Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

BACKGROUND:The allochimeric MHC class I molecule [alpha1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells. METHODOLOGY/PRINCIPAL FINDINGS:To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs). CONCLUSIONS:These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation

Osteocalcin Effect on Human β-Cells Mass and Function

The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes

Allochimeric molecules and mechanisms in abrogation of cardiac allograft rejection.

International audienceBACKGROUND: Dendritic cells are professional antigen presenting cells that perform antigen processing and antigen presentation functions and rely on the proper functioning and distribution of the endoplasmic reticulum (ER) and Golgi apparatus and of vesicular trafficking pathways. We previously developed a model system to study the mechanisms governing inhibition of chronic rejection of heart allografts. METHODS: Heterotopic cardiac transplants were placed intra-abdominally and the major histocompatibility class (MHC) class I allochimeric molecule, [α1h1/u]-RT1.Aa, which contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) sequences, was delivered by portal vein to the recipients of heterotopic hearts. Dendritic cells were isolated from the recipient bone marrow at 1 and 3 days after transplantation and were immunostained or processed for Western blotting with anti-RhoB, translationally controlled tumor protein (TCTP), Sprouty-related (Spred1) protein, ER, and Golgi antibodies. RESULTS: Western blotting analyses showed the downregulation of RhoB GTPase, TCTP, and Spred1 in dendritic cells isolated from allochimeric molecule-treated rats. Immunostaining showed that in these cells, Spred 1 was shifted to the base of cellular processes, Rho B formed nonvesicular band in the cell equator, and TCTP was highly enriched in the cell nucleus. The Golgi apparatus was drastically reduced in size and formed a tiny nonvesicular aggregate, and the ER partially lost vesicular appearance. CONCLUSIONS: The function of allochimeric molecule in the abrogation of heart allograft rejection may rely on the downregulation of RhoB pathway components that regulate the structure and function of the ER/Golgi/vesicular trafficking pathways involved in antigen processing and presentation by dendritic cells

Clinico-Radiological Outcomes in WNT-Subgroup Medulloblastoma

Medulloblastoma (MB) comprises four broad molecular subgroups, namely wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4, respectively, with subgroup-specific developmental origins, unique genetic profiles, distinct clinico-demographic characteristics, and diverse clinical outcomes. This is a retrospective audit of clinical outcomes in molecularly confirmed WNT-MB patients treated with maximal safe resection followed by postoperative standard-of-care risk-stratified adjuvant radio(chemo)therapy at a tertiary-care comprehensive cancer centre. Of the 74 WNT-MB patients registered in a neuro-oncology unit between 2004 to 2020, 7 patients accrued on a prospective clinical trial of treatment deintensification were excluded, leaving 67 patients that constitute the present study cohort. The median age at presentation was 12 years, with a male preponderance (2:1). The survival analysis was restricted to 61 patients and excluded 6 patients (1 postoperative mortality plus 5 without adequate details of treatment or outcomes). At a median follow-up of 72 months, Kaplan–Meier estimates of 5-year progression-free survival and overall survival were 87.7% and 91.2%, respectively. Traditional high-risk features, large residual tumour (≥1.5 cm2), and leptomeningeal metastases (M+) did not significantly impact upon survival in this molecularly characterized WNT-MB cohort treated with risk-stratified contemporary multimodality therapy. The lack of a prognostic impact of conventional high-risk features suggests the need for refined risk stratification and potential deintensification of therapy

Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells

The translationally controlled tumor protein (TCTP) plays a role in cell growth, cell cycle and cancer&lt;br /&gt;progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the&lt;br /&gt;cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased&lt;br /&gt;migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,&lt;br /&gt;a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA&lt;br /&gt;expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,&lt;br /&gt;cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing&lt;br /&gt;low level of inducible p53) ovarian epithelial cancer cells with different metastatic potential. Immunostaining&lt;br /&gt;and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin&lt;br /&gt;filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between&lt;br /&gt;the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified&lt;br /&gt;negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked&lt;br /&gt;with the high aggressiveness of ovarian cancers.<br>The translationally controlled tumor protein (TCTP) plays a role in cell growth, cell cycle and cancer&lt;br /&gt;progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the&lt;br /&gt;cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased&lt;br /&gt;migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,&lt;br /&gt;a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA&lt;br /&gt;expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,&lt;br /&gt;cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing&lt;br /&gt;low level of inducible p53) ovarian epithelial cancer cells with different metastatic potential. Immunostaining&lt;br /&gt;and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin&lt;br /&gt;filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between&lt;br /&gt;the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified&lt;br /&gt;negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked&lt;br /&gt;with the high aggressiveness of ovarian cancers

Downregulation of miR-204 expression defines a highly aggressive subset of Group 3/Group 4 medulloblastomas

Abstract Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells