WO1990013027A1

WO1990013027A1 - Process for detecting cells with foreign antigens or a modified surface structure

Info

Publication number: WO1990013027A1
Application number: PCT/AT1990/000029
Authority: WO
Inventors: Ursula Keplinger
Original assignee: Keplinger, Klaus
Priority date: 1989-04-18
Filing date: 1990-04-12
Publication date: 1990-11-01
Also published as: AT392481B; ATA91489A; AU5423390A

Abstract

In order to detect cells with foreign antigens or a modified surface structure in the human or animal organism, the activity and morphological properties of the immunological cells in a blood sample taken from the organism are examined. An activating substance is then added to the blood sample. If the cells to be detected are present, typical changes in the activity and possibly the morphological properties of the immunological cells are found after a reaction period.

Description

Method for the detection of cells with an unruly or altered surface structure »

The invention relates to a method for the detection of cells with foreign antigen or with an altered surface structure in the human or animal organism.

When describing and understanding immune reactions, there are great difficulties, which is due to the fact that the multilayered control system, which represents an immune reaction, can only be described in a highly simplified manner. This linear representation, which is absolutely necessary to provide an overview, hardly does justice to the complicated control loop, which consists of a large number of sub-control loops.

One has to be aware that the course of an immune reaction does not consist of chains of causes and effects, but of a dense network of reactions, in which every event is both cause and effect and influences itself as well as other events.

If an otherwise healthy organism is exposed to a constantly more or less constant amount of antigen over a longer period of time, the antigen is both represented on the surface of cells and freely available in the body as a soluble agent. The primary immune response has already taken place, that is, specific antibodies have already been formed. These bind antigens, so soluble immune complexes are formed. The first fraction of the complement, Clq, attaches to the H2 region of the Fc piece of these complexes. Thereby ice esterase activity gains and binds C2 and C4. As a C3 convertase, C2C4 has the ability to activate C3. Soluble C3a is formed, which serves as a chemotactic factor, along the gradients of which macrophages and granulocytes migrate to the place where it happened. C3b is bound to the immune complex. It has a short The other hydrophobic site with which it can bind to CRI receptors.

CRl receptors occur on several cell types, firstly on erythrocytes: if the immune complex is bound to them, these cells are broken down in the liver and spleen, secondly on the surface of granulocytes: complexes bound here are phagocytosed and degraded lysoso al, and third, on macrophages: like receptors for Fc of immunoglobulins, these receptors on these cells have the task of binding immune complexes from the blood. However, the total binding capacity is never achieved without a functioning complement system.

Macrophages not only phagocytize immune complexes, they also present the antigen on their surface together with MHC II. At the same time they produce ~ interferon (IFN-y ~) and interleukin-1 (IL-1). Through IFN- the cells stimulate themselves in a positive feedback and natural killer cells (NK cells), NK cells lyse cells whose surface appears, and IL-1 leads to a stimula ¬ tion of the T cell population. The T cells begin to release IL-2 and at the same time form IL-2 receptors on their surface. As a result, they stimulate themselves and form clones of mature effector cells: TH cells release even more IL-2 and IFN-f, as well as a number of lymphokines that are able to stimulate B cells. TC cells attack and lyse cells, on the surface of which antigen-antibody complexes appear together with MHC I. TS cells also form effector cells, ^'but different by ver¬. Mechanisms, inter alia by free antigen, which is able to bind to a determinant of its receptors, are still prevented from being released by suppressor factors. The influence of TH cells leads to the formation of B effector cell populations which form antibodies. The antibodies bind to both free and antigen bound to cell surfaces, which in turn results in complement activation. In the cell-bound antigen-antibody complexes, higher complement fractions form the lytic complex C8C9, which results in the lysis of the ar. self-infected cells.

In this way there is a constant increase in the formation of antibodies and the removal of antigens made vulnerable until the immune system takes over the damaging agent and can completely eliminate it from the body. After the end of the immune reaction, new control loops are activated in which both the specific TS cells and the idiotype networks play a role, which lead to the switching off of the immune reaction.

