Immune Mediator Profile in Aqueous Humor Differs in Patients with Primary Acquired Ocular Toxoplasmosis and Recurrent Acute Ocular Toxoplasmosis.

PURPOSE: To compare the intraocular cytokine and chemokine profiles in patients with acute primary acquired ocular toxoplasmosis (pOT) or recurrent ocular toxoplasmosis (rOT) and to correlate them with their clinical characteristics. METHODS: Aqueous humor samples were collected from 62 consecutive patients (21 pOT, 30 rOT, and 11 noninfected controls) and analyzed by multiplex assay. Correlations were assessed between cytokine/chemokine levels, type of inflammatory response (Th1, Th2, and Th17), and clinical characteristics. In all OT patients, the clinical diagnosis of either pOT or rOT was confirmed by positive intraocular Goldmann/Witmer-Desmonts coefficient. Correlations were assessed between a preselected panel of immune mediators and the clinical characteristics of OT. RESULTS: In pOT patients, increased levels of IL-2, IFN-γ, TNF-α, IL-15, IL-4, IL-5, IL-9, IL-13, IL-17, IL-1Rα, IL-6, IL-1ß, and chemokines MIP-1α, MIP-1ß, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, GM-CSF, G-CSF, and MCP-1 were found in comparison to those in controls (p < 0.05). Patients with rOT showed elevated levels of IL-2, IFN-γ, TNF-α, IL-15, IL-4, IL-5, IL-9, IL-17, IL-1Rα, IL-6, IL-1ß, and chemokines MIP-1α, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, G-CSF, and MCP-1 compared to controls (p < 0.05). In addition, IL-7 (p = 0.028) differed between pOT and rOT; IL-9 (p = 0.054) and IL-13 (p = 0.051) showed a tendency of higher concentration in pOT than in rOT. A negative correlation was found between IL-7 (p = 0.017) as well as IL-9 (p = 0.008) and the number of recurrences. Cytokine ratios showed no difference between pOT and rOT, indicating a dominant Th1-type response in both infectious groups. Moreover, a positive correlation was detected between IL-7, VEGF, IL-13 and age at aqueous humor sampling (p < 0.05). CONCLUSIONS: This study for the first time shows subtle differences between the intraocular cytokine profiles in patients with either acute pOT or rOT.

T cell immunoregulation in active ocular toxoplasmosis.

Toxoplasma gondii infection is an important cause of infectious ocular disease. The physiopathology of retinochoroidal lesions associated with this infection is not completely understood. The present study was undertaken to investigate cytokine production by T cells from individuals with active toxoplasmic retinochoroiditis (TR) comparing with controls. Eighteen patients with active TR and 15 healthy controls (6 controls IgG+ to Toxoplasma and 9 negative controls) were included in the study. Peripheral blood mononuclear cells were incubated in the presence or absence of T. gondii antigen (STAg), and stained against CD4, CD8, TNF, IL-10 and IFN-γ. Baseline expression of cytokines was higher in TR/IgG+ patients in comparison with controls. Cytokine expression was not increased by STAg in vitro stimulation in controls. After stimulation, TR/IgG+ patients' lymphocytes increased cytokine as compared to cultures from both controls. While T cells were the main source of IL-10, but also IFN-γ and TNF, other lymphocyte populations were relevant source of inflammatory cytokines. Interestingly, it was observed a negative correlation between ocular lesion size and IL-10 expression by CD4+ lymphocytes. This study showed that T cells are the main lymphocyte populations expressing IL-10 in patients with TR. Moreover, expression of IL-10 plays a protective role in active TR.

Subretinal fluid in eyes with active ocular toxoplasmosis observed using spectral domain optical coherence tomography.

