Human embryonic and neuronal stem cell markers in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin. Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively. Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype. Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties

LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin, augments the capacity of a ricin toxin subunit vaccine to evoke neutralizing antibodies and protective immunity.

Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense

Novel mechanism of cytotoxicity of triple negative breast cancer cells by the type II heat-labile enterotoxin LT-IIc through ganglioside ligation

No abstract available

LT-IIb(T13I) is minimally inflammatory in comparison to LT-IIb.

<p>Mice were immunized by the i.d. route with 5.0 µg of RiVax, RiVax adsorbed to Imject®(alum), or RiVax combined with 1.0 µg of either LT-IIb or LT-IIb(T13I). (A) Gross morphologic photos and H&E stained micrographs of typical inflammation at the site of injection on day 7 post-immunization (n = 5). (B) Level of inflammatory induration at the injection site (n = 5). (C) Percent of CD45⁺ immune cells normalized to the total number of cells counted using Axiovision and ImageJ software from 15 random fluorescent micrographs per skin section (n = 3). Data are presented as the arithmetic mean with error bars denoting one S.E.M. Key: significance in comparison to LT-IIb(T13I) *, p<0.05; **, p<0.01; ***, p<0.001. Data in B were compared using an unpaired Student’s t-test; data in C were compared using ANOVA; #, p<0.05 - significance in comparison to LT-IIb; **, p<0.01 - significance in comparison to RiVax.</p

Administration of LT-IIb(T13I) by the i.d. route enhances protective immunity to ricin.

<p>Mice were immunized intradermally on days 0, 10, and 20 with 0.5 µg of RiVax in the presence or absence of 1.0 µg of LT-IIb(T13I) and challenged 14 days after the final immunization with 10 LD₅₀ of ricin. (A) Survival of immunized mice after i.p. challenge with ricin. Data were compared by the Logrank test. (B) Blood glucose levels of immunized mice during challenge with ricin. Data presented as the arithmetic mean with error bars denoting one S.E.M. (n = 5). Key: *, p<0.05; **, p<0.01 compared to RiVax; †<b>,</b> p<0.05 compared to 0 h. Data were compared using an unpaired Student’s t-test.</p

LT-IIb(T13I) is superior to alum (Imject®) at enhancing immune responses to RiVax when administered intradermally.

<p>Mice were immunized intradermally on days 0, 10, and 20 with 0.5 µg of RiVax, RiVax adsorbed to Imject®, or RiVax combined with 1.0 µg of either LT-IIb or LT-IIb(T13I). (A) Level of RiVax-specific serum IgG Ab from days 17, 27, 60, 90, and 120. (B) RiVax-specific IgG subclass analysis from the sera of immunized mice taken on day 90. Data are presented as the arithmetic mean with error bars denoting one S.E.M. (n = 5). Key: (A) *, p<0.05; **, p<0.01; ***, p<0.001 compared to RiVax; #, p<0.05 compared to RiVax+alum. (B) *, p<0.001 compared to IgG2a and IgG2b from the same group. Data were compared using ANOVA.</p

I.d. Immunization of RiVax with LT-IIb or LT-IIb(T13I) enhances ricin-neutralizing Ab production.

<p>Sera from immunized mice were assessed for the capacity to neutralize ricin in a Vero cell cytotoxicity assay. Ricin (10 ng/mL) was incubated with serum for 30 min and the mixture was applied in triplicate to Vero cells grown in 96-well microtiter plates for 2 h at 37°C. After washing, fresh media was applied and cell viability was assessed 48 h later. Data shown is representative for each group of immunized animals (n = 5) with the error bars denoting one S.E.M. of the average of three replicate wells of treated Vero cells.</p

Immunization with LT-IIb(T13I) by the i.n. route enhances RiVax-specific Ab production.

<p>Mice were immunized intranasally with 5.0 µg of RiVax alone or with 1.0 µg of LT-IIb or LT-IIb(T13I) on days 0, 10, and 20. (A) Level of RiVax-specific IgG Ab in sera from immunized mice on days 7, 17, and 27. (B) Level of RiVax-specific IgG and IgA Ab obtained on day 27 from lung lavage, saliva, and fecal samples from immunized mice. Data (n = 5) are presented as the arithmetic mean with error bars denoting one S.E.M on a logarithmic scale (Y-axis). Key: *, p<0.05; **, p<0.01; ***, p<0.001 compared to RiVax. Data were compared using an unpaired Student’s t-test.</p

I.n. immunization of RiVax with LT-IIb(T13I) enhances protective immunity to ricin.

<p>Mice were immunized intranasally on days 0, 10, 20, and 34 with 0.5 µg of RiVax in the presence or absence of 1.0 µg of LT-IIb or LT-IIb(T13I) and challenged 14 days after the final immunization with 10 LD₅₀ of ricin. (A) Survival of mice after i.p. challenge with ricin. Data were compared by the Logrank test. (B) Blood glucose levels of immunized mice during ricin challenge. Data are presented as the arithmetic mean with error bars denoting one S.E.M. (n = 4 or 5) Key: **, p<0.01 compared to RiVax; †, p<0.01 compared to 0 h. Data were compared using an unpaired Students t-test.</p