Cyst Burden Research Articles

Sex dichotomy in the course of experimental latent toxoplasmosis

Toxoplasma gondii is an opportunistic zoonotic protozoan that exceeds neurological and congenital impact sequence to reactivating latent toxoplasmosis especially under immunosuppression. Sex-associated hormones influence the severity of Toxoplasma infection. Thus, our study aimed to compare toxoplasmosis associated morbidity in both male and female mice and to monitor the response to anti-Toxoplasma therapeutics fortified with sex hormones in comparison to presently used drugs. Twenty male and 20 female mice were infected with ME49 Toxoplasma strain. The morbidity was assessed in the chronic stage in both sexes by estimating brain cyst burden, brain histopathological examination and monitoring serum anti-Toxoplasma IL-12 using ELISA method. Another 40 male and 40 female mice were infected with ME49 Toxoplasma strain then after 6 weeks received different treatment regimens including Atovaquone, Spiramycin, Metronidazole, Estradiol benzoate and Testoserone propionate either as a monotherapy or as a combination. Treatment response was monitored by scoring mice activity and brain cyst burden. This study showed that female mice demonstrated higher cyst burden and manifested more pathological reactions than male mice. While, the IL-12 serum level was significantly higher in male than female mice. Also, it is proved that the Toxoplasma cyst number was reduced significantly when used testosterone/atovaquone, or testosterone/spiramycin/metronidazole combined regimen in female mice groups. While for male mice, the combined therapy of spiramycin/metronidazole was the superior one. Accordingly, combined therapy with sex hormones is a promising strategy for discovering new therapeutic regimens for treating latent toxoplasmosis especially in female.

Guanabenz Reverses a Key Behavioral Change Caused by Latent Toxoplasmosis in Mice by Reducing Neuroinflammation.

Toxoplasma gondii is an intracellular parasite that has infected one-third of humans. The infection is permanent because the replicative form (tachyzoite) converts into a latent tissue cyst form (bradyzoite) that evades host immunity and is impervious to current drugs. The continued presence of these parasitic cysts hinders treatment and leads to chronic infection that has been linked to behavioral changes in rodents and neurological disease in humans. How these behavioral changes occur, and whether they are due to parasite manipulation or the host response to infection, remains an outstanding question. We previously showed that guanabenz possesses antiparasitic activity; here, we show that guanabenz reproducibly lowers brain cyst burden up to 80% in chronically infected male and female BALB/cJ mice when given intraperitoneally but not when administered by gavage or in food. Regardless of the administration route, guanabenz reverses Toxoplasma-induced hyperactivity in latently infected mice. In contrast, guanabenz increases cyst burden when given to chronically infected C57BL/6J mice yet still reverses Toxoplasma-induced hyperactivity. Examination of the brains from chronically infected BALB/cJ and C57BL/6J mice shows that guanabenz decreases inflammation and perivascular cuffing in each strain. Our study establishes a robust model for cyst reduction in BALB/cJ mice and shows for the first time that it is possible to reverse a key behavioral change associated with latent toxoplasmosis. The rescue from parasite-induced hyperactivity correlates with a decrease in neuroinflammation rather than reduced cyst counts, suggesting that some behavioral changes arise from host responses to infection.IMPORTANCEToxoplasma gondii is a common parasite of animals, including up to one-third of humans. The single-celled parasite persists within hosts for the duration of their life as tissue cysts, giving rise to chronic infection. Latent toxoplasmosis is correlated with neurological dysfunction in humans and results in dramatic behavioral changes in rodents. When infected, mice and rats adapt behaviors that make them more likely to be devoured by cats, the only host that supports the sexual stage of the parasite. In this study, we establish a new mouse model of tissue cyst depletion using a drug called guanabenz and show that it is possible to reverse a key behavior change back to normal in infected animals. We also show that the mechanism appears to have nothing to do with parasite cyst burden but rather the degree of neuroinflammation produced by chronic infection.

Enrichment and Proteomic Characterization of the Cyst Wall from In Vitro Toxoplasma gondii Cysts.

The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface.IMPORTANCEToxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.

