Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs), which are artificial molecules containing antibody-derived fragments (to bind the specific target), joined with potent signalling T-Cell Receptor (TCR)-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future. Chimeric T-cell Antigen Receptors (CARs) are a fascinating bio-technologic step in the field of immunotherapy to orient the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumours, including haematological malignancies. CARs are artificial T-cell receptors constituted by an antigen-recognizing extracellular domain derived from an antibody molecule linked to a T-cell triggering domain -4. CARs The CAR approach permits to overcome the major limitations associated with the use of a "classical" TCR transgenic molecule. In fact, target recognition by CAR is non-MHC restricted and independent of antigen processing, therefore allowing its use in patients with different haplotypes and bypassing tumour escape due to MHC-molecules down-regulation. In addition, CARs can be targeted toward molecules other than peptides, like carbohydrates and glycolipids, and there is no risk to trigger unpredictable and potentially harmful specificities, as it may happen with transduced TCR, that could form hybrids with the endogenous TCR -4.Different CARs have been generated so far ,3,5, agahttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1160141/. This software calculates the overlapping oligonucleotides following the putative sequence with specific values of T melting (Tm), thus avoiding non-specific and random annealing possibilities. The overlapping oligonucleotides are then joined together exploiting a basic PCR reaction in order to generate the putative template. A second reaction of PCR is then needed to amplify the new synthetic template using flanking primers Figure The ectodomain represents the extracellular part of the artificial T-cell receptor. It is generally composed by a signal peptide, an antigen recognition region and a spacer sequence. The signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor needs to be glycosylated and anchored in the cell membrane leading to the CAR expression. The antigen recognition domain is usually a scFv, obtained by the fusion of the variable regions of the heavy (VH) and light (VL) chains of Immunoglobulins (Igs), joined together with a short linker. The latter is usually a serine-glycine rich motif, whose flexibility assures a high mobility capacity to the molecule Figure http://www.childhope.eu/contents.php have been developed, funded in the contest of a STREP (6th framework) to target high-risk B-ALL in molecular relapse after transplantation using donor-derived anti-CD19. CAR-expressing EBV-CTLs. The preclinical in vivo data, targeting primary patient-derived high-risk B-ALL [Haematological malignancies represent optimal targets for the exploitation of CARs, because of the bright expression of specific antigens on the surface of tumour cells. Previous studies from our group demonstrated that B-lineage acute lymphoblastic leukaemia can be efficiently targeted by a CAR directed against the CD19 antigen . We havesk B-ALL , demonstin vivo superior effect of second generation CARs containing, beyond the zeta-chain, a costimulatory molecule, specifically the CD28 domain. Patients with B cell lymphomas were infused with two different autologous T-cell products expressing CARs with the same specificity for the CD19 antigen: one CAR encoded both the costimulatory CD28 and the zeta endodomains, while the other presented the zeta endodomain alone. CAR-expressing T cells containing the CD28 endodomain showed markedly enhanced growth and persistence compared with CAR-expressing T cells encoding the zeta endodomain alone [Similarly, four other main groups are developing CAR-based strategies for targeting B-origin leukaemia-lymphoma neoplasm, by using CD19 as the selected target. Indeed, the MK Brenner's group has recently published interesting even though preliminary observations concerning the in alone . Similarin alone ,14. Bothin alone .Other potential optimal targets of CAR-gene transfer are represented by Acute Myeloid Leukaemia (AML) and Chronic Lymphocytic Leukaemia (B-CLL), because of the selective expression of tumour-associated surface antigens, possible targets of CAR recognition.+ myeloid leukaemia using gemtuzumab ozogamicin (Mylotarg) [in vitro, has been successful for AML [in vitro model that strongly demonstrates the efficiency of anti-CD33. CARs towards myeloid cell lines and primary blasts, by using gene modified CIK cells [+ progenitor cells was measured, their residual haematopoietic activity was anyway preserved [In vivo experiments are currently ongoing in order to measure the safety and efficacy profile of anti-CD33. CAR-expressing cells in an immunodeficient mouse model.Immunotoxin-based therapy against CD33ylotarg)  had someylotarg) . With thylotarg) . Immunotylotarg) . The ide for AML . HoweverIK cells . The infIK cells . To imprreserved , thus opin vitro and in vivo model that corroborates this hypothesis [+ tumour cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without a significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also physiologically expressed by normal B lymphocytes. Moreover, autologous CLL-derived-CD23. CAR-expressing T cells significantly released inflammatory cytokines and IL-2 was also produced to sustain the CD23-driven proliferation of such modified T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γc(-/-) xenograft mouse model [+ leukaemia cells in patients affected by B-CLL.Similarly to ALL and AML, B-CLL may represent as well an interesting target for CAR strategy. B-CLL cells expressing CD19 and CD20dim are suitable to be targeted with T cells redirected against these pan-B markers. In fact, CARs have been generated to target these self tissue-restricted B-cell antigens -15. Howepothesis . We clonse model . Indeed,in vivo [in vitro stimulated T cells: their blockade can accelerate tumour eradication [Despite the promising results obtained so far, CAR strategy might be further optimized. Several reports indicate that the addition of costimulatory molecules , either as single domain or as multiple combinations, to the signalling CAR endodomain induces a higher rate of proliferation, higher levels of IL-2 secretion and prolonged effector cells survival with a subsequent better activity in vivo . An addidication . A diffedication . In thisdication .in vivo use of chimeric EBV-CTLs in paediatric relapsed/refractory ALL patients, as planned in a Phase I trial in the context of the "Childhope" STREP-funded network. However, additional preclinical experiments are crucial to compare the respective anti-tumour efficacy of chimeric EBV-CTLs, chimeric CIK cells and chimeric gamma9delta2 T cells (GDT) for future clinical applications. CIK cells are T cells with natural killer cell phenotype and function, enriched in CD3+CD56+ cells [in vivo potent anti-tumour activity, while having reduced propensity to induce GvHD [in vivo or in vitro in the presence of aminobisphosphonates without prior antigen priming [in vitro model, of the above mentioned populations as putative effectors of CAR-mediated immunotherapy [in vitro analysis. Moreover, these results are consistent with recent observations that have emerged from clinical trials with CAR-modified T cells, suggesting the need to perform such type of studies in the human setting.Alternatively to the use of EBV-CTLs, other T-cell subsets have shown some potential for future adoptive immunotherapeutic approaches. In line with previous considerations, there is enough evidence to support the 6+ cells , that ca6+ cells . It has uce GvHD . Phase Iuce GvHD -27. Simiuce GvHD . GDT cel priming . Recentl priming . To unraotherapy . Our resunpublished data) confirm a substantial equivalence of the HSV-TK and Casp9-CID systems, having the latter the double advantage of not being immunogenic and exerting its activity in a more rapid timeframe.An important consideration for the clinical applicability of CAR strategy is represented by the safety issues concerning the use of integrating vector for gene transfer, as well as the potential reactivity of CAR-transduced cells against host-normal cells expressing the target antigen. Furthermore, the potential hazards associated with the use of CAR containing costimulatory or growth-promoting molecules that may improve the survival and the proliferation of manipulated T cells need to be investigated -4. Thesehttp://ClinicalTrials.gov, and there is some encouraging preliminary evidence of their clinical efficacy. On the other side, two serious adverse events have been reported. The first event [10 T cells expressing a third-generation CAR targeting HER2, after being pre-treated with an intensive lymphodepletion regimen. The patient rapidly presented pulmonary toxicity within few minutes, followed by cardiac arrest and finally died few days later. The investigators concluded that the toxicity was due to targeting of low levels of HER2 on pulmonary endothelium by transgenic T cells. In the second clinical case, Brentjens and colleagues described a patient with bulky CLL and extensive previous chemotherapy treatment who received autologous T cells expressing a second generation anti-CD19. CAR at a dose of 3 × 107 cells/kg after being lymphodepleted with cyclophosphamide [in vivo activation process of modified T cells.In conclusion, CARs represent a potential useful tool to fight various resistant forms of leukaemia. As recently discussed by Helen Heslop , more thst event  occurredsphamide . This paAll these aspects have been currently widely considered and discussed by many researchers and regulatory experts in the field of cell and gene therapy. Different schedules of administration  with a reduced cell numbers or fractionated doses, together with a better choice of pre-administration lympho-depletive regimens, in patients with minimal residual disease, would probably diminish the possibility of observing severe adverse events. Withal, the use of CARs containing suicide-gene switch-off mechanisms could be helpful in case of off-target side events ,40.The authors declare that they have no competing interests.EB and AB conceived and wrote the article. VM and GMPGA performed and wrote the literature search. VM and EC edited the parts (and performed literature search) concerning applications of CARs in B-cell malignancies. VM, IP and ST edited the parts (and performed literature search) concerning applications of CARs in AML. GMPGA edited the parts (and performed literature search) concerning applications of CARs in CLL. GMPGA, IP, ST, EC contributed to figures preparation. VM edited the article for publication. All authors have read and approved the script.


