Restoration of immune system function is accelerated in immunocompromised mice by the B-cell-tropic isoxazole R-11

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Restoration of immune system function is accelerated in immunocompromised mice by the B-cell-tropic isoxazole R-11

Micha³ Zimecki¹, Jolanta Artym¹, Maja Kociêba¹, Bo¿ena Obmiñska-Mrukowicz², Marcin M¹czyñski³, Stanis³aw Ryng³

1Laboratory of Immunobiology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, PL 53-114 Wroc³aw, Poland

2Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroc³aw University of Environmental and Life Sciences, Norwida 25/27, PL 50-375 Wroc³aw, Poland

3Department of Organic Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Grodzka 9, PL 51-351 Wroc³aw, Poland

Correspondence: Micha³ Zimecki, e-mail: zimecki@iitd.pan.wroc.pl

Abstract:

Background: Restoration of impaired immune response in immunocompromised patients is a crucial problem. In this study we evaluated the efficacy of isoxazole R-11 in reconstitution of the immune response in immunosuppressed mice.

Methods: Mice were given a sublethal dose (250 mg/kg b.w.) of cyclophosphamide (CP). The cellular immune response to ovalbu- min (OVA) and the humoral immune response to sheep erythrocytes (SRBC) were generated. R-11 was administered at repetitive, intraperitoneal doses (20 µg/mouse) until determination of the immune responses: 7 and 15 doses on alternate days for cellular and humoral immune response, respectively. For phenotypic studies R-11 was given per os, at a single dose of 20 µg/mouse. The ability of R-11 to affect interleukin- 6 (IL-6) production was determined in the whole human blood cell culture.

Results: R-11 increased the content of CD19+ cells in the spleens and lymph nodes with a concomitant decrease of CD3+ and CD4+

cells. The compound significantly accelerated restoration of both cellular and humoral immune responses, elevated the numbers of circulating leukocytes and splenocytes and normalized the blood cell picture. Supplementary experiments showed that R-11 was not toxic with regard to human peripheral blood mononuclear cells (PBMC) and that it upregulated IL-6 production in blood cell culture stimulated with lipopolysaccharide (LPS).

Conclusions: We demonstrated that R-11 is likely a B-cell tropic agent which can restore both cellular and humoral immune re- sponses in immunocompromised mice and may have a potential to be applied in therapy of immunocompromised patients.

Key words:

isoxazoles, mice, immune response, cyclophosphamide

Introduction

Advances in medicine and pharmacology have led to a turning point in therapy of neoplastic diseases and in

management of organ transplantation. However, therapeutic protocols are often associated with application of antimetabolites and immunosuppressors, leading to transient but significant impairment of the

Pharmacological Reports 2012, 64, 403411 ISSN 1734-1140

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immune functions. In the first period following chemotherapy, the main phagocytic cell compartment (neutrophils) involved in the innate immune response is destroyed [4]. Although this cell type undergoes a rapid, spontaneous renewal due to a short life span [1], the T- and B-cell pool, responsible for development of an adaptive immune response, is restored much later [7, 9, 18, 20]. At that time of recovery, a patient requires particular protection against a potential infection and the hospitalization period extends significantly. In order to accelerate renewal of the myelocytic lineage, a recombinant granulocyte colony stimulating factor (Filgrastim®) has been applied for the last two decades [16]. However, that preparation is expensive, not stable, requires intravenous administration, and its activity in stimulation of lymphopoiesis is limited and results from cooperation with other cytokines [23].

In the experimental model of CP-immunocompromised mice, several strategies were explored to protect or renovate the impaired immune system, including application of thymic factors [15], cytokines [14], syn- thetic compounds [11], fungal [22], bacterial [24] and milk [2, 3] fractions. Until now, however, none of the mentioned agents has been applied in therapy. There is, therefore, a constant need for introduction into therapy of new compounds that are non-toxic, stable, bio-accessible by the oral route and able to promote differentiation of T and B cells.

In the course of our search for immunoregulatory heterocyclic structures, we described an isoxazole derivative, RM-11 (Fig. 1), which exhibited potent stimulatory actions in the humoral and cellular immune response [17]. More recently, we found that RM-11 was also able to accelerate restoration of the immune response in CP-immunocompromised mice and to counteract a suppressive action of methotrexate in the immune response in vitro [25]. Beside its ability

the immunotropic activity of another isoxazole derivative, R-11 (shown in Fig. 1), in a model of mice subjected to administration of a sublethal dose of cyclophosphamide (CP) (250 mg/kg) since preliminary phenotypic experiments suggested its B-cell-tropic nature.

