| In=20
Vitro Studies of Cytotoxic Activity of Natural Killer (NK) and=20
Lymphokine-Activated Killer (LAK) Cells Obtained from Blood of = Dogs |
| Prof. =
Dr. E.=20
Burkhardt DVM, Doctorate of veterinary medicine, Specialist in = Pathology, PhD, Professor Justus-Liebig-University - Giessen, = Germany,=20 Diplomate of the European College of Veterinary = Pathology |
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This is a combination of Prof. Dr. Burkhardt's synopsis, and = Pat Long's=20 notes during his presentation October 8th, 2006 in Como Italy at = the=20 Health Symposium held by the SIBB, the Societ` Italiana Bovaro del = Bernese The immune system produces cells to mount an attack against any = cells=20 seen as foreign, or antigens. Especially two different types of=20 lymphocytes the NK or natural killer cells, and Tc or cytotoxic = T-cells (-=20 where cytotoxic means it is toxic to cells) show this capability. = The=20 immune system is complex, and mounts a number of different types = of=20 responses to antigens. One specific type of response involves = T-cells (T=20 helper cells) producing cytokines (a signaling compound for = inter-cell=20 communication formerly named interleukines or lymphokines) which = then=20 helps activate natural killer cells and cytotoxic T-cells which = will=20 destroy the cells with the specifically identified antigen. Tumor cells have specific surface antigens which can be = recognized by=20 the immune system which will then mount an immune response. In = humans (and=20 also in animals) with compromised immune systems such as people = with AIDs=20 this response is suppressed which makes the person more = susceptible to=20 cancers that a person with a healthy immune system would not = normally=20 acquire. In 1985 an article published in the New England Journal of = Medicine=20 313(23):1485-92 reported cases of people with malignant tumors = where=20 standard treatments had failed, but remission of some of the = tumors was=20 achieved by an immunotherapy consisting of lymphokine activated = killer=20 (LAK) cells. The advantage of this type of therapy would be that = it allows=20 controlled manipulation of effector cells outside the body to = avoid=20 uncontrolled and possible negative side effects. The natural killer cells are suppressed in their killing = activity by an=20 unaltered MHC (major histocopatibility complex)I molecule which is = expressed on the surface of all normal nucleated cells of the body = and=20 which allows the immune system to recognize it as friendly. For = use in=20 attacking cancer cells, which often have an altered formation of = this MHCI=20 molecule, the natural killer (NK) cells mount an unspecific and=20 immediately starting response, while the cytotoxic T-cells (Tc) = mount a=20 response against specific tumor antigens, but with a delay. NK cells are a special subpopulation of lymphocytes (like T- = and=20 B-cells). They are a medium size cell found mainly in the blood = and=20 spleen. NK cells will attack tumors or virus infected cells = without prior=20 sensitization. For experimentation, the NK cells can be enriched from whole = blood=20 using a centrifuge. The cell weights will force them into separate = layers=20 using a special gradient medium. The gradient of choice is 58.% = density of=20 Percoll=C2=AE. The centrifuge is used to separate a whitish band = of lymphocytes=20 from a healthy dog which can then be used for testing on different = cancer=20 cell lines. To demonstrate cytotoxic activity of the isolated cells the = test=20 described used a cancer cell line derived from a thyroid = adenocarcinoma=20 (CTAC) cell line. S. Krakawka at Ohio State University had = demonstrated=20 that CTAC is susceptible to lymphocytes. After 12 - 14 hours of = incubation=20 there was a strong killer response and a spontaneous killer = effect. Most=20 of the cancer cells died from necrosis caused by perforin, a toxic = substance within the granules of NK cells. But cells are also programmed to die, which is apoptosis. = Apoptosis is=20 triggered by genes, but the process can be stopped if the genes = are=20 disturbed. The genes which regulate apoptosis can be affected by a = withdrawal of growth hormones, or by injury from things such as = radiation,=20 toxins, or free radicals. NK cells induce apoptosis by binding to = the Fas=20 receptor of target cells. Therefore they use both of these = processes, this=20 is the "deadly kiss" of killer cells. The methods used to measure the spontaneous cytotoxic activity =
of=20
effector cells include: RBA was used in his study to measure spontaneous cytotoxic = activity of=20 isolated peripheral blood lymphocytes (PBL). They demonstrated a = ten-fold=20 increase in the cytotoxic activity of PBL after cultivation in the = presence of recombinant human interleukin-2 (rh IL-2) for seven = days. They=20 also demonstrated a direct binding of effector cells to the = surface of the=20 tumor targets and both, necrosis and apoptosis of the tumor cells = was the=20 result. A comparison was also made of the spontaneous cytotoxic = activity of PBL=20 isolated from the blood of tumor bearing dogs and healthy control=20 dogs. They found a statistically significant decrease of spontaneous=20 cytotoxic activity from dogs with malignant mammary = adenocarcinomas, while=20 in dogs with other types of carcinomas or benign mammary tumors no = significant decrease was observed. In the literature three possible factors for the down =
regulation of an=20
immune response against tumor cells is dicussed: These effects may be counteracted by reactivating the effector = cells in=20 vitro by IL-2 and IL-12 outside the patient=E2=80=99s body, thus = generating=20 lymphokine activated killer cells LAK and reperfusing them into = the=20 patient=E2=80=99s body (adoptive immunotherapy). Cytokines are small protein molecules which regulate the immune = response. They are used for short distance response and for a = short=20 duration. Interleukin is a type of cytokine. IL-2 induces = proliferation in=20 activated T- and B-cells. It also activates NK cells, IL-12 leads = to=20 proliferation of these cells. IL-2 is produced by helper T-cells. = From=20 humans, recombinant IL-2 is commercially available. A study measuring the IL-2 activity level found that IL-2 or = IL-12 had=20 to be added every 3 days. Lack of IL-2 leads to cell death. Currently they are studying means to improve the production of = LAK=20 cells. They have generated a cell line which continuously secretes = IL-2 by=20 transfection of the gene encoding for canine IL-2 into BHK cells = (baby=20 hamster kidney cells - it is a special mesenchymal, commercially = available=20 cell line). Similar experiments are desired to achieve the = permanent=20 secretion of canine IL-12. Conclusion: In the dog there is spontaneous cytotoxic activity =
against=20
tumor cells by NK cells which can be activated and induced to =
proliferate=20
by IL-2 secreting cells and enhancement of their killing activity =
by=20
additional incubation with =
IL-12. |