WO2004045630A1 - An application of bacteriophages in transplantation - Google Patents
An application of bacteriophages in transplantation Download PDFInfo
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- WO2004045630A1 WO2004045630A1 PCT/PL2003/000122 PL0300122W WO2004045630A1 WO 2004045630 A1 WO2004045630 A1 WO 2004045630A1 PL 0300122 W PL0300122 W PL 0300122W WO 2004045630 A1 WO2004045630 A1 WO 2004045630A1
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- Prior art keywords
- bacteriophages
- cells
- influence
- transplantation
- bps
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
Definitions
- the object of the invention is a new medical application of bacteriophages.
- the goal of the invention is to provide medications which can be applied in the treatment or prevention of bacterial infection, especially of drug-resistant bacteria, which appear in transplant patients without increasing the risk of graft rejection.
- the medication should optimally also possess immunosuppressive properties.
- the goal of the invention is to provide medications which may be applied in combating graft rejection, especially the process of acute and chronic allograft rejection.
- An object of the invention is the application of bacteriophages in the preparation of medication to be administered to patients who have undergone transplantation. This medication serves advantageously in the treatment and prevention of bacterial infection and/or increasing graft survival and/or combating the process of acute and chronic allograft rejection.
- a further object of the invention is the application of bacteriophages in evoking immunosuppression.
- bacteriophages are employed advantageously in inhibiting the activity of NK (Natural Killer) cells and/or inhibiting the production of immunoglobulin by B lymphocytes.
- Bacteriophages used in accordance with this invention must be free of adverse impurities, such as bacterial endotoxin. Suitable bacterial strains may be obtained, for example, according to the methods described in the Polish patents held by the Institute of Immunology and Experimental Therapy, PAN, in Wroclaw, e.g. P.348740 of 18 July 2001, P 354822 of 30 June 2002, and P.355355 of 5 August 2002, or the international patent application PCT/PL02/000053 of 18 July 2002. It was unexpectedly shown that bacteriophage medications, besides their advantageous antibacterial properties may display immunosuppressive properties.
- bacteriophages may be safely administered to patients to treat drug-resistant bacterial infections arising post transplantation, because such treatment is not connected with a risk of allograft rejection.
- bacteriophages may inhibit humoral response and exert significant immunosuppressive activity. This was evident in both primary and secondary anti-SRBC antibody production. The amount of bacteriophages needed to produce the observed immunosuppressive effect was low (below million PFU/ ouse). This establishes rational grounds for applying bacteriophages as immunosuppressants, which possess the additional advantage of being bactericidal. At the same time it was unexpectedly ascertained that they induce a significant increase in skin- graft survival in mice. Again, the immunosuppressive effect of bacteriophages causing a significant prolongation of allograft survival was evident in both normal and pre-sensitized individuals, which has a clear clinical significance.
- the effect could be produced by administering bacteriophages prior to transplantation and, in some instances, a single administration of phages produced the immunosuppressive effect.
- strikingly low amounts of phages proved to be an efficient means of extending allograft survival (see above).
- no harmful or any side effects of bacteriophage administration were observed even when high amounts of phage were injected intraperitoneally or intravenously (eg 0.5 ml of phage preparation 10x8 PFU/ml).
- Bacteriophages may therefore be employed in, for example, combating the process of acute and chronic allograft rejection.
- Figure 1 presents the influence of preparations containing the bacteriophage T4 on skin allograft survival in mice.
- Figure 2 presents the influence of preparations containing the bacteriophage Pseudomonas F8 on skin allograft survival in mice.
- Figure 3 presents the influence of preparations containing the bacteriophage Pseudomonas F8 on skin allograft survival in mice.
- Figure 4 presents the influence of Pseudomonas bacteriophages on the cytotoxic activity of NK cells.
- Figure 5 presents the influence of Pseudomonas bacteriophages on the proliferative response of T lymphocytes induced by OKT3.
- Figure 6 presents the influence of Pseudomonas bacteriophages on proliferative response of T lymphocytes induced by PHA.