Materials and Methods

Animals

CBA and BALB/c female mice, 8–12 weeks old, were used for the studies. The mice were kept under con- ventional conditions. The animals were fed a com- mercial, granulated food and water ad libitum. The Local Ethics Committee approved the studies.

Reagents

Ovalbumin (OVA), lipopolysaccharide (LPS) from Escherichia coliserotype O111:B4, dimethyl sulfoxide (DMSO) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) were from Sigma, complete (cFa) and incomplete (iFa) Freund’s adjuvants and fetal calf serum (FCS) were from Difco (USA), bovine serum albumin (BSA) from Sigma, RPMI- 1640 medium was purchased from Cibi/Life Tech- nologies (UK), cyclosporin A (CsA) in ampoules (Sandimmun) from Novartis (Germany) and CP from ASTA Medica (Germany). Sheep red blood cells (SRBC) were from Wroc³aw University of Life and Environmental Sciences, Wroc³aw, Poland. SRBC were stored in Alsever’s solution at 4^oC until use.

R-11 was synthesized and delivered by Prof. Ryng from the Department of Organic Chemistry, Wroc³aw Medical University, Poland. For the experiments, R-11 was initially dissolved in DMSO (5 mg in 0.3 ml of DMSO) followed by 20 min incubation in an ultra- sonic bath. Then, the compound was further diluted with 0.9% NaCl (for in vivo experiments) or in the culture medium for in vitro experiments. The mice in in vivoexperiments were treated with appropriate dilutions of DMSO.

Fig. 1. The structures of R-11 and RM-11 compounds

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Humoral immune response to sheep erythrocytes

CBA mice were immunized intraperitoneally (ip) with 0.2 ml of 5% SRBC suspension in 0.9% NaCl (see Fig. 2). CP, at a single dose of 250 mg/kg body weight, was given ip 35 days before immunization.

R-11 (20 µg/mouse) was administered ip in 15 doses, on alternate days, before immunization, beginning from the next day after CP administration. Four days after immunization, the splenocytes were isolated and the number of antibody-forming cells (AFC) was determined by the local hemolysis assay [13]. The results are shown as the mean AFC values of 5 mice/group calculated per 10⁶ viable splenocytes

± SE.

Delayed type hypersensitivity to OVA

CBA mice were sensitized with 5 µg of OVA emulsi- fied in Freund’s complete adjuvant, subcutaneously (sc), into the tail base. After 4 days the mice were challenged with 50 µg of OVA in Freund’s incomplete adjuvant (sc) into both hind foot pads (Fig. 2). On the next day the delayed type hypersensitivity reaction was measured as the foot pad edema using a spring caliper with 0.05 mm accuracy. The background, non- specific inflammatory response was induced by administration of an eliciting dose of OVA to naive mice and was subtracted from the response of sensitized mice. CP was administered ip at a dose of 250 mg/kg body weight, 14 days before sensitization of mice with OVA. One day following CP, mice were given 7

intraperitoneal doses of R-11 (20 µg/mouse) on alternate days. The results are presented as the mean values of 5 mice/group (10 determinations) and ex- pressed in DTH units (one unit = 0.1 mm) [12].

Determination of splenocyte and leukocyte numbers

CBA mice were given CP ip (250 mg/kg body weight). One day following CP, mice were given 7 intraperitoneal doses of R-11 (20 µg/mouse) on alternate days (Fig. 2). The splenocyte and leukocyte numbers were analyzed on day 21 after CP administration.

The spleens were homogenized by pressing the organs through plastic screens into 0.83% NH₄Cl solution to lyse erythrocytes. Then, the cells were washed 2 × with PBS and filtered through cotton wool col- umns to dispose of cell debris. Finally, the cells were resuspended in PBS containing 0.1% trypan blue to visualize viable cells. For determination of leukocyte number the circulating blood was diluted (20 ×) in Türk’s solution. Both splenocytes and leukocytes were counted in a Bürker hemocytometer.

Preparation of blood smears

CBA mice were given CP ip (250 mg/kg body weight), followed by ip R-11 administration (20 µg/ mouse/dose) on alternate days (7 doses, Fig. 2). Changes in the composition of the major blood cell types were analyzed on day 21 after CP administration. Blood smears were prepared on microscopic glasses and stained with Giemsa and May-Grünwald reagents. The prepara-

Restoration of immune system function by isoxazole

Micha³ Zimecki et al.