If an AIDS virus encounters a physiologically functioning immune system when an individual is infected, the disease penetration of practically 100% shows that the human organism is unable to successfully combat the damaging agent. This inability to cope with AIDS viruses is due to specific virus properties. After infection with HIV, a physiological primary response first takes place, ie there is primarily an increase in specific IgM, and subsequently an increase in IgG titers in the serum. The mononucleosis-like clinical picture 1 to 2 weeks after virus contact is the clinical expression of this immune reaction. However, this rise in antibodies is not sufficient for two reasons to destroy all viruses "on the first strike": on the one hand, the immunogenicity of HIV is so weak that the primary antibody response is not as massive as it would be to completely remove the antigen would be necessary. On the other hand, the virus itself has a protective mechanism against antibody attacks: the surface glycoproteins (gp 110-120) are so weakly attached to the virus that a cross-linking with antibodies leads to a shedding process, ie immune complexes from antibodies and gp 110-120 detach from the virus. Di ^• ser operation may even lead to a virulence of the virus. It can be assumed that p 41, a transmembrane protein which is stereotactically hindered by gp 110-120, facilitates adhesion to cells. The target cells that have T4 receptors on their surface are infected. These are cell groups of the central nervous system on the one hand, and cells of the white blood series on the other. The leukocytes primarily attack TH cells and macrophages. The first discrete immunological changes occur when antigenic material appears on the surface of these cells and leads to the lysis of the cells. The sharp drop in the TH-TS quotient and the breakdown of the entire immune system are, however, not due to this: on the contrary, it occurs in a very slowly progressing process in which the virulence of HIV is only slightly overweight over the counteracting processes of the immune reaction, for further infestation of cells. In particular, the involvement of stem cells in the bone marrow subsequently plays an essential role in the fatal course of the disease. This phase of the disease is clinically expressed in the latency phase, which lasts up to 10 years and precedes the development of specific symptoms of the disease. As a result of hematopoietic stem cells, but also of dentritic cells and other macrophages in the whole body, the disease enters its acute stage, which begins with the lymphadenopathy syndrome (LAS) and ends with the full picture of AIDS. Essential for the increasingly rapid deterioration of the immune system during this time is a loss of CRI receptors in erythrocytes, granulocytes and macrophages caused by cell infestation and possibly by an inhibitory factor derived from the virus.

This reduced expression of CRI receptors is not only visible in AIDS, even in the LAS stage, but also in the case of an autoimmune disease. In AIDS, the number of receptors is reduced by up to 50% during the ARC stage; in the full picture of AIDS, up to 80% fewer receptors are pronounced on the surface of the cells.

In general, with every poor immune situation, this loss of the receptors has far-reaching consequences for the clinical picture of the disease as well as for its further course. In the case of an autoimmune disease, the freely circulating antigen-antibody complexes cannot be adequately bound and thus eliminated from the body. The soluble immune complexes therefore multiply, even excessively strongly in the case of HIV infection, since they also increase due to the multiplication of the viruses. However, gp 110/120 does not become detached from the viruses themselves, but also shedding processes from the surface of the virus-infected cells. The soluble complexes are therefore deposited in predestined places in the body in the same way as is the case with an autoimmune disease, such as systemic lupus erythematosus.

The symptoms of the disease that are visible in every bad immune situation are vasculopathies, rheumatoid muscle and joint pain, sclerotizing glomerulopathies and myopathies and pericarditis of unclear origin. These pathological processes subsequently lead to further autoimmunological processes which can further worsen the clinical picture, and on the other hand also lead to activation as an alternative pathway of the complement system.

A further consequence of the reduction in the CRI receptors is that the macrophages are less able to bind immune complexes and present antigen, the more virus material floods the organism. In addition to the infection of the TH cells by HIV and the resulting cell death, the massively disturbed communication between macrophages and TH cells is another reason for the poor TH / TS

Quotients: on the one hand the macrophages are no longer able to adequately present antigens, on the other hand there are also strong quantitative changes in the receptors on the TH cells. The T4 receptors are significantly reduced, presumably also the receptors for interleukin-1, since this substance is produced by macrophages to a large extent without causing effects on the TH cells. A fundamental property of the AIDS virus appears to be to lead to a substantial change in the cell surface structure in the case of infected cells, as is also visible in the case of an autoimmune disease. The change leads to a breakdown of the interaction between essentials

Components of the immune system. The cutaneous anergy and the missing de novo syrithesis of antibodies are an expression of this lack of cell communication. For this reason, there is no adequate increase in antibody production against HIV, but even a decrease in antibodies, which in the most extreme case can lead to a conversion of the serum. '-

With every poor immune situation, another major effect of the greatly reduced CR1 receptors affects the complement system. Due to the lack of response of the macrophages and other CRI receptor-carrying cells to the formation of C3, the entire complement cascade runs at idle. This uneconomically high complement consumption is significantly increased by activating the alternative pathway, which takes place in the course of the autoimmunological processes mentioned above.

