Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: Histological, biochemical and immunological perspectives

International Immunopharmacology 15 (2013) 73–83

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Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: Histological, biochemical and immunological perspectives Atanu Mallick a, 1, Sarbari Ghosh a, 1, Saptak Banerjee a, Sayantani Majumder a, Arnab Das b, Bipasha Mondal a, Subhasis Barik a, Kuntal K. Goswami a, Smarajit Pal b, Subrata Laskar c, Koustav Sarkar d, 2, Anamika Bose e, 2, Rathindranath Baral a,⁎ a

Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), 37, S. P. Mukherjee Road, Kolkata 700026, India Clinical Biochemistry Unit, Chittaranjan National Cancer Institute (CNCI), 37, S. P. Mukherjee Road, Kolkata 700026, India c Department of Chemistry, University of Burdwan, Burdwan, West Bengal, India d Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA e Department of Molecular Medicine, Bose Institute, C.I.T. Scheme, Kolkata, India b

a r t i c l e

i n f o

Article history: Received 20 August 2012 Received in revised form 5 November 2012 Accepted 5 November 2012 Available online 21 November 2012 Keywords: Neem leaf glycoprotein Toxicity Immunomodulation

a b s t r a c t We have evaluated the toxicity profile of a unique immunomodulator, neem leaf glycoprotein (NLGP) on different physiological systems of Swiss mice and Sprague Dawley rats. NLGP injection, even in higher doses than effective concentration caused no behavioral changes in animals and no death. NLGP injection increased the body weights of mice slightly without any change in organ weights. NLGP showed no adverse effect on the hematological system. Moreover, little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Histological assessment of different organs revealed no alterations in the organ microstructure of the NLGP treated mice and rats. Histological normalcy of liver and kidney was further confirmed by the assessment of liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine. NLGP has no apoptotic effect on immune cells but induces proliferation of mononuclear cells collected from mice and rats. Number of CD4+, CD8+ T cells, DX5+ NK cells, CD11b+ macrophages and CD11c+ dendritic cells is upregulated by NLGP without a significant change in CD4+CD25+Foxp3+ regulatory T cells. Type 1 cytokines, like IFNγ also increased in serum with a decrease in type 2 cytokines. Total IgG content, especially IgG2a increased in NLGP treated mice. These type 1 directed changes help to create an anti-tumor immune environment that results in the restriction of carcinoma growth in mice. Accumulated evidence strongly suggests the non-toxic nature of NLGP. Thus, it can be recommended for human use in anti-cancer therapy. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Plant derived medicines always occupied a vital position in the remedy of a variety of ailments [1]. Systemic toxicity and drug-resistance are major problems of chemically synthesized drug therapy that can be reduced by the use of natural medicine [2]. Among several medicinal plants, neem (Azadirachta indica) is well known for its medicinal properties having a wide spectrum of biological activity and recognized as a cynosure of modern medicine [3]. The Sanskrit name of neem tree is “Arishtha” meaning “reliever of sickness” and hence is considered as “Sarbaroganibarani” [4]. The importance of the neem tree has been

⁎ Corresponding author at: Department of Immunoregulation and Immunodiagnostics, Chittaranjan National Cancer Institute (CNCI), 37, S. P. Mookherjee Road, Kolkata-700026, India. Tel.: +91 33 2476 5101x334. E-mail addresses: [email protected], [email protected] (R. Baral). 1 Both authors contributed equally. 2 Present address. 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2012.11.006

recognized by the US National Academy of Sciences. It published a report in 1992 entitled, “Neem — a tree for solving global problem” [5]. The components of the neem tree like bark, seed, leaf, fruit, gum, oil etc. contain compounds offering some impressive therapeutic applications including anti-viral, anti-bacterial, anti-tumor, anti-microbial, anti-pyretic, analgesic, anti-fungal, anti-inflammatory and immune stimulatory functions [6]. There are several reported active compounds in neem plant like nimbin, azadirachtin, nimbidiol, quercetin and nimbidin [7–10]. In spite of all these efforts, knowledge is too preliminary and no systematic effort has been taken to unravel the myth behind neem tree and human health. We have selected neem leaves for the subject of our study in relation to murine tumor growth restriction. An aqueous preparation from neem leaf (NLP), proved to be nontoxic, hematostimulatory and immunostimulatory [11], showed immunoprophylactic function by preventing the growth of murine Ehrlich's carcinoma and B16 melanoma [12]. NLP also acts as an adjuvant by inducing an active antitumor immunity in murine model against B16 melanoma [13] and breast tumor associated antigen [14]. Moreover, NLP mediated