0 15 19 20

CP

21 33 37

R-11

OVA in cFa (immunization)

OVA in incFa (elicitation)

DTH

Leukocyte and splenocyte numbers, blood picture

SRBC ip

AFC number R-11

DTHAFC

Fig. 2. Scheme of tests

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per group). The results are presented as the mean values (in percentage) for each cell type (immature lymphocytes, lymphocytes, neutrophil precursors – band forms, neutrophils and eosinophils).

Induction and determination of cytokines in the whole blood cell cultures

Venous blood from a single donor was diluted 10 × with RPMI-1640 medium and distributed in 1 ml aliquots in 24-well culture plates (Nunc). The cultures were stimulated by addition of 1 µg/ml of LPS from E. coli. The compounds (R-11 and CsA) were added to the cultures at concentrations of 10 and 100 µg/ml.

Appropriate dilutions of DMSO served as controls.

After overnight incubation, the supernatants were harvested and frozen at –20^oC until cytokine determination.

Determination of TNF-a activity

The assay was performed according to Espevik et al.

[6]. TNF-a concentration was measured by the WEHI 164.13 bioassay. Briefly, WEHI 164.13 cells were seeded at a density of 2 × 10⁴cells/well in quadruplicate in 96-well plates. Increasing dilutions of the assayed supernatant were mixed with the target cells in the presence of actinomycin D (1 µg/ml). After 20 h of incubation, MTT [8] was added into the wells and the cultures were incubated for additional 4 h. Next, a lysing buffer was added and the optical density (OD) at 550 nm (Dynatech 5000) was measured. The results are presented as TNF-a activity units per milli- liter of the studied supernatants. One unit of TNF-a activity was defined as an inverse of supernatant dilu- tion where 50% of cell death took place.

Determination of interleukin-6 (IL-6) activity

The assay was performed according to Van Snick et al. [21]. Briefly, IL-6-dependent murine B cell hybri- doma (7TD1 line) was incubated (2 × 10³cells/well) in 96-well plates with serial dilutions of assayed supernatants in quadruplicate. Three days later the rate of cell proliferation was measured by MTT colorimetric method [8]. The results are presented as IL-6 activity units per milliliter of the studied supernatants.

One unit of IL-6 activity was defined as an inverse of

Determination of cell toxicity in the PBMC cultures

Venous blood from a single donor was taken into hepar- inized syringes, diluted 2 × with PBS and overlaid on a Ficoll-Uropoline gradient (density 1.077 g/ml). After centrifugation for 20 min at 800 × g, the cells from the interphase were harvested and washed 2 × with PBS by centrifugation. Eventually, the cells were resuspended in a culture medium consisting of RPMI-1640 medium supplemented with FCS (final concentration 10%), L-glutamine, sodium pyruvate, 2-mercapto- ethanol and antibiotics (penicillin and streptomycin) at a density of 2 × 10⁶/ml. The cells were then distributed into 96-well plates (Nunc) in 100 µl aliquots.

R-11 was added to the cultures at 6.25–100 µg/ml concentration range. Appropriate DMSO dilutions served as control cultures. After 2-day incubation in a cell culture incubator, cell viability was measured by MTT colorimetric assay [8].

Determination of cell phenotypes

BALB/c mice were given a single, oral 20 µg dose of R-11. After 24 h the spleens and mesenteric lymph nodes were isolated and pressed through a nylon screen into cold PBS. The cells were subsequently separated on a discontinuous Ficoll-Uropoline gradient (density 1.071 g/ml), washed and resuspended in PBS with addition of 1% BSA. The following antibodies were used for cell staining: rat monoclonal anti-mouse CD4:FITC/CD8:RPE dual color reagent, clone YTS 191.1/KT15 (Serotec, Kidlington, UK) and rat monoclonal anti-mouse CD19:FITC/CD3:RPE dual color reagent, clone 6D5/KT3 (Serotec, Kidlington, UK). The antibodies were used at dilutions suggested by the manufacturer. The stained cell suspensions were subjected to phenotypic analysis using a flow cytometer (FACS Calibur, Becton Dickinson Biosci- ences, USA) according to Cell Quest version 3.1.f software. The content of respective cell phenotypes in the spleens and mesenteric lymph nodes are given in percentages (mean values from 7 mice) ± SE. Mice treated with appropriate DMSO dilutions served as controls.

