Abstract:
A panel of monoclonal antibodies produced from human gastrointestinal tumors as immunogen is used to diagnose the presence of colon cancer. The antibody panel subsets the human gastrointestinal tract in its reactivity vis-a-vis esophagus, stomach, small intestine and colon. The panel is useful as a diagnostic probe for cancer.

Description:
This invention was made in-part with government support under Grant No. CA08748 awarded by The National Cancer Institute. The government has certain rights in this invention. 
    
    
     BACKGROUND 
     This invention concerns monoclonal antibodies recognizing human gastrointestinal (GI) cells. The monoclonal antibodies recognize antigenic markers on normal as well as cancerous GI cells. Capable of distinguishing among normal GI cells as well as colon carcinomas, these mAbs are useful in diagnosis and prognosis of colon and gastrointestinal cancer. Examination of human both wastes, exudates, and fluids with these mAbs is a diagnostic procedure to probe for cancer of the gastrointestinal tract and especially colon cancer. These mAbs are of special importance because of the widespread occurrence of colon cancer. 
     In 1975 Kohler and Millstein introduced a procedure for the production of monoclonal antibodies (mAbs) using hybrid cells (hybridomas) which allows the production of almost unlimited quantities of antibodies of precise and reproducible specificity. Conventional antisera, produced by immunizing animals with tumor cells or other antigens, contain a myriad of different antibodies differing in their specificity and properties, whereas hybridomas produce a single antibody with uniform characteristics. The Kohler-Millstein procedure entails the fusion of spleen cells from an immunized animal with an immortal myeloma cell line. From the fused cells (hybridomas), clones are selected that produce antibody of the desired specificity. Each clone continues to produce only that one antibody. As hybridoma cells can be cultured indefinitely (or stored frozen in liquid nitrogen), a constant supply of antibody is assured. 
     Antibodies are proteins that have the ability to combine with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies except that they are very uniform in their properties and recognize only one antigen or a portion of an antigen known as a determinant. 
     In the case of cells, the determinant recognized is an antigen on or in the cell which reacts with the antibody. It is through these cell antigens that a particular antibody recognizes, i.e. reacts with, a particular kind of cell. Thus the cell antigens are markers by which the cell is identified. 
     These antigenic markers may be used to observe the normal process of cell differentiation and to locate abnormalities within a given cell system. The process of differentiation is accompanied by changes in the cell surface antigenic phenotype, and antigens that distinguish cells belonging to distinct differentiation lineages or distinguish cells at different phases in the same differentiation lineage may be observed if the correct antibody is available. Initial recognition of differentiation antigens came about through analysis of surface antigens of T-cell leukemias of the mouse and the description of the TL, Thy-1, and Lyt series of antigens. (Old, Lloyd J., Cancer Research, 41, 361-375, February 1981) The analysis of these T-cell differentiation antigens was greatly simplified by the availability of normal T cells and B cells of mouse and man and is relatively advanced. (See U.S. Pat. Nos. 4,361,549-550; 4,364,932-37 and 4,363,799 concerning mAb to Human T-cell antigens). Little is known about differentiation antigens displayed on normal and neoplastic cells belonging to other lineages. 
     This is due to the difficulty of obtaining a ready source of the appropriate normal cell type as well as the vagaries of the art of monoclonal antibodies. The preparation of hybrid cell lines can be successful or not depending on such experimental factors as nature of the innoculant, cell growth conditions, hybridization conditions etc. Thus it is not always possible to predict successful hybridoma preparation of one cell line although success may have been achieved with another cell line. 