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immune activation protects mice from leucopenia, caused by cancer chemotherapy [15,16]. This neem directed immune response against tumor growth might be associated with the generation of type1 immune response as evidenced by our earlier observations related to the increased release of IFNγ, TNFα [17] and IL-12 [18]. Subsequently, we have identified a glycoprotein as the active principle of NLP and termed, neem leaf glycoprotein (NLGP) [19]. NLGP augments macrophage mediated presentation of CEA for better antitumor action [20], promotes the migration of CXCR3 + effector T cells [19], CCR5 + monocytes [21], and restricts the movement of CCR4 + Treg cells at tumor site [22]. NLGP activates NK and NKT cells [18,23], promotes the generation of tumor antigen specific CTLs to induce cytotoxicity to antigen positive tumor cells [24] and directs the T-bet dependent immune response towards type 1 [25]. NLGP helps in the maturation of dendritic cells (DCs) [26], especially in tumor microenvironment it helps to transform immature DCs to mature DCs, thereby increasing the antigen presenting ability [27]. By converting immature DC2 to mature DC1, NLGP may downregulate the tumor induced propagation of Treg pool [22] by preventing the acquisition of Treg character (CD4+CD25− to CD4+CD25+). NLGP has appeared as a downregulator of Foxp3, a functional regulator of Treg functions and CTLA4, and a gateway of signal transduction for Treg cells [28]. Recently, therapeutic efficacy of NLGP has been demonstrated in murine carcinoma, melanoma and sarcoma [29] models. The obtained promising results prompted us to evaluate the toxicological profile of effective dose of NLGP before stepping forward into preclinical and clinical applications. Accordingly, the investigation is initiated to know the effect of biological dose of NLGP on various physiological systems of Swiss mice and Sprague Dawley rats including hematological, hepatic, excretory and immunological systems. 2. Materials and methods 2.1. Neem leaf glycoprotein Neem leaf preparation was prepared by the method described previously [12,13]. Briefly, matured neem leaves of similar size and color were procured from a standard source, shade dried and pulverized. Neem leaf powder (0.5 mg) was soaked with phosphate-buffered saline (PBS) at pH 7.4 overnight, centrifuged at 1500 g and supernatant was collected. NLGP was purified from this crude preparation by extensive dialysis against PBS (pH 7.4) and ammonium sulfate precipitation, as described [19,20]. Finally, NLGP was concentrated with Centricon Membrane Filtration (Millipore Corporation, Bedford, MA, USA) with a 10 kDa molecular weight cut-off. The purity of NLGP was checked by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and protein concentration of the purified preparation containing NLGP was measured by Lowry's method using Folin's phenol reagent. The purity of NLGP was confirmed by HPLC before use and a standard NLGP concentration (1.5 μg/ml) was used in most experiments. Endotoxin content of the NLGP preparation was determined by the Limulus Amebocyte Lysate test as instructed by the manufacturer (Salesworth India, Bangalore, India). The endotoxin content of all the batches of NLGP was found to be less than 6 pg/ml.

2.3. Mice survival Four groups of mice (n = 8 in each group) and four groups of rats (n = 4 in each group) were treated with four different doses of NLGP (up to 4 times greater than effective dose, 25 μg for mice and 100 μg for rats) weekly for four weeks in total. Survival of mice and rats was recorded by regular observation. 2.4. Behavioral pattern Two groups of mice were injected with PBS and NLGP (n = 4) weekly for four weeks. Their activity like stretching and/or jumping attitude was observed at an interval of three days, taking these mice under a bell jar for a specific time (15 min) of the day in a quiet room, starting from the next day of the first immunization. Stretching and/or jumping of each group of mice were noted carefully. 2.5. Organ weights Mice were weighed at an interval of three days for four weeks starting from the next day of first immunization. After the completion of the immunization, both mice and rats were sacrificed and different organs, e.g., liver, spleen, kidney, lungs, lymph node and brain were collected and weighed. 2.6. Hematological assessment Blood was collected weekly by retro-orbital puncture after anesthesia from both mice and rats and used for hematological assessment. Total RBC, WBC, platelet count and hemoglobin content were assessed in an autoanalyzer (Sysmex, Japan). Neutrophils and lymphocytes were counted from the blood smear after Leishman's staining. 2.7. Hepatic functions Alkaline phosphatase, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) (IFCC method, Kinetic) were estimated by collecting the serum from both mice and rats in Clinical Biochemistry Autoanalyzer (Olympus, AU-400, Japan) using standard kit (Beckman Coulter, Inc. CA, USA). 2.8. Kidney functions The level of serum urea and creatinine for both mice and rats was estimated in Clinical Biochemistry Autoanalyzer (Olympus, AU-400, Japan) using standard kit (Beckman Coulter, Inc. CA, USA). 2.9. Organ histologies Liver, kidney, spleen, brain, lymph node and lungs were collected from both sacrificed mice and rats and fixed in 10% formal saline for histological analysis. Tissues were dehydrated by graded alcohol. Paraffin embedded sections were prepared and stained with hematoxylin– eosin for microscopical observation. 2.10. Immune functions

