Microsatellite instability (MSI) is a unique molecular abnormality, indicative of a deficient DNA mismatch repair (MMR) system. Described and characterized in the colorectal cancer literature, the MSI-positive phenotype is predictive of disease susceptibility, pathogenesis, and prognosis. The clinical relevance of MSI in colorectal cancer has inspired similar inquisition within the sarcoma literature, although unfortunately, with very heterogeneous results. Evolving detection techniques, ill-defined sarcoma-specific microsatellite loci and small study numbers have hampered succinct conclusions. The literature does suggest that MSI in sarcoma is observed at a frequency similar to that of sporadic colorectal cancers, although there is little evidence to suggest that MSI-positive tumors share distinct biological attributes. Emerging evidence in Ewing sarcoma has demonstrated an intriguing mechanistic role of microsatellite DNA in the activation of key EWS/FLI-target genes. These findings provide an alternative perspective to the biological implications of microsatellite instability in sarcoma and warrant further investigation using sophisticated detection techniques, sensitive microsatellite loci, and appropriately powered study designs. The majority of microsatellite DNA is comprised of mono-, di-, tri- and tetra-nucleotide repeats, and these repetitive elements constitute ~3% of the human genome [−4 to 5 × 10−5 [The biological precedence of tandem nucleotide repeats scattered throughout the human genome has intrigued scientific inquiry since these genetic elements were first characterized in the early 1980s. More precisely, the term n genome . The polThe mechanism by which microsatellite DNA undergoes a length mutation is commonly believed to occur via “replication slippage,” where the replicating DNA strand transiently dissociates from the DNA template and reanneals out of frame in denominations of the repeat motif . In geneMicrosatellite instability was discovered and characterized nearly 20 years ago in patient-derived colorectal tumors , 9. ThesThe Bethesda Panel) of mono- and di-nucleotide microsatellite loci sensitive to the development of microsatellite instability [This important molecular signature has lead to the development of standardized screening guidelines, diagnostic protocols and even a reference panel 10, which is commonly mutated in MSI-positive tumors [βRIITGF signalling is known to inhibit cell growth and proliferation and therefore is considered to have a tumor-suppressor role in oncogenesis [βIIRTGF gene expression has been implicated in the process of oncogenic transformation in Ewing sarcoma cells [BAX, also harbors a coding microsatellite region [BAX is downstream target of p53-mediated apoptosis [The determination of MSI and defects of the MMR system in sarcoma has gained increasing attention for several evolving reasons. Firstly, there remains a paucity of clinically relevant and easily assayed molecular biomarkers in sarcoma. The characterization of MSI in colorectal cancer and the informative nature of this mutator phenotype have provided optimism that similar findings may be observed in a distinct subset of sarcomas. Secondly, although microsatellite DNA is primary in noncoding regions of the genome, clusters of microsatellite DNA are found in coding and flanking regions of genes identified in sarcomagenesis. For example, the e tumors . TGFβRIIogenesis , 25. EWSma cells , 26, 27.e region . BAX is poptosis  and dysrpoptosis –31. Intepoptosis . Other spoptosis –35. Finapoptosis , which hp53 and RB loci on chromosomes 13q and 17p, respectively. Many studies assessing these sarcoma-specific microsatellite loci were often simultaneously assessing MSI and loss of heterozygosity (LOH) as a collective phenotype of allelic imbalance.Shortly after the characterization of MSI in CRC, numerous studies emerged addressing the issue of MSI in sarcoma. Not surprisingly, these studies do not provide conclusive evidence for or against the presence of microsatellite instability in sarcoma. Five studies observed a high frequency of MSI –41, rangThe accurate detection and interpretation of MSI is a complex and persistently evolving subject. Consequently, a variety of methodological issues must be appreciated when interpreting these discordant results. The accuracy of MSI detection is highly dependent on the molecular techniques used for assessment. Firstly, Taq polymerase is prone to slippage errors during PCR amplification. The slippage rate of Taq polymerase increases with an increasing number of repeats and is inversely proportional to the length of the microsatellite repeat unit , 51. The The advent of fluorescent-labeled PCR and automated sequencing methods negates many of the shortcomings of traditional electrophoresis and autoradiographic detection systems. Fluorescent-labeled nucleotides are added to the PCR condition and synthesized amplicons from independent samples are coelectrophoresed and quantitatively assessed using a laser scanner . Using fA very important technical consideration pertains to the microsatellite loci assessed in determining the MSI-positive phenotype. Based on the plethora of literature detailing this topic in colorectal cancer, the ideal microsatellite panel should consist of the following: microsatellite harboring mono- and di-nucleotide repeats, a panel of ≥5 informative loci, microsatellite loci sensitive to mismatch repair deficiency and avoidance of microsatellite loci in regions highly susceptible to copy number deletion (loss of heterozygosity) , 19, 58.Given the discrepancy of results and the methodological heterogeneity used to assess MSI, it remains difficult to ascertain whether MSI is a prominent molecular phenotype in common sarcomas. The incidence of MSI in sporadic colorectal carcinomas is routinely reported around 15% –23, 58 aOptimistically, it was hoped that the identification of the MSI phenotype in sarcoma could similarly predict tumor behavior and clinical outcomes. From the available studies in sarcoma, there is no compelling evidence to suggest a predictive value of microsatellite instability in sarcoma. The clinical conclusions from the sarcoma literature should be interpreted cautiously though as in contrast to the colorectal cancer literature, which is comprised of clinical studies and meta-analysis assessing 100s–1000s of patients , 17–21,  EWS gene is fused with the DNA-binding C-terminus of a member of the ETS family of transcription factors. The most common translocation, EWS/FLI1 (t22 : 11) is observed in roughly 85%–90% of Ewing tumors [CAV1 and NR0B1, are necessary for oncogenic transformation [GSTM4, is associated with therapeutic resistance and inferior clinical outcomes [NR0B1, which contains a 102bp microsatellite, consisting of 25 GGAA motifs and 2 single-base insertions [NR0B1 promoter has demonstrated that a minimum of four GGAA motifs are needed for EWS/FLI-mediated gene activation, which increases exponentially with an increasing number of GGAA repeats [NR0B1 GGAA microsatellite have also been observed across various Ewing sarcoma cell lines, where a significant correlation between overall microsatellite length and NR0B1 gene expression is observed [Recent evidence in the Ewing sarcoma literature has demonstrated a novel, mechanistic necessity of microsatellite DNA during EWS/FLI-mediated oncogenesis , 59. Likg tumors . Genomewg tumors , 61. Addormation , 63, whioutcomes . The mossertions . Charact repeats , 59. Lenobserved . These dP = 0.13 and P = 0.28, resp.). On the contrary, studies by Alldinger et al. [The importance of microsatellite DNA in Ewing sarcoma oncogenesis has stimulated renewed interest in the determination of microsatellite instability in this subtype of sarcoma. If an increasing number of GGAA repeat motifs enhances EWS/FLI binding and gene activation, microsatellite instability within these response elements has potential for significant biological ramifications in Ewing sarcoma cells. Three studies listed in r et al.  and Ebinr et al.  documentNR0B1 microsatellite is quite polymorphic across populations, with extremely large repeats, containing as many as 70 GGAA motifs, observed in both African and European subjects (unpublished data). The shear magnitude of these 70 repeat microsatellites would be more intrinsically unstable than the 20–25 GGAA repeats observed in many of the Ewing sarcoma cell lines.  Despite the discordant data in Ewing sarcoma, the discovery of GGAA microsatellites as EWS/FLI-response elements recapitulates the need for a more detailed assessment of MSI in Ewing sarcoma. Certainly, instability within these GGAA response elements has potential to significantly affect gene expression of key EWS/FLI targets. Given the tetranucleotide composition of the GGAA repeat motif, these microsatellites may theoretically be more intrinsically stable than simple mono- and di-nucleotide repeats. However, the In summary, microsatellite instability is a captivating oncological observation, highlighting the contribution of microsatellite DNA and mismatch repair system in the process of oncogenesis. Despite regular observations in the colorectal literature, the detection and clinical ramifications of MSI in sarcoma are less consistent. Certainly there is evidence to suggest that many sarcomas display the MSI-positive phenotype although this finding may relate more to generalized genomic instability than a deficient mismatch repair system. The observation of microsatellite response elements in Ewing sarcoma provides an alternative motivation to reassess microsatellite instability in this context. Future efforts in this topic must ensure that studies are appropriately designed; employing advanced sequencing techniques, sensitive microsatellite markers, and adequately powered to accurately measure clinical outcomes.


