DOI 10.1515/jpem-2012-0079 

 J Pediatr Endocr Met 2012; 25(7-8): 747–752

Daiva Gorczyca*, Barbara Basiewicz-Worsztynowicz, Daria Augustyniak, Wieslawa Karnas-Kalemba, Hanna Cebula and Adam Jankowski

Relation among ghrelin, nutritional status, and immunity in children Adam Jankowski: Third Department and Clinic of Paediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland

Abstract Background: Ghrelin is an important mediator of energy balance and metabolism. The aim of this study was to investigate the relationship among ghrelin concentration, growth patterns, and immunological parameters in children with an impairment or inefficiency in functioning of the immune system. Methods: Twenty patients with primary immunodeficiency diseases (PIDs), 20 patients with recurrent respiratory tract infections (RRTIs), and 20 healthy children (control group) were included. The anthropometric measurements, ghrelin plasma levels, and selected immunological parameters were measured. Results: Ghrelin levels and nutritional status parameters (weight, height, and body mass index) values were negatively correlated only in the control group. Ghrelin negatively correlates with complement hemolytic activity in the PID group and with IgA serum level in the RRTI group. Conclusion: Our results show evidence that there is a relationship between ghrelin and nutritional status of healthy children but not in children with PID or RRTI. Keywords: children; ghrelin; immunological parameters; nutritional status.

*Corresponding author: Daiva Gorczyca, MD, PhD, Third Department and Clinic of Paediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, al. Kasprowicza 64/66, 51-137 Wroclaw, Poland, Phone: +48 71 3236450, Fax: +48 71 3236446, E-mail: [email protected] Barbara Basiewicz-Worsztynowicz: Third Department and Clinic of Paediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland Daria Augustyniak: Institute of Genetics and Microbiology, Department of Pathogen Biology and Immunology, Wroclaw University, Wroclaw, Poland Wieslawa Karnas-Kalemba: Third Department and Clinic of Paediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland Hanna Cebula: Third Department and Clinic of Paediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland

Introduction Research done over the last two decades provides more evidence about the existence of the bidirectional connection between the neuroendocrine and the immune systems (1, 2). Ghrelin, a peptide produced in the stomach and in the hypothalamus, stimulates not only food intake and growth hormone (GH) secretion but also participates in inflammatory processes (3–5). Previous studies focused on this hormone as a regulator of food intake and as the best indicator of nutritional status (6–8). However, ghrelin receptor, the GH secretagogue receptor (GHS-R), is expressed in various lymphoid organs (9), T and B cells, monocytes (10, 11), and dendritic cells. This suggests that ghrelin may also influence and modulate the humoral and cellular immunity (11–13) and mediate anti-inflammatory signals (1, 14). Moreover, immune cells are sensitive to energy-dependent processes that show the interaction between the innate and acquired immune responses and the metabolic system. Among the primary immunodeficiency diseases (PIDs), antibody defects are the most frequent (15). The impairment of the immune system in patients with antibody deficiencies and inefficiency in immune response function in children with severe recurrent infections are the common causes of growth and development disturbances (16–18). The possible relationship between ghrelin and growth of this group of children has not been clarified. There was not enough research conducted concerning this problem, and the majority of studies were performed on children with obesity or anorexia nervosa (12, 19, 20). In the current study, we examined the relation of serum ghrelin concentration to different growth patterns, such as weight, height, and body mass index (BMI), and to selected parameters of individual immune status in children with PID or with recurrent respiratory tract infections (RRTIs).

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 Gorczyca et al.: Ghrelin, nutritional status, and immunity in children

Subjects and methods Subjects Forty patients of both sexes, aged 4–18 years, were enrolled in this study. They were diagnosed and treated in the Department of Paediatric Immunology and Rheumatology between 2006 and 2010. Two groups of patients were identified: – The PID group consisted of 20 children with PIDs. The inclusion criteria into this group were defined according to the guidelines of the International Union of Immunological Societies Expert Committee (21). – The RRTI group consisted of 20 children with severe recurrent infections. The inclusion criterion into this group is eight or more incidents of acute respiratory tract infections per year. Children with diagnosis of primary or secondary immunodeficiency disease were excluded. The control group consisted of 20 healthy children. The children were outpatients who visited the hospital for a routine medical follow-up. Physical examinations were performed, and medical history was obtained to ensure the child was healthy and in good physical condition. The children’s parents or legal representatives gave written informed consent. Exclusion criteria for children of the three groups were any of the following: – Systemic diseases or chronic inflammatory diseases – History of any illness that affected growth – Acute febrile illness The study protocol was approved by the Research Ethics Committee of the Wroclaw Medical University.

Anthropometric measurements Height and weight were measured by the same person using standardized methods and equipment (22, 23). The measurements were taken in the morning before breakfast. BMI values were calculated according to this formula: weight (kg)/height (m2). The most current recommendations for the evaluation of the nutritional status of children in a clinical setting show that the use of body weight, height, and BMI percentiles is the most useful method (24). According to this method, the results of our anthropometric measurements were recorded on the current Polish growth charts, and then the height, weight, and BMI parameters were compared with the relevant percentile curves.

Laboratory tests All blood samples were drawn in the early morning between 8 and 9 a.m. at the same time with the laboratory tests necessary for the diagnostic and therapeutic processes. The received plasma was then placed into the Eppendorf test tube and stored at a temperature of –80°C until the start of the procedure. Human non-acylated ghrelin plasma levels were analyzed with ELISA kits produced by the

BioVendor Company (BioVendor-Laboratorni medicina a.s., Brno, Czech Republic). The sensitivity of the method was 0.2 pg/mL. The following were examined in children from the PID and the RRTI groups: ghrelin concentration in plasma; complete blood count (CBC); IgG, IgA, and IgM concentrations in the serum; subpopulations of lymphocytes CD3+, CD3+CD4+, CD3+CD8+, CD3–56+, and CD19+; the phagocytic activity (phagocytic index) of polymorphonuclear (PMN) leukocytes in the blood; and complement hemolytic activity of human serum (CH50). The following were examined in children from the control group: ghrelin concentration in plasma; CBC; IgG, IgA, and IgM concentrations in serum; the phagocytic index of PMN leukocytes in the blood; and CH50. Serum immunoglobulin concentrations were measured by the turbidimetry method; lymphocyte phenotypes were identified by using the Beckman EPICS XL-MCL flow cytometer (Beckman Coulter, USA); the phagocytic index was defined as the mean values of Staphylococcus aureus 209P bacteria ingested per 100 PMN cells; and the complement hemolytic activity of human serum against IgG-opsonized sheep red blood cells (the intensity of hemolysis) was estimated on a spectrophotometer (DYNATECH MR 5000, Dynex Technologies, Chantilly, Virginia, USA) at λ = 540 nm and quantitatively calculated according to the standard hemolytic curve. The subpopulations of lymphocytes were not determined the control group.

Statistical analysis For each parameter, the mean, median, and standard deviation (SD) were calculated. The statistical significance between the means for the different groups was calculated by the non-parametric KruskalWallis test, as the number of cases was too small to use parametric tests. Statistical significance between frequencies was calculated by the χ2-test with Yate’s correction or, if the expected value was   75 25–75

Percentile

RRTI (n = 20)

  75 25–75

Control (n =20)

  75 25–75

p-Value