Rathindranath Baral

Dopamine regulates endothelial progenitor cell mobilization from mouse bone marrow in tumor vascularization

Mobilization of endothelial progenitor cells (EPCs) from the bone marrow and their subsequent participation in neovessel formation are implicated in tumor growth and neovascularization. As the neurotransmitter dopamine (DA) modulates adult endothelial cell function, we hypothesized that DA might have a regulatory role in mobilization of EPCs from the bone marrow niche. We show that there was a significant decrease in bone marrow DA content and an increase in EPC mobilization in tumor-bearing mice associated with tumor neovascularization. DA treatment of tumor-bearing mice inhibited EPC mobilization and tumor growth through its D2 receptors, as DA treatment failed to inhibit EPC mobilization in tumor-bearing mice treated with a specific DA D2 receptor antagonist and in tumor-bearing mice lacking the D2 receptor. In addition, we found that DA, through D2 receptors, exerted its inhibitory effect on EPC mobilization through suppression of VEGFA-induced ERK1/ERK2 phosphorylation and MMP-9 synthesis. These findings reveal a new link between DA and EPC mobilization and suggest a novel use for DA and D2 agents in the treatment of cancer and other diseases involving neovessel formation.

Prophylactic Dose of Neem (Azadirachta indica) Leaf Preparation Restricting Murine Tumor Growth is Nontoxic, Hematostimulatory and Immunostimulatory

Significant restriction of growth of Ehrlichs carcinoma was observed following prophylactic treatment on Swiss albino mice with neem leaf preparation (NLP-1 unit) once weekly for four weeks. Toxic effects of this particular dose (1 unit), along with 0.5 unit and 2 units of NLP doses, were evaluated on different murine physiological systems. One hundred percent of mice could tolerate 4 injections of 0.5 and 1 unit NLP doses. Body weight, different organ-body weight ratios and physical behavior of treated mice remained completely unchanged during treatment with different NLP doses. All of these NLP doses were observed to stimulate hematological systems as evidenced by the increase in total count of RBC, WBC and platelets and hemoglobin percentage. As histological changes as well as elevation in serum alkaline phosphatase, SGOT, SGPT were not observed in mice treated with three different doses of NLP, the nonhepatotoxic nature of NLP was proved. The level of serum urea remained unaltered and normal architecture of the cortical and medullary parts of the kidney were also preserved after NLP treatment. Increased antibody production against B16 melanoma antigen was detected in mice immunized with 0.5 unit and 1 unit of NLP. Number of splenic T lymphocytes (CD4+ and CD8+) and NK cells were also observed to be increased in mice injected with 0.5 unit and 1 unit of NLP. However, NLP dose of 2 units could not exhibit such immunostimulatory changes; NLP mediated immunostimulation was correlated well with the growth restriction of murine carcinoma. In other words, tumor growth restriction was observed only when mice were injected with immunostimulatory doses of NLP (0.5 unit and 1 unit).

Neem leaf glycoprotein restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance

Interaction between CXCL10 and CXCR3 is dysregulated in head and neck squamous cell carcinoma (HNSCC) and hampers chemotaxis of cytotoxic cells at tumor site. In continuation of the demonstration of significant immunomodulatory effects of neem leaf preparation (NLP), the active ingredient of NLP is characterized as a glycoprotein (NLGP). NLGP is responsible for in vivo immunomodulation to restrict the growth of mice tumors. Effect of NLGP in rectification of the dysregulated IFN gamma dependent chemokine and its receptor CXCR3 splice variants was investigated. Upregulated expression of CXCR3B in HNSCC-PBMC were downregulated following in vitro NLGP treatment. Unchanged expression of CXCR3A+B by NLGP with downregulation of the CXCR3B indirectly suggests the upregulation of the CXCR3A, responsible for cellular migration. However, stimulation of healthy-PBMC with NLGP maintains physiological homeostasis of CXCL10 and increases IFN gamma secretion. The suppressed chemotaxis of HNSCC-PBMC could be restored either by in vitro treatment with NLGP or during use of NLGP stimulated PBMC supernatant as a chemoattractant. Neutralization studies confirmed that the chemoattraction process is guided by both receptor (CXCR3A) and its ligand (CXCL10). Neutralization of the IFN gamma in PBMC culture in presence of NLGP unexpectedly increases the intracellular release of CXCL10, suggesting the NLGP mediated IFN gamma independent release of CXCL10. Interestingly, downregulation of the CXCL10 release was detected after IFN gamma neutralization in absence of NLGP and IFN gamma receptor neutralization in presence of NLGP. Efficacy of NLGP in restoration of the dysregulation of the chemokine signaling may be utilized to design new immunotherapeutic protocol.

Neem leaf glycoprotein helps to generate carcinoembryonic antigen specific anti-tumor immune responses utilizing macrophage-mediated antigen presentation

In an objective to generate effective carcinoembryonic antigen (CEA) specific anti-tumor immune response in Swiss mice, CEA was presented using macrophages with adjuvant help from neem leaf glycoprotein (NLGP). Such vaccination generates significantly higher antibody (IgG2a) and T cell response than immunization protocol without NLGP. NLGP controls the function of both B cells and macrophages by altering the expressions of various regulatory molecules, like, CD19, CD11b, etc. NLGP also directs CEA vaccination towards Th1 bias, by modulating cytokine secretion. This NLGP-generated anti-CEA immune response would be effective as a vaccine to lyse CEA(+) tumors in vitro and in vivo.