Yet specific immunotherapy risks allergic reactions including anaphylaxis as a consequence of preformed allergen-specific IgE antibodies binding to the protein, subsequent cross-linking and mast cell degranulation. CD4+ T cells direct their responses to short "immunodominant" peptides within the allergen. Such peptides can be given therapeutically to induce T cell tolerance without facilitating IgE cross-linking. Peptide immunotherapy (PIT) offers attractive treatment potential for allergic disease. However, PIT has not yet been shown to be effective in children. This review discusses the immunological mechanisms implicated in PIT and briefly covers outcomes from adult PIT trials. This provides a context for discussion of the challenges for the application of PIT, both generally and more specifically in relation to children.Allergic sensitisation usually begins early in life. The number of allergens a patient is sensitised to can increase over time and the development of additional allergic conditions is increasingly recognised. Targeting allergic disease in childhood is thus likely to be the most efficacious means of reducing the overall burden of allergic disease. Specific immunotherapy involves administering protein allergen to tolerise allergen reactive CD4 Allergic disease including atopic eczema, allergic rhinitis, allergic asthma and food allergy causes significant patient morbidity and economic costs to healthcare systems ,2. Curre+ T cells, particularly the T helper (Th) 2 subset, in allergy. These cells express the transcription factor GATA-binding protein 3 (GATA-3) and can produce allergy-associated cytokines such as IL-4, IL-5 and IL-13 which are implicated in a host of allergic responses such as eosinophil recruitment and airway hyperreactivity [reviewed in [+ T cells therefore has the capacity to abrogate downstream allergic responses [+ T cells via the administration of protein allergen. First used over a century ago [+ T cells so they no longer mount an allergic response to the allergen. This can occur either through direct effects on allergen-reactive T cells and/or through the actions of T regulatory cells [Evidence suggests a key role for CD4iewed in ]. Th2 ceiewed in . Less coiewed in ]. Therapesponses . One wayry cells . SIT canry cells , therapery cells  and SIT ry cells ]. Yet SIry cells -20.+ T cell response is directed . Genera+ T regulatory cells. That said, peptide-reactive Foxp3+ T regulatory cells were generated in a model using a peptide complex designed to specifically target peptide to DCs, ensuring peptide uptake and presentation , and/orolecules ]. Additi+ cells in some PIT studies [in vitro on T cells with a limited TCR repertoire may behave differently in vivo on encountering a more diverse T cell repertoire [There are advantages to altering the sequence of a therapeutic peptide. APLs have defined amino acid substitutions which can change MHC and/or TCR binding properties of the peptide . Differe studies . The sti studies ,74. Therpertoire . Using Apertoire .Leech et al altered a myelin-based peptide reducing its IgG1 binding affinity while maintaining its tolerising capacity [Other applications of APLs could be more amenable to clinical translation. For example, APLs can be generated to reduce the allergenicity of a peptide. capacity . Hence, The most desirable route of delivery of PIT for children would be oral or intranasal as they are minimally distressing. PIT delivered in these ways can be effective in inducing tolerance ,77, raisThere remains no clear consensus as to the optimal peptide dose or regimen which have varied considerably in different trials -51,79. DA key question is how long PIT needs to be given to induce long-term tolerance? Limited data on this exist from PIT allergy studies which to date have focused on short-term clinical outcomes. However, tolerance following SIT has been demonstrated up to 7 years after completion of treatment . AlthougConcurrent illness may hold particular relevance for application of PIT to children. Data concerning this do not exist for PIT. However, giving SIT during concurrent systemic illness has been associated with fatalities  and currPIT holds promise for the treatment and modification of allergic disease. There have been significant advances in the application of PIT to adult allergic patients, and there is substantial scope for future opportunities for the application of PIT. Yet to maximise therapeutic efficacy necessitates translation to allergic children. For this, further understanding of the underlying mechanisms involved in PIT, potential confounding factors such as viral infection and the interplay between dose, regimen and route need to be further elucidated. Mechanistic studies utilising experimental models together with carefully structured, appropriately powered clinical trials looking at a host of outcome measures should aid future translation to the paediatric clinic.APL: altered peptide ligand; GATA-3: GATA-binding protein 3; HLA: human leukocyte antigen; LARs: late asthmatic reactions; LPS: lipopolysaccharide; MHC: major histocompatibility complex; PBMC: peripheral blood mononuclear cells; PIT: peptide immunotherapy; SIT: specific immunotherapy; TCR: T cell receptor.The authors declare that they have no competing interests.All 3 authors framed the scope of the article, KJM wrote the article and SMA and JS contributed to refine the substance. All authors read and approved the final manuscript.


Repair of plasma membrane tears is an important normal physiological process that enables the cells to survive a variety of physiological and pathological membrane lesions. Dysferlin was the first protein reported to play a crucial role in this repair process in muscle, and recently, several other proteins including Mitsugumin 53 (MG53), annexin and calpain were also found to participate. These findings have now established the framework of the membrane repair mechanism. Defective membrane repair in dysferlin-deficient muscle leads to the development of muscular dystrophy associated with remarkable muscle inflammation. Recent studies have demonstrated a crosstalk between defective membrane repair and immunological attack, thus unveiling a new pathophysiological mechanism of dysferlinopathy. Here I summarize and discuss the latest progress in the molecular mechanisms of membrane repair and the pathogenesis of dysferlinopathy. Discussion about potential therapeutic applications of these findings is also provided. Damage to the plasma membrane induces entry of toxic agents such as calcium and oxidants into the cells, releases intracellular molecules producing inflammatory responses, and threatens the afflicted cells with an immediate cell death. Recent studies reveal a rapid membrane repair response that is conserved in many different types of cells to restore the plasma membrane integrity and enables the cells to survive following a limited level of membrane disruptions -7. Defec2+-triggered vesicular exocytosis [2+-independent, but rather mediated by cholesterol exposure and oxidation-induced oligomerization in skeletal and cardiac muscle [2+-triggered vesicle-membrane fusion are two distinct steps in the membrane resealing process. Thus, Ca2+, cholesterol and oxidation can trigger different components of the membrane repair machinery to initiate the emergency response. It is interesting what other signals could be involved in the initiation of the membrane repair responses. In addition to Ca2+ influx, cholesterol exposure and oxidation, membrane damage can result in other changes such as Na+ influx and membrane potential depolarization, which may also be involved in membrane repair.It is known that the membrane repair process requires intracellular vesicles  which deocytosis ,17 similocytosis   proteins [2+-dependent repair of membranes in plants [2+-sensitive phospholipid binding activities [2+ sensor and directly regulates the SNARE-mediated vesicle-membrane fusion during membrane repair. Direct interactions between dysferlin and SNARE proteins have not been established yet. However, otoferlin, a mammalian homologue of dysferlin, has been demonstrated to bind synaptosomal-associated protein 25 (SNAP25) and syntaxin-1 and directly regulate synaptic vesicle exocytosis in inner hair cells [2 (PLA2) may also regulate the membrane repair process. Annexin A1 was shown to concentrate at the site of membrane damage and ablation of annexin A1 effectively inhibits membrane repair [2 and the generation of arachidonic acid promote membrane fusion mediating neutrophil degranulation [2 [After vesicle translocation, Caproteins  and synaproteins . Previouproteins ,29-31. Sproteins . Geneticproteins . Moreoven plants ,35. Thestivities ,37. Dysfir cells ,39. Thuse repair . The actnulation . Moreoveation [2 ,43. Whetation [2  was showation [2 ,46. ThisExcept for dysferlin, Syt7 and MG53, the current knowledge regarding membrane repair is largely from non-muscle studies. Future studies are necessary to bridge our knowledge gap in skeletal muscle membrane repair. Since membrane repair is highly conserved during evolution, it is no wonder that skeletal muscle may use some of the common mechanisms as identified in other cells to repair membrane damage. However, skeletal muscle cells are quite special in that they undergo frequent mechanical stress, which causes frequent membrane disruption and thus may require skeletal muscle cells to use some unique mechanisms to satisfy the high demand of membrane repair.Recent studies have revealed new functions of dysferlin which may be linked to membrane repair and/or inflammatory activation. Dysferlin was observed to associate with developing T-tubules ,48 and iDysferlin may coordinate cytoskeleton remodeling through its interaction with focal adhesion components. Previous work showed that dysferlin interacts with β-parvin , a prote2+-triggered intercellular signaling in response to membrane wounding in fertilized sea urchin embryo [2+ signaling [Recent data found that dysferlin may also be involved in cytokine and/or chemokine secretion. Cultured myoblasts from dysferlin mutant mice showed impaired secretion of cytokine MCP-1 when stimulated with IFN-γ or damaged with saponin . The autn embryo . Disruptignaling . Interesignaling , implicaignaling . Future Finally, dysferlin was found to play a role in endothelial cell adhesion and angiogenesis ,61. Exprin vivo [It has been known that the immune system is able to produce remarkable responses in the absence of infectious organisms. A "danger" hypothesis has been put forward to explain how the immunological responses occur in these situations ,13,62,63in vivo , and comin vivo . Thus, iin vivo .Since plasma membrane integrity is important for preventing the release of endogenous molecules, a defective membrane repair in animal cells is inevitably immunologically dangerous. Several proteins including Syt7 ,33, dysfin vivo [Recent studies have begun to unveil the pathophysiological mechanisms underlying muscle inflammation in dysferlinopathy. Dysferlin-deficient monocytes from SJL/J mice and dysferlinopathy patients were reported to have increased phagocytic activity  and dysfin vivo . Howeverin vivo  commentein vivo ,79, sugg2+-dependent endocytic and exocytic processes [Previously, dysferlin-deficient muscle cells were reported to be susceptible to complement attack, which was attributed to the down-regulation of the complement regulator CD55 on the plasma membrane without dysferlin . Activatrocesses .2+ release from the sarcoplasmic reticulum [Thus, the compromised membrane repair in the absence of dysferlin results in the prolonged release of endogenous "danger" molecules which lead to the local activation of the complement system  and upreeticulum . The ideCurrently, there is no effective therapeutic treatment for dysferlinopathy patients. The primary defect lies in the defective membrane repair caused by dysferlin deficiency. Thus, gene replacement therapy to restore the expression of functional dysferlin and membrane repair represents a great promise. However, it is still far away from any clinical application in dysferlinopathy. Pharmacological interventions implicated from recent studies represent novel avenues for the treatment of this disease. Although the efficacy of anti-inflammatory corticosteroids in dysferlinopathy is still controversial , our recA protective membrane resealing mechanism at the cell level is highly conserved among different species and cell types. It is mediated by exocytosis of intracellular vesicles forming a membrane "patch" at the disruption site. This process requires the participation and coordination of a large group of proteins involving cytoskeleton remodeling, vesicle translocation, and membrane fusion. Some of these proteins have recently been discovered and linked to human diseases, emphasizing the importance of the membrane repair proteins in life. An acute membrane repair mechanism not only prevents damaged cells from necrosis, but also reduces the exposure of "danger" signals to the immune system, which otherwise amplify the signals and cause massive tissue injury.The authors declare that they have no competing interests.