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Statistics

The results of one representative experiment from three independent experiments were presented. The results are presented as the mean values ± standard er- ror (SE). Levene’s test was used to determine the ho- mogeneity of variance between groups. Analysis of variance (ANOVA with post-hoc Tukey’s test) was applied to estimate the significance of the difference between groups. Significance was determined at p <

0.05. The statistical analysis was performed using STATISTICA for Windows statistical package.

Results

Phenotypic changes of splenocytes and lymph node cells induced by oral administration of R-11

The preliminary investigations aimed at establishing effect of R-11 on B- and T-cell phenotypes were con- ducted on BALB/c mice. The results presented in Fig- ure 3 show alterations of the cell phenotype in the spleen (Fig. 3A) and lymph node populations (Fig.

3B) of mice pretreated with a single oral dose of R-11 (20 µg/mouse) 24 h before phenotype determination.

In the spleen the percentage of CD3+ and CD4+ cells decreased from 24.3 to 16.6% and from 21.5 to 15.7%, respectively. In contrast, the content of CD19+ cells rose significantly both in the spleen (from 64.0 to 74.9%) and in the lymph nodes (47.6 to 57.0%). The changes in the content of CD3+ and CD4+ cells in the lymph nodes were not statistically significant.

Lack of toxicity of R-11 against human PBMC

Figure 4 presents the effects of R-11 on viability of PBMC in 2-day culture, at the concentration range of the compound from 6.25 to 100 µg/ml. Cell cultures containing DMSO at appropriate dilutions served as controls. The results (Fig. 4) showed that DMSO alone, at the tested dilutions, did not affect cell survival. R-11 also had no influence on cell survival.

0 10 20 30 40 50 60 70 80 90

CD19+ CD3+ CD4+

Phenotype of cells

%ofcells

DMSO R-11

B

0 10 20 30 40 50 60 70

CD19+ CD3+ CD4+

Phenotype of cells

%ofcells

DMSO R-11

A

Fig. 3. Alterations in cell phenotype in lymphoid organs of mice treated with a single oral dose of R-11. BALB/c mice were given 20 µg of R-11, po. After 24 h the spleens (A) and mesenteric lymph nodes (B) were isolated and the phenothype of T and B lymphocytes was determined in fluorocytometer. The content of respective cell phenotypes in the spleens and mesenteric lymph nodes is given in percentage (mean values from 5 mice/group) ± SE. Statistics (DMSO vs.R-11): (A) CD19+: NS (p = 0.0198); CD3+: p = 0.0117; CD4+: NS (p = 0.2876); (B) CD19+: p = 0.0150; CD3+: NS (p = 0.8456); CD4+:

NS (p = 0.9978)

0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500

(–) 6.25 12.5 25 50 100 6.25 12.5 25 50 100

DMSO R-11

OD(550/630nm)

Fig. 4. R-11 is devoid of cell toxicity against human PBMC. R-11 was added to the human PBMC cultures at 6.25–100 µg/ml concentration range. Appropriate DMSO dilutions served as control cultures. After a 2-day incubation, the cell viability was measured by MTT colorimetric assay. The results are presented as the mean optical density values from quadruplicate wells ± SE. (–) – no additions. Statistics (DMSO vs.R-11): 6.25 µg/ml: NS (p = 0.9999); 12.5 µg/ml: NS (p = 1.0000);

25 µg/ml: NS (p = 0.7561); 50 µg/ml: NS (p = 0.9805); 100 µg/ml: NS (p = 0.9999)

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Regulatory effects of R-11 on LPS-induced production of IL-6 and TNF-a in human whole blood cultures

The effects of R-11 on cytokine production by LPS- stimulated human whole blood cultures are shown in Figure 5. The compound was used at concentrations of 10 and 100 µg/ml. It appeared that (Fig. 5A) R-11 stimulated IL-6 production, particularly at the dose of 100 µg/ml. The action of CsA was deeply inhibitory at that concentration. On the other hand (Fig. 5B), the production of TNF-a was slightly inhibited.

Stimulatory effects of R-11 on splenocyte and blood leukocyte numbers in CP-treated mice

Figure 6 illustrates effects of R-11 administration on splenocyte numbers in normal and CP-treated mice.

As shown, the splenocyte numbers strongly declined (from about 4 × 10⁷to 2.5 × 10⁷cells in CP-treated mice). R-11 alone did not change the cell numbers in normal mice but elevated cell numbers in CP-treated mice. R-11 had also a similar, regulatory effect on the circulating leukocyte numbers (Fig. 7).

The changes in blood cell composition in mice treated with CP and R-11 are presented in Table 1.

