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Helicobacter pylori infection and serum IgG avidity
ELSEVIER
Clinica Chimica Acta 245 (1996) 129-132
Letter to the editor
Helicobacter pylori infection and serum IgG avidity D. Basso a, L. Brigato a, F. Di Mario b, Mario Plebani *a aDepartment of Laboratory Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy bDepartment of Gastroenterology, University of Padova, Via Giustiniani 2, 35128 Padova, Italy Received 4 May 1995; revision received 11 September 1995; accepted 15 September 1995
Keyword~: Helicobacter pylori; Serum anti-Hp IgG; PBS; Urea
Dear Editor, It is known that Helicobacter pylori (Hp) infection can cause chronic gastritis and duodenal ulcer, and recently it was suggested that it also causes gastric adenocarcinoma and MALToma [I-4]. Pathologic gastric alterations may be attributable to various microbial-associated pathogenetic factors, including the ability of Hp to produce a vacuolating cytotoxin [5] and chemotactic agents for neutrophils and monocytes [6,7]. Once diagnosed, it is recommended that Hp infection should be treated. Antimicrobial therapy, however, is sometimes ineffective. This may be because Hp are becoming resistant to antibiotics, but it may depend on other pathophysiological mechanisms, in particular the induction by Hp of an altered host immune response. Although Hp can stimulate the immune system to produce antibodies, it seems to have a suppressive effect on the cellular immune response [8]. The serum measurement of anti-Hp antibodies can be of help in diagnosing and monitoring infection [9]. However, as Hp may interfere with the cellular immune response, it cannot be ruled out that it also modifies properties of anti-Hp antibodies, such as their avidity for Hp antigen. The aims of * Corresponding author, Tel.: 49 8212792; Fax: 49 663240. 0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0009-8981 (95)06169-E
130
D. Basso et al. / Clinica Chimica Acta 245 (1996) 129-132
our study were to ascertain (1) whether the avidity of serum anti-Hp IgG for Hp antigen varies in infected patients, and (2) whether unsuccessful treatment is associated with variations in the avidity of antibodies for Hp. Anti-Hp IgG titres were assayed by means of an ELISA procedure (Menarini Diagnostics, Firenze, Italy). To assess anti-Hp IgG avidity we used an indirect solid-phase enzyme immunoassay with alkaline phosphatase as the marker enzyme. Each serum sample was run in parallel (series 1 and 2): it was submitted to four serial dilutions and each diluted sample was incubated with Hp antigen coated onto the polystyrene surface of microstrip wells. The wells were then washed with PBS (series 1) or urea solution 6 mold (series 2), a denaturating agent for the antigen-antibody complex. Both series were then incubated with the second antibody anti-human IgG conjugated with alkaline phosphatase. After appropriate incubation periods and washing procedures, a colorless substrate, p-nitrophenylphosphate (pNPP) was added and the colored end-product, p-nitrophenol, was measured at 405 nm. Two absorbance curves were obtained, one from series 1 and another
62 rnm : 11.7%avidit
1.5.
I
E_ 1.0. in 0
\
I,i,I
(,,1 z,<
:\
;:o,-
m 0.5. n, o (z)
< 0.2 4'0
12.5
8~3
50 SAMPLE
1t0
200
1~i0
800
260 m m
3200
DILUTIONS
Fig. 1. Anti-Helicobacter pylori IgG avidity. The figure shows the two dilution curves obtained from a serum sample after washing the antigen-antibody complex with PBS or with urea 6 mol/1.