2.2. Animals Female Swiss mice (age, 6–8 weeks, body weight, 24–27 g) and female Sprague Dawley rats (age, 6–8 weeks; body weight, 280–300 g) were obtained from the Institutional Animal Facilities, CNCI, Kolkata, India. Autoclaved dry pellet diet (Epic Laboratory Animal Feed, Kalyani, India) and water were given ad libitum. Maintenance and treatment of animals were done according to the guidelines established by the Institutional Animal Care and Ethics Committee.

2.10.1. Apoptosis of immune cells Mouse T, B and NK cells were purified using magnetic sorting technology, as described earlier [22,28]. Macrophages were isolated by plastic adherence technique [21]. Dendritic cells were prepared from monocytes using GMCSF + IL-4 and matured with LPS [26]. All of these cells were exposed to active dose of NLGP for 72 h. Apoptotic death of cells was measured by Annexin V–Propidium Iodide staining, followed by flow cytometric analysis of gated population [18].

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2.10.2. Immune phenotypic analysis Flow cytometric analysis for surface phenotypic markers for T cells, suppressor T cells, NK cells, neutrophils and macrophages was performed after labeling with 0.5 μl (for 1 × 10 6 cells) of different anti-mouse fluorescence labeled antibodies (CD3, CD4, CD8, CD19, DX5, CD11b, CD11c, CD25) for 30 min as per manufacturer's recommendation. After labeling, cells were washed in FACSCAN buffer. Similarly, intracellular transcription factor Foxp3 was stained with anti-mouse fluorescence labeled antibodies after addition of cytofix– cytoperm buffer (200 μl) at 4 °C for 30 min as per manufacturer's recommendation. Before labeling cells were washed by adding 500 μl permwash buffer. Cells were fixed in 1% paraformaldehyde in PBS and cytometry was performed using Cell Quest software on a FACScan flow cytometer (Becton Dickinson, Mountain view, CA). Suitable negative isotype controls were used to rule out the background fluorescence. The data was generated by cytofluorometric analyses of 10,000 events. Percentage of each positive population and mean fluorescence intensity (MFI) were determined by using quadrant statistics. 2.10.3. Lymphocyte proliferation Spleen cells from immunized and control mice and rats after the removal of RBC by lysing buffer were taken in RPMI-1640 medium containing 10% FBS, penicillin (50 U/ml) and streptomycin (50 μg/ml). Cells (2× 105/well) were then plated in each well of a 96 well microtitre plate and stimulated with NLGP (1.5 μg/ml) and nonspecific stimulatorConcanavalin A (ConA) (1 μg/well) for 72 h. Cell proliferation was checked by MTT assay as described [13]. In brief, 20-μl aliquot of MTT solution (5 mg/ml) was added in cell culture and incubated for 4 h at 37 °C. The medium was removed by aspiration and the purple colored formazon precipitate was dissolved in DMSO (100 μl) and the absorbance was measured at 550 nm using a microplate reader (BioTek, UK). The absorbance obtained was directly proportional to the number of viable cells. 2.10.4. Cytokine secretion The extracellular release of cytokines (IFNγ, IL-12, IL-4, IL-10) from spleen cell culture was determined by analyzing culture supernatants by ELISA. In brief, 96-well microtiter plates were coated with 100 μl of cell free supernatant, incubated overnight at 4 °C and blocked with 5% bovine serum albumin for 2 h. After washing, primary antibody (anti-mouse IFNγ/IL-12/IL-4/IL-10) was added to each well and incubated overnight. Washing was performed with PBS with Tween 20. Bound cytokines were detected by staining with peroxidase conjugated secondary antibody (anti-mouse IgG). Color was developed with a tetramethylbenzidine (TMB) substrate solution. The reaction was stopped with 1N H2SO4 and absorbance was measured at 450 nm using a microplate reader (BioTek, UK).

