Alipogene tiparvovec contains the human LPL naturally occurring gene variant LPLS447X in a non-replicating viral vector based on AAV1. Such virus-derived vectors administered to humans elicit immune responses against the viral capsid protein and immune responses, especially cellular, mounted against the protein expressed from the administered gene have been linked to attenuated transgene expression and loss of efficacy. Therefore, a potential concern about the use of AAV-based vectors for gene therapy is that they may induce humoral and cellular immune responses in the recipient that may impact on efficacy and safety. In this paper, we review the current understanding of immune responses against AAV-based vectors and their impact on clinical efficacy and safety. In particular, the immunogenicity findings from the clinical development of alipogene tiparvovec up to licensing in Europe will be discussed demonstrating that systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec have no deleterious effects on clinical efficacy and safety. These findings show that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.Alipogene tiparvovec (Glybera Glybera® has received marketing authorization under exceptional circumstances in Europe in 2012. Alipogene tiparvovec is an AAV-based gene therapy vector that has been developed for the treatment of patients with lipoprotein lipase deficiency (LPLD). It contains the gene of the naturally occurring gain-of-function variant LPLS447X of the human lipoprotein lipase (LPL) in a non-replicating viral vector based on adeno-associated virus serotype 1 (AAV1). The immunogenicity findings from the clinical development of alipogene tiparvovec will be discussed, demonstrating that systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec have not shown deleterious effects on clinical efficacy and safety. Alipogene tiparvovec is an example that muscle-directed AAV-based gene therapy is a promising approach for the treatment of human diseases.Adeno-associated virus (AAV) is a naturally occurring virus that is known to infect humans and other primates. It is expected to interact at multiple levels with the innate and adaptive immune system and elicit immune responses when injected in man. AAV-based vectors are nowadays often chosen for the development of new, promising gene therapy approaches because of several interesting features such as their inability to self-replicate. However, a potential concern about the use of such virus-derived vectors is the potential to induce humoral and cellular immune responses in the recipient that may impact on efficacy and safety. In this report, we review the current understanding of immune responses against AAV-based vectors and their impact on clinical efficacy and safety using alipogene tiparvovec (Glyberain vitro to have the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19 with minimal risk for random incorporations into the genome. For these reasons, AAV has attracted considerable interest because of its potential as a gene therapy vector. The use of AAV as gene therapy vectors has required the elimination of the rep gene from the vector, since it is coding for the protein that is involved in replication of the viral DNA and site-specific integration. In the vector genome, the rep and cap genes are replaced by the transgene, in the case of alipogene tiparvovec the gene for LPL, together with a promoter that is necessary to drive transcription. This cassette is flanked by inverted terminal repeats (ITRs) that are necessary for the formation of so-called concatemers in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. These episomal concatemers remain intact in the nucleus of non-dividing host cells. Hence, transferred genomes tend to persist inside the cells mainly in this episomal, non-integrated form  antibodies against AAV and/or the expressed therapeutic protein , 28–37. The eye and central nervous system are known to be immune-privileged compartments of the body due to adaptations that limit immune responses. Delivery of AAV-vectors directly into the brain has been tested in a number of studies , 38–40. Cellular immune responses against the AAV-vector have been found in only some of the clinical trials performed. The first observation of a cellular immune response induced by AAV-gene therapy to our knowledge was in patients with hemophilia B who were treated with an AAV-vector to deliver human coagulation factor IX , 41, 42.S447X in the early phases of clinical development) is an AAV1 vector expressing a naturally occurring variant of the LPL transgene, LPLS447X, associated with improved lipid profile and carried by approximately 20% of the general population . The patients did not receive immunosuppressants pre- or post-exposure to AAV1–LPLS447X, and were followed-up for up to 18 months after administration in the active phase of the study.Eight patients with LPL-deficiency were first monitored for several months, and then treated once with multiple intra-muscular injections of AAV1–LPLAfter an observation period of a few months, 14 patients with LPL-deficiency were treated with alipogene tiparvovec. Twelve of these patients received immunosuppressants. The immunosuppressant regimen consisted of cyclosporine A at a dose of 3 mg/kg/day and mycophenolate mofetil at a dose of 2 g/day and was maintained until 12 weeks after administration of alipogene tiparvovec. The patients were followed-up for 5 years after administration.After a run-in phase of a few months, five patients with LPL-deficiency were treated with alipogene tiparvovec. All patients have received immunosuppressants, starting shortly before exposure to alipogene tiparvovec. The immunosuppressant regimen consisted of 3 mg/kg/day cyclosporine A, 2 g/day mycophenolate mofetil, and a bolus injection of methylprednisolone was given 30 min prior to alipogene tiparvovec administration. The immunosuppressant regimen with cyclosporine A and mycophenolate mofetil was maintained until 12 weeks after exposure. The patients were followed-up for a year after administration in the active phase of the study.A summary of the clinical analyses schedule with alipogene tiparvovec is given in Figure Humoral  responses against AAV1 capsid proteins were measured with an ELISA assay. Briefly, AAV1 capsid proteins were fixed to polystyrene ELISA plates and incubated with the serum samples to be tested. Bound antibodies were detected by a subsequent incubation with conjugated antibodies against human immunoglobulins. The ELISA did not discriminate between IgG subclass antibodies. To discriminate between samples with normal and with elevated levels of anti-AAV antibodies, a cut-off level was established using serum samples from 30 healthy volunteers. The test results of the samples to be tested were scored by comparison with those of the negative control, which was a serum sample from a healthy human control. To this end, algorithms were developed to convey the optical density results into a semi-quantitative scoring system. Based on the algorithms, samples were said to be strongly positive (++), weakly positive (+), or negative (−) for AAV1 total antibodies.S447X were assessed by measuring total antibody levels in pre- and post-exposure serum samples with an ELISA assay. This ELISA was similar to that described above for antibodies against AAV1 proteins, except that recombinant LPL was used to coat plates. The recombinant LPLS447X was isolated from medium of stably transfected CHO cells that express LPLS447X. The ELISA did not discriminate between IgG subclass. A cut-off level of the assay was established in a similar way as described in the previous paragraph for anti-AAV1 antibody ELISA. Also for this ELISA, an algorithm was developed to convey the optical density results into a semi-quantitative scoring system. Based on the algorithms, samples were said to be positive (+) or negative (−) for anti-LPL antibodies.Antibody responses against LPLIn order to monitor the T-cell-mediated immune response in the patients treated with alipogene tiparvovec, an ELISpot assay was developed. This assay is based on the detection and quantification of interferon gamma (IFN-γ) secreting cells upon stimulation with AAV1 capsid antigens. To this end, PBMCs were obtained from the patients and incubated with AAV1 capsid antigens. A one color ELISpot assay was used for this purpose as previously described , 24 and Two criteria are widely used to evaluate test results of ELISpot assays, which are the number of spot forming unit (SFU) per million cells upon stimulation with antigen and the increase of SFU number compared to that in medium only. Generally, samples are said to be positive for T-cells when upon stimulation with antigen they contain >50 SFU per million cells, and when that number is at least