- Figure 7 presents the influence of Pseudomonas bacteriophages on the in vitro synthesis of antibody induced by PWM.
- Example 1 The influence of bacteriophages on skin allograft rejection in mice
- H-2 a system of strong tissue incompatibility
- mice were performed according to the method described by J. J. Mond (2) modified by Z. agodzinski (3).
- Tissue donors were males of the C57B16 strain, and recipients males of the Balb/c strain (aged 10-12 weeks).
- the animals were given general anesthesia using 3.6% chlorine hydrate (0.1 ml/lOg body weight).
- the donor skin was prepared and placed on the previously prepared recipient's flank.
- the dried graft was stabilized with Acutol and a dressing was placed over it.
- the dressing was removed after 7 days and the degree of the graft rejection reaction was determined.
- the skin graft recipient mice were given 0.1 lml intraperitoneal injections of BPs (bacterial lysates of S. aureus, E. Coli and Pseudomonas, and highly purified Pseudomonas bacteriophages). Ultrasonically disrupted bacteria were used as controls, and 0.9% NaCl in additional control groups.
- BPs bacterial lysates of S. aureus, E. Coli and Pseudomonas, and highly purified Pseudomonas bacteriophages.
- BPs react with integrins of the ⁇ 3 family (which play an important role in tumor growth and metastasis) and in this way block tumoral spread. Recent research indicates an important role of these integrins in graft rejection. It is therefore possible that the lengthening of graft survival depends on BP blockage of ⁇ 3 integrins, as these enable the recipient's attacking T lymphocytes influx to the graft and initiation of the process of its rejection.
- BPs Natural Killer
- NK lymphocytes The influence of BPs on the cytotoxic activity of NK lymphocytes was determined by evaluating their ability to kill cancer cells of the K562 line using a method based on the literature (5, 6), in short: mononuclear cells (MNCs) were isolated from heparinized peripheral blood by separation on gradisol L (AquaMedica). K562 leukemia erythrocytes were used as the target cells. The K562 cells were rinsed and suspended in 1 ml PBS.
- % Td (Td / T) x 100%), while cell lysis was evaluated as: % Td (incubated with MNCs) - % Td (incubated w/o MNCs).
- Intracellular cytokine synthesis was evaluated on a method based on literature (7, 8, 9).
- Mononuclear cells were suspended in culture medium (based on Parker's medium) at a concentration of lxl 0 6 cells/ml, and PMA activator (50 ng/ml), ionomycine (l ⁇ g/ml) and brefeldine A ⁇ g/ml) were added. This was cultured for ca. 18 hrs. at 37°C in 5% CO 2 (on 6- flat bottom plates of the company Nunc). The cells were collected from the plates and rinsed in Staining Buffer (SB: PBS with an additional 1% FCS solution and 0.1% sodium azide). The cells were suspended in 500 ⁇ l SB.
- SB Staining Buffer
- lOO ⁇ l of the cell solution in SB was added to each of the five samples. From one culture five samples were designated as CD69, control, IL-2, IFN- ⁇ , and TNF- ⁇ . To each of these samples 15 ⁇ l of CD3-PE (a marker of the T lymphocyte population) was added. They were incubated for 15 min. at room temperature in darkness and the SB was rinsed off. 500 ⁇ l of Cytofix fixing solution (in the PharMinger kit) was slowly added to the cell trays and the cells were incubated for 20 min. at room temperature in the dark. Then the cells were centrifuged, suspended in Perm Wash (in the PharMingen kit) and incubated for 10 min. at room temperature in the dark.
- CD3-PE a marker of the T lymphocyte population
- the influence of BPs on T lymphocyte proliferation induced by the OKT3 monoclonal antibody and mitogen (PHA) was determined by estimating the level of response by a standard method utilizing a thymidine isotope marker (10,11).
- the mononuclear cells were stimulated with a phytohemagglutinin solution (PHA, Sigma) at a concentration of 10 ⁇ g/ml or cultured on plates previously coated with a solution of OKT3 (ORTHO) at a concentration of 1 ⁇ g/ml.
