IMMUNOPATHOGENESIS OF ENTAMOEBA HISTOLYTICA AT NEWLY DIAGNOSED MALE PATIENTS, IN THE EASTERN REGION OF IRAQ.

Zaid Habeeb Abd-Alkareem 1 , Jaber Hameed hussien 1 , Qays Ahmed Al-Kafaji 2 and Mohammed Al-Aaraji 1 . 1. Department of Clinical Laboratory Sciences, College of Pharmacy, AL-Mustansiriyah University. Baghdad, Iraq. 2. Kamal Al-Samarraee Teaching Hospital for Infertility and IVF. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Entamoeba histolytica is an anaerobic parasitic protozoa, it infects a large part of gastrointestinal tract of human causing amebiasis, human is the initial reservoir of Entamoeba histolytica, and the infection is mainly transmitted via contaminated fresh water and food (Khan and Jahan, 2017). Entamoeba histolytica infection is distributed worldwide, it causes colitis, and extraintestinal amebiasis to 50 million with 100.000 deaths per year (Bercu et al, 2007). Patients with intestinal amebiasis may suffer from severe abdominal pain, and diarrhea. However, endoscopic assessment reveals the scattered colonic ulcerations and liver abscess in some cases. Entamoeba histolytica has a two-stage life cycle, which is represented by cysts and trophozoites (Peterson et al 2011). It is an intracellular protozoan, it attaches to the host tissue in a manner of contact-dependent, this process and E histolytica execratorysecretory products (ESP) is reported to play a key role in cellular invasion (Ahn et al 2018). Moreover, E histolytica is found to initiate a local, secondary immune reaction, an anti-amoebic secretary IgA (sIgA) is diagnosed in saliva, faeces, breast milk, and bile from infected patients (Carrero et al, 2007). The pathogenesis of E histolytica is still not fully understood; however, many studies revealed, that the pathogenicity of E histolytica is a multifactorial tactic (Espinosa-Cantellano M and Martínez-Palomo, 2011). Some manifestations of E histolytica`s pathogenesis are proposed, they are generally grouped according to the involved mechanisms in to the following: (a) interaction of E histolytica with the intestinal normal flora, (b) E histolytica lytic effect on the host cell via direct adhesion or by cytotoxic products (c) E histolytica phagocytic activity of target cell (Espinosa-Cantellano, 2000). The microscopic examination of rectal smears is the common diagnostic test, to detect the trophozoites of E histolytica that are found in the mucus and exudates of mucosal ulcers, laboratory test depends on detecting anti E histolytica is recently used (Abbas et al 1997). Cytokines are small secreted proteins released by immune cells have a specific effect on the 1412 interactions and communications between cells, Interleukin-1-alpha, one form of interleukin-1, which is made mainly by white blood cell to fight infections. It also helps leukocytes pass through blood vessel walls to sites of infection and causes fever by affecting areas of the brain that control body temperature (Choi et al, 2006).
The IL-1α precursor is constitutively present in epithelial layers of the entire gastrointestinal tract, lung, liver, kidney, endothelial cells, and astrocytes ( Upon cell death by necrosis, as occurs in ischemic diseases such as myocardial infarction, stroke, acute renal failure and tumor necrosis, the IL-1α precursor is released (Garcia-zepeda et al 2007). Interleukin 2 (IL-2) is a monomeric glycoprotein, it is produced by activated CD4+ T-cells, CD8+ Tcells and dendritic cells . It is characterized as a pro-inflammatory cytokine that is secreted by Th1 cells (Gaffen and Liu, 2004). IL-8 is a chemoattractant cytokine produced by a variety of tissue and blood cells; unlike many other cytokines, it has distinct target specificity for the neutrophil, with weak effect on other blood cells (Begum et al, 2015).

Patients and methods:-
This study included 110 cases, 55 were newly diagnosed with acute Entamoeba histolytica infection, and 55 cases were the healthy control. All studied cases were located at Wassit province, and male only were selected. Stool Samples were mixed with 0.9% sodium chloride solution (wet amount), then samples were subjected to direct microscopic examination; to detect the motile trophozoites of Entamoeba histolytica. Also, stool samples were stained with Lugol's iodine solution to identify trophozoites and cysts. Enzyme Linked Immunosorbent Assay (ELISA) technique was conducted to measure the levels of IL-1α, IL-2 and IL-8 in serum.

Statistical analysis:-
The statistical analysis was applied using Anderson Darling test to assess the distribution of continuous variables. Discrete variables presented using there number and percentage used to present data, chi square test was used to analyse the discrete variable, and statistical difference between studied groups was rated using one way ANOVA and Tukey`s multiple comparison. All experiments were carried out three times independently: N=3, and data was expressed as ±SE. Binary logistic regression analysis used to calculate the odd ratio (OR) and their 95% confidence intervals. SPSS 20.0.0 software package was used to make the statistical analysis; p <0.05.

Interleukin-1 alpha (IL-1α)
A significant increase was seen in IL-1α levels in patients compared to the healthy control, (6.143± 1.476) for the infected patients and (2.383± 0.71) for the healthy control group respectively, p<0.05, data represented as Mean± SD, as in table 1 and figure 1.

Interleukin 8
The study results involve significant elevated in IL8 in patients groups than control groups P <0.001 with confidence interval (3.512-4.881), t-test (12.211) and Mean± SD (5.439± 1.545), 9.636± 1.367 respectively. Table 3 and figure 3.  IL-2 is a monomeric glycoprotein produced by activated CD4+ T-cells, CD8+ T-cells and dendritic cells (Gaffen and Liu 2004). IL-2 is a pro-inflammatory cytokine, that is secreted by Th-1 cells, and it effectively participates in the activation of T cells to produce the cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFNγ); IL-2 can also enhance the 3 cytolytic activity of natural killer cells (NK) ( ). Therefore, IL-2 is used therapeutically to stimulate the immune system (Al-Oumashi 2012). IL-2 also contributes to the development of regulatory T-cells, which controls the apoptosis of activated T cells, Furthermore, IL-2 influences cell survival, differentiation (Campbell et al 1999) and the formation of immune memory cells and acts as a negative regulator of immune activation (Gaffen and Liu 2004 IL-8 also known as NAP-1 for Neutrophil-activating peptide is a chemoattractant protein for neutrophils. IL-8 is a pro-inflammatory mediator secreted by different cells such as monocytes, neutrophils, endothelial cells, fibroblast after activation via mitogen-stimulated T-lymphocytes (Bickel 1993). IL-8 is a key cytokine, which is expressed in scales of psoriasis patients, in synovial fluid of patients suffering from rheumatoid arthritis and gout (Diaz-Valencia et al 2015). The current study revealed a significant increase in IL-8 in patients diagnosed with E histolytica compared with the healthy control group, P <0.005 as in Table 3 and figure 3.
Our results were coincident with YU et al, 1997 stated that tissue infiltration by neutrophils during the acute inflammatory response is dependent on the local release of soluble chemoattractants such as IL-8, we hypothesize that E. histolytica can cause the induction of IL-8 by colonic epithelial cells in the absence of cell-cell contact. The secretory response (IL-8 secretion) to E. histolytica may play a role in initiation of an acute inflammatory response before amebic invasion. (Dey and Chadee 2008). Based on the obtained results, it is possible to reach the following conclusions: IL-1α, IL-2 and IL-8 levels in patient's revealed a significant increase in patient group in comparison to healthy control group.