Fig. 1. Behavioral changes in NLGP treated Swiss mice. Two groups of mice were treated with NLGP and PBS (n = 6, in each group), weekly for four weeks in total. Their stretching (A) and jumping (B) behaviors were monitored weekly under a bell jar at a specific time of the day in a quiet room.

2.10.5. Immunoglobulin profile The microtiter plates were coated with anti-Ig (5μg/ml) for overnight and blocked with 5% BSA. Diluted serum samples (1:50) were added in wells in triplicate and incubated for 2 h. The plates were washed with PBS containing Tween-20 and goat anti-mouse Ig labeled with peroxidase was added (1:1000 dilution). Color was developed with TMB substrate solution and absorbance was measured at 450 nm using microplate reader (BioTek, UK). For the determination of immunoglobulin isotypes in the test sera by ELISA goat anti-mouse IgG, IgM, IgA antibodies labeled with alkaline phosphatase were used (1:1500 dilutions in 1% BSA-PBS). Immune reaction was detected by using pNPP. Optical density of the color reaction was measured at 405 nm.

2.10.6. Tumor growth restriction Mice (n = 6, in each group) were immunized with NLGP (25 μg/mice/injection) weekly for four times in total. Seven days after the completion of immunization, mice were inoculated with Ehrlich's carcinoma (1 ×106 cells/mice) on right hind leg quarter. Tumor growth was monitored regularly by caliper measurement and tumor volume was calculated by the formula: (width)2 × length / 2.

Table 1 Survival of mice and rats after different doses of NLGP administration. NLGP (μg)

1×

2×

3×

4×

Swiss mice 25a 50 100 200

8/8 8/8 8/8 8/8

8/8 8/8 8/8 8/8

8/8 7/8 8/8 8/8

8/8 7/8 8/8 8/8

Sprague Dawley rats 25a 50 100 200

4/4 4/4 4/4 4/4

4/4 4/4 4/4 4/4

4/4 4/4 4/4 4/4

4/4 4/4 4/4 4/4

Mentioned dose is given for each mice/rat per week. a Effective dose.

Fig. 2. Body weight monitoring of Swiss mice in the course of NLGP treatment. Two groups of mice were treated with NLGP and PBS (n = 6, in each group), weekly for four weeks in total. Body weight of each mouse was measured weekly.

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Table 2 Organ weights in mice and rats after NLGP injection. Liver

Lung

Kidney

Spleen

Ly node

Thymus

Swiss mice (n = 8) PBSa 0.49 ± 0.02 NLGP 0.49 ± 0.03

Brain

1.17 ± 0.04 1.19 ± 0.08

0.21 ± 0.02 0.18 ± 0.02

0.17 ± 0.02 0.21 ± 0.02

0.14 ± 0.03 0.20 ± 0.04

0.04 ± 0.00 0.13 ± 0.03

0.08 ± 0.00 0.12 ± 0.02

Sprague Dawley rats (n = 4) PBSa 1.79 ± 0.03 NLGP 1.78 ± 0.13

9.12 ± 1.13 9.11 ± 1.22

1.34 ± 0.18 1.62 ± 0.33

1.01 ± 0.10 1.00 ± 0.14

0.34 ± 0.02 0.46 ± 0.05

0.08 ± 0.05 0.17 ± 0.01

0.19 ± 0.07 0.26 ± 0.04

Organs are collected after the completion of 4 NLGP injections. a All weights are in gram.

3. Results 3.1. NLGP has no adverse effect on animal survival even in higher doses In addition to effective NLGP doses for mice (25 μg/injection) and rats (100 μg/injection), 3 additional doses were administered to these animals to note their survival status. As presented in Table 1, no death of mice or rats was recorded after the administration of 8 times higher doses. Moreover, no behavioral abnormality was noticed in any animal even after the injection of NLGP in higher doses (data not shown). 3.2. NLGP treated mice exhibited active gesture Two groups of mice were injected with PBS and NLGP for four weeks. Their activity like, stretching and/or jumping was recorded at an interval of three days for four weeks from the first day of

immunization. It was found that NLGP injected mice showed more stretching and/or jumping gesture that was significantly greater than that of PBS-control mice, indicating physically active state of mice (Fig. 1A, B). 3.3. NLGP maintains normal body and organ weights in treated mice and rats The mice/rats were treated with 25 μg/100 μg of NLGP weekly for 4 weeks. Body weights were taken at an interval of 7 days for 4 weeks from the first date of NLGP treatment. No significant changes in body weight of mice (Fig. 2) and rats (data not shown) were noticed after NLGP treatment, except a little increase in the mean body weight of animals in NLGP treated groups. At the termination of experiments, different organs like brains, livers, spleens, lymph nodes, lungs and kidneys were collected from each mice/rats and weighed.