In mammals, hematopoietic stem cells (HSCs), which give rise to all blood cells and their progenies, including immune cells are controlled by special microenvironments, termed niches in the bone marrow during homeostasis and infection. However, the identity, nature and function of these niches remain unclear. It has been reported that HSCs are in contact with osteoblasts lining the bone surface and osteoblasts act as niches for HSCs . However, recent studies suggest that only a small number of HSCs reside in the endosteal niche. In contrast, many HSCs are shown to be in contact with endothelial cells in the marrow. In addition, recent studies suggest that primitive mesenchymal cells, including CXCL12-abundant reticular (CAR) cells and Nestin-expressing cells, which have the ability to differentiate into adipocytes as well as osteoblasts act as niches for HSCs. Here we review candidate niches for HSCs in the bone marrow controlling hematopoiesis and chronic inflammation. Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells, including immune cells within the bone marrow. The bone marrow is surrounded by bone surface and occupies the medullary cavities of bones throughout the skeleton and contains a dense network of medullary vascular sinuses, and blood cells and their precursors are packed in the extravascular spaces between the sinuses. It has been assumed that HSCs and hematopoietic progenitors reside in the microenvironments in the bone marrow, known as niches, which provide HSCs and hematopoietic progenitors with regulatory signals essential for their maintenance, proliferation and differentiation to produce the appropriate numbers of mature blood cells throughout life -3. Howevin vitro for several months, was reported. This suggests that adherent stromal cells in the culture, which might include osteoblasts, endothelial cells and macrophages, support hematopoiesis [in vivo.In 1977, a liquid culture system, in which primitive hematopoietic cells proliferated opoiesis , 5. On topoiesis . Howeverin vitro, raising the possibility that osteoblasts are involved in hematopoiesis [in vivo [+CD45- osteoblastic (SNO) cells, which express a high level of N-cadherin [+ and N-cadherin were identified as HSCs and found in contact with bone-lining osteoblasts expressing osteocalcin in mice myelosuppressed with 5-FU [In 1994, primary human osteoblasts were shown to maintain the proliferation of primitive hematopoietic progenitors opoiesis . Osteobl[in vivo . In addiith 5-FU . On the ith 5-FU , 12. The+CD48-CD41-Lin- HSCs were adjacent to the bone surface in bone marrow sections [However, recent studies complicate the data presented above. It was reported that only 4.6% of HSCs retained BrdU after 10 days treatment and a 70-day chase , few CD1sections , and thasections . Additiosections . Furthersections , 14 and sections . Thus, iOsteoclasts, which are generated from HSCs and resorb the mineralized bone matrix formed by chondrocytes or osteoblasts, are located in endosteal niches. The role of osteoclasts in hematopoiesis remains a controversial issue. It has been reported that osteoclasts degrade endosteal niche components and enhance mobilization of hematopoietic progenitor cells . In contThe studies focusing on the chemokine CXCL12 revealed that the specialized reticular cells in marrow cavities function as a niche for hematopoiesis. Chemokines are a large family of structurally related chemoattractive cytokines, which act through seven-transmembrane-spanning receptors coupled to heterotrimeric GTP-binding proteins (G-protein-coupled receptors) . CXCL12 1a). In addition, most CD150+CD48-CD41- HSCs (97%), earliest B cell precursors, plasma cells, pDCs and NK cells were in contact with the processes of CAR cells in the marrow, suggesting that CAR cells function as a niche for HSCs and all immune cells produced in bone marrow (reticular niches) [1).Immunohistochemical analysis of mice containing the green fluorescent protein (GFP) gene knocked into the CXCL12 locus (CXCL12-GFP mice) revealed that high CXCL12-GFP expression was detected in a small population of reticular cells, termed CXCL12-abundant reticular (CAR) cells, which are uniformly scattered throughout the bone marrow and have the long processes creating a network , 30  , 29, 30 1a); however, CAR cells do not express the pan-endothelial marker platelet/endothelial cell-adhesion molecule 1 (PECAM-1)/CD31 or the smooth muscle cell maker and smooth muscle α-actin (SMαA), suggesting that they are different from endothelial cells and smooth muscle cells [-CD31-Sca-1-PDGFRβ+ cells [Histochemical analysis revealed that all bone marrow sinusoidal endothelial cells are surrounded by a proportion of CAR cells   mouse model selectively ablating CAR cells has been generated, in which the numbers of CAR cells are severely reduced but other candidate niches, including bone-lining osteoblasts or endothelial cells are not affected 2 days after DT administration [in vivo impaired SCF and CXCL12 production, indicating that CAR cells are the major producer of CXCL12 and SCF in the bone marrow [in vivo impaired the adipogenic and osteogenic differentiation potential of bone marrow cells [1a) although it remains unclear what determines the specificity of interaction between certain hematopoietic cells and CAR cells.To clarify the stration . HSCs fre marrow . It has ow cells . These row cells   gene, GFP expressing cells, termed Nes-GFP+ cells showed an exclusive perivascular distribution, and that many CD150+CD48-Lin- HSCs (60%) were adjacent to Nes-GFP+ cells. In addition, mRNA expression of CXCL12 and SCF was much higher in sorted CD45-Nes-GFP+ cells, compared with sorted CD45-Nes-GFP- non-hematopoietic cells. Furthermore, HSC numbers were reduced about 2-fold in the marrow but increased in the spleen 16 days after in vivo depletion of nestin-Cre-expressing cells using a DTR-mediated cell knockout technique, suggesting that nestin-expressing cells are involved in retaining HSCs in the bone marrow.Nestin is an intermediate filament protein that was originally identified as a marker of neural progenitors . Its exp+ cells contained cells that formed mesenchymal spheres and differentiate into adipocytes and osteoblasts, and colony-forming units-fibroblasts (CFU-F). Based on these findings, it was concluded that Nes-GFP+ cells are mesenchymal stem cells [1). However, the frequencies of Nes-GFP+ cells with ability to give rise to multiple cell types is unclear, and only about 6.9% or 0.7% of Nes-GFP+ cells possess the ability to form mesenchymal spheres or CFU-F, respectively [+ cells are not mesenchymal stem cells. In addition, a recent study has shown that nestin-expressing cells in the marrow contain some neural cells [+ cells suggests that nestin-expressing cells contain CAR cells [+CD45-Ter119-PDGFRα+CD34+ cells, which had much lower levels of CXCL12 mRNA expression than Sca-1-PDGFRα+ non-hematopoietic cells (including CAR cells) have been reported as a candidate for bone marrow mesenchymal stem cell population [+ mesenchymal stem cell population as well as CAR cells and neural cells in the marrow.Sorted Nes-GFPem cells  ectively , raisingal cells . AbundanAR cells ; howeverAR cells . ConsistAR cells . On the pulation . Thus, iListeria monocytogenes, an intracellular bacterial pathogen requires the chemokine receptor CCR2 [+ inflammatory monocytes in the bone marrow cavities migrate to vascular sinuses and emigrate from the marrow into the circulation following low concentrations of the Toll-like receptor (TLR) ligands LPS [-Ter119-CD31-Sca1-PDGFRβ+ cells after administration of low concentrations of LPS and after infection with Listeria monocytogenes. Sorted MCP1-expressing cells expressed a high amount of CXCL12 mRNA following LPS administration. These results strongly suggest that the majority of MCP1-expressing cells following LPS administration are CAR cells [hi inflammatory monocytes but not neutrophils was significantly reduced after administration of low concentrations of LPS [2). In addition, the results that some MCP1-expressing CAR cells were located in intersinusoidal spaces following the administration of low dose LPS are consistent with the fact that not all inflammatory monocytes exit the marrow and raise the possibility that MCP1-expressing intersinusoidal CAR cells retain some monocytes in the bone marrow during infection [2).Hematopoietic niches are thought to control hematopoiesis not only during homeostasis but also in inflammation. Recruitment of inflammatory monocytes as well as neutrophils from bone marrow to sites of infection is critical for innate immune defense against microbial pathogens. It has been shown previously that emigration of inflammatory monocytes from bone marrow into the circulation during infection with tor CCR2 . The recands LPS . In tranAR cells , 38, 39.Recently, mice containing the GFP gene knocked into the SCF locus (SCF-GFP mice), in which GFP was mainly expressed in cells surrounding sinusoidal endothelial cells, have been generated . SCF-GFP+CD48-CD41-Lin- cells and imaged in tissue sections [+CD48-CD41-Lin- HSCs are associated with sinusoidal endothelium, suggesting that endothelial cells function as a niche for HSCs (vascular niches) [1). After myeloablation, selective activation of Akt1 in the endothelial cells of adult mice increases the number of HSCs and accelerates hematopoietic recovery [The arteries enter the bone marrow through the cortical bone  and contrecovery . These rrecovery . Of noterecovery , 17, thein vivo roles for some mesenchymal cells in bone marrow cavities as HSC niches have been recently reported [Although HSCs have been reported to be in contact with bone-lining osteoblasts and osteoblasts act as niches for HSCs, recent studies suggest that only a small subpopulation of HSCs reside in the endosteal niche. In contrast, reported , 34. Selreported  althoughreported  suggestireported .+, cells seem to be relatively rare non-hematopoietic cells that function as niches unique for HSCs. In contrast, CAR cells are relatively abundant and might function as a niche for both HSCs and more differentiated progenitors, including immune cells. In addition, CAR cells have long processes and form the reticular network within the bone marrow cavity. Considering that osteocytes, the most abundant cells in bone, communicate with each other through their processes, this raises the possibility that processes of CAR cells play a vital role in spatial and temporal regulation of their niche function in the bone marrow cavities. In this context, it will be important to address in future work what determines the specificity of niche-hematopoietic cell interaction, and to identify molecular mechanisms by which hematopoietic niches regulate maintenance, production and exit of various types of immune cells during homeostasis, microbial infection and inflammatory and myeloproliferative diseases. Furthermore, in addition to known PTH and its receptor PPR [Of the candidate cellular niches for HSCs, SNO and Nes-GFPptor PPR , and LPSptor PPR , molecul