In the serum of AIDS patients in the ARC stage, all complement fractions and the fractions of the alternative pathway are reduced by 30 to 50%. This results in a reduction in the complement activity of the classic type by 50%, the alternative pathway even by 70 to 80%. The complement cleavage products in the serum of these patients are also greatly increased. Similar reductions in complement are also observed in the systemic lupus erythematosus mentioned, as well as when the complement is exhausted in the case of genetically immunological disorders, for example in the absence of the C3b inactivator and in the rare, genetically fixed Clr / C2 / C4 deficiency. In these cases, too, patients are prone to severe recurrent infections, such as those observed with HIV infection. In the case of these massive disturbances in essential control circuit systems involved in the immune reaction, not only does the function of the macrophages, nulocytes and TH cells, but ultimately. all cells that play a role in the immune system are no longer able to react against the AIDS virus and cells infected by it. The logical consequence of this complete breakdown of all possible reactions of the organism directed against harmful substances is the death of the individual concerned. So far, the following clinical picture can be outlined:

After an incubation period of 1 to 2 weeks, the first signs of infection appear as mononucleosis-like disease pictures which subside after a few days up to a maximum of three weeks. As a rule, seroconversion to HIV antigens occurs 3 to 8 weeks after these first symptoms. This initial infection pattern is followed by a phase that lasts up to 10 years, during which the patient is practically symptom-free. During this time, the patient's immunological situation slowly and continuously deteriorates. After this completely symptom-free phase, there follows a time when swollen lymph nodes are almost the only clinical change. At this point in time, however, the immunological parameters begin to change significantly. After the development of the lymphadenopathy stage (LAS), all patients already within 18 months at least to the next stage. The formation of the AIDS-related complex (ARC) is the next disease stage following LAS. There are already clear clinical symptoms: fever for several months, a weight loss of more than 10%, lymphadenopathy, diarrhea, fatigue and night sweats. Laboratory findings show a reduction in the TH cells, a reduced TH / TS quotient, increased serum globulin values, a reduced blastogenesis and cutaneous anergy.

ARC is followed by the full picture of AIDS. It is accompanied by recurrent opportunistic infections caused by bacteria, viruses and fungi, neurological deficiency symptoms and the development of malignancies. The laboratory parameters continue to deteriorate rapidly during this time, the serum is converted. Finally, the patients become cachectic and die either from one of the opportunistic infections or from multiple malignancies.

Antibody tests are currently being carried out for the detection of HIV infections, which only provide information as to whether the patient has been infected with virus fragments, especially with envelope proteins. However, the evidence is unable to provide information as to whether the patient is infected with the virus genome and is therefore actually ill. For example, an HIV antibody carrier that showed no signs of AIDS was found to have a normal immune response, and the positive HIV body diagnosis was a result of rubella infection. Although direct virus detection is possible, it is so complex and expensive that it cannot be used as a broad diagnostic method.

As experience with AIDS shows, it is by no means the rule that with every positive detection of HIV antibodies AIDS actually breaks out. Occasionally, cases have become known in which, after positive evidence, negative evidence could later be provided if the cause for this was only a one-time infection. This is because it is much more likely that in the case of a single infection, similar to a vaccination, only coat proteins of the virus will be transmitted. It is therefore reasonable to assume that a large number of HIV antibody carriers are not infected and will not become ill, so that the disease cannot be passed on. From a human and epidemiological point of view, it is of great importance to be able to recognize whether an actual virus infection is present in the case of a positive HIV antibody detection.

The object of the invention is therefore to develop a detection method of the type mentioned at the outset which is suitable for a wide range of uses and should therefore be simple and quick to carry out and inexpensive. According to the invention, this is achieved by first determining the activity and the morphological properties of the immunological cells, in particular the monocytes and the lymphocytes, in a blood sample taken from the organism, then adding an activating substance to the blood sample, and after expiry of a Response time for the presence of the cells to be detected, typical changes in the activity and, if appropriate, in the morphological properties of the immunological cells can be determined.

