p53 associates with and targets ΔNp63 into a protein degradation pathway

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation.

Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.

The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.

Identification and characterization of an alternative cytotoxic T lymphocyte-associated protein 4 binding molecule on B cells.

To determine whether alternative cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins exist on B cells, we constructed (i) mCTLA4hIgG consisting of the extracellular region of a mouse CTLA4 molecule and the Fc portion of a human IgG1 molecule and (ii) PYAAhIgG, a mutant mCTLA4hIgG, having two amino acid substitutions on the conserved MYPPPY motif in the complementarity-determining region 3-like region and lacking detectable binding to both B7-1 and B7-2 molecules. Using these fusion proteins (mCTLA4hIgG and PYAAhIgG), we demonstrated that a mouse immature B-cell line, WEHI231 cells, expressed alternative CTLA4 binding molecules (ACBMs) that were distinct from both B7-1 and B7-2. ACBMs were 130-kDa disulfide-linked proteins. More importantly, ACBMs were able to provide costimulatory signal for T-cell proliferation in the presence of anti-CD3 monoclonal antibodies. In addition, we demonstrated that more than 20% of B220+ cells obtained from normal mouse spleen expressed ACBMs.

Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H.

Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.

Novel particulate BCG-loaded delivery system for mucosal immunization against tuberculosis

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Administration routes affect the quality of immune responses:a cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Obesity And Inflammation And The Effect On The Hematopoietic System.

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Intermittent Fasting Induces Hypothalamic Modifications Resulting In Low Feeding Efficiency, Low Body Mass And Overeating.

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.