threefold higher than that of the medium control. These criteria were also used to assess T-cells against AAV1.Open muscle biopsies were collected between 10 and 52 weeks after intra-muscular injection of AAV1–LPL from both an injected (tattooed) muscle  and a non-injected control muscle site . The specimens were analyzed according to routine evaluations, including muscle histology and immunohistochemical characterization of infiltrating cells when present. The biopsy procedures were performed at variable time after injection mainly dependent on the availability of the patients. The predefined criteria for the collection of tissue specimens in the protocol were 14 weeks for the first biopsy and 52 weeks for the follow-up biopsy, independently of any clinical indication. However, due to the availability of the patients, deviations from the protocol occurred and the real time of collection of the biopsy specimens is reported for each patient in Figure An overview of the systemic as well as local humoral and cellular immune responses observed in all patients participating in the clinical studies is shown in Figures Fifteen of the 26 subject had pre-existing antibodies against AAV1. Among the 19 patients of whom a post-exposure biopsy was available, 11 had pre-existing anti-AAV antibodies. No apparent relationship was found between pre-existing AAV1 antibodies and LPL-expression after administration of alipogene tiparvovec: 7 of the 11 patients with pre-existing anti-AAV1 antibodies had LPL-expression in the biopsy versus 4 of the 7 patients with no such antibodies Figure . As expeNone of the patients had anti-LPL antibodies prior to the administration of alipogene tiparvovec nor developed those after delivery of the product. The baseline levels of anti-LPL antibodies were below the cut-off level of detection in all patients prior to administration of alipogene tiparvovec. Post-administration levels remained below the cut-off level, demonstrating that no antibody responses were mounted against the expressed LPL protein after administration of alipogene tiparvovec even in the long-term.Enzyme-linked immunospot assays were used to assess the course of numbers of T-cells in peripheral blood that were directed against AAV1 epitopes. The results obtained across studies and per subjects are given in Figures Enzyme-linked immunospot assays were also used to evaluate whether there was any T-cell responses against the expressed LPL protein product. In none of the patients, such immune responses were observed upon administration of alipogene tiparvovec at any dose.Biopsy specimens of the injected muscle from 19 patients were available for immunohistochemical analyses. As described previously, these specimens were assessed for the presence of inflammatory cell infiltrate by qualified histopathologists. Infiltrates of varying intensity consisting of B cells, macrophages, and mainly T-cells, were found in 15 patients whereas in the other 4, no inflammatory cell infiltrate was observed. However, as illustrated in study AMT-011-02 with subject 02-002 for which no infiltrates in the injected muscle was detected, the biopsy of some subjects was taken weeks after the peak of systemic T-cells response reached baseline, compared to a few weeks for others subjects. Therefore, the time of the biopsy should be taken into consideration when putting in parallel local and systemic T-cell responses.As none of the patients developed a T-cell response against LPL, the relation between an immune response to the transgene and the presence of a local T-cell infiltrate was not further investigated. The presence of cytotoxic T-lymphocytes (CTL) in the inflammatory cell infiltrates was investigated in the muscle tissues injected with alipogene tiparvovec as well as in muscle biopsies from non-injected muscle. As a parameter for cellular cytotoxicity, granzyme B and Fas ligand expression by the cells was measured. CD8-positive T-cells in the infiltrates were negative for granzyme B and Fas ligand expression, which suggest that the majority of the T-lymphocytes present in the injected muscle tissue lack cytotoxic properties. Furthermore, CD4-positive T-cells observed in muscle biopsies from LPLD subjects injected with alipogene tiparvovec were further assessed for the expression of the transcription factor FoxP3, as a marker for regulatory T-cells. FoxP3-positive/CD4-positive T-cells were also found in the infiltrates suggesting that multiple mechanisms contribute to the local immune tolerance to alipogene tiparvovec administration .The presence of LPL protein in the muscle biopsies and the improved clearance of post-prandial chylomicrons levels in plasma were used to measure local and systemic activity of alipogene tiparvovec and were considered as efficacy markers , 51. HowFifteen of the 26 patients, with registered data for presence of pre-administration anti-AAV1 antibodies, had pre-existing antibodies against AAV1. Among the 15 patients with pre-existing antibodies against AAV1, biopsies were obtained from 11 patients. Among those 11 patients, 7 had LPL-expression in the biopsy. In comparison, from the 11 patients without pre-existing antibodies, 8 muscle biopsies were obtained; and from those, 5 were stained positive for LPL-expression. This distribution was very similar across the three studies CT-AMT-010, CT-AMT-011-01, and CT-AMT-011-02. Our results strongly indicate that there was no apparent relationship between the presence of pre-existing AAV1 antibodies in LPLD patients and LPL-expression after administration of alipogene tiparvovec.After the administration of alipogene tiparvovec in all 27 patients, the development of treatment-emergent antibodies against AAV1 capsid proteins was observed, independently of whether pre-existing antibodies were present or not. In relation with efficacy, those treatment-emergent antibody responses against the AAV1 capsid proteins upon treatment with alipogene tiparvovec, did not seem to affect expression of the transgene.11 gc/kg having a positive response, 40% (4/10) of the patients treated with 3 × 1011 gc/kg, and 69% (9/13) of the patients treated with 1 × 1012 gc/kg, which suggest an AAV1-dose-dependent kinetics of T-cell response appearance  of the subjects treated with 1 × 10pearance –50. Of tHowever, as mentioned previously, the variability of the biopsy procedure and by consequence, the difficulty for quantification and comparison between patients has to be considered in the interpretation of the data. The differences in the results obtained with the biopsies of patient 01-001 (in study CT-AMT-011-02), collected at 18 and 52 weeks after administration of alipogene tiparvovec, illustrate this issue. The biopsy taken at week 18 yielded no detectable LPL-expression, whereas the biopsy taken at week 52 yielded a strong expression of LPL.The administration of alipogene tiparvovec resulted in functional LPL activity levels sufficient to achieve a beneficial clinical effect in patients with LPLD. This conclusion is supported by the evidence that levels of plasma TG decreased in LPLD patients after administration of alipogene tiparvovec. The data obtained in studies CT-AMT-010, without immunosuppression, and CT-AMT-011-01 and AMT-011-02, with immunosuppression, are considered comparable in terms of the decrease in TG levels Figure . HoweverFurthermore, an ongoing study has shown a reduction in acute pancreatitis events in a series of more than 25 affected subjects . The anaS447X, was performed without treating the patients with immunosuppressants. In this study, no antibody or T-cells responses against LPL were found. However, antibodies against AAV1 capsid epitopes were observed in all patients whereas a T-cell response against AAV1 was detected only in four of the eight subjects.Study CT-AMT-010, the first clinical study with AAV1–LPLAs discussed, none of these immune responses raised specific safety concerns. However, they triggered more a concern about the efficacy of the product; especially the development of any AAV-specific T-cells  that werThese discussions heavily influenced the decision to include an immunosuppressant regimen in the clinical study protocols for CT-AMT-011-01 as well as CT-AMT-011-02.S447X vector used cannot induce expression of viral proteins in host cells. Hence, AAV1 capsid proteins are expected to be only transiently presented to the immune system of the recipient after injection of AAV1–LPLS447X  treated with alipogene tiparvovec received a concomitant administration of immunosuppressants. Treatment-emergent anti-AAV1 antibody responses were observed in all the patients and were not affected by the immunosuppressants, neither during the time of administration nor after cessation of the administration. The AAV1–LPLLPLS447X , 19, 53.S447X was used . In this subject, at 