The activities of the macrophages (Ma) and lymphocytes (Ly) formed from the monocytes are defined as the activation levels 0, +, ++ and +++ as follows:

1) monocytes:

Stage population morphology activity

Ma 0 homogeneous nucleus: small, kidney-inactive shape, chromatin homogeneously distributed no nucleoli Plasma: relatively narrow border, no vacuoles visible Cell size: 10 - 15 ju

Ma + heterogeneous nucleus: larger, recruit. approx. 20% of the multiform chromatin cells are active

Cells recruited, 1 - 2 nucleotides of plasma: wider border, partly vacuoles. Cell size: -15 - 17

Ma ++ heterogeneous core: large multiform recruit. approx., 50 ^ -60.% Chromatin cloddy. Cells, the cells active several nucleoli

2) LYMPHOCYTE;

Level population I morphology activity Ly 0 homogeneous nucleus: small, inactive chromatin homogeneously dark, no nucleoli plasma: practically invisible narrow border cell size: 7- 10

Ly + heterogeneous, core: medium-sized recruit. approx. 20% chromatin homogeneous cells recruited brighter loosened active 1-2 nucleoli plasma: narrow but visible border cell size: 10,

Ly heterogeneous nucleus: large recruitment, approx. 50% of the chromatin wheel storage cells cells well heterogeneous ordered active recruitment of several nucleoli Plasma: wide border with vacuoles Cell size: 15 _

Example 1) Macrophages (Ma) and lymphocytes (Ly) with the activation values Ma + and Ly 0 are determined in the blood sample of a clinically healthy patient who is therefore in good immune condition and is exposed to a foreign antigen . After the activation substance (AS) has been added and the reaction time has elapsed, activation values Ma +++ and Ly ++ • are determined.

with AS control (without AS) initial values Ma + Ly 0 after 20 'Ma +++ Ly + Ma + Ly 0 after 40' Ma +++ Ly ++ Ma + Ly 0

Anomalies: none

Example 2)

In the blood sample of a patient with a small cell

Bronchial carcinoma with the macrophages and lymphocytes

Activation values Ma (+) and Ly 0 determined. After the reaction time has elapsed, the activation values Ma ++ and Ly + are obtained, which are therefore lower than in Example 1. There is therefore a moderate immune system.

with AS control (without AS) initial values Ma (+) Ly 0 after 20 'Ma ++ Ly (+) Ma (+) Ly 0 after 40 ^» Ma ++ Ly + Ma (+) Ly 0

Anomalies: none

Example 3

In the blood sample of a patient with atopic dermatitis, the initial determination gave activation values as in Example 2, that is Ma (+) and Ly 0. After the reaction time, the addition of the activation substance leads to values as in Example 2, that is also Ma ++ and Ly +. The immune situation is again to be rated as medium, and the autoimmune disease is easily classifiable. with AS control (without AS)

Initial values Ma (+) Ly 0 to 20 • Ma ++ Ly (+) Ma (+) Ly 0 to 40 • Ma ++ Ly + Ma (+) Ly 0

Abnormalities: some plasma cells in both specimens.

HIV-infected cells, especially the number of macrophages reduced in number, have typical properties, for example a changed (approximately "withered") appearance, a vacuolized plasma border which is narrower than it corresponds to the core state, in part white inclusions in the nucleus and plasma, differently colored vukuoles, etc. Here, the difference in immunosuppression is used by checking the activity after a reaction time which is sufficient for a clear indication of activity for other causes of immunosuppression. If there is actually an HIV infection, then there are hardly any signs of activation, but instead an intensification of the infection-typical properties is often recognized, so that a clear and rapid diagnosis is achieved. Furthermore, depending on the extent of the infection, there are changes in the appearance of the lymphocytes.

Example 4)

Macrophages and lymphocytes with the same activation values as in Examples 2 and 3, ie Ma (+) and Ly, were found in the blood sample of a patient with HIV infection before the activation substance was added. However, after the reaction time had elapsed, only activated up to the values Ma + and Ly (+), so that there was a clear difference to the values of Examples 2 and 3. In addition, the macrophages in particular have the typical properties in an intense form. The immune system is bad.

with AS control (without AS)

Initial values Ma (+) Ly 0 to 20 * Ma + Ly 0 Ma (+) Ly 0 to 40 'Ma + Ly (+) Ma (+) Ly 0 Abnormalities: typical properties, as described above.

