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Pernicious Anemia; an Examination of Gastrointestinal Tissues Using Immunehistochemisty

To determine whether samples of patient sera contain antibodies to the gastric proton pump by immunehistochemistry

Introduction

Pernicious anemia is a type of megaloblastic anemia caused by the failure of gastric parietal cells responsible for sections of proteins essential to absorption of Vitamin B12 in the gastric walls1. This leads to insufficiency of absorption of Vitamin B12 which is dependent on the intrinsic factor for absorption. The specific deficiency of Vitamin B12 as a result of lack of sufficient parietal cells to influence availability of intrinsic factor is what is called pernicious anemia and not any other cause of Vitamin B12 deficiency2.

This report gives details on the symptoms showed by gastric tissues of suspected pernicious anemia patients. It will detail the method used in preparation of samples for view, the observations made on the tissues and the relevance of the findings to the conclusion that the patient suffers PA. The tissues will be subjected to different sera examinations to determine if they would react with the antibodies of gastric proton pump. This would be indicative of possible pernicious anemia.

The method used involved the separation of protein extracts of gastric tissues by using SDS polyacrylamide gel electrophoresis. The proteins were incubated in a nitrocellulose membrane to determine the immunological reaction. A stain mouse gastrointestinal tissues was done by using heamatoxylin and eosin was also done as control.

The method used in this research is the immunohistochemistry. Alternative methods are such as ELISA which uses the color change of antibodies to identify substances. The application simply applies antigens to a surface and antibodies linked to enzymes are also attached to the surface so they can bind. The reaction produces a color change to signal the presence of specific chemicals in the substrate.

In the research I used sheep anti human lg HRP conjugate which is specific to the human lg classes’ lgA, lgD, lgG, lgM3. The antibody is also specific to light chain. It however has no cross reactivity with sera of rabbits, horse and calf, while it shows weak reactivity with sera of mouse54. The reagent is a human anti body obtained from sheep through pappaine cleavage and peroxidase. The antibody was conjugate with the enzyme peroxidase.

Electrophoresis of gastrointestinal tissues

200µL of protein was placed in a microfuge tube and 50µL 5*SDS sample buffer was added and allowed to sit at room temperature. The microfuge tubes containing the protein MW markers were then subjected to spinning for few seconds before being used to ensure the liquid was at the botton of the tube. The MW markers and protein samples were loaded into the gel using special gel loading tips. The gel was then run at 200v until the dye reached the bottom of the gel.

  • Transfer of the gel; the apparatus was powered off and the position of the protein marker on the gel was noted. The glass plates were then separated and the corner of the bottom gel cut off and labeled. Glass plates containing the gel were put in a tray with enough buffers to remove the gel. The container was gently rocked to weaken the gel after which the glass plate was set aside from the gel.

  • Transfer the proteins to a nitrocellulose membrane; 2 pieces of filter paper, the nitrocellulose membrane, gel and another 2 pieces of filter paper were all soaked in the transfer buffer and arranged in the apparatus in the order in which they appear.

Immunehistochemistry of mouse gastro intestinal wall

The sample was incubated in xylene for 2 minutes then incubated in ethanol for another 2 minutes. The samples were rinsed in tap water for 30 seconds. After this the slides were incubated in 0.3% hydrogen peroxide in PBS for 30 minutes to remove endogenous activity. The slides were then washed in PBS for 2 minutes and incubated in 1% BSA in PBS for 30 minutes to block non specific binding of proteins. The block solution was removed and 100µL of the patients’ serum was added and incubated for 16 minutes. The slides were washed in PBS. 50 µL of the sheep anti-human Lg HRPO conjugate was added and incubated for 20 minutes. The slides were then washed in PBS and briefly rinsed in water. 100µL of DAB substrate was added to the section and incubated for 4 minutes after which the slides were rinsed in PBS and dipped in heamatoxylin for 3 seconds. The slides were then washed in water for 30 seconds and incubated in ethanol for 2 minutes. Finally, a drop of DPX mounting medium was put on the section and the slides examined under the microscope.

