurrence of a single or of multiple seizures. The seizure recurrence was found to be more common in patients with EEG abnormalities, and those with high TNF-α and IL-1β levels. The Kaplan Meier analysis revealed a non-significant higher cumulative risk for seizure recurrence in the calcification group [Figure 3]. A high baseline serum TNF-α level was independently associated with seizure recurrence (P = 0.021, OR = 1.041, CI = 1.006 to 1.078).
A high baseline CSF MMP-2 and a high TNF-α level were associated with persistence of the granuloma. Complete resolution of the lesion was associated with a high baseline serum MMP-2, IL-6 levels and a low baseline CSF IL-10 level. In the "calcified" lesion group, only IL-1β was found to be significantly elevated [Table 3] and [Table 4].
We observed that serum and CSF levels of the cytokines (IL-1β, IL-5, IL-6, IL-10 and TNF-α) and MMP-2 and MMP-9 were markedly elevated in our patients with a solitary cysticercus granuloma when compared with controls. Our findings, in fact, indicated that both systemic as well as local inflammatory changes were involved in the pathogenesis of the seizures precipitated by a solitary cysticercus granuloma. Histopathologically, the cysticercus granuloma comprises of a dying cyst surrounded by fibrosis, angiogenesis, and an infiltration of inflammatory cells. The most abundant cell types are the plasma cells, B and T lymphocytes, macrophages, and mast cells. The proinflammatory cytokines (TNF-alpha and IL-1) play a crucial role in the granuloma formation. In a cysticercus granuloma, plenty of both T helper type 1 cytokines and T helper type 2 cytokines have been demonstrated.  Viable parasites induce T helper type 2 cells and regulatory cytokines, and suppress the host T helper type 1 response. In contrast, death of the cysticerci is associated predominantly with T helper type 1 cytokines.  The brain tissue surrounding the cyst also demonstrates inflammatory changes. Brain specimens were demonstrated to be having a proinflammatory activity as reflected by the altered blood-brain barrier permeability, secretion of pro-inflammatory cytokines, and the up-regulation of molecules responsible for antigenic presentation. There were anti-inflammatory cytokines, and an active wound-healing process, as reflected by angiogenesis, collagen deposition and glial scar formation.  Perhaps, some genetic factors trigger the inflammatory reaction in patients with neurocysticercosis. In a study, Toll-like receptor 4 polymorphisms were found to be associated with symptomatic neurocysticercosis. 
We observed that MMP-2 and MMP-9 were markedly elevated in patients with a solitary cysticercus granuloma. MMPs also play a significant role in the generation or perpetuation of these inflammatory changes. MMPs accomplish tissue remodelling via degradation of the extracellular matrix. Integrity or disruption of the blood brain barrier plays a crucial role in the pathogenesis of inflammatory changes seen in the cysticercus granuloma. MMP-2 and MMP-9 are two of the most widely studied enzymes that have been shown to be critical in regulating blood-brain barrier permeability. Several studies in animals have suggested that breakdown of the blood-brain barrier occurs because of the high activity of MMP-9.  In humans, too, MMP-2 and MMP-9 have been shown to play an important role in the development of symptoms. 
We observed that higher levels CSF IL-1β and serum TNF-α were associated with seizure recurrence in patients with a solitary cysticercus granuloma who present with an acute seizure disorder. Recurrent seizures and Todd's paralysis (that quickly resolves), are frequently observed. The seizures in neurocysticercosis are thought to result from the inflammatory response to the release of parasitic antigens at the time of transformation of the vesicular stage to the inflammatory colloidal stage. Is it possible that an inflammatory substance produced by the cysticercus granuloma initiates the epileptiform activity? In a study, several pieces of granulomas (Taenia crassiceps) were removed from the peritoneal cavity of mice. A piece of the granuloma was used for histological staging of the dying parasite and the second piece was used to generate extracts, which were injected into the hippocampus of rat. Seizures were noted after injection of the extracts prepared from the early-stage granulomas, but not with those from the late-stage granulomas. This observation suggested that there was an epileptogenic element in the early-stage granulomas.  It was later shown that the levels of cytokines were not significantly different between the early- and the late- stage granulomas. Thus, it was thought that proinflammatory cytokines were possibly not responsible for seizures.  Substance P evokes epileptiform activity, whereas, somatostatin has anticonvulsant properties. Both possibly play a role in the pathogenesis of seizures in neurocysticercosis. The neuropeptide substance P also stimulates growth of the granuloma and the production of T helper 1 type cytokines. Somatostatin stimulates T helper type 2 cytokine production and impairs the granuloma formation. Pretreatment with substance P receptor antagonist inhibited the seizures induced by extracts from the early granulomas. Similarly, injection of substance P into the rat brain induced seizures and altered the hippocampal activity that was blocked by substance P receptor antagonist or somatostatin. , We suggest that, in humans too, marked elevation of TNF-α and other cytokines may be responsible for the development of seizures.
The reason why some cysticercus granulomas resolve completely while others get calcified is not precisely known. We also explored if the inflammatory profile observed, helped in predicting the disappearance or calcification of the lesion. This fact is important because if a lesion persists or gets calcified, it may act as a source of epileptogenic activity. We found that a lower serum MMP-2, a lower serum IL-6 and a higher CSF TNF-α level were associated with an unresolved cysticercus granuloma. We observed a significant association of resolution of the lesion with serum IL-6 and CSF IL-10, while CSF IL-1β correlated with calcification of the lesion. Our findings indicate that a balance in the pro-inflammatory and regulatory inflammatory response is crucial for the complete resolution of the granuloma. Increased blood-brain barrier permeability in a calcified lesion is probably secondary to the perilesional inflammation. This impaired blood-brain barrier, secondary to inflammation, may result in penetration of plasma proteins into the brain parenchyma and may result in seizure recurrence in conjunction with a calcified lesion. 
Our study had certain limitations. The group showing resolution of the parasite was small. A follow-up cytokine and MMP profile in the serum and CSF might have helped us in understanding the variability in the radiological outcome. This would have also eliminated the influence of seizures on the cytokine and MMP levels as all the enrolled patients were seizure-free by the end of 6 months of follow-up. Another limitation in our study was that, due to ethical reasons, we did not include as controls, patients who were suffering from epilepsy but did not have the presence of a solitary cysticercus granuloma. Seizures by themselves can cause an increase of inflammatory factors.
In patients with a solitary cysticercus granuloma, cytokines and matrix metalloproteinases in CSF and serum are elevated. Different patterns of immunological changes were observed in patients following resolution or calcification of the lesion.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]