     Progress in defining surface antigens on melanocytes was made possible by the recently discovered technique of culturing melanocytes from normal skin (Eisinger, et al., Proc. Nat&#39;l. Acad. Sci. USA, 79 2018 (March 1982). This method provides a renewable source of proliferating cells for the analysis of melanocyte differentiation antigens. Likewise, a large number of cell lines derived from melanomas have now been established and these have facilitated the analysis of melanoma surface antigens. The advent of mAbs has greatly accelerated knowledge about the surface antigens of malignant melanoma. Cell markers on both melanomas and melanocytes have been identified. A panel of typing monoclonal antibodies has been selected which recognizes differentiation antigen characteristics at each stage of development in both melanocytes and melanomas. These differentiation antigens may be used to classify melanocytes and melanomas and to group them into characteristic sub-sets. Dippold et al. Proc. Nat&#39;l. Acad. Sci. U.S.A. 77, 6114 (1980) and Houghton, et al. J. Exp. Med. 156, 1755 (1982). Immunoassay of melanocytes and melanoma cells within sub-sets is thus made possible. 
     SUMMARY 
     Cancers of the gastrointestinal tract are especially widespread; stomach cancer in Japan, colon cancer in the west and U.S.A. Early diagnosis would be desireable to prevent drastic surgery and loss of life. Clearly positive diagnosis help to support the surgical decision. Cytohistological methods to date are not always successful. A panel group of twelve mAbs of the present invention recognizing cancerous colon cells enables such a distinction for the first time. In addition, the panel distinguishes normal from cancerous cells. 
     The invention thus comprises hybridoma cell lines producing mAbs recognizing human colon cancer cells, from the group of CLH6, CLT85, CLT479, CLT174, CLH68, CLT152, CLH70, HT29/15, HT29/26, HT29/36, CLT218, CLT15, CLT307 and CLT86. These mAbs of the invention recognize colon glycoprotein (gp) antigens molecular weights 25 kd, 29 kd and 95 kd (mAbs CLH70, HT29/26 and CLT479 respectively). mAb CLT152 recognizes a protein antigen of 52 kd. The antigens for CLH6, CLT85, CLT174 and HT29/36 are heat stable and therefore probably glycolipids. CLT85, CLT479, CLT174, HT29/36, CLH68, CLT152 and HT29/15 are gamma sub one (γ 1 ) immunoglobulins. HT29/26 is a gamma sub 2A (γ 2A ) immunoglobulin. HT29/36 is a gamma sub 3 (γ 3 ) immunoglobulin and CLT218, CLT307, CLT86 and CLH6 are mu (μ) immunoglobulins. (HT 29/36 is the same mAb as HT 29-36 HT 29/15 is the same mAb as HT 29-15 and HT 29/26 is the same mAb as HT 29-26). 
    
    
     DESCRIPTION 
     A preferred embodiment of the present invention is to test a human speciman as for example human body wastes, fluids and exudates with each of the monoclonal antibodies of the panel. The cells are tested or contacted separately with each of the monoclonal antibodies in a series of dilutions. Thus, an assay for cancer is possible with minimal patent disruption. Indeed, the present invention permits testing of human urine specimans for cell fragments containing antigenic markers for the monoclonal antibodies. Entire cells need not be present. Cytohistological methods requiring whole cells are not always successful. 
     The monoclonal antibodies of the present invention were prepared by an improved Kohler-Millstein procedure wherein spleen cells from a mouse (or other mammal) immunized with human cancerous colon cells from established human tumor cell lines were fused with mouse myeloma to form hybridomas. By serological screening, antibodies from these hybridomas were found which recognize differentiation antigens on normal bladder and/or cancerous bladder. Other tissues, both normal and cancerous, may be recognized as well by some of these monoclonal antibodies. A system of classification of normal as well as cancerous colon based on these differentiation antigens is now possible, and serological assays for tumors of the colon have been developed. These assays are of special use in the early diagnosis of gastrointestinal cancer especially colon cancer. 
     The assay of the present invention comprises contacting a tissue containing colon cells with the antibody recognizing colon cell antigens, preferably monoclonal antibodies to one or more cell antigens of the gastrointestinal antigenic system, and observing the immunoserological or immunopathological antigenic reaction between said monoclonal antibody and said antigen. In a preferred embodiment of the invention, the tissue sample to be contacted is gastrointestinal tissue and the antigenic reaction of the contacted tissue is observed by well known techniques such as immunofluorescence, Rosette formation with sheep or human red blood cells linked to Protein A or anti-Ig direct absorption and the like. In another embodiment of the present invention, the tissue to be assayed is first excised and is then either freshly, or after being frozen or embedded in paraffin by methods well-known in the art, contacted with the monoclonal antibodies of the invention. Observation of the reaction is as before. 
     In another preferred embodiment of the present invention, the tissue to be assayed comprises the intact body of an individual or a whole portion thereof. The antibody, tagged with a radioactive or other energy-producing element, is administered to the individual, and the whole body or part thereof is scanned externally for localization of radioactivity at the site of cancerous gastrointestinal cells. In another preferred embodiment cancerous colon cells are located. 
     The present invention also makes possible the treatment of gastrointestinal tumors in a patient wherein the monoclonal antibody recognizing the cell antigen of cancerous colon or other cancerous GI cells, is administered to the patient in an amount effective to inhibit the growth or proliferation of cancer cells. In a preferred embodiment of this method, the antibody is tagged with a potentially tissue destructive agent which causes destruction of the cancer cells. Examples of tissue destructive agents comprise chemotoxic agents, chemotherapeutic agents including vaccines, radionuclides, toxins, complement activators, clotting activators and the like. 
     The above examples are for illustrative purposes only and are not meant to limit the scope of the invention. 
     EXAMPLES OF ANTIBODIES, AND ANTIGENS RECOGNIZED 
     The following examples are intended to illustrate the invention without limiting same in any manner especially with respect to substantially functional equivalents of hybridomas, monoclonal antibodies and cell lines described and claimed herein which equivalents can be produced in accordance with the invention following the procedures outlined in the specification of this application. 
     The monoclonal antibodies selected for use in the present invention were derived from spleen cells of mice immunized with whole cells of colon carcinoma cell lines such as Tallevi and HT-29 by fusion methods well known in the art. 
     A group of monoclonal antibodies which were found to recognize specific cell antigens of gastrointestinal cells, was selected as the grastrointestinal panel. This panel and the antigenic systems recognized are given in Tables I &amp; II. Heterogeneity of human colon carcinoma is therein noted. The table data point out and define the heterogeneity of colon carcinomas. Gastrointestinal antigenic systems are defined by these mAbs as determined by serological analysis with over 70 tumor cell lines; 18 colon cancers, over 50 non-gastrointestinal cancers as well as immunopathology on frozen sections of normal adult and normal fetal tissue. (See Table I &amp; II) 
     Several of the antigens, as defined by the monoclonal antibodies of the panel, are expressed differentially by cell populations within the adult GI system. CLT152 antigen is expressed by epithelial cells of the GI mucosa of esophagus, stomach, small intestine and colon, but is not found in other adult tissues. CLH70, CLT307, CLT86 and CLH68 antigens are expressed by adult stomach, small intestine and colon. CLT218 is expressed by adult small intestine and colon. HT29/26 is expressed by colon and some cells of small intestine in the adult. CLT15 also is expressed by normal colon epithelium as well as some upper GI cells except stomach in adult tissues. Thus the mAbs antigens HT29/26, CLT15, CLT218, CLH70, CLT307, CLT86 and CLH68 occur in adult colon epithelial cells; they vary among themselves in their pattern of distribution within the rest of the GI tract. There is some limited expression of these antigens in epithelial cells of other tissues as well See Table II. Thus, for example, CLT218, CLT86, HT29/26 antigens are expressed on bronchial epithelium whereas CLH6, HT29/36 and HT29/15 are not. Thus, the panel antibodies differ in their expression on normal cells even as to similar cells of other tissues. 
     It is important that the mAbs CLH6, CLT85, and 29/36 do not react with normal adult tissue in immunopathology of frozen tissue sections but do react with distinct subsets of colon adenocarcenomas. 
     Serologically CLT85 reacts with approximately 11 of 17 colon cancer lines, and CLH6 with approximately 8 out of 17 colon cancer cell lines. CLT85 and CLH6 show no reaction with normal adult cells in serology. 
