Serological Biomarkers for the Prediction and Detection of Human Papillomavirus Associated Cancers

High-risk human papillomavirus (HPV) types are not only associated to uterine cervical cancer, but also to a fraction of cancers of the vulva, vagina, penis, anus, head and neck. An HPV infection generates a protective humoral immune response against the capsid proteins L1 and L2; however, an immune response against other HPV early proteins is also generated. This latter is not a protective response, but those antibodies can be useful as biomarkers of the status of the infection and/or the stage of the cancer lesion. Until now, there are no conclusive results regarding the use of anti-HPV antibodies as biomarkers in diagnosis. In this review, we hereby summarized the actual panorama of the humoral immune response against different HPV early proteins during the development of the disease as possible biomarkers for the prediction and detection of HPV-associated cancers

The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27Kip1

AbstractE5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27Kip1 expression at protein level

The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination

CD4+ T cells are major players in the immune response against several diseases; including AIDS, leishmaniasis, tuberculosis, influenza and cancer. Their activation has been successfully achieved by administering antigen coupled with antibodies, against DC-specific receptors in combination with adjuvants. Unfortunately, most of the adjuvants used so far in experimental models are unsuitable for human use. Therefore, human DC-targeted vaccination awaits the description of potent, yet nontoxic adjuvants. The nontoxic cholera B subunit (CTB) can be safely used in humans and it has the potential to activate CD4+ T cell responses. However, it remains unclear whether CTB can promote DC activation and can act as an adjuvant for DC-targeted antigens. Here, we evaluated the CTB's capacity to activate DCs and CD4+ T cell responses, and to generate long-lasting protective immunity. Intradermal (i.d.) administration of CTB promoted late and prolonged activation and accumulation of skin and lymphoid-resident DCs. When CTB was co-administered with anti-DEC205-OVA, it promoted CD4+ T cell expansion, differentiation, and infiltration to peripheral nonlymphoid tissues, i.e., the skin, lungs and intestine. Indeed, CTB promoted a polyfunctional CD4+ T cell response, including the priming of Th1 and Th17 cells, as well as resident memory T (RM) cell differentiation in peripheral nonlymphoid tissues. It is worth noting that CTB together with a DC-targeted antigen promoted local and systemic protection against experimental melanoma and murine rotavirus. We conclude that CTB administered i.d. can be used as an adjuvant to DC-targeted antigens for the induction of broad CD4+ T cell responses as well as for promoting long-lasting protective immunity

CD43 Promotes Cells Transformation by Preventing Merlin-Mediated Contact Inhibition of Growth

<div><p>In normal tissues, strict control of tissue size is achieved by regulating cell numbers. The mechanism that controls total cell number is known as contact inhibition of growth and it depends on the NF2/Merlin pathway. Negative regulation of this pathway by deleterious mutations or by oncogenes results in cell transformation and tumor progression. Here we provide evidence that the CD43 sialomucin cooperates with oncogenic signals to promote cell transformation by abrogating the contact inhibition of growth through a molecular mechanism that involves AKT-dependent Merlin phosphorylation and degradation. Accordingly, inhibition of endogenous CD43 expression by RNA interference in lung, cervix and colon human cancer cells impaired tumor growth <i>in vivo</i>. These data underscore a previously unidentified role for CD43 in non-hematopoietic tumor progression. </p> </div

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>In epithelial cells, upon interaction with putative ligand(s) present on the cell surface of neighboring cells or in the extracellular matrix, CD43 activates the PI3K/AKT pathway that results in the inhibition of the Hippo pathway by a mechanism involving Merlin phosphorylation and degradation, thus favoring cell survival and proliferation. Depending of the cellular context, signaling from intracellular CD43 located either on membrane vesicles or the nuclear membrane may also contribute to cellular transformation. Nonetheless, this might not be sufficient to overcome the anti-proliferative and death effects resulting from the activation of the ARF-p53 pathway also induced by CD43 [24]. However, in transformed cells with an impaired p53 pathway resulting from oncogenic signals like those provided by the E6 oncoprotein from HPV16, CD43 signaling promotes cells proliferation and tumor formation. </p

CD43 expression confers tumoral fitness to human tumor-derived cells.