Manduca sexta, the projection neurons (PNs) arborizating in the MGC exhibit two types of responses to pheromone stimulation [+, K+, Ca2+, A, SK) and leak current but different parameter values for PNs and LNs. First, we examined the effect of inhibition from LNs. In PN responses, the after-hyperpolarization (AHP) was preserved when the LN-IIa were removed but prior inhibition vanishes so that triphasic patterns (-/+/-) are changed to biphasic (+/-). However, removing LN-IIb made the duration of the AHP phase significantly shorter. The synchrony rate was also disturbed by removing one or another of LNs but loss in synchrony remained more important without LN-IIa (fig.The macroglomerular complex (MGC) is known as the olfactory sub-system processing pheromonal information. In the moth mulation : a simplmulation . Here wemulation . LN-IIa’-IIa fig.. Then we-IIa fig. and late-IIa fig.. As LNs


C-MYC, IGH, BCL-2), various copy number variations, DNA mutations , and polymorphisms in both the host  and the EBV genome to B-cell PTLD development. Furthermore, the tumor microenvironment seems to play an important role in the course of disease representing a local niche that can allow antitumor immune responses even in an immunocompromised host. Taken together, B-cell PTLD pathogenesis is very complex due to the interplay of many different (patient-dependent) factors and requires thorough molecular analysis for the development of novel tailored therapies. This review aims at giving a global overview of the currently known parameters that contribute to the development of B-cell PTLD.Posttransplant lymphoproliferative disorder (PTLD) is a potentially fatal disease that arises in 2%–10% of solid organ and hematopoietic stem cell transplants and is most frequently of B-cell origin. This very heterogeneous disorder ranges from benign lymphoproliferations to malignant lymphomas, and despite the clear association with Epstein-Barr Virus (EBV) infection, its etiology is still obscure. Although a number of risk factors have been identified , it is currently not possible to predict which transplant patient will eventually develop PTLD. Genetic studies have linked translocations (involving  Posttransplant lymphoproliferative disorder (PTLD) is the most severe complication of solid organ and hematopoietic stem cell transplantation and occurs in 2%–10% of posttransplant patients. The presentation of PTLD is highly variable and ranges from benign lymphoproliferations to overt lymphoma. In the majority of cases, PTLD is of B-cell origin , 4), areThis review aims at giving a general overview of the pathogenetic mechanisms that underlie B-cell PTLD. Because of the rarity of PTLD, insight into disease development and progression is currently limited. Most probably, EBV predisposes infected B-cells to uncontrolled proliferation which may result in the accumulation of (epi)genetic aberrations. Furthermore, the microenvironment of the transformed lymphocyte together with the genetic background of the individual provide a particular environment that could further promote lymphomagenesis.HLA-matched noncomplicated transplants) to 25% (for T-cell-depleted highly immunosuppressed transplants that lack T-cell-mediated anti-EBV immune responses) [Despite its obscure pathogenesis, a number of commonly accepted risk factors for PTLD development have been identified. EBV-naive individuals lacking cellular immunity to EBV are susceptible to graft-mediated EBV infection resulting in early-onset PTLD (within 12 months following transplantation). This population mainly comprises children . Analogosponses) . Furthersponses) , 15. Folsponses) . Overallsponses) , 17. Thesponses) , 19. Lucsponses) . 90% of PTLD arising following SOT are derived from the postgerminal center host lymphocytes suggesting a role for chronic B-cell stimulation by the graft and endogenous EBV reactivation . In caseReported mortality rates of PTLD range from 25% to 60% –29 due tThe World Health Organisation (WHO) discriminates three types of morphological lesions assumed to represent different stages in the pathogenesis of PTLD: benign polyclonal early lesions, polymorphic PTLD, and malignant monoclonal monomorphic PTLD . It is aBCL-6 is mutated in 50% of P-PTLD and associated with aggressive disease [In polymorphic PTLD (P-PTLD) the underlying lymphoid structure is effaced by an extensive proliferation of stromal immune cells  and few transformed cells, similarly to classical Hodgkin lymphoma. Although clonal genomic alterations are rare in P-PTLD,  disease . Most P- disease , 33.Monomorphic PTLD (M-PTLD) represents a group of neoplastic lymphoproliferations corresponding to similar pathologic entities in immunocompetent individuals . More thE-PTLD and P-PTLD lesions generally arise earlier following transplantation than M-PTLD. A possible explanation is that E- and P-PTLD develop due to an aberrant response to EBV that may be introduced via the graft. This also explains why the vast majority of E- and P-PTLD are EBV-positive although exact percentages vary. One study detected EBV-positive lymphocytes in only 67% of FFH . This obThe onset of P-PTLD and M-PTLD may overlap but is variable, partly because diagnosis depends on when these lesions become clinically detectable. Because M-PTLD is thought to arise from E- and P-PTLD, the majority of M-PTLD is EBV-positive. Within the M-PTLD group, EBV-positive lymphomas arise earlier following transplantation than EBV-negative lymphomas . This obin vitro. In contrast, it was shown that EBV infection in vivo induces transient proliferation and latent persistence in nonpathogenic memory B-cells rather than (immediate) transformation. This explains why EBV can persist benignly for a lifetime in over 90% of human hosts and also why not every infected individual develops cancer, even when immunocompromised. Additional aberrations in B-cell biology or viral infective mechanisms are required to prevent latent infection from being established and allow cancer to develop [EBV is the first virus that has been associated with oncogenesis and is one of the most effectively transforming viruses  develop . Upon B-cell infection, the linear viral genome circularizes and is maintained as a nuclear episome that can integrate into the host genome . The lifThe coordinated expression of viral proteins has been associated with specific methylation patterns at their promoter sites .C-MYC, IL-18 receptor, etc.) [c-FLIP, an apoptosis inhibitor, BCL-2) and cytokine expression. It upregulates IL-10 that functions as an autocrine B-cell growth factor, and via induction of IFN-gamma, LMP1 indirectly upregulates STAT1 resulting in a sustained expression of CXCL9 and p21 [Upon EBV infection, the first viral protein to be expressed is EBNA2, a potent inducer of viral (LMP1 and LMP2A) as well as cellular proteins  . LMP1 anr, etc.) , 38. Fur and p21 .LMP2A is localized in the B-cell membrane and binds tyrosine kinases eventually impairing BCR-mediated activation and entry of the lytic cycle. To ensure survival of the infected cells LMP2A can induce the vital signals normally provided by BCR signaling . Together, LMP1 and 2A stimulate the infected B-cell to become a proliferating blast and guide it throughout the germinal center reaction ultimately driving the infected lymphocyte towards the memory cell stage where EBV can persist. At this stage, viral expression is shut down minimizing immunogenicity of the infected cells. Only during memory cell division, EBNA1 is expressed to ensure replication of the viral episome. When the memory cell differentiates into a plasma cell in response to antigen stimulation, EBV enters the lytic cycle producing new infectious particles that are shed in the saliva. From this infectious mechanism, it becomes clear that the oncogenic potential of EBV becomes substantial when the B-cell lymphoblast is unable to differentiate or when the growth program is aberrantly expressed in the absence of an effective cytotoxic T-lymphocytes (CTLs) response. Under normal conditions CTLs recognize and kill EBV-infected lymphoblasts, but in case of immunodeficiency (like posttransplantation), infected cells can proliferate uncontrollably. Nevertheless, this is still a rare event as only 1 or 2 of millions of EBV-infected cells eventually develop into tumors . IL-10 and BCL-2) and mediate the pathogenic, and oncogenic effects of EBV [Other gene products encoded by the EBV genome include EBV-encoded RNAs (EBERs), miRNAs , and a s of EBV , 49. C-MYC translocation and induce IL-10 expression resulting in B-cell survival and immunosuppression [The 2 EBERs are expressed in all latency programs but their function remains unclear although they represent the most abundantly expressed viral products in most infected cells . It is apression , 52. BART clusters (in the introns of the BART gene) and BHRF-1 clusters (in the 3′ UTR of the BHRF-1 open reading frame) and can regulate cellular genes, possibly conferring resistance to apoptosis [BHRF-1-encoded protein is the viral homolog of BCL-2. Furthermore, EBV miRNAs can downregulate viral proteins such as LMP1 and 2A representing a possible mechanism for immune escape . Studieiewed in . Expressiewed in . Aside fiewed in ) and lymiewed in ).So far, no genetic aberrations have been associated exclusively with EBV-positive or -negative PTLD suggesting that EBV infection alone is not sufficient for lymphomagenesis but that  preceding and/or subsequent genetic events are required for full transformation of a B-cell. However, increased expression of BCL-6, decreased expression of MUM1 (multiple myeloma oncogene 1), and a more frequent germinal center-derived cell of origin have been associated with EBV-negative PTLD . FurtherGenomic profiling is warranted to shed light on the molecular pathogenesis of PTLD, but the 2 small gene expression profiling (GEP) studies that have been performed on PTLD yielded somewhat conflicting results. Craig et al. performed a microarray analysis on a group of 4 EBV-positive and 4 EBV-negative M-PTLD patients and demonstrated segregation of PTLD cases based on the EBV status. Antiviral immune responses and cell cycle proteins were upregulated in EBV-positive PTLD, whereas components of the BCR and their downstream signaling on the other hand were downregulated . The groClearly, no consensus exists on the role of EBV and the molecular features of PTLD. Based on a microarray experiment performed by our lab and comprising 48 DLBCL cases of which 33 occurred following transplantation (72% EBV-positive) and 15 in immunocompetent hosts (none EBV-positive), we concluded that EBV-positive and EBV-negative PTLD are characterized by a distinct gene expression profile. Innate and tolerogenic immune responses played a central role in EBV-positive posttransplant DLBCL as opposed to EBV-negative posttransplant DLBCL. In addition, except for decreased T-cell signaling, these latter cases coincided with EBV-negative DLBCL occurring in immunocompetent individuals suggesting that EBV-negative posttransplant lymphomas are coincidental cases of lymphoma biologically similar to lymphoma in the immunocompetent population . C-MYC), 3q27 (BCL-6), and 14q32  [Multiple genetic alterations that have been associated with PTLD  suggest H, TCL1) . Two othH, TCL1) , 57% of H, TCL1) ). These H, TCL1) –63.TP53; gains of 3q27: BLC-6, 5p, 7q, 8q24: C-MYC, 11p, 12q, 12p, 18q21: BCL-2 and MALT, and 21q) [FOXP1, BCL-6, and NFKBIZ) and 18q (BCL-2 and NFATC1) together with losses of 6q (PRDM1 and TNFAIP3) in posttransplant DLBCL despite the postgerminal origin of most of their reported cases [Comparative genomic hybridization analysis has confirmed the occurrence of these alterations and has shown that PTLD is characterized by distinct genetic aberrations  as well as changes that are also common in lymphoma arising in immunocompetent patients but with different frequencies  . Rinaldied cases . Furthered cases . These fFRA2E as the most common lesion in posttransplant DLBCL [FRA2E is very similar to an EBV insertion site discovered in a Burkitt's lymphoma cell line suggesting that the genomic instability of PTLD might be due to integration of viral DNA upon infection. FRA2E contains FANCL (an ubiquitin ligase important in DNA repair) and VRK2 (a negative regulator of the MAPK pathway) which may play a role in oncogenesis [Comparative high-density genome-wide analysis identified del(2p16.1) targeting the fragile site nt DLBCL . Interesogenesis , 67. Comparison of EBV-positive and EBV-negative post-transplant DLBCL showed that the latter contain more recurrent genomic lesions among which del(4q25-q35), gains of 7p, 7q and 11q24-q25. It has been hypothesized that in EBV-negative PTLD (or after hit-and-run infection), genetic alterations accumulate in order to substitute for the oncogenic effects of EBV .Loss of heterozygosity  occurs commonly in cancer –70. InteIn general, posttransplant lymphoma demonstrates a lower frequency of unbalanced genomic aberrations than DLBCL in immunocompetent hosts. This can be explained by the mutator phenotype that is a unique feature of a number of immunosuppression-related lymphomas and which may result in microsatellite instability. The mutator phenotype is induced when loss of a gene, for example, involved in DNA repair, accelerates the accumulation of mutations in numerous other genes with potentially deleterious consequences , 75. It is unclear whether different grafts are linked to particular genetic alterations but, remarkably, DLBCL following heart transplantation has been associated with a high prevalence of 6p gains .IgV) genes in an attempt to generate high-affinity antibodies [PIM-1, PAX-5, C-MYC, and RhoH/TTF and has been reported in lymphoma to be independent of the immune and EBV status of the host [Ig genes resulting in crippled BCR. In these cells, LMP2A may function as a BCR substitute providing the necessary survival signals [Nucleotide-level variations can be caused by aberrant somatic hypermutation (SHM) during the germinal center reaction. SHM normally targets the immunoglobulin variable  and βIL-1 alleles producing more severe inflammatory responses were found to protect patients against EBV viremia [IFN-gamma that results in decreased IFN-gamma synthesis has been associated with early-onset and pediatric PTLD [TNF-alpha promoter has been described to affect transcriptional regulation by NF-KB, potentially resulting in higher TNF-alpha levels [Despite the ubiquity of EBV, cancer driven by EBV is still a relatively rare phenomenon, even in  immunocompromised individuals. Part of the explanation probably lies in the numerous (single-nucleotide) polymorphisms present in the human as well as the viral genome that may affect disease progression at the level of the immune responses and behavior of EBV . Recent  viremia . Analogoric PTLD , 81. Furric PTLD . A polyma levels  and TNF-a levels , 84. Inta levels . IL-10 and TGF-beta have protumoral (immunosuppressive) as well as antitumoral characteristics , and low expression of both has been linked to late-onset EBV-positive PTLD [IFN-gamma, IL-10, and TGF-beta is decreased in PTLD suggests that a shift of the T-helper 1/T-helper 2 (Th1/Th2) balance towards the Th2 pathway plays a role in PTLD development. ive PTLD . AltogetHLA) system comprises 2 classes of antigen-presenting proteins of which polymorphic variants have been associated with virus-associated cancers [HLA and PTLD involve interactions of natural killer cells and CTLs with HLA proteins. It is also possible that inefficient antigen presentation of EBV proteins contributes to decreased immunoreactivity towards EBV. One study showed that the HLA-A26 variant conferred a threefold increased risk for PTLD development if it was present in the transplant recipient or donor. It was hypothesized that the latter represents donor-derived PTLD, the prevalence of which might currently be underestimated. The same study also identified a protective HLA haplotype linked with a “hyperactive immune system” suggesting that carriers of this haplotype have better immunological defenses against EBV [HLA alleles, HLA haplotypes, HLA mismatches, and preexisting HLA antibodies in the development of PTLD in transplant recipients. Clearly, larger studies are needed to further clarify this complex association.The human leukocyte antigen  which would decrease the chance of malignant transformation [At the level of the viral genome, variations that may influence viral load and PTLD development have also been described. The 30-bp deleted LMP1 variant has been associated with lower EBV serum levels compared to wild-type LMP1 . Other slication . Gutiérrlication . It is pormation . This hyormation .DAPK1 (proapoptotic), MGMT (involved in gene repair) and SHP1 (antiproliferative) are a few examples of tumor suppressor genes that are hypermethylated in the majority of M-PTLD [Apart from genetic, also epigenetic alterations and more specifically DNA hypermethylation have been implicated in the pathogenesis of PTLD. DNA methylation is carried out by DNA methyltransferases  counteracted by DNA methylases as a mechanism to fine-tune gene expression. Excess methylation of tumor suppressor genes results in significant downregulation of gene expression and may contribute to cancer development. f M-PTLD , 62, 102EBV is known to modulate DNA methylation in germinal center B-cells via downregulation of DNMT1 (by LMP1) and DNMT3B and upregulation of DNMT3A resulting in clustered changes in methylation status of cellular genes depending on the CpG content of the promoter region. It has been hypothesized that DNMT3A silences the viral Wp promoter by methylation inducing a switch to the viral Cp promoter resulting in expression of more viral proteins. These observations indicate that EBV infection alters methylation of the host as well as its own genome . Importain vitro EBV-positive lymphoma was more sensitive to inhibitors of PI3K/mTOR and HPS90 than EBV-negative lymphoma [Ideally, data regarding gene expression and genetic alterations are correlated with evidence at protein level, but proteomic analysis of PTLD is very rarely reported. A study comprising 6 monomorphic PTLD of which 5 were EBV-positive demonstrated upregulation of NFKB, PI3K, Akt, mTOR, MAPK and PKC pathways, cell cycle regulation, endoplasmic reticulum homeostasis (HSP90), and apoptosis-related proteins (caspase 7-8 and MAP2K4). Furthermore, lymphoma . The diflymphoma . A compllymphoma . It is plymphoma , and apolymphoma .The tumor microenvironment refers to the local niche in which tumor cells reside and consists of stromal as well as inflammatory cells. Recent studies have highlighted its importance in oncogenesis, tumor progression, and prognosis, and during the past years, the role of the microenvironment in tumor development has gained significant importance , 110. DeRegulatory T-cell (Treg) infiltration is thought to be consistently restricted in PTLD limiting immunosuppressive effects but also suppression of B-cell proliferation which could potentially contribute to PTLD development . This waEBV interacts intensely with the innate immune system (reviewed in ) suggestIL-10 is an anti-inflammatory cytokine and a B-cell growth factor. Elevated serum concentration of human IL-10 has been put forward as a marker for detection of early PTLD development [IFN-gamma/IL-2 negative; IL-4/IL-10 positive; [Only few data are available concerning the cytokine profile of the PTLD microenvironment. elopment , 121 andelopment . One stuositive; ). Howeveositive;  potentiaIL-10 analogue encoded by BCRF-1. Apart from B-cells, EBV can also infect monocytes and macrophages, one of the first cell types to arrive at the site of the viral infection [IL-10 is expressed earlier than human IL-10 following infection, it can efficiently prevent IFN-gamma-induced upregulation of primary (MHC-antigen complex) as well as costimulatory  signals in myeloid cells eventually impairing antiviral T-cell activation and inducing anergy [Matters are further complicated by expression of the viral nfection , 126. Beg anergy , 128.IL-10, IL-6 is a B-cell growth factor of which serum concentration is increased at diagnosis of PTLD. Possibly, IL-6 is expressed by the tumoral cells as part of an autocrine feedback loop providing an explanation for the therapeutic success of an IL-6 antibody [Like antibody , 130.The current options for treatment of B-cell PTLD are largely limited to chemotherapeutic regimens  and immunotherapy with Rituximab, an anti-CD20 antibody (as monotherapy or combination therapy with CHOP) . HoweverBecause most PTLD cases are EBV-related, different strategies have been developed to decrease the EBV viral load. Nucleoside inhibitors that inhibit viral replication have little effect due to limited lytic replication of EBV in PTLD . HoweverOther types of immunotherapy, namely, cytokine therapy and antibody therapy have also been developed. However, administration of antiviral/antitumoral IFN-alpha was poorly tolerated and associated with graft rejection , whereasIn vitro studies have shown that activation of apoptosis by small molecules is effective against EBV-associated lymphoma which could represent a novel way to treat PTLD [In recent years, increasing molecular insight in PTLD pathogenesis has been translated in new potential therapeutics. Inhibitors of mTOR have been studied in small clinical trials but their efficacy is debated –140. In eat PTLD . An EBV eat PTLD  and may Although PTLD pathogenesis is still not well understood, current knowledge of disease development at the DNA, RNA, and protein level underlines its complex etiology. It is important to keep in mind that malignant PTLD comprises many different lymphoma subtypes  of which the disease mechanisms can still differ considerably warranting the development of patient-specific therapies.


HCV is one of the most relevant co-morbidities seen in HIV-infected individuals as evidenced by the negative impact that HIV exerts on the course of HCV infection. Despite remarkable results on HIV infection alone, the impact of highly active antiretroviral therapy (HAART) on liver disease in co-infection remains unknown. We sought to explore the impact of Raltegravir (RAL) on amino transferase (ALT) in HIV/HCV co-infected individuals.HIV-infected individuals co-infected with HCV within the last 5 years receiving non-integrase inhibitor containing HAART with a subsequent switch to RAL-containing HAART were identified from a retrospectively maintained outpatient database. Patient demographics were extracted. Biochemical, virological and immunological parameters were collated and individuals received pegylated interferon with ribavirin were excluded. ALT levels at switch and post switch were compared using Kruskal-Wallis test. Spearman’s Rank correlation was used to assess the relationship between ALT and HCV-RNA.Twenty seven HIV-HCV co-infected individuals were identified between January 2007 and January 2012 and seven individuals were excluded. Median age was 44 years (range: 31-68). Five had acute and fifteen had chronic HCV infection during the switch. Twenty (100%) had HIV-RNA-1.In our study, RAL had a favourable effect on the liver up to 24 weeks after switch in HIV/HCV infected individuals.