     Thus normal versus neoplastic cells of the colon can be differentiated and assayed by contacting a speciman from a human patient with each of the 14 monoclonal antibodies of the panel in serial dilution, and observing any antigen antibody reaction by any of the methods cited. Although 14 hybridomas producing monoclonal antibody against gastrointestinal cell antigens are presented, it is obvious that the present invention encompasses all the mAbs exhibiting the characteristics described therein, especially the embodiment describing reaction with normal as well as tumor cell antigens of the GI tract. 
     Changes in cell antigens are associated with different stages of differentiation and different stages of cancer. Thus this invention technique defined cell antigens associated with differentiation and cancer of the GI tract. 
     The following hybridoma cell lines are maintained on deposit at Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021 under designations corresponding to the mAb produced by each hybridoma as follows: 
     CLH 6, CLT 85, CLT 479, CLT 174, CLH 68, CLT 152, CLH 70, HT 29/15, HT 29/26, HT 29/36, CLT 218, CLT 15, CLT 307 and CLT 86. 
     Said hybridoma cell lines have been deposited on Mar. 11, 1983 with the American Type Culture Collection, 1230 Parklawn Drive, Rockville, Md. 20852 under ATCC designations corresponding to the above Sloan-Kettering designations as follows: 
     
         ______________________________________Sloan-Kettering         Corresponding ATCCdeposit #     deposit accession #______________________________________CLH 6         HB 8232CLT 85        HB 8240CLT 479       HB 8241CLT 174       HB 8242CLT 68        HB 8243CLT 152       HB 8244CLH 70        HB 8245HT 29/15      HB 8246HT 29/26      HB 8247HT 29/36      HB 8248CLT 218       HB 8249CLT 15        HB 8250CLT 307       HB 8251CLT 86        HB 8252______________________________________ 
    
     LEGEND TO TABLE I 
     Serological reaction of colon panel monoclonal antibodies with human tumor cell lines of various tissues by rosette formation with human red blood cells conjugated with rabbit anti-Ig, Dippold Supra 
     where 
     0=no reaction by rosette formation or absorption 
     2=positive rosette reaction &lt;1,000 fold dilution antibody supernatant 
     3=positive rosette reaction at &gt;1,000 fold dilution antibody supernatant 
     1=positive reaction by absorption test only. 
     If there is no rosette reaction, the absorption test was done. Thus if a mAb was negative for the rosette reaction but absorbed onto the test antigen system it was deemed to be a positive reaction such that 
     1=positive reaction by the absorption test though mAb gives a negative test for rosette formation i.e. 0 test for rosette reaction is further tested by the absorption test. Therefore 0 on this table indicates no reaction by either absorption or rosette reactions. For comparison, mAb 19.9 was obtained from H. Kaprowski and assayed as well alongside the mAbs of the present invention Atkinson, B. F. et al., Cancer Research, 42:4820-4823(1982). 
     In Table I actual titers are included. 
     Immunogen for CLT series is Tallevi, for HT and CLH antibodies the immunogen is HT-29. 
     LEGEND TO TABLE II 
     Immunopathological reaction of colon panel monoclonal antibodies with fetal and adult normal human tissue in frozen section by indirect immunofluorescence. 
     0=no reaction 
     +=positive reaction 
     ±=heterogeneous reaction within the tissue 
     For comparison, mAb 19.9 was obtained from H. Kaprowski and assayed as well alongside the mAbs of the present invention Atkinson, B. F. et al., Cancer Research, 42:4820-4823(1982). 