Fig. 3. Histological profile of different organs of Swiss mice after NLGP treatment. Two groups of mice (n = 6) were treated with NLGP and PBS, weekly for four weeks in total. After the completion of the treatment, animals were sacrificed and various organs were collected to be analyzed histopathologically. Organs of mice. A. Spleen; B. Brain; C. Lung; D. Liver; E. Kidney; and F. Lymph node. ×400; inset, ×100.

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Fig. 4. Hematological profile of Swiss mice with NLGP treatment. Two groups of Swiss mice (n = 6) were treated with NLGP and PBS, weekly for four weeks in total. Blood was collected from mice following 7 days of each NLGP injection. Total count of RBC, WBC, platelet and hemoglobin percents was determined.

Weights of different organs revealed no significant alterations between NLGP and PBS treated groups (Table 2). However, in some cases a little increment was noticed in NLGP treated groups.

3.4. NLGP treated mice and rats showed normal histology Histological evaluation of brain, liver, lung, kidney, spleen and lymph nodes from both mice (Fig. 3A) and rats (data not shown) revealed no abnormality. Livers maintained their lobular structure with completely normal parenchymal cells. Rightly distributed malpighian corpuscles in cortex of kidney were seen in NLGP treated mice and rats. Healthy appearance of tissue architecture was demonstrated in lung and brain from NLGP treated animals. Mononuclear cells were properly distributed in spleen and lymph nodes and no difference was noticed in organs from NLGP and PBS treated animals.

3.5. NLGP has no adverse effect on hematological profile Blood was collected from mice (Fig. 4) and rats (data not shown) with NLGP treatment to assess the total count of RBC, WBC, platelet and hemoglobin percentages. Number of RBC was unaltered in the NLGP treated animals in comparison to PBS controls. Total number of WBC increased in mice after NLGP treatment from day 14 onwards. The hemoglobin content increased in NLGP treated mice than PBS treated controls. The platelet count also increased on NLGP treatment from day 7 onwards. However, the difference is not statistically significant. Evaluation of blood smear from mice and rats at an interval of 7 days for 4 weeks with or without NLGP treatment revealed that lymphocyte count increased from day 7 onwards after NLGP treatment. On the other hand, there is a significant decrease in the percentage of neutrophils after NLGP treatment in comparison to the mice treated with PBS (data not shown).

Fig. 5. Assessment of hepatic functions of Swiss mice with NLGP treatment. Two groups of Swiss mice (n = 6) were treated with NLGP and PBS, weekly for four weeks in total. Blood was collected from mice following 7 days of each NLGP injection and serum was collected. Content of SGOT, SGPT and alkaline phosphatase was determined from mice serum.

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3.8. NLGP is not cytotoxic to immune cells As a result of exposure of T, B, NK cells, macrophages and dendritic cells to NLGP, insignificant number of proapoptotic and apoptotic cells was detected by Annexin V–PI staining (Fig. 7). Doubling the dose of NLGP also had no apoptotic effect on immune cells (data not shown). 3.9. NLGP alters immunophenotypic profile in mice

Fig. 6. Assessment of kidney functions of Swiss mice with NLGP treatment. Two groups of Swiss mice (n = 6) were treated with NLGP and PBS, weekly for four weeks in total. Blood was collected from mice following 7 days of each NLGP injection and serum was collected. Content of urea and creatinine was determined.

3.6. NLGP has no adverse effect on hepatic functions Heparinized blood was collected from the NLGP and PBS treated mice and rats in every 7 day intervals for 4 weeks and sera were analyzed for liver enzymes, e.g., alkaline phosphatase, SGOT and SGPT. No significant difference was observed in NLGP treated animals in comparison to the PBS treated controls (Fig. 5).