15 weeks post-administration, a transient rise in CPK, accompanied by a rise in CRP Figure , was corFrom the clinical data obtained across studies, we conclude that there is no clinical untoward impact of the delivery of alipogene tiparvovec.To evaluate the immunogenicity of alipogene tiparvovec, an extensive testing program was performed that included the analyses of antibody and T-cell responses to LPL as well as to AAV1. Antibody and cellular responses were measured pre-exposure to alipogene tiparvovec, and at various occasions post-exposure. In addition, biopsies were taken from the injected muscle and non-injected muscle as control, to evaluate local immune and inflammatory processes. Finally, CPK and CRP levels and neutrophil counts were measured, and patients were clinically evaluated for local and systemic symptoms indicative for inflammatory or immune reactions. Expression of LPL in the injected muscle as well as the improvement of post-prandial chylomicrons clearance in plasma was used as biochemical markers for efficacy.in vivo gene therapy not only depends on the ability to control the immune response toward the vector, but also to monitor any potential reaction to the therapeutic protein expressed from the transgene. The data obtained from patients with LPL-deficiency who received a single treatment with multiple injections of alipogene tiparvovec, support the initial expectation that the protein product is minimally immunogenic, if at all: neither treatment-emergent antibody responses against LPL nor T-cells responses against LPL were found. Thus, the expressed protein itself appears to be non-immunogenic.The success of Concerning anti-AAV immunity, the majority of healthy individuals are exposed to AAV during lifetime , 54. HenThe markers that were used to demonstrate LPL-expression and functionality after the delivery of alipogene tiparvovec are complex. Therefore, the present report is focusing on LPL-expression in muscle biopsies and post-prandial chylomicron clearance as marker for systemic LPL activity. For the direct measurement of LPL-expression, we made use of biopsies that were obtained at different time points up to 12 months after drug administration. However, and as described above, not all patients gave their consent to this procedure which can present some variability in the execution. Nineteen patients provided their consent and in total 20 biopsies were obtained, which all were examined for LPL-expression. In 12 of the 20 biopsies, LPL-expression was found irrespective of the presence of antibodies against AAV1. The second biochemical marker for successful gene delivery in this review is the level of clearance of post-prandial chylomicrons in plasma, which reflect the systemic activity of LPL . This maAltogether, these observations demonstrate that anti-AAV1 antibody responses did not exclude sustained transgene expression nor did impair the systemic biological activity of the expressed protein. Our results further support that treatment-emergent anti-AAV1 antibody responses do not necessarily have any influence on the long-term efficacy and safety of AAV-based gene therapy.12 gc/kg, were more prompt to develop an AAV1-specific T-cells responses. Somewhat higher T-cell responses were noticed in some patients upon cessation of the immunosuppressants, pointing to a suppressive effect on the T-cell responses. It is therefore not possible to make definitive conclusions regarding the effect of immunosuppressants on T-cell responses to AAV1. The immunosuppressants did not affect antibody levels as was expected since the regimen was mainly aiming at reducing potential T-cell responses. MMF and cyclosporine are both described to be effective in suppressing cytotoxic T-cell responses. MMF has an effect on B-cell proliferation, because it inhibits de novo guanosine nucleotide synthesis, a pathway commonly required for both T- and B-cell proliferation. Cyclosporine specifically inhibits T-cell activation by inhibiting IL-2 production and exerts limited, if any, effect on B-cell proliferation. Thus, the increased anti-AAV1 titers are not surprising. Similar observations were made in a study in monkeys  and impurities profile of the vectors used in the various clinical trials described in the literature may be very divergent and therefore may lead to very different immune responses. Nonetheless, the scientific debate has been powerfully influenced by previous findings with other vectors and by the hypothesis that the vector sensitizes transduced cells to become targets for CTL-mediated clearance. Therefore, since the safety data on alipogene tiparvovec have been collected in a small number of patients, their clinical relevance and possible interpretations were considered not fully unequivocal and further data collection has been requested by the European Medicine Agency post-approval of GlyberaThe funding body (uniQure) was involved in all aspects of the study. The authors  are employes of uniQure.http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00082/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCFSkp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type.In  Drosophila hematopoiesis has been an emerging field in recent years, as the fly hematopoietic system has many parallels with that of vertebrates. Among these similarities are the myeloid cell lineage, biphasic nature of hematopoiesis and conserved genes important for proper hematopoietic development [The study of elopment ,2. TheseDrosophila occurs in two distinct spatiotemporal waves, embryonic and larval. Important to this study is the embryonic wave, which occurs in the head mesoderm and generates mature hemocytes that are present throughout larval development and maintained into the adult stage [Hematopoiesis in lt stage ,4. The llt stage .Drosophila is that they both have the evolutionarily-conserved myeloid blood cell lineage. Within this lineage in Drosophila, there are two types of hemocytes, which are known to arise from a common precursor. These cell types are the plasmatocyte and the crystal cell. Plasmatocytes compose 95% of the cells in the Drosophila hemolymph, are involved in phagocytosis of foreign particles, and considered homologous to mammalian macrophages [Another similarity between vertebrates and rophages ,6,7. Cryrophages -10. Therrophages ,12. Lamerophages -15.Both plasmatocytes and crystal cells divide exactly four times during embryonic stages, until there are approximately 700 plasmatocytes and 36 crystal cells . During eater blood cell-specific transcriptional enhancer was utilized as a driver for gene function knockdown experiments. Eater is an EGF-rich phagocytic receptor expressed solely in mature plasmatocytes. The receptor is known to be involved in antigen recognition and its expression is regulated by the GATA factor Serpent [eaterGal4>UAS-GFP line allowed us to identify, by directed RNAi knockdown, the SCF complex members that function in plasmatocyte development. The SCF complex is a ubiquitin ligase complex that has one of each of the core family proteins: Skp, Cullin, and F-Box, each of which have multiple members, and are important in substrate specificity [Skp2 complex is necessary for the degradation of Dup, allowing for proper progression of plasmatocytes through the cell cycle.In order to study the importance of individual genes for the production and proliferation of circulating hemocytes, an  Serpent ,17. Genecificity ,19. Ideneater enhancer necessary to direct GFP reporter expression in mature plasmatocytes [eaterGal4 line of flies for use within the UAS-Gal4 system, to selectively knockdown gene function using a UAS-RNAi strategy [UAS-GFP was crossed into the eaterGal4 background. Hemolymph samples of RNAi knockdown animals were obtained and compared to eaterGal4>UAS-GFP>w1118 control samples for any aberrations in plasmatocytes, including cell size and cell number  development [lin19 might affect plasmatocytes in circulation. lin19 was identified as a regulator of cell size in circulating hemocytes, as evidenced by the approximately 5% of cells which are vastly enlarged in hemolymph samples obtained from eaterGal4>UAS-GFP>UAS-lin19 RNAiHM05197, Roc1a (Roc), Skp2 (F-box) and Nedd8, as knockdown of these genes, similar to knockdown of lin19, also caused approximately 5% of hemocytes to become 2-6 times the size of control cells as shown in eaterGal4>UAS-GFP>UAS-SCF component RNAi blood cell samples , a similar but stronger driver than eaterGal4 , with an increase in medium (43/100 under 22 μm) or slightly larger cells (49/100 were 22.1-25 μm) as assayed by anti-P1 plasmatocyte-specific antibody staining using our method of cell size counting . This is indicative that the size of the large cells caused by knockdown of the SCF complex, and lack of ubiquitination