Interferon-alpha2b restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by modulating CXC receptor ligand interaction.

We have studied the immunomodulatory effects of interferon-alpha2b (IFN-alpha2b) in rectification of the dysregulated IFN-gamma-dependent chemokines and their receptor CXCR3 splice variants in head and neck squamous cell carcinoma-peripheral blood mononuclear cells (HNSCC-PBMC). CXCR3 expression was upregulated in HNSCC-PBMC, with the demonstration of poor chemotactic function. CXCR3 upregulation possibly represents the increased synthesis of CXCR3B splice variant, without significant change in CXCR3A. Upregulated expression of CXCR3B was downregulated following in vitro IFN-alpha2b treatment of HNSCC-PBMC. Upregulation of CXCR3A+B by IFN-alpha2b with downregulation of the CXCR3B indirectly suggests that the upregulation of the CXCR3A splice variant induces cellular migration. On the other hand, the stimulation of PBMC with IFN-alpha2b maintains physiological homeostasis of CXCR3 ligands, CXCL10 and CXCL9, and increases the secretion of IFN-gamma. The suppressed chemotactic ability of HNSCC-PBMC could be restored either by in vitro treatment of PBMC with IFN-alpha2b or during the use of IFN-alpha2b stimulated PBMC supernatant as a chemoattractant. Chemoattraction process is guided at the level of both receptor and its ligands, as confirmed by neutralization studies. IFN-alpha2b possibly controls chemotaxis by regulating the interaction between CXCL10 and CXCR3A. Neutralization of IFN-gamma downregulates the IFN-alpha2b mediated CXCL10 release, suggesting the active role of IFN-gamma in the transduction of chemotactic signal for the migration of cytotoxic T/NK cells at the tumor site.

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.

Tumor associated release of interleukin-10 alters the prolactin receptor and down-regulates prolactin responsiveness of immature cortical thymocytes.

The soluble factors produced either by Ehrlich's ascites carcinoma (EAC) or thymic adherent cells (TAC) of tumor-bearing mice comprising of CD11b(+) and CD11c(+) antigen-presenting cells caused a sharp decrease in prolactin (PRL)-induced ConA-mediated effect on survival of PNA(+) thymocytes. Similar suppression of PRL-induced effect was observed when the cells were cocultured with TAC of EAC-bearing mice. Anti-IL-10 antibody could reverse the PRL inability to induce ConA-mediated effect on PNA(+) thymocyte survival, indicating the presence of IL-10 in EAC culture supernatant (EAC sup) and thymic microenvironment. IL-10 could block PRL-induced proliferation of PNA(+) thymocytes without affecting spontaneous apoptosis. IL-10 altered the expression of the long-form (LF) of PRL-R and reduced the PRL binding of the cells, suggesting down-regulation of the PRL effect on PNA(+) thymocyte by the cytokine. Induction of tumor, which was found to increase the IL-10 secretion by TAC, also modified the PRL-R (LF) to PRL-R (SF). Since PRL plays a role in survival, proliferation and differentiation of lymphoid progenitor cells, the tuning of PRL action by IL-10 may be a possible mechanism of depletion of immature cortical thymocytes and thymic atrophy in tumor-bearing mice.

Neem leaf preparation enhances Th1 type immune response and anti-tumor immunity against breast tumor associated antigen.

An 85-kDa breast tumor associated antigen (BTAA) has been identified and partially characterized from human breast tumors. As BTAA is poorly immunogenic, enhancement of the anti-tumor immunity induced by BTAA is required to obtain an objective clinical response. The potent immune activation by an aqueous preparation of neem (Azadirachta indica) leaf (NLP) suggests its possible utility for enhancing immune responses to tumor vaccines. Mice (Swiss and Balb/c) and rats (Sprague Dawley) immunized with BTAA and NLP have a higher IgG antibody response and a lower IgM response than mice immunized with BTAA alone. Antibody generated by immunization with BTAA and NLP can induce antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T cell (CTL) response towards BTAA-expressing MCF-7 cells. Antibody produced by vaccination with BTAA alone generated little cytotoxic response. The occurrence of ADCC and CTL response induced by BTAA plus NLP vaccination was possibly assisted by the induction of a Th1 response, as evidenced by the enhanced secretion of IFN-gamma and decreased release of IL-10 from spleen cells and the greater production of IgG2a antibody in immunized mice. As NLP is nontoxic, abundantly available in the Indian subcontinent and can be extracted by a cost-effective method, this preparation may be considered a promising immune enhancer for BTAA vaccine.

Macrophage inflammatory protein (MIP)1alpha and MIP1beta differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy.

