Endothelial dysfunction in acute acquired toxoplasmosis.

BACKGROUND: Acute toxoplasmosis (AT) which is caused by Toxoplasma gondii (T. gondii) leads to induction of pro-inflammatory and/or oxidative stress changes through activation of host immune response. Therefore, the endeavor of the present study was to assess endothelial dysfunction(ED) and oxidative stress in patients with acute toxoplasmosis.
METHODS: This study involved 21 patients with AT compared with 20 healthy controls. Serum immunoglobulin levels [IgG], IgM, IgA), Interleukin-6 (IL-6), endothelin-1 (ET-1), and human malondialdehyde (MDA) serum levels were evaluated.
RESULTS: IgM, IgG, and IgA levels were high patients with AT as compared with the control (P < 0.01). IL-6, MDA, and ET-1 serum levels were high in patients with AT compared with control (P < 0.01). In patients with AT, IgM serum level was significantly correlated with other immunoglobulin, and with the biomarker of oxidative stress, lipid peroxidation, and ED (P = 0.0001).
CONCLUSION: AT is linked with oxidative stress and pro-inflammatory changes which together provoke ED. Copyright:

INTRODUCTION

Toxoplasma gondii (T. gondii) is one of the most well studied parasites because of its medical and veterinary importance. It is used extensively as a model for cell biology of apicomplexan organisms. Historically, T. gondii originated probably as a coccidian parasite of cats with a fecal-oral cycle. With domestication, it adapted transmission by several modes, including transmission by fecal-oral cycle, by carnivorism, and transplacentally. There are three infectious stages of T. gondii: the tachyzoites, the bradyzoites, and oocysts.[1] People can be tainted by eating under-cooked meat of tainted creatures, devouring nourishment or water polluted with feline defecation, blood transfusion, organ transplantation, or trans-placentally from mother to fetus, which is the most genuine one. Toxoplasmosis in right on time pregnancy is normally joined by extreme harm to the fetus prompting premature birth and congenital malformations.[2]

The infective stage of T. gondii including three stages, sporozoite, tachyzoite which is rapidly multiplying form, and bradyzoite which is tissue cystic form. Bradyzoite is less vulnerable and more resistance to the drugs and host immunity, while tachyzoite is concerned with the clinical manifestation of acute toxoplasmosis. Tachyzoite is more vulnerable to the consequence and effect of host immunity.[3]

The frequency of seropositive for T. gondii is highly fluctuated depending on age, hygiene, eating habits, and geographical area. The risk of infection with T. gondii is higher in warm and humid climate. The rapid intracellular proliferation of T. gondii leads to damage of the reticuloendothelial system during the acute phase of infection.[4]

Moreover, host immunity against T. gondii infection depends on the activation of cellular and humoral immunity. Specific immunoglobulin G (IgG) antibody can lyse extracellular trophozoites, but the activation of T cells and natural killer cells appear to be more important in preventing disease progression.[5]

T. gondii infection provokes the release of different cytokines and pro-inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interferon-gamma(INF-g) from activated T-helper cells.[6] In addition, nitric oxide (NO) inhibits the growth of T. gondii; in acute toxoplasmosis.[7]

Normally, vascular endothelial cells preserve vascular tone via regulation of vasodilar and vasoconstrictor mediators, such as nitric oxide (NO), endothelin-1(ET-1), and prostocyclin. Any injury these cells due to inflammatory or oxidative injury lead to disturbances in the endothelial function, which called endothelial dysfunction (ED).[8]

The most important biomarker of ED is ET-1, which is a potent vasoconstrictor produced by the vascular endothelial cells. Serum level of ET-1 is linked and correlated with ED of different etiologies.[910]

Oxidative stress which causes the overproduction of free radicals and/or reduction of endogenous antioxidant capacity leads to severe endothelial dysfunction. The pathogenesis of T. gondii is linked with the induction of oxidative stress.[11]

Lipid peroxidation and oxidative stress have been regarded as the main factors responsible for the generation of free radicals which leads to platelets and leukocytes adhesion to the vascular endothelium causing vasoconstriction and augmentation of peripheral vascular resistance. Malondialdehyde (MDA) is a biomarker of lipid peroxidation and oxidative stress and increased in chronic acquired toxoplasmosis.[1213]

Therefore, the endeavor of the present study was to assess ED and oxidative stress in patients with acute toxoplasmosis.