- the cells were cultured for 3 days at 37°C in an atmosphere of 5% CO in culture medium (Parker's medium from Wytw ⁇ rnia Surowic i Szczepionek[Serum and Vaccine Manufacture], Lublin) supplemented by 10% inactivated FCS (Sigma), a 20mM solution of Hepes (Sigma), 2mM-L-glutamin (Sigma), and 5xl0 "5 M 2-mercapto-ethanol (Sigma).
- culture medium Parker's medium from Wytw ⁇ rnia Surowic i Szczepionek[Serum and Vaccine Manufacture], Lublin
- DNA synthesis in the dividing cells was measured by the degree of integration of tritium-marked thymidine (Polatom), which was added 17 hours before the end of culturing.
- Nucleic acid freed from the cells was transferred with the aid of a harvester (Skatron) onto glass-wool filter paper (LKB Wallac).
- ⁇ -ray emission of the tritium bound to the DNA was measured in a scintillation counter (Microbeta plus, Wallac). ⁇
- Mononuclear cells were activated by pokeweed mitogen (10 ⁇ g/ml) (PWM, Sigma) in RPMI culture medium (Wytw ⁇ rnia Surowic i Szczepionek, Lublin) supplemented by 10% pooled human serum derived from at least 25 donors, 20mM of Hepes, and 2mM L- glutamin (Sigma) as well as 2-mercapto-ethanol (5xl0 "5 M, Sigma). The cells were incubated at a concentration of 10 "5 cells/0.2 ml/well at 37°C in 5% CO 2 for 7 days.
- Sheep erythrocytes (SRBC: sheep red blood cells) were incubated for 60 min. in a warm bath at 37°C in a solution of 0.9% physiological saline supplemented with 0.5 mg/ml of A protein of Staphylococcus aureus (SpA, Sigma) and 55 ⁇ g/ml chromium chloride
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
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Abstract
A new application of bacteriophages in the preparation of pharmaceuticals for administration to patients undergoing transplantation and/or to invoke immunosuppression was revealed.
Description
APPLICATION OF BACTERIOPHAGES IN TRANSPLANTATION
The object of the invention is a new medical application of bacteriophages.
Despite significant progress in transplantology, allograft rejection remains a serious clinical problem. Chronic rejection is especially dangerous. Although incidents of acute rejection can usually be brought under control, we have at our disposal practically no effective treatment of chronic rejection, the progress of which inevitably leads to loss of the graft (1).
Another serious problem in transplantology are infections, including those of bacteria resistant to all available antibiotics. The weight of this problem was recently emphasized at a separate session organized during the international Congress of The Transplantation Society (USA, August 2002) devoted to precisely these bacterial infections.
Within the context of the growing problem of infections by antibiotic-resistant bacteria, we have been observing a increasingly large interest in recent years in the potential use of bacteriophages (BPs, viruses which destroy bacteria) in such situations. One could also, then, consider the possibility of bacteriophage therapy of infections of the graft recipient, but in this situation such treatment must not have a negative effect on the fate of the graft (e.g. lead to an escalation of its rejection).
The goal of the invention is to provide medications which can be applied in the treatment or prevention of bacterial infection, especially of drug-resistant bacteria, which appear in transplant patients without increasing the risk of graft rejection. In accordance with the goal of the invention, the medication should optimally also possess immunosuppressive properties. In particular, the goal of the invention is to provide
medications which may be applied in combating graft rejection, especially the process of acute and chronic allograft rejection.
This invention unexpectedly proved to be a realization of the goal defined in this manner. An object of the invention is the application of bacteriophages in the preparation of medication to be administered to patients who have undergone transplantation. This medication serves advantageously in the treatment and prevention of bacterial infection and/or increasing graft survival and/or combating the process of acute and chronic allograft rejection. A further object of the invention is the application of bacteriophages in evoking immunosuppression. In accordance with this object of the invention, bacteriophages are employed advantageously in inhibiting the activity of NK (Natural Killer) cells and/or inhibiting the production of immunoglobulin by B lymphocytes.
Bacteriophages used in accordance with this invention must be free of adverse impurities, such as bacterial endotoxin. Suitable bacterial strains may be obtained, for example, according to the methods described in the Polish patents held by the Institute of Immunology and Experimental Therapy, PAN, in Wroclaw, e.g. P.348740 of 18 July 2001, P 354822 of 30 June 2002, and P.355355 of 5 August 2002, or the international patent application PCT/PL02/000053 of 18 July 2002. It was unexpectedly shown that bacteriophage medications, besides their advantageous antibacterial properties may display immunosuppressive properties.
It was unexpectedly shown, as the results presented below prove, that bacteriophages may be successfully applied in clinical transplantology.
First, bacteriophages may be safely administered to patients to treat drug-resistant bacterial infections arising post transplantation, because such treatment is not connected with a risk of allograft rejection.
Secondly, it was determined unexpectedly that bacteriophages may inhibit humoral response and exert significant immunosuppressive activity. This was evident in both
primary and secondary anti-SRBC antibody production. The amount of bacteriophages needed to produce the observed immunosuppressive effect was low (below million PFU/ ouse). This establishes rational grounds for applying bacteriophages as immunosuppressants, which possess the additional advantage of being bactericidal. At the same time it was unexpectedly ascertained that they induce a significant increase in skin- graft survival in mice. Again, the immunosuppressive effect of bacteriophages causing a significant prolongation of allograft survival was evident in both normal and pre-sensitized individuals, which has a clear clinical significance. The effect could be produced by administering bacteriophages prior to transplantation and, in some instances, a single administration of phages produced the immunosuppressive effect. Again, strikingly low amounts of phages proved to be an efficient means of extending allograft survival (see above). Importantly, no harmful or any side effects of bacteriophage administration were observed even when high amounts of phage were injected intraperitoneally or intravenously (eg 0.5 ml of phage preparation 10x8 PFU/ml). Bacteriophages may therefore be employed in, for example, combating the process of acute and chronic allograft rejection. This is of great clinical significance especially in cases of chronic rejection, where, as mentioned above, clinical transplantology does not currently have any effective medications to combat this serious complication at its disposal. In summary, this invention opens new perspectives in the therapeutic application of bacteriophages, among others in clinical transplantology.
This description of the invention is augmented by the following figures: Figure 1 presents the influence of preparations containing the bacteriophage T4 on skin allograft survival in mice. Figure 2 presents the influence of preparations containing the bacteriophage Pseudomonas F8 on skin allograft survival in mice. Figure 3 presents the influence of preparations containing the bacteriophage Pseudomonas F8 on skin allograft survival in mice. Figure 4 presents the influence of Pseudomonas bacteriophages on the cytotoxic activity of NK cells. Figure 5 presents the influence of Pseudomonas bacteriophages on the proliferative response of T lymphocytes induced by OKT3.
Figure 6 presents the influence of Pseudomonas bacteriophages on proliferative response of T lymphocytes induced by PHA. Figure 7 presents the influence of Pseudomonas bacteriophages on the in vitro synthesis of antibody induced by PWM.
To better understand the essence of the invention, it has been illustrated by the examples below. However, it would be a mistake to limit the scope of the invention to these sample realizations.
Example 1. The influence of bacteriophages on skin allograft rejection in mice In order to exclude the possibility of a negative influence of BPs on graft fate, we conducted a study on the influence of BPs on skin allograft survival in mice in a system of strong tissue incompatibility (H-2, Balb/c mice and C57B1). Skin grafts were performed according to the method described by J. J. Mond (2) modified by Z. agodzinski (3).
Tissue donors were males of the C57B16 strain, and recipients males of the Balb/c strain (aged 10-12 weeks).
The animals were given general anesthesia using 3.6% chlorine hydrate (0.1 ml/lOg body weight). The donor skin was prepared and placed on the previously prepared recipient's flank. The dried graft was stabilized with Acutol and a dressing was placed over it. The dressing was removed after 7 days and the degree of the graft rejection reaction was determined. The skin graft recipient mice were given 0.1 lml intraperitoneal injections of BPs (bacterial lysates of S. aureus, E. Coli and Pseudomonas, and highly purified Pseudomonas bacteriophages). Ultrasonically disrupted bacteria were used as controls, and 0.9% NaCl in additional control groups.
Subsequent series of experiments showed that BP administration not only does not shorten graft survival, but causes a significant increase of it. This effect was evident with administration of BPs from the moment of transplantation to the time when rejection appeared (i.e. during the first 7-10 days after transplantation) (Figs. 1, 2), but also in the cases of single administration on the day prior to transplantation and on the day of transplantation (Fig. 3). Later administration of BPs (the day after skin transplantation) no longer brought about this effect.
These results indicate that BPs exert immunonsuppressive activity and influence the lengthening of allogenic graft survival. We suggest the mechanism of BP activity may be similar to the effect we recently described of BP arrest of tumor metastases in mice. (4). It is probable that BPs react with integrins of the β3 family (which play an important role in tumor growth and metastasis) and in this way block tumoral spread. Recent research indicates an important role of these integrins in graft rejection. It is therefore possible that the lengthening of graft survival depends on BP blockage of β3 integrins, as these enable the recipient's attacking T lymphocytes influx to the graft and initiation of the process of its rejection. The influence of BPs on human immunity in vitro
As the in vivo study above showed that BPs do not stimulate an uncontrolled immune response in mice, it was therefore of interest to check the influence of BPs on parameters of the human immune system. Example 2. Investigation of NK (Natural Killer) cell activity The influence of BPs on the cytotoxic activity of NK lymphocytes was determined by evaluating their ability to kill cancer cells of the K562 line using a method based on the literature (5, 6), in short: mononuclear cells (MNCs) were isolated from heparinized peripheral blood by separation on gradisol L (AquaMedica). K562 leukemia erythrocytes were used as the target cells. The K562 cells were rinsed and suspended in 1 ml PBS. They were then marked with 1.2μl DIO (3,3 'dioctadecyloxacarbocyxacarbocyanine perchlorate, Sigma) - (fluorochrome, which becomes imbedded in the membranes of K562 cells) and incubated for 20 min. at 37°C in an atmosphere of 5% CO2. The cells were then rinsed twice and suspended in 1ml RPMI with 1% fetal calf serum (FCS), and brought to a concentration of 1.2 x 106 cells/ml. The mononuclear cells were incubated together with the K562 target cells for 4 hrs. at 37°C in 5% CO2 at a ratio of 50:1 and 12:1 (control: MNCs in RPMI 10% FCS and K562 in RPMI 10% FCS). 25μl of a propidine iodide solution (O.lμg/ml PI, Sigma) was added to each sample.
After incubation, acquisition and analysis was carried out using the Cell Quest program (Becton Dickinson). Live K562 cells (T) were identified on the basis of their intensive fluorescence in the green band (FL1), and dead cells (Td) on the basis of their intensive fluorescence in the green and red bands (FL1, FL2). The proportion of dead K562 target cells was evaluated according to the equation: % Td = (Td / T) x 100%), while cell lysis was evaluated as: % Td (incubated with MNCs) - % Td (incubated w/o MNCs).
In the above investigations, inhibition of NK activity by the Pseudomonas BP was ascertained (fig. 4). Example 3. Investigation of the influence of BPs on the intracellular synthesis of cytokines by lymphocytes and monocytes in vitro
Intracellular cytokine synthesis was evaluated on a method based on literature (7, 8, 9). Mononuclear cells were suspended in culture medium (based on Parker's medium) at a concentration of lxl 06 cells/ml, and PMA activator (50 ng/ml), ionomycine (lμg/ml) and brefeldine Aμg/ml) were added. This was cultured for ca. 18 hrs. at 37°C in 5% CO2 (on 6- flat bottom plates of the company Nunc).The cells were collected from the plates and rinsed in Staining Buffer (SB: PBS with an additional 1% FCS solution and 0.1% sodium azide). The cells were suspended in 500μl SB. Then lOOμl of the cell solution in SB was added to each of the five samples. From one culture five samples were designated as CD69, control, IL-2, IFN-γ, and TNF-α. To each of these samples 15μl of CD3-PE (a marker of the T lymphocyte population) was added. They were incubated for 15 min. at room temperature in darkness and the SB was rinsed off. 500μl of Cytofix fixing solution (in the PharMinger kit) was slowly added to the cell trays and the cells were incubated for 20 min. at room temperature in the dark. Then the cells were centrifuged, suspended in Perm Wash (in the PharMingen kit) and incubated for 10 min. at room temperature in the dark. lOOμl Perm Wash was added to each sample, followed by the respective monoclonal antibody (MoAb) in quantities of 20μl for CD69, 2μl for the control, 2μl for IL-2, 2μl for IFN-γ, and 2μl for TNF-α. The cells were then incubated for 30 min. at room temperature in the dark, then rinsed of the Perm Wash. 500μl of 2% formalin in PBS was added to each
sample, and cell acquisition and analysis was performed using an FACS Calibur (Becton- Dickinson) flow cytometer. No significant increase in cytokine synthesis was shown under the influence of BPs in both types of cells (the stimulation level of 0-3% was no different from background stimulation). Example 4. Investigation of the influence of BPs on cellular and humoral immune response
The influence of BPs on T lymphocyte proliferation induced by the OKT3 monoclonal antibody and mitogen (PHA) was determined by estimating the level of response by a standard method utilizing a thymidine isotope marker (10,11). The mononuclear cells were stimulated with a phytohemagglutinin solution (PHA, Sigma) at a concentration of 10 μg/ml or cultured on plates previously coated with a solution of OKT3 (ORTHO) at a concentration of 1 μg/ml. The cells were cultured for 3 days at 37°C in an atmosphere of 5% CO in culture medium (Parker's medium from Wytwόrnia Surowic i Szczepionek[Serum and Vaccine Manufacture], Lublin) supplemented by 10% inactivated FCS (Sigma), a 20mM solution of Hepes (Sigma), 2mM-L-glutamin (Sigma), and 5xl0"5 M 2-mercapto-ethanol (Sigma).
DNA synthesis in the dividing cells was measured by the degree of integration of tritium-marked thymidine (Polatom), which was added 17 hours before the end of culturing. Nucleic acid freed from the cells was transferred with the aid of a harvester (Skatron) onto glass-wool filter paper (LKB Wallac). β-ray emission of the tritium bound to the DNA was measured in a scintillation counter (Microbeta plus, Wallac). \
No significant influence of BPs on immune response was observed (Figs. 5, 6). Research into the influence of BPs on humoral response showed that they may inhibit B-lymphocyte immunoglobulin production in vitro (fig. 7) as evaluated in the standard manner, i.e. by the so-called reverse plaque forming test of Jerne (12).
Mononuclear cells were activated by pokeweed mitogen (10 μg/ml) (PWM, Sigma) in RPMI culture medium (Wytwόrnia Surowic i Szczepionek, Lublin) supplemented by 10% pooled human serum derived from at least 25 donors, 20mM of Hepes, and 2mM L-
glutamin (Sigma) as well as 2-mercapto-ethanol (5xl0"5M, Sigma). The cells were incubated at a concentration of 10"5 cells/0.2 ml/well at 37°C in 5% CO2 for 7 days.
Sheep erythrocytes (SRBC: sheep red blood cells) were incubated for 60 min. in a warm bath at 37°C in a solution of 0.9% physiological saline supplemented with 0.5 mg/ml of A protein of Staphylococcus aureus (SpA, Sigma) and 55 μg/ml chromium chloride
(Polskie Odczynniki Chemiczne [Polish Chemical Reagents]) in 0.9% physiological saline.
