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

Source:Nongkhai Provincial office

Data of intestinal helminths in Nongkhai

year Positive of common parasite

by stool exam.

All age School age

2000 22.0% 19.3%

2002 33.3% 28.2%

Taenia,Hook worm,Opisthochis,Ascaris,Strongyloid

Long-term infection intestinal parasites :

-Malnutrition

-Retardation of the intelectual

-Impair physical growth of children

Oberhelman et al.,1998:

Helminth infection :

-Impair immune response to virus,bacteria protozoa and even oral vaccine

( Cooper et al, 2001)

Ascaris and Hook worm induce expansion of the Th2 lymphocyte subset and regulate IgE antibody production, eosinophilia and mastocytosis

(Pritchard et al,1995)

Th2 cytokines have antagonistic effects on the expansion of the Th1 lymphocyte subset

(Cooper et al,2001)

Anisakiasis

Clinical : Urticaria,Angioedema,

Anaphylaxis

Immunological response :

IgE , mast cell, basophil

Th 2 interleukin 4,predominant

• Nippostrongylus brasiliensis

Villus pathology induced by the rodent

nematode Nippostrongylus brasiliensis

E-cadherin expression decreases at villus tips after infection

days after infection

281410701

2

3

4

Day 0 Day 10

E-cadherin immunohistochemistry

E-cadherin score

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)

**

glucose

glucose

glucose

Na

Na

Na

K

SGLT1 (Na-driven glucose cotransporter)

Na-K ATPase

Lumen

Lamina propria

Absorptivecell

Glucose transport in the small intestine

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)

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)

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

Materials:

Duodenal biopsy specimens

Methods :

Semi-quantitative RT-PCR for IL-5 ,IL-13, IFN- , GLUT-1 and GLUT-5

Laboratory study:

At central lab. of Kyoto Prefectural University of Medicine

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

Table1 The primer pairs used for the PCR amplifications Size of expected product(bp)

IFN- 5'-GGGTTCTCTTGGCTGTTACTG-3‘5'-GACAGTTCAGCCATCACTTGGA-3'

 

384

IL-5 5'-GAAATTCCCACAAGTGCATTGG-3'5'-CTTTCTATTATCCACTCGGTGTTC-3'

335

IL-13 5'-AGGAGCTGGTCAACATCACC-3'5'-GTTGAACCGTCCCTCGCGAA-3'

296

FceRI1 5'-TCAGTGACTGGCTGCTCCTT-3'5'-GGGGTTTGGCTTAGGATGTG-3'

437

McTr2 5'-TCAGCAGGATCATCGTGCAC-3'5'-TGGGGACATAGTGGTGGATC-3'

507

CLC3 5'-TACCCGTGCCATACACAGAG-3'5'-CTCTCCACACTTGCACCATC-3'

375

GLUT-1 

5'-ATCGTCAACACGGCCTTCAC-3'5'-AAGCCGGAAGCGATCTCATC-3'

458

GLUT-5 5'-GGTACAACGTGGCTGCTGTC-3'5'-CATGGGGACCACGTTGGAAG-3'

347

-Actin 5'-TCAGAAGGATTCCTATGTGGGC-3'5'-CCATCACGATGCCAGTGGTA-3'

317

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/female 22/9 9/0 13/9 5/1 17/8

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)

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

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

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

Table 3 Expression levels of IL-5, IFN-, CLC, FcRI, McTr, GLUT1 and GLUT5 in the duodenal mucosa.

Non-eosinophilics Eosinophilics

Total

(N=21)

Uninfected

(N=16)

Infected

(N=5)

Total

(N=10)

Uninfected

(N=6)

Infected

(N=4)

IL-5 0.21(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)

CLC 0.69(0.10) 0.66(0.11) 0.78(0.31) 0.74(0.19) 0.92(0.27) 0.48(0.21)

FcRI 0.52(0.11) 0.42(0.09) 0.82(0.32) 0.58(0.09) 0.56(0.12) 0.60(0.14)

McTr 2.16(0.19) 1.98(0.18) 2.76(0.52) 2.79(0.44) 3.41(0.57) 1.87(0.13)

FcRI/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)‡

GLUT1 1.17(0.15) 1.13(0.20) 1.23(0.17) 0.90(0.23) 1.01(0.32) 0.74(0.32)

GLUT5 1.17(0.09) 1.12(0.10) 1.32(0.25) 1.25(0.13) 1.41(0.17) 1.00(0.12)

Table 4 The results of the principal-components analysis

Parameter Value

Eigenvalue(and percentage of variation explained) 2.13966(42.8)

Unrotated facter loadings

Eosinophils

Interleukin-5

Interferon- IgE receptor type (FcRI)

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)

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

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