METHODS

In this observational study, 21 patients with acute toxoplasmosis (AT) aged 23-51 years were recruited The National Center of Infectious Diseases, Ibn-Akateeb Hospital, compared with 20 healthy controls that recruited from patient relatives. This study was done in the Department of Clinical Pharmacology and Therapeutic in Cooperation with the Department of Medical Microbiology in College of Medicine, Al-Mustansiriyia University, Iraq Baghdad, 2019. This l study was approved by Medical Editorial Board and Ethical Committee with respect to the Declaration of Helsinki. Full medical history, clinical examination, and stereological tests were done for all patients and healthy controls, to confirm the chronic acquired infection in the patients, and to exclude healthy control with asymptomatic infections.

Exclusion criteria included psychological diseases, neurological diseases, hypothyroidism, end-stage kidney disease, hepatic dysfunction, connective tissue disorders, malignant disorders, and immunodeficiency.

The assessment of biochemical variables

5 mLs of venous blood were obtained from the antecubital area of each patient and healthy control; the blood was centrifuged at 3000/rpm and stored at −20°C for further analysis.

Serological tests

Anti-T. gondii antibody was determined by direct antigen-antibody reaction (CTK biotech. Inc., USA).

Assessment the biomarkers of endothelial dysfunction

IL-6 serum level was measured by ELISA kit method (Human IL-6 Kit, high sensitive, ab46o42, Abcam, USA). The ET-1 serum level was measured by ELISA kit method (ET-1 ELISA Kit, ab133030, Abcam, USA). Human MDA was measured by ELISA kit method (Colorimetric/Fluorometric, ab118970, Abcam, USA).

Statistical analysis

Data analysis was done by using SPSS (IBM SPSS Statistics for Windows version 20.0, 2014 Armonk, NY, IBM, Corp, USA). Unpaired student t test was used to test the level of significance between two study groups. Pearson correlation was used to detect the of correlations. The level of significance was regarded when P<0.05.

RESULTS

Regarding the demographic characterstics, this study, illustrated that the age, and history of contact with animals were not differed significantly compared with the controls (P>0.05). As well, smoking history was low in the infected patients compared with control (P = 0.04). In this study, 100% of infected patients with T. gondii illustrated positive for IgM and 90.32% for IgG. 10% of controls showed a positive test for IgG. Furthermore, 23.80% and 80.95% of infected patients with T. gondii were treated with spiramycin and clindamycin, respectively. Other characteristics of the present study are presented in Table 1.

Table 1

Demographic characteristics of the present study

The characteristics	Control (n=20)	Patients (n=31)	P
Age (years)	31.78±9.28	33.67±8.41	0.45
Gender (male:female ratio)	6:14	4:27	-
Male	4 (20)	4 (12.90)	0.50
Female	14 (70)	27 (87.09)	0.13
Marital status
Married	11 (78.57)	21 (77.78)	0.94
Unmarried	3 (21.43)	6 (22.21)	0.93
History of recurrent abortion	1 (7.14)	18 (66.67)	0.0001*
History of contact with animals	13 (65.00)	25 (80.64)	0.21
Smoking	7 (35.00)	3 (9.68)	0.02†
Associated diseases
Hypertension	-	9 (29.03)	-
Asthma	-	3 (9.67)	-
Dyslipidemia	-	8 (25.80)	-
Peptic ulcer	-	3 (9.67)	-
Current therapy
Aspirin	6 (30.00)	14 (45.16)	0.28
Theophylline	-	3 (9.67)	-
Statins	-	8 (25.80)	-
Lansoprazole	-	3 (9.67)	-
IgM positive	-	31 (100)	-
IgG positive	3 (15.00)	28 (90.32)	0.0001*
Anti-toxoplasmosis agents
Spiramycin	-	8 (25.80)	-
Clindamycin	-	23 (74.19)	-