Transcriptional Analysis Shows a Robust Host Response toToxoplasma gondiiduring Early and Late Chronic Infection in Both Male and Female Mice

The long-term host effects caused by the protozoan parasite Toxoplasma gondii are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10 days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21 days), early (28 days), mid (3 months), and late (6 months) chronic infection. We found that a majority of the host genes which increase in abundance at day 21 postinfection are still increased at 6 months postinfection for both male and female mice. While most of the differentially expressed genes were similar between sexes, females had far fewer genes that were significantly less abundant, which may have led to the slightly increased cyst burden in males. Transcripts for C-X-C motif chemokine ligand 13 and a C-C motif chemokine receptor 2 (CCR2) were significantly higher in females than in males during infection. As T. gondii chronic infection and profilin (PRF) confer resistance to Listeria monocytogenes infection in a CCR2-dependent manner, the differences in CCR2 expression led us to retest the protection of PRF in both sexes. Male mice were nearly as effective as female mice at reducing the bacterial burden either with a chronic infection or when treated with PRF. These data show that most of the host genes differentially expressed in response to T. gondii infection are similar between males and females. While differences in transcript abundance exist between the sexes, the infection phenotypes tested here did not show significant differences.

362 Necklace sign as a marker of pulmonary disease severity in tuberous sclerosis complex

Acrochordons are prevalent in the general population and may signify insulin resistance. Multiple acrochordons on the neck, known as the necklace sign, have been observed in tuberous sclerosis complex (TSC), a genetic disorder characterized by tumors in multiple organs. Tumorigenesis in TSC involves activation of mechanistic target of rapamycin (mTOR) signaling with concomitant disruption of insulin sensitivity. We sought to determine if the necklace sign is associated with increased phenotypic severity of TSC, reasoning that presence of acrochordons in some individuals may indicate heightened susceptibility to mTOR activation. We performed a retrospective chart review of a cohort of 113 adult women with TSC enrolled for studies of pulmonary lymphangioleiomyomatosis (LAM), a female-predominant manifestation of TSC. Twenty-four patients were excluded due to inadequate photos and 7 due to segmental disease. Of the 82 patients analyzed, 20 (24.3%) showed the necklace sign, defined as 2 or more acrochordons on the neck. The frequency of LAM was greater in patients with the necklace sign (20/20, 100%) compared to those without (48/62, 77.4%) (p=0.017). LAM symptom onset age was earlier in patients with the necklace sign (30.3 years, n=19) than those without (35.6 years, n=43) (p=0.023). In patients with the necklace sign, pulmonary function measured by % predicted forced expiratory volume in 1 second (80.5, n=20) and % predicted diffusing capacity for carbon monoxide (85.6, n=20) were lower compared to those without (89.7, n=48 (p=0.12); 69.3, n=48 (p=0.010), respectively). Initial cyst burden, determined computationally using chest CT scans, was higher in patients with the necklace sign (13.2%, n=16) versus those without (7.21%, n=43) (p=0.044). These results suggest that the necklace sign may aid in identification of TSC patients at risk for developing more severe LAM earlier in life and that interindividual variability in mTOR signaling may influence TSC-associated tumorigenesis.

Polyphasic Rhabdomyositis in California Sea Lions (Zalophus Californianus): Pathology and Potential Causes.

A myositis syndrome has been recognized for more than a decade in California sea lions (CSLs; Zalophus californianus) but a detailed description of the lesions and potential causes of this condition is lacking. The tissues of 136 stranded CSLs with rhabdomyositis were examined. Rhabdomyositis was considered incidental in 67% (91/136) of the CSLs, and a factor contributing to the animal stranding (significant rhabdomyositis) in 33% (45/136). Of the 91 cases with incidental rhabdomyositis, lesions consisted of a few small foci of lymphohistiocytic inflammation. Of the 45 cases with significant rhabdomyositis, 28 (62%) also presented with major comorbidities such as leptospirosis (2 animals) and domoic acid toxicosis (6 animals), whereas 17 (38%) had severe polyphasic rhabdomyositis as the only major disease process associated with mortality. In these animals, most striated muscles had multiple white streaks and diffuse atrophy. Microscopically, there was myofiber necrosis surrounded by lymphocytes and histiocytes admixed with areas of myofiber regeneration, and/or moderate to severe rhabdomyocyte atrophy usually adjacent to intact Sarcocystis neurona cysts. At the interface of affected and normal muscle, occasional T lymphocytes infiltrated the sarcoplasm of intact myocytes, and occasional myofibers expressed MHCII proteins in the sarcoplasm. S. neurona antibody titers and cyst burden were higher in animals with significant polymyositis antibody titers of (26125 ± 2164, 4.5 ± 1.2 cysts per section) and active myonecrosis than animals with incidental rhabdomyositis antibody titers of (7612 ± 1042, 1.7 ± 0.82 cysts per section). The presented findings suggest that S. neurona infection and immune-mediated mechanisms could be associated with significant polyphasic rhabdomyositis in CSLs.