Example 5)

The activity values Ma + and Ly 0 are determined in the blood sample of a patient with advanced HIV infection, whose initial values correspond approximately to those in Example 4, after the reaction time. Lymphocytes, some of which were initially enlarged, had disappeared and the typical properties were much more pronounced.

with AS control

1. Initial values Ma (+) Ly 0

2. after 20 'Ma + Ly 0 Ma (+) Ly 0

3. after 40 "Ma + Ly 0 Ma (+) Ly 0

Abnormalities: enhanced typical properties

Example 6

In the blood sample of a patient with a severe genetic immune disorder, activation values similar to those in Examples 4 and 5, namely Ma + and Ly (+), are shown after the reaction time. However, a mix-up is not possible due to their normal appearance.

(+) means a half step between the activation values.

The activation values in Examples 2 to 6 show a medium to poor immune situation. In a preferred embodiment of the invention, the diagnosis can be refined by applying activation substance to the organism, and after an organism-specific waiting period, the activity and the morphological properties of the immunological cells, in particular the monocytes, in a second blood sample taken from the organism and the lymphocytes are detected, then the blood sample is mixed with the activating substance and after the lapse of a reaction time for the presence of those to be detected Typical changes in the activity of the cells and, if appropriate, the morphological properties of the immunological cells are ascertained. A reaction of the organism to the activating substance is thus awaited, and the detection method is then repeated.

In the case of a medium immune situation (examples 2 and 3), the activation values Ma + -H- and Ly ++ are found in the blood sample taken after the reaction time if there is a carcinoma or an "ordinary" virus infection and the activation values Ma + + and Ly + found if there is a slight autoimmune disease or an allergy.

If, on the other hand, the immune system is poor (Example 4-6), worse activation levels are achieved in the blood sample after the reaction time. Thus, activation values of Ma ++ (+) and Ly ++ occur in the case of an infection with leukotropic viruses, apart from HIV, activation values of Ma ++ and Ly 0 (+) result in the case of an HIV infection, the additional written typical properties of the immunological cells are given. After weaker activation values, namely Ma + (+) and Ly 0, are achieved in the case of severe autoimmune disease and in the case of a genetic immune disorder.

in a preferred embodiment it is provided that a root extract from Uncaria tomentosa (WILLD) is used as the activating substance. C. is used. As is known from EP-B-219 491, oxindole alkaloids, in particular isotropopodine and pteropodine, which can also be obtained from Uncaria tomentosa, also have an immune-stimulating effect, so that they can also be used for the process according to the invention are. This effect is briefly explained below.

An essential property of the extract from Uncaria tom- tosa or the entire oxindole alkaloids is the ability to bring saturated and unsaturated fatty acids into aqueous solution without having toxic effects on cells. A second property responsible for the therapeutic effect is that this has a positive effect on its polar part. charged uncaria alkaloid isopteropodin is attached to cell membranes. In particular, the ion distribution in the vicinity of virus-occupied cells, of degenerate cells and of cells in permanent excitation, for example of activated cells of the immune system, leads to a high concentration of isopteropodin selectively on the membrane of these cells. There is therefore a normally unattainable high concentration of free fatty acids in the vicinity of the surface of these cells. In this way, the cells can incorporate fatty acids into their lipid matrix much more effectively and with better selection. On the one hand this results in a higher stability of the cell surface, on the other hand the dynamic functionality of the membrane is increased. This means that the exchange between the surface membrane and the membranes inside the cell is greatly accelerated. Since the entire communication of a cell takes place via its membranes, the information exchange between the cell and its surroundings is significantly improved. Due to the accelerated receptor exchange, the cell can quickly perceive even minor changes in its environment. The reaction to changed external conditions due to changes in the cell metabolism and in the membrane structure is also quicker and more effective. The consequences for the organism are far-reaching:

1) Increase in phagocytosis:

The significantly accelerated clearance of injected carbon particles under the influence of extract in the phagocytosis test according to Biozzi shows that macrophages and granulocytes have greatly improved phagocytosis properties. At the same time that the granulocytes of the control animals were able to phagocytize an average of two particles, the granulocytes of the test animals phagocytized an average of four to six particles.

2) Improved communication between the cells of the immune system:

If one incubates blood with the extract in vitro with the addition of an antigenic substance at room temperature, one can Observe an activation of the macrophages by light microscopy after 20 minutes and an activation of the lymphocytes after 40 minutes. Without extract, both macrophages and lymphocytes are not activated. Without additional incubation with antigen, a slight increase in the activity of the macrophages can be seen, but the lymphocytes show no change in their appearance. This means that the influence of extract not only increases the phagocytosis activity of the macrophages, but also improves their ability to present antigen.

