1. Immunological characteristics of patients infected with common intestinal helminths : result of a study based on reverse-transcriptase PCR S.Lertanekawattana,T.Wichatrong,K.Chaisari, R.Uchikawa and N.Arizono Nongkhai Hospital and Department of Medical Zoology, KyotoPrefectural University of Medicine

2. Source:Nongkhai Provincial office Data of intestinal helminths in Nongkhai yearPositive of common parasite by stool exam. All ageSchool age 200022.0%19.3% 200233.3%28.2% Taenia,Hook worm,Opisthochis,Ascaris,Strongyloid

3. Long-term infection intestinal parasites : -Malnutrition -Retardation of the intelectual -Impair physical growth of children Oberhelman et al.,1998:

4. Helminth infection : -Impair immune response to virus,bacteria protozoa and even oral vaccine ( Cooper et al, 2001)

5. Ascaris and Hook worm induce expansion of the Th2 lymphocyte subset and regulate IgE antibody production, eosinophilia and mastocytosis (Pritchard et al,1995)

6. Th2 cytokines have antagonistic effects on the expansion of the Th1 lymphocyte subset (Cooper et al,2001)

10. Anisakiasis Clinical : Urticaria,Angioedema, Anaphylaxis Immunological response : IgE, mast cell, basophil Th 2 interleukin 4,predominant

11. Nippostrongylus brasiliensis Villus pathology induced by the rodent nematode Nippostrongylus brasiliensis

12. E-cadherin expression decreases at villus tips after infection days after infection 28 14107 0 1 2 3 4 Day 0Day 10 E-cadherin immunohistochemistry E-cadherin score

13. Pathological changes develop in the intestinal mucosa,including partial villus atrophy and crypt hyperplasia,decreasing of sucrase and alkaline phosphatase activity,deterioration of epithelial permeability and barrier function (Hyoh et al,2002 ; Sekikawa et al;2003)

14. * * glucose Na K SGLT1 (Na-driven glucose cotransporter) Na-K ATPase Lumen Lamina propria Absorptive cell Glucose transport in the small intestine

15. Infection with intestinal nematodes in rodents causes induction Th2 cytokines such as IL-4.IL-5and IL-13 and induce IgE antibody responses, eosinophilia and intestinal mastocytosis (Matsuda et al, 1995 ; Garside et al,2000)

16. Aim of study : To determine whether common helminth infections could modify the intestinal immunopathological status of the host,by the expression in the human duodenal mucosa of cytokines,eosinophil,mast cell specific molecule,monosaccharide transporters of the glucose- transporter (GLUT)

17. Subjects : 31 patients with gastrointestinal problems and indicated for gastroduodenal fiberscopy as diagnotic measurement and did not have dysenteric disease and gave their written,informed consent for the study

18. Materials: Duodenal biopsy specimens

19. Methods : Semi-quantitative RT-PCR for IL-5,IL-13, IFN- , GLUT-1 and GLUT-5

20. Laboratory study: At central lab. of Kyoto Prefectural University of Medicine

21. Density analysis of PCR products Electrophoresis on agar each band was evaluated using NIH Image software, the band densities were normalized relative to those of ß-actin

22. Table1 The primer pairs used for the PCR amplificationsSize of expected product(bp) IFN-  5'-GGGTTCTCTTGGCTGTTACTG-3 ‘ 5'-GACAGTTCAGCCATCACTTGGA-3' 384 IL-55'-GAAATTCCCACAAGTGCATTGG-3' 5'-CTTTCTATTATCCACTCGGTGTTC-3' 335 IL-135'-AGGAGCTGGTCAACATCACC-3' 5'-GTTGAACCGTCCCTCGCGAA-3' 296 FceRI 1 5'- TCAGTGACTGGCTGCTCCTT -3' 5'- GGGGTTTGGCTTAGGATGTG -3' 437 McTr 2 5'- TCAGCAGGATCATCGTGCAC -3' 5'- TGGGGACATAGTGGTGGATC -3' 507 CLC 3 5'- TACCCGTGCCATACACAGAG -3' 5'- CTCTCCACACTTGCACCATC -3' 375 GLUT-1 5'-ATCGTCAACACGGCCTTCAC-3' 5'-AAGCCGGAAGCGATCTCATC-3' 458 GLUT-55'-GGTACAACGTGGCTGCTGTC-3' 5'-CATGGGGACCACGTTGGAAG-3' 347  -Actin 5'-TCAGAAGGATTCCTATGTGGGC-3' 5'-CCATCACGATGCCAGTGGTA-3' 317

