Additional file 1: of Current challenges in treatment options for visceral leishmaniasis in India: a public health perspective

Multilingual abstracts in the six official working languages of the United Nations. (PDF 277 kb

Cytokine recall responses.

<p><i>In vitro</i> cytokine levels induced by hCG, βhCG or TT in splenocytes derived from βhCG-TT or βhCG-TT + <i>MIP</i> immunized BALB/c and C57BL/6 mice. Bars represent arithmetic mean ± standard error. *p≤0.05 versus animals immunized with βhCG-TT. n = 6.</p

Inflammatory activity of MIP.

<p>(A) BMDC (derived from BALB/c and C57BL/6 mice) were treated with <i>MIP</i> (or LPS a control) and indicated cytokines were quantified. Bars represent arithmetic mean ± standard error. (B) IL-6 and TNF-α secretion upon incubation of MIP or LPS with CD11b⁺ and CD11b⁻ splenic cells derived from C57BL/6 mice.</p

Reactivity of anti-hCG antibodies towards tumor cells.

<p>(A) Flow cytometric analysis on permeabilized and non-permeabilized (i) SP2/O and (ii) B16F10 cells using pooled antisera from βhCG-TT (black profiles) and βhCG-TT + <i>MIP</i> (green profiles) immunized mice; pre-immune sera (blue profiles) served as control. Filled red profiles: Secondary antibody controls. (B) Right panels: Immunofluorescence analysis on permeabilized COLO205 cells depicting reactivity of (i) pre-immune sera, (ii) antisera derived from βhCG-TT + <i>MIP</i> immunized mice and (iii) antisera derived from βhCG-TT immunized mice. Left panels: Corresponding phase contrast images. Bars = 30 µm.</p

Synergistic Activation of Innate and Adaptive Immune Mechanisms in the Treatment of Gonadotropin-Sensitive Tumors

<div><p>Human chorionic gonadotropin (hCG) prolongs the secretion of progesterone from the corpus luteum, providing a critical stimulus for the sustenance of pregnancy. hCG (or individual subunits) is also secreted by a variety of trophoblastic and non-trophoblastic cancers and has been associated with poor prognosis. Early clinical studies have indicated merit in anti-hCG vaccination as potential immunotherapy, but anti-tumor efficacy is believed to be compromised by sub-optimal immunogenecity. In the present study, enhanced tumorigenesis was observed when SP2/O cells were subcutaneously injected in either male or female BALB/c x FVB/J^βhCG/- F1 transgenic mice, establishing the growth-promoting effects of the gonadotropin for implanted tumors <i>in vivo</i>. The utility of <i>Mycobacterium indicus pranii</i> (<i>MIP</i>) was evaluated, as an innate anti-tumor immunomodulator as well as adjuvant in mice. <i>MIP</i> elicited the secretion of the inflammatory cytokines IFNγ, IL-6, IL-12p40, KC and TNFα from murine antigen presenting cells. When <i>MIP</i> was incorporated into an anti-hCG vaccine formulation previously employed in humans (a βhCG-TT conjugate adsorbed on alum), elevated T cell recall proliferative and cytokine responses to hCG, βhCG and TT were observed. <i>MIP</i> increased vaccine immunogenicity in mice of diverse genetic background (including in traditionally low-responder murine strains), leading to enhanced titres of bioneutralizing anti-hCG antibodies which exhibited cytotoxicity towards tumor cells. Individual administration of <i>MIP</i> and βhCG-TT to BALB/c mice subcutaneously implanted with SP2/O cells resulted in anti-tumor effects; significantly, immunization with βhCG-TT supplemented with <i>MIP</i> invoked synergistic benefits in terms of tumor volume, incidence and survival. The development of novel vaccine formulations stimulating both adaptive and innate anti-tumor immunity to induce collaborative beneficial effects may fill a niche in the adjunct treatment of hCG-sensitive tumors that are resistant to conventional therapy.</p> </div

Bioneutralization potential of elicited antisera.

<p>hCG neutralization capacity of pooled antisera obtained from animals immunized with alum-adsorbed βhCG-TT or βhCG-TT + <i>MIP</i>, as determined by radio-receptor assay. (A) BALB/c; (B) FVB/J; (C) C57BL/6; (D) CBA/Cat and (E) C3H/HeJ. Data is presented as percentage inhibition (over maximal binding obtained in the absence of serum) as a function of antiserum dilution. *p≤0.05 versus corresponding value for βhCG-TT elicited antisera.</p

Anti-hCG antibody isotype analysis.

<p>Comparative anti-hCG immunoglobulin isotypic analysis of antibodies in five murine strains immunized with βhCG-TT and βhCG-TT + <i>MIP</i>. (A) BALB/c; (B) FVB/J; (C) C57BL/6; (D) CBA/Cat; (E) C3H/HeJ. Relative Units: Relative end-point titres. Pre-immune sera served as control. (F) Fold-increases in hCG specific IgG1, IgG2a and IgG2b antibody isotypes in different strains upon the inclusion of <i>MIP</i> in the vaccine formulation.</p

<i>In vivo</i> tumor-promoting potential of βhCG. SP2/O cells were implanted in BALB/c x FVB/J^βhCG/- F1 transgenic and non-transgenic male (A) and female (B) mice.

<p>Left panels depict tumor volumes in individual animals and right panels mean ± standard error, at different times post-cell implantation. *p≤0.05; **p≤0.01 versus non-transgenic littermates. n = 6.</p

Immunogenecity studies.

<p>Immunogenicity of alum-adsorbed βhCG-TT and βhCG-TT + <i>MIP</i> in (A) BALB/c; (B) FVB/J; (C) C57BL/6; (D) CBA/Cat and (E) C3H/HeJ mice. Each point represents hCG binding capacity of an individual animal and bars denote geometric means. ***p≤0.001, **p≤0.01, *p≤0.05 versus βhCG-TT immunized animals. n = 16.</p

Proliferative T cell recall responses.

<p><i>In vitro</i> proliferative responses to hCG, βhCG, TT or <i>MIP</i> of splenocytes derived from naive, βhCG-TT or βhCG-TT + <i>MIP</i> immunized (A) BALB/c and (B) C57BL/6 mice. Bars represent arithmetic mean ± standard error. *p≤0.05 versus vs animals immunized with βhCG-TT. n = 6.</p