     
                                           TABLE I__________________________________________________________________________ SerologySerological Reaction of Monoclonal AntibodiesProduced from Human Colon Tumor Immunogen With VariousHuman Cancer Cell LinesIMMUNIZING TUMOR: COLON     CLH        CLT           CLT              CLT                 CLH                    CLT                       CLH                          HT29                              HT29                                  HT29                                      CLT                                         CLT                                            CLT                                               CLTAntibody  6  85 479              174                 68 152                       70 -15 -26 -36 218                                         15 307                                               86 19.9__________________________________________________________________________IG class of antibody:      μ        γ1           γ1              γ1                 γ1                    γ1                          γ1                              γ2a                                  γ3                                      μ                                         μ                                            μ                                               μgp Antigen     gp95           p52                       gp25   gp29detectedCELLS TESTEDColon Ca.:HT 29     3  3  3  3  3  3  3  3   3   3   0  0  3  3  10.sup.3SW 48     3  3  2  3  0  3  0  0   3   3   2  2  0  0  10.sup.3SW 403    0  3  3  3  2  3  0  3   3   2   3  0  3  3  10.sup.6SW 480    3  3  3  3  3  3  3  3   3       3  3  3  3  10SW 620    3  3  2  0  0  3  3  3   3   3   2  3  2  0  10.sup.2SW 837    0  0  0  1  0  0  0  0   3       2  0  0  0  0SW 1083   2  0  0  0  0  0  0  0   3   0   3  0  0  0  0SW 1116   0  1  3  3  3  3  0  3   3   3   3  0  3  3  10.sup.2SW 1222   0  3  3  3  3  3  3  3   3   3   0  0  3  3  10.sup.3SW 1417   1  1  3  3  3  3  3  3   3       0  0  2  3  0CACO2     2  3  0  0  0  2  3  3   3   0   3  0  0  0  0SK-CO-1   0  0  0  0  0  0  2  3   3       0  0  0     0Tallevi   0  3  3  3  3  3  3  3   3   0   3  3  3  0  10.sup.3Moutsiatzos     1  0  0  0  0  0  0  0   0   0   0  0  0     0Kolraga   0  3  3  3  3  3  3  3   3       3  2  0  3  10Friedland 0  0  0  3  3  2  3  0   3   0   3  0  3  0  0Redmond   1  0  0  0  0  0  0  3   3   0   2  0  0  0  1HSO7O3TPancreas Ca.:CAPAN 1   3  0  0  1  0  0  3  0           0  0  3  0  0CAPAN 2   0  3  3  3  3  3  0  3           3  3  0  3  10.sup.2SW 850    0  0  0  0  0  0  0  0           0  0  0  0SW 979    0  0  0  0  0  0  0  0           0  0Liver Ca.:SK-HEP-1  1  0  0  0  0  0  2  3   3   0   0  0  0  0  0Biliary duct:Charles   0  0  2  0  0  0  0  3   3       0  0     0  0Astrocytoma:Goodstein 0  0  0  0  0  0  0  0           0  0  0  0  0U251      0  0  0  0  0  0  0  0           0  0  0     0Becker    0  0  0  0  0  0  0  0           0  0  0     0Machino   0  0  0  0  0  0  0  0   0   0   0  0  0  0  0Jones     0  0  0  0  0  0  0  0           0  0  0     0AJ        0  0  0  0  0  0  0  0   0       0  0  0     0Lear      0  0  0  0  0  0  0  0   0   0   0  0  0  0  0Healy     0  0  0  0  0  0  0  0   0       0  0  0  0  0T98       0  0  0  0  0  0  0  0           0  0  0  0  0U373      0  0  0  0  0  0  0  0   0   0   0  0  0  0  0Melanoma:SK-MEL-31(3)     0  0  0  0  0  0  0  2   0   0   0  0  0  0  023        0  0  0  0  0  0  0  0   0       0  0  0  0  013        0  0  0  0  0  0  0  0   0   0   0  0  0  0  037        0  0  0  0  0  0  0  2   0   0   0  0  0  0  093(2)     0  0  0  0  3  0  0  3   2   0   0  0  0  0  028        0  0  0  0  0  0  0  0   0   3   0  0  0  0  0MeWo      0  0  0  0  0  0  0  0   0   0   0  0  0  0  0Neuroblastoma:SK-N-MC   0  0  0  0  0  0  3  0   0   0   0  0  0  0  0SH        0  0  0  0  0  0  3  0   0   0   0  0  0  0  0BE2       0  0  0  0  0  0  3  0   0   0   0  0  0  0  0LAN-1-5   0  0  0  0  0  0  0  0   0   0   0  0  0  0  0CHP-234NBreast Ca.:MDA MB 361     0  0  0  0  0  0  3  3   0   0   0  0  0  0  0MDA MB 231     0  0  0  0  0  0  2  3   3   0   0  0  3  0  0BT 20     0  0  0  0  0  0  0  3   3   0   0  0  0  0  0CAMA      0  0  1  1  0  0  0  3   0       3  3  3  3  0SK-BR-3   0  0  0  0  0  0  0  3   0       0  0  0  0  0ALAb      0  0  0  0  0  0  3  3           0  0  0  0  0MCF-7     0  0  0  0  0  0  0  3   3   3   3  0  0  3  0Kidney Ca:SK-RC-6   0  0  0  0  0  0  0  0   3   0   0  0  0  07         0  0  0  0  0  0  0  0   3   0               029        0  0  0  0  