Stem Cells 2011;29:163–168Solid tumors are complex “organs” of cancer cells and a heterogeneous population of hematopoietic cells, mesenchymal cells, and endothelial cells. The cancer stem cell model proposes that tumor growth and progression is driven by rare populations of cancer stem cells; however, nontumor-forming stem and progenitor cells are also present within the tumor microenvironment. These adult stem cells do not form tumors when injected into experimental animals, but they may augment tumor growth through juxtacrine and paracrine regulation of tumor cells and by contributing to neovascularization. Thus, cancer cells may actively co-opt nontumor-forming stem cells distally from the bone marrow or proximally from nearby tissue and subvert their abilities to differentiate and maintain tissue growth, repair, and angiogenesis. This review will cover the roles of nontumor-forming vascular stem cells in tumor growth and angiogenesis.  The suggestion that tumors may be characterized by hierarchies of organized stem cells was a paradigm shift in our understanding of tumor biology . PutativFirst, a distinction should be made between bona fide stem cells that have the capacity for unlimited self-renewal and multilineage differentiation  and progenitor cells or “precursor cells” that have limited self-renewal and differentiation abilities. The generation of progenitor cells is hierarchical with an undifferentiated parental cell lying at the apex that generates progressively more differentiated progeny. For example, hematopoietic stem cells, which reside in the bone marrow, generate an array of more committed hematopoietic progenitor cells (HPC) that act as specific precursors to different blood cell lineages. Mesenchymal stem cells  although often referred to as stem cells, are also a type of progenitor or “precursor” cell. MPCs are found throughout the body, they can differentiate to form multiple cell types  and they have a limited ability to self-renew. Endothelial progenitor cells (EPCs) are another type of lineage-restricted progenitor cell. Although there is still debate about the definitive site(s) where EPC reside in the body, the progeny of circulating EPC  circulate in peripheral blood, can be ex vivo expanded into clonal populations and will form vessel lumens when injected into mice. EPCs, MPCs, and HPCs are often identified in blood or tissues using the markers listed in EPCs may be defined as circulating precursor cells with the ability to differentiate to form functional blood vessels at sites of neovascularization. However, there has been a lack of consensus regarding EPC definition, origin, and function in humans and animal models of disease. As a consequence, the contribution of these cells to the vasculature of solid tumors is still the subject of lively debate .A seminal article identified EPC as cells that circulate in peripheral blood and express markers such as CD34 and vascular endothelial growth factor receptor-2 (VEGF-R2) (kinase insert domain receptor [KDR]) . HoweverThe discrepancies surrounding the definition of EPC have made it difficult to identify these elusive cells in vivo, especially in tumors. Bone marrow has been proposed as a depot for circulating EPC in humans and in mouse tumor models although it is not always clear whether the identified cells are ECFC or hematopoietic cells located close to the blood vessel wall (reviewed in ). The abRecently, the vascular wall itself has been proposed as a source for EPC because it contains subpopulations of endothelial cells with properties similar to blood-derived ECFC . BecauseMPC and their progeny are present in most if not all postnatal organs. Their major function is to maintain and regenerate connective tissues and to replenish damaged tissue following injury or inflammation. MPC differentiation is dependent on the local production of cytokines/growth factors resulting in tissue specificity. Therefore, MPC may differentiate into the type of tissue they engraft . Because+ progenitor cells were shown to localize in a perivascular position raising the possibility that circulating MPC or MPC-like cells “seek out” blood vessels in the tissue they engraft [MPC may form a large proportion of the reactive stroma in solid tumors and share properties with carcinoma-activated fibroblasts, which express alpha smooth muscle actin (α-SMA), however, it is difficult to distinguish bona fide MPC from fibroblasts due to overlapping expression of several surface markers. Recently, Hoechst dye efflux was used to isolate and culture expand MPC from prostate carcinoma . One una engraft . On the  engraft  showed tTRAIL [The effect of MPC on primary tumor growth is equivocal with studies showing enhancement , inhibitTRAIL . Some ofThe tumor-promoting properties of MPC might be related to angiogenesis stimulation. MPC differentiate to form pericytes and are a major source of VEGF secretion . Althoug+ T lymphocytes may promote tumor cell invasion and metastasis by altering the function of tumor-associated macrophages (TAMs) [The role of hematopoietic cells in tumor angiogenesis has recently received great attention. HPC from the bone marrow emigrate to the blood and tissues and differentiate to form cells of the innate and adaptive immune system. Tumor-infiltrating leukocytes can either protect against or promote tumor formation. The simplest example of the former is loss of immune surveillance and spontaneous tumor formation in some strains of immunodeficient mice. On the other hand, CD4s (TAMs) . Myeloids (TAMs) ). Althou+-circulating myeloid cells impaired blood vessel development and anastomosis with host vasculature [One of the first clues that HPC might be required for angiogenesis came from studies in the developing embryo. For example, acute myeloid leukemia 1 (AML1)–deficient embryos, which lack definitive hematopoiesis, showed impaired angiogenesis in the head and pericardium that was rescued by addition of HPC-expressing angiopoietin 1 (ANG-1) . Thus, eculature . Patholoculature ). For exculature . SelectiThe catalytic function of hematopoietic cells during tumor progression may be related to their expression of proangiogenic and tissue-remodeling factors including MMPs and VEGF. Furthermore, tumor-derived factors may activate or change the phenotype of the recruited pool of hematopoietic cells. A good example is the M2 skewing of TAMs as tumors become more vascular and invasive . Macroph+ macrophages, for example, were recently shown to guide tip-cell fusion, a critical step during blood vessel anastomosis [+ myeloid cells to form endothelial-like cells was described by Bailey [+/CD11b+ cells acquired properties of endothelial cells in tumors, expressed VE-cadherin and lined blood vessels. Similarly, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)+ macrophages were shown to differentiate to form lymphatic endothelium [In addition to their paracrine functions, myeloid-lineage cells may act as “vascular bridges” by guiding and connecting nascent blood vessels. Proangiogenic TIE-2stomosis . The abiy Bailey . Yang ety Bailey  found thothelium , a featuSolid tumors are caricatures of a dysfunctional “organ.” In the shadow of malignant cells is the resident population of mesenchymal, hematopoietic, and endothelial cells that constitutes the tumor stroma. More than innocent bystanders, these stromal cells provide a growth advantage, for example, by forming the building blocks of tumor blood vessels . ProlongA clue that malignant cells might impinge on the differentiation state in the stroma comes from studies identifying aberrant DNA methylation and chromatin modification patterns in cancer-associated fibroblasts. Multiple tumor types, for example, breast and prostate are characterized by stromal alterations in methylated DNA (reviewed in ). BecausPathologists have long noted the heterogeneous and disorganized nature of parenchymal cells in tumors. As recently suggested, these stromal cells may coevolve with tumor cells during tumor progression and acquire characteristics of tumor cells nearby . “NormalSolid tumors may be characterized by two populations of stem/progenitor cells: CSC that may ultimately drive tumor growth and progression and nontumor/forming progenitors, some of which comprise the building blocks of tumor blood vessels. Malignant cells and their supporting stromal cells are held together by an extracellular matrix. Tumors may thus resemble dysfunctional organs able to recapitulate features of development while malignant cells provide corrupt instructions to surrounding tissues . Underst


A 30-year old woman was referred to our center because of suspicion of a primary lung tumor of the right upper lobe. Histological examination of the lung lesion revealed lung metastasis of a previously treated alveolar soft part sarcoma of the musculus vastus medialis of the right femur, which was resected 20 years ago. Alveolar soft-part sarcoma is a rare malignant tumor that occurs most often in the soft tissue of lower limbs. It is a slow-growing malignant soft tissue tumor arising in muscle tissue, usually in young adults. Due to pleural and extensive mediastinal infiltration with bilateral lung metastases, a systemic treatment with chemotherapy doxorubicin and ifosfamide was initiated. Late metastases from previously treated alveolar part sarcoma should be considered in patients with suspicious lung lesions even if surgical treatment was performed a long time ago. Alveolar soft-part sarcoma is a rare malignant tumor that occurs most often in the soft tissue of lower limbs. Alveolar soft-part sarcoma is a very uncommon soft tissue tumor known for late metastases to lung, bone, and brain. Lillehei and coworkers reported an interval of 21 years between primary presentation and development of lung metastasis and 33 years between primary presentation and development of brain and renal masses. Our case represents the second longest interval of 20 years between primary tumor presentation and development of a pulmonary recurrence.A 30-year old female patient was referred to our institution because of a large lung mass of the right upper lobe. The patient complained about increasing dyspnea for 2 months. Her initial physical examination was unremarkable and laboratory tests were all within normal limits. Spirometry revealed a mild restrictive pattern (TLC 88% soll) and reduced diffusion capacity (DLCO-SB 52% soll). CT thorax scan  revealedContrast-enhanced computed tomography scan  also revThe patient, interestingly, had a history of alveolar soft-part sarcoma of the musculus vastus medialis of the right femur successfully resected 20 years ago. The primary alveolar soft-part sarcoma had been initially staged as IA (T1 N0 M0) 20 years ago. All regular follow-up evaluations were unremarkable. However, the last chest X-ray evaluation was performed 8 years ago. Clinical workup revealed bilateral pulmonary metastases with ipsilateral pleural infiltration.Histological workup of material obtained during bronchoscopy showed a solid tumor composed of medium-sized to large cells arranged in compartmentalizing cords and nests, which were separated by delicate septa with capillaries. The cytoplasm of the tumor cells was broad and clear or eosinophilic. The nuclei were round and uniform, frequently with a single nucleolus .After immunohistochemistry demonstrating reactivity of the tumor cells for protein S100, but not for melanocytic markers or keratin markers, and comparison with the primary tumor, a diagnosis of metastatic alveolar soft-part sarcoma was rendered.Alveolar soft-part sarcoma was initially described in 1952 by Christopherson et al. . There iPulmonary metastases are encountered in 42–65% of patients, and the lung is most commonly involved. The brain and the skeleton are the two next most commonly involved metastatic sites . At firsChemotherapy is widely used in the treatment of nonresectable advanced disease, primarily with palliative intention. Initial standard chemotherapy for advanced or metastatic soft tissue sarcoma consists of single-agent anthracycline (mainly doxorubicin) or an anthracycline-based combination with, for example, ifosfamide and dacarbazine. The use of doxorubicin is limited, due to its cumulative cardiac toxicity . RegimenOur patient had been diagnosed with alveolar soft-part sarcoma of the lower extremity 20 years ago and was initially been successfully treated with complete resection of the tumor and adjuvant radiochemotherapy. Due to the extensive tumor manifestation at actual presentation with pleural infiltrations and bilateral lung metastases, a surgical option was not considered. Systemic chemotherapy with doxorubicin and ifosfamide was initiated.Alveolar soft-part sarcoma is an uncommon soft tissue tumor known for late metastases to lung, bone, and brain. Only Lillehei and coworkers reported an interval of 21 years between primary presentation and development of lung metastasis and 33 years between primary presentation and development of brain and renal masses .To the best of our knowledge of the current literature, our case represents the second documented patient with such an extremely late tumor recurrence of alveolar soft-part sarcoma.


