JP4666284B2 - Glucan sensitivity prediction method, glucan sensitivity enhancer and screening method thereof - Google Patents

Description

  The present invention relates to a glucan sensitivity prediction method, a glucan sensitivity enhancer, and a screening method thereof.

Immunostimulatory polysaccharides such as β-glucan such as lentinan are known to be effective as immunostimulants and antineoplastic agents, but it is also known that there are large individual differences in the expression of the effect (non-patent) Reference 1).
On the other hand, it is known that immunostimulatory polysaccharides bind to the surface of mammalian peripheral blood mononuclear cells such as humans and mice, for example, monocytes, granulocytes and lymphocytes (Non-patent Documents 2 and 3). And it has been shown that there are individual differences and mouse strain differences in the binding ability of immunostimulatory polysaccharides to peripheral blood mononuclear cells, and it is highly possible that the expression of effects and binding ability of immunostimulatory polysaccharides are correlated.
The ability of immunostimulatory polysaccharides to bind to peripheral blood mononuclear cells is known to use antibodies of immunostimulatory polysaccharides such as anti-lentinan antibodies, but this method is difficult to use because anti-lentinan antibodies are IgM. Anti-lentinan antibodies are self-produced, and there is a problem that it is difficult to constantly supply those with a constant titer. Furthermore, since the leukocyte fraction is separated from the blood and subjected to the reaction in the presence of serum, there is a problem that the operation is complicated.

In contrast, fluorescence-labeled immunostimulatory polysaccharides are added to peripheral blood mononuclear cells (whole blood) and cultured, and the fluorescence intensity is measured (Non-patent Documents 4 and 5). And it has been shown that the lentinan binding rate of CD14 positive monocytes correlates with lentinan sensitivity (Non-patent Document 6).
However, this method is expensive, and there is a demand for a method for measuring glucan sensitivity more easily at a lower cost.

Cancer Res. 44: 5132 (1984) 50th Annual Mtg. Proc. Jpn. Cancer Assoc. P259 (1991) Int. J. Immunopharmac. 18: 211 (1996) J. Clin. Invest. Vol. 98, No. 1, July 1996, pp50-61 J. Exp. Med. Vol. 196, No. 3, August 5, 2002, pp407-412 Biotherapy19 (suppl. I): 65, October. 2005, W-1-5

An object of the present invention is to provide a method for measuring glucan sensitivity more easily at a lower cost.
Another object of the present invention is to provide a glucan sensitivity enhancer and a screening method thereof.

The present invention measures at least one complement concentration and / or complement value of blood serum or plasma collected from an individual, and compares the measured complement concentration and complement value with a reference value thereof. This is based on the knowledge that the sensitivity to glucan can be predicted with high accuracy, and according to this, the above problem can be effectively solved.
That is, the present invention measures at least one complement concentration and / or complement value of blood serum or plasma collected from an individual, and compares the measured complement concentration and complement value with a reference value thereof. Provides a glucan sensitivity prediction method for predicting sensitivity to glucan.
Further, the present invention measures at least one complement concentration and / or complement value of blood serum or plasma collected from an individual, and compares the measured complement concentration and complement value with a reference value thereof. Provides a glucan-binding ability prediction method for CD14-positive monocytes, which predicts the glucan-binding ability of CD14-positive monocytes.
Furthermore, the present invention provides a glucan sensitivity enhancer comprising a substance that induces a complement activation pathway and a glucan binding ability enhancer of CD14 positive monocytes.
Furthermore, the present invention provides a glucan sensitivity enhancer comprising a complement production enhancing substance and a glucan binding ability enhancer for CD14 positive monocytes.
Furthermore, the present invention measures at least one complement concentration and / or complement value of blood serum or plasma collected from an individual before and after administration of the candidate substance, and complement concentration and / or before and after administration of the candidate substance. A screening method for a glucan sensitivity-enhancing substance including comparing complement values and a screening method for a glucan-binding ability-enhancing substance of CD14-positive monocytes are provided.
Add candidate substance to blood in a test tube, isolate serum or plasma after culture, measure at least one complement concentration and / or complement titer, and supplement the serum and plasma complement concentration and / or complement without adding candidate substance A method for screening a substance for enhancing glucan sensitivity and a method for screening a substance for enhancing glucan-binding ability of CD14-positive monocytes, which comprises comparing with a titer.