3) Growth Inhibition of Virus-Transformed Cells:

It was demonstrated on SV-40-transformed fibroblasts and on HIV-transformed H9 cells that the growth of both cell lines could be significantly reduced by incubation with extract. Since transformed cells release IFN and the growth-inhibiting effect of IFN on transformed cells is known, it can be concluded that the cells, owing to their improved membrane properties, react much more sensitively to the IFN they have formed.

4) Inhibition of cytopathic effects:

Two cell lines were infected with HIV, incubated with extract in different doses and drawn over six days. Compared to control groups not incubated with extract, cytopathic effects were reduced by up to 50%. On the one hand, this can be attributed to the effect of ° C-IFN, on the other hand to an improved membrane stability. Another property of the extract, which is also due to the attachment of isopteropodin to membranes, is also responsible for the reduction of cytopathic effects.

5) The infectivity of viruses is inhibited:

With HSV-1 and -2 it could be shown that these viruses are modified in the presence of extract into non-infectious particles. "This effect can be eliminated by washing out with fetal calf serum. The reaction time of the activation substance in the blood sample is in particular between 20 and 60 minutes. In particular when using an uncariae extract which contains the oxindole alkaloids mentioned, a preferred embodiment provides that in each blood sample taken the change in the activity and the morphological properties of the monocytes after a reaction time of 20 minutes and the change in the activity and the morphological properties of the lymphocytes can be determined after a reaction time of 40 minutes.

Specifically, 1 ml of blood is incubated with 25 L1 HCl-sourced extract of unaria. As a control, 25 μl of 0.9% NaCl solution (0.14 m) is added to 1 ml of blood. After 20 to 40 minutes, blood smears are made, which are stained according to Pappenheim and can be evaluated by light microscopy. In the event that there is a good immune situation, the activity display becomes even more clearly visible when the blood sample is mixed with Buri-Kohletusche (BK). If Buri-Kohletusche (BK) is added, this is advantageously done after 20 minutes

Withdrawal, the first blood smear being made 10 to 20 minutes and the second blood smear 30 to 40 minutes after the addition of coal ink.

Example 7 good immune system:

with AS control

Initial values Ma (+) Ly 0

+ BK + BK after 20 'Ma + Ly 0 Ma (+) Ly 0 after 40 * Ma +++ Ly -Hi- Ma + Ly 0 Abnormalities: increased phagocytosis

The evaluation under the light microscope shows at most a poor reaction in the sense of that immunological activation which occurs particularly with a good but also with a moderate immune situation in the case of a poor immune situation. Example 8, poor immune system due to advanced HIV infection according to Example 6

with AS control starting Ma (+) Ly 0

+ BK + BK after 20 ^» Ma + Ly 0 Ma (+) Ly 0 after 40 'a + Ly 0 Ma (+) Ly 0 Abnormalities: typical properties, no increase in phagocytosis

In comparison to Example 6, no appreciable difference can be seen despite the addition of Buri coal ink.

Laboratory tests with the oxindole alkaloids isopteropodine and pteropodine led to similar results.

Damage to the immune system due to chemotherapy can also be recognized from the activity values, the first Ma (+), Ly 0, 20 'after the addition of the activating substance. Ma + and Ly (+), and appear after 40 'as Ma ++ and Ly +.

The figures of the accompanying drawings show flow diagrams of the detection method.

Show it:

1 shows an overview of the first process stage, FIG. 2 shows an overview of the second process stage in the case of a middle immune position determined in the first stage, and FIG. 3 shows the overview of the second process stage in the case of poor immune position.

Investigations on persons under chemotherapy have shown that signs of activation can be detected in the cellular area of the immune system in the cellular area of the immune system after just 7 days of oral intake of 20 mg extract from Uncaria tomentosa. The studies on an effect of extract from Uncaria tomentosa in the therapy of HIV infections were carried out on patients in the disease stages CDC III (ARC) and CDC IV a (AIDS). The observation period extends for a 28-year-old patient over 22 months, for the other patients over six to twelve months.

The drug is administered orally: the patients were given 20 mg extract from Uncaria tomentosa, 200 mg ascorbic acid and 130 mg lyctose in capsule form per day.