Cases with subcutaneous metastasis of differentiated hepatocellular carcinoma to the abdominal wall without prior seeding as a consequence of local interventions with a negative or normal alpha-fetoprotein level in the serum are extremely rare.This is the first report of a case with AFP-negative, differentiated hepatocellular carcinoma metastasis to the abdominal wall within a pre-existing subcutaneous lipoma since childhood after antiandrogen therapy with leuprorelin and buserelin acetate for prostate cancer without seeding.Clinical features including histology, immunohistochemistry, clinical course and surgical approach are presented.Histological examination revealed a hepatocellular carcinoma with a trabecular and pseudoglandular growth pattern with moderately atypical hepatocytes with multifocal bile formation within a lipoma. The postoperative course of abdominal wall reconstruction with a monocryl-prolene mesh and a local flap after potentially curative resection was uncomplicated.It may be that previous antiandrogen treatment for prostate carcinoma contributed to the fact that our patient developed alpha-fetoprotein-negative and androgen receptor-negative subcutaneous abdominal wall metastasis within a pre-existing lipoma since childhood. Hepatocellular carcinoma (HCC) remains one of the most common malignant diseases and the incidence is increasing worldwide -5. TodayCases with subcutaneous differentiated metastasis of hepatocellular carcinoma with a negative or normal alpha-fetoprotein (AFP) level in the serum are extremely rare , cytology, radiofrequency ablation or percutaneous ethanol injection (PEI). In the current literature during the last 10 years, only few cases have been reported with abdominal wall metastasis without prior seeding as a consequence of local interventions . He was in good general condition with an obese nutritional status (Body Mass Index 30.4 kg/m²) and an impression of fatty liver parenchyma in ultrasound and magnetic resonance imaging. He was reported to drink two glasses of whisky per day. The previous history included multiple subcutaneous lipomas within the abdominal wall since childhood and prostate carcinoma, which was diagnosed in 2005 and removed by prostatectomy  in the same year, followed by antiandrogen therapy with leuprorelin and buserelin acetate until August 2008. The multiple lipomas showed a marked progression in size under antiandrogen therapy from August 2007 until August 2008. Final diagnostic evaluation, including ultrasound-guided FNAB of the liver mass and magnetic resonance imaging, resulted in the diagnosis of a multilocular, well-differentiated and AFP-negative hepatocellular carcinoma in liver segments II/III, VI, and VIII in April 2010. AFP levels stayed within normal range at all times during further follow-up. Viral hepatitis or other risk factors for hepatocellular carcinoma like hemochromatosis, liver fibrosis or liver cirrhosis could be ruled out. After interdisciplinary tumor board consultation  transarterial chemoembolisation (TACE) was performed from July 2010 to January 2011 every two months leading to a complete remission.Consultation with our surgical service was initiated in November 2011 due to a hard palpable subcutaneous mass with a diameter of approximately 7 cm that had developed recently in the left upper abdominal wall, exactly where a soft subcutaneous lipoma manifestation has been, in the consciousness of the patient, since his childhood  subcutaneously with suction for a total of five days -related hepatocarcinogenesis and hepatocyte proliferation are increased by androgenic stimulation in TGF-alpha transgenic mice [It could be demonstrated that the majority of hepatocellular carcinoma cells express androgen receptors in higher levels as compared to normal hepatocytes -42. Takenic mice . Severalnic mice -47. Intenic mice .Androgen receptors have been demonstrated in HCC in 74 % of males and 37 % of females, and their presence is associated with a more rapid progression of disease . ArgumenMore recent studies with Cre-Lox conditional knockout mice model who lack androgen receptor (AR) expression demonstrated that the androgen receptor, but not androgens, may be a potential new and better therapeutic target for the treatment of HCC . A new aDue to the low incidence of subcutaneous abdominal wall metastasis of HCC, therapeutic algorithms are widely missing. In the current literature, surgical resection is the favored therapeutic option ,23,32. Het al. reported 21 patients with a subcutaneous HCC metastasis without seeding who were treated by radiotherapy , encompassing up-to-date interventional, chemotherapeutic, and surgical approaches, including liver resection and liver transplantation, in one of the largest academic treatment centres for HCC in Germany.


