Introduction: The immunity against cervical cancers is many unknown. Major histocompatability complex class I 

(MHC-I) with the alpha domain has binding site for Cytotoxic T cells (CD8+) to target cells. The objective of this ex-

periment is to known the expression of CD8+ and MHC-I in cervical cancer with HPV infection. Method: DNA was 

isolated from seventeen sample cervical cancer tissues frozen section. Diagnose related with HPV was made by PCR. 

Paraffin block of the tissues was cut  in thoroughly cleaned cryotome and place in glass plate that covered with 

poly-elysine. The immunohistochemistry is done with monoclonal antibody again MHC-I and CD8+ with TSA-indirect 

method. The power of expression was counted the number of positive cells to express the MHC-I and CD8+ every 100 

cells per one view look. Result: The Result of this experiment show that expression of MHC-I and CD8+ in cervical 

cancer with HPV infection is in mild category (30% - 70%). Conclusion: Immunity against uterine cancer that played 

by MHC-I and CD8+ are in milt category. The experiment that related with uterine cancer immunology is suggested

Asnar,  Elyana

Astirin, Okid Parama

Prayitno, Adi

Putra, Suhartono Taat

Sebelas Maret Institutional Repository

Journal of Cancer Therapy, 2013, 4, 15-18 doi:10.4236/jct.2013.45A003 Published Online May 2013 (http://www.scirp.org/journal/jct) 15The Expression of CD8+ and MHC-I in Cervical Cancer with HPV Infection Adi Prayitno1, Elyana Asnar2, Okid Parama Astirin3, Suhartono Taat Putra2  1Medical School of Sebelas Maret University, Surakarta, Indonesia; 2Medical School of Airlangga University, Surabaya, Indonesia; 3Mathematical and Science Faculty of Sebelas Maret University, Surakarta, Indonesia. Email: drgadiprayitno@yahoo.com  Received March 17th, 2013; revised April 16th, 2013; accepted April 23rd, 2013  Copyright © 2013 Adi Prayitno et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Introduction: The immunity against cervical cancers is many unknown. Major histocompatability complex class I (MHC-I) with the alpha domain has binding site for Cytotoxic T cells (CD8+) to target cells. The objective of this ex-periment is to known the expression of CD8+ and MHC-I in cervical cancer with HPV infection. Method: DNA was isolated from seventeen sample cervical cancer tissues frozen section. Diagnose related with HPV was made by PCR. Paraffin block of the tissues was cut in thoroughly cleaned cryotome and place in glass plate that covered with poly-elysine. The immunohistochemistry is done with monoclonal antibody again MHC-I and CD8+ with TSA-indirect method. The power of expression was counted the number of positive cells to express the MHC-I and CD8+ every 100 cells per one view look. Result: The Result of this experiment show that expression of MHC-I and CD8+ in cervical cancer with HPV infection is in mild category (30% - 70%). Conclusion: Immunity against uterine cancer that played by MHC-I and CD8+ are in milt category. The experiment that related with uterine cancer immunology is suggested.  Keywords: CD8+; MHC-I; HPV; Cervical Cancer 1. Introduction Cervical cancer is the most common form neoplasm in women. This neoplasm is found in developing state as leading cause of neoplasm-related deaths in women in the world as a whole. Now the pathogenesis of cervical cancer is focused to human papilloma virus (HPV) [1-3]. Immunity to neoplasm or cancer as a whole are decided by Cytotoxic T Lymphocyte cells (CD8+, CTL, Tc), natural killer (NK) cells and specific antibody [4-10]. Immunity to destroy viruses to be many ways like in-terferon, secretory Immunoglobulin A (s-Ig A) of epithe-lial surface, antibody dependent cellular cytotoxicity (ADCC), and NK-cells, and more difficult to be sure which one are important [8-13]. Identity of TSAs (tu-mor-specific antigens) in experimentally induced tumors and their existence on human tumors remained elusive until the molecular basis of T lymphocyte cell recogni-tion was understood. With realization that T lymphocyte cell receptors recognize peptides bound to the antigen- binding cleft of major histocompatability complex mole-cules class I [4-7]. CD8+ is a lymphocyte that kills other cells. CD8+ rec-ognized class I alloantigens or conventional antigens (e.g., viruses) associated with class I histocompatability molecules [5-8]. MHC-I molecules are integral membrane proteins found on all nucleated cell and platelets. The extracellu-lar portion has three domains. The alpha-3 domain has a binding site for CD8+. CD8+ T lymphocytes kill target cells when they recognize antigen in associated with self MHC-I [5-8]. 2. Material and Method Frozen section tissues are collected from obstetric and gynecologic part of doctor Muwardi Hospital Surakarta patient with cervical cancer from January 2001 to Janu-ary 2002. DNA isolation was made by Henk Schmits method with some modifications [9]. Cut up to 25 mgr of tissue into small pieces, place in 1.5 ml a microfuge tube vol-ume, and add 200 ul of DNA extraction buffer. Add 20 ul of Proteinase K stock solution, mix by vortexing, and incubate at 55˚C overnight. Diagnose related with HPV infections are made by Henk Schmits and/or Nigel McMillan and Nina Fowler Copyright © 2013 SciRes.                                                                                  