Burden Of Cysts And Tumors Around Impacted Third Molars

Objective: To assess the frequency of cysts and tumors around impacted third molars. Study Design and Setting: Cross-sectional study was conducted at the OMFS department, Lahore Medical & Dental College, from August 2015 to January 2018. Methodology: A total of 2057 patients were operated for the removal of 2354 impacted third molars and analyzed for their associated pathological lesions(cysts/tumors). Patients were evaluated with thorough history, clinical and radiological examination. Panoramic and periapical radiographs were primarily used to assess the site of third molar impactions (maxilla/mandible) and their associated pathologies confirmed with histopathological examination. Data was analyzed using SPSS version 20. Results: The patients’ ages were between 17 to 62 years (Mean±SD, 28.12±8.585). There were n=709 (34.47%) females and n=1348 (65.53%) male with female to male ratio of 1.9:1. The mandibular to maxillary impacted third molars ratio was 1.66:1. An overall frequency of 1.36% for cysts and 0.72% for tumor associated with impacted third molars was demonstrated in the current study. The most frequently diagnosed cyst was dentigerous whereas ameloblastoma was the most commonly identified tumor. Conclusion: A relatively lower frequency (2.08%) of pathological lesions was found around third molars. The study reported a smaller number of pathological lesions affecting a significant minority of patients. It is recommended that a dentist/oral maxillofacial surgeon should be consulted at the earliest if any symptoms in the third molar region arise along with imaging of the area.

Plasma metabolites and lipids associate with kidney function and kidney volume in hypertensive ADPKD patients early in the disease course

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity.MethodsTo investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey’s false discovery rate (FDR) q-values to correct for multiple testing.ResultsTwelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value < 0.05. Specific significant metabolites, including pseudo-uridine, indole-3-lactate, uric acid, isothreonic acid, and creatinine, have been previously shown to accumulate in plasma and/or urine in both diabetic and cystic renal diseases with advanced renal insufficiency.ConclusionsThis study identifies metabolic derangements in early ADPKD which may be prognostic for ADPKD disease progression.Clinical trialHALT Progression of Polycystic Kidney Disease (HALT PKD) Study A; Clinical www.clinicaltrials.gov identifier: NCT00283686; first posted January 30, 2006, last update posted March 19, 2015.

A systematic review on efficiency of microneme proteins to induce protective immunity against Toxoplasma gondii.

Toxoplasma gondii is an intracellular parasite infecting almost all warm-blooded animals. Many studies on vaccination have been performed previously, and micronemal proteins (MICs) have crucial importance in this regard. The current review aims to reveal the efficiency of MICs as target antigen, adjuvants, animal models (species/strain), T. gondii strains for challenge infection, and routes of vaccine to prevent Toxoplasma infection. A comprehensive literature search was performed on April 18, 2018, in several known databases. Studies were included when evaluating vaccines based on MIC against T. gondii compared to that of a control group. Two independent researchers done the search process, study choice, and data extraction. A total of 28 articles published were selected for further analysis. Among them, 57.03% of the studies focused on MIC3 and its epitopes. SAG1 was further used in cocktail vaccines compared to other antigens. GM-CSF and Freund's complete were the predominant adjuvants used. BALB/c mice have been introduced as a proper model for lethal challenge. Virulent T. gondii (RH) was utilized more than other strains for challenge. Among MICs, the results of vaccination with MIC1-4, MIC6, and PLP1 demonstrated significantly strong humoral and cellular immunity, increased survival time, and reduced cyst burden in the mice. This review summarizes the latest results on MIC-based vaccines and presents that the most effective vaccination procedure is the administration of the cocktail vaccines. Our survey can serve as a basis for further studies to develop more efficient novel vaccines against T. gondii for animals and humans.

Nonselective Cyclooxygenase Inhibition Retards Cyst Progression in a Murine Model of Autosomal Dominant Polycystic Kidney Disease.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease.Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume.Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p<0.05). Basal Cyclooxygenase-1 levels were unchanged in both immunohistochemistry and real-time PCR. Administration of Sulindac to Pkd2WS25/- mice and to control mice for 8 months resulted in reduced kidney weights and volume in cystic mice. Renal function and electrolytes were not significantly different between groups.Conclusion: Thus treatment of a murine model of polycystic kidney disease with Sulindac results in decreased kidney cyst burden. These findings provide additional implications for the use of Cyclooxygenase inhibition as treatment to slow the progression of cyst burden in patients with polycystic kidney disease.