The erythrocytes, coated with A protein, were rinsed twice with PBS and a 30% suspension was prepared in RPMI 1640 culture medium (Wytwόrnia Surowic i
Szczepionek, Lublin). A 1% agar solution (Serva) in Hank's solution (Wytwόrnia Surowic i Szczepionek, Lublin) was poured onto Petri dishes of 40 mm diameter, and then activated mononuclear cells were suspended in a 0.08% low-fusible agar solution (Seaplaque, Serva) together with the 30% suspension of A protein-coated sheep erythrocytes and a 1 :20 solution of rabbit anti-human-immunoglobulin antibody (Dako). The cultures were incubated for 60 min. at 37°C in 5% CO , and then 1:10 diluted guinea pig complement (Biomed) was added which had previously been absorbed by sheep erythrocytes in a proportion of 1:1 incubated for 90 min. at 37°C in 5% CO . Then the supernatant was removed from the Petri dishes and the areas caused by hemolysis ("bald spots") were counted. The number of bald spots per 105 mononuclear cells was calculated.
References: 1. Gourishaiikar S., Halloran P. Late deterioration of organ transplants: problems in injury and homeostasis. Curr. Opin. Immunol. 2002,14,576.
2. Mond J. J., Brunswick M.: Assays for B cell function. In vitro antibody production. In:
Cologan J. E., Kruisbcek A. M., Margulies D. H., Shevach E. M., Strober W., eds.
Current protocols in immunology. New York: Wiley, 1992, Ch. 7,1. 3. Lagodziήski A., Gόrski A., Wasik M.: Immunosuppressive action of low-dose heparin.
Effect on skin allograft survival. Transplantation, 1990, 50, 4: 714.
4. Gόrski A., Dabrowska K., Wietrzyk J., et al.: "Bacteriophage inhibits metastasis in mouse transplantable model", report presented at the American Association for Cancer
Research conference "Proteases, Extracellular Matrix and Cancer". 9-13 Oct. 2002, The Westin Resort, Hilton Head Island, South Carolina, USA.
5. Johann Sabine, Gϋnther Blumel, Martin Lipp, Reinhold Forster, A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity, J. Immunol. Meth., 1995, 185: 209.
6. Kimberly L. Kane, Faith A. Ashton, Jolm L. Schmitz, James D. Folds, Determination of Natural Killer Cell Function by Flow Cytometry, Clinical and Diagnostic Laboratory Immunology, 1996, Vol.3: 295.
7. Gagro Alenka, Rabatic Sabina, Bendelja Kresimir, Jelacic Jasenka, Gotovac Katja et al., Detection of intracellular cytokines by flow cytometry, Central-European Journal of Immunology 1999, 24: 125.
8. Carrock Sewell W. A., North Margaret, Webster A., David B., Farrant John, Determination of intracellular cytokines by flow-cytometry following whole-blood culture, Journal of Immunological Methods 1997, 209: 67. 9. O'Mahony L., Holland J., Jackson J., Feighery C, Hennessy T. P. J., Mealy K., Quantitative intracellular cytokine measurement: age-related changes in proinflammatory cytokine production, Clin. Exp. Immunol. 1998, 113: 213.
10. A. Gόrski, N. Castronovo, B. Stςpieή-Sopniewska, et al.: Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions, Cell Adhesion and Communication, 1994, 2: 225.
11. M.W^sik, B. Stζpieή-Sopniewska, Z. Lagodziήski, A. Gόrski: "Effects of FK-506 and cyclosporine on human T and B lymphoprolifarative responses", Immunopharmacology, 20 (1990) 57.
12. A. Gόrski, M. W^sik, B. Stςpieή-Sopniewska, T. Glapiήski: "Heparin and platelet interactions influence alloantihen-induced proliferative responses of human T lymphocytes and immunoglobulin synthesis in vitro", Immunology Letters, 28 (1991): 161.