23. Table 2 The baseline demographic and laboratory data for the 31 subjects Total N=31 Helminth infected 9 Uninfected 22 Ulcer 6 Non-ulcer 25 Male/female22/99/013/95/117/8 Age(years) 45.8 ( 13.5)(31) 43.3 (10.8)(9)46.8(14.5)(22 ) 47.8(8.9)(6)45.3 (14.6)(25) Albumin(g/dl)3.9(0.7)(23)4.1(0.6)(8)3.8(0.7)(15)3.5(1.0)(5)4.0(0.6)(18) Haemoglobin (g/dl) 13.3(2.8)(29)13.5(2.2)(8)13.0(3.0)(21)12.3(4.1)(6)13.4(2.4)(23) Eosinophils(% 0f leucocyte) 4.8(4.6)(31)5.3(4.6)(9)4.6(4.7)(22)3.1(3.2)(6)5.2(4.9)(25)

25. Figure 1 Some of PCR results,showing the expression of IL-13,IL- 5,IFN- ,andfor reference,  -actin in the duodenal mucosa of the 31 patients

26. Figure 2 (a) The frequencies of expression of IL-5( )and IL-13 ( ) In the duodenal mucosa of the 10 patients with eosinophilia and the 21 without

27. Figure 3 (b) In the 9 patients with helminth infection(on presentation or in the previous 3 months ) and the 22 without P=0.041

28. Table 3 Expression levels of IL-5, IFN- , CLC, Fc  RI, McTr, GLUT1 and GLUT5 in the duodenal mucosa. Non-eosinophilicsEosinophilics Total (N=21) Uninfected (N=16) Infected (N=5) Total (N=10) Uninfected (N=6) Infected (N=4) IL-50.21(0.09)0.14(0.08)0.45(0.33)0.36(0.10)0.33(0.11)0.39(0.24) IFN-  0.65(0.11)0.66(0.14)0.62(0.17)1.20(0.22)*1.32(0.28)*1.02(0.42) IL-5/ IFN-  0.27(0.09)0.15(0.06)0.65(0.29) † 0.41(0.17)0.39(0.24)0.45(0.25) CLC0.69(0.10)0.66(0.11)0.78(0.31)0.74(0.19)0.92(0.27)0.48(0.21) Fc  RI 0.52(0.11)0.42(0.09)0.82(0.32)0.58(0.09)0.56(0.12)0.60(0.14) McTr2.16(0.19)1.98(0.18)2.76(0.52)2.79(0.44)3.41(0.57)1.87(0.13) Fc  RI/McTr 0.21(0.04)0.20(0.04)0.26(0.06)0.23(0.05)0.16(0.03)0.33(0.09) ‡ GLUT11.17(0.15)1.13(0.20)1.23(0.17)0.90(0.23)1.01(0.32)0.74(0.32) GLUT51.17(0.09)1.12(0.10)1.32(0.25)1.25(0.13)1.41(0.17)1.00(0.12)

29. Table 4 The results of the principal-components analysis ParameterValue Eigenvalue(and percentage of variation explained)2.13966(42.8) Unrotated facter loadings Eosinophils Interleukin-5 Interferon-  IgE receptor type ( Fc  RI ) Mast cell tryptase ( McTr ) -0.55202 -0.43733 -0.72827 -0.61319 -0.85866 Mean (S.E.) component scores Others (uninfected patients without eosinophilia;N=16) Helminth-infected (N=9) Eosinophilia (uninfected patients with eosinophilia; N=6) 0.761(0.283) -0.327(0.375) -1.538(0.733) With intestinal helminth infection on presentation or within previous 3 months. Significantly different to other component scores (p<0.05) Significantly different to other component scores (p<0.01)

30. Summary: The present findings indicate that infections with common intestinal helminths, including Taeniasis are likely to modify mucosal immunology so that it shifts to a Th2-predominant status. SGUT-1 and GLUT-5 no significant decrease in patients with helminth infection

31. Acknowledgment: This work was supported by Grants-in-Aid for Research from the Japan Society for the Promotion of Science and the Japanese Ministry of Health,Labour and Welfare