Blood from NLGP and PBS treated mice was stained with various antibodies for T cells (CD4, CD8) B cells (CD19), Treg cells (CD4, CD25, Foxp3), NK cells (DX5), macrophages (CD11b), DCs (CD11c) etc. It was found that all cell types increased in the circulating blood of NLGP treated mice, except a little decrease (statistically insignificant) in Treg cells. Results of these flow cytometric analyses are presented in Fig. 8A, B and C. 3.10. NLGP enhances circulating immunoglobulins in mice In addition to the observation on changes in various cell types, we evaluated whether immunization with the effective dose of NLGP alters total immunoglobulin pool. Results demonstrated that 4 injections of NLGP with 25 μg dose generated a good amount of antibody (Fig. 9A), predominantly IgG type with proportionate amount of IgM and IgA (Fig. 9B). Again, IgG subclass analysis revealed the presence of higher amount of IgG2a, indicating type 1 directed immune response (Fig. 9C). 3.11. NLGP induces splenocytic proliferation in mice and rats

3.7. NLGP has no adverse effect on kidney functions Heparinized blood was collected from the PBS and NLGP treated animals in every 7 days interval for 4 weeks and sera were analyzed for urea and creatinin content. Serum urea and creatinine levels remained unchanged in NLGP treated mice (Fig. 6) and rats (data not shown) in comparison to those having PBS treatment.

Mononuclear cells were purified from blood of mouse and rats. These cells were stimulated in vitro with NLGP and ConA. It appeared that cells from NLGP treated animals proliferated more against the immunizing component, NLGP and non-specific stimulator, ConA. This observation is true for both mice (Fig. 10A) and rats (Fig. 10B).

Fig. 7. Assessment of apoptotic effect of NLGP on different immune cells of Swiss mice. B cells, T cells, NK cells, macrophages and dendritic cells were purified from blood of normal mice (n = 4), incubated with NLGP for 72 h and apoptosis was analyzed by AnnexinV–PI staining. Proapoptotic cells as denoted by AnnexinV staining and apoptotic cells by both AnnexinV–PI staining are presented.

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Fig. 8. Immunophenotypic changes in different immune cells of Swiss mice following NLGP treatment. Two groups of mice were treated with NLGP and PBS (n = 4, in each group), weekly for four weeks in total. After the completion of the treatment, spleen was isolated from mice of both groups. Splenic mononuclear cells were stained with antibodies specific for T cells (CD4, CD8), B cells (CD19) (A), Treg cells (CD4, CD25, Foxp3) (B), NK cells (DX5), macrophages (CD11b) and dendritic cells (CD11c) (C). Stained cells were analyzed by flow cytometry. *p b 0.013; *p = 0.001; +p = 0.048; and ++p = 0.001.

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Fig. 9. Analysis of immunoglobulin isotypes and subclasses in serum from Swiss mice following NLGP treatment. Two groups of mice were treated with NLGP and PBS (n = 4, in each group), weekly for four weeks in total. After the completion of the treatment, blood was collected from each mice and serum was collected. Total content of immunoglobulins (A), immunoglobulin isotypes (B) and subclasses (C) was determined by ELISA by using specific antibodies.

3.12. NLGP induces greater release of type 1 cytokines Mononuclear cells were obtained from blood of NLGP and PBS treated mice and cultured in vitro for 72 h. Supernatants were analyzed for the content of type 1 cytokines, IFNγ/IL-12 and type 2

cytokines, IL-4/IL-10. It is apparent from Fig. 11 that level of type 1 cytokines is significantly higher in NLGP treated groups than PBS treated controls. However, IL-4 and IL-10 levels are low in mice with NLGP treatment (Fig. 11). 3.13. Immunization with NLGP result in restriction of murine carcinoma growth Two groups of mice were either immunized with NLGP or PBS, once weekly for 4 weeks in total. Seven days after the completion of NLGP treatment both groups of mice were inoculated with Ehrlich's carcinoma and tumor growth was monitored regularly. It is apparent from Fig. 12 that tumor volume of mice, pretreated with NLGP, was significantly less than that of control mice (p b 0.001). 4. Discussion

Fig. 10. Proliferation of blood mononuclear cells from Swiss mice and Sprague Dawley rats after NLGP treatment. Two groups of either Swiss mice (n = 6) or Sprague Dawley rats (n = 4) were treated with NLGP and PBS, weekly for four weeks in total. After the completion of the treatment, blood was collected from each mice and rats. Mononuclear cells were prepared from the collected heparinized blood and cultured in vitro with media, NLGP and ConA for 72 h. Proliferation was assessed by MTT assay for mice (A) and rats (B). *pb 0.001.