Normal mice typically contained about 20% mature neutrophils in the blood and administration of CP caused (after 14 days) significant changes in the cell composition. The percentage of neutrophils increased about 3-fold. This phenomenon was also accompa-

0 500 1000 1500

(-) 10 100 10 100 10 100

DMSO CsA R-11

IL- 6

(units/m

B

0 50 100 150 200 250

(-) 10 100 10 100 10 100

DMSO CsA R-11

TNF-(units/ml)

Fig. 5. Effects of R-11 on LPS-induced IL-6 (A) and TNF-a (B) pro- duction. R-11 was added to the LPS-stimulated human whole blood cell cultures at concentrations of 10 and 100 µg/ml. After an overnight incubation, the activities of IL-6 and TNF-a were measured in the su- pernatants using bioassays; (–) – no additions. CsA and appropriate dilutions of DMSO served as controls. The results are presented in units/ml

0 5 10 15 20 25 30 35 40 45 50

Control R-11 CP CP/R-11

Cells/spleen(x106)

Fig. 6. The effect of R-11 on splenocyte numbers. CBA mice were given CP ip (250 mg/kg body weight), followed by ip R-11 administra- tion (20 µg/mouse/dose) on alternate days (7 doses). The splenocyte numbers were analyzed on day 21 after CP administration. The data are presented as the mean values from 5 mice/group (cell number/

spleen) ± SE. Control – mice treated with appropriate dilutions of DMSO. Statistics: control vs. CP p = 0.0015; control vs. R-11 NS (p = 0.9866); control vs. CP/R-11 NS (p = 0.2771); CP vs. CP/R-11 NS (p = 0.0634)

0 1000 2000 3000 4000 5000 6000

Cells/mm

Fig. 7. The effect of R-11 on circulating leukocyte numbers. CBA mice were treated with CP and R-11 as described in Figure 6. The leukocyte numbers were analyzed on day 21 after CP administration.

The data are presented as the mean values from 5 mice/group (cell number/mm³) ± SE. Control – mice treated with appropriate dilutions of DMSO. Statistics: control vs. CP p = 0.0001; control vs. R-11 NS (p = 0.9223); control vs. CP/R-11 NS (p = 0.0535); CP vs. CP/R-11 p = 0.0027

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nied by appearance of band forms, eosinophils and immature lymphocyte forms. The treatment of normal mice with R-11 resulted in some decrease in mature neutrophil content with the concomitant appearance of immature lymphocytes and a small fraction of eosinophils. The administration of R-11 in CP-treated mice led to a significant diminution of the neutrophil content. On the other hand, the proportion of eosinophils and immature lymphocytes slightly increased.

Restoring effects of R-11 on the magnitude of cellular and humoral immune responses in CP- treated mice

The effects of R-11 treatment in CP-immunocompromised mice were studied on days 14 and 35 following CP treatment (cellular and humoral immune responses, respectively). Figure 8 shows that the deeply suppressed cellular immune response to OVA

0 2 4 6 8 10 12 14 16 18

DTHunits

Fig. 8. Restoration of suppressed cellular immune response by R-11.

CBA mice were given CP (250 mg/kg b.w.) ip and treated with 7 doses of R-11 (20 µg ip on alternate days). Mice were sensitized with OVA after 14 days after CP administration. Four days later the eliciting dose of OVA was administered and after 24 h the DTH reaction was measured as foot pad swelling. The data are presented as the mean values from 5 mice/group (DTH units) ± SE. Control – mice treated with appropriate dilutions of DMSO. Statistics: control vs. CP p = 0.0001; control vs. R-11 NS (p = 0.9658); control vs.

CP/R-11 p = 0.0316; CP vs. CP/R-11 p = 0.0001

Tab. 1. Alteration in blood cell picture after administration of CP and R-11. CBA mice were treated with CP and R-11 as described in Figure 6.