of Dup, is being partially rescued by the overexpression of Gem.Noticeably, hemolymph samples with overexpression of counting . All celeaterGal4; UAS-GFP strain allowed us to identify the functional importance of SCF complex members for the plasmatocyte blood cell lineage by a RNAi knockdown approach. Using this technique, we obtained hemolymph samples and were able to identify several genes belonging to the core SCF complex that, when knocked-down, caused a very distinctive giant cell phenotype. Importantly, as we were using eater as a driver to identify complex components, it was confirmed that these enlarged cells were plasmatocytes by anti-P1 plasmatocyte-specific antibody staining. This suggested to us, as proof-of-principle, that knockdown of gene function in mature plasmatocytes could elicit aberrant phenotypes dependent on the functional requirement of an essential gene/gene complex.The generation of an Drosophila genes that may be involved in SCF complexes in order to determine specificity for a substrate. The F-box is thought to convey specificity of this complex by recruiting the substrate, however activation of the Cullin by neddylation factors also plays a role in ubiquitation of the substrate [Drosophila hematopoiesis, as knockdown of only one of each of the core components caused enlarged plasmatocytes  [Skp2 complex plays a role in murine hematopoiesis, by ubiquitinating proteins necessary for proper cell cycle, such as Cyclin E. There are still many questions to be answered about SCF regulation in blood cells, as some of these results are contradictory [Using fluorescence microscopy, it was noted that the enlarged cells caused by the SCF knockdown had a significant excess of DNA in the nuclear region. To investigate the hypothesis that DNA re-replication was occurring in plasmatocytes with the SCF complex knockdown, anti-gamma- Tubulin staining of centrioles was performed. Previously, it was shown that knockdown of Gem elicits DNA re-replication, therefore we used it as a positive control . It was lication ,27,32-34lication . In our lication . As the lication . ConversCyclin E . This isadictory -38. In addition to these data, protein localization in the knockdown of Cyclin E showed that Gem had been removed from the nucleus, again consistent with the notion that it was titrated away from the nucleus by binding Dup. This is plausible because the SCF complex can recognize its substrates due to phosphorylation state ,40. SincDrosophila plasmatocytes. Further, Drosophila plasmatocytes are most similar to mammalian macrophages, which also do not endoreplicate [Although re-replication is one mechanism to explain the SCF loss-of-function phenotype, a similar non-canonical process, known as endoreplication, could also account for the over-replicative system in these cells. Endoreplication is a cycle in which cells undergo S phases that are separated only by gap phases but not an intervening mitosis ,47. Howeeplicate . Since seplicate ,32,34,45eaterGal4 is activated during second instar, however there is likely a latent period between activation of Gal4 and protein knockdown by the RNAi. This is consistent with only five percent of cells having an active cell cycle, and becoming enlarged through re-replication. A final possibility is that there are partially redundant mechanisms for the regulation of Dup. As previously described, the SCF complex has been shown to be involved in the ubiquitination and subsequent degradation of Dup, and Gem will inhibit the remainder of the Dup that may be in the nucleus [It is intriguing that only five percent of the cells display the re-replication phenotype, and we have several hypotheses on this point. One explanation is that the smaller cells have arrested. There are many intrinsic mechanisms to ensure proper cell cycle progression preventing re-replication and ultimately cancer. It is possible these enlarged cells have escaped these mechanisms, causing the cell to replicate their DNA many times without going through mitosis, while the smaller cells arrest, to prevent this phenotype. It is also possible that only five percent of these cells are going through cell division during the time the RNAi is functionally knocking down the gene. Previous research has suggested that during mid-to-late third instar larval stages, only one to two percent of cells are going through mitosis at a given time . eaterGa nucleus ,27,34. TpxnGal4>UAS-Gem43>UAS-lin19 RNAiHM05197 compared with pxnGal4>UAS-lin19 RNAiHM05197. There is a drastic decrease in the number of giant cells, which are larger than 25.1 μm, in pxn>UAS-Gem43>UAS-lin19 RNAiHM05197 (8/100) plasmatocytes compared with SCF knockdown hemocytes (45/100). It was also noted that there was a significant decrease in the average size of plasmatocytes in hemolymph samples from Gem overexpression in the SCF knockdown background (p<0.001). These lines of evidence are all suggestive that knockdown of the SCF complex increased nuclear Dup leading to re-replication. By over-expressing its inhibitor, Gem, it is possible to partially rescue this enlarged cell phenotype generated by excess nuclear Dup. These data suggest the regulation of Dup is important in the proper cell cycle progression of plasmatocytes. Furthermore, these data support the hypothesis that the SCFSkp2 complex is responsible for the ubiquitination of Dup, allowing plasmatocytes to proliferate properly. Although this study provides substantial genetic evidence that the SCFSkp2 complex is necessary to ubiquitinate Dup allowing for proper hematopoietic cell cycle progression, future studies using biochemical techniques to show physical interactions are needed to support the model proposed here.To further implicate the necessity of Dup regulation in the proper cell cycle of plasmatocytes, a rescue experiment was performed by overexpressing the Dup inhibitor, Gem. By overexpressing this inhibitory protein, it was hypothesized that the nuclear localization of Gem would increase, the protein would bind Dup, and therefore decrease the re-replication that is observed in SCF complex knockdown. Performing immunohistochemistry experiments identified that there was an increase in nuclear Gem and a decrease in Dup. Additional experimental evidence supports this hypothesis as there is a decrease in size of plasmatocytes with genotype Skp2 complex, described in this manuscript, and the Cul4-DDB1-CDT2-PCNA (Cul4CDT2) complex [CDT2 complex was responsible for the enlarged cell phenotype, both DDB1 and PCNA were knocked-down via RNAi and Cul4 mutants were also analyzed. Although DDB1 functional knockdown elicited a small number of enlarged cells, these cells had a different morphology than the SCFSkp2 knockdown (data not shown). Additionally, none of the other analyses elicited any enlarged cells as observed when the SCFSkp2 complex was knocked-down. This further implicates the necessity of the SCFSkp2 complex in the proper plasmatocyte cell cycle.Furthermore, there are two ubiquitin ligase complexes known to be involved in the ultimate degradation of Dup: The SCF complex ,49,50. TSkp2 complex , 2-1 UA. Calvi; ) and UAShardson; ). eaterGi stocks ,20.2 software . Larval hemocyte samples were obtained as described previously . The folBrdU was diluted in standard fly food at 1 mg/ml (Sigma Item #B5002). Flies were maintained on this media for 3 days, when the adults were removed, and vials placed at 25°C. Hemocytes were obtained as previously described and stained with DAPI (1:500) and anti-BrdU  .Hemocyte samples were obtained as previously described . SamplesFigure S1DAPI staining of enlarged cells induced by SCF component RNAi knockdown.Hemocytes from Figure 4 were DAPI stained to identify the nuclear region. (A-L) Visualization of the nucleus using DAPI staining of the remaining components of the SCF complex. The results recapitulate the data observed in pxnGal4>UAS-lin19 RNAi knockdown showing enlarged cells have increased DNA content. (M-O) DAPI staining of pxnGal4>UAS-CyclinERNAiJF02473 indicating knockdown of Cyclin E, cytoplasmic staining of anti-Gem and nuclear staining of anti-Dup.(TIF)Click here for additional data file.Table S1RNAi stocks used to analyze knockdown of SCF complex function. A list of RNAi stocks used to determine and verify knockdown of the SCF complex that caused the enlarged plasmatocyte phenotype. (XLSX)Click here for additional data file.Table S2Drosophila hematopoiesis.List of stocks used to determine members of the SCF complex functioning in  The majority of possible SCF complex members were knocked-down via RNAi, with the remaining possible members screened by mutant analysis. The analysis identified knockdown of one member of each of the core components induced the enlarged cell phenotype, shown in bold.(XLSX)Click here for additional data file.