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Fig 1: stomach tissue sample (high power)

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Fig 2: stomach tissue sample (low power)

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Fig 3: stomach tissue with antibodies

Our western blot results indicated the presence of a mix of protein present in the preparation at concentrations of 0.02g/l. The proteins showed visible bands with heamotoxylin staining. Of the proteins were two pernicious anemia specific antigens which were detected in immune-blots of the PA sera compared to the mouse sera. The first antigen detected is a protein with molecular weight of 65-70 kD that was present in the patient sera. However, none of the control sera reacted with this antigen; it was only antigenic in the sulphydryl non reduced state in which it manifested as a blurred band on the blot. The richest preparation of this protein was from the bench 4 results in which the microsomal fraction was from >90% of mouse parietal cells.

The second protein was of molecular weight of 85-90 kD. The reactivity of this antigen correlates with the parietal cell microsomal auto antibody. It is only present in the bench 3 results of the AM examinations. The antigen is a co-purifier with the microsomal fraction and is active in heamotoxylin reduced form. It is manifested as a sharp discrete band on the immunological blots of bench 3 results.

Discussion

The aims of the practical were met as the immunological reactions to different sera indicated presence of antibodies to pernicious anemia. From the results above it is proven that tissues with pernicious anemia produce immunoflourescent when reacted with mouse gastric mucosa. This corresponds with findings of D’elios et al6. The presence of the protein with molecular weight of 65-70 kD in the gastric mucosal microsomes and their correlation with the reactivity of PCMA suggest that the 65-70 kD protein is the parietal cell microsoma autoantigen.

The blurring of the bands is not a result of the non reducing conditions because the other bands that were stained with heamotoxylin were sharp. The other possible explanation as to the blurring of the band is the insufficiency of the protein antigen; the available quantities may have not been enough to cause a sharper band.

The reactivity of the second protein with molecular weight of 85-90 KD of bench 4 has significant correlation with the microsomal autoantibody in PA tissues. This is visible as a band in heamotoxylin stained microsomes prepared from the mouse mucosa. Their absence in the other tissues and in the microsomes suggests that the protein may be present in the microsomal fraction of non parietal cells of the other mucosa. The protein may also be less antigenic compared to the 65-70KD of bench 3 which seems to be present in only small quantities.

The results obtained may have been compromised by the denaturing of the gastric antigens by the use of SDS in the immunoblotting tests. This may have caused one or more of the antigens not to react on the blot; the fluorescence of the parietal cells may have therefore resulted from only the 65-70KD antigen. The multiple bands observed in the bench 3 results may be indicative of secondary autoimmunization of the autoantibodies to the proteins.

These results had problems such as possible inverted membrane during observation, the marker also failed to fit and the poor methods of transfer of proteins which may have reduced the credibility of the results. An intense approach at protein transfer should be adopted to ensure accuracy of the results.

1
Davidson RJ, Atrah HI, Sewell HF. “Longitudinal study of circulating gastric antibodies in pernicious anaemia.” J Clin Pathol 1989; 42: 1096

2
Carmel R. “Reassessment of the relative prevalences of antibodies to gastric parietal cell and to intrinsic factor in patients with pernicious anaemia: influence of patient age and race.” Clin Exp Immunol 1992; 89: 74-77

3
Shoenfeld Y, Gershwin ME, Meroni PL,editors. “Autoantibodies.” 2nd ed. Amsterdam: Elsevier, 2007:479-486

4
Davidson RJ, Atrah HI, Sewell HF. “Longitudinal study of circulating gastric antibodies in pernicious anaemia.” J Clin Pathol 1989; 42: 1092-1095

5
Field J, Biondo MA, Murphy K, Alderuccio F, Toh BH. “Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease.” Int Rev Immunol 2005; 24: 93-110

6
D’Elios MM, Appelmelk BJ, Amedei A, Bergman MP, Del Prete G. “Gastric autoimmunity: the role of Helicobacter pylori and molecular mimicry.”Trends Mol Med 2004; 10: 316-323