The method for predicting glucan sensitivity and the method for predicting glucan binding ability of CD14-positive monocytes of the present invention include measuring at least one complement concentration and / or complement value of blood serum or plasma collected from an individual.
The glucan of the present invention may be either α-glucan or β-glucan, but β-glucan is particularly preferable, and β-1,3-glucan is more preferable. Examples of β-glucan include lentinan, zymozan, pahimaran, schizophyllan, pustulan, scleroglucan, and lichenan. Especially effective against lentinan.
Examples of complements for measuring the concentration include 9 types of complements C1 to C9. If one type of complement concentration is measured, β-glucan sensitivity and β-glucan binding ability of CD14-positive monocytes can be predicted. However, the accuracy can be increased by measuring two or more complement concentrations. Of these complements, it is preferable to measure the concentration of C3 and / or C4.
Complement concentration can be measured using any method known to those of skill in the art. Examples thereof include immunoturbidimetry, single radial immunodiffusion (SRID), laser nephelometry, and latex agglutination turbidimetry. The complement concentration can also be determined by measuring the degradation product of the complement.
Complement value is an index representing the overall activity of C1 to C9 remaining without being activated, and can be measured using any method known to those skilled in the art. For example, when a certain amount of sheep-sensitized red blood cells (EA) obtained by reacting sheep erythrocytes with an antibody (hemolysin) is reacted with serum, the degree of hemolysis changes depending on the amount of complement in the serum. Complement titer is determined with the amount of complement required to hemolyze 50% of 5 × 10 ⁸ EA as 1 unit (1 CH50) when reacted at 7.5 ° C. for 60 minutes at 37 ° C. (Mayer MM: Complement and complement fixation. In: Experimental Immunochemistry (ed by Kabat EA, Mayer MM), p133-240, Charles C Thomas Publisher, Springerfield, 1961).

Next, the present invention includes comparing the measured complement concentration and complement value with their reference values. According to our study, complement concentration and / or complement value correlate well with glucan sensitivity or glucan binding rate of CD14 positive monocytes, and complement concentration and / or complement value is high. In an individual, glucan sensitivity is strong, or the glucan binding rate of CD14 positive monocytes is high. Therefore, the glucan sensitivity of an individual or the glucan binding ability of CD14 positive monocytes can be predicted by comparing the measured complement concentration and complement value with the reference value.
The reference value of complement concentration and complement value can be determined from the correlation between β glucan sensitivity or glucan binding rate of CD14 positive monocytes and complement concentration or complement value. A reference value defined as an inspection item can be used. For example, for C3, the reference value is 50 mg / ml and for C4 it is 11 mg / dl. The complement value is 25 CH50 / ml.
When at least one of the measured complement concentration and complement value is equal to or higher than the reference value, it can be predicted that there is sensitivity to glucan or glucan binding ability of CD14-positive monocytes. Moreover, when at least one of the measured complement concentration and complement value is less than the reference value, it can be predicted that the sensitivity to glucan or the glucan binding ability of CD14-positive monocytes is weak.

In addition, by increasing the complement concentration and / or complement value, glucan sensitivity becomes stronger, or the glucan binding rate of CD14-positive monocytes increases, so that a substance that induces the complement activation pathway and complement production The potentiator enhances glucan sensitivity and the glucan binding ability of CD14 positive monocytes.
Examples of substances that induce the complement activation pathway include immunoglobulins such as IgA, IgD, IgE, IgM, IgG1, IgG2, and IgG3, C polysaccharide-CRP conjugate, polymer DNA, bacterial lipopolysaccharide, dextran, and dextran. Sulfuric acid, polyanion-polycation conjugate, zymosan (membranous membrane of yeast / gram-negative bacteria), mannan, bacterial wall peptidoglycan, polymeric substances such as inulin, proteolytic enzymes such as trypsin and plasmin, trypanosoma, dysentery amoeba, cystosomula And microorganisms such as viruses, cells such as virus-infected cells, tumor cells, uric acid crystals, myocardial mitochondria, retrovirus, cobra venom, dialysis membrane, and C3 nephritic factor (anti-C3b / Bb autoantibodies).
Examples of the complement production enhancing substance include LPS, vitamins A, C, D, E, IFN-γ, immune complexes, and the like.
The substance that induces the complement activation pathway or the substance that enhances complement production is administered in an amount effective to induce the complement activation pathway or an amount effective to enhance complement. The effective amount for inducing the complement activation pathway per day varies from substance to substance, and is appropriately selected by those skilled in the art.