The C-C chemokines, macrophage inflammatory protein (MIP)1alpha and MIP1beta are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1alpha and MIP1beta in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) alpha release by the PBM was markedly stimulated by MIP1alpha in CaBr patients, but only marginally so in healthy women. In contrast, MIP1beta stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1alpha, but not MIP1beta, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1alpha and MIP1beta augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1alpha being more effective in CaBr patients and MIP1beta in healthy women. IFN-gamma co-stimulated and IL-4 suppressed MIP1alpha and beta-induced cytotoxicity in PBM. The synergy of IFN-gamma was more marked with MIP1alpha than with MIP1beta. The differential effects of MIP1alpha and MIP1beta on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5.

Prolactin induced reversal of glucocorticoid mediated apoptosis of immature cortical thymocytes is abrogated by induction of tumor.

Glucocorticoid (GC) and prolactin (PRL) are the two neuroendocrines that regulate thymocyte differentiation and maintain the immune homeostasis during stress. We found that dexamethasone (Dex), a synthetic GC, induced apoptosis in normal immature cortical thymocytes which remained unaltered in the presence of Elrich's ascitic carcinoma (EAC). PRL protected the normal CD4+ CD8+ cortical thymocytes from Dex-induced apoptosis but failed to alter the effect of Dex in tumor-bearing mice. Dex-treated normal thymocytes became unresponsive to PRL in presence of tumor cell culture supernatant. Low binding affinity of the microsomal membranes of thymocytes to PRL and absence of the mRNA of a particular form of prolactin receptor (PRL-R) suggest the presence of a different PRL-R in CD4+ CD8+ thymocytes of EAC-bearing mice. The induction of tumor may alter the PRL-R that can be correlated with the failure of PRL in rescuing CD4+ CD8+ immature cortical thymocytes from GC induced death.

Cervical screening by visual inspection with acetic acid (VIA) is well accepted by women--results from a community-based study in rural India.

OBJECTIVE: Among the low cost alternative screening tests Visual Inspection after Acetic Acid Application (VIA) has been found to be most promising. The objective of the present study was to evaluate the safety and acceptability of VIA done by health workers among rural Indian women. We also evaluated the level of women's satisfaction with the screening program. METHODS: Women residing in a defined geographic area were offered cervical screening using VIA by trained health workers. Women testing positive were colposcoped by a medical officer at the same sitting. Based on the feedback from a few focus group discussions a structured questionnaire was designed to interview the women after screening. A total of 498 women were selected randomly from the screened women for interview by a social worker. Besides enquiring about any discomfort they faced during or within seven days after screening, the women were also asked to indicate their level of satisfaction with the service. Their opinions to improve the quality of service were also sought. RESULTS: Most women reported no pain or only slight discomfort during screening (94.2%). The most common complaint after screening was vaginal discharge (12%). A burning sensation in the vagina was experienced by some of the women (5.8%). These complaints were mild and short-lasting in majority of cases. Most of the women were satisfied with the screening service (94.6% selected the top three of a six-point response scale) and 97% said they would recommend the test to others. The most common reasons for dissatisfaction with screening were discomfort during or after screening, long waiting time and failure to get treatment for other medical problems. CONCLUSION: VIA by trained health workers followed by colposcopy at the same sitting is an acceptable screening algorithm for Indian women. A VIA based screening program has to be integrated to the existing primary health care facility in developing countries.

Immunostimulatory neem leaf preparation acts as an adjuvant to enhance the efficacy of poorly immunogenic B16 melanoma surface antigen vaccine.

Immunogenecity of the poorly immunogenic B16 melanoma cell surface antigen (B16MelSAg) was enhanced by combining B16MelSAg with NLP in C57BL/6 mice, as evidenced by ELISA and flow cytometry. NLP was as effective as Freund's complete and incomplete adjuvant to generate antibodies recognizing the B16MelSAg. The NLP generated antibody was a gamma globulin with a subtype of IgG1. Splenic lymphocytes from B16MelSAg+NLP treated mice proliferated more rapidly in vitro when stimulated by specific (B16MelSAg) and nonspecific (ConA) stimulators, in comparison to the proliferation detected in B16MelSAg and NLP treated groups. Vaccination of mice with B16MelSAg+NLP more efficiently prevented the growth of B16 melanoma tumor than mice immunized with B16MelSAg or NLP alone. In another experiment, the immune sera (B16MelSAg+NLP) was mixed with B16Mel tumors and injected subcutaneously into syngenic C57BL/6 mice. Tumor burden was less in mice receiving a tumor along with B16MelSAg+NLP generated immune sera than other groups. The B16MelSAg+NLP generated immune sera induced antibody dependent cellular cytotoxicity specifically towards B16Mel tumor cells in vitro. We concluded that NLP might be a potential immune adjuvant for inducing active immunity towards tumor antigens.