D. Basso et al./ Clinica Chimica Acta 245 (1996) 129-132
131
from series 2. After washing with urea the antigen-antibody complexes dissociate faster as the avidity is lower, giving lower absorbances in comparison with those obtained after PBS washing. On this basis, avidity results were obtained as a ratio of IgG titres from the two dilution curves (Fig. 1), as already suggested for diagnosis of hantavirus disease [10]. In particular, the ratio between the serum dilutions for PBS and urea corresponding to a 405 nm absorbance cut-off of 0.2 was calculated and expressed as a percentage. Intra-assay coefficients of variation were 8.8%, 5.7% and 9.2% for low, medium and high avidity values (4.3%, 11.1% and 17.1%, respectively). Twenty-six Hp positive patients were studied. All had histologically confirmed Hp infection (Giemsa and/or Whartin-Starry). A serum sample was obtained from each patient immediately before and 2 months after eradication treatment. In 15 patients treatment was effective (group 1) while in the remaining I 1, Hp infection persisted (group 2). In each serum sample, total anti-Hp IgG and their avidity for Hp antigen were measured as described above. No variations were found in IgG titres before and after therapy, either in group 1 (Mann-Whitney U-test = 131.0, P = NS) or group 2 ( U = 62.5, P = NS). This finding confirms data reported elsewhere on the slow decrement of IgG anti-Hp [9]. Basal IgG avidity for Hp antigen, which ranged from a minimum of 5.1% to a maximum of 28.7%, was correlated neither with the anti-Hp IgG titre (Spearman's r = -0.079, P = NS), nor with the histologically assessed bacterial load (Spearman's r =-0.082, P = NS). In group 1, after therapy, anti-Hp IgG avidity increased in 10/15 patients, it decreased in 4/15 and did not change in 1 case, while in group 2 it increased in 2/11, decreased in 7/11 and did not change in 2/11 cases (x 2 = 6.01, P < 0.05). The differences of anti-Hp IgG avidities before and after therapy significantly differed between groups 1 and 2 (U = 123.5, P < 0.05). These results suggest that the Hp infection triggers the production of antibodies with a different avidity towards the Hp antigen itself. Some Hp strains might interfere with the host immune response, lowering anti-Hp IgG avidity and thus contributing to unsuccessful therapy. Hp could interfere with the host immune response inducing specific cytokines production, as demonstrated for IL-8 [11]. However, the exact mechanism by which Hp alters anti-Hp IgG avidity has yet to be clarified. References [1] Biasco G, Paganelli GM, Santucci R, Brandi G, De Santis F, Barbara L. The relationship between type A and B gastritis and Helicobacter pylori. Eur J Gastroenterol Hepatol 1993;5:$67-S69. [2] Saita H, Murakami M, Yoo JK, et al. Link between Helicobacter pylori-associated gastritis and duodenal ulcer. Dig Dis Sci 1993;38:117-122.
132
D. Basso et al. /Clinica Chimica Acta 245 (1996) 129-132
[3] Hansson L-E, Engstrand L, Nyren O, et al. Helicobacter pylori infection: independent risk indicator of gastric adenocarcinoma. Gastroenterology 1993;105:1098-1103. [4] Calvert R, Randerson J, Evans P, et al. Genetic abnormalities during transition from Helicobacter-pylori-associated gastritis to low-grade MALToma. Lancet 1995;345:26-27. [5] Cover TL, Reddy LY, Blaser MJ. Effects of ATPase inhibitors on the response of HeLa cells to Helicobacter pyiori vacuolating toxin. Infect Immun 1993;61:1427-1431. [6] Yoshida N, Granger DN, Evans DJ, et al. Mechanisms involved in Helicobacter pyloriinduced inflammation. Gastroenterology 1993;105:1431-1440. [7] Craig PM, Territo MC, Kames WE, Walsh JH. Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut 1992;33:1020-1023. [8] Knipp U, Birkholz S, Kaup W, Opferkuch W. Immunosuppressive effect of Helicobacter pylori on human peripheral blood mononuclear cells. Med Microbiol Immunol 1993;182:63-76. [9] Kosunen TU, Seppala K, Sama S, Sipponen P. Diagnostic value of decreasing IgG, IgA and IgM antibody titres after eradication of Helicobacter pylori. Lancet 1992;339:893-895. [lO] Hedman K, Vaheri A, Brummer-Korvenkontio M. Rapid diagnosis of hantavirus disease with an IgG-avidity assay. Lancet 1991;338:1353-1356. [ll] Crabtree JE, Farmery SM, Lindley IJD, Fignra N, Peichl P, Tompkins DS. CagA/cytotoxic strains of Hehcobacter pylori and interleukin-8 in gastric epithelial cell lines. J Clin Pathol 1994;47:945-950.