PURPOSE: To describe the clinical finding of subretinal fluid (SRF) in the posterior pole by spectral domain optical coherence tomography (SD-OCT) in eyes with active ocular toxoplasmosis (OT). DESIGN: Retrospective case series. PARTICIPANTS: Thirty-nine eyes from 38 patients with active OT [corrected].. METHODS: Eyes with active OT which underwent SD-OCT were reviewed. SRFs in the posterior pole were further analyzed. MAIN OUTCOME MEASURES: Presence of SRF; its accompanying features, e.g. retinal necrosis, cystoid macular edema (CME), choroidal neovascularization (CNV); and longitudinal changes of SRF, including maximum height and total volume before and after treatment. RESULTS: SRF presented in 45.5% (or 15/33) of eyes with typical active OT and in 51.3% (or 20/39) of eyes with active OT. The mean maximum height and total volume of SRF were 161.0 (range: 23-478) µm and 0.47 (range: 0.005-4.12) mm3, respectively. For 12 eyes with SRF related to active retinal necrosis, SRF was observed with complete absorption after conventional anti-toxoplasmosis treatment. The mean duration for observation of SRF clearance was 33.8 (range: 7-84) days. The mean rate of SRF clearance was 0.0128 (range: 0.0002-0.0665) mm3/day. CONCLUSIONS: SRF (i.e., serous retinal detachment) is a common feature in patients with active OT when SD-OCT is performed. The majority of SRF was associated with retinal necrosis and reacted well to conventional therapy, regardless of total fluid volume. However, SRF accompanying with CME or CNV responded less favorably or remained refractory to conventional or combined intravitreal treatment, even when the SRF was small in size.

Ocular cytokinome is linked to clinical characteristics in ocular toxoplasmosis.

PURPOSE: To determine the cytokine levels in aqueous humor (AH) of Colombian patients with active ocular toxoplasmosis (OT), and to correlate them with their clinical characteristics. METHODS: 27 Cytokines/chemokines were assayed in 15 AH samples (nine patients with diagnosis of OT biologically-confirmed and six controls that underwent cataract surgery). Correlations were assessed between cytokine/chemokine levels, type of inflammatory response (Th1, Th2, Th17, Treg), and clinical characteristics. RESULTS: Th2 predominant response was related to more severe clinical features. The presence of VEGF and IL-5 was related to higher number of recurrences. Growth factors (VEGF, FGF, PDGF-ß), were related to higher number of lesions. Patients infected by type-I/III strains had a particular intraocular cytokine-pattern. CONCLUSIONS: Th2 response was related to more severe clinical characteristics in patients infected by Type I/III strains. IL-5 and VEGF were associated with recurrences. We correlate for the first time, specific cytokine-patterns with clinical characteristics and with the infecting Toxoplasma strain.

Interleukin-6 gene polymorphism (-174 G/C) is associated with toxoplasmic retinochoroiditis.

PURPOSE: Experimental data have demonstrated a relevant role for IL-6 in the modulation of acute ocular toxoplasmosis. Therefore, we aim to investigate the possible association between the IL-6 gene polymorphism at position -174 and toxoplasmic retinochoroiditis (TR) in humans. METHODS: Ninety-seven patients with diagnosed TR were recruited from the Uveitis Section, Federal University of Minas Gerais. For comparison, 83 healthy blood donors with positive serology for toxoplasmosis and without retinal signs of previous TR were included in the study. Genomic DNA was obtained from oral swabs of individuals and amplified using polymerase chain reaction (PCR) with specific primers flanking the locus -174 of IL-6 (-174G/C). PCR products were submitted to restriction endonuclease digestion and analysed by polyacrylamide gel electrophoresis to distinguish allele G and C of the IL-6 gene, allowing the detection of the polymorphism and determination of genotypes. RESULTS: There was a significant difference in the genotype (χ(2) = 12.9, p = 0.001) and allele (χ(2) = 6.62, p = 0.01) distribution between TR patients and control subjects. In a subgroup analysis, there was no significant difference in genotypes and allele frequencies regarding TR recurrence. CONCLUSIONS: This study suggests that the genotypes related with a lower production of IL-6 may be associated with the occurrence of TR.

Migration of toxoplasma gondii-infected dendritic cells across human retinal vascular endothelium.