Neem (Azadirachta indica) leaf mediated immune activation causes prophylactic growth inhibition of murine Ehrlich carcinoma and B16 melanoma.

Conditional growth inhibition of murine Ehrlich carcinoma (EC) and B16 melanoma (B16Mel) was observed, following treatment of mice (Swiss and C57BL/6) with aqueous extract of neem (Azadirachta indica) (1 unit/mice/week for 4 weeks) either before or after inoculation of 1 x 10(6) tumor cells. Tumor inoculation after weekly injections for 4 weeks with neem leaf preparation (NLP) induced significant reduction of tumor growth (both EC and B16Mel) and increased survivability of mice. On the other hand, NLP treatment after tumor inoculation demonstrated no tumor growth inhibition in the NLP treated group in comparison to the PBS treated control. No direct cytotoxic effect of NLP towards EC and B16Mel tumor cells was observed in vitro. The spleen cells of NLP treated mice when mixed with inoculum of B16Mel tumor cells and injected into a group of mice, tumor growth was found to be significantly reduced and survivability of the tumor hosts increased remarkably in comparison to mice inoculated with tumor along with normal spleen cells. Concanavalin A (ConA) induced proliferation of lymphocytes from NLP treated mice was significantly higher than the lymphocytes of untreated mice. In in vitro, NLP by itself had no proliferative effects on lymphocytes but it co-stimulated ConA induced mitogenesis. NLP induced lymphocytosis as evidenced by increased lymphocyte count in blood as well as spleen. Flow cytometric evidence suggested that increase in CD4+ and CD8+ T cells accounted for lymphocytosis. The conditional tumor growth retardation, observed in mice treated with NLP before tumor inoculation, may be regulated by NLP mediated immune activation, having prominent role in the cellular immune function of the tumor host.

Detection and purification of a novel 72 kDa glycoprotein male breast tumor associated antigen.

A male breast tumor associated antigen (MBTAA) was purified and partially characterized from human male breast tumor. Three protein peaks were obtained by DEAE-cellulose column chromatography of a crude extract of human male breast tumor tissues. Circulating antibodies against one of these peaks, MF1, which contained MBTAA, were observed in male breast cancer patients but not in normal male or male patients with carcinoma of other organs (stomach, colon, lung). The MBTAA was partially purified from MF1 by subjecting the fraction to SDS-PAGE and eluting the protein from band 3 (MB-3) and by subjecting MF1 to size exclusion-high performance liquid chromatography (SE-HPLC). The MBTAA was characterized as a glycoprotein with MW of approximately 72 kDa. It showed no immunological relatedness with TAG-72, a tumor associated antigen expressed in breast epithelial cells. A 72 kDa protein, immunologically related to MBTAA, was detected and partially purified from female breast tumor. The female breast cancer patients did not have circulating antibodies against this 72 kDa protein or MBTAA. Presence of 72 kDa glycoprotein MBTAA in MF1 and specificity of the anti-MBTAA antibodies in the sera of male breast cancer patients were further confirmed by Western blot analysis. Absence of anti-MBTAA antibodies in healthy men and in patients with other cancers suggested that expression of MBTAA may be malignancy-associated and is highly overexpressed in male breast cancer.

Prolactin regulates antitumor immune response through induction of tumoricidal macrophages and release of IL-12.

The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL-Rs in these cells. Here, we show that PRL, though it failed to activate mouse peritoneal resident macrophages (RMs), acted as a second signal and activated mouse peritoneal inflammatory macrophages (EMs) to a tumoricidal state. The cytotoxicity of mouse tumor-associated macrophages (TAMs) isolated at day 1 of tumor (Ehrlich ascites carcinoma, EAC) growth was enhanced by PRL. However, with progression of tumor growth, TAMs became nonresponsive to the hormone. PRL-induced killing of P815 target cells by EMs and TAMs was independent of TNF but correlated with the hormone-induced augmentation of NO2(-) and O2(-) release in these macrophages. Administration of PRL in vivo inhibited EAC growth and augmented NO2(-) release by TAMs. PRL synergized with the TH1 cytokine IFN-gamma, a known activator of macrophages, in inducing tumor killing and release of NO2(-) from EMs and TAMs. The hormone might activate macrophages at least partially, through the release of IFN-gamma as anti-IFN-gamma blocked IFN-gamma- as well as PRL-induced cytotoxicity in EMs. The TH2 cytokine IL-4 suppressed PRL-induced activation of macrophages. PRL induced release of IL-12 from EMs also, which suggested that the hormone might drive the TH1 response through IL-12. Our observations further suggest that PRL alone and in synergy with IFN-gamma, released through induction of IL-12, may generate tumoricidal macrophages and thus regulate the antitumor immune response of tumor hosts.