Clinica Chimica Acta 245 (1996) 129-132
Letter to the editor
Helicobacter pylori infection and serum IgG avidity D. Basso a, L. Brigato a, F. Di Mario b, Mario Plebani *a aDepartment of Laboratory Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy bDepartment of Gastroenterology, University of Padova, Via Giustiniani 2, 35128 Padova, Italy Received 4 May 1995; revision received 11 September 1995; accepted 15 September 1995
Keyword~: Helicobacter pylori; Serum anti-Hp IgG; PBS; Urea
Dear Editor, It is known that Helicobacter pylori (Hp) infection can cause chronic gastritis and duodenal ulcer, and recently it was suggested that it also causes gastric adenocarcinoma and MALToma [I-4]. Pathologic gastric alterations may be attributable to various microbial-associated pathogenetic factors, including the ability of Hp to produce a vacuolating cytotoxin [5] and chemotactic agents for neutrophils and monocytes [6,7]. Once diagnosed, it is recommended that Hp infection should be treated. Antimicrobial therapy, however, is sometimes ineffective. This may be because Hp are becoming resistant to antibiotics, but it may depend on other pathophysiological mechanisms, in particular the induction by Hp of an altered host immune response. Although Hp can stimulate the immune system to produce antibodies, it seems to have a suppressive effect on the cellular immune response [8]. The serum measurement of anti-Hp antibodies can be of help in diagnosing and monitoring infection [9]. However, as Hp may interfere with the cellular immune response, it cannot be ruled out that it also modifies properties of anti-Hp antibodies, such as their avidity for Hp antigen. The aims of * Corresponding author, Tel.: 49 8212792; Fax: 49 663240. 0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0009-8981 (95)06169-E
130
D. Basso et al. / Clinica Chimica Acta 245 (1996) 129-132
our study were to ascertain (1) whether the avidity of serum anti-Hp IgG for Hp antigen varies in infected patients, and (2) whether unsuccessful treatment is associated with variations in the avidity of antibodies for Hp. Anti-Hp IgG titres were assayed by means of an ELISA procedure (Menarini Diagnostics, Firenze, Italy). To assess anti-Hp IgG avidity we used an indirect solid-phase enzyme immunoassay with alkaline phosphatase as the marker enzyme. Each serum sample was run in parallel (series 1 and 2): it was submitted to four serial dilutions and each diluted sample was incubated with Hp antigen coated onto the polystyrene surface of microstrip wells. The wells were then washed with PBS (series 1) or urea solution 6 mold (series 2), a denaturating agent for the antigen-antibody complex. Both series were then incubated with the second antibody anti-human IgG conjugated with alkaline phosphatase. After appropriate incubation periods and washing procedures, a colorless substrate, p-nitrophenylphosphate (pNPP) was added and the colored end-product, p-nitrophenol, was measured at 405 nm. Two absorbance curves were obtained, one from series 1 and another
62 rnm : 11.7%avidit
1.5.
I
E_ 1.0. in 0
\
I,i,I
(,,1 z,<
:\
;:o,-
m 0.5. n, o (z)
< 0.2 4'0
12.5
8~3
50 SAMPLE
1t0
200
1~i0
800
260 m m
3200
DILUTIONS
Fig. 1. Anti-Helicobacter pylori IgG avidity. The figure shows the two dilution curves obtained from a serum sample after washing the antigen-antibody complex with PBS or with urea 6 mol/1.
