High hopes for RANKL: will the mouse model live up to its promise?

The steroid hormones, estrogens and progesterone are key drivers of postnatal breast development and are linked to breast carcinogenesis. Experiments in the mouse mammary gland have revealed that they rely on paracrine factors to relegate their signal locally and to amplify it. In particular, RANKL is a key mediator of progesterone action. Systemic inhibition of RANKL blocked proliferation in the mammary epithelium with potential clinical implications: a RANKL-inhibiting antibody, Denosumab (Amgen), has been approved by the US Food and Drug Administration for osteoporosis treatment. Two publications now provide evidence that progestin-driven mouse mammary tumorigenesis can be blocked by ablating RANK signaling. Can the osteoporosis drug help breast cancer patients? The burning question now is whether the role of this pathway is conserved in the human breast and whether RANKL signaling has a role in the pathogenesis of one or more subtypes of breast cancer

The small GTP-binding protein RhoA regulates c-Jun by a ROCK-JNK signaling axis

RhoA regulates the actin cytoskeleton and the expres- sion of genes associated with cell proliferation. This includes c-fos and c-jun, which are members of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth. Whereas RhoA stimulates the c-fos SRE by a recently elucidated mechanism that is dependent on actin treadmilling, how RhoA regulates c-jun is still poorly understood. We found that RhoA stimulates c-jun expression through ROCK, but independently from the ability of ROCK to promote actin polymerization. Instead, we found that ROCK activates JNK, which then phosphor- ylates c-Jun and ATF2 when bound to the c-jun pro- moter. Thus, ROCK represents a point of signal diver- gence downstream from RhoA, as it promotes actin reorganization and the consequent expression from the c-fos SRE, while a parallel pathway connects ROCK to JNK, thereby stimulating c-jun expression. Ultimately, these pathways converge in the nucleus to regulate AP1 activity

Resting cells rely on the DNA helicase component MCM2 to build cilia

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues

Isolation of putative stem cells present in human adult olfactory mucosa

The olfactory mucosa (OM) has the unique characteristic of performing an almost continuous and lifelong neurogenesis in response to external injuries, due to the presence of olfactory stem cells that guarantee the maintenance of the olfactory function. The easy accessibility of the OM in humans makes these stem cells feasible candidates for the development of regenerative therapies. In this report we present a detailed characterization of a patient-derived OM, together with a description of cell cultures obtained from the OM. In addition, we present a method for the enrichment and isolation of OM stem cells that might be used for future translational studies dealing with neuronal plasticity, neuro-regeneration or disease modeling

Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase

Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis

Isolation of putative stem cells present in human adult olfactory mucosa

<div><p>The olfactory mucosa (OM) has the unique characteristic of performing an almost continuous and lifelong neurogenesis in response to external injuries, due to the presence of olfactory stem cells that guarantee the maintenance of the olfactory function. The easy accessibility of the OM in humans makes these stem cells feasible candidates for the development of regenerative therapies. In this report we present a detailed characterization of a patient-derived OM, together with a description of cell cultures obtained from the OM. In addition, we present a method for the enrichment and isolation of OM stem cells that might be used for future translational studies dealing with neuronal plasticity, neuro-regeneration or disease modeling.</p></div

Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis

Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Supanitsky, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Echeverria, Pablo C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Tanos, Tamara. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant

Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis

Fil: Martin, Valentina. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Supanitsky, Alicia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Echeverria, Pablo C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Litwin, Silvana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Tanos, Tamara. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant

Characterization of the olfactory lamina propria.

<p>Immunostaining of the olfactory mucosa for S100 and S100β in A, Vimentin (Vim) and CK8 in B, SMA and CK8 in C, Fibronectin (FN) and CK8 in D. Hematoxylin-Eosin staining of nerve fascicles (E) and immunostaining for Tuj1 and Nestin in F, for Synapto and NSE in G, for Vimentin (Vim) in H, for Fibronectin (FN) in I and for S100 and S100β in J. In all the immunofluorescence panels, Dapi was used as nuclear counterstaining. In OM, dashed lines indicate basement membrane and yellow asterisks indicate Bowman´s glands. In E-J, dashed lines indicate the limits of nerve fascicles. Scale bar, 20 μm.</p

Antigenic markers of OM cells in culture.

<p>A. Representative FACS data of OM cells, SK-N-BE neuroblastoma cell line, U87MG glioblastoma cell line, primary fibroblasts (Fib) and primary epithelial cells. The antibodies used were anti-CD31 PE Cy7 and CD45 FITC in upper panels, and CD56 PE and Epcam FITC in lower panels. Data are presented as dot plots to visualize the expression of each marker. The percentage of the gated population is included. B. Western blots to detect Fibronectin (FN), Tuj1, Epcam, CK14, Vimentin (Vim l.e. and h.e., low and high exposure), p63, CK8 and CK5. Vinculin (Vinc) was used as loading control. C. Immunostaining for Tuj1, NSE, Nestin, S100β, FN, Vim and CK8. Dapi was used to stain nuclei. Bar, 50 μm.</p