RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

Background: NADPH-cytochrome- P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling. Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Functional CD40-ligand is expressed by T cells in rheumatoid arthritis

CD40-1igand (CD40-L), a member of the tumour necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. CD40 is expressed by B cells, monocytes and dendritic cells. Interactions between CD40-L and CD40 induce B cell proliferation, differentiation, immunoglobulin production and isotype switching as well as monocyte activation and dendritic cell differentiation. Since the rheumatoid synovium is characterized by T cell activation, B cell immunoglobulin production, monocyte cytokine production and dendritic cell differentiation, the expression and function of CD40-L in RA was examined. RA synovial fluid (SF) T ceils expressed CD40-L mRNA, as well as low level cell surface CD40-L. A subset of CD4+ RA synovial fluid T cells could express cell surface CD40-L within 15 rain of in vitro activation even in the presence of cycloheximide. CD40-L expressed by RA SF T cells was functional, since RA SF T cells, but not normal PB T cells, stimulated CD40-L dependent B cell immunoglobulin production in the absence of in vitro T cell activation. These data indicate that SF T cells express functionally significant levels of surface CD40-L, and have the potential for rapid upregulation of surface expression from preformed CD40-L stores. Thus, CD40-L is likely to play a central role in the perpetuation of RA by induction of Ig synthesis, cytokine production and dendritic cell differentiation. Moreover, the data provide important evidence of recent activation of RA synovial T cells. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF)T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Decreased AIRE Expression and Global Thymic Hypofunction in Down Syndrome

The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 +/- 49.59 AIRE-positive cells/mm(2) in DS versus 154.70 +/- 61.16 AIRE-positive cells/mm(2) in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm(2) was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-related genes included those involved in 1) Ag processing and presentation (HLA-DQB1, HLA-DRB3, CD1A, CD1B, CD1C, ERAP) and 2) thymic T cell differentiation (IL2RG, RAG2, CD3D, CD3E, PRDX2, CDK6) and selection (SH2D1A, CD74). It is noteworthy that relevant AIRE-partner genes, such as TOP2A, LAMNB1, and NUP93, were found hypoexpressed in DNA microarrays and quantitative real-time PCR analyses. These findings on global thymic hypofunction in DS revealed molecular mechanisms underlying DS immune phenotype and strongly suggest that DS immune abnormalities are present since early development, rather than being a consequence of precocious aging, as widely hypothesized. Thus, DS should be considered as a non-monogenic primary immunodeficiency. The Journal of Immunology, 2011, 187: 3422-3430.

Prognostic relevance of TTF-1 and MMP-9 expression in advanced lung adenocarcinoma

Background: The thyroid transcription factor-1 (TTF-1) is a tissue-specific transcription factor that Could playan important role in cell differentiation and morphogenesis of lung tumors. Matrix metalloproteinase-9 (MMP-9) is a protease commonly expressed in non-small cell lung cancer, conferring angiogenic and metastatic potential. Methods: We assessed TTF-1 and MMP-9 tumor expression by immunohistochemistry in 51 patients with lung adenocarcinoma, stage 11113 or IV, treated with platinum regimens. A bicategorical prognostic model was obtained using the Kaplan-Meier method, COX regression, and conjunctive consolidation. Results: The median expression of TTF-1 was 30.0% (range: 0-85.9%). All tumors expressed MMP-9 (median: 78.7%: range: 15.2-96.1%). Median survival was 41.6 weeks, with estimated 1- and 2-year survival rates of 45.0% and 22.0%, respectively. Poor performance status (Karnofsky scale) - hazards ratio(HR): 1.03. 95% confidence interval (CI): 1.01-1.06: low TTF-1 expression (<40%) - FIR: 4.00, 95% CI: 1.75-9.09: and high MMP-9 expression (>= 80%) - HR: 2.82, 95% CI: 1.30-6.08 were independent prognostic factors. Patients could be stratified in three death risk groups according to markers expression: low risk (high TTF-1 and low MMP-9; median survival: 127.6 weeks), intermediate risk (low TTF-1 OF high MMP-9; median survival: 39.0 weeks): and high risk (low TTF-1 and high MMP-9: median survival: 16.4 weeks). Conclusion: TTF-1 and MMP-9 tumor expression as detected by immunohistochemistry may allow identification of different, clinically meaningful, prognostic groups of advanced lung adenocarcinoma patients treated with platinum regimens. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1(+/-) and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploin-sufficient mice, a process that apparently depends on a relative deficiency of p2l activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.