Recently, much scientific attention has been focused on resting brain activity and its investigation through such methods as the analysis of functional connectivity during rest . However, investigation of the magnitude of brain activity during rest has focused on the relative decrease of brain activity during a task, rather than on the absolute resting brain activity. It is thus necessary to investigate the association between cognitive factors and measures of absolute resting brain activity, such as cerebral blood flow (CBF), during rest (rest-CBF). In this study, we examined this association using multiple regression analyses. Rest-CBF was the dependent variable and the independent variables included two essential components of cognitive functions, psychometric general intelligence and creativity. CBF was measured using arterial spin labeling and there were three analyses for rest-CBF; namely mean gray matter rest-CBF, mean white matter rest-CBF, and regional rest-CBF. The results showed that mean gray and white matter rest-CBF were significantly and positively correlated with individual psychometric intelligence. Furthermore, mean white matter rest-CBF was significantly and positively correlated with creativity. After correcting the effect of mean gray matter rest-CBF the significant and positive correlation between regional rest-CBF in the perisylvian anatomical cluster that includes the left superior temporal gyrus and insula and individual psychometric intelligence was found. Also, regional rest-CBF in the precuneus was significantly and negatively correlated with individual creativity. Significance of these results of regional rest-CBF did not change when the effect of regional gray matter density was corrected. The findings showed mean and regional rest-CBF in healthy young subjects to be correlated with cognitive functions. The findings also suggest that, even in young cognitively intact subjects, resting brain activity (possibly underlain by default cognitive activity or metabolic demand from developed brain structures) is associated with cognitive functions. Cerebral blood flow (CBF) during rest (rest-CBF) and cognitive functions among people with cognitive decline have been reported to be associated Many recent neuroimaging studies have focused on a particular set of brain regions – the so called default mode network (DMN). These regions are deactivated during a wide range of externally directed, attention-demanding tasks , rather than being deactivated during rest TID in the DMN is associated with various cognitive factors and pathological states. The magnitude of TID in the DMN positively correlates with working memory capacity and decreases with aging It is thought that investigating the association between cognitive factors and absolute resting brain activity, instead of TID, gives new insights into the association between default brain functions and cognition for the following reasons. First, regions that show relatively increased resting brain activity (measured by rest-CBF) and regions that show TID are partly similar, yet different In order to examine the association between cognitive factors and measures of absolute resting brain activity, mean and voxel-by-voxel rest-CBF analyses were performed using arterial spin labeling ASL; see . The assIn accordance with the Declaration of Helsinki (1991), Written informed consent was obtained from each subject. This study was approved by the Ethics Committee of Tohoku University.Sixty-three healthy, right-handed individuals (32 men and 31 women) participated in this study as part of our ongoing project to investigate the association between brain imaging, cognitive functions, and aging g factor Raven's advanced progressive matrix (RAPM) The S-A creativity test In addition to cognitive functions, subject mood on the experiment day was also evaluated using the shortened Japanese version All MRI data acquisition was conducted with a 3-T Philips Intera Achieva scanner. ASL is an MR imaging method that allows noninvasive measurement of regional CBF. ASL was performed with quantitative signal-intensity targeting by alternating radio-frequency pulse labeling of arterial regions, which is a pulsed ASL method developed by Petersen et al.3 voxels, 70 slices, 2.0 mm slice thickness. Scan duration was 6 min 39 sec. Finally, using a magnetization-prepared rapid gradient-echo Imaging sequence, high-resolution T1-weighted structural images  were collected. These MTR maps and T1-weighted structural images were used for the normalization of the regional CBF maps. These scans, as well as other scans used in another study In addition to ASL, to obtain magnetization transfer ratio (MTR) maps, which represent a loss of signal due to the magnetization transfer effect, two successive scans, both with and without an additional RF magnetization transfer pulse, were acquired. The scan parameters were: TE = 3.1 ms, TR = 66 ms. FOV = 24 cm, 1.25×1.25×2 mmMaps of rest-CBF and the longitudinal relaxivity (R1) of each subject were obtained by using dedicated software running on IDL , which was developed and provided by Petersen et al. Preprocessing and data analysis were performed using Statistical Parametric Mapping software  implemented in Matlab . Prior to the normalization of the regional CBF map, an original template of the T1-weighted structural image was created as follows. (a) The T1-weighted structural image of each subject was spatially normalized to the T1 template in SPM5. (b) The normalized T1-weighted structural image of each subject was then smoothed using a Gaussian kernel of 8 mm FWHM and finally averaged across subjects to make an original template of the T1-weighted structural image. Previous voxel-based morphometry (VBM) and fMRI studies have shown the usefulness of making a study-specific template for the normalizing procedure from the normal subject group 3 voxels. The MTR image was then segmented using SPM5's segmentation option. Next, a gray matter mask image that consisted of voxels whose values were higher than 0.2 in the gray matter density image, resulting from the segmentation of the MTR image, was generated. This gray matter mask image was applied to the regional CBF map to limit the analysis so as to generate the gray matter regional CBF map. Also, this gray matter mask image was applied to the segmented gray matter (density) image to generate the regional gray matter density (rGMD) map, which has the same gray matter area as the gray matter regional CBF map generated above. These processed normalized gray matter regional CBF maps and (masked) rGMD maps were then spatially smoothed using a Gaussian kernel of 8 mm FWHM. The resulting maps, representing the gray matter regional CBF and (masked) rGMD maps , were forwarded to the group regression analysis described below.Prior to MTR calculation, the magnetization transfer image volume acquired with the saturation pulse (Sat) was linearly registered to the magnetization transfer image volume acquired without the saturation pulse (NoSat). The MTR image volume was calculated by: 100 * (NoSat−Sat)/NoSat. Then, the R1 map of each subject, which maintained its alignment with the regional CBF map of each subject, was coregistered to the MTR image with the within-subject registration method (the coregisteration option in SPM) Both the MTR image and the T1-weighted structural image were used for preprocessing for the following reasons, (a) The T1-weighted structural image has the most refined solution and is suited for normalizing procedures. However, the contrast of our T1 weighted structural image is not close to the R1 image. It is probably for this reason that the within-subject registration between the T1 weighted structural image and the R1 image did not work in several cases, as was confirmed by visual inspection. Furthermore, our T1 weighted structural image is not compatible with SPM5/VBM5's segmentation. The segmentation of this image with SPM5/VBM5 sometimes results in apparent segmentation errors, which are visible on first sight from visual inspection. (b)The R1 image's resolution was not good and the R1 image taken with our ASL sequence does not show the whole brain structure. It is best suited for neither normalization nor segmentation. We