PURPOSE: Toxoplasma gondii, the parasite responsible for ocular toxoplasmosis, accesses the retina from the bloodstream. We investigated the dendritic cell as a potential taxi for T. gondii tachyzoites moving across the human retinal endothelium, and examined the participation of adhesion molecules and chemokines in this process. METHODS: CD14-positive monocytes were isolated from human peripheral blood by antibody-mediated cell enrichment, and cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate dendritic cells. Transmigration assays were performed over 18 hours in transwells seeded with human retinal endothelial cells and using dendritic cells exposed to laboratory or natural strains of T. gondii tachyzoites. Parasites were tagged with yellow fluorescent protein to verify infection. In some experiments, endothelial monolayers were preincubated with antibody directed against adhesion molecules, or chemokine was added to lower chambers of transwells. RESULTS: Human monocyte-derived dendritic cell preparations infected with laboratory or natural strain T. gondii tachyzoites transmigrated in larger numbers across simulated human retinal endothelium than uninfected dendritic cells (P ≤ 0.0004 in 5 of 6 experiments). Antibody blockade of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and activated leukocyte cell adhesion molecule (ALCAM) inhibited transmigration (P ≤ 0.007), and CCL21 or CXCL10 increased transmigration (P ≤ 0.031). CONCLUSIONS: Transmigration of human dendritic cells across retinal endothelium is increased following infection with T. gondii. Movement may be impacted by locally produced chemokines and is mediated in part by ICAM-1, VCAM-1, and ALCAM. These findings have implications for development of novel therapeutics aimed at preventing retinal infection by T. gondii.

Kinetic analysis of cytokines, chemokines, chemokine receptors and adhesion molecules in murine ocular toxoplasmosis.

BACKGROUND/AIMS: To investigate the molecules possibly influencing the recruitment and migration of leucocytes in murine ocular toxoplasmosis, the kinetics of the messenger RNA expression levels of cytokines, chemokines, chemokine receptors and adhesion molecules in the retina were analysed. METHODS: Retina and brain were obtained sequentially from Toxoplasma gondii Fukaya strain-infected wild-type (WT) C57BL/6 and interferon gamma (IFN-γ) knockout (GKO) mice of the same background. The mRNA expression levels of these molecules were analysed by real-time PCR assay. RESULTS: In the retina of WT mice the expression levels of IFN-γ, interleukin 17A, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL10, CCR5, CCR7, CXCR2, CXCR3 and intracellular adhesion molecule 1 increased, reaching peaks approximately 14-28 days after infection. The expression levels of CXCR4 and CXCR5 were absent and very low, respectively, during the infection. In the brain of WT mice, the kinetic patterns of these expression levels tended to be the same as in the retina except CXCR4. On the other hand, in GKO mice these molecules, except CXCL1, CXCL2 and CXCR2, remained at basal levels. CONCLUSION: In murine ocular toxoplasmosis, cytokines, chemokines, chemokine receptors and adhesion molecules were involved in the pathogenesis, and IFN-γ played a pivotal role.

Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro.

Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

IFN-gamma-regulated Toxoplasma gondii distribution and load in the murine eye.

PURPOSE: To establish a mouse model of ocular toxoplasmosis in both wild type (WT) and immunocompromised hosts and to clarify the effects of interferon (IFN)-gamma on the infectivity of Toxoplasma gondii in various parts of the eye. METHODS: Susceptible WT C57BL/6, resistant WT BALB/c, and IFN-gamma knockout (GKO) mice were infected with cysts of T. gondii perorally. The tissues were harvested for molecular and histopathologic studies. Analysis included a quantitative competitive polymerase chain reaction (QC-PCR) assay and reverse transcription (RT)-PCR for IFN-gamma and stage conversion markers. All animals underwent ophthalmic examinations including fluorescein angiography (FA). RESULTS: In WT C57BL/6 mice, T. gondii was detected in tissue in the following order: brain, retina, choroid, sclera, and optic nerve (ON). The highest T. gondii load was observed in the posterior retina, and was much greater than that in WT BALB/c mice. In GKO mice, disseminated infection was evident, and the T. gondii load was highest in the choroid and ON. IFN-gamma mRNA expression in WT C57BL/6 mice was higher than that in WT BALB/c mice after infection. Tachyzoites existed in GKO mice, whereas bradyzoites existed in WT C57BL/6 mice. FA showed dye leakage from the retinal capillaries of GKO mice. CONCLUSIONS: The T. gondii load in the retina in the susceptible WT strain continued to increase, unlike in the resistant WT strain. IFN-gamma was shown to regulate the T. gondii load and interconversion in the eye. A toxoplasmic vasculitis model was established with GKO mice and assay systems with QC-PCR and FA.

