The Tests For the Drug RESAN

The determination of the cytokines confirms the switching-on of the cellular immune answer after the introduction of vaccine RESAN

Under the action of RESAN the cells of the immune system get activated. Fig. 1 illustrates a classical scheme showing how the T-helpers function out.

Fig. 1. Development and interaction of the T- helpers of 1 and 2 types, production of cytokines

The prevailing production of the T- helpers of type 1 (Th1) shows an activation of the cellular immune answer, where as the prevailing production of T- helpers of 2 type (Th2) shows an activation of the humoral immune answer. On a basis of the contents of the cytokines in vivo (in blood serum) one can make a conclusion whether the cellular or the humoral immune answer is prevailing. After the administration of the vaccine RESAN, an authentic rise in the level of TNF-α (a factor of necrosis of tumour-alpha) and IFN-γ (gamma-interferon) take place, showing the activation of the cellular immune answer.

Determination of these cytokines in blood serum is carried out before and in 7-14 days after the administration of the drug.

In normal, the blood serum contents 0-50 pg/mls (picogram per ml) of TNF-α and IFN-γ.

A simple test proving the immune responses of the drug based on the general blood test
The general blood test is taken prior to the injection of the drug RESAN , and through 1,7,14 days after the injection.

On the next day of the injection of RESAN, the growth of the leucocytes (mainly phagocytes) is observed. An increase of segmented and rod nuclear cells and a decrease of the lymphocytes are observed in the leucocyte formula.

After 7 days of injection, the decrease in the number of leucocytes in compared with the prior analysis is observed. A decrease in the percentage of segmentated cells, rod nuclear cells and increase of the lymphocytes are observed in the leucocyte formula.

After 14 days of the injection, the number of leucocytes becomes same as was prior the injection, where as in the leucocyte formula an increase of lymphocytes is observed.

The rosette-forming test, which shows the antitumoral responses of the drug
The blood tests are done as in the case of marking the immune status.The tests are carried out before and after the 10-12 days of the injection.

It is necessary to grow a tumor cell culture from the tumoral tissue obtained during biopsy (in a sterile condition). The tumor cell suspension containing 6·10⁷ cells/1 ml is prepared. From the blood plasma a leucocyte suspension of 2·10⁶ per ml is prepared. 0.1 ml each of leucocyte suspension and tumor cell suspension are mixed and stirred in a "U"- shaped plate. The mixer is incubated for 30 minutes in 370 C and centrifuged for 3 minutes in 1000 rev/min. The undescended liquid part is removed and 0.1 ml of 0.05% of glutaraldihyde is added, carefully stirred.

0.1 ml of the solution is replaced in an object- plate, dried out, fixed up with alcohol and is colored with the solution of Romanovski-Hinza. The number of rosettes is counted up through a microscope. The increase in the rosette formation of lymphocytes with tumor cells states the antitumoral immune responses.

The tumor cell lysis test (in a sterile condition)
The culture of tumoral cells from the tumoral tissues obtained during the biopsy is grown up. The test is carried out before and 10-12 days after the injection. A suspension of 2.5·10³ cells per ml is prepared from the tumor cell culture. 20 ml of this suspension is replaced in a (falcon No. 3034, USA). Another suspension containing T-lymphocytes of 5·10⁵ cells per ml is prepared from the patient's blood plasma after 24 hrs. The liquid part above the precipitated layer from the microtitre- plate is removed out. Around 200 tumor cells are left adhesed on the surface of the plate. About 20 mcl (microlitre) suspension of T- lymphocytes is added to them, incubated in 1 atmosphere with 10% CO2 for 48 hrs. After washing 2 times, it is fixed up by alcohol and colored with solution- Hinza. The number of live tumor cells left are counted up. The result is calculated using the formula- tumor cell lysis (TCL) in %, it shows the % lysis of tumoral cells in 48 hrs.

TCL = ( NTCC - NTCE)/ NTCC·100%.

Where NTCC- the number of tumor cells in control, NTCE- number of tumor cells in experiment. If TCL = 30% and above, it shows that the antitumor immune system has been switched on.

A conlusion is made after comparing the results obtained before and after the injection.

The monitoring of the immunotherapy based on the level of the tumor-associated antigens (tumor markers)
For the verification of effectiveness of the administered immunotherapy, a monitoring of the tumor-associated antigens (tumor markers) may be used.

- The maintenance of the dynamic growth of the tumor-associated antigens in the patient's blood shows the ineffectiveness of the immunotherapy.
- The slow dinamic growth of the tumor-associated antigens shows a weak immune responses.
- A halt in the growth or a decrease in the level of the tumor-associated antigens shows an effective antitumor immune responses.

Here are some accepted blood tumor markers for common cancers:

If you have any questions concerning the cancer diagnosis, monitorings you may contact us.