Relationship between Helicobacter pylori virulence factors and regulatory cytokines as predictors of clinical outcome

Abstract

Helicobacter pylori infection is highly prevalent in Chile (73%). Usually a minority of infected patients develops complications such as ulcers and gastric cancer that have been associated with the presence of virulence factors (cagA, vacA) and host T helper response (Th1/Th2). Our aim was to evaluate the relationship between strain virulence and host immune response, using a multiple regression approach for the development of a model based on data collected from H. pylori infected patients in Chile. We analyzed levels of selected cytokines determined by ELISA (interleukin (IL)-12, IL-10, interferon (IFN)-gamma and IL-4) and the presence of cagA and vacA alleles polymorphisms determined by PCR in antral biopsies of 41 patients referred to endoscopy. By multiple regression analysis we established a correlation between bacterial and host factors using clinical outcome (gastritis and duodenal ulcer) as dependent variables. The selected model was described by: clinical outcome=0.867491 (cagA)+0.0131847 (IL-12/IL-10)+0.0103503 (IFN-gamma/IL-4) and it was able to explain over 90% of clinical outcomes observations (R(2)=96.4). This model considers that clinical outcomes are better explained by the interaction of host immune factors and strain virulence as a complex and interdependent mechanism.

Figure 1

cagA detection. A. Scheme showing the amplified region of cagA gene. The primers were designed according to the 26695 strain as described in the Materials and Methods section. B. Amplification using A17BN1-Cag7 primers. Lane 1: 1kb DNA ladder Gibco. Lane 2: strain cagA -. Lanes 3–6 positive samples for this reaction. Lane 7: negative control with template DNA isolated from Campylobacter jejuni. Lane 8: positive control with chromosomal DNA isolated from strain 26695. C. Amplification using A17BN1-Cag6 primers. Lane 1: 1kb DNA ladder Gibco. Lanes 2–7: positive samples for this reaction. Lane 8: positive control with chromosomal DNA isolated from strain 26695. D. Amplification using A17BN1 and A17BH2 primers. Lane 1: 1kb DNA ladder Gibco. Lanes 2–7: positive strains for this reaction. Lane 8 positive control with chromosomal DNA isolated from strain 26695. Arrows indicate the expected size for each fragment and the size of the nearest band in DNA ladder.

Figure 2

vacA amplification. A. PCR amplification of m1 allele. Lane 1: 100 bp DNA ladder standard, Lanes 2 through 13 samples obtained from patients living in IQ. Lane 14: negative control with template DNA isolated from Campylobacter jejuni. Lanes 15: positive control with chromosomal DNA isolated from strain J99. The arrow indicates the 290 bp expected PCR product. All PCR assays were repeated at least 3 times. B. PCR amplifications for m2 VacA allele. PCR fragments were separated in a 3% agarose gel. Lane 1: 100 bp ladder DNA standard, Lane 2: positive sample from a patient living in SA. Lane 3: positive control with chromosomal DNA isolated from strain 8822. Lanes 4 and 5: negative samples. Lane 6: strain 26695 as a negative control. The arrow indicates electrophoretic migration in a 3% agarose gel of the 352 bp expected fragment. C. Size differences in s1 and s2 PCR fragments derived from vacA gene from Chilean H. pylori clinical isolates. Fragments were separated in a 3% agarose gel. Lane 1: 100 bp ladder DNA standard. Lanes 2 and 3: s2 positive strains isolated from SA. Lane 4: a s1 positive strain from TE. Lane 5: a S1 positive control corresponding to H. pylori strain 26695. Arrows indicate the expected size for each fragment.

Figure 3

A. IFN-γ/IL-4 ratio showed no statistical difference among patients with different clinical outcomes. B. IL-12/IL-10 ratio was higher in patients with duodenal ulcer compared to non-infected patients. Data are presented as individual points plus median. p<0.05, Kruscall-Wallis, Dunn post test.

Figure 4

Observed and predicted values of clinical outcomes explained by the equation Clinical outcome: 0.867491 (cagA) + 0.0131847 (IL-12/IL-10) + 0.0103503 (IFN-γ/IL-4) that relates virulence factors such as CagA and host immune response defined as Th1/Th2 cytokine ratios. Titers of equation predicted values, obtained by fitting to the equation measured parameters, increase with severity of endoscopy observed outcome. Data are presented as individual points plus median. p<0.05, Mann-Whitney test.