Leukocyte functions are linked to their migratory responses, which, in turn, are largely determined by the expression profile of classical chemokine receptors. Upon binding their cognate chemokines, these G-protein-coupled receptors (GPCRs) initiate signaling cascades and downstream molecular and cellular responses, including integrin activation and cell locomotion. Chemokines also bind to an alternative subset of chemokine receptors, which have serpentine structure characteristic for GPCRs but lack DRYLAIV consensus motive required for coupling to G-proteins. Duffy antigen receptor for chemokines (DARC) is a member of this atypical receptor subfamily. DARC binds a broad range of inflammatory CXC and CC chemokines and is expressed by erythrocytes, venular endothelial cells, and cerebellar neurons. Erythrocyte DARC serves as blood reservoir of cognate chemokines but also as a chemokine sink, buffering potential surges in plasma chemokine levels. Endothelial cell DARC internalizes chemokines on the basolateral cell surface resulting in subsequent transcytosis of chemokines and their immobilization on the tips of apical microvilli. These DARC-mediated endothelial cell interactions allow chemokines produced in the extravascular tissues to optimally function as arrest chemokines on the luminal endothelial cell surface. Duffy, was found to cause a delayed hemolytic transfusion reaction. In the following year, an antibody to the antithetic antigen, Fyb, was found in a multigravida exposed to fetal Fyb erythrocytes . This phenotype results from a polymorphic form of the FYB gene, FYB(ES) “erythroid silent”, present in up to 99% of West Africans and the majority of African Americans  in codon 42 in the NH2 extracellular domain, encoding glycine in Fya and aspartic acid in Fyb (FYB(ES) allele has a single T to C substitution at nucleotide –67 within the erythroid GATA-1 promoter region, 33 bp upstream from the erythroid transcription starting point and 46 bp upstream from the start of the major transcript translation codon, thus preventing DARC transcription in erythroid cells only (The Duffy antigen receptor for chemokines (DARC) has recently become the focus of studies investigating interactions of inflammatory chemokines with erythrocytes during systemic inflammatory responses as well as with venular endothelial cells during chemokine-induced leukocyte adhesion and emigration. These studies uncovered new functional facets of this rather “old” molecule. DARC was first described in 1950 as the Duffy blood group antigen . An antihrocytes . SubsequYB genes . Howeverans 68%; . The DARn humans . The twod in Fyb . The FYBlls only .FYB(ES) Fy(a–b–) individuals still express DARC at non-erythroid sites, e.g., on endothelial cells and possibly other cells  phenotype of almost 100%  and was first predicted to have nine trans-membrane domains , but lat, and 33 , and epi and Fy6 . The Fy6P. vivax .in situ binding mirroring exactly the ligand specificity of DARC  DARC “negative” polymorphism. Future work should uncover molecular and cellular mechanisms explaining how the lack of erythrocyte chemokine sink and depot functions in these DARC-negative individuals affects pathomechanisms in various inflammatory diseases and cancer. In endothelial cells DARC functions as a transcytosis receptor leading to correct patterning of chemokines on the tissue–blood interface in venules and veins, thus supporting optimal chemokine-induced leukocyte endothelial cell adhesion and subsequent leukocyte emigration.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zinniaelegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from  Deconstruction of the major plant cell wall polymers to small molecules is the essential first step in converting biomass to liquid biofuels. Biomass efficiently decomposes in nature through the synergistic activity of enzymes from microbial communities, which attack different components of the plant cell wall, ultimately resulting in carbon recycling . The celGiven the natural recalcitrance of the cell wall, much research focused on improving the efficiency of lignocellulose degradation towards the cost-effective production of biofuels ,4. RecalZinniaelegans mesophyll cells during in vitro xylogenesis [Zinnia xylem cells are easily recognized by microscopy [Zinnia xylem cells are simpler than plant tissue, but more importantly, they possess the natural complexity of cell wall structures, making them suitable for in vitro studies of cell wall degradation. Using these cells, we focused on examining cellulose digestion, a process catalyzed by a variety of functionally distinct microbial endoglucanases and cellobiohydrolases, collectively known as cellulases [Studies examining the activity of cellulases have often relied on purified substrates that have dubious predictive value for the kinetic efficiency of enzymatic digestion of plant biomass during industrial biofuel production . In contogenesis ,16. Zinncroscopy ,18 due tcroscopy ,19. Thescroscopy ,18,20. Cllulases .Clostridium thermocellum (CtCBM3-GFP) [To determine how cell wall deconstruction was influenced by lignin content, we examined the digestibility of cellulose in xylem cells following two different methods to deplete lignin: treatment with an oxidative chemical or with inhibitors of lignin biosynthesis. We then used microscopy to monitor cell wall degradation and a fluorescent cellulose-specific probe from BM3-GFP)  to perfoBM3-GFP) –25. The Zinnia xylem cells, which exhibit prominent wall thickenings , which has been shown to bind crystalline cellulose [CtCBM3-GFP exhibited similar fluorescence levels after a 3-hour incubation , indicating an increase in cellulose content with larger cell size. This relationship was more pronounced in ASC-treated cells , suggesting that cellulose content can be more accurately measured by CtCBM3-GFP after removal of cell wall components using ASC.Given that the cellulose levels of different cells cannot be directly and accurately compared at a specific time using polarized light, we applied a fluorescence-based labeling strategy to quantify the degradation of cellulose in populations of xylem cells. To specifically label and quantify cellulose content, we used a fluorescently tagged family 3 carbohydrate-binding module (ellulose . Based oCtCBM3-GFP (p<0.001) when compared to corresponding cells incubated in buffer without cellulase  added 3 days after inducing their differentiation into xylem cells in culture. To evaluate the extent of lignification in treated cells, we measured their autofluorescence between 512 and 542 nm, which mainly originates from lignin in the secondary wall thickenings. We found that cell autofluoresce nce was significantly reduced by at least 70% (p<0.0001) following treatment with DPI, GSH, or DPI combined with GSH (data not shown), which is consistent with the reduction in cell autofluorescence observed.We observed that pretreatment with ASC was necessary to accomplish efficient enzymatic degradation of xylem cells using cellulase. Since ASC is a classical oxidative chemical treatment that primarily removes lignin from the cell wall, we decided to use an alternate approach that would reduce the amount of lignin deposited in xylem cells during their development. Accordingly, we cultured isolated CtCBM3-GFP to measure cellulose content; however, as this protein is partly derived from a non-catalytic domain that binds directly to cellulose, it can also be used to examine cell wall accessibility to enzymes of similar size or smaller. Therefore, we compared the binding of CtCBM3-GFP between undigested xylem cells treated with chemical inhibitors of lignin biosynthesis and control samples. We found that inhibitor-treated cells were significantly more fluorescent (p<0.01) than the corresponding controls by 73% (DPI), 40% (GSH) and 101% (DPI combined with GSH), showing that treated cells had increased binding of the cellulose probe . This result indicates that xylem cells with lower lignin content have increased cellulose accessibility, i.e. more cellulose vulnerable to a potential enzymatic attack.After confirming that we could generate xylem cell cultures with reduced lignification, we investigated how the accessibility and digestibility of the cell wall were affected. In the experiments described we used CtCBM3-GFP fluorescence between cells incubated with or without cellulase for each inhibitor treatment. In the following experiments, cells were incubated with cellulase for a longer time period (24 hours) in order to clearly detect changes in the cell wall. First, we examined control cells and found that, qualitatively, cells incubated with cellulase maintained similar labeling by CtCBM3-GFP compared to those incubated without any enzyme   and chemical inhibition of lignin biosynthesis during xylem cell differentiation in culture. ASC is a classical treatment that may be the most prevalent laboratory approach for the removal of lignin from biomass ,35,36. TTo specifically measure changes in cellulose accessibility, fluorescently labeled probes derived from bacterial cellulases have been successfully used in "pure" cellulosic substrates as well as in corn stover biomass –40. DireZinnia xylem cells correlates with a striking increase in binding of the cellulose-specific probe CtCBM3-GFP [CtCBM3-GFP binding and thus, accessibility to cellulose, in cells whose lignification was blocked during development. Inhibitors of lignin polymerization do not directly alter hemicellulose content, so the increase in CtCBM3-GFP binding found was substantial, yet not as striking as previously observed in ASC-pretreated cells [We have previously shown that significant loss of lignin in ASC-pretreated CBM3-GFP . Furthered cells .It has been suggested that even when lignification is selectively targeted in plants, compensatory mechanisms may ensue and affect the characteristics of the cell wall so that there may not be a measurable effect in digestibility . For exaZinnia xylem cells. Our results also support the idea that common pretreatments that solubilize polymers coating cellulose fibrils within the cell wall render natural plant substrates more digestible and thus require the use of less enzyme for efficient hydrolysis [Given that the inhibition of lignin biosynthesis alters the normal progression of secondary wall assembly, other cell wall matrix components that normally associate with lignin could also become modified or delocalized in inhibitor-treated xylem cells. Hence, we cannot rule out the possible contribution of such factors in reducing the accessibility to cellulose. Nonetheless, our observations collectively showed that cellulose accessibility is a key factor affecting the enzymatic digestibility of the cell wall in drolysis ,46.The digestion of pretreated xylem cells by cellulase can occur quite rapidly, and the loss of cell wall material is unmistakable using brightfield microscopy. However, this type of microscopic examination is limited to qualitative observations lacking a precise grasp of the dynamics that take place. To quantify the amount of cellulose present in cells we used polarization microscopy, which takes advantage of the birefringent nature of the secondary wall thickenings of xylem cells  and alloin situ [Clostridium thermocellum cipA gene, which encodes the scaffoldin subunit of its cellulosome [Zinnia xylem cells [CtCBM3-GFP labeling strategy used in this study represents a desirable approach, because we could specifically and conveniently detect the accessible cellulose present in the cell wall of individual cells in a single step.Non-catalytic carbohydrate-binding modules (CBMs), which originate from microbial enzymatic complexes, have become attractive probes for the detection of polysaccharides within cell wall structures, particularly because of their intrinsic ability to recognize their targets in situ . Currentin situ ,48–50. Wlulosome . By fusilulosome  were unnlulosome ,53, direlulosome –41,51,54em cells , but thiem cells . Taking Zinnia xylem cells, we observed that these cells became more easily digested by cellulase and concluded that this effect was due to increased access of the enzyme to its target. For decades, Zinnia xylem cells or tracheary elements have been used successfully as an in vitro cell culture system to understand the process of secondary wall patterning and xylogenesis. Here we show that these cells, which represent a simple lignocellulosic substrate compared to biomass, can be exploited to make quantitative measurements of cell wall deconstruction rather than biogenesis. Finally, fluorescent CBM probes, such as the cellulose-specific CtCBM3-GFP probe used in this study, constitute powerful tools to identify cell wall polymers as well as provide a measure of content and accessibility. Hence, the quantification of accessible polysaccharides in large populations of cultured xylem cells using CBMs is an approach that can be valuable to lignocellulosic biofuel research, particularly if coupled to analytical techniques that may take advantage of the single-cell nature of these Zinnia cell cultures.In summary, after using two different approaches to decrease lignin content in Zinnia seedlings for 2 weeks, the first true leaves were removed and surface sterilized. Mesophyll cells were harvested and cultured as detailed previously [5 cells/ml in 6-well plates containing 1 µg/ml of 6-benzylaminopurine and 1 µg/ml of alpha-naphthaleneacetic acid in S-medium. Xylem cells from 7- to 10-day old cultures were separated from undifferentiated and dead cells by density-fractionation using a gradient of plant-tested 72% Percoll . The lowest of three distinct bands, which contained the largest number of xylem cells, was collected. Cells were then washed 3 times with double distilled H2O by low speed centrifugation using a tabletop Nanofuge  before additional experimental use.Cells were cultured and isolated as previously described . Brieflyeviously , except Tviride.   were enzymatically digested using 10 U/ml in sodium citrate buffer by incubation in Eppendorf tubes at 200 rpm, 50°C for 1-24 hours. Aliquots were taken at the specified times and washed 3 times with double distilled H2O before labeling with CtCBM3-GFP. Experiments were repeated at least twice, and a single representative experiment was selected for data presentation.Cells were enzymatically digested with a purified endoglucanase from the fungus de , and Leica software . Time-lapse images were acquired using a 40X oil-immersion objective, whereas we used a 20X air objective to acquire fluorescence images.CtCBM3-GFP labeling or cell autofluorescence in images, we used ImageJ software (http://rsb.info.nih.gov/ij/) to draw close-fitting polygons around single cells and measured their area and mean fluorescence intensity. The total fluorescence was calculated by multiplying the mean intensity by the total number of pixels encompassing each cell. The average of the mean fluorescence of cells was also reported. At least 20 cells were analyzed for each population. In r).To analyze For analysis of polarization signal, we used the same polygon-drawing method and quantified the intensity in each image of the time-lapse sequences. These values were then normalized to the intensity of each cell at time 0.CtCBM3-GFP [-1 of CtCBM3-GFP in 200 µl of 1% BSA, PBS buffer [CBM3-GFP  was usedCBM3-GFP ,57. BrieS buffer  at room 2O before additional manipulation. To inhibit lignification during the differentiation of mesophyll cells into xylem cells, we added diphenyleneiodonium  and/or reduced glutathione  to cultures in 6-well plates after 3 days in culture [2O) to control cultures. Culturing was allowed to proceed normally, and xylem cells were enriched by density-fractionation as described above.To chemically extract lignin, xylem cells were incubated in acidified sodium chlorite  at 70°C for 20 hours  in a sin culture . We addeVideo S1Loss of secondary wall thickenings is evident in ASC-pretreated xylem cells (right), while untreated cells appear unaltered (left) during incubation with cellulase. Time-lapse images were collected using brightfield microscopy at 15 min intervals for 3 hours. Also see (AVI)Click here for additional data file.Video S2The polarized light signal from an ASC-pretreated cell rapidly decreases during incubation with cellulase (right), while the signal from an undigested cell remains stable (left). Time-lapse images were collected at 15 min intervals for 6 hours. Also see (AVI)Click here for additional data file.