Involvement of apoptosis and interferon-gamma in murine toxoplasmosis.

PURPOSE: A murine toxoplasmosis model has been developed that results in central nervous system (CNS) and ocular inflammation characterized by encephalitis with numerous brain tissue cysts and milder inflammation with rare tissue cysts in the eye after 4 weeks of Toxoplasma gondii infection. In this model IFN gamma and inducible nitric oxide (iNO) are protective against T. gondii infection. In this study, the role of apoptosis in the pathogenesis of toxoplasmosis was investigated. METHODS: C57BL/6 (wild-type mice), B6MRL/lpr, and B6MRL/gld (defective Fas or FasL expression, respectively) mice were infected intraperitoneally with 20 to 30 tissue cysts of the ME-49 strain of T. gondii. Mice were killed at days 0, 14, or 28 after infection. The eyes and brains were harvested for histologic, immunohistochemical, and molecular studies. Analysis included immunostaining for Fas, FasL, Bcl-2, and Bax; in situ apoptosis detection (TUNEL assay); RT-PCR amplification for IFN gamma; and measurement of ocular nitrite levels. The control mice were naïve mice of each strain that received no inoculation or injection. RESULTS: Wild-type mice appeared to constitutively express apoptotic molecules at higher levels in the eye than in the brain. Consequently, during T. gondii infection, apoptosis was greater in the eyes than in the brain. Untreated naïve lpr and gld mice showed no expression of Fas and FasL, respectively. After infection, a slightly higher number of tissue cysts (lpr, 11.8 +/- 2.4; gld, 10.3 +/- 3.4) were found in the brains of the mutants than in the control animals (8.8 +/- 2.9). However, no significant differences between the number of apoptotic cells, inflammatory scores, or number of tissue cysts were noted in the eyes. IFN gamma mRNA in control mice was detected at day 28 after infection, whereas in both mutants, mRNA production occurred earlier, at day 14. Ocular nitrite levels were higher in lpr and gld mice than in wild-type mice. CONCLUSIONS: No significant difference in the degree of ocular inflammation and apoptosis was detected between the wild-type and Fas or FasL mutant mice. However, there was an earlier and subjectively greater expression of IFN gamma in the brain and eye and a higher level of nitrite in the ocular tissue of mutant strains than in the wild type. Multiple factors are likely to be involved in the pathogenesis of ocular toxoplasmosis.

Inhibition of nitric oxide production exacerbates chronic ocular toxoplasmosis.

There is considerable controversy as to the roles of parasite proliferation and the inflammatory response in destruction of the retina during Toxoplasma gondii infection. A murine model was used to investigate the role of nitric oxide in pathogenesis of chronic ocular toxoplasmosis. Increased quantities of messenger RNA (mRNA) transcripts for iNOS were detected in the eyes of chronically infected C57BL/6 mice compared with noninfected control mice. Inhibition of nitric oxide (NO) by the addition of Lomega-nitro-L-arginine methyl ester (L-NAME) to the drinking water of infected mice between weeks 4-6 of infection, exacerbated ocular inflammation. The amount of inflammation was assessed semiquantitatively in histological sections of the eye. Eyes from L-NAME treated mice showed a significant increase in inflammation of the retina (P = 0.02), choroid (P = 0.03), and vitreous (P = 0.02) compared with control mice. These results demonstrate a protective role for NO in the control of chronic, ocular toxoplasmosis.

Analysis of immunoregulatory cytokines in ocular fluid samples from patients with uveitis.