confirmed that these processes  using R1 images often failed through visual inspection. (c) The MTR image is close to both the R1 image and the T1-weighted structural image and allows itself to be coregistered with both the R1 image and the T1 weighted structural image. Additionally, it is compatible with SPM5's segmentation. We confirmed these processes did not fail (at least the failures were not apparent at first sight) in all subjects, through visual inspections.The behavioral data and data of the mean rest-CBF of the gray and white matter were analyzed using the statistic software SPSS 18.0 . Associations among psychological data  were analyzed using simple regression analyses. We used a multiple linear regression analysis to investigate whether each of the mean rest-CBFs of the gray and white matter was significantly related to RAPM score, total creativity score in the S-A creativity test, total mood disturbance score of the POMS, age, and sex. In these analyses, dependent variables were the mean rest-CBF value of gray and white matter, respectively. The independent variables were RAPM score, total creativity score in the S-A creativity test, total mood disturbance score of the POMS, age, and sex.At the group level analysis, we tested for a relationship between cognitive functions  and regional rest-CBF. In the whole brain analysis, we used a multiple linear regression analysis to look for areas where the regional rest-CBF was significantly related to individual general intelligence (RAPM score) and individual creativity as measured by the divergent thinking test . The analysis was performed with sex, age, and the score of POMS as additional covariates. By using SPM5's ANCOVA option of global normalization and setting the grand mean scale value at 50 ml/dl/min, we corrected the confounding effect of differences in mean (gray matter) CBF.In the abovementioned whole brain regional rest-CBF analysis, the effect of the regional gray matter structure on regional-rest CBF was not corrected. This was done so that our studies could be associated with relevant previous studies. Gray matter distributions were also not taken into account in PET studies, nor in the other functional MRI studies P<0.05, after correction for multiple comparisons at cluster size with a voxel-level cluster-determining threshold of P<0.005 uncorrected, were considered statistically significant in this analysis.Regions with significance were inferred using cluster-level statistics P>0.05, simple regression analysis).A multiple regression analysis with mean gray mater rest-CBF as the dependent variable and five other variables  as independent variables was performed. The analysis revealed that the RAPM score was significantly and positively correlated with mean gray matter rest-CBF . HoweverThen, a multiple regression analysis with mean white mater rest-CBF as the dependent variable and five other variables  as independent variables was performed. The analysis revealed that the RAPM score was significantly and positively correlated with mean white matter rest-CBF . Also, tThe association between negative mood and increased rest-CBF in healthy subjects is consistent with previous studies x, y, z = −53, −16, −1; t = 3.62; P = 0.036, corrected for multiple comparisons at cluster size with a voxel-level cluster-determining threshold of P<0.005, uncorrected. A whole brain multiple regression analysis was then performed. After controlling for the total S-A creativity test score, the POMS score, age, sex, and mean gray matter rest-CBF, it became evident that the RAPM score was significantly and positively correlated with regional rest-CBF in a perisylvian cluster. This perisylvian cluster included the superior temporal gyrus, insula, and contingent regions . In this analysis in which the effect of rGMD was corrected at every voxel, there were no other significant results.The significance of this cluster did not change when the effect of rGMD was corrected at every voxel . In this analysis, there were no other significant results.The significance of this cluster did not change when the effect of rGMD was corrected at every voxel  is associated with cognitive functions. Consistent with our hypothesis, our findings showed that mean gray and white matter rest-CBF were significantly and positively correlated with individual psychometric intelligence as measured by RAPM. Furthermore, after correcting the effect of mean rest-CBF, regional rest-CBF in the precuneus showed a significant negative correlation with individual creativity. This negative correlation between creativity and regional rest-CBF in the precuneus may at least partly underlie the association between creativity and reduced TID in the DMN In this study, increased mean gray and white matter rest-CBF were associated with higher general intelligence. This finding is congruent with the notion that areas distributed across the brain are associated with general intelligence. Increased rest-CBF may be necessary for maintaining cortical structures rich in cell components in subjects with higher general intelligence. Recent studies There was a significant positive correlation between general intelligence and increased regional rest-CBF in the left-side perisylvian region. This results is congruent with some previous studies on the neural basis of psychometric intelligence and may indicate high facilitation of language processes in highly intelligent subjects. Increased regional rest-CBF in the left perisylvian region,  is associated with higher general intelligence after the effect of mean rest-CBF has been removed. This finding is congruent with the report that higher psychometric intelligence is associated with functional activity in the left-side perisylvian region during a language-related task Increased mean white matter rest-CBF (but not mean gray matter rest-CBF) in creative subjects may be necessary for maintaining the myelinated white matter structures of creative subjects. As for creativity, a positive correlation between mean white matter rest-CBF (and not mean gray matter rest-CBF) was observed. The correlation between cognitive functions and rest-CBF in white matter observed in this study is congruent with that of people with cognitive decline Decreased regional rest-CBF in the precuneus in creative subjects is congruent with (a) clinical studies that reported an association between resting brain activity and pathology and (b) studies that reported an association between creativity and pathology. Additionally, it may have roots in creative subjects' poor ability in social functioning. This pattern of rest-CBF in creative subjects is distinct from the pattern of rest-CBF in the highly intelligent subjects observed in this study, supporting the distinction between creativity and psychometric intelligence. Previous studies have reported reduced TID in the DMN in patients with schizophrenia and their relatives, and also in patients with autism spectrum disorders Alternatively, reduced regional rest-CBF in the precuneus may have the same mechanism which was suggested to explain the reduced TID in this region during a working memory task Our previous study reported that reduced TID in the precuneus during a working memory task is associated with higher creativity a priori hypothesis. Our statistical procedures are standard and widely used in analyses of mean rest-CBF and of regional rest-CBF . As was the case with our previous studies This is the first study to investigate the association between rest-CBF and cognitive functions among healthy young subjects. Recent neuroimaging studies focused on the associations between resting brain activity and cognition using the paradigm of TID and functional connectivity between different brain regions during rest. Our findings showed that mean and regionally specific rest-CBF during rest in healthy young subjects correlated with cognitive functions. The significance of the regional rest-CBF results did not change when the effect of rGMD was corrected. The findings suggest that, even in young cognitively intact subjects, resting brain activity (possibly underlain by default cognitive activity or metabolic demand from developed brain structures) is associated with cognitive functions.


