Abstract

It is acknowledged that the most common autoimmune enteropathy in children is celiac disease (CD). In people who are genetically sensitive, gluten causes it. The genetic foundation of CD is well understood.

This study is project of graduated students. ELISA and PCR-sequencing method were done for (35) patients and (35) control samples groups.Each participant had a vein puncture to extract three milliliters of blood. Blood samples were taken in EDTA tubes, and two milliliters of blood were placed in a gel tube for serum separation. The study comprised 20 depression patients in total. Samples were obtained from private laboratories visited by the patients, who were between the ages of 13 and 18. The results above about age indicate that there is no significance value with the disease, p value= 0.372. According to results of gender obtained from the current study, male has more frequency than female. The results above about residence indicate that there is differences between residence in rural and urban. The result above indicates some variation in genetic relationship between patients samples.The results above about age indicate that there is no significance value with the disease, p value= 0.372. According to results of gender obtained from the current study, male has more frequency than female. The results above about residence indicate that there is differences between residence in rural and urban. The result above indicates some variation in genetic relationship between patients samples.

Keywords: Biological parameters, IL-15, Celiac disease

Introduction

            Iran constitutes one of the 88 nations where visceral leishmaniasis (VL) is endemic. The parasites within cells of the Leishmania (L.) donovani complex, which includes L. donovani, L. infantum, and L. chagassi, are the cause of vector-borne infectious illness known as vector-borne lympholysis (VL) (Mahajan and Mohan , 1996).

            It is being discovered that L. infantum is the main causal factor in Iran’s northwestern and southwest (Mazloumi et al., 2004; Alborzi et. al. , 2006).

            The VL is fatal if left untreated and affects several organs, including the liver, spleen, and bone marrow. Since leishmania parasites can multiply in macrophages, nearly all immune cells are involved in the complicated immunological response to VL. It has been demonstrated that the outcome of the VL in the leishmaniasis mice study is determined by the responses of the T cells; T helper 1 (Th) reactions are linked to obstacle, whereas Th2 reactions are linked to vulnerability or chronicity (Manoochehr et. al., 2013).

            Although the immune system’s reactions in the human VL version remain unclear compared to the mouse method, it has been demonstrated that Th1 cytokines, notably interleukin -2 (IL) and gamma interferon (IFN-c), may entirely regulate and resolve VL disease (Cillari  et. al., 1991). IFN-c can also be generated by cytotoxic T lymphocytes (CTLs) and engaged NK cells in along with Th1 cells  (Bach et. al., 1995).

            Another of the cytokines that stimulates NK cells to create IFN-c is IL-15, a 14–18 KDa pleiotropic cytokine that has biological properties comparable to IL-2 (Gin  et al., 1995), mainly made by monocytes and that enhances Th1 and Th2 replies (Carson  et al. 1994; Manoochehr et. al., 2013).

            Nevertheless, it has been shown that this cytokine can alter the ratio of Th1/Th2 to Th1 reactions (Takeuchi  et. al., 2001). For the best generation of IFN-c, which triggers adaptive responses versus infections caused by bacteria, particularly that brought on by parasites within cells, IL-15 and IL-12 must work in concert (Yoshikai and Nishimura,  2000).

            An analysis of the relationship involving this cytokine and VL showed that the bone marrow and sera of VL sufferers had higher levels of IL-15, transforming growth factor (TGF)-b, and IL-10 (Milano et. al., 2002). In a different study, the symptomatic VL patients had considerably greater levels of IL-15, IFN-c, and IL-12 than the asymptomatic patients as well as the general population in general (Hailu  et. al., 2004).

            Furthermore, it has been demonstrated that IL-15 can boost the leishmanicidal activity of VL patient-derived macrophages by lowering IL-4 production and raising IL-12 output (Milano  et. al., 2002; D’Agostino et. al., 2004).

Material and Methods

Samples collection

            Each participant had a vein puncture to extract three milliliters of blood. Blood samples were taken in EDTA tubes, and two milliliters of blood were placed in a gel tube for serum separation. The study comprised 20 depression patients in total. Samples were obtained from private laboratories visited by the patients, who were between the ages of 13 and 18. Prior to the collection of any data or samples, participants signed an informed consent form and were given a proper explanation of the research project. Questionnaires were used to gather pertinent data regarding the participants’ sociodemographic characteristics and medical history. Medical professionals assessed the clinical evaluations of depressed patients.