D. Basso et al./ Clinica Chimica Acta 245 (1996) 129-132
131
from series 2. After washing with urea the antigen-antibody complexes dissociate faster as the avidity is lower, giving lower absorbances in comparison with those obtained after PBS washing. On this basis, avidity results were obtained as a ratio of IgG titres from the two dilution curves (Fig. 1), as already suggested for diagnosis of hantavirus disease [10]. In particular, the ratio between the serum dilutions for PBS and urea corresponding to a 405 nm absorbance cut-off of 0.2 was calculated and expressed as a percentage. Intra-assay coefficients of variation were 8.8%, 5.7% and 9.2% for low, medium and high avidity values (4.3%, 11.1% and 17.1%, respectively). Twenty-six Hp positive patients were studied. All had histologically confirmed Hp infection (Giemsa and/or Whartin-Starry). A serum sample was obtained from each patient immediately before and 2 months after eradication treatment. In 15 patients treatment was effective (group 1) while in the remaining I 1, Hp infection persisted (group 2). In each serum sample, total anti-Hp IgG and their avidity for Hp antigen were measured as described above. No variations were found in IgG titres before and after therapy, either in group 1 (Mann-Whitney U-test = 131.0, P = NS) or group 2 ( U = 62.5, P = NS). This finding confirms data reported elsewhere on the slow decrement of IgG anti-Hp [9]. Basal IgG avidity for Hp antigen, which ranged from a minimum of 5.1% to a maximum of 28.7%, was correlated neither with the anti-Hp IgG titre (Spearman's r = -0.079, P = NS), nor with the histologically assessed bacterial load (Spearman's r =-0.082, P = NS). In group 1, after therapy, anti-Hp IgG avidity increased in 10/15 patients, it decreased in 4/15 and did not change in 1 case, while in group 2 it increased in 2/11, decreased in 7/11 and did not change in 2/11 cases (x 2 = 6.01, P < 0.05). The differences of anti-Hp IgG avidities before and after therapy significantly differed between groups 1 and 2 (U = 123.5, P < 0.05). These results suggest that the Hp infection triggers the production of antibodies with a different avidity towards the Hp antigen itself. Some Hp strains might interfere with the host immune response, lowering anti-Hp IgG avidity and thus contributing to unsuccessful therapy. Hp could interfere with the host immune response inducing specific cytokines production, as demonstrated for IL-8 [11]. However, the exact mechanism by which Hp alters anti-Hp IgG avidity has yet to be clarified. References [1] Biasco G, Paganelli GM, Santucci R, Brandi G, De Santis F, Barbara L. The relationship between type A and B gastritis and Helicobacter pylori. Eur J Gastroenterol Hepatol 1993;5:$67-S69. [2] Saita H, Murakami M, Yoo JK, et al. Link between Helicobacter pylori-associated gastritis and duodenal ulcer. Dig Dis Sci 1993;38:117-122.
132
D. Basso et al. /Clinica Chimica Acta 245 (1996) 129-132
[3] Hansson L-E, Engstrand L, Nyren O, et al. Helicobacter pylori infection: independent risk indicator of gastric adenocarcinoma. Gastroenterology 1993;105:1098-1103. [4] Calvert R, Randerson J, Evans P, et al. Genetic abnormalities during transition from Helicobacter-pylori-associated gastritis to low-grade MALToma. Lancet 1995;345:26-27. [5] Cover TL, Reddy LY, Blaser MJ. Effects of ATPase inhibitors on the response of HeLa cells to Helicobacter pyiori vacuolating toxin. Infect Immun 1993;61:1427-1431. [6] Yoshida N, Granger DN, Evans DJ, et al. Mechanisms involved in Helicobacter pyloriinduced inflammation. Gastroenterology 1993;105:1431-1440. [7] Craig PM, Territo MC, Kames WE, Walsh JH. Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut 1992;33:1020-1023. [8] Knipp U, Birkholz S, Kaup W, Opferkuch W. Immunosuppressive effect of Helicobacter pylori on human peripheral blood mononuclear cells. Med Microbiol Immunol 1993;182:63-76. [9] Kosunen TU, Seppala K, Sama S, Sipponen P. Diagnostic value of decreasing IgG, IgA and IgM antibody titres after eradication of Helicobacter pylori. Lancet 1992;339:893-895. [lO] Hedman K, Vaheri A, Brummer-Korvenkontio M. Rapid diagnosis of hantavirus disease with an IgG-avidity assay. Lancet 1991;338:1353-1356. [ll] Crabtree JE, Farmery SM, Lindley IJD, Fignra N, Peichl P, Tompkins DS. CagA/cytotoxic strains of Hehcobacter pylori and interleukin-8 in gastric epithelial cell lines. J Clin Pathol 1994;47:945-950.