The Drosophila GAGA factor (GAF) participates in nucleosome remodeling to activate genes, acts as an antirepressor and is associated with heterochromatin, contributing to gene repression. GAF functions are intimately associated to chromatin-based epigenetic control, linking basic transcriptional regulation to heritable long-term maintenance of gene expression. These diverse functions require GAF to interact with different partners in different multiprotein complexes. The two isoforms of GAF depict highly conserved glutamine-rich C-terminal domains (Q domain), which have been implicated in complex formation.Here we show that the Q domains exhibit prion-like properties. In an established yeast test system the two GAF Q domains convey prion activities comparable to well known yeast prions. The Q domains stably maintain two distinct conformational states imposing functional constraints on the fused yeast reporter protein. The prion-like phenotype can be reversibly cured in the presence of guanidine HCl or by over-expression of the Hsp104 chaperone protein. Additionally, when fused to GFP, the Q domains form aggregates in yeast cells.Drosophila.We conclude that prion-like behavior of the GAF Q domain suggests that this C-terminal structure may perform stable conformational switches. Such a self-perpetuating change in the conformation could assist GAF executing its diverse epigenetic functions of gene control in  Drosophila is a ubiquitous transcription factor that plays important roles in multiple processes ranging from regulation of gene expression to the structural organization of heterochromatin and chromatin remodeling   .psi-] soluble, functional state and a [PSI+] aggregated, non-functional state [psi-] cells, the Sup35 protein acts as a translation termination factor that, together with Sup45, recognizes stop codons and terminates translation. In contrast, in [PSI+] cells the Sup35 is unable to efficiently participate in translation termination as most of Sup35 protein is sequestered in self-replicating prion multimers ,2120,21]ultimers ,23. The ultimers ,23. Althultimers -26, onlyultimers ,28.Saccharomyces cerevisiae. We replaced the N-term prion-forming domain of Sup35 with the GAF519 and GAF582 Q domains (GAF-Q) and fused it to the C-terminal portion of the Sup35 in order to test whether the chimeric proteins were able to induce a prion-like state as demonstrated for a variety of other potential prion forming domains [PSI+] in cells over-expressing Sup35. This prion-like phenotype of the GAFQ-SupC expressing cells could be reversibly cured in the same manner as the [PSI+] prion by growing the cells in the presence of guanidine HCl (GuHCl) or by over-expression of the Hsp104 chaperone protein. Furthermore, when fused to GFP, the GAF519 and GAF582 polyQ domains formed aggregates in yeast cells. In corroboration, sedimentation analysis using differential centrifugation revealed that GAFQ-SupC fusions are aggregated in the same manner as the [PSI+] prion. These aggregates were cured by growing the cells in the presence of GuHCl. Finally, meiotic segregation analysis through tetrad dissection also revealed that the prion-like phenotype of GAF582SupC segregated in a non-Mendelian fashion. Importantly, prion-like behavior exhibited by meiotic progeny was also cured when grown in the presence of GuHCl. Such prion-like behavior of GAF polyQ domains in yeast suggest that polyQ domains in GAF may render a conformational switch which may help GAF perform its versatile functions in maintaining different epigenetic states of gene expression.Expansion of polyQ domains is known to contribute to heritable alterations of protein conformation which is associated with prion proteins ,21. Henc domains -32. The PSI+] reporter system [psi-] and [PSI+] states of Sup35 can be employed to reproducibly monitor the prion-like behavior of Q-rich regions [psi-] state, cells that carry a premature stop codon in their ADE1 gene (mutant designated ade1-14) do not make functional Ade1 and accumulate a metabolite making the colonies appear red on complete medium. In contrast, the [PSI+] cells are characterized by a read-through of the premature stop codon (nonsense suppression) in ade1-14. Functional Ade1 is produced as most of Sup35 protein is sequestered in self-replicating prion aggregates and is unable to participate in translation termination. [PSI+] cells therefore produce white colonies and can grow on adenine-deficient medium [The GAF isoforms GAF519 and GAF582 share the same N terminal domain but have distinct glutamine (Q)-rich C termini of different lengths. The C terminal 80 amino acids of the GAF519 contains 39% Q residues whereas the C terminal 178 amino acids of the GAF582 comprises of 41% of Q residues in multiple repeats   and grow without adenine supplementation. In contrast [psi-] cells do not form colonies on adenine-deficient medium (−Ade) and are red on YPD  phenotype [psi-] phenotype  ,38. The henotype , the GAFe Figure D, which e Figure E. The coPSI+] like phenotype  where each haploid stably exhibiting nonsense suppression phenotype showed high frequency of metastable behavior i.e. appearance of many red colonies in the progeny after streaking  [PSI+] or [PIN+]. The inducible expression of zeste-GFP (Z-GFP) in any of the yeast strains mentioned above showed no aggregation  was demonstrated by mating 582q + MATa (pink/white) haploid with a 582q- MATα haploid, i.e. 582q- represents a red colony indicating non-prion form of 582q  but also when sporulated produced haploid (1n) progeny. These exhibited prion like behavior displaying meiotic inheritance of 582q + phenotype in a dominant, non-Mendelian manner. All 4 haploid (1n) products of meiosis showed stable non-sense suppression phenotype by their growth on -ADE plates, which was curable when grown in the presence of GuHCl . We fused the GAF519 and GAF582 Q domains to green fluorescent protein (GFP) under an inducible promoter   fused toPSI+]. Importantly, the nonsense suppression prion-like state exhibited by the GAFQ-SupC fusion was cured by growth in the presence of GuHCl. Similar to the [PSI+] prion state of Sup35p, the nonsense suppression phenotype by GAFQ-SupC could also be cured by the over-expression of Hsp104. The GAF-Q domains fused to GFP also formed visible aggregates resembling those of GFP labeled Sup35p in [PSI+], which also depended on [PIN+]  . Many sen [PIN+] ,45. Howew levels ,29,34,40w levels . The priw levels .Drosophila have extended Q-rich tracts, remarkably similar to those found in the prion-forming domains of yeast proteins  sup35Δ diploid yeast strain Y133 and transformants were selected at –His plates. Individual diploids were spotted on medium lacking His and replicated plated on plates with YPD and -Ade to monitor prion phenotype. In addition, GAF519-Q and GAF582-Q PCR products with Bam H I and Sac II restriction sites, described above, were also cloned in frame with GFP at the C terminus under a copper inducible promoter in the pCUP-GFP plasmid  strain as described  and [PIN+] or both prions. Both 519Q-GFP and 582Q-GFP fusions showed aggregation pattern similar to NM-GFP as their aggregation seem to depend on the presence of [PIN+], the prion form of the RNQ1 protein of yeast required for Sup35 aggregation and prion formation. Visualization of protein aggregates with GFP fused to GAF-Q domains (GAFQ-GFP). (A) Schematic illustration of GAF519 and GAF582 Q domains (GAF-Q) fused to GFP under a promoter inducible with copper (Click here for fileS. cerevisiae used in this study. Strains of Click here for file


However, the molecular and functional consequences of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function.The goal of the present study was to investigate if targeted inhibition of upregulated expression of e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.These data provide the first evidence that a moderate inhibition ( Despite diagnostic and therapeutic advances in clinical cardiology, heart failure (HF), both systolic and diastolic, remains a leading cause of morbidity and mortality in developed countries. The precise stimuli for and mechanisms of ventricular remodeling in acquired HF are not yet clearly delineated. Early studies used a candidate gene approach focused mainly on factors within adrenergic and renin-angiotensin pathways. A recent trend, based on a gene expression topology of the developing and diseased heart, has resulted in the re-interpretation of pathological ventricular remodeling in terms of rearrangement of key gene regulatory networks and downstream signaling pathways that are imbalanced, attenuated, or abnormally activated in failing myocardium myocd expression/function in Xenopusmyocd gene does not alter heart development. However, after birth mutant mice with a conditionally inactivated myocd gene develop dilated cardiomyopathy accompanied by impaired cardiomyocyte structural organization and severely depressed systolic function. In chimeric myocd knockout mice, generated by injection of myocd−/− embryonic stem cells into myocd+/+ blastocysts, myocd−/− cells almost completely fail to contribute to formation of ventricular myocardium, although myocd−/−-derived myocytes were phenotypically normal myocd is specifically required for functional differentiation of ventricular cardiomyocytes.The ability of MYOCD to contribute to heart development and cardiomyocyte differentiation is conserved, although to a different extent, in frogs, chickens, and mammals. Inhibition of endogenous myocd activation appears to be involved in the adaptive hypertrophic response of the heart during early postnatal development myocd expression and activity are enhanced in cardiomyocytes upon hypertrophic stimuli in vitro forced expression of myocd in mouse neonatal cardiomyocytes increases cell size and activates transcription of established myocd targets, such as smooth muscle (SM) α-actin (ACTA2) and SM myosin heavy chain (MYH11), along with a set of cardiac hypertrophy-associated genes Myocd inactivation, by dominant-negative mutant in vivo forced myocd expression upregulates genes for SM22α/transgelin (TAGLN) and fetal muscle light chain 3f myosin associated with transiently impaired systolic function myocd leads to the rapid-onset of HF due to dilated cardiomyopathy which is associated with attenuated expression of SRF/MYOCD-regulated cardiac genes and activation of pro-apoptotic factors in failing myocardium Despite its importance in heart development and cardiomyocyte differentiation, myocd has also been implicated in the response of the postnatal/adult heart to pathological stresses during hypertrophic remodeling myocd expression in failing left ventricular (LV) myocardium. In addition, myocd targets, such as ACTA2 myocd gene activation plays in HF conditions in vivo has yet to be determined.In addition to its role in adaptive gene expression and maintenance of cardiac function, myocd overexpression might represent a maladaptive response of ventricular myocardium to pathological remodeling myocd in failing ventricular myocardium could normalize dysregulated MYOCD signaling pathways and restore, at least in part, impaired cardiac function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which upregulation of myocd was established as a HF-related feature myocd but also myocd-dependent SM-marker genes are highly activated in failing LV-myocardium of Dox-injected piglets.A provocative hypothesis was that In vivo silencing of endogenous myocd expression by short-hairpin (sh) interfering RNAs at advanced stages of DHF in piglets resulted in downregulation of myocd-dependent SM-gene expression in failing myocardium. Such adjusting of MYOCD and SM-target levels to the scores