Dual origin of mesenchymal stem cells contributing to organ growth and repair

In many adult tissues, mesenchymal stem cells (MSCs) are closely associated with perivascular niches and coexpress many markers in common with pericytes. The ability of pericytes to act as MSCs, however, remains controversial. By using genetic lineage tracing, we show that some pericytes differentiate into specialized tooth mesenchyme-derived cells-odontoblasts-during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSC-like properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. Thus, although pericytes are capable of acting as a source of MSCs and differentiating into cells of mesenchymal origin, they do so alongside other MSCs of a nonpericyte origin. This study identifies a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSC-derived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity.

Peroxiredoxin I is differentially expressed in multiple myelomas and in plasmablastic lymphomas

BACKGROUND: Plasmablastic lymphoma (PBL) and multiple myeloma (MM) are B cell-derived malignancies that share many morphologic and immunophenotypic traits, making the differential diagnosis particularly complicated. We have recently demonstrated that peroxiredoxin I (PrdxI) is expressed in plasma cells but not in B lymphocytes, suggesting that its expression is development-associated. AIM: To analyze PrdxI expression in PBL and in MM in order to study its utilization as an additional diagnostic molecular tool. METHODS AND RESULTS: Eight cases of PBL and nine of MM were studied by immunohistochemistry. We have demonstrated that PrdxI expression is closely connected with the immunoglobulin production capacity of the cells, which means high in MM, but absent in PBL cases, except one, wherein few cells were stained. CONCLUSIONS: We hypothesize PrdxI as a component of the unfolded protein response (UPR), an adaptive pathway essential for plasma cell differentiation. As we have not detected immunoglobulin in our PBL cases, we suggest that UPR was not activated in the cells, accounting for the impediment of the developmental process, and for the inhibition of PrdxI expression observed. PrdxI could be considered an additional plasma cell functional marker and could also be speculated as a therapeutic target in the treatment of MM. Oral Diseases (2008) 14, 741-746

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Stem cells in the aetiopathogenesis and therapy of rheumatic disease

Animal models of autoimmune disease and case reports of patients with these diseases who have been involved in bone marrow transplants have provided important data implicating the haemopoietic stem cell in rheumatic disease pathogenesis. Animal and human examples exist for both cure and transfer of rheumatoid arthritis, systemic lupus erythematosus (SLE) and other organ-specific diseases using allogeneic haemopoietic stem cell transplantation. This would suggest that the stem cell in these diseases is abnormal and could be cured by replacement of a normal stem cell although more in vitro data are required in this area. Given the morbidity and increased mortality in some patients with severe autoimmune diseases and the increasing safety of autologous haemopoietic stem cell transplantation (HSCT), pilot studies have been conducted using HSCT in rheumatic diseases. It is still unclear whether an autologous graft will cure these diseases but significant remissions have been obtained which have provided important data for the design of randomized trials of HSCT versus more conventional therapy. Several trials are now open to accrual under the auspices of the European Bone Marrow Transplant Group/European League Against Rheumatism (EBMT/EULAR) registry. Future clinical and laboratory research will need to document the abnormalities of the stem cell of a rheumatic patient because new therapies based on gene therapy or stem cell differentiation could be apllied to these diseases. With increasing safety of allogeneic HSCT it is not unreasonable to predict cure of some rheumatic diseases in the near future.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase

Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane microlocalization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC 12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains similar to16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.