The agent for enhancing glucan sensitivity and the glucan-binding ability of CD14-positive monocytes containing a substance that induces the complement activation pathway of the present invention includes the activity of these substances in addition to the substance that induces the complement activation pathway. Additives such as excipients may be added as long as they do not affect the above.
The glucan-sensitive and CD14-positive monocyte glucan-binding ability enhancer of the present invention may be in an appropriate dosage form, and is prepared, for example, as a powder, fine granule, granule, tablet, capsule or liquid.
As additives to be added to powders, fine granules, granules, tablets, capsules, etc., excipients (for example, lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, light anhydrous silicic acid, Trehalose, etc.), binders (eg starch paste, gelatin solution, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, ethanol, etc.), disintegrants (eg starch, gelatin powder, carboxymethylcellulose, carboxymethylcellulose calcium salt, etc.) , Lubricants (eg, magnesium stearate, talc, etc.), coating agents (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, acetylcellulose, sucrose, titanium oxide, etc.) Colorants, flavoring agents and the addition in accordance with the. Additives added to the internal solution include preservatives (for example, benzoic acid, paraoxybenzoic acid ester, sodium dehydroacetate, etc.), suspending agents or emulsifiers (for example, gum arabic, tragacanth, carboxymethylcellulose sodium salt) , Methylcellulose, egg yolk, surfactant, etc.), sweet and sour agents (for example, trehalose, citric acid, etc.), and colorants, stabilizers, etc. are added as necessary. Although it is mainly purified water, ethanol, glycerin, propylene glycol or the like can also be used.

The agent for enhancing glucan-binding ability of glucan-sensitive and CD14-positive monocytes according to the present invention is a pharmaceutically and pharmaceutically acceptable additive as it is depending on the dosage form, as it is a substance that induces the complement activation pathway And can be prepared by a conventional method by mixing and granulating, or emulsifying or suspending by dissolving in a suitable solvent and further mixing with a suitable base.
The agent for enhancing glucan binding ability of glucan sensitivity and CD14 positive monocytes of the present invention can be administered orally or parenterally. The content of the substance that induces the complement activation pathway is adjusted so that the above-mentioned daily dose can be achieved by taking once or several times a day. Preferable administration methods include oral, enteral (transgastric using a tube, transduodenum, gastric fistula (PEG), enema is preferred), and intravenous administration.

In addition, glucan sensitivity-enhancing substances other than the above and substances that enhance the glucan binding ability of CD14-positive monocytes (ie, substances that induce the complement activation pathway or complement production-enhancing substances) are collected from individuals before and after administration of candidate substances. The serum can be screened by measuring at least one complement concentration and / or complement value of serum or plasma, and comparing the complement concentration and / or complement value before and after administration of the candidate substance. Alternatively, at least one complement concentration and / or complement value of serum or plasma separated after adding the candidate substance to blood and culturing in vitro is measured, and the complement concentration of serum or plasma not containing the candidate substance and / or Or it can screen by comparing with a complement value. If the complement concentration and / or complement value after administration (addition) of the candidate substance is higher than that before administration (no addition) of the candidate substance, the candidate substance is a glucan sensitivity-enhancing substance or a CD14 positive single substance. It functions as a substance that enhances glucan binding ability of spheres.
In culturing the blood to which the candidate substance is added in a test tube, any commonly used medium such as DMEM or RPMI 1640 can be used. Further, the culture may be performed with whole blood without using a medium. Cultivation is performed by diluting blood 0 to 100 times with the above medium. Culture conditions are performed at 4 to 37 ° C. for 15 minutes to 24 hours.

(Measuring method of lentinan binding ability of CD14 positive monocytes)
Heparin was added as an anticoagulant to blood collected from 5 healthy people. 100 μl of blood was dispensed into a polystyrene tube, and Fluorescein-labeled lentinan was added to 100 μg / ml. After reacting at 37 ° C. for 45 minutes, a PE-labeled anti-CD14 antibody was added and reacted for another 30 minutes. Add 4 ml of hemolytic agent (0.826% ammonium chloride + 0.1% potassium bicarbonate + 0.0037% EDTA / 4Na / distilled water pH = 7.0) to hemolyze at room temperature and wash buffer (0.1% BSA + 0.1% sodium azide) Washed once with 1 ml of / PBS (−), suspended in a washing buffer, passed through a mesh, and then subjected to 2-color analysis (Fluorescein / PE) using FACS Calibur.
Data analysis was performed by fractionating CD14-positive monocytes with SSC / FL2 (PE), creating a FL1 (FITC) histogram of CD14-positive monocytes, and measuring the percentage of cells present in channel 2763 or higher.