Claims
1. The application of bacteriophages to prepare medications for administration to patients undergoing transplantation.
2. The application according to provision 1 is characterized in that the medication serves in the treatment and prevention of bacterial infections.
3. The application according to provision 1 is characterized in that the medication serves to lengthen graft survival.
4. The application according to provision 3 is characterized in that the medication serves to counteract the process of acute and chronic allograft rejection.
5. The application of bacteriophages to evoke immunosuppression.
6. The application according to provision 5 is characterized in that bacteriophages are used to inhibit the activity of NK cells.
7. The application according to provision 5 is characterized in that bacteriophages are used to inhibit the production of immunoglobulin by B lymphocytes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003286983A AU2003286983A1 (en) | 2002-11-15 | 2003-11-15 | An application of bacteriophages in transplantation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PLP-356853 | 2002-11-15 | ||
| PL356853A PL196148B1 (en) | 2002-11-15 | 2002-11-15 | Application of bacteriphages |
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| WO2004045630A1 true WO2004045630A1 (en) | 2004-06-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/PL2003/000122 WO2004045630A1 (en) | 2002-11-15 | 2003-11-15 | An application of bacteriophages in transplantation |
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| Country | Link |
|---|---|
| AU (1) | AU2003286983A1 (en) |
| PL (1) | PL196148B1 (en) |
| WO (1) | WO2004045630A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110563815A (en) * | 2019-08-06 | 2019-12-13 | 天津科技大学 | Pseudomonas aeruginosa bacteriophage K8 putative protein GP075, and mutant strain, mutant protein and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0870505A1 (en) * | 1994-08-15 | 1998-10-14 | Uop | Use of d-allose as an immunosuppressive agent |
| DE19828596A1 (en) * | 1997-06-26 | 1999-02-11 | A Daniela Dr Nodar | Use of polyvalent bacteriophages |
-
2002
- 2002-11-15 PL PL356853A patent/PL196148B1/en unknown
-
2003
- 2003-11-15 WO PCT/PL2003/000122 patent/WO2004045630A1/en not_active Application Discontinuation
- 2003-11-15 AU AU2003286983A patent/AU2003286983A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0870505A1 (en) * | 1994-08-15 | 1998-10-14 | Uop | Use of d-allose as an immunosuppressive agent |
| DE19828596A1 (en) * | 1997-06-26 | 1999-02-11 | A Daniela Dr Nodar | Use of polyvalent bacteriophages |
Non-Patent Citations (4)
| Title |
|---|
| DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 11 October 2002 (2002-10-11), WEBER-DABROWSKA BEATA ET AL: "Effect of phage therapy on the turnover and function of peripheral neutrophils.", XP002275640, Database accession no. PREV200300005865 * |
| FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 34, no. 2, 11 October 2002 (2002-10-11), pages 135 - 138, ISSN: 0928-8244 (ISSN print) * |
| GORSKI A ET AL: "New insights into the possible role of bacteriophages in transplantation.", TRANSPLANTATION PROCEEDINGS, vol. 35, no. 6, September 2003 (2003-09-01), pages 2372 - 2373, XP002275639, ISSN: 0041-1345 * |
| SULAKVELIDZE A ET AL: "BACTERIOPHAGE THERAPY", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC, US, vol. 45, no. 3, March 2001 (2001-03-01), pages 649 - 659, XP001016193, ISSN: 0066-4804 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110563815A (en) * | 2019-08-06 | 2019-12-13 | 天津科技大学 | Pseudomonas aeruginosa bacteriophage K8 putative protein GP075, and mutant strain, mutant protein and application thereof |
| CN110563815B (en) * | 2019-08-06 | 2022-04-08 | 天津科技大学 | Pseudomonas aeruginosa bacteriophage K8 putative protein GP075, and mutant strain, mutant protein and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003286983A1 (en) | 2004-06-15 |
| PL196148B1 (en) | 2007-12-31 |
| PL356853A1 (en) | 2004-05-17 |
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