Testicular lymphoma was first reported by Malassez and Curling in 1866. Primary testicular lymphoma constitutes only 1 - 7% of all testicular neoplasms and less than 1% of all non Hodgkin lymphoma. The authors report a new case of primary testicular lymphoma and highlight its diagnostic and therapeutic challenge. We report the case of a 26-year old man without a particular past medical history, who presented with a painful right testicular swelling that he has noticed for several weeks. Radiological findings consisted in multiple hypoechoic masses that corresponded in histological examination to a diffuse intratubular lymphomatous infiltration situated away from the spermatic cord, the epididymis, ductuli efferentes and rete testis. Immunohistochemical study showed positivity for leukocytic common antigen (CD45), B-cell marker (CD20) and bcl 6. The patient underwent full staging for lymphoma showing no evidence of extra-testicular involvement by lymphoma and no lymph nodes. The diagnosis of stage I primary testicular large B-cell lymphoma of germinal center B-cell-like group was made. The patient is now treated by chemotherapy. Primary testicular lymphoma is a rare tumour whose diagnosis is based on histological findings. There are non consensual etiological or predisposing factors. Treatment modalities consist in surgical excision, chemotherapy and radiation therapy but the accurate procedures are not standardized. Factors that have been linked to more favorable outcomes include younger patient age, localized disease, presence of sclerosis at pathologic analysis, smaller tumour size, lower histological tumor grade and lack of epididymal or spermatic cord involvement.Testicular lymphoma; Germinal center; B-cell Primary testicular lymphoma is a rare tumor accounting for 1% of all testicular non Hodgkin lymphoma [We report the case of a 26-year old man without a particular past medical history, who presented with a painful right testicular swelling that he has noticed for several weeks. There was no reported history of trauma, night sweets, fever or chills. Scrotal examination revealed a firm and enlarged testis with a homolateral hydrocele. The remainder of the clinical examination was noncontributory. The ultra-sound examination showed an enlarged, heterogeneous testis with multiple hypoechoic masses . LaboratTesticular lymphoma was first reported by Malassez and Curling in 1866 ,3. PrimaHistopathological differentiation of testicular lymphomas from germinal tumors is usually a challenge but these lymphomas generally appear more lobulated with well defined borders at ultra-sound examination . Other c


In the past decade cancer research has recognized the importance of tumorstroma interactions for the progression of primary tumors to an aggressive and invasive phenotype and for colonization of new organs in the context of metastasis. The dialogue between tumor cells and the surrounding stroma is a complex and dynamic phenomenon, as many cell types and soluble factors are involved. While the function of many of the players involved in this cross talk have been studied, the regulatory mechanisms and signaling pathways that control their expression haven't been investigated in depth. By using a novel, interdisciplinary approach applied to the mechanism of action of the metastasis suppressor, Raf kinase inhibitory protein (RKIP), we identified a signaling pathway that suppresses invasion and metastasis through regulation of stroma-associatedgenes. Conceptually, the approach we developed uses a master regulator and expression arrays from breast cancer patients to formulate hypotheses based on clinical data. Experimental validation is followed by further bioinformatic analysis to establish the clinical significance of discoveries. Using RKIP as an example we show here that this multi-step approach can be used to identify gene regulatory mechanisms that affect tumor-stroma interactions that in turn influence metastasis to the bone or other organs. Under normal physiological conditions, the stromal compartment of epithelial tissue regulates homeostasis by maintaining the proper architecture and nutrient levels required for epithelial function. It also serves as an important barrier to cell transformation. However in response to lesions  the stromal compartment undergoes changes including the recruitment and activation of fibroblasts, immune, and endothelial cells that in turn provide growth, and matrix remodeling factors, as well as a new blood supply. Similar changes in the stromal compartment have been shown to occur during tumor growth and the importance of the stromal compartment, called “the tumor microenvironment,” in modulating and driving cancer progression has become increasingly evident . The tumα-smooth muscle actin (α-SMA), are frequently found in the stroma of human breast carcinoma and are referred to as carcinoma-associated fibroblasts (CAFs). The precise cellular origin of these activated myofibroblasts is not clear but it has been shown that when inoculated with carcinoma cells, CAFs can promote tumor growth in mouse xenograft models [It is believed that during the first phase of carcinogenesis the tumor microenvironment initially reacts to suppress malignant transformation by maintaining tissue architecture and differentiation. As cancer progresses, however, the local stromal compartment shifts to an activated, growth-promoting state, in many ways similar to an inflammatory state, which is initiated and maintained by continuous paracrine communication between stromal and tumor cells. Stromal components engage in a dynamic signaling circuit with primary tumor cells and coevolve with tumor cells to promote tumor progression to an invasive phenotype . Varioust models . CAFs set models .β (TGF-β), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and osteopontin (OPN) [A number of other cytokines, chemokines, and growth factors secreted by cancer cells themselves or by tumor-associated stromal cells have been shown to sustain tumor cell proliferation and progression through different mechanisms. The list of these autocrine/paracrine factors is constantly growing and includes vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-in (OPN) . An impoin (OPN) . BMDCs cMetastasis is the primary cause of mortality in breast cancer patients and can emerge many years after the removal of the primary tumor. Metastastic progression is a complicated multistep process which includes at least three discrete stages: (1) epithelial-mesenchymal transition (EMT) leading to migration, invasion, and intravasation; (2) circulation, transportation, and extravasation of cells, which then undergo mesenchymal-epithelial transition (MET); (3) colonization of tumor cells within distal tissues including bone and lung . The effMany studies have highlighted the concept of tissue tropism: although the blood flow pattern certainly contributes to preferred metastatic sites of specific carcinomas, the complex molecular mechanism of homing metastatic cells is also determined by interactions with the microenvironment at target organs. A number of molecular mediators of this interaction have been revealed by recent publications, and gene expression profiling studies have generated distinct gene expression signatures for organ-specific metastatic variants –13. A maβ and TNFα), and mediators of fibroblasts activation (FGF and TGFβ). Moreover cancer cells promote bone degradation by direct secretion of metalloproteinases (such as MMP1) and collagenase I or through indirect mechanisms by activating osteoclasts. Other tumor-derived cytokines and cell surface/ECM proteins like bone morphogenetic protein (BMP), interleukin-11 (IL-11), osteopontin (OPN), and endothelin-1 participate and feed this vicious cycle. In this scenario bone reabsorption by osteoclasts releases a number of growth factors embedded in the bone matrix including insulin-like growth factors (IGFs), TGF-β, platelet-derived growth factor (PDGF), and BMP which become part of this signaling circuit that push osteolytic lesions.Breast cancers metastasize to lung, liver, bone, and brain. Bone metastasis is very common among late-stage breast cancer patients but current treatment methods for bone metastasis are mainly palliative, and more effective disease-modifying therapies are needed. Breast cancer frequently generates osteolytic bone metastasis by secreting a series of growth factors that influence bone matrix and bone stromal cells, tipping the balance to osteolytic bone destruction. In this context tumor-derived factors include angiogenic factors (FGF and VEGF), mediators of immune cell recruitment and activation  . As expeTo understand the mechanisms by which metastasis is regulated, we have focused on identifying key signaling pathways that can inhibit breast cancer metastasis to the bone. Metastasis suppressors define a class of proteins that do not affect primary tumor growth but instead regulate one or more steps in the process leading to metastasis: invasion, intravasation, circulation, extravasation, and colonization of the secondary site . Raf kinin vitro using breast tumor cell lines and in vivo using a xenograft mouse model. Finally, having determined which genes regulate metastasis in experimental breast tumor models, we validated their clinical significance by further bioinfomatic analysis using independent breast tumor data.As a means of identifying signaling pathways downstream of a key metastasis regulator in cancer, the Rosner and Minn groups developed a novel interdisciplinary approach that utilizes clinical data from breast tumors to generate and test hypotheses . The basUsing this approach, we identified a number of RKIP-regulated let-7 targets including HMGA2 and a novel target, BTB-and-CNC homology 1 (BACH1). A leucine zipper transcription factor, BACH1, has been linked previously to senescence and heme oxidation but has never been correlated to cancer progression . ExperimTo test the hypothesis that RKIP is a potential regulator of genes implicated in the development of bone metastasis, we performed a similar bioinformatic analysis. We initially determined whether RKIP expression inversely correlates to the expression of bone metastasis signature (BMS) genes . We asseFinally, we determined experimentally the relationship between RKIP, let-7, the two let-7 targets, HMGA2 and BACH1, and the three BMS genes. Interestingly, knockdown of BACH1 suppressed the BMS genes MMP1 and CXCR4 but not OPN while HMGA2 knockdown suppressed CXCR4, and OPN but not MMP1. Additionally, we could partially reverse the effects of HMGA2 and BACH1 knockdown on invasion and metastasis by overexpressing their target BMS genes MMP1, CXCR4 and OPN. The simultaneous overexpression of the three BMS genes together showed a more profound effect on the metastatic phenotype compared to the overexpression of a single gene. These results suggest that the coordinate regulation of genes with different metastasis-promoting functions is a prerequisite for efficient metastatic spread.Having determined which genes regulate metastasis in experimental breast tumor models, we defined a signaling pathway signature termed the RKIP pathway metastasis signature (RPMS) that we could use to further validate the clinical significance of our findings . While tThe results described here reveal a novel regulatory mechanism, controlled by the RKIP signaling pathway, that modulates the dialogue between breast tumor cells and the microenvironment and affects metastatic progression to the bone . Specifi


RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed. In temperate regions, annual influenza epidemics usually start in November and last until March. Influenza viruses can affect persons at any age, but the elderly (>65 years), young children (<2 years) and persons with underlying chronic diseases or weakened immune systems are particularly susceptible to severe and sometimes fatal respiratory distress symptoms [Influenza viruses are highly contagious, rapidly evolving respiratory pathogens with established lineages in avian and mammalian species. Human influenza viruses circulate worldwide, causing annual epidemics associated with three to five million cases of severe illness and 250,000 to 500,000 deaths  that had either adapted to infect human cells or had generated reassortants with mammalian influenza viruses. Pandemic viruses usually show major antigenic differences from the current circulating human influenza viruses (“antigenic shift”). Accordingly, they will encounter a human population with little pre-existing immunity, allowing for rapid global virus spread. The epidemic of highly pathogenic AIV H5N1 which started 2003 in Southeast Asia and spread to Europe and Africa exemplifies the zoonotic potential of AIV . FortunaCurrent human influenza virus vaccines rely on inactivated split vaccines containing the major viral surface antigens HA and NA. The existing seasonal vaccines employ antigens of the most prevalent human influenza viruses, namely influenza A virus subtypes H3N2 and H1N1 together with an influenza B virus strain, present as a triple vaccine for parenteral application. According to WHO recommendations, the vaccine composition is updated every year to match the rapid “antigenic drift” of the viruses. However, vaccine efficacy is strongly influenced by the age and immunocompetence of the recipient, as well as the degree of similarity between the vaccine strains and the viruses circulating during an influenza season [The annual production cycle for conventional influenza vaccines must be initiated at least 6 months before the vaccine can become available . It begiGenerally, the current split vaccines for influenza induce good serum antibody responses, but are less efficient in triggering mucosal IgA antibodies. Vaccination is most often done by the parenteral route, which is not ideal for inducing mucosal immunity. In addition, these split vaccines may be poor at eliciting the cellular immune responses which are probably important in the context of cross-protectivity ,9. Thus,Pandemic influenza virus vaccines encounter additional obstacles. They often have to be supplied at a particularly short notice in sufficiently large quantities to cover the immunization of several millions of people. However, the limited availability of specific pathogen-free embryonated chicken eggs may hinder meeting this demand, and can even prove insufficient for production of the required vaccine doses to control large seasonal influenza epidemics. Moreover, one cannot guarantee that all influenza viruses will be equally efficient at producing the required amounts of antigen. For example, HA of certain potential pandemic H5N1 strains were found to be less immunogenic, making high antigen doses necessary to achieve appreciable antibody responses . CurrentAnother approach taken for influenza virus vaccine production was that of cold-adapted influenza virus vaccines, extensively employed in Russia in the past . A cold-None of the classical vaccine concepts (inactivated or live attenuated viruses) perfectly satisfies the demands for seasonal and pandemic influenza virus vaccines. Ideally, a high-quality influenza virus vaccine would integrate both the safety of inactivated vaccines and the efficacy of live attenuated vaccines. An ideal vaccine is expected to provide long-lasting and heterosubtypic protection, and should be available at short notice in sufficient quantities to face an influenza pandemic. The recent race to produce adequate doses of vaccine against the new H1N1 2009 virus exemplifies the critical nature of this problem. While the virus was continuing to induce disease during the spring and summer months of 2009, vaccines were not available before November 2009. In the future, it is important to identify means of producing influenza virus vaccines more rapidly, and increasing the potential for producing efficacious vaccines with the capacity of inducing both humoral and cellular immunity. In this context, RNA replicons offer major advantages. They meet several of the aforementioned criteria, and readily offer themselves to the generation of high-quality influenza virus vaccines. The remainder of this review will focus on introducing the RNA replicon concept, summarizing previous work on replicon-based influenza vaccines, and presenting the future perspectives for this type of vaccine.2.Most RNA viruses are relatively simple and have small genomes (<15 kb). They are classified into positive- and negative-strand RNA viruses (single-stranded RNA genomes), or viruses with a double-stranded RNA genome. The genome of positive-strand RNA viruses (PSV) can directly function as mRNA when delivered into the host cell, and is therefore regarded as infectious. Translation of this RNA results in the generation of one or more polyproteins, which are proteolytically processed to produce a set of structural and non-structural proteins including the RNA-dependent RNA polymerase. This viral polymerase is essential for the transcription and replication of the viral RNA, which usually occurs in the infected cell cytosol independently of host nuclear functions (except for retroviruses).i.e., transfection of in vitro-transcribed genomic RNA will not result in the generation of infectious virus). In contrast to PSV, the RNA genome of NSV has to be transcribed by a pre-existing viral RNA polymerase into positive-sense RNA. For this reason, the viral RNA polymerase protein complex forms an essential part of the virion, and remains associated with the viral RNA after infection to ensure initiation of transcription and replication. Although the majority of NSV replicate in the cytosol, some also require host nuclear functions . A further contrast to PSV is seen with the organization of non-segmented NSV genomes which are arranged in a modular, non-overlapping fashion, with each gene flanked by conserved transcription start and stop signals. A typical feature of non-segmented NSV is the gradient of transcription; that is, genes at the 3′ end of the genome are transcribed at higher levels than genes located further downstream. NSV with segmented RNA genomes, such as influenza virus, usually encode for one or two proteins per segment. Again contrasting with PSV, neither non-segmented nor segmented NSV genomes encode for polyproteins.Negative-strand RNA viruses (NSV) comprise viruses with segmented and non-segmented genomes. The genomic RNA of NSV is non-infectious , the proteasomes can function as immunoproteasomes which are physically and functionally linked to TAP transporters (transporter associated with antigen presentation), promoting transport of the resulting peptides into the ER for loading on to MHC class I molecules. These are in turn transported to the APC surface for presenting the peptides to CD8+ T lymphocytes. Of the APC, dendritic cells (DC) are particularly well designated for triggering cellular immunity in this manner. A number of RNA viruses can directly infect DCs, but there are also viruses with a different tropism.An alternative mode of antigen presentation relies on the induction of apoptosis in RNA replicon-infected cells (distinct from DCs). The antigen-loaded apoptotic vesicles can be efficiently taken up by DCs (or other antigen-presenting cells), which proteolytically degrade the antigens in acidified endocytic vesicles. These endocytic vesicles subsequently fuse with vesicles containing MHC class II molecules, the MHC class II compartment (MIIC). Stable association of the antigenic peptide with the MHC class II molecules leads to transportation of the complexes to the cell surface for presentation of the peptides to CD4+ T lymphocytes. In addition, DCs can present on their surfaces peptides derived from proteolytic degradation of internalized extracellular antigens via MHC class I molecules – a process known as cross-presentation . With an2.3.Today, the most frequently used positive-strand RNA replicon vaccines are derived from (i) alphaviruses such as Sindbis virus, Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis (VEE) virus ,27, or . VRPs based on alphaviruses are produced in mammalian cell lines that are transfected with a set of in vitro transcribed RNAs  or vesicular stomatitis virus (VSV). With the genomic RNA of these viruses being non-infectious es virus . The sames virus –46. Simies virus ,48.in trans. If this protein proves to be cytotoxic, a conditional expression system may be advantageous for generation of the helper cell line [in vivo delivery of naked DNA or RNA.Once the VRPs have been generated, they can be propagated on transgenic helper cell lines providing the deleted structural protein ell line –43,49. Tell line . Anotherell line . This prell line ,48,50. AAlthough NSV-based replicons have been less frequently used as vaccine vectors than replicons based on PSV, they reveal some features that make them particularly promising vectors. For example, non-segmented NSV have a module-like organization, which has high potential for hosting several recombinant antigens. Thanks to the typical transcriptional gradient of non-segmented NSV, different antigen expression levels can be achieved by using transcriptional units close to or remote from the 3′end of the genome. In contrast, PSV usually express large polyproteins that have to be proteolytically processed into individual viral components, which would be less favourable for the expression of multiple antigens. Moreover, RNA recombination seems to be more rare with non-segmented NSV ,52 compa2.4.Influenza