(Inhibition rate of lentinan binding ability of antibody to complement receptor)
Heparin was added as an anticoagulant to blood collected from 5 healthy people. 200 μl of blood was dispensed into a polystyrene tube, and anti-CD11b antibody, anti-CD35 antibody, or control antibody was added. After reaction at 37 ° C. for 30 minutes, Fluorescein-labeled lentinan was added to 100 μg / ml. After reacting at 37 ° C. for 45 minutes, a PE-labeled anti-CD14 antibody was added and reacted for another 30 minutes. Add 4 ml of hemolytic agent (0.826% ammonium chloride + 0.1% potassium bicarbonate + 0.0037% EDTA / 4Na / distilled water pH = 7.0) to hemolyze at room temperature and wash buffer (0.1% BSA + 0.1% sodium azide) Washed once with 1 ml of / PBS (−), suspended in a washing buffer, passed through a mesh, and then subjected to 2-color analysis (Fluorescein / PE) using FACS Calibur.
Data analysis was performed by fractionating CD14-positive monocytes with SSC / FL2 (PE), creating a FL1 (FITC) histogram of CD14-positive monocytes, and measuring the percentage of cells present in channel 2763 or higher. The percentage of cells when the control antibody was added was compared with the percentage of cells when the antibody was added to the complement receptor, and the inhibition rate was calculated.
Inhibition rate (%) = (1−ratio of cells upon addition of antibody to complement receptor / ratio of cells at the time of addition of control antibody) × 100

(Complement receptor expression rate of CD14 positive monocytes)
Heparin was added as an anticoagulant to blood collected from 3 healthy people. Blood 200μl dispensed into polystyrene tubes, addition of FITC-labeled anti-CD14 antibody and PE-labeled anti-CD 11 b antibodies or PE-labeled anti-CD35 antibody, and reacted at 37 ° C. 30 min. Thereafter, 10 ml of a hemolyzing agent was added, hemolysis was performed at room temperature, washed once with 3 ml of the washing buffer, suspended in the washing buffer, passed through the mesh, and then subjected to 2-color (Fluorescein / PE) analysis by FACS Calibur. .
Data analysis was performed by fractionating monocytes with SSC / FSC, expanding to FL1 / FL2, and determining the proportion of FL2-positive cells in FL1-positive cells.

(Effect of plasma components on lentinan binding ability of CD14 positive monocytes)
Heparin was added as an anticoagulant to blood collected from 5 healthy people. 2 ml of blood was centrifuged at 3000 rpm for 10 minutes, and 1 ml of plasma was collected. 1 ml of hemolytic agent (0.826% ammonium chloride + 0.1% potassium bicarbonate + 0.0037% EDTA · 4Na / distilled water pH = 7.0) was added to the blood cells, and dispensed into two polystyrene tubes at 200 μl / tube. Furthermore, 10 ml of a hemolyzing agent was added to hemolyze at room temperature, washed once with a washing buffer solution, and the volume was adjusted to 100 μl. The collected plasma was divided into 0.5 ml portions, and one was inactivated by heating at 56 ° C. for 30 minutes. 100 μl of plasma was added to one tube and deactivated plasma to the other tube. In another polystyrene tube, blood was dispensed at 200 μl / tube. Fluorescein-labeled lentinan was added to these three tubes at 100 μg / ml. After reacting at 37 ° C. for 45 minutes, a PE-labeled anti-CD14 antibody was added and reacted for another 30 minutes. The tube containing the blood was hemolyzed at room temperature by adding 4 ml of a hemolyzing agent. All samples were washed once with 1 ml of wash buffer (0.1% BSA + 0.1% sodium azide / PBS (-)), suspended in wash buffer, passed through mesh, then 2 colors by FACS Calibur. (Fluorescein / PE) analysis was performed.
Data analysis was performed by fractionating CD14-positive monocytes with SSC / FL2 (PE), creating a FL1 (FITC) histogram of CD14-positive monocytes, and measuring the percentage of cells present in channel 2763 or higher.