measured in non-failing controls resulted in restoring diastolic function and extending the survival of failing animals. These data provide the first evidence that a moderate inhibition  of activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.myocd-silencing (Group III and V) or sh-scrambled  vectors before the planned end of the study. At the end of each experimental run , maintained in a conventional Nürtinger nursery system for days 8 after birth, and randomized in seven groups according to experimental design . A diast run see , ECGs an2O, and kept at −80°C. RNA yield and purity was determined spectrophotometrically at 260–280 nm and RNA integrity was verified by running samples on 1.2% agarose gels and staining with ethidium bromide.For total RNA isolation, deep-frozen tissue/cell samples were directly disrupted in RLT buffer  using a high-speed rotor-stator homogenizer , digested with Proteinase K (Qiagen), loaded onto RNeasy Mini/Midi columns (Qiagen), subjected to on-column digestion of DNA with RNase-free DNase (Qiagen), and processed in accordance with the manufacturer's recommendations. Resulting RNA preparations were ethanol-precipitated, resolved in RNase-free HS. scrofa. Sample processing, array hybridization, scanning, and quantification were performed at the Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” . Genes were considered to be differentially expressed if a change of at least 2-fold was observed for evaluated (failing versus non-failing) comparisons at a p-value cutoff of 0.05. The microarray data have been deposited in the NCBI's Gene Expression Omnibus .Total RNAs isolated from LV biopsies of three failing  and three non-failing  piglets were independently hybridized on the Affymetrix GeneChip® Porcine Genome Array , which contains 23,937 probe sets interrogating 23,256 transcripts and representing 20,201 genes of bus GEO;  and are myocd-B mRNA . Resulting plasmids were verified by sequencing as a service provided by Secugen . For transfections, plasmids were purified using a PureLink HiPure plasmid filter purification kit  according to the manufacturer's protocol.Nine short-hairpin (sh) 19-mer sequences located throughout the full-length of the porcine d-B mRNA  were desmyocd and ankrd1 (ankyrin repeat domain 1 protein) were amplified from piglet LV oligo-dT-primed cDNA, cloned into p3XFLAG-CMV-14  vectors, verified by sequencing, and purified as previously described Silencer 2.1-U6 puro myocd sh-RNA or pSilencer 2.1-U6 puro scramble sh-RNA vectors, (2) FLAG-tagged pig myocd-B, and (3) FLAG-tagged pig ankrd1 constructions. All transfections were carried out with Lipofectamine LTX and PLUS Reagents  following the manufacturer's instructions. For each combination of plasmids, two separate transfection assays were employed, and in each assay, transfections were performed in duplicate. Transfected cells were cultured for 9 hours, then culture media were replaced with fresh media supplemented with 10% FBS. The cells were harvested at 24–48 hours after transfection and processed for RNA and protein extraction myocd-silencing or scramble sh-vectors essentially as described above for COS-7 cells. The sh-RNA-expressing populations were selected with puromycin  at the concentration of 1.0 µg/ml. Stable transfectants were grown in the presence of 1.0 µg/ml of puromycin for 6 days and then processed for RNA and protein extraction.COS-7 and porcine aortic smooth muscle cells (PAOSMCs) were purchased from the European Collection of Cell Cultures . COS-7 cells were cultured in Dulbecco's modified Eagle's Medium  supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin–glutamine (Gibco) under standard tissue culture conditions at 37°C. Cells were trypsinized at 70–80% confluence, cell numbers were determined using an automated cell counter  and 80,000 cells were plated in each of the 12-well plates and allowed to attach overnight. Porcine myocd-1554 or sh-scr1 constructions were delivered into the LV free wall (LVFW) of Dox- and PBS-injected piglets. Transcatheter, intramyocardial delivery was performed using a protocol developed and validated in our laboratory In accordance with experimental design see , naked pSilencer 2.1-U6 puro vector that amplify a fragment of predicted size . The level of plasmid DNA in extracted total DNAs was quantified using semiquantitative PCR with primers surrounding the sh-insert in the prpl19  gene was amplified to normalize expression. For each RNA sample, genomic DNA contamination was determined by PCR on a no-RT control for the rpl19 gene. Within each experiment, PCR reactions were done in duplicate. Each PCR reaction was evaluated using melting curve analysis and checking the PCR products on 8% SYBR Green-stained polyacrylamide gels. Fold changes were calculated using the CT method. Data were analyzed using IQ5 optical system software 2.0 and CT comparative analysis. For primer sequences used in qPCR analysis, please see Four µg of RNA were reverse transcribed using SuperScript III reverse transcriptase  and oligo-dT primer according to the manufacturer's instructions. Two-step quantitative real-time RT-PCR (qPCR) was performed on a Bio-Rad IQ5 detection system  with SYBR Green I mix following conditions previously described Rabbit polyclonal antibodies to transgelin , phospholamban , SM-α-actin , SM-myosin heavy chain 2 , SM-calponin , cardiac troponin I , cardiac calsequestrin-2 , and glyceraldehyde 3 phosphate dehydrogenase  were purchased from Abcam . Mouse polyclonal antibodies to SM-γ-actin 2  were purchased from Novus Biologicals . Rabbit polyclonal antibodies to MYOCD  according to the manufacturer's protocol. All samples were stored at −80°C before electrophoresis. Protein supernatants (5–10 µg/run or 25–30 µg/run for MYOCD detection) were subjected to SDS-PAGE , stained with Coomassie R-250  or blotted onto PVDF-membranes . Molecular weight (MW) standards (Precision Plus Protein WesternC Standards from Bio-Rad or SeeBlue Plus2 Pre-Stained Standard from Invitrogen) were included on each gel. Blots were probed with the antibodies indicated above and visualized by the Super-Signal West Pico chemiluminescent substrate  as described Tissue/cell samples were homogenized and solubilized in standard 2× Laemmli buffer  supplemented with complete protease inhibitor cocktail  as previously described t-test. Statistical analyses were performed with SPSS 13 software. A value of p≤0.05 was considered statistically significant.Results are expressed as mean±SEM. Statistical significance was evaluated by Student's Neonatal piglets injected with Dox see ; Group IMyocd mRNA levels, measured by microarray followed by qPCR analyses, were 2-3-fold higher in failing versus non-failing LV myocardium, whereas srf expression levels remained unchanged  in failing versus non-failing myocardium. In addition, the relative level of phospholamban  controls. FLAG-tagged ANKRD1 levels were uniform in all samples, indicating efficient transfection and the absence of nonspecific inhibitory or toxic effects on overall transgene expression. Two sh-myocd vectors (sh-424 and sh-1554) provoked a nearly complete inhibition of MYOCD protein expression in co-transfected COS7 cells.Nine sh-RNAs were designed against the porcine ndicated  and the myocd vectors was then evaluated by using a primary PAOSMC line, because we found that these proliferating cells exhibit a proper expression of myocd mRNA and protein at levels sufficient for analysis. Results of transient transfection of PAOSMCs revealed that sh-1554 exhibits more inhibitory activity on myocd expression than sh-424 (data not shown). The sh-1554 vector was therefore used for permanent transfection of PAOSMCs. Stable sh-1554 and sh-scr1 transfectants were screened for MYOCD and its SM-target proteins by immunoblotting. As shown in myocd sh-1554 transfectants displayed a nearly complete knockdown of MYOCD protein expression. Although none of the sh-1554 transfectants showed complete MYOCD depletion, the average inhibition of MYOCD protein expression was more than 80% from the sh-scr1 transfectant level. This MYOCD inhibition was associated with decreased levels of ACTA2 (the average inhibition – 88%) and MYH11 (the average inhibition – 60%) proteins in sh-1554 versus sh-scr1 transfectants, being expression of TAGLN unaffected   finding was that LV-ESP (an accepted marker for end-systolic performance) was slightly but significantly increased in sh-1554-transfected piglets as compared to controls (The sh-1554 vector was used in downstream experiments on lets see  were intlets see  has beenNA (40%) . In addiocardium . These icontrols .in vivo test-systems, the interfering sh-1554 vector was chosen as the most promising shRNA for silencing myocd expression in diastolic failing heart in vivo.On the basis of results obtained from cell-based and myocd inhibition were examined in experimental versus control groups two and seven days after transfection.To test whether inhibition of MYOCD signaling could influence the evolution of diastolic dysfunction in the piglet model of DHF, the following experimental design was used see . First, myocd-A (80%) and myocd-B (75%) mRNAs in failing LV-myocardium of the Dox/sh-1554 piglets as compared to that in the Dox/sh-scr1 animals (myocd transcript expression in failing myocardium of the Dox/sh-1554 group was lower (50% the average decrease) than that in the control PBS/crb1 group . Even more importantly, the reduced levels of these SM-marker proteins in Dox/sh-1554 piglets became comparable to those in control animals (PBS/sh-scr1 group). These effects of myocd silencing were not the consequence of a general, non-specific impairment of protein expression in transfected myocardium, because the protein content of TNNI, CASQ2 and GAPDH was equal in all groups studied.Next we measured expression of ctor see . Comparemyocd silencing on heart function, we used hemodynamic, ECG and intracardiac pressure measurements 2 days after transfection (myocd-silencing vector (Dox/sh-1554 group) to values similar to those in hearts of PBS-treated piglets (PBS/sh-scr1 group). The re-establishment of impaired diastolic function was associated with lowering the rate of ECG-indicators of myocardial ischemia  as well as with the trend toward amelioration of bradycardia in Dox/sh-1554 compared to Dox/sh-scr1 transfected piglets  in Dox-failing piglets , rols see .in vivomyocd expression also drops, leading to the restoration of upregulated levels of myocd in failing myocardium at distant time points after transfection.The efficacy of plasmid-based  vectors can be compromised by a rapid degradation of delivered plasmid constructions myocd mRNA expression in the Dox/sh-1554 group was re-activated  as compared with the same experimental run at day 2 post-delivery  are elevated in DHF piglets  was found to induce dilated cardiomyopathy and fatal HF in postnatal mice myocd-transfected and genetically myocd-depleted postnatal hearts can be explained by many factors on multiple levels. As compared to myocd gene ablation, a moderate sh-mediated inhibition of myocd expression: (1) did not lead to the almost complete loss of the targeted mRNA  in the LV-myocardium and (2) did not affect myocd activity in other cardiac chambers (data not shown). In addition, species-specific differences in MYOCD requirements for postnatal LV-remodeling, in pigs as compared to mice, could not be excluded, although the underlying mechanisms remain elusive at present.The moderate reduction (60–70%) of ventricular myocd were reported to be expressed in mammalian heart myocd B transcript being more abundant in pig ventricular myocardium myocd-B mRNA was generated, and after validation in three distinct experimental setups, the