Data presented as mean±SD, n, %, *P<0.01, †P<0.05. SD: Standard deviation, IgM: Immunoglobulin M, IgG: Immunoglobulin G

On the other hand, immunoglobulin concentrations were higher in patients with acute toxoplasmosis, IgM, IgG, and IgA levels were high in the infected patients compared with control (P < 0.01). As well, IL-6 serum level was high in the infected patients (6.25±2.84 pg/mL) compared with control (1.21±0.54 pg/mL) (P < 0.01) [Table 2]. In addition, biomarkers of endothelial dysfunction were augmented in patients with acute toxoplasmosis, ET-1 level was high in acute toxoplasmosis (7.29 ± 4.59 pg/mL) compared with control (3.11 ± 1.69 pg/mL) (P < 0.01). As well, MDA serum level was high (9.34 ± 4.17 nmol/mL) compared with control (2.87 ± 1.13 nmol/mL) (P < 0.01), [Figure 1].

Table 2

Changes in the immunoglobulin levels, pro-inflammatory, and oxidative stress biomarkers in patients with acute toxoplasmosis

Variables	Control (n=20)	Patients (n=31)	95% CI	P
IgM (g/L)	1.2±0.8	3.6±2.99	1.0213-3.778	0.001*
IgG (g/L)	4.31±2.95	22.96±9.57	10.1073-19.192	0.0001*
IgA (g/L)	1.99±1.34	4.72±2.54	1.4877-3.9723	0.0001*
IL-6 (pg/mL)	1.88±0.51	3.22±1.61	0.5912-2.088	0.0007*

*P<0.01. IL-6: Interluekin-6, CI: Confidence interval, IgM: Immunoglobulin M, IgG: Immunoglobulin G, IgA: Immunoglobulin A

Figure 1

Biomarkers of endothelial dysfunction and oxidative stress in acute toxoplasmosis compared with the healthy controls

Regarding the correlation of Igs in patients with acute toxoplasmosis, IgM serum level was significantly correlated with IgG, IgA, IL-6 and MDA, [Table 3].

Table 3

Correlations of immunoglobulin M levels with immunoglobulin levels, pro-inflammatory, and oxidative stress biomarkers in patients with acute toxoplasmosis

Variables	Control (n=20) (r, P)	Patients (n=31) (r, P)
IgG (g/L)	0.26, 0.26	0.55, 0.001*
IgA (g/L)	0.22, 0.35	0.57, 0.0008*
IL-6 (pg/mL)	0.19, 0.42	0.45, 0.01†
ET-1 (pg/mL)	0.28, 0.23	0.51, 0.003*
MDA (nmol/mL	0.20, 0.39	0.85, 0.0001*

*P<0.01, †P<0.05. r: Correlation, P: Significant level, IL-6: Interluekin-6, ET-1: Endothelin, MDA: Malondialdehyde, IgG: Immunoglobulin G, IgA: Immunoglobulin A

DISCUSSION

T gondii is an intracellular pathogen affecting approximately one-third of the human population. It exists in nature as oocysts, bradzyzoites (contained in latent tissue cysts), and replicating tachyzoites, with the last form being the hallmark of active disease. For clinical purposes, toxoplasmosis can be divided for convenience into three infection categories including, acquired, congenital, and reactivated one in immunodeficient patients. In any category, the clinical presentations are not specific for toxoplasmosis, and a wide differential diagnosis must be considered. Furthermore, methods of diagnosis and their interpretations may differ for each clinical category.[14]

In clinical presentation AT is typically asymptomatic in immunocompetent individuals, but cervical lymphadenopathy or ocular disease can occur. Infection of immunocompetent individuals with more virulent strains of T. gondii, which are prevalent and, can result in severe pneumonia and disseminated disease, including death.[15] In the present study, all of the recruited patients were immunocompetent that explains the mild clinical presentation of acquired toxoplasmosis in the recruited patients.