<p>A549 lung (<b>A</b>), CasKi cervix (<b>B</b>) or DLD-1 colon (<b>C</b>) tumor cells containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were cultured to confluence, the monolayer was then wounded and healing was evaluated (upper panel). Cells were also cultured in soft agar as indicated in material and methods, after three weeks colonies were counted (middle panel). Cells (1X10⁶ for A549, 3X10⁶ for CasKi or DLD-1) were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor weight was evaluated (lower panel). Data represents the average ± SD of four independent experiments performed with four independent pSup or RNAi clones for each cell line. *p < 0.05, **p < 0.01 vs pSup.</p

CD43 intracellular domain is required to promote cell transformation.

<p>Total cell extracts from A549 lung tumor cells stably containing the empty pFNeo vector (pFNeo) or expressing CD43 lacking the intracellular domain (ΔIC) were resolved on SDS-PAGE and transferred to nitrocellulose membranes. CD43 expression levels were determined by immunoblot using anti-CD43 specific antibodies (<b>A</b>). The arrowhead indicates the endogenous wild-type CD43 molecule; the bracket, the CD43 mutant lacking the intracellular domain. A549 lung tumor cells were grown to confluence, the monolayer was then wounded and healing was evaluated by light microscopy (<b>B</b>) or were grown in soft agar as described in material and methods and three weeks later colonies formed were counted (<b>C</b>). The data represents the average ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs pFNeo.</p

CD43 signaling cooperates with oncogenic signals to promote cell transformation.

<p>NIH-3T3-hEGFR (<b>A</b>) or E6/E7 transgenic mouse fibroblasts (<b>C</b>) carrying the pFNeo empty vector (pFNeo), expressing wild-type CD43 (Wt) or CD43 lacking the intracellular domain (ΔIC) were grown to confluence; the monolayer was then wounded (t=0) and healing was evaluated by light microscopy at the indicated time points. NIH-3T3-hEGFR fibroblasts were grown in soft agar as described in material and methods; after three weeks, colonies were counted (<b>B</b>). E6 transgenic mouse fibroblasts stably transfected with the indicated constructs were grown to confluence for three weeks; foci were stained with Giemsa and counted (<b>D</b>). 3X10⁶ E6/E7 fibroblasts stably transfected were injected subcutaneously into nu/nu mice; one month later, animals were sacrificed and tumor mass was weighed (<b>E</b>). Data shown are representative of at least four independent experiments performed with at least four independent pFNeo, Wt or ΔIC clones from each cell line. Graphs represent the average cell number ± SD of three independent experiments using at least 3 independent clones. *p < 0.05 vs pFNeo.</p

Rotavirus Infection Activates Dendritic Cells from Peyer's Patches in Adult Mice ▿ †

This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIMwt). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-α), and beta interferon (IFN-β), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response

Merlin mediates the inhibition of A549 cell proliferation by cell-cell contact.

<p><b>A</b>) A549 clones expressing the CD43 specific RNAi (RNAi) were transfected with non-specific (Ctl) or Merlin specific siRNAs (Merlin). 48 hrs after cells reached confluence total cell extract were prepared and Merlin protein levels (Merlin) as well as phosphorylated Yap levels (p-YAP) were evaluated by immunobloting using specific antibodies. Actin levels were used as loading control. <b>B</b>) The proliferation capacity of A549 clones expressing the CD43 specific RNAi (RNAi) transfected with non-specific (Ctl) or Merlin specific siRNA (Merlin) was evaluated at the indicated time points after cells reached confluence. Data represent the average of three independent experiments using two different clones.</p