sh-interfering RNA1554 was chosen as the most promising sequence for silencing myocd in DHF, because it effectively targets the endogenous pool of myocd-A/B mRNAs in both pig aortic SM-cells in vitro  this decrease of MYOCD signaling activity in failing myocardium to the level comparable with that in non-failing animals resulted in improvement of impaired diastolic function and amelioration of myocardial ischemic conditions, (3) the posterior restoration of elevated myocd expression led to activation of myocd-dependent SM-marker genes in failing LV-myocardium associated with a return to altered diastolic function, and (4) the transient inhibition of MYOCD signaling at advanced stages of DHF delayed the progression of diastolic dysfunction and extended the survival of failing piglets. In rats, myocardial ischemia and bradycardia are two ECG-manifestations of the acute Dox-induced cardiotoxicity, embodied with decreased heart rhythm myocd targeting was associated with amelioration of such ECG-manifestations suggesting an involvement of MYOCD signaling in the regulation of cardiac function.We explored the potential role of srf expression because srf transcript levels were not changed in failing as compared to non-failing piglet myocardium. Thus, it is tempting to speculate that the activation of SM-marker genes in DHF myocardium can be attributed, at least in part, to selective upregulation of MYOCD. In cardiomyocytes, the level and functional activity of MYOCD is regulated by a balance of positive It is not unreasonable to suggest that impaired diastolic function in Dox-treated piglets is conditioned, at least in part, by overexpression of SM-marker genes  in failing myocardium. The induction of these SM-marker genes seems not to be associated with activation of Myocd overexpression correlates with a wide array of pathological conditions. Upregulation of myocd enforces cardiac hypertrophic response myocd is a frequent event in HF conditions myocd suppression was sufficient to downregulate the elevated expression of MYOCD-dependent SM-marker genes, ameliorate diastolic chamber dysfunction, and prevent pre-mature mortality of DHF animals.myocd expression to the range of physiological variation could be essential in order to reduce and normalize the expression of SRF/MYOCD-dependent SM-marker genes, that are upregulated in advanced HF of diverse etiologies (More broadly, the results of the present work give a clear indication that adjusting altered


An in vivo study using SIV-infected monkeys has been planned to explore this new therapeutic concept.Carbohydrate-binding agents (CBA) represent a broad family of agents that recognize specific glycan conformations. Several members of this compound family including peptidicprokaryotic, plant, invertebrate and vertebrate lectins, but also the well-defined non-peptidicpradimicin and benanomicin antibiotics have shown to be endowed with anti-HIV activity. These compounds inhibit entry of virus particles in susceptible T-lymphocytes and macrophages, block syncytia formation between HIV-infected and non-infected T-lymphocytes, prevent capture of HIV by DC-SIGN-expressing cells and by the macrophage mannose receptor and prevent subsequent transmission of captured HIV to susceptible T-lymphocytes. No other antiviral class of compounds have been demonstrated to block these four important ways of HIV infection/transmission, which make the CBAs an interesting new family of potential anti-HIV drugs that may be particularly useful for both systemic and topical  applications. CBAs bind to the glycans of the HIV envelope gpl 20 and block viral entry, most likely by freezing the envelope conformation during viral entry. Escalating drug pressure in HIV-infected cell cultures result in the selection of mutant virus strains predominantly having one or several N-glycan deletions in their envelope gp120.ln this respect, many CBAs have a high genetic barrier since several N-glycan deletions seem to be required for significant phenotypic drug resistance. Interestingly, mutant virus strains containing an increasing amount of N-glycan deletions in gp120 tend to show a compromised ínfectivity potential. Moreover, we have found one specific, highly conserved N-glycan in HIV-1 gp120, whose deletion results in the production of noninfectious virus particles lacking gp41gp120 in their envelope. This N-glycan may be regarded as a hot-spot for a targeted therapeutic intervention. Also, it is expected that if mutant virus particles lacking N-glycans in gp120 are selected under CBA pressure


Despite recent advancement in medicine, nearly 50% of patients with colorectal cancer show recurrence of the disease. Although the reasons for the high relapse are not fully understood, the presence of chemo- and radiotherapy-resistant cancer stem/stem-like cells, where many oncomirs like microRNA-21 (miR-21) are upregulated, could be one of the underlying causes. miR-21 regulates the processes of invasion and metastasis by downregulating multiple tumor/metastatic suppressor genes including PTEN (phosphatase and tensin homolog). Tumor suppressor protein PTEN controls self-renewal of stem cells. Indeed, our current data demonstrate a marked downregulation of PTEN in SCID mice xenografts of miR-21 over-expressing colon cancer HCT116 cells. Colonospheres that are highly enriched in cancer stem/stem like cells reveal increased miR-21 expression and decreased PTEN. Difluorinated curcumin (CDF), a novel analog of the dietary ingredient curcumin, which has been shown to inhibit the growth of 5-Flurouracil + Oxaliplatin resistant colon cancer cells, downregulated miR-21 in chemo-resistant colon cancer HCT116 and HT-29 cells and restored PTEN levels with subsequent reduction in Akt phosphorylation. Similar results were also observed in metastatic colon cancer SW620 cells. Since PTEN-Akt confers drug resistance to different malignancies including colorectal cancer, our observation of normalization of miR-21-PTEN-Akt pathway by CDF suggests that the compound could be a potential therapeutic agent for chemotherapy-resistant colorectal cancer. The emerging challenge in the treatment of colorectal cancer (CRC), the third most common cancer, is the relapse of the disease. Nearly 50% of CRC patients show recurrence of the disease. The presence of chemo- and radio-therapy resistant cancer stem/stem-like cells (CSCs/CSLCs) could be one of the underlying causes PTEN is one of the target proteins of miR-21, which is upregulated in many malignancies, including colorectal cancer Difluorinated curcumin (CDF), a nontoxic analog of the dietary ingredient curcumin with a much greater bioavailability than the parent compound K-ras mutant), HCT-116 , HT-29  and SW620 were obtained from the American Type Culture Collection . Chemo-resistant (CR) colon cancer cells were generated in our laboratory as described previously The human colon cancer cells HCT-116  media supplemented with B27 , as reported previously CDF was dissolved in DMSO. Where applicable, CR HCT116, CR HT29 or SW620 cells were treated with 100 nM CDF for 72 hours. Control cells received 0.05% DMSO. After 72 h, the cells were subjected to DNA, RNA and protein isolation.The animal research protocol was approved by Wayne State University Institutional Animal Care and Use Committee (IACUC). Xenograft derived from HCT116 cells, stably transfected either with miR-21 or the vector (pcmv), were generated in SCID mice as reported earlier ® Green PCR Reagents (Applied Biosystems). The relative amount of miR-21 and PTEN-mRNA were normalized to RNU1α (Exiqon) and human β-actin gene respectively.RNA was isolated from FFPE tissues using miRNeasy FFPE Kit (Qiagen) and from cultured cells using miREasy kit (Qiagen). Twenty nanogram of total RNA were reverse transcribed into cDNA using Universal cDNA Synthesis Kit  according to the manufacturer's protocol. To estimate the relative levels of PTEN-mRNA, 500 ng of RNA were reverse transcribed into cDNA using Gene Amp Gold RNA PCR Kit (Applied Biosystems). Real time PCR was performed using specific primers for miR-21 (Exiqon) and PTEN Western blot analysis was performed according to our standard protocol TM 2000 according to the manufaturer's protocol. Six hours post transfection, the media was changed and the cells transfected with NC were incubated with 100 nM CDF for 24 h. Luciferase activity was determined using Promega luciferase kit following manufaturer's protocol. β-galactosidase assays were performed to normalize the transfection variability. The data were presented as relative luciferase activity per microgram protein.HCT116-CR cells were transfected either with anti-sense miR-21 or negative control [NC] ( Ambion) along with the luciferase reporter plasmid having PTEN 3'UTR seed sequence for miR-21 The results were expressed as means ± SD. Comparisons of the continuous variables between two independent groups were calculated using two-tailed student's t test. The p value of <0.05 was considered to be statistically significant.PTEN is a target of oncomir miR-21. To examine the relationship between miR-21 and PTEN, SCID mice xenografts derived from miR-21 over-expressing HCT116 cells (miR-21) and the vector-transfected control cells (pcmv) were analyzed for the expression of miR-21 and PTEN. As expected, the data from qRT-PCR analysis revealed that the relative levels of miR-21 were significantly higher in xenografts from miR-21 over-expressing HCT-116 cells, compared to the controls . In contColonospheres, considered to be surrogate tumors, are highly enriched in CSCs/CSLCs Earlier, we demonstrated that CR colon cancer cells are not only enriched in CSCs/CSLCs but also express significantly higher levels of miR-21 than their parental counterparts To further examine whether CDF would affect the expression of miR-21- PTEN – Akt axis in metastatic colon cancer, we carried out the same experiment with SW620 cell line that was generated from metastasis colon cancer tissue. As has been observed for CR-HCT116 and HT29 cells, CDF caused a marked reduction in miR-21 expression in SW620 cells . This waDespite recent advancement in medicine, nearly 50% of patients with CRC show tumor recurrence. Although the reasons for this are not fully understood, it may partly be related to the inability to target CSCs/CSLCs that express cancer stem cell markers, retain the limitless potential to regenerate and are resistant to conventional therapies Although it is widely believed that cancer stem cells arise from normal stem or progenitor cells upon mutation Results of our current investigation demonstrate that the colonospheres, that are highly enriched in CSCs/CSLCs with increased expression of miR-21 miR-21 the potential “oncomir” invoked us to search for agent(s) that could be effectively used to downregulate miR-21 in colon cancer cells and thus reducing their tumorigenic potential. Rationale for pursuing this line of investigation comes from our recent observation that downregulation of miR-21 in chemo-resistant colon cancer cells causes differentiation and rendering them susceptible to anti-cancer agents, specifically difluorinated curcumin (CDF), an analog of the dietary ingredient curcumin with greater bioavailability than the parent compound In conclusion our current data demonstrate that miR-21 and PTEN are inversely related in that an increased expression of miR-21 results in reduction in PTEN that leads to activation of Akt signaling. These events may play pivotal roles in tumor progression. Furthermore, our observation that miR-21 mediated PTEN-Akt signaling could be normalized by CDF in chemo-resistant colon cancer cells suggests that CDF could be a potential therapeutic agent for recurrent colon cancer.


BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the  Cell-cell junctions, which include adherens junctions, desmosomes, and tight junctions, are fundamental to the intricate cellular architecture of all epithelial tissues In seeking to elucidate the functions of folliculin (FLCN), the protein product of the Birt-Hogg-Dube (BHD) gene, we used a yeast two-hybrid approach to uncover a physical interaction between the junctional protein p0071  and FLCN. p0071 is a relatively understudied member of the armadillo repeat-containing protein family, which includes p120-catenin (p0071’s closest homolog) and beta-catenin BHD gene, which is on chromosome 17p11.2 and was cloned in 2002 BHD mutations are truncating, and RCC from BHD patients exhibit loss of heterozygosity of chromosome 17p FLCN is a 64 kDa highly conserved protein with no obvious functional domains. Germline mutations of the BHD mutations lead to renal and skin tumorigenesis and cystic lung disease are incompletely understood.FLCN is known to exist in a complex with AMP-associated protein kinase (AMPK) via two interacting proteins: Folliculin-interacting protein 1 (FNIP1) and FNIP2 flox/flox mice have delayed eyelid opening, wavy fur, hair loss, and epidermal hyperplasia, phenotypes that resemble inactivation of other cell adhesion proteins, including p120-catenin, using the same promoter We found that downregulation of either FLCN or p0071 results in increased cell-cell adhesion, which is surprising given the conventional view that decreased cell-cell adhesion is associated with tumorigenesis. These results implicate enhanced cell-cell adhesion as a contributing factor in tumorigenesis. To examine this in vivo, we utilized a classic model of cell-cell adhesion integrity: keratin-14 cre-recombinase (K14-cre) To elucidate the molecular functions of FLCN, we used a yeast two-hybrid approach to discover novel FLCN interacting partners. Full-length myc-FLCN was used as bait and screened against a human fetal brain protein library see . The topTo confirm that FLCN and p0071 interact, we performed a GST-pulldown assay in which Madine-Darby canine kidney (MDCK) cell lysates were incubated with either GST-FLCN, or empty GST. p0071 interacts with GST-FLCN, but not GST  and UOK257-2 (FLCN-expressing) cells to determine the impact of FLCN-deficiency on cell junction formation. As shown in To confirm that the hanging drop results were FLCN-dependent and not cell-type specific to the UOK cells, we grew T84 colon carcinoma cells stably expressing non-targeting or FLCN shRNA in adherence-free conditions using a rotary shaker. T84 cells were chosen because they are often used as an experimental model for studies of cellular junctions To determine whether FLCN is necessary for the expression of cell junction proteins, we measured the levels of p0071, plakoglobin (JUP), zonula occludens-1 (ZO-1), and p120 catenin in UOK257 cells compared to UOK257-2 cells. JUP is a desmosomal and adherens junction protein that forms complexes with catenins and cadherins , p0071 levels are decreased in FLCN-null UOK257 cells, while JUP, ZO-1, and p120 catenin levels are unaltered by FLCN-deficiency.As shown in To determine whether the increased cell-cell adhesion observed in FLCN-deficient cells is mediated via the FLCN-p0071 interaction, HEK293 cells that stably express non-targeting, FLCN, or p0071 shRNA were grown in adherence-free conditions as in 3F. After a standardized shearing protocol see  the HEK2Keratins are the major structural component of intermediate filaments in desmosomes, and play a critical role in cell-cell and cell-matrix adhesion deficiency of either FLCN or p0071 leads to increased cell-cell adhesion and defects in cell polarity.The establishment of proper apical-basal polarity is another important component of cellular homeostasis and defects in cell polarity are commonly observed in tumorigenesis. Therefore, we next examined the impact of FLCN and p0071 downregulation on cell polarity. T84 cells stably expressing either non-targeting, FLCN, or p0071 shRNA were grown in mixed extracellular matrix proteins. These 3-dimensional matrices cause epithelial cells to form spherical cysts consisting of a monolayer of epithelial cells around a hollow lumen. The individual cells are connected to each other by cell-cell contacts and form a highly polarized structure where the apical surface is oriented to the lumen of the cyst in vivoBhd in the mouse skin, in order to evaluate the effects of FLCN-deficiency on cell adhesion in vivo. Bhdflox/flox (Bhdf/f) mice f/f K14-cre mice. By three weeks of age, the Bhdf/f K14-cre mice were significantly smaller than their littermate controls, developed coarse and wavy hair, and displayed erythematous patches of skin and hair loss, which progressed with age (f/f K14-cre mice had done so (representative image shown in ).Inactivation of junctional proteins with keratin-14 driven cre-recombinase (K14-cre) is used frequently to study cell-cell adhesion f/f K14-cre mouse skin revealed a thickened epidermis involving the spinous layer (acanthosis), granular layer (hypergranulosis) and stratum corneum (hyperkeratosis)  Figure 6BHD mutations also occur in families with hereditary lung collapse (pneumothorax) without renal or skin disease BHD mutations occur in ∼10% of sporadic chromophobe RCC BHD is an autosomal dominant disease associated with lung cysts, skin tumors, and kidney cancer, primarily of the chromophobe subtype These data provide a critical new component to our emerging understanding of the pathogenesis of BHD. BHD is one of the least studied monogenic tumor suppressor gene disorders, with fewer than 12 publications on the functions of FLCN. FLCN has been linked to AMPK signaling via a direct interaction with FNIP1/2, to HIF signaling and mitochondrial dysfunction, to aberrant TGF-beta signaling, and to mTOR complex 1 signaling CDH1 gene, which encodes E-cadherin, cause human gastric cancer Our finding that FLCN-deficient and p0071-deficient cells have enhanced cell-cell adhesion is unexpected, given that cell-cell junctions are critical for epithelial tissue homeostasis and loss of the integrity of these junctions is associated with cancer progression In addition to their localization to adherens junctions, both p120-catenin and p0071 localize to the cytoplasm where they regulate RhoA activity BHD gene develop renal cell carcinomas (RCC), the majority of which are chromophobe and chromophobe/oncocytic hybrid tumors, and somatic BHD mutations have been found in sporadic chromophobe RCC, which is a rare disease affecting ∼1500 individuals in the United States each year. There appear to be at least two potential links between defective cell junctions and sporadic RCC. First, we found that FLCN-deficient cells have more desmosomes than FLCN-expressing cells, and Kim et al. found that desmosomes are increased in RCC compared to normal kidney tissue and identified an association between desmosome number and tumor grade Patients with mutations in the The cellular and physiologic mechanisms that lead to cystic and emphysematous airspace enlargement in BHD are not known. Respiration exposes the lung to unique mechanical forces. Cell-cell junctions allow the lung to expand in response to these forces and then “snap back” to their original shape and organization. Mechanical-induced stretch of pulmonary alveolar type II cells leads to widened cell-cell gaps and is speculated to be a protective mechanism in response to lung injury, likely due to a flexibility effect Bhd is inactivated in the epidermis via the Keratin-14 (K14) promoter. K14-cre driven inactivation of adhesion proteins has been used to study other junctional proteins in vivoFinally, we have developed a novel mouse model in which In conclusion, we have uncovered a physical interaction between FLCN and p0071 that is critical for the proper regulation of cell-cell adhesion, RhoA activity, and epithelial cell polarity. A mouse model of FLCN inactivation in the epidermis leads to wavy hair and whiskers, delays in eyelid opening, hair loss, and increased epidermal thickness, phenotypes which resemble the inactivation of p120-catenin, the closest homolog of p0071. Taken together, these findings reveal a novel paradigm in which loss of the FLCN-p0071 interaction and consequent defects in cell-cell adhesion, cell polarity and RhoA activity participates in the pathogenesis of renal cell carcinoma, skin tumors, and cystic airspace enlargement .The yeast two-hybrid assay was performed by Hybrigenics . Full-length human FLCN coding sequence was PCR amplified and inserted into pB29 vector as an N-terminal fusion to LexA (N-FLCN-LexA-C). The construct was used as a bait to screen the Hybrigenics Human Fetal Brain_RP1 library. The DNA insert of the positive clones was sequenced to identify the corresponding gene in the GenBank database.Human BHD cDNA was cloned into the pGEX4T-2 expression vector  and transformed into BL-21 bacteria (Stratagene). Isolation and purification of GST-FLCN or empty GST control was performed according to a previously published protocol The T84, HEK293 and MDCK cell lines were purchased from ATCC; FLCN-null UOK257 cells and FLCN re-expressing UOK257-2 cells were a gift from Drs. Laura Schmidt and Marston Linehan  Cells were lysed on ice in urea lysis buffer  and centrifuged at 14,000 rpm for 15 minutes. The supernatants were boiled, proteins separated by SDS-PAGE, and transferred onto PVDF membrane. Membranes were incubated with the following antibodies: anti-FLCN, anti-zonula occludens-1, anti-pan-keratin, and anti-keratin-18 ; anti-actin, anti-JUP, and anti-tubulin (Sigma); and anti-p0071 ; and anti-p120-catenin (Life Technologies). Chemiluminescent signals were captured using a Syngene G-BOX iChemi XT imager and quantified with Syngene GeneTools software.Cell aggregates were prepared using a previously described approach 100,000 HEK293 cells were seeded on a 60 mm suspension culture dish. Cells were incubated at 37°C on an orbital shaker at 70 rpm for 4 days, and allowed to form aggregates. Aggregate suspensions were harvested and sheared by pipetting 15 times using a P200 pipet. The sheared cell suspension was then transferred to a 35 mm dish and imaged under bright field using an Olympus IX-81 microscope. Aggregate size was quantified using ImageJ and the Integrated Morphometry Analysis feature in Metamorph .3 cells were trypsinized to a single cell suspension and re-suspended in 500 uL medium with 2.5% Matrigel  and plated on coverglass chambers  precoated with 5 uL of 100% Matrigel. Cysts were allowed to form over 5–7 days at 37°C. For visualization of F-actin with phalloidin Alexa 546  the fixation and staining procedure of Chu et al. was followed T84 cells with stable knock down  were maintained in DMEM supplemented with 10% FBS and Puromycin (5 ug/mL). For 3D culture, approximately 4.5×10Cells were plated at 75% confluency in three 35 mm dishes, and cultured until fully confluent. Linear wounds were created by scraping a P200 pipette tips across the surface of each confluent monolayer. Bright field images of wound migration were acquired at 0, 8, 24, 48 80 and 92 hours at 4×magnification using an Olympus IX-81 microscope. For each time point, the wound size in pixels was calculated for each dish, and the average of the 3 dishes was used as a single data point.The phosphorylation of the myosin binding subunit (MBS) was measured to analyze Rho-associated kinase (ROCK) activity using a modification from a previously published method Rho activity was examined using the Active Rho Pull-Down and Detection Kit (Pierce Biotechnology) as previously described Bhd were generated by crossing Bhdf/f mice (a gift from Drs. Laura Schmidt and Marston Linehan) with Keratin14-Cre transgenic mice (Jackson Laboratories) to establish Bhdf/+ K14-Cre animals. Bhdf/+ K14-Cre mice were intercrossed to generate Bhdf/f K14-Cre mice. Tissue processing and staining was performed by the Rodent Histopathology Core . Epidermal thickness was measured using Olympus FSX100 integrated software.All mouse experiments were carried out under guidelines approved by the ARCH Institutional Animal Care and Use Committee (IACUC). The ARCH IACUC specifically approved this study. Transgenic mice with skin-specific inactivation of mouse