0  3  0  0   3   0   0  0  0     04         0  0  0  0  0  0  0  0   3   0   0  0  0Ovary Ca:SK-OV-3   0  0  0  0  0  0  0  0   3   0   0  0  0  0  0ROAC      0  0  0  0  0  0  3  0   3       0  0  0  0  02774      0  0  0  0  0  0  0  0   3       0  0  0  0  0SW 626    0  0  3  3  0  0  0  3   3       3  0  3  3  0Shustak   0  0  0  0  0  3  0  3   3       3  3  3  3  0Turanek   0     2  0  0  3  2  3   3   0   0  0  0  3  10Uterine Ca:ME180     0  0  0  0  0  0  0  0   3   3   0  0  0  0  0Chorioepithelium:SVCC      0  0  2     0  0  0  0   3       0  0  3  0  0Lung Ca:SK-LC-3   0  0  2  0  0  0  3  3   3   0   0  0  0  0  04         0  0  0  0  0  0  0  3   3   2   0  0  0  0  105         1  0  0  0  0  0  0  3   3   0   0  0  0  0  06         0  0  0  0  0  0  3  2   0   0   0  0  2  0  07         0  0  0  0  0  0  0  0   3   2   0  0  0  0  08         0  0  0  2  0  0  0  0   3   3   0  0  0  0  013        0  0  0  0  0  0  2  0   3   2   0  0  0  0  0Lawson    0  1  1  1  0  3  2  3   3   2   3  0  0  3  10.sup.3Bladder Ca:T-24      0  0  0  0  0  0  0  0   0   3   0  0  0  0  0TCC SUP   0  0  0  0  0  0  0  0   0   0   0  0  0  0  0253J      0  0  0  0  0  0  0  0   0   0   0  3  0  0  0639V      0  0  0  0  0  0  0  0   3   3   0  0  0  0  0486P      0  0  0  0  0  0  3  3   3   0   0  0  0  0  0__________________________________________________________________________ 
    
     
                                           TABLE II__________________________________________________________________________ ImmunopathologyNormal Tissue Distribution of the MonoclonalAntibodies Produced from HumanColon Tumor Immunogen      CLT  CLH              HT  HT  CLT                         CLT                            CLT                               CLT                                  CLH CLT   CLT                                               CLT                                                  HT  CLH      85 28           6  29/36                  29/15                      479                         15 174                               86 70  152                                         19.9                                            307                                               218                                                  29/26                                                      68__________________________________________________________________________A.FETAL TISSUESLUNG       ± 0  0   0   +  +  +  +  +   +     +  +  +   0Bronchial  ± 0  0   0   ±                         ±                            ±                               +  +   +     +  +  +   0EpitheliumCartilage  0    0  0   0   0  0  0  0  0   0     0  0  0   0Pneumocytes      0    0  0   0   0  0  0  0  0   0     0  0  0   0Connect. Tis      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0HEART      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0THYMUS     0    0  0   0   +  0  0  0  0   +  +  0  0  0   +Hassal&#39;s C.      0    0  0   0   +  0  0  0  0   +  +  0  0  0   +Thymocytes 0    0  0   0   0  0  0  0  0   0  0  0  0  0   0SPLEEN     0    0  0   0   0  0  0  0  0   0  0  0  0  0   0White Pulp 0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Red Pulp   0    0  0   0   0  0  0  0  0   0  0  0  0  0   0LIVER      0    0  0   0   0  0  0  +  +   +  0  +  +  +   +Hepatocytes      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Biliary Epi      0    0  0   0   0  0  0  +  +   +  0  +  +  +   +CellsGALLBLAD.  0    0  0   0   0  0  0  +  +   +  0  +  +  +   +ESOPHAGUS  0    0  0   0   ±                         0  ±                               ±                                  0   ±                                         ±                                            +  ±                                                  ±                                                      ±STOMACH    0    ±              0   0   0  0  0  ±                                  +   ±                                         ±                                            ±                                               +  0   +SMALL INT. 0    0  0   0   0  0  0  0  0   0  0  0  0  ±                                                      +COLON      ± ±              0   0   ±                         ±                            ±                               +  +   +  ±                                            +  +  +   +PANCREAS   0    +  0   +   +  +  +  +  +   +  +  +  +  +   0Exocrine   0    ±              0   +   +  +  +  +  +   +  +  +  +  +   0Endocrine  0    0  0   0   0  0  0  0  0   0  0  0  0  0   0KIDNEY     0    +  0   0   0  