Exclusion criteria

            Study participants with chronic diseases that can affect the digestive system were excluded from the study.

Genomic DNA Mini Kit (Blood)

            Genomic DNA from white blood cells (WBCs) for both depressed patients and control group were extracted by using DNA extraction kit (fovergene kit)

PCR Amplification of IL-15 gene

For IL-15 genotyping , a set of primers as in table (1)

Table 1: IL-15 primers

Table 2: Amplification conditions of IL-15 gene

            The conditions of the PCR and annealing temperature was determined as reported in Yalcın Kara, et. al. (2020). Amplified DNA fragments were electrophoresis on 1% agaros(0.5x) TBE buffer (40 min at 75v) and the bands visualized after staining with ethidium bromide under UV light , A 100 base- pair ladder were as a size marker for estimation of fragment sizes.

DNA Sequencing- PCR

            PCR- Sequences of IL-15 gene were performed by MACROGEN company, for confirming the presence of  mutations through the genomic DNA in the blood of the patients for IL-15 gene. The samples of the patients were primed and amplified for the mentioned genes above by using  primers. The samples were sent for analytic sequences.

Statistical Analysis

            The statistical analysis of the results was performed using GraphPad Prism7 software.T-tests were used to find P-value. A difference of P<0.05 was considered statistically significant.

Results and discussion

Demographic data

            The results of the current study have been  shown in table (3).

Table 3: Demographic data

            The results above about age indicate that there is no significance value with the disease, this may be due to two reasons.

First reason is due to race and the second is due to the low sample number of the graduated project.This result was accepted with Yalcın et. al. (2020).According to results of gender obtained from the current study, male has more frequency than female, table(3).

            This result accepted with Gloria et. al.) 2022( which indicates Biological sex is genetically determined, while gender regards the social context. In the current study, biological sex and gender overlapped in all patients. The term “gender” was used for sociodemographic and lifestyle features, such as age, symptoms, comorbidities, body mass index, and family history, while “sex” was used for immunologic and genetic features.

            The results above about residence indicate that there is differences between residence in rural and urban table(3).

            This result not accepted with Posterick  and  Ayars, (2023) revealed those living in urban areas had, on average, good adherence to a gluten-free diet, while those living in nonurban areas had, on average, poor adherence to the diet. Those in nonurban areas face an increased risk for ongoing physical damage and disease progression due to the dietary adherence rates found. The study findings provide support to public health and healthcare practitioners in their efforts to intervene at both the community and individual levels to support those with celiac disease. By directing their efforts and allocating resources to nonurban areas, among younger patients and those with lower incomes, there is potential to enhance gluten-free dietary adherence and positively impact symptoms of celiac disease.

Relationship of H. pylori infection and disease development

            The results of current study have been  shown in table (4).

Table(4) Relationship of H. pylori infection and disease development

            The results above about age indicate that there is no significance value with the disease, this may be due to the low sample number of the graduated project.

            This result not accepted with Min et. al. (2022), which indicates that this study confirmed that there is a correlation between H. pylori and CD, but the causality cannot be clarified. A demonstration of a causal role of H. pylori in CD in future prospective studies could have important therapeutic implications.

Relationship of IL-15 gene and disease development

            PCR product of IL-15 gene (250). Figure (1) indicates the genetic tree of IL-15 gene between patients and control groups.

Figure 1:The genetic tree of IL-15 gene between patients and control groups

            The result above indicates some variation in genetic relationship between patients samples, and this result accepted with Manoochehr et. al. (2013).This study may be speculated that these gene variants with probable effects on the IL-15 production can serve as the factors influencing VL among Iranian population. However, to clarify the association of these variants with the level of IL-15, further studies are recommended.

Conclusions

          The results above about age indicate that there is no significance value with the disease, p value= 0.372. According to results of gender obtained from the current study, male has more frequency than female. The results above about residence indicate that there is differences between residence in rural and urban. The result above indicates some variation in genetic relationship between patients samples

