Experimental Animals. Research Places. Procedure.

Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015 ISBN: 978-602-74204-0-3 Chemistry Page 149 increasing of ROS will further increase epithelial necrosis, which led to increasing Hsp70 Nasronudin, 2005. Another study states that increasing of ROS will further enhance T cell apoptosis CD4 + and APC which have an impact on the decrease of T cells CD4 + and expression of IgA Prasad, 2010. Immunoglobulin A IgA produced by lymphocytes is the primary antibody involved in local immune system in the small intestine. IgA have ability to agglutinate infectious agents and facilitate the clearance of microbial processes through peristalsis and movement of small intestine mucosa Mestecky et al, 1999. Production and function IgA are expressed by plasma cells is strongly influenced by the presence of T lymphocytes CD4 + in the intestinal mucosa. Indications of reduced expression of IgA plasma cells in the intestinal mucosa can be caused by reduced T cell activation and decreased content of CD4 + T lymphocytes expression on Peyers patches. Rey et al, 2007. The lymphocytes histologic score ratio of CD4 + CD8 + is a comparison between lymphocytes histologic score of CD4 + with CD8 + . The role CD4 + is a profound effect on lymphocyte ratio histologic score. In several studies about ratio of Cell T CD4 + CD8 + , as in the healing process lymphocytes cell, CD8 + and CD4 + are joined regulate the healing process. CD8 + is downregulator of wound healing, while CD4 + is upregulator of wound healing Boice and Davis, 2001. An increasing of ratio of CD4 + CD8 + can describe the repair process damage the intestinal villi Boice and Davis, 2001. But to find out and explain how the description of the presence of IgA and balanced ratio of CD4 + CD8 + in the intestinal mucosa in the gastrointestinal tract malnourished after getting Anadaragranosasupplementation in the immune system of the digestive tract has not been scientifically reported. Scientific information about the expression of IgA and the ratio of CD4 + CD8 + can be studied through research with immunohistochemically methods. This method is considered to be able to describe reaction mechanism of antibody with an immunoenzyme principle. 2. Materials and Methods 2.1. Research Design. This research is an experimental research laboratory with the Randomized Post Test Only Control Group Design on 30 rats as subjects. The sample was selected by simple random sampling, were divided into five treatment groups:i normal controls [KN], ii and iii were a group of malnourished subjected to non-protein food as a positive control [K.Kg+] and standard food with casein 20 as a negative control [K.Kg-], iv and v as a group of malnourished thas was treated with casein protein diet 10 combined Anadara granosa flour 10 [P.Kg1] and were given Anadara granosa flour 20 P.Kg2].Treatment for 45 days. Malnourished condition Albumin2.7 gdL, obtained by non-protein diet. Water were given ad libitum for 14 days. End of the study, the rats were sacrificed for the intestinal tissue. Further examination of immunoglobulin A IgA and histologic score ratio of CD4 + T cellsCD8 + in mucosal tissues of the intestine jejunum and ileum by immunohistochemically methods. Final step, all of samples be operated for making bowel tissue. Examination of immunoglobulin A IgA and balanced ratio of cells T CD4 + CD8 + in the mucosal tissues of the intestine jejunum and ileum by immunohistochemically methods.

2.2. Experimental Animals.

Experimental animals used were rat Rattus norvegicus spraque Dawley strain aged 3 weeks, weight 75-105 gr, in good health and there is no anatomical abnormalities. The use of Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015 ISBN: 978-602-74204-0-3 Chemistry Page 150 experimental animals have received a certificate of Conduct Feasibility from Feasibility Ethics Committee on College. 2.3. Anadara granosa as Samples. Blood Cockle Species of Anadara granosa Linn as main material obtained from Tomini gulf coast, Pohuwato, Gorontalo Province. Anadara granosa flour manufacture material supplementation include: washing, steaming, and separation of intact meat from shell, draining, drying and milling. Drying use sunlight to reduce the water content. Grinding with disc mill with size 40 mesh, and produced Anadara granosa flour.

2.4. Research Places.

Research conducted at the Chemistry Laboratory, Chemistry Dept., State University of Gorontalo. Making the histology and immunohistochemical methods performed at the Laboratorium Primata Bogor.

2.5. Procedure.

The level of IgA. Immunohistochemically staining includes several stages of preparation, beginning preparation glass object, plating coating glass object with poly-L lysine, pasting preparations slices on glass objects and immunohistochemically staining procedure itself. Immunohistochemically staining procedures include: deparaffination, rehydration, endogenous peroxidation, washing with distilled water and PBS, the provision of primary antibody anti- rat polyclonal antibody IgA, washing with PBS, giving one step secondary antibody HRP Polymer, washing, visualization with DAB 3,3-diaminobenzidine, washed or put in the DWMQ stopping point, counterstain with hematoxylin-DWMQ, and dehydration, Xilol and mounting. For further histological preparations ready observed under a microscope. Histology Score determination of CD4 + , CD8 + , and ratio CD4 + CD8 + . Histologic score performed through immunohistochemical examination procedure is the same as the expression of IgA antibodies that differ only anti primary CD4 + and CD8 + , anti-rat monoclonal with a streptavidin-biotin staining method in intestinal mucosal tissue that can be seen in the light microscope magnification of 400 times. Selected visual field most positive cells with 400x magnification, then calculated the number of positive cells in five visual fields clockwise. Histologic score was calculated and compared between the control and treatment. 3. Results and Discussion 3.1. Result.