0  0  +  +   0  0  0  +  +   0Glomerulus 0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Prox. Tub. 0    0  0   0   0  0  0  0  +   0  0  0  0  0   0Distal Tub.      0    +  0   0   0  0  0  +  0   0  0  0  +  +   0Collec. Tub      0    +  0   0   0  0  0  +  0   0  0  0  +  +   0URETER     0    +  0   ±                      0  0  0  ±                                  0   +  0  +  +  +   +UR. BLAD.  0    +  0   ±                      0  0  0  ±                                  0   +  0  +  +  +   +ADRENAL    0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Cortex     0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Medulla    0    0  0   0   0  0  0  0  0   0  0  0  0  0   0TESTES     0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Germ. Cells      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Endoc. Cel.      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0Connect. T.      0    0  0   0   0  0  0  0  0   0  0  0  0  0   0OVARY      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Germ. Cells      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Connect. T.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0FALLOP. T. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  +   0UTERUS     0  0 0  0   0   0  0  0  +  +   0  0  0  +  +   0Endometrium      0  0 0  0   0   0  0  0  +  +   0  0  0  +  +   0Myometrium 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0CERVIX     0  0 0  0   0   0  0  0  +  0   0  0  0  0  +   +Endocervix 0  0 0  0   0   0  0  0  +  0   0  0  0  0  +   0Exocervix  0  0 0  0   0   0  0  0  ±                                  0   0  0  0  0  0   +SKIN       0  0 0  0   0   0  ±                            0  +  0   +  0  +  +  +   +Epidermis  0  0 0  0   0   0  ±                            0  ±                                  0   ±                                         0  ±                                               ±                                                  ±                                                      ±Melanocytes      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Sweat Gland      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Sebac. Gld.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  +   0Hair Fol.  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Dermis C.T.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0BRAIN      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Neurons    0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Glial Cells      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Dendrites  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0LYMPH NODE 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0BLOOD VES. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Endoth. Cel.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Smooth Ms. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SOFT TIS.  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SECRETION  ±         0 0  0   0   +  0  +  +  +   +  +  +  +  +   +B. ADULT TISSUESLUNG       0  0 0  0   0   0  0  0  +  0   0  +  0  +  +   0Bronchial  0  0 0  0   0   0  0  0  ±                                  0   0  ±                                            0  +  +   0EpitheliumCartilage  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Glandular  0  0 0  0   0   0  0  0  +  0   0  +  0  +  +   0EpitheliumPneumocytes      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Connect. Tis      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0HEART (ms) 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SPLEEN     0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0White pulp 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Red pulp   0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0LIVER      0  0 0  0   0   0  0  +  0  +   +  +  +  +  +   0Hepatocyte 0  0 0  0   0   0  0  0  0  0   0  0  0  ±                                                  0   0Bil. Epit. 0  0 0  0   0   0  0  +  0  +   +  +  +  +  +   0Sinusoids  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0GALLBLADDER      0  0 0  0   0   0  0  +  0  +   +  +  +  +  +   0ESOPHAGUS  0  0 0  0   0   0  +  ±                               0  0   +  ±                                            +  +  0   0STOMACH    0  0 0  0   0   0  0  0  ±                                  +   +  +  ±                                               0  0   +SM. INTEST.      