We have reported an array of immunomodulatory activities of NLGP in relation to the activation of T cells [24,25], B cells [20], NK cells [18,23], monocytes/macrophages [21,28] and dendritic cells [26,27] and suppression of regulatory T cells [22,28]. Immune modulation by neem leaf preparation (precursor of NLGP) has shown significant tumor growth restriction in prophylactic [11–13] settings. NLGP also therapeutically restricted the sarcoma growth [29]. Mode of action of this glycoprotein on various immune cells was discussed in several manuscripts from our group [18–29]. Although, the receptor of NLGP is not identified yet, different immune cells are targeted by this glycoprotein. It has the ability to bind on membranes of several immune cells. NLGP phosphorylates STAT1, STAT4, but dephosphorylates STAT3 [25]. Accordingly, Th1 cytokines are produced by T cells, NK cells, dendritic cells, macrophages etc. and proliferative responses are triggered. It activates p38MAPK pathway, but inhibits ERK [21]. After obtaining promising views on immunomodulatory functions of NLGP and tumor growth restriction in mice, we are looking forward towards preclinical and clinical trials on cancer patients. Indian civilization knows the unique medicinal and nonmedicinal (in agriculture, in industry etc.) uses of different parts of neem tree [4] and it is added as a food constituent in diet of various Indian communities. No

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Fig. 11. Assessment on cytokine secretory profile from blood mononuclear cells of Swiss mice after NLGP treatment. Two groups of mice were treated with NLGP and PBS (n = 4, in each group), weekly for four weeks in total. After the completion of the treatment, blood was collected from each mouse, and mononuclear cells were prepared and cultured in vitro for 72 h. Supernatant was collected after 72 h and secreted cytokines (IL-12, IFNγ, IL-4 and IL-10) were assessed by ELISA. *p b 0.01; and **p b 0.001.

toxicities of effective dose of any neem products are reported in literature. However, to translate the bioimmunomodulatory functions of NLGP in clinical settings, it is important to evaluate the toxicity profile of NLGP on different physiological systems. In order to check the safety of this preparation, Swiss albino mice and Sprague Dawley rats were injected with NLGP in three different concentrations which are higher than the effective dose. No death was recorded in either mice or rats even after four weekly injections of 8 fold higher dose of NLGP than the effective dose. During this period no behavioral change was detected in any species. We categorically analyzed the activity of mice under bell jar, with no exposure of any external stimulus. An active gesture was noted in NLGP treated mice, as they moved or jumped within bell jar throughout the NLGP injection period. No hyperactivity was demonstrated in any of these mice. Body weight of these NLGP treated mice and rats was recorded regularly and no significant change was noted in comparison to PBS treated controls. Mice with NLGP treatment have shown little increase in body weight indicating optimization of physiological functions in rodents by NLGP. After the completion of the NLGP treatment as per protocol used for immunoprophylactic studies, autopsies of

Fig. 12. Tumor growth restriction in NLGP pretreated mice. Two groups of mice were treated with NLGP and PBS (n = 6, in each group), weekly for four weeks in total. Seven days after completion of the treatment, Ehrlich's carcinoma cells were inoculated on right hind leg quarter. Tumor growth was monitored regularly by caliper measurement and mean tumor volume is presented. *pb 0.001.

various organs (brain, liver, kidney, spleen, lymph nodes and thymus) were collected from mice and rats of both groups. No visual abnormalities were noted in any organs and organ weights were maintained perfectly as PBS treated animals. A little increase in weight of spleen and thymus was noted, indicating stimulated immune activity in these vital immune organs. Using aqueous neem leaf extract, Beuth J et al. (2004) [30] reported an increase in spleen weight, however, no change in thymus weight was noticed. We consistently observed an increase in thymus weight after NLGP treatment. Difference in the composition of the extract might be the possible reason of such difference [29]. Apart from these, no other NLGP induced changes in any organ weight were detected. Histological assessment of all of these organs was also performed. Microscopical observations revealed no alterations in the histology of any of these organs in NLGP treated mice and rats. Characteristic features of all organs like, hepatic lobule of liver, malpighian corpuscles of kidney etc. are maintained in their normal architecture perfectly after NLGP treatment. We have also tested the effect of neem leaf preparation, precursor of NLGP, on histological profile of different organs. No abnormalities were observed in such investigation [11]. We then investigated the hematological profile following NLGP administration. Although, no significant changes were noted in hematological parameters, the overall result suggests hematostimulatory behavior of NLGP. Increment in WBC, platelet count and hemoglobin percentage was noted in blood from NLGP treated mice and rats. In addition, percentage of lymphocytes also increased in NLGP treated mice. Similar to our observation, an aqueous extract of neem leaf enhances lymphocyte count in Balb/c mice [29]. Observed hematostimulation definitely improves the general health of animals. Therefore, NLGP may provide additional beneficial effect for cancer control apart from immunomodulation. In agreement with our observations with NLGP, another preparation from neem leaf was reported to increase the hemoglobin concentration and lymphocyte differential count significantly by 24%, and 20% respectively [30]. As mentioned earlier, we observed normal histology of liver and kidney of NLGP treated mice and rats. In continuation of this observation, we have tested enzymes related to liver and kidney functions. No alteration in these enzyme levels was noted in serum of NLGP treated animals, in comparison to PBS control. Thus, the possibility of generation of hepatotoxicity or nephrotoxicity is negligible after