Clinical significance of out-pouching structures of the left atrium (LA) as potential embolic sources remains unclear. We sought to evaluate the association between stroke and LA out-pouching structures.A case-control study was conducted to assess the prevalence of LA out-pouching structures in subjects with and without stroke. Case subjects were 270 stroke patients who had undergone cardiac CT. Control subjects were 270 age- and sex-matched patients without a history of stroke and who had undergone cardiac CT. Presence of LA out-pouching structures was determined by ECG-gated cardiac CT. The location of out-pouching structures was categorized as near Bachmann bundle, anterior, inferoseptal, inferior, and lateral. The prevalence, number and location of out-pouching structures and clinical characteristics were compared between the two groups.One hundred sixty eight out-pouching structures were identified in 139 stroke patients (51%), while a total of 169 out-pouching structures were found in 155 control patients (57%) (p=0.1949). The prevalence of LA out-pouching structures with different locations was not significantly different between the stroke group and control group. In the stroke group, the prevalence of out-pouching structures was not significantly different by subtypes of ischemic stroke and the prevalence of LA out-pouching structures was not different between patients with atrial fibrillation (AF) and without AF.The left atrial out-pouching structures are commonly seen in a population with and without stroke with similar prevalence. Our study suggests that LA out-pouching structures are not significant risk factors of stroke. Cardiogenic emboli have been considered to be the main causal factor in 20-40% of all stroke cases . Stroke The ongoing development of cardiac computed tomography (CT) techniques has resulted in better visualization of cardiac anatomy and cardiac CT has become a potential noninvasive imaging modality in the detection of cardiac sources of embolism in stroke patients -4. RecenIn this study, we aimed to evaluate the prevalence, number, and location of LA out-pouching structures in patients with stroke in comparison to those of patients without stroke using ECG-gated cardiac CT and to reveal the relationship between these variant structures and stroke.This retrospective study was approved by our institutional review board. Informed consent was given by the patients for their information to be stored in the hospital database and used for research.We reviewed the records of consecutive patients who were included in the Yonsei stroke registry. Yonsei stroke registry is a prospective hospital-based registry for patients with acute ischemic stroke or transient ischemic attack . The patFrom April 2009 to September 2011, 270 consecutive patients who had been admitted to our hospital with an episode of stroke and had undergone cardiac CT were included in our study and classified as the stoke group. The stroke group consisted of 179 men  and 91 women . For a control group, we selected 270 patients who had no history of stroke or transient ischemic attack from our cardiac CT database and who were age- and sex-matched with the stoke group. The indication for cardiac CT in the control group was clinically suspected coronary artery disease.Clinical information including diabetes mellitus, hypertension, dyslipidemia, atrial fibrillation, and smoking status were obtained from electronic medical records. The subtypes of ischemic stroke in the stroke group were classified according to the Trial of Org 10172 in Acute Stroke Treatment criteria (TOAST criteria) . The claCT scans were performed using a dual-source CT scanner . In the absence of contraindications, an oral β-blocker  was administered 1 hour prior to examination to reduce the heart rate in patients with heart rates above 65 bpm and a 0.3mg sublingual dose of nitroglycerin was administered just before the scan.Scan delay times between the start of contrast agent injection and of scanning were determined by the timing bolus technique. After a bolus injection of 10 mL of iopamidol  followed by 20 mL of saline with 5 mL/s, optimal delay times were determined by automatic evaluation of the contrast enhancement in the ascending aorta. All CTs were performed with the triple-phase injection method .The data acquisition mode was determined by heart rate (HR). In cases of a regular HR < 60 bpm, scans were performed with the prospectively electrocardiogram (ECG)-triggered high-pitch spiral mode. In cases of a regular HR between 60 and 75 bpm, scans were performed with the prospectively ECG-triggered axial mode targeting mid-diastole, and in cases of a regular HR ≥ 75 bpm, with the prospectively ECG-triggered axial mode targeting end-systole . If freqImages were reconstructed with a slice thickness of 0.75 mm and an increment of 0.5 mm using a medium kernel of B36f and sent to a workstation . Images were evaluated with axial reconstruction images, volume-rendering images, and interactive multiplanar reformat images.Two experienced radiologists  reviewed the images via consensus reading. Each reader was blinded to the patient’s group and other clinical information. Evaluation of the LA out-pouching structures was based on the axial and short axis CT images. In general, an accessory LA appendage was defined if the structure had a discernible ostium and irregular contours suggestive of the presence of pectinate muscles, and LA diverticulum was defined if the structure had a saclike shape with a broad-based ostium and a smooth contour at the body portion. However, the differentiation of an accessory LA appendage from LA diverticulum can be difficult because the defining features may be subtle and some cases may express features of both appendages and diverticula ,16. ThusThe presence, number and location of LA out-pouching structures were recorded in all subjects, regardless of them being either an accessory LA appendage or LA diverticulum. The location of the out-pouching structures was divided into five categories: 1) near Bachmann Bundle, 2) anterior (excluding 1), 3) inferoseptal , 4) inferior , 5) lateral  .Under the assumption that the prevalence of LA out-pouching structures was 40% in the control group and 55% in the stroke group, the sample size was calculated as 462 to have a 90% power and to detect a difference between arms with a two-sided type I error rate of 5%. The calculated power in an unmatched sample size of 270 in each group with a 1:1 comparison  was 94%.The prevalence and location of out-pouching structures were compared between the stroke group and control group. In addition, we investigated whether the prevalence of out-pouching structures were statistically different according to the presence of atrial fibrillation and stroke subtypes based on the TOAST criteria. These categorical prevalence data are presented as percentages, and comparisons were performed with the chi-square test.All statistical analyses were performed using MedCalc for Windows, version 12.3.0.0 .The clinical characteristics of the stroke group and control group are summarized in The prevalence of hypertension, diabetes mellitus, and smoking status were significantly higher in the stroke group than in the control group. However, atrial fibrillation was observed with similar prevalence in both groups.A total of 168 out-pouching structures were found in 139 out of 270 (51%) patients in the stroke group, whereas 169 out-pouching structures were found in 155 out of 270 patients (57%) in the control group. There was no statistically significant difference in the prevalence of out-pouching structures (p=0.1949). No thrombi were found within these structures. Thirty-four of 270 stroke patients had atrial fibrillation. The prevalence of out-pouching structures was not significantly different between patients with and without atrial fibrillation (p=0.9989) . In the To our knowledge, this is the first study which assesses the prevalence and characteristics of LA out-pouching structures using ECG-gated cardiac CT in a cohort with stroke and compares them with a control group of patients without stroke. This study showed no difference in the prevalence or location of LA out-pouching structures in patients with stroke compared to control patients without stroke. Also, the prevalence of LA out-pouching structures had no relationship with subtypes of ischemic stroke. In addition, this study showed that LA out-pouching structures was not significantly associated with AF.In this study, the prevalence of out-pouching structures was high for both groups . The prevalence of LA out-pouching structures was slightly higher than previously reported prevalence values of accessory LA appendages and LA diverticula that were around 10%-46% ,17. The There is still debate about the clinical significance of LA out-pouching structures as a potential cause of cardioembolic stroke. Previous reports regarding accessory LA appendages or diverticula suggested that these out-pouching structures might be associated with an increased risk of thromboembolic disease because of low flow and turbulence within the sac-like structure ,16. TherIn our study, when patients with stroke were divided into patients with AF and without AF, there was no significant difference in the presence of LA out-pouching structures. Several recent studies evaluated the prevalence of LA out-pouching structures between patients with AF and those with sinus rhythm ,9,20. ThThe limitations of this study should be mentioned. First, we did not evaluate the accessory LA appendages and diverticula separately but evaluated together as “LA out-pouching structures” because the differentiation between the two structures is difficult to do with CT appearance. An example is the presence of pectinate muscles in structures of small size. Therefore, the possible difference in clinical significance between the accessory LA appendage and diverticulum could not be evaluated in this study. Second, we could not reveal how the size of LA out-pouching structures affected the patients because most of the LA out-pouching structures detected in our study population were very small (less than 1cm) and the largest one measured just 1.2cm in diameter. In previous reports, the thrombogenic or arrhythmogenic potential of LA out-pouching structures were initially suggested in patient cases with large accessory LA appendages or diverticula ,19,21. TIn conclusion, LA out-pouching structures are commonly seen in the population with and without stroke with similar prevalence. We found no association between the prevalence and location of LA out-pouching structures and stroke in this case-control study.