vaccines based on RNA replicons still represent experimental vaccines, which have not been evaluated in humans yet. Most studies have been performed in small animal systems, in particular in mice.2.4.1.in vitro transcribed RNA or packaged VRPs [Probably, the first experimental alphavirus replicon vaccine for protection against influenza was based on SFV. The replicon was engineered to express the influenza virus nucleoprotein (NP), and was applied to mice by intramuscular injection of either ged VRPs . Both veged VRPs . A DNA-lged VRPs . In thisged VRPs .Some attempts have been made to improve the immunogenicity of HA antigen derived from a human H5N1 isolate by genetically fusing the antigen with the herpes simplex virus 1 VP22 protein then expressed by SFV VRPs . Comparein trans. This significantly improved the safety of the system, because regeneration of propagation-competent viruses was not detected. Two subcutaneous inoculations with VRPs expressing HA antigen of A/PR/8/1934 (H1N1) protected mice against intranasal challenge with the homologous influenza virus strain.A DNA-layered Sindbis virus replicon vaccine has also been used for expression of influenza virus HA antigen of A/PR/8/1934 (H1N1). In addition, replicon vaccines based on Venezuelan equine encephalitis (VEE) virus have been generated , in analIn contrast to previous studies evaluating alphavirus replicon vaccines in mice, a recent report provided evidence that alphaviral replicon particles expressing HA and NA antigens of A/Wyoming/3/2003 (H3N2) induced significant humoral and cellular immune responses in mice, rabbits and rhesus macaques with no adverse side effects . However2.4.2.Rhabdoviridae and is endemic in parts of America. The virus is transmitted by insects to livestock such as cattle, horses, and pigs, causing a mild vesicular disease. Infections of man have been rarely reported, being described as being Flu-like [9 pfu/ml) and a pronounced cytopathogenicity. The VSV genome consists of about 11,200 nucleotides encoding five genes. Deletion of the G gene which encodes for the single envelope glycoprotein G resulted in a propagation-incompetent VSVΔG replicon that was initially used for pseudotyping experiments to characterize heterologous viral glycoproteins [VSV is a member of the family Flu-like . In cellproteins . Later, proteins ,61, HIV-proteins , respiraproteins , and infproteins ,50,64. Rproteins . Two int2.5.In many developed countries general vaccination of poultry against highly pathogenic avian influenza viruses (HPAIV) is not allowed, a policy based in principal on two arguments. Firstly, the currently available inactivated influenza virus vaccines may protect poultry in the sense that they do not show signs of diseases. However, inactivated vaccines are inefficient in providing sterilizing immunity, such that vaccinated animals may shed significant amounts of virus. Such vaccines may even expedite viral antigenic drift by selection of escape mutants . SecondlExperimental RNA replicon vaccines have demonstrated potential as novel marker vaccines for poultry. Importantly, alphaviruses such as VEE or rhabdoviruses such as VSV are not natural avian pathogens, thus excluding that birds would possess any pre-existing immunity against the viral vectors. Immunization with a single dose of VEE VRPs expressing the HA antigen of an H5N1 isolate resulted in complete protection of chickens against lethal challenge with the homologous virus .The HA protein is certainly a most important influenza virus antigen for inducing neutralizing antibodies. Accordingly, in most studies this antigen has been used for immunization. Nevertheless, the role of the NA antigen in protection against avian influenza viruses has also been studied. Sylte and co-workers immunized chickens with VEE VRPs driving the expression of the NA antigen from A/pheasant/Maryland/4457/93 . They foVSVΔG replicons encoding either HA or NP antigen of an HPAIV (H7N1) strain were also evaluated as vaccines for chickens . HA was 2.6.The immune correlates of protection against influenza virus infection and disease have been only partially addressed over recent years, leaving an important gap in our current knowledge on what is required . An intrAnother aspect of immune correlates which is also particularly poorly studied is the importance of local immunity in resistance to influenza virus induced disease. Being a respiratory infection, one would consider that mucosal immunity would be of prime importance in the ability of the host to resist the virus and the disease it can induce. Influenza viruses replicate predominantly in the epithelial cells of the respiratory tract. Therefore, mucosal antibodies (IgA) are believed to represent an important line of defence. Although antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, VEE VRPs have been reported to induce mucosal immunity even when applied parenterally . Both mu3.Presently, inactivated subunit vaccines are commonly used for immunoprophylaxis of influenza virus infection. While clear advantage of subunit vaccines is their safety, their usage has come with a number of drawbacks including low efficacy (in particular in the elderly), low cross-protective immunity, and short-term immunity . In prinRNA replicons may show a way out of the above dilemma. They combine the safety of inactivated vaccines with the superior immunogenicity of live, attenuated vaccines . Neverth3.1.Most studies on RNA replicon vaccines so far were based on vectors driving single expression of HA, the major influenza virus antigen particularly subject to antigenic drift. In order to approach the efficacy of live attenuated vaccines with respect to cross-protection against drift and heterosubtypic viruses, RNA replicons have to be generated that mediate the expression of multiple influenza antigens. One particular element therein would be the highly conserved internal virus antigens, which contain dominant T cell epitopes ,77. The 3.2.The respiratory tract is lined by epithelial cells organized in a polar fashion . Influen3.3.The inability of RNA replicons to produce infectious progeny is a clear plus with respect to biosafety. However, it limits the dissemination of antigen and thus the number of dendritic cells having access to the antigen. This lack in antigen dissemination may be overcome by expression of recombinant soluble antigens ,84 that This review has referenced our current knowledge on RNA replicon vaccines, which have been mostly evaluated in mice to date. In future, it will be necessary to demonstrate that RNA replicons also provide protection of larger non-rodent mammals such as pigs and ferrets. Protection of vaccinated animals has tended to analyse challenge with homologous virus, but this should now be elaborated to include challenge analyses using heterologous virus. Such challenge infections are clearly needed to test the quality and value of a modern influenza virus vaccine. The induction of cellular immune responses providing long-lasting immunity against heterosubtypic influenza viruses will certainly be an important aspect in future vaccine developments, an element which can be offered by the RNA replicon vaccine.The rapid availability of any influenza virus vaccine is a crucial aspect, particularly in face of an influenza virus pandemic as we recently experienced when the new H1N1 virus emerged in 2009. Production of alphavirus replicon vaccines by transfecting approved cell lines with naked RNA or cDNA may be straightforward, because these procedures can be easily performed under conditions of Good Manufacturing Practice (GMP). However, the use of specialised packaging cell lines to generate VRP holds considerable promise for generating effective vaccines, because such VRPs are based on the inherent viral delivery systems for their own genetic material. A crucial aspect here is the generation of approved transgenic helper or packaging cell lines for propagation of RNA replicons. These cell lines must allow high yield propagation of RNA replicons, and comply with the highest biosafety standards. In particular, the regeneration of propagation-competent viruses by RNA recombination must be excluded. These problems are likely to be solved in the very near future so that VRPs can be produced in approved mammalian cell lines akin to other biologicals.


BMC Medicine by Kenneth Ross, the author uses existing datasets to look at somatic gene conversion and deletion in human disease. Here, we describe how Ross's recent efforts to detect such occurrences could impact the field going forward.The hunt for the genetic contributors to complex disease has used a number of strategies, resulting in the identification of variants associated with many of the common diseases affecting society. However most of the genetic variants detected to date are single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) and fall far short of explaining the full genetic component of any given disease. An as yet untapped genomic mechanism is somatic gene conversion and deletion, which could be complicit in disease risk but has been challenging to detect in genome-wide datasets. In a recent publication in http://www.biomedcentral.com/1741-7015/9/12/abstractSee research article:  However, it has turned out to be challenging to isolate the genes underlying the genetic component conferring susceptibility to most complex disorders. The genetic underpinnings of such traits have remained largely unsolved until relatively recently, where the advent of array-based technologies and large population cohorts have enabled investigators to leverage genetic variation across the entire genome to pinpoint major contributing genetic factors. These discoveries have been primarily driven by genome-wide association studies (GWAS) using single nucleotide polymorphism (SNP) markers, which have revealed compelling evidence, including robust replication, for genetic variants associated with a broad range of phenotypes  -3, whichNonetheless, these approaches to date have generally only captured a small proportion of the predicted genetic component of various complex traits . It is whttp://www.ncbi.nlm.nih.gov/gap). The Wellcome Trust Case Control Consortium (WTCCC) has also made its datasets available to the wider scientific community and has been a key leader in whole genome genetic approaches [While advances are taking place on these fronts, the question remains of whether there are ways that the existing genome-wide SNP datasets could be mined further. After all, many datasets have been deposited in the public domain, most notably those found on dbGaP The authors declare that they have no competing interests.All authors contributed equally to this work.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/13/prepub