The composition of major blood cell types was determined on day 21 after CP administration. Control – mice treated with appropriate dilutions of DMSO. The content of respective cell types in the blood is given in percentage (mean values from 5 mice) ± SE. Statistics: Bands (B): control vs.CP NS (p = 0.2843); control vs. R-11 NS (p = 0.9935); control vs. CP/R-11 NS (p = 0.3054); CP vs. CP/R-11 NS (p = 0.9994); Segments (S):

control vs. CP p = 0.0001; control vs. R-11 NS (p = 0.5487); control vs. CP/R-11 p = 0.0003; CP vs. CP/R-11 p = 0.0001; Eosinophils (Eo): con- trol vs. CP NS (p = 0.4791); control vs. R-11 NS (p = 0.8843); control vs. CP/R-11 p = 0.0132; CP vs. CP/R-11 NS (p = 0.2616); Lymphocytes (L): control vs. CP p = 0.0001; control vs. R-11 NS (p = 0.9203); control vs. CP/R-11 p = 0.0002; CP vs. CP/R-11 p = 0.0002; Immature lympho- cyte forms (L immat.): control vs. CP NS (p = 0.5111); control vs. R-11 p = 0.0021; control vs. CP/R-11 p = 0.0005; CP vs. CP/R-11 p = 0.0010;

Monocytes (Mono)

Experimental group Cell types in the blood

B S Eo L L immat. Mono

Control 0.33 ± 0.21 19.00 ± 1.15 0.50 ± 0.22 80.00 ± 1.24 0.17 ± 0.17 0.00 ± 0.00 R-11 0.17 ± 0.17 14.33 ± 1.31 0.83 ± 0.31 82.50 ± 1.61 2.17 ± 0.40 0.00 ± 0.00

CP 1.50 ± 0.67 59.17 ± 4.30 1.17 ± 0.31 37.17 ± 4.83 0.83 ± 0.31 0.00 ± 0.00

CP/R-11 1.43 ± 0.48 36.29 ± 1.67 2.00 ± 0.38 57.86 ± 1.94 2.43 ± 0.37 0.00 ± 0.00

0 500 1000 1500 2000 2500

AFC/106cells

Fig. 9. Partial restoration of the humoral immune response by R-11.

CBA mice were given a single ip dose of CP (250 mg/kg b.w.) and treated with 15 doses of R-11 (20 µg ip on alternate days). On day 35 after CP administration mice were immunized with ip injection of SRBC suspension in PBS. After 4 days the spleens were isolated and determination of AFC was performed. The results were presented as the mean AFC number per 10⁶viable splenocytes from 5 mice/group ± SE. Control – mice treated with appropriate dilutions of DMSO.

Statistics: control vs. CP p = 0.0001; control vs. R-11 NS (p = 0.1012);

control vs. CP/R-11 p = 0.0123; CP vs. CP/R-11 p = 0.0008

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though not to the same degree.

Discussion

In this investigation we demonstrated that a new isoxazole derivative, R-11, is a nontoxic compound, promoting B-cell differentiation by up-regulation of CD19, a pan B-cell marker, on splenocytes and lymph node cells, and able to accelerate restoration of the cellular and humoral immune response in mice following sublethal administration of CP. We suggest that R-11 accelerates the ability of CP-treated mice to develop the immune response to SRBC by a selective action on B-cell precursors which acquire CD19 marker of immunocompetent B cells. Supplementary experiments in the human model, revealing the ability of R-11 to increase IL-6 production, a B-cell differentiation cytokine [10], are in agreement with the B- cell-tropic nature of R-11. The immunorestoring activity of R-11 was similar to but more potent than that of RM-11 [25] and lactoferrin [2, 3]. In addition, the action of R-11 on the blood cell composition seemed to be more beneficial than that of RM-11. In the case of RM-11, the rebound in numbers of neutrophils and their precursors, 11 days following CP administration, was further augmented by the compound, with no concomitant appearance of immature forms of lymphocytes. In contrast, R-11 lowered the percentage of neutrophils, this phenomenon being accompanied by the appearance of immature forms of lymphocytes, which suggests that the action of R-11 is shifted to- wards lymphopoiesis.

Although the restoring effect of the B-cell-tropic R-11 on the cellular immune response, mediated by T cells, seems intriguing, B cells also play a role in gen- eration of the cellular response [19]. In conclusion, promotion of B-cell development by R-11 would also be profitable for acceleration of the cellular immune response.

It is also conceivable that besides a potential bene- fit of R-11 in restoration of the immune response in immunocompromised patients, the compound could find application in treatment of B-cell immunodeficiencies [5]. To fully assess the value of R-11, we plan

bone marrow cells.

Acknowledgments:

The authors thank Ms. Krystyna Spiegel for her excellent technical assistance. The study was supported by a statutory grant from the Polish Ministry of Education, No. 4/2009, for the Institute of Immunology, Polish Academy of Sciences, Wroc³aw, Poland and by the State Committee for Scientific Research, Grant No. KBN 3P05F 01224.

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Received: April 26, 2011; in the revised form: November 3, 2011;

accepted: December 5, 2011.