Systematic review and meta-analysis of variation in Toxoplasma gondii cyst burden in the murine model

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects approximately 30% of the population of the United States, with worldwide distribution. The chronic (latent) infection, mediated by the bradyzoite parasite life stage, has attracted attention due to possible links to host behavioral alteration and psychomotor effects. Mice are a common model organism for studying the chronic stage, as they are natural hosts of infection. Notably, published studies demonstrate vast ranges of measured cyst burden within the murine brain tissue. The inconsistency of measured cyst burden within and between experiments makes interpretation of statistical significance difficult, potentially confounding studies of experimental anti-parasitic approaches. This review analyzes variation in measured cyst burden in a wide array of experimental mouse infections across published literature. Factors such as parasite infection strain, mouse strain, mode of infection, and infectious dose were all examined. The lowest variation in measured cyst burden occurred with the commonly available Balb/c and CBA mice undergoing infection by the ME49 strain of T. gondii. A summary of cyst variation and average cyst counts in T. gondii mouse models is presented, which may be useful for designing future experiments.

Chronic infection with Toxoplasma gondii does not prevent acute disease after virulent strain reinfection in experimental mice

Background: Toxoplasmosis is a wide spread protozoan disease. It was generally believed that primary infection by T. gondii protects from reinfection, however, multiple cases of reinfection have been detected in immune mothers. This work reports results of re-infection of Swiss Webster (SW) mice with different strains of T. gondii. Objective: To simulate the impact of reinfection in experimentally infected mice with lethal strain of T. gondii after prime infection with non-virulent genotype. Methodology: The study was conducted on 36 female SW mice which were divided into four groups: 6 mice were infected with ME49 only (GI); 18 mice were infected with ME49 and re-challenged with RH on day 65, i.e. 8 weeks post ME49 infection (GII); 6 mice were infected with RH only (GIII); and 6 non-infected control mice (G1V). Toxoplasma RH strain re-challenged mice (GII) were monitored over two weeks observation period for mortality, clinical signs of acute illness (scoredgradesI-II), presence of intraperitoneal tachyzoites, brain cyst burden, and compared to chronically infected non-challenged mice (GI) and mice infected with RH only (GIII). Results: Prolonged survival rate of re-challenged group of mice thanin RH only infected group was the only significant result. Cyst number and diameter were higher in re-challenged group than in mice infected only with ME49. Tachyzoites were recovered from peritoneal lavage of all mice that received RH whether primarily infected with ME49 or not. Clinical grading (I-II) was the same for both groups and both reached grade II. Conclusion: These results highlighted that mice with chronic toxoplasmosis developed acute disease when re-challenged with another virulent strain. Therefore,chronic infection with T. gondii apparently neither prevents acute disease nor impairs colonization of the brain with tissue cysts after virulent strain superinfection. The present work supports and explains the possibility of congenital toxoplasmosis in immune pregnant mothers when re-infected by a virulent strain of T. gondii.

Impact of Engineered Expression of Mitochondrial Association Factor 1b on Toxoplasma gondii Infection and the Host Response in a Mouse Model.