JCT The Expression of CD8+ and MHC-I in Cervical Cancer with HPV Infection 16 PCR-method with some modifications [9,10]. Twenty five ul microfuge tube Ready To Go PCR Bead (Amer-sham Pharmacia Biotech) mixed with 2 ul HPV consen-sus primers (MY09 and MY11) (CYBERGENE AB) and 2 ul DNA template. PCR protocol for both amplifications are 94˚C for 50 seconds, 59˚C for 50 seconds, 72˚C for 50 seconds and 4˚C soak. Paraffin-embedded tissue cervical cancer with HPV infection are cut using a thoroughly cleaned microtome and placed in glass plate that covered with poly-elisen. Immunohistochemistry stain is done with TSA-indirect method (NEN Life Science Products, RENAISSENCE) used monoclonal antibody again MHC-I (1:500) and CD8+ (1:500) produced by Santa Cruz. Photo micro-scopic collected via aX100 objective lens. 3. Result and Discussion From gel electrophoresis (Figure 1) can look the 450 band of DNA L1-HPV virus. Neoplasm of the cervical uterine is the most common form neoplasm in women. This neoplasm is found in developing state as leading cause of neoplasm-related deaths in women in the world as a whole. The mortality rate has been estimated at 2.8 deaths per 100,000 women, with a total of 252 deaths reported in 1993. Now the pathogenesis of cervical cancer is pointed to human papilloma virus (HPV). HPV infection associated with malignancies of urogenital tract and anus and also thought to be related to disorder of skin and the up-per-respiratory tract [1-3]. Immunity to cancer as a whole is decided by Cytotoxic T cells, NK-cells and specific antibody [4-10], otherwise to destroy viruses of human have many ways, and more difficult to be sure which one is important. Number ways are Interferon, s-Ig A of epithelial surface, antibody and antibody dependent cytotoxic cell (ADCC), and NK-cells [5-8,11-17]. Cytokines are included in the mechanism of immunity to cancer. From immunohistochemistry stain can look in Figures 2 and 3 and Table 1 for CD8+ expression and Figures 4 and 5 and Table 2 for MHC-I expression. Cytotoxic T cell (CTL, Ts, CD8+) is a T lymphocyte cell that kills other cells. Most CTL are CD8+; these cell recognize class I alloantigens or conventional antigens (e.g. viruses) associated with self class I histocompata-  500 kDa 450 kDaM 1 2 3 4 5 Figure 1. Gel electrophoresis the 5 sample with HPV posi-tive. The band about 450 bp long are the DNA HPV from L1 gene amplified (arrow). M: Marker. CD8+ (100X) CD8+ (400X) Figure 2. Immunohistochemistry staining of CD8+. The yellow-dark show that the CD8+ (black arrow) are ex-pressed in this tissue with cervical cancer.  010203040506070801 2 3 4 5 6 7 8 9 10CD8+Expression(percent)Number of sample  Figure 3. Expression of CD8+ from 10 sample cervical can-cer with HPV infection.  MHC-I (100X)MHC-I (400X) Figure 4. Immunohistochemistry staining of MHC-I. The yellow-dark show that the MHC-I (black arrow) are ex-pressed in this tissue with cervical cancer.  010203040506070801 2 3 4 5 6 7 8 9 10MHC‐ IExpression(percent)Number of sample  Figure 5. Expression of MHC-I from 10 sample cervical cancer with HPV infection.  bility molecule. Some CTL are CD4+; these cells recog-nize antigens associated with class II alloantigens or self class II histocompatability molecule. The mechanism of killing has been studied in detail with CD8+ T lympho-cytes. Once a cytotoxic T lymphocyte has recognized antigen, it binds to the target cell and kills it within a few hours. The mechanism of taget killing is not well under-stood and appears to include release of trypsin-like serine proteases, and cytotoxic lymphokine (e.g. perforin) [5-8, 11-19]. Genetic loci involved in the rejection of foreign tissue or non-self-forming region known as MHC. Structure is highly polymorphic cell surface that are involved in the  Copyright © 2013 SciRes.                                                                                  JCT The Expression of CD8+ and MHC-I in Cervical Cancer with HPV Infection 17Table 1. Expression of CD8+ (in percent) from 10 sample cervical cancer with HPV infection. No. of sample CD8+ (%) 1. 40 2. 70 3. 40 4. 30* 5. 50 6. 60 7. 50 8. 50 9. 40 10. 40 Mean 47 Percentage is got from cells positif/100 cells/one view look 5% - 30%: light expression category (*); 31% - 70%: mild expression category; 71% - 100%: hard expression category.  Table 2. Expression of MHC-I (in percent) from 10 sample cervical cancer with HPV infection. No. of sample MHC-I (%) 1. 40 2. 70 3. 50 4. 60 5. 50 6. 30* 7. 70 8. 50 9. 70 10. 70 Mean 57 Percentage is got from cells positif/100 cells/one view look 5% - 30%: light expression category (*); 31% - 70%: mild expression category; 71% - 100%: hard expression category.  rejection, which is called MHC antigen, which was ini-tially characterized by alloantibodies produced in one strain of inbred mice immunized with cells from other types only differ in the MHC. The same technique is used to determine the human MHC, which is known as antigen (HLA) system of human leukocytes. Three classes have been identified as encoded in murine and human MHCs. MHC-I consists of a glycosylated heavy chain (45 kDa) non-covalent assosiated with microglobu-line beta-2 (12 kDa) polypeptide that is also found in the serum-free [12-14,16]. MHC-I molecules are integral membrane proteins found in all nucleated cells and platelet. The alpha-3 domain has a binding site for Cyto-toxic T cells. Cytotoxic T cells kill target cells when they recognize antigen in association with self MHC-I mole-cules [5-8,14,20-25]. 4. Conclution The result of this experiment shows that expression of CD8+ and MHC-I in cervical cancer with HPV infection are in mild category (30% - 70%). 5. 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The Expression of CD8+ and MHC-I in Cervical Cancer 

with HPV Infection

Asnar, Elyana

Putra,  Suhartono Taat

http://eprints.uns.ac.id/15379/1/Publikasi_Jurnal_168.pdf

The Expression of CD8+ and MHC-I in Cervical Cancer with HPV Infection

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Sebelas Maret Institutional Repository