One week after the start of the therapy, the first signs of an improvement in the immune situation become apparent: in the light microscopic assessment, one sees in the blood smear an increase in active blood macrophages with loosened chromatin, nucleoli and a wide plasma margin. The improved phagocytosis performance of these cells can be demonstrated if a blood sample is incubated for 20 minutes with Buri carbon ink. It can be seen that the macrophages of the patients phagocytize better one week after the start of therapy than before the start of therapy.

After another two weeks, light microscopy shows an increase in large lymphocytes with loosened chromatin and a wide border. At this point in time, communication between macrophages and T-lymphocytes, which make up about 67% of the lymphocytes in peripheral blood, is reactivated.

The reactivation of the T lymphocytes is already clearly visible five weeks after the start of therapy: The following picture emerges when cells are subtyped with monoclonal antibodies:

The number of total T lymphocytes increases, the T4 cell population (TI and TH cells) only increasing by 25%, while the number of T8 cell population (TC and TS cells almost doubles there is a sharp drop in the T4 / T8 ratio, but this occurs when therapy occurs, that is, if it does not indicate a drop in the T4 cell len, but is due to the rise in T8 cells, is by no means to be assessed negatively. It is rather the case that the sharp rise in T8 cells is only possible if the T cells are still able to react to the antigen increasingly presented by macrophages and subsequently produce interleukin-2. Since the receptor matrix of the T8-2 cells is not disturbed in AIDS, but the receptors of the T4 cells show strong pathological changes in their number, the T8 cells react much more strongly to stimulation than T4 cells. In addition, freely circulating gp 110/120 bind to the T4 receptors of the TH cells. The appearance of the antigen on the surface of the cells leads to the destruction of these cells by the immune system. Nevertheless, the lower stimulation of the T4 cells is already sufficient, because an increase in the B cells can also be found in the peripheral blood as well as a regular de novo synthesis of antibodies: when the Patients with a new antigen experience an increase in IgM shortly after infection, which in turn leads to an increase in specific IgG. At the same time that changes in T cells are observed, the number of NK cells increases by approximately 30% due to the y-IFN formed by macrophages and TH cells. This increase in both the NK and TC effector cells, which attack and lyse virus-infected cells, leads to a first substantial relief of the complement system.

The immune system changed in this way remains practically constant during the first six months of therapy. The corresponding clinical changes during this period are profound: the size of the lymph nodes normalizes just one month after the start of therapy, diarrhea and anorexia disappear, the body temperature increased before the start of the therapy to normal values, the body weight begins to increase, patient performance is increased. As a result, the oppor- tunistic infections disappear, even severe generalized herpes lesions heal without complications. Patients are hardly exposed to infection when in contact with a new anti Again, a denovo synthesis of antibodies takes place. The complete cutaneous anergy that existed before the start of therapy regresses, the tuberculin skin test is positive again.

After six months of therapy, the correlation of all immunocompetent cells is optimized, which is expressed in the increased lysis activity of NK and TC cells, as well as in the increased formation and constant updating of antibodies which correspond to the antigen drift counteracts the virus, considerably advanced. Due to the improved defense situation, infected stem cells can now be destroyed in the bone marrow before they can form daughter generations of infected effector cells. So only healthy stem cells, that is, those that are not infected with viruses, form mature blood cells. In this way, on the one hand, the viral pool is severely restricted, and on the other hand, cells that generate from healthy progenitor cells again have a physiological receptor pattern. The consequences of this normalization of the receptor numbers are again very extensive: increasing the number of CRI receptors on erythrocytes and granulocytes leads to a massive clearing of circulating immune complexes with simultaneous further substantial relief of the complement system. Both the classic complement activation path and the alternative pathway are greatly reduced. Clinically, the massive phagocytosis of attached antigen-antibody complexes by the granulocytes with the subsequent breakdown of granulocytes and the formation of pus bodies in the form of a severe acute illness is expressed: Six to eight months after the start of therapy, the patients have a fever of up to 39 for two weeks. 6 o C without significant daily fluctuations. The patients suffer from vomiting and excrete large amounts of yolk yellow pus through the nose and through the skin. Despite this severe clinical picture, the body weight does not decrease during this time, but continues to increase. After two weeks, the symptoms of the disease subside and the patients now offer the clinical picture of a healthy person. The increase in the number of CRI receptors on the macrophages leads to a significant improvement in antigen opsonization and consequently to an even better presentation of antigen and an even stronger release of J ^ - interferon. At the same time, the pathologically high interleukin - 1 level in the serum drops to normal values, as does the serum neopterin level. When measuring the activity of the leukocytes, up to 65% large cells, that is to say involved in the immune response, are found in the blood.