The opportunistic intracellular parasite Toxoplasma gondii causes a lifelong chronic infection capable of reactivating in immunocompromised individuals, which can lead to life-threatening complications. Following invasion of the host cell, host mitochondria associate with the parasitophorous vacuole membrane. This phenotype is T. gondii strain specific and is mediated by expression of a host mitochondrial association-competent (HMA+) paralog of the parasite protein mitochondrial association factor 1 (MAF1b). Previous work demonstrated that expression of MAF1b in strains that do not normally associate with host mitochondria increases their fitness during acute infection in vivo However, the impact of MAF1b expression during chronic T. gondii infection is unclear. In this study, we assess the impact of MAF1b expression on cyst formation and cytokine production in mice. Despite generally low numbers of cysts generated by the in vitro culture-adapted strains used in this study, we find that parasites expressing MAF1b have higher numbers of cysts in the brains of chronically infected mice and that MAF1b+ cyst burden significantly increases during the course of chronic infection. Consistent with this, mice infected with MAF1b+ parasites have higher levels of the serum cytokines RANTES and VEGF (vascular endothelial growth factor) at day 57 postinfection, although this could be due to higher parasite burden at this time point rather than direct manipulation of these cytokines by MAF1b. Overall these data indicate that MAF1b expression may also be important in determining infection outcome during the chronic phase, either by directly altering the cytokine/signaling environment or by increasing proliferation during the acute and/or chronic phase.IMPORTANCE The parasite Toxoplasma gondii currently infects approximately one-third of the world's population and causes life-threatening toxoplasmosis in individuals with undeveloped or weakened immune systems. Current treatments are unable to cure T. gondii infection, leaving infected individuals with slow-growing tissue cysts for the remainder of their lives. Previous work has shown that expression of the parasite protein mitochondrial association factor 1 (MAF1b) is responsible for the association of T. gondii parasites with host mitochondria and provides a selective advantage during acute infection. Here we examine the impact of MAF1b expression during chronic T. gondii infection. We find that mice infected with MAF1b-expressing parasites have higher cyst burden and cytokine levels than their wild-type counterparts. A better understanding of the genes involved in establishing and maintaining chronic infection will aid in discovering effective therapeutics for chronically infected individuals.

Could Araucaria heterophylla resin extract be used as a new treatment for toxoplasmosis?

Toxoplasmosis is a worldwide parasitic disease responsible for serious health problems to human. The currently available drugs used for toxoplasmosis treatment showed a limited efficacy and cause serious host toxicity. The in vitro screening for toxoplasmicidal activity of Araucaria heterophylla resin (AHR) extract and its major component 13-epi-cupressic acid (CUP) showed that both AHR (EC50 = 3.90) and CUP (EC50 = 3.69) have high toxoplasmicidal activity in comparison with standard cotrimoxazole (EC50 = 4.28). The antiprotozoal effects of AHR and CUP were investigated against acute and chronic toxoplasmosis using mice models. Two groups of Swiss albino mice were infected by RH Toxoplasma strain intraperitoneally and by Me49 strain orally. Both groups were treated with AHR and CUP in different doses. Their effects were evaluated by survival rate, peritoneal, spleen and liver parasite burdens, brain cyst burden, NO serum level and histopathological lesions. The ultrastructural changes of tachyzoites of acutely infected mice were studied using scanning electron microscopy (SEM). There is an evidence of toxoplasmicidal activity of AHR and CUP in acute and chronic experimental toxoplasmosis. In the acute model, mice treated with AHR and CUP showed prolonged survival rates, a significant decrease in the parasite density in peritoneal lavage and pathological insult in both liver and spleen compared with that of untreated ones. SEM results denote evident morphological alterations of treated tachyzoites. In chronic experimental toxoplasmosis, AHR and CUP treated groups could significantly reduce brain cyst burden by 96.05% and 98.02% respectively. This study indicates that AHR and CUP showed potent toxoplasmicidal activities experimentally and could be used as a potential natural nontoxic agent for treatment of toxoplasmosis.

Chronic Toxoplasma gondii Infection Induces Anti-N-Methyl-d-Aspartate Receptor Autoantibodies and Associated Behavioral Changes and Neuropathology.

Anti-NMDA receptor (NMDAR) autoantibodies have been postulated to play a role in the pathogenesis of NMDAR hypofunction, which contributes to the etiology of psychotic symptoms. Toxoplasma gondii is a pathogen implicated in psychiatric disorders and associated with elevation of NMDAR autoantibodies. However, it remains unclear whether parasite infection is the cause of NMDAR autoantibodies. By using mouse models, we found that NMDAR autoantibody generation had a strong temporal association with tissue cyst formation, as determined by MAG1 antibody seroreactivity (r = 0.96; P < 0.0001), which is a serologic marker for the cyst burden. The presence of MAG1 antibody response, but not T. gondii IgG response, was required for NMDAR autoantibody production. The pathogenic relevance of NMDAR autoantibodies to behavioral abnormalities (blunted response to amphetamine-triggered activity and decreased locomotor activity and exploration) and reduced expression of synaptic proteins (the GLUN2B subtype of NMDAR and PSD-95) has been demonstrated in infected mice. Our study suggests that NMDAR autoantibodies are specifically induced by persistent T. gondii infection and are most likely triggered by tissue cysts. NMDAR autoantibody seroreactivity may be a novel pathological hallmark of chronic toxoplasmosis, which raises questions about NMDAR hypofunction and neurodegeneration in the infected brain.

Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic disease caused by the intracellular protozoan Toxoplasma gondii; and a major source of infection in humans is via ingestion of T. gondii tissue cysts. Ultimately, the goal of anti-toxoplasmosis vaccines is to elicit a sustainable immune response, capable of preventing formation of the parasite tissue cysts—or, at least, to restrain its growth. In this study, we formulated a cocktail DNA vaccine and investigated its immunologic efficacy as a protection against the establishment of T. gondii cysts in the mouse brain. This multicomponent DNA vaccine, encoded the TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, which play key roles in the pathogenesis of T. gondii infection. Results showed that mice immunized via intramuscular injection three times, at 2-week intervals with this multicomponent DNA vaccine, mounted a strong humoral and cellular immune response, indicated by significantly high levels of total IgG, CD4+ and CD8+ T lymphocytes, and antigen-specific lymphocyte proliferation when compared with non-immunized mice. Immunization also induced a mixed Th1/Th2 response, with a slightly elevated IgG2a to IgG1 ratio. The increased production of proinflammatory cytokines gamma-interferon, interleukin-2, and interleukin-12 (p < 0.0001) correlated with increased expression of p65/RelA and T-bet genes of the NF-κB pathway. However, no significant difference was detected in level of interleukin-4 (p > 0.05). The number of brain cysts in immunized mice was significantly less than those in non-immunized mice (643.33 ± 89.63 versus 3,244.33 ± 96.42, p < 0.0001), resulting in an 80.22% reduction in the parasite cyst burden. These findings indicate that a multicomponent DNA vaccine, encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, shows promise as an immunization strategy against chronic toxoplasmosis in mice, and calls for a further evaluation in food-producing animals.

Carotid Taenia solium Oncosphere Infection: A Novel Porcine Neurocysticercosis Model.

Neurocysticercosis (NCC), the infection of the human central nervous system (CNS) with larval cysts of Taenia solium causes widespread neurological morbidity. Animal models are crucial for studying the pathophysiology and treatment of NCC. Some drawbacks of current NCC models include differences in the pathogenesis of the model and wild-type parasite, low rates of infection efficiency and lack of reproducibility. We describe a novel porcine model that recreates infection in the CNS with high efficiency. Activated oncospheres, either in a high (45,000-50,000) or low (10,000) dose were inoculated in the common carotid artery of 12 pigs by ultrasound-guided catheterization. Following oncosphere injection, either a high (30 mL) or low (1-3 mL) volume of saline flush was also administered. Cyst burden in the CNS was evaluated independently according to oncosphere dose and flush volume. Neurocysticercosis was achieved in 8/12 (66.7%) pigs. Cyst burden in the CNS of pigs was higher in the high versus the low oncosphere dose category (median: 4.5; interquartile ranges [IQR]: 1-8 and median: 1; IQR: 0-4, respectively) and in the high versus the low flush volume category (median 5.5; IQR: 1-8 and median: 1; IQR: 0-2, respectively), although not statistically different. All cysts in the CNS were viable, whereas both viable and degenerated cysts were found in the musculature. Carotid injection of activated oncospheres in pigs is effective in reproducing NCC. Oncosphere entry into the CNS by way of vasculature mimics wild-type infection, and provides a useful alternative for future investigations on the pathogenesis and antiparasitic treatment of NCC.

Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis.

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.

Immuno-efficacy of DNA vaccines encoding PLP1 and ROP18 against experimental Toxoplasma gondii infection in mice

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.

PD-1 immune checkpoint blockade promotes brain leukocyte infiltration and diminishes cyst burden in a mouse model of Toxoplasma infection

Tissue cysts, the hallmark of chronic Toxoplasma gondii infection, are predominantly located in the brain making clearance of the parasite difficult. Currently available anti-T. gondii drugs are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. We examined whether abrogation of inhibitory signaling pathways that maintain T cells in an exhausted state can be exploited for treating T. gondii tissue cysts. By using a mouse model of chronic toxoplasmosis, we showed immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway results in a significant reduction in brain cyst number (77% lower). We showed leukocyte infiltration (CD3+ T cells, CD8+ T cells, and CD11b + cells) in the leptomeninges, choroid plexus, and subependymal tissue, which are known routes of entry of immune cells into the brain, and in proximal brain parenchyma. Our study provides proof of concept for blockade of immune checkpoint inhibitors as a therapy for chronic toxoplasmosis and potentially for other brain pathogens.