(Measurement of serum and plasma complement values and concentration)
Half of the blood collected from 3 healthy people was dispensed into two tubes, and heparin was added as an anticoagulant to one of the tubes. Serum or plasma was collected by centrifugation at 3000 rpm for 10 minutes. The collected serum and plasma were stored at −80 ° C., and SRL was requested to measure complement values and complement concentrations (C3, C4).

(Experimental result)
(Lentinan-binding ability of 5 healthy human CD14 positive monocytes)
The ratio of lentinan binding cells in CD14 positive monocytes is shown in FIG. There were individual differences in the lentinan binding ability of 5 people, A and D had high binding ability, and B and C had low binding ability.

(Inhibition rate of lentinan binding ability of antibody to complement receptor)
FIG. 2 shows the inhibition rate of lentinan binding ability of antibodies against complement receptors. Regardless of individual differences in lentinan binding ability, addition of CD11b and CD35 antibodies inhibited lentinan binding to monocytes, indicating that lentinan binds via the monocyte complement receptor.

(Complement receptor expression rate of CD14 positive monocytes)
FIG. 3 shows the complement receptor expression rate in CD14 positive monocytes of three healthy individuals. Regardless of individual differences in lentinan binding ability, both CD11b and CD35 were expressed in most monocytes of CD14 positive monocytes.

(Effect of plasma components on lentinan binding ability of CD14 positive monocytes)
Since the cause of individual differences in lentinan binding ability was not on the monocyte side, we focused on plasma components and examined whether lentinan binding decreased when inactivated plasma was used. Regardless of individual differences in lentinan binding ability, plasma inactivation significantly reduced lentinan binding to monocytes (FIG. 4). FIG. 5 also plots the results of five cases with the vertical axis representing the difference between the binding ability measured with whole blood and the binding ability measured with leukocytes + inactivated plasma and the horizontal axis representing the binding ability measured with whole blood. The results are shown. There is a significant correlation between these values, and individual differences in binding ability are considered to be regulated by plasma components.

(Measurement of serum and plasma complement values and concentration)
6 to 8 show serum and plasma complement values (FIG. 8) and complement concentrations (C3 (FIG. 6) and C4 (FIG. 7)). There was no difference between serum and plasma. In addition, although individual differences were observed for any of the items, the complement value and C3 concentration were A> C ≧ B, C4 was A>B> C, and A was a high value. The lentinan binding ability of monocytes was also the strongest in A, suggesting the possibility that complement value and complement concentration correlate with lentinan binding ability.

It is a graph which shows the lentinan binding ability of CD14 positive monocyte. It is a graph which shows the lentinan binding ability inhibition rate of the antibody with respect to a complement receptor. It is a graph which shows the complement receptor expression rate of a CD14 positive monocyte. It is a graph which shows the influence of the plasma component which gives the lentinan binding ability of CD14 positive monocyte. It is a graph which shows the correlation with the binding ability measured with whole blood, and the binding ability measured with leukocyte + inactivated plasma. It is a graph which shows C3 density | concentration of serum and plasma. It is a graph which shows C4 density | concentration of serum and plasma. It is a graph which shows the complement value of serum and plasma.

Claims (6)

Complement concentration and / or complement value of blood serum or plasma collected from an individual is measured, and an individual whose measured complement concentration and / or higher complement value is higher is the measured complement concentration and / or complement A β-1,3- glucan sensitivity prediction method that predicts a higher sensitivity to β-1,3- glucan compared to a lower titer individual , wherein the measured complement is C3 and / or A β-1,3-glucan sensitivity prediction method, which is C4 . The β-1,3- glucan sensitivity prediction method according to claim 1, wherein the measured complement concentration and / or complement value is a complement concentration of C4. The β-1,3 - glucan sensitivity prediction method according to claim 1 or 2 , wherein β-1,3- glucan is lentinan . Complement concentration and / or complement value of blood serum or plasma collected from an individual is measured, and an individual whose measured complement concentration and / or higher complement value is higher is the measured complement concentration and / or complement value is compared to the lower individual, beta-1,3-glucan binding ability of the CD14-positive monocytes is predicted that a higher, a beta-1,3-glucan binding ability prediction method of CD14 + The β-1,3-glucan binding ability prediction method, wherein the measured complement is C3 and / or C4 . The method for predicting β-1,3- glucan binding ability of CD14-positive monocytes according to claim 4 , wherein the measured complement concentration and / or complement value is a complement concentration of C4. The method for predicting β-1,3- glucan binding ability of CD14-positive monocytes according to claim 4 or 5 , wherein β-1,3- glucan is lentinan .

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