[Show abstract][Hide abstract] ABSTRACT: BACKGROUND Hematopoietic stem cells transplantation has high clinical potential against a wide variety of hematologic, metabolic, and autoimmune diseases and solid tumors. Clinically, hematopoietic stem cells derived from peripheral blood are currently used more than those obtained from sources such as bone marrow. However, mobilizing agents used in the clinic tend to fail in high rates, making the number of mobilized cells insufficient for transplantation. We investigated whether sodium caseinate induces functional mobilization of hematopoietic stem cells into peripheral blood of Balb/c mice. MATERIAL AND METHODS Using a mouse model, we administrated sodium caseinate or Plerixafor, a commercial mobilizing agent, and analyzed counts of hematopoietic stem cells in peripheral blood, and then cells were transplanted into lethally irradiated mice to restore hematopoiesis. All assays were performed at least twice. RESULTS We found that sodium caseinate increases the number of mononuclear cells in peripheral blood with the immunophenotype of hematopoietic stem cells (0.2 to 0.5% LSK cells), allowing them to form colonies of various cell lineages in semisolid medium (p<0.05). This effect is similar to that of Plerixafor, and cells transplanted into lethally irradiated mice can restore hematopoiesis at higher percentages than mononuclear cells mobilized by Plerixafor (40% vs. 20%, respectively). Further, a secondary transplant rescued a separate group of irradiated mice from death, proving definitive evidence of hematopoietic reconstitution after hematopoietic stem cells transplantation. Data are presented as mean ± standard deviation. To determine significant differences between the data, one-way ANOVA and the Tukey test were used. CONCLUSIONS Collectively these results show the utility of sodium caseinate as a mobilizer of hematopoietic stem cells and its potential clinical application in transplantation settings.
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer (CeCa) tumors are characterized by increased expression of TGF-β1 and IL-10, which are correlated with downregulated expression of major histocompatibility complex class I antigens (HLA-I) on cancer cells and a reduced immune response mediated by cytotoxic T lymphocytes (CTLs). Mesenchymal stromal cells (MSCs) are important components in the tumor microenvironment that have been suggested to contribute to cancer progression through the induction of TGF-β1 and IL-10. In this study, we provided evidence that MSCs derived from cervical tumors (CeCa-MSCs) cocultured with CeCa cells induced significant expression of TGF-β1 and secretion of IL-10 by CeCa cells compared to MSCs derived from the normal cervix (NCx-MSCs) and normal bone marrow (BM-MSCs; gold standard). This increase in expression was associated with a significant downregulation of HLA-I molecules and protection of the cells against specific CTL lysis. Interestingly, the addition of the neutralizing antibody anti-TGF-β to the CeCa/CeCa-MSCs coculture strongly inhibited the expression and production of IL-10 by CeCa cells. Anti-TGF-β as well as anti-IL-10 also abolished HLA-I downregulation, and reversed the inhibition of CTL cytotoxicity. These results provide evidence that TGF-β1 and IL-10 could play an important role in the downregulation of HLA-I molecules on CeCa cells induced by tumor MSCs. Our findings suggest a novel mechanism through which MSCs may protect tumor cells from immune recognition by specific CTLs.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein, we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSCs populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papiloma virus (HPV), indicating that these cells are not infected by such a viral agent. Also interesting, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with production of IL-10 in cell co-cultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing down-regulation of HLA class I molecules. This mechanism may have important implications in tumor growth.
Stem cells and development 05/2013; 22(18). DOI:10.1089/scd.2013.0084 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Most infections with human papillomavirus (HPV) are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL) from low-grade squamous intraepithelial lesions (LSIL) patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors. Results: We found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonists in vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PBL from patients infected by HPV-16 and ?18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells.
Infectious Agents and Cancer 05/2012; 7(1):12. DOI:10.1186/1750-9378-7-12 · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presence of IgG antibodies to HPV-16 L1-virus like particles (VLPs) in serum has been reported as a result of persistent exposure to the virus and as a marker of disease progression. However, detection of VLP-specific antibodies in sera does not always indicate a malignant lesion as positive results may also be due to a nonmalignant viral infection. Furthermore, malignant lesions are associated with an increased antibody titer for E6 and E7 proteins. The aim of this study was to develop an ELISA using a novel chimeric virus-like particle (cVLP) encoding an L1 protein fused with a string of HPV-16 E6 and E7 seroreactive epitopes to its C-terminus to be used for detection of HPV-16 specific antibodies in patients with cervical intraepithelial lesion grade 1 (CIN 1).