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NLGP treatment. In accordance to our observation, Mbah et al. (2007) [31] reported no adverse effects and no abnormalities in kidney and liver functions by using another preparation of neem, i.e., acetonewater neem leaf extract. NLGP is an immunomodulator and it modulates immune system in such a way that anti-tumor cytotoxic efficacy is enhanced [20,24,26] and suppressive functions are reduced [22,28]. It is a pertinent question to know whether immunomodulation by NLGP is associated with the death of immune cells including T cells. It was reported earlier that NLP [17] and NLGP [22] have no cytotoxic effect on tumor cells. Thus, it is expected that NLGP might have no killing effect on immune cells too. To clarify this point, mouse T, B, NK cells, macrophages and dendritic cells were incubated with NLGP and cell death was monitored by Annexin V–Propidium Iodide staining. In both experiments, no cellular cytotoxicity was noticed even after addition of 4 times higher concentration of NLGP for 72 h. This result confirms that NLGP would not be the cause of death of immune cells after immune activation/modulation, if administrated in vivo. Phenotypic analysis of MNCs from NLGP treated mice indicated an increase in CD4+ helper, and CD8+ cytotoxic T cells, but a decrease in CD4+CD25+Foxp3+ regulatory T cells. Acetone-water neem leaf extract, enhances CD4+ cell count in 50 HIV/AIDS patients among 60 [31]. In addition, upregulation of NK cells (DX5+), macrophages (CD11b+), and dendritic cells (CD11c+) was detected in NLGP treated mice. These observations reconfirmed the non-toxicity of NLGP on different immune cells. Cancer is an immunosuppressive disease, thus, the application of NLGP for treatment of cancer patients definitely improves the suppressed immune state of patients. This further helps in immune maintenance of patients under standard therapy like, chemotherapy. Moreover, MNCs from NLGP treated mice have shown greater mitogenic proliferation and in vitro stimulation of MNCs with NLGP also demonstrated more proliferation, although not significant statistically. NLGP maintains the immune functions in activated state, but no hyperactivation was experienced. Similar treatment of MNCs with NLGP, caused enhanced release of type 1 cytokines, IFNγ, and IL-12, but diminished the secretion of IL-4 and IL-10. Observed type 1 bias would be helpful for anti-tumor immunity, but it never exceeded the limit to cause auto-immunity. Histological studies of different organs also exhibited no signature of auto-immunity. In addition to the studies on cellular immune system, we also assessed the immunoglobulin content in the serum of NLGP treated mice in comparison to PBS controls. IgG content increased in mice after NLGP treatment and this IgG is chiefly IgG2a, which is a good indicator of type 1 immune response. Thus, evidence indicates generation of type 1 immunity from studies on both cellular and humoral immune functions. Generation of type 1 immune response by means of immunomodulation with NLGP helped in anti-tumor immunity, as we have demonstrated significant tumor growth restriction in mice pretreated with NLGP (prophylactic settings). NLGP is also effective to reduce established tumor by activating CD8 + T cell mediated anti-tumor immune response (therapeutic settings) [29]. As the investigations are objected to confirm the non-toxic nature of NLGP before its proposition for clinical use, convincing evidence from mice and rat studies is accumulated in favor of non-toxic nature of NLGP in every physiological aspect. Moreover, hematostimulatory and immunostimulatory property of NLGP will provide additional mileage for tumor growth restriction, if it is administrated alone or in combination with chemotherapy. Based on these data clinical trial with NLGP may be proposed. Conflict of interest statement None. Acknowledgments We acknowledge Dr. Jaydip Biswas, Director, CNCI, India for providing the necessary facilities. Thanks to Dr. Abhijit Rakshit for

providing the experimental animals. The work was partially supported by the Indian Council of Medical Research, New Delhi (grant nos. 3/2/ 2/188/2009/NCD-III; 61/5/2008-BMS-2008), the Department of Science and Technology, New Delhi (DST/INSPIRE Fellowship/2011/188); the Council of Scientific and Industrial Research, New Delhi (09/030 (0050)/2008-EMR-I); and the University Grant Commission, New Delhi (grant no. F.2-3/2000 (SA-1) to A. B. and K.G.).

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