Throughout the following year the number of NK cells continues to increase, as does the number of TC cells. This leads to an increasingly effective destruction of virus-infected cells. Since the pathologically high complement consumption is stopped by the normalization of the CRI receptors, the complement fractions in the serum can normalize again. This leads to an increased lysis of infected cells by complement. The number of TH cells is also slowly increasing, but is still below the norm. The improved correspondence between macrophages and TH cells and, subsequently, between TH cells and B cells leads to a further increase in antibodies.

18 months after the start of therapy, the 28-year-old patient offers the following picture:

No opportunistic infections occurred during the entire year, all attempts to grow opportunistic germs from various body fluids were negative. A total of four times during the year after the elimination was attempted to detect HIV viruses from the serum. The tests were carried out at two different institutes using different methods (Abbott and Bender) and were always negative. The differential blood count has normalized, as have serum neopterin, interleukin-1, TNF-c * and y-IFN. IgA and IgM are in the normal range, IgG is still increased with 2810 mg%. In the Western blot, after 18 months of therapy, the patient shows the clearly most intense picture, that is to say unusual, in comparison with 30 other patients reactive antibodies. This unusually strong approach, even to gangs that are normally undetectable (45 and 75), is probably the first indication of a cure. Because this sudden, intensive western blot image is certainly only due to an increased formation of antibodies, since in this case it should have developed continuously over a longer period of time, that is to say the blot images would have lasted for several months continuously have to reinforce, which was not the case. Rather, this sudden, sharp increase in reactive, ie free, antibodies indicates a strong loss or disappearance of antigen.

Four months later, that is, 22 months after the start of therapy, the suspicion of healing cures: the TH cells, which, despite a slight increase in their number 18 months after the start of therapy, were still below 400, rose to 632. This doubling of the TH population within four months can only be based on the cessation of its destruction, this in turn only on the disappearance of antigenic material from the cell surface. This means that, on the one hand, there are no virus-infected TH cells, and, on the other hand, that there are no more free gp 110/120 cells that bind to T4 receptors and can thus shape the cell antigen. Furthermore, one sees an approximation of the IgG to the upper normal values (2090).

22 months after the start of therapy, not only all clinical symptoms of the disease have regressed, but also all - both cellular and humoral - parameters are back in the normal range. Only the strong reaction in the Western blot and ELISA testify - still - of an infection with HIV.

Claims

PATENT CLAIMS

1. A method for the detection of cells with foreign antigen or with an altered surface structure in the human or animal organism, characterized in that first the activity and the morphological properties of the immunological cells, in particular the monocytes and the lymphocytes, in a blood sample taken from the organism , it is found that an activating substance is then added to the blood sample, and that after a reaction time for the presence of the cells to be detected, typical changes in the activity and, if appropriate, in the morphological properties of the immunological cells are found.

2. The method according to claim 1, characterized in that activating substance is applied to the organism, and that after an organism-specific waiting time, the activity and ie the morphological properties of the immunological cells, in particular the monocytes and in particular in a second blood sample taken from the organism of the lymphocytes are determined, then the blood sample is mixed with the activating substance and after a reaction time typical changes in the presence of the cells to be detected are detected in the activity and possibly the morphological properties of the immunological cells.

3. The method according to claim 1 or 2, characterized in that a root extract from Uncaria tomentosa (WILLD.) DC as the activation substance. is used.

4. The method according to claim 1 or 2, characterized in that an oxindole alkaloid, in particular an isopteropodine or a pteropodine is used as the activation substance.

5. The method according to claim 1 and 3, characterized in that 1/40 ml of the root extract is added to each blood sample per milliliter.

6. The method according to claim 1 or 2, characterized in that in each blood sample taken the changes in the activity and the morphological properties of the monocytes after a reaction time of 20 minutes and the changes in the activity and the morphological properties of the lymphocytes after a Response time of 40 minutes can be determined.

7. The method according to claim 2, characterized in that the activating substance is administered orally to the human organism in daily doses, and for the second determination of the changes in activity and morphological properties, the blood sample after a waiting time of 10 to Is removed 20 days after the first application.

8. The method according to claim 1, characterized in that each blood sample is mixed with Buri coal ink.

9. The method according to claim 1 or 2, characterized in that for each determination of the changes in activity and morphological properties, a blood smear is made and colored, which is assessed under an immersion light microscope.