PURPOSE: To investigate the T-helper cell cytokine profiles in two well-defined clinical uveitis entities caused by an infectious mechanism. METHODS: Cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and interferon [IFN]-gamma) were measured in ocular fluid samples obtained from patients with herpes simplex- or varicella-zoster virus-induced acute retinal necrosis (ARN; n = 17) and toxoplasma chorioretinitis (n = 27) using enzyme-linked immunosorbent assay techniques. The data were compared with data for 51 control samples taken during cataract surgery (n = 10), vitrectomy in diabetic retinopathy (n = 10), eye bank eyes (n = 10) and with samples from patients with "autoimmune" uveitis (n = 21). RESULTS: Interleukin-6 was detected in 44 of 51 control samples and 43 of 44 eyes of patients with uveitis. The highest levels in the control samples were detected in 9 of 10 vitreous samples from patients with diabetic retinopathy (mean, 648 pg/ml). In 8 of 10 samples taken from patients during cataract surgery and in 7 of 10 eye bank eyes the amount of IL-6 was significantly lower (mean, 10 pg/ml and 136 pg/ml, respectively). Interleukin-6 levels in patients with ARN (mean, 1436 pg/ml) were significantly higher than in those with toxoplasma chorioretinitis (mean, 272 pg/ml). Interleukin-2 was detected in one of the samples from patients with toxoplasma chorioretinitis (1105 pg/ml) and in three samples from the control subjects suffering from Fuchs' heterochromic anterior uveitis (mean, 752 pg/ml). No IL-4 (<2 pg/ml) was detected either in patient or control samples. Interferon-gamma could be detected in 7 of 17 ARN patients (range, 277-3483 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 12-250 pg/ml), and in 1 of 21 of the samples from control subjects with uveitis (31 pg/ml) but was absent in nonuveitic control samples. Interleukin-10 was detected in 10 of 17 ARN patients (range, 29-3927 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 4-67 pg/ml), and in only 3 of 51 control samples (6 pg/ml, 16 pg/ml, and 20 pg/ml). CONCLUSIONS: Various immunoregulatory cytokines (IL-6, IL-10, and IFN-gamma) were detected in ocular fluid samples from patients with uveitis. A separate role for either a T-helper type 1 or T-helper type 2 response in the pathogenesis of clinical uveitis could not be proven.

Protective role of nitric oxide in ocular toxoplasmosis.

AIMS: To evaluate the role of nitric oxide (NO) in ocular involvement during systemic toxoplasmosis. METHODS: C57B1/6 mice were infected with Toxoplasma gondii strain ME49. The synthesis of NO was inhibited by an intraperitoneal injection of aminoguanidine every 8 hours, starting on the day of infection. Control infected mice received phosphate buffered saline vehicle alone. After 14 days, the ocular lesions were evaluated by histopathological examination. The expression of NO synthase induced in the spleen by toxoplasma infection was evaluated by immunostaining. The production of NO by the spleen cells of infected mice was measured by the colorimetric assay of Griess in the supernatant of cultures stimulated with toxoplasma antigen or concanavalin A. RESULTS: The inhibition of NO production in T gondii infected mice resulted in a marked increase in the symptoms of ocular inflammation. We observed a strong induction of NO synthase expression in the spleen of infected animals. In culture, the spleen cells from these mice produced high levels of NO in response to T gondii antigens. This elevation of NO synthesis was suppressed in the presence of aminoguanidine. CONCLUSION: This study indicates that NO plays a crucial role in the protection against T gondii infection as reflected by the severity of the ocular involvement.

Paradoxical effect of clindamycin in experimental, acute toxoplasmosis in cats.

Cats were experimentally inoculated parenterally with the ME49 strain of Toxoplasma gondii to characterize the efficacies of two different dosages of orally administered clindamycin hydrochloride in the treatment of ocular toxoplasmosis. Concentrations of clindamycin hydrochloride at levels previously suggested to be inhibitory to T. gondii replication in vitro were achieved in the serum and aqueous humor but not in the cerebrospinal fluid. Antibiotic therapy, initiated 7 days after inoculation, resulted in no significant difference in the morphometric severity of ocular posterior segment lesions compared with that in the control groups. Treatment appeared to blunt T. gondii-specific immunoglobulin M production but had no significant effect on immunoglobulin G titers. Paradoxically, clindamycin administration was associated with increased morbidity and mortality from hepatitis and interstitial pneumonia, which are characteristic of generalized toxoplasmosis. Serum tumor necrosis factor alpha activity was detected at moderate levels in all groups of cats and correlated with the severity of clinical disease. The results of the study suggest that clindamycin, when administered at this specific time interval following inoculation, does not ameliorate ocular lesions and has a detrimental effect on the clinical course of acute, experimental toxoplasmosis in cats. The factors responsible for and the relevance of this detrimental effect to naturally occurring toxoplasmosis in humans and pet cats were not clear from the study but may relate to an antibiotic-associated decrease in the antitoxoplasmic activity of phagocytic cells responsible for the control of T. gondii.