References

• Alborzi A, Rasouli M, Shamsizadeh A (2006) Leishmania tropica- isolated patient with visceral leishmaniasis in southern Iran. Am J Trop Med Hyg 74:306–307
• Amy Posterick  and Candace L. Ayars, (2023). Celiac Disease Dietary Adherence on the Rural–Urban Continuum. Nutrients 2023, 15, 4535. https://doi.org/10.3390/nu15214535,https://www.mdpi.com/journal/nutrients
• Bach EA, Szabo SJ, Dighe AS, Ashkenazi A, Aguet M, Murphy KM, Schreiber RD (1995) Ligand-induced autoregulation of IFNc receptor beta chain expression in T helper cell subsets. Science 270:1215–1217
• Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M (1994) Interleukin-15(IL) is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180:1395–1403
• Cillari E, Milano S, Dieli M, Maltese E, Di Rosa S, Mansueto S, Salerno A, Liew FY (1991) Reduction in the number of UCHL- 1? cells and IL-2 production in the peripheral blood of patients with visceral leishmaniasis. J Immunol 146:1026–1030
• D’Agostino P, Milano S, Arcoleo F, Di Bella G, La Rosa M, Ferlazzo V, Caruso R, Chifari N, Vitale G, Mansueto S, Cillari E (2004) Interleukin-15, as interferon-gamma, induces the killing of Leishmania infantum in phorbol-myristate-acetate-activated macrophages increasing interleukin-12. Scand J Immunol60:609–61
• Gin JG, Anderson DM, Kumaki S, Park LS, Grabstein KH, Cosman D (1995) IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol 57:763–766
• Gloria Galli  , Giulia Amici, Laura Conti, Edith Lahner, Bruno Annibale and Marilia Carabotti, (2022). Sex–Gender Differences in Adult Coeliac Disease at Diagnosis and Gluten-Free-Diet Follow-Up. Nutrients 2022, 14, 3192. https://doi.org/10.3390/nu14153192 https://www.mdpi.com/journal/nutrients
• Hailu A, Vander Poll T, Berhe N, Kager PA (2004) Elevated plasma level of interferon (IFN)-gamma, IFN-gamma inducing cytokines, and IFN-gamma inducible CXC chemokines in visceral leishmaniasis. Am J Trop Med Hyg 71:561–565
• Mahajan RC, Mohan K (1996) Epidemiology of visceral leishmaniasis and its control. In: Ozcel MA, Alkan MZ (eds) Parasitology for the 21st century. Cab International, Oxon, pp 41–49
• Manoochehr Rasouli , Mehdi Kalani ,  Simin Kiany, (2013). The role of IL15 gene variants in visceral leishmaniasis among Iranian patients. Mol Biol Rep DOI 10.1007/s11033-013-2617-0
• Manoochehr Rasouli , Mehdi Kalani , Simin Kiany. 2013. The role of IL15 gene variants in visceral leishmaniasis among Iranian patients. Mol Biol Rep DOI 10.1007/s11033-013-2617-0
• Mazloumi AS, Esmaeili H, Davies C (2004) Species and strains identification of Leishmania isolated from kalaazar patients in northwest of Iran. Urmia Med J 1:39–46
• Milano S, Di Bella G, D’Agostino P, Barbera C, Caruso R, La Rosa M, Ferlazzo V, Vitale G, La Russa C, Gambino G, Chifari N, Mansueto S, Cillari E (2002) IL-15 in human visceral leishmaniasis caused by Leishmania infantum. Clin Exp Immunol 127:360–365
• Min Yue , Qiang Chen , Xinxin Zhou , Lan Li , Chao Lu, (2022). Is Helicobacter pylori Infection Associated with Celiac Disease? A Meta-analysis. Turk J Gastroenterol 2022; 33(3): 205-212 Yue et al. Helicobacter pylori and Celiac Disease
• Takeuchi E, Yanagawa H, Suzuki Y, Shinkawa K, Bando H, Sone S (2001) Interleukin (IL-15) has less activity than IL-2 to promote type 2 cytokine predominance in tumor-associated mononuclear cells from lung cancer patients. Cytokine 13:119–123
•  Yalcın Kara , Makbule Eren , Serap Arslan , Oğuz Cilingir (2020). IL-15 Gene Polymorphism in Celiac Disease Patients andTheir Siblings. Turk J Gastroenterol 2021; 32(4): 349-356 Kara et al. IL-15 Effect on Celiac Disease.
• Yalcın Kara , Makbule Eren , Serap Arslan , Oğuz Cilingir (2020). IL-15 Gene Polymorphism in Celiac Disease Patients andTheir Siblings. Turk J Gastroenterol 2021; 32(4): 349-356 Kara et al. IL-15 Effect on Celiac Disease.
• Yoshikai Y, Nishimura H (2000) The role of interleukin-15 in mounting an immune response against microbial infection. Microbes Infect 2:381–389