Neem leaf glycoprotein matures myeloid derived dendritic cells and optimizes anti-tumor T cell functions

In an objective to find out an effective, nontoxic dendritic cell (DC) maturating agent for human use, CD14(+) monocytes were differentiated with GMCSF/IL-4 and matured with neem leaf glycoprotein (NLGP). NLGP matured DCs (NLGP-DCs) show upregulated expression of CD83, CD80, CD86, CD40 and MHCs, in a comparable extent of control, LPS. NLGP-DCs secrete high amount of IL-12p70 with low IL-10. NLGP upregulates the expression of crucial transcription factor, ikaros, indicating maturation towards DC1 phenotype. Increased expression of CD28 and CD40L on T cells following co-culture with NLGP-DCs was noticed to promote DC-T interactions. As a result, T cells secrete high amount of IFN gamma with low IL-4 and generates anti-tumor type 1 immune microenvironment. Such NLGP-DCs present carcinoembryonic antigen (CEA) effectively to T cells to increase T cell mediated cytotoxicity of CEA(+) tumor cells in vitro and in vivo. With emergence of the NLGP as a promising DC maturating agent, NLGP-DCs can be used as a candidate vaccine tool for antigen specific cancer immunotherapy.

Neem Leaf Glycoprotein Activates CD8 + T Cells to Promote Therapeutic Anti-Tumor Immunity Inhibiting the Growth of Mouse Sarcoma

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8+ T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8+ T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8+ T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3+ and CCR5+ T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8+ T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44hiCD62Lhi central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8+ T cells into established tumors to operate significant tumor cell lysis.

Neem leaf glycoprotein enhances carcinoembryonic antigen presentation of dendritic cells to T and B cells for induction of anti-tumor immunity by allowing generation of immune effector/memory response.

Vaccination with neem leaf glycoprotein matured carcinoembryonic antigen (CEA) pulsed dendritic cells (DCs) enhances antigen-specific humoral and cellular immunity against CEA and restricts the growth of CEA(+) murine tumors. NLGP helps better CEA uptake, processing and presentation to T/B cells. This vaccination (DCNLGPCEA) elicits mitogen induced and CEA specific T cell proliferation, IFN gamma secretion and induces specific cytotoxic reactions to CEA(+) colon tumor cells. In addition to T cell response, DCNLGPCEA vaccine generates anti-CEA antibody response, which is principally IgG2a in nature. This antibody participates in cytotoxicity of CEA(+) cells in antibody-dependent manner. This strong anti-CEA cellular and humoral immunity protects mice from tumor development and these mice remained tumor free following second tumor inoculation, indicating generation of effector memory response. Evaluation of underlying mechanism suggests vaccination generates strong CEA specific CTL and antibody response that can completely prevent the tumor growth following adoptive transfer. In support, significant upregulation of CD44 on the surface of lymphocytes from DCNLGPCEA immunized mice was noticed with a substantial reduction in L-selectin (CD62L).

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mφ) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1α and MIP-1β) of this chemokine receptor were also secreted in lesser quantity from MO/Mφ of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor–ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mφ of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1α and MIP-1β was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor–ligand signaling seen in MO/Mφ was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mφ after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mφ by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mφ-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mφ.

Neem Leaf Glycoprotein Induces Perforin-mediated Tumor Cell Killing by T and NK Cells Through Differential Regulation of IFNγ Signaling

We have demonstrated augmentation of the CD3−CD56+ natural killer (NK) and CD8+CD56− T-cell–mediated tumor cell cytotoxicity by neem leaf glycoprotein (NLGP). These NK and T cells were isolated from the peripheral blood of head and neck squamous cell carcinoma patients with a state of immunosuppression. NLGP induces TCRαβ-associated cytotoxic T lymphocyte (CTL) reaction to kill oral cancer (KB) cells. This CTL reaction is assisted by NLGP-mediated up-regulation of CD28 on T cells and HLA-ABC, CD80/86 on monocytes. CTL-mediated killing of KB cells and NK-cell–mediated killing of K562 (erythroleukemic) cells are associated with activation of these cells by NLGP. This activation is evidenced by increased expression of early activation marker CD69 with altered expression of CD45RO/CD45RA. NLGP is a strong inducer of IFNγ from both T and NK cells; however, IFNγ regulates the T-cell–mediated cytotoxicity only without affecting NK-cell–mediated one. Reason of this differential regulation may lie within up-regulated expression of IFNγ-receptor on T-cell surface, not on NK cells. This NLGP-induced cytotoxicity is dependent on up-regulated perforin/granzyme B expression in killer cells, which is again IFNγ dependent in T cells and independent in NK cells. Although, FasL expression is increased by NLGP, it may not be truly linked with the cytotoxic functions, as brefeldin A could not block such NLGP-mediated cytotoxicity, like, concanamycin A, a perforin inhibitor. On the basis of these results, we conclude that NLGP might be effective to recover the suppressed cytotoxic functions of NK and T cells from head and neck squamous cell carcinoma patients.

Neem leaf glycoprotein directs T-bet–associated type 1 immune commitment

Neem leaf glycoprotein (NLGP)-mediated immune activation and associated immune polarization was studied. NLGP-induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-gamma and a lower amount of the Th2 cytokine interleukin (IL)-4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor -alpha and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3(+) T-Reg cells. A type 1-specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.