Genotyping of HLA class I and II allele and some immunological markers in Hydatidosis patients

number: 
3282
إنجليزية
Degree: 
Imprint: 
medicine-Medical Microbiology
Author: 
Samir Sabaa Raheem Al-Jawahiry
Supervisor: 
Dr. Lazim Al-Taie
Dr. Batool Al-Ghurabi
year: 
2013
Abstract:

Echinococcosis or hydatid disease results from infection with the larvae (a developing phase) of the tapeworms Echinococcus granulosus which is found most commonly in dogs that consume the viscera of infected sheep, , Infection with E. granulosus results in the formation of cysts in the liver, lungs, kidney and spleen, and nearly every location . Hydatidosis is a worldwide problem, and Iraq is an endemic region for the disease .The most important determinants of genetic susceptibility to cystic echinococcosis are located on HLA gene area on short arm of chromosome 6 . The host immune status appear to control the susceptibility to or resistance against the parasite it triggers humoral and cellular response characterized by increased serum antibodies and concurrent intervention of Th I and Th2 cytokines (Shcherbakove and Monje 19989 Shcherbakove,1993). Since the HLA-disease association might vary among ethnic groups the main objective of this study was to detect the association of HLA-class class I (A ,B) and Class II (DR and DQ) alleles with Cystic Echinococcosis by using Polymerase Chain Reaction-Sequence Specific Oligonucleutide PCR-SSO in Iraqi patients , as well as to provide information about genotypes that confer susceptibility or resistance to develop the disease. and to investigate the levels of cytokines (TNF-α, IL- 4 and IL-10) ,and the levels of IgE and IgG4 in hydatid patients with different cyst localizations (before and after surgery) , in addition to the correlation between the presence of specific HLA-alleles with antibody titers and the presence of cytokines . Also to assess a possible relationship of serum cytokines with immunoglobulin expression and cyst characteristics (location and number). A total of (33 )patients of both sexes ( 13 males and 20 females) with confirmed cystic echinococcosis (CE), aged from (11–57) years, and 20 healthy control were enrolled , venous blood withdrawn used for detection of serum cytokines and antibodies. the anticoagulated blood were used for HLA typing first by DNA extraction ,then DNA amplification by PCR-SSO (polymerase Chain Reaction –Sequence Specific Oligonucleotide) ,then using the allele - specific probing technique by Automatic Line probe assay (Auto-Lipa) for HLA typing. All the patients’ and control s subjects ’ HLA class I And II alleles typing and allele frequencies were calculated, . The immunological markers(IL-4, IL-10, TNF-a, IgE , IgG4) were estimated for both control and patients group by ELISA test using the separated serum. A follow up study were enrolled for 14 patients before and after surgery to evaluate the differences in the cytokines and immunoglobulin's level and their role in the course of the disease. , There were a significant differences in frequency of HLA-A *0273 0% in Patients group compared to 20% healthy control group , HLA-DR *0403 with 6.67% in patients group compare to 35% in control group ,and HLA-DR *0701 30% in patients compared to 0% in control groups with P value less than < 0.021 , < 0.020, < 0.007 respectively. The majority of infected individuals were females 60.6% in comparison to 39.4% males. The majority of cysts were located in the liver (76%)and the rest (24%) in the lunge. with few number(3 cases )of combined infections(liver +lung).An increase in IL-4 levels in hydatidosis patients. A highly increase in IL-10 levels and P value was <0.05 , with significant decrease after three months from the surgery. Non Significant increase in TNF-a level during infection with non significant decrease after surgery, P value more than 0.05. A significant elevation in serum IgE levels during infection with the disease . A highly significant decrease in IgG4 levels in hydatidosis patients in post-operational samples ,withdrawn after 3 months with non-significant increase in IgG4 levels due to infection . There were no significant association was reported for the HLADR* 0701 allele with the gender type of patients , cyst location or numbers and the family history . A significant association between the occurrence of this allele and levels of IgE formation , IL-4 and IL-10 . Non significant association with levels of IgG4 and TNF-a formation were reported, In conclusion a unique alleles profile HLA-A*0273, HLA-DR*0403 might confer protection against Hydatid Disease ,whereas HLADR* 0701 alelle might confer susceptibility to the infection in Iraqi hydatid patients as compare to other studies in various regions and ethnic groups. A significant correlation between HLA-DR*0701 allele with levels of IgE , IL-4 , and IL-10 .IgE, IL-4, IL-10 could be a valuable diagnostic and confirmative tool for diagnosis of hydatid disease.