The diagnosis of acute acquired infection of T. gondii depends on the detection of specific antibodies against antigens of T. gondii. Serology for the detection of IgM and IgG should be done in recognition of toxoplasmosis while IgA test gives a supplementary evidence concerning reactivation or acute infection. Increase of IgM, IgG, and IgA levels occurs in an acute infection, while high IgG and IgA with negative tests for IgM are occurring in reactivation but not in acute infection.[16] These findings explain the high levels of IgG, IgM, and IgA in the present study since all of the recruited patients were with acute infection of T. gondii. During AT IgM is early appear in plasma and rapid decline, but IgG may persist for months-years following acute infection; therefore, a negative test for IgG excludes AT. Therefore, the diagnoses of acute infection based on a four-fold increase in IgG with or without a positive test for IgM as both IgG and IgM are highly sensitive and specific.[17] In the present study, there was a significant five-fold increase in IgG level that confirms AT as well, the IgA serum level was increased significantly compared with healthy controls. These findings are in agreement with a recent study that illustrated high IgA serum level is correlated with the accuracy and sensitivity of the serological panel for the diagnosis of acquired acute toxoplasmosis.[18]

As well, the present study illustrated that MDA serum level was high in patients with acute toxoplasmosis compared with control due to the induction of oxidative stress and lipid peroxidation. Dincel and Atmaca study confirmed that a high level of MDA is associated with acute toxoplasmosis due to the induction of oxidative stress and reduction of endogenous antioxidant capacity.[19]

On the other hand, the present study demonstrated a significant increase in the biomarkers of endothelial dysfunction, as both ET-1 and IL-6 were elevated in patients with acute toxoplasmosis compared with healthy controls. These findings are in concurrence with Knight et al.'s study which illustrated significant endothelial damage caused T. gondii during acute infection.[20]

Moreover, different studies illustrated that T. gondii induces endothelial inflammatory changes due to the activation of pro-inflammatory cytokines. As well, chronic toxoplasmosis leads to cholesterol esterification, endothelial foam cell formation, and development of endothelial dysfunction. Foam cells secrete IL-6 which causes leukocyte adhesion and provoke the expression of adhesion molecules on the endothelial cell.[21]

Moreover, intercellular adhesion molecule-1 (ICAM-1) is used by T. gondii for migration across vascular endothelial cells, so ICAM-1 inhibitors attenuate T. gondii-induced endothelial dysfunction in acute toxoplasmosis. Similarly, T. gondii provokes the adhesions of dentritic, monocyte, and other inflammatory cells to the vascular endothelial cells, thereby causing endothelial injury and dysfunction.[22]

Hence, endothelial cells react to these incendiary changes by release of IL-6, which down regulates and constrict endothelial harm; in this manner, IL-6 is viewed as a biomarker of endothelial dysfunction.[23] This finding affirms our outcomes that portrayed high IL-6 in acute toxoplasmosis.

It is well-known MDA is a marker of lipid peroxidation and oxidized low-density lipoprotein (LDL) which are increased during toxoplasmosis induced-oxidative stress. Acute oxidative stress leads to endothelial intracellular lysosomal membrane damage. Bahrami et al.'s study illustrated that T. gondii acquired cholesterol for their replication from the host LDL receptor pathway and also scavenge different lipids from the host cell.[24]

Our findings revealed that high levels of ET-1 and IL-6 were increased during AT. Recent study showed that infected mice with T. gondii illustrated an exaggeration of leukocyte adhesion to the endothelial cells that causing endothelial dysfunction. Furthermore, therapy with sulfadiazine improves endothelial dysfunction in acute toxoplasmosis through the reduction of parasitic load and related endothelial inflammations.[25]

Therefore, statins therapy in AT may improve endothelial dysfunction in patients with toxoplasmosis due to the protective effect of statins. Beside, statins inhibit adhesion and proliferation of T. gondii.[2627]

The present study has different limitations; the sample size was relatively small, the severity of AT was not taken into account, baseline co-morbidities were not estimated, doses of used drugs were not considered precisely, and finally patient outcomes were not followed-up after the study period. Despite these limitations, this study is regarded as a base study discussing the relationship between AT and ED. Therefore, further studies are needed to confirm the association reported in this study.

CONCLUSION

Acute toxoplasmosis is associated with significant oxidative stress and pro-inflammatory changes which contribute in the development of ED.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.