On July 24, 2024, the Doctoral Program in Medical Science of FMUB celebrated the graduation of one of its students, Dr. Ovi Sofia, Sp.M(K). The presentation of her dissertation results and future perspectives by Dr. Ovi Sofia, Sp.M(K) took place in the FMUB Auditorium, and the newly appointed Doctor has successfully published two articles in reputable international journals. Dr. Ovi serves as a lecturer in the Infection and Immunology Division and is part of the teaching staff at the Department of Ophthalmology, FMUB/RSSA.
Summary Disertas Dr Ovi:
Ocular toxoplasmosis (OT) is a manifestation of Toxoplasma gondii (T. gondii) infection in the eye, which is the most common cause of posterior veitis in the world. The most common clinical manifestation of OT is retinochoroiditis with the predominant location in the macula, thus contributing to decreased vision. Factors contributing to the occurrence of OT infection include host factors, parasitic factors, and immune responses. The Th1-type immune response is the dominant immune response in OT, but the Th17-type immune response is also thought to play an important role in the occurrence of OT, even though the previous reports are limited. Interferon-gamma (IFN-γ) is a key cytokine in TO infection, while interferon
type I, namely interferon-alpha (IFN-α) and interferon-beta (IFN-β), which show anti-Toxoplasma activity in in vitro and in vivo studies in experimental animals, has not been studied for its role in OT patients. Interleukin-17 as the main proinflammatory cytokine resulting from the Th17 immune response
has a pleiotropic effect. Interleukin-17 levels is regulated by Transforming Growth Factor-β (TGF-β), produced by Treg, which can also exert different effects on toxoplasmosis. The balance of Th1 and Th17 immune responses, as the primary immune responses in OT, is important to evaluate, because
it affects the replication of tachyzoites in the retina which results in tissue damage. The cytokines ratio may provide a better description of the balance of immune response than the measurement of a single cytokine level. The ratio of cytokines that play a dominant role in the immune response of Th1 and Th17, namely the IL-17/IFN-γ ratio; the ratio of pro-inflammatory cytokine that are dominant in the Th17 immune response and Treg, namely the IL- 17/TGF-β ratio; and the ratio of important cytokines in Th1 immune response
to TGF-β, namely the IFN-γ/TGF-β ratio, IFN-α/TGF-β ratio, and IFN-β/TGF- β ratio, are very important to analyze in OT patients, because studies measuring cytokine ratios in OT patients have not been widely conducted. There were limited studies measuring cytokine ratios in ocular toxoplasmosis. Existing studies measured cytokine ratios in aqueous humor, and compared it between primary and recurrent active OT patients. The immune response in OT also occurs systemically, so further research is needed to analyze the activity of cytokines involved in OT infection in serum.
Peripheral blood sampling and tear collection are non-invasive methods compared to aqueous tap. Further research comparing cytokine ratios between OT patients and seropositive individuals is essential. Toxoplasma gondii infection also causes tear protein changes in experimental animals but has not been further studied in OT patients. Tears are part of the natural defense mechanism, and lactoferrin (LF), the biggest component of tears, is thought to have antiparasitic activity through several mechanisms. Measurements of tear LF levels of OT patients have never been carried out, but the results of an experimental study in animals infected with T. gondii oocysts showed an increase in tear LF levels.
This study aims to compare the levels of IFN-α, IFN-β, IFN-γ, IL-17, and TGF-β, the ratio of IL-17/IFN-γ, IL-17/IFN-α, IL-17/IFN-β, IL-17/TGF-β, IFN-γ/TGF-β, IFN-α/TGF-β, IFN-β/TGF-β, and tear LF levels between OT patients and seropositive individuals without ocular lesions. The sampling method used is consecutive sampling for 14 months. The diagnosis of typical OT lesion is made clinically, and confirmed by serological titers. The research group was categorized into OT group and the seropositive group without
ocular lesion (SP) group. Ophthalmological examinations performed include Best Corrected Visual Acuity (BCVA), anterior segment examination, intraocular pressure, and posterior segment using a binocular indirect ophthalmoscope. Tear collection is carried out using a Schirmer strip. Examination of tear LF levels is conducted by the ELISA method. IgM and IgG anti-Toxoplasma gondii titers are measured using the CLIA method. IgG avidity test is performed on subjects with positive IgM and IgG titers using
the ELFA method. Cytokine levels are measured by the ELISA method. The differences between groups are analyzed using an independent T-test, if the data met the assumption of normality and homogeneity of variances, with sig > 0.05. Binary logistic regression determines the significance of risk factors
to OT. It is expected that this study will find risk factors for the occurrence of ocular toxoplasmosis in plasma and tears so that it can help determine groups of seropositive individuals who have the potential to experienceocular manifestations.
This research involved 34 subjects, 16 subjects in the OT group and 18 subjects in the SP group. There were 21 eyes, either with active or inactive lesions, from 16 subjects in the OT group. There were no significant
differences in age and gender between the two groups. Based on the Mann- Whitney test, the results showed that the median IFN-α level in the OT group (123.205 pg/mL) was lower than in the SP group (165.333 pg/mL) with pvalue= 0.015. There was no significant difference between the median IFN-β
levels in the OT group (147.013 pg/mL) and the SP group (146.233 pg/mL) with p-value=0.059. The IL-17/IFN-γ ratio in the OT group (1.833) was not significantly different from the SP group (3.146) with p-value=0.126, likewise with the ratio of IL-17/IFN-α between TO group (0.767) and SP group (0.645)
with p-value = 0.528; and the ratio of IL-17/IFN-β between TO group (0.582) and SP group (0.469) with p-value = 0.297. The ratio of IL-17/TGF-β in the OT group (0.158) was significantly smaller than in seropositive individuals without ocular lesions (0.244) with p-value=0.018. There was no significant
difference between the IFN-γ/TGF-β ratio in the OT group (0.077) and the SP group (0.142) with p-value=0.126. The ratio of IFN-α/TGF-β in the OT group (0.231) was significantly smaller than in the SP group (0.325) with pvalue= 0.012. The IFN-β/TGF-β ratio in the OT group (0.304) was significantly
smaller than in seropositive individuals without ocular lesions (0.424) with pvalue=0.02. Based on the independent sample t-test, there was no significant difference between tear LF levels in the OT group (166 ± 19.11 ng/mL) and the SP group (171 ± 25.87 ng/mL) with p-value = 0.515. Based on the multiple binary logistic regression test using the Backward Stepwise (Likelihood Ratio) method, the factor that played the most role in the risk of OT occurring among the variables studied was IFN-α levels (p-value = 0.014). It can be concluded that there are significant differences in IFN-α levels, IL-17/TGF-β ratio, IFN-α/TGF-β ratio, and IFN-β/TGF-β ratio between OT patients and seropositive individuals without ocular lesions, where the level of IFN-α is the factor that plays the most role in the occurrence of OT among the variables studied. The novelty of this study is discovering that type I interferons, especially IFN-α, and the ratio of key cytokines in Th1 and Th17 immune responses play a role in the occurrence of OT, which these results have not been reported in previous studies. The division of groups in this study is also a highlight of this study, because the control group selected is seropositive individuals who do not experience clinical manifestations, in contrast to previous studies that mostly used healthy controls or seronegative individuals. The results of this study have important clinical implications, which demonstrate that levels of IFN-α, whose role has only been proven in experimental animals, have an effect on the occurrence of OT in human. The cytokine ratio has been proven to also influence the occurrence of OT and is an objective parameter to describe the balance of the immune response.