The sera of 30 patients with CIN 1 who also tested positive for HPV-16 DNA and of 30 age-matched normal donors negative for HPV infection were tested for the presence of IgG antibodies specific for either VLP-L1 (HPV-16 L1), gVLP (derived from Gardasil), or cVLP by ELISA. The cVLP-reactive sera yielded two distinct groups of results: (H) reactivity levels that presented very strong cVLP-specific titers, and (L) reactivity levels with significantly lower titers similar to those obtained with VLP-L1 and gVLP antigens. Additionally, the sera that presented the higher cVLP titers closely matched those that had significantly stronger reactivity to E6 and E7 epitopes. Interestingly, the samples with the highest titers corresponded to patients with the higher numbers of sexual partners and pregnancies. On the other hand only 4 out of the 12 sera that harbored antibodies with VLP neutralizing ability corresponded to the group with high cVLP antibody titers.
We report for the first time that chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes enable much better detection of IgG antibodies in the sera of CIN 1 patients positive for HPV-16 infection than those obtained with VLPs containing only the HPV-16 L1 protein. We also found that the sera with higher cVLP antibody titers corresponded to patients with more sexual partners and pregnancies, and not always with to those with a high neutralizing activity. This novel assay could help in the development of a tool to evaluate cervical cancer risk.
[Show abstract][Hide abstract] ABSTRACT: Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility.
We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes.
In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing prophylactic/therapeutic vaccines. The fact that they are produced in plants, may lower production costs considerably.
[Show abstract][Hide abstract] ABSTRACT: Introducción: La hipermetilación del ADN y la desacetilación de histonas en tumores malignos, son eventos epigenéticos que contribuyen a la pérdida o disminución de moléculas clase I del complejo principal de histocompatibilidad (HLA-I) y del proce-samiento de antígenos, lo cual afecta el reconocimiento inmune mediado por linfocitos T citotóxicos (LTC). 1 En cáncer cérvico-uterino (CaCu), más del 90% de los tumores presentan pérdida o disminución de la expresión de estas moléculas. 2 Por otro lado, se tiene probado que hidralazina (H), funciona como una droga que inhibe la metilación del ADN; mientras que el ácido valproico (AV), causa hiperacetilación de histonas en tumores sólidos, lo cual se relaciona con la inducción de la expresión de proteínas supresoras de tumor y del reconocimiento inmune. 3 Objetivo: Analizar la expresión de moléculas HLA clase I y del procesamiento antigénico en células tumorales de CaCu tratadas con el agente desmetilante hidrlazina (H), el inhibidor de desacetilasas de histonas ácido valproico (AV) e interferón gama (IFN-γ).
[Show abstract][Hide abstract] ABSTRACT: DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells.
Cell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 microM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid.
Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma.
These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.
Journal of Translational Medicine 02/2006; 4(1):55. DOI:10.1186/1479-5876-4-55 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A nonapeptide (16L1) was derived from the human papillomavirus type 16 (HPV-16) major capsid protein and tested for detection of potential cross-reactive serum IgG and cervical IgA antibodies in low- and high-risk HPV-associated low-grade squamous intraepithelial lesions (LSIL) and cervical cancer patients by ELISA. The IgG response was similar in women with low-risk HPV-associated LSIL and controls (P=0.1). In contrast, more than 90 % of patients with high-risk HPV-associated LSIL were seropositive. Although tumours from cancer patients were all positive for the presence of high-risk HPV DNA, the level of seropositivity decreased significantly in this group (P<0.0001). Cervical IgA antibodies were also detected in a significantly high proportion of women with high-risk HPV-associated LSIL compared with controls. However, the proportion of IgA-positive patients was lower than the proportion of IgG seropositives. In conclusion, the 16L1 peptide appears to be a high-risk type-common epitope that induces cross-reactive antibodies in high-risk, but not low-risk, HPV-associated LSIL patients, allowing differentiation of high- and low-risk infected women at this stage of infection.
Journal of General Virology 09/2004; 85(Pt 9):2643-50. DOI:10.1099/vir.0.80077-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work we present evidence that the homologous peptides IHSMNSTIL and IHSMNSSIL derived from L1 HPV-16 and 18 proteins respectively, and with high specificity for the allele HLA-B*3901, according with an algorithm prediction program, induced T cell stimulation in patients with advanced cervical cancer positive for HPV-16 or 18 infection and for the HLA-B*3901 allele. Interestingly, T lymphocytes derived from a patient with HPV-18 infection and stimulated with the peptide IHSMNSTIL were capable to kill a cervical cancer cell line named Rova, derived from the tumor of the same patient. In addition, the cytotoxic activity was strongly increased when this cell line was previously treated with hrIFN-gamma. These results suggest that the CTL immune response to L1 HPV-16 and 18 protein derived epitopes is maintained in patients with advanced cervical cancer within specific alleles, and opens the possibility that homologous epitopes may be used in the generation of prophylactic vaccines for cervical tumors bearing different HPV-types.
Archives of Virology 11/2002; 147(10):1933-42. DOI:10.1007/s00705-002-0854-y · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.