0  0 0  0   0   0  ±                            0  +  ±                                      +  0  +  ±                                                  0   +COLON      0  0 0  0   0   0  +  0  +  +   +  0  +  +  +   +G.I. Smc   0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0PANCREAS   0  0 0  0   +   +  +  +  +  +   +  +  +  +  +   0Exocrine   0  0 0  0   +   +  +  +  +  +   +  +  +  +  +   0Endocrine  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0KIDNEY     0  0 0  0   0   0  0  0  0  0   +  0  0  0  +   0Glomerulus 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Prox. Tub. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Henle&#39;s L. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  ±                                                      0Distal Tub.      0  0 0  0   0   0  0  0  0  0   +  0  0  0  +   0Collec. Tub      0  0 0  0   0   0  0  0  +  0   +  0  0  0  +   0URETER     0  0 0  0   0   0  0  ±                               0  0   0  0  +  +  +   +URI. BLAD. 0  0 0  0   0   0  0  ±                               0  0   0  0  +  +  +   +ADRENAL    0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Cortex     0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Medulla    0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0THYROID    0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Epithelium 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Colloid    0  0 0  0   0   0  0  0  0  0   0  0  +  +  +   +BREAST     0  0 0  0   +   0  0  +  0  0   +  +  +  +  +   +Duct Cells 0  0 0  0   +   0  0  +  +  0   +  +  ±                                               +  +   0Acinar Cel.      0  0 0  0   +   0  0  0  0  0   +  ±                                            0  0  0   0Connec. Tis.      0  0 0  0   0   0  0  0  +  0   0  0  0  +  +   +PROSTATE   0  0 0  0   0   0  0  0  0  +   0  +  0  +  +   +Epithelium 0  0 0  0   0   0  0  0  0  +   0  +  0  0  0   0Stroma     0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0TESTES     0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Germ Cells 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Endocrine Cel      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Connec. Tis.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0OVARY      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Germ Cells 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Connec. Tis.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0FALLOP. TUB.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  +   0UTERUS     0  0 0  0   0   0  0  0  0  0   0  0  0  0  +   0Endometrium      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Myometrium 0  0 0  0   0   0  0  0  0  0   0  0  0  0  +   +CERVIX     0  0 0  0   0   0  0  0  0  +   0  +  0  0  +   0Endocervix 0  0 0  0   0   0  0  0  0  +   0  +  0  0  +   +Exocervix  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0PLACENTA   0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Cytotrophb.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Syncytotrb.      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Sinusoids  0  0 0  0   0   0  0  0  0  0   0  0  0  +  +   0SKIN       0  0 0  0   +   ±                         0  ±                               ±                                  +   +  0  0  0  ±                                                      0Epidermis  0  0 0  0   0   0  0  0  0  0   0  0  0  +  +   0Melanocytes      0  0 0  0   0   0  0  0  0  0   0  0  0  ±                                                  ±                                                      0Sweat Gld. 0  0 ±              0   +   ±                         0  ±                               +  +   +  0  0  0  0   0Sebaceous G.      0  0 0  0   +   0  0  0  0  +   0  0  0  0  0   0Dermis CT  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0BRAIN      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Neurons    0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Glial Cell 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Dendrites  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0LYMPH NODE 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Fol/Modul  0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0BLOOD VES. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Endothelium      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0Smooth Ms. 0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0CAPILLARIES      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SKELETAL MS      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SOFT TISSUE      0  0 0  0   0   0  0  0  0  0   0  0  0  0  0   0SECRETIONS 0  0 ±              +   +   +  +  +  +  +   +  +  +  +  +   +__________________________________________________________________________