The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC-) in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs) show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1+ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the  Due to this fact, the identification of specific markers for the osteogenic progenitors from the jaw periosteum is of paramount importance for the selection of these cells from the whole heterogeneous cell population. Screenings for cell surface markers revealed that two mesenchymal stem cell markers were significantly induced during the osteogenesis of jaw periosteal cells. We were able to show that the stem cell marker CD271 (LNGFR – low affinity nerve growth factor receptor) was significantly more induced in mineralizing than in non-mineralizing periosteal cells during osteogenic induction. However, we demonstrated that this antigen does not designate the subpopulation with the highest osteogenic potential. In contrast, the cell surface antigen mesenchymal stem cell antigen-1 (MSCA-1) was shown to be significantly more induced in mineralizing compared to non-mineralizing jaw periosteal cells, both in the undifferentiated and differentiated states. Additionally, we could prove that the MSCA-1-positive subpopulation exhibits a significant higher osteogenic capacity than the respective MSCA-1-negative subpopulation; therefore, it most likely represents the osteoprogenitor fraction from the entire cell population in vitro expansion of the entire population is required before magnetic separation of the MSCA-1+ subpopulation can be performed. However, the long in vitro passaging of the cells is accompanied by undesirable increases in the occurrence of cellular senescence. Therefore, the shortening of the in vitro expansion procedure, as well as the use of serum-free culture conditions, should be achieved to ensure the success of future tissue engineering applications using cell-based constructs.In previous studies, we characterized the phenotype, osteogenic potential and features of jaw periosteal cells (JPCs) grown in two- and three-dimensional culture conditions We focused the present study on the comparison of the JPC phenotype under FCS-containing and FCS-free culture conditions and analyzed in detail the proliferation and mineralization capacities of the cells, as well as their expression of common stem cell and osteogenesis-relevant markers.2 culture flasks. For JPC expansion under FCS-containing and animal–free culture conditions, cells were cultured in DMEM/F-12  containing 10% FCS  and 1% fungicide and penicillin/streptomycin  and/or in MesenCult-XF medium  containing 1% glutamine and 1% fungicide and penicillin/streptomycin. DMEM-cultured cells were passaged using trypsin-versene EDTA , and MC-XF-cultured cells were passaged using the MesenCult-ACF dissociation kit. Furthermore, for MC-XF culture conditions, culture dishes or flasks were coated overnight with MesenCult-XF attachment substrate provided from the same company.Human jaw periosteum biopsies were obtained during routine oral and maxillofacial surgery after obtaining written informed consent. Samples from 6 donors  were included in this study in accordance with the local ethical committee . After breaking down the jaw periosteal tissue followed by a main digestion step using type XI collagenase  for 90 min, the JPCs were plated into 75 cmFor the calculation of the population doubling times, the individual constant for each fraction/patient was taken into consideration and firstly determined according the following formula:On the basis of an expected exponential cell growth, the population doubling time (t′) was calculated according following formula:The obtained values  for the population doubling times are summarized in 4 cells per well in 6-well plates) were treated with osteogenic medium  for 30 days. The medium was replaced three times per week. Untreated cells that were cultivated without any osteogenic compounds for the same period served as undifferentiated controls (co).DMEM-cultured JPCs  for the same time period as DMEM-cultured cells. In this case as well, untreated MC-XF-cultured JPCs served as undifferentiated controls (co).Additionally, 6×10The xCELLigence system  is suitable for the real-time analysis of cell proliferation, migration and invasion. Measurements are based on electric cell impedance-dependent signals (micro-electrodes are integrated onto the bottoms of culture E-plates) due to cell adherence and spreading for label-free cell analysis. The cell index (CI) was derived to represent cell status based on the measured electric cell impedance.3 cells (of passage 4–5) were plated per well into uncoated special E-plates with a total volume of 200 µl per well.For life-monitoring measurements, 2×10For the comparison of cell proliferation under both media conditions, cells were first plated in DMEM/F-12 containing 10% FCS. Approximately 2 days later , resuspended in 20 µl of 10% Gamunex  and incubated for 15 min at 4°C. After adding 100 µl of FACS buffer , the cells were incubated with specific phycoerythrin (PE)-labeled mouse anti-human CD29, CD45, CD73, CD90 , CD105 (AbD Serotec) and MSCA-1  for 15 min at 4°C. The cells were centrifuged  and washed two times with FACS buffer and resuspended in 200 µl of FACS buffer for flow cytometric analysis. The measurements were carried out with the guava easyCyte 6–2 L flow cytometer . Cells incubated with polyclonal goat anti-mouse immunoglobulins served as negative controls. For data evaluation, guavaSoft 2.2.3 (InCyte 2.2.2) software was used.Unseparated JPCs were detached from culture dishes, centrifuged  was carried out using the NucleoSpin RNA XS kit  following the manufacturer's instructions. The amount of isolated RNA was photometrically measured and quantified  and 15 ng of RNA was used for cDNA synthesis using the QuantiTect Whole Transcriptome Kit  following the manufacturer's instructions.The quantification of mRNA expression levels was performed using the real-time LightCycler System . For the PCR reactions, commercial primer kits  and DNA Master Sybr Green 1 (Roche) were used. The amplification was performed with 35 cycles. The transcript levels were normalized to those of the housekeeping gene glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), and the induction factors of the osteogenic induced samples in relation to those of untreated samples were calculated.The data are expressed as the mean ± standard deviation. For the statistical analysis, Student's t-test was used. A p-value <0.05 was considered significant.For the explicit comparison of both culture conditions (DMEM/F-12 and MC-XF), cell morphology was first analyzed by light microscopy. Compared to DMEM-cultured JPCs that showed an elongate and fibroblastoid shape, MC-XF-cultured cells significantly diminished their cell size. Additionally, we detected an earlier formation of calcium phosphate precipitates in MC-XF-cultured JPCs at day 12 of osteogenesis. Furthermore, the cell population seemed to be more homogenous, due to appearance of nodules derived from almost each cell of the entire monolayer. In contrast, the mineral deposition of DMEM-cultured cells originated from individual cells; these mineralization centers increased continuously.The examination of the population doubling times revealed higher proliferation rates of MC-XF- in comparison to DMEM-cultured JPCs as determined for four passages (passage 4–7). An overview of the calculated population doubling times (PDT) at indicated passages is given in Cell proliferation was analyzed by life-monitoring measurements using the xCELLigence system (ACEA Biosciences). In the first step, the cell impedance of unseparated JPCs derived from 2 patients was analyzed, as shown in positive fraction was favored, whereas the proliferation of the MSCA-1negative fraction was rather abolished. We reproduced these results in 3 independent experiments with MSCA-1-separated cells derived from 3 different donors.In successive experiments, unseparated and MSCA-1-separated JPCs were life-monitored using the same system . Serial + cells, we performed the experiments exactly the same as for the life-monitoring measurements by xCELLigence and plated 4×104 cells  per well of an uncoated 6-well plate in DMEM medium. Afterwards, parallel experiments were conducted. For the first series, cells were maintained in DMEM medium, whereas the second series underwent stepwise FCS reduction and convertion to MC-XF medium. At the same time points , cells were detached from the dishes and the percentages of MSCA-1+ cells were determined by FACS analysis as shown in In our experience, the selection of the MSCA-1+ cells takes place within the first 10 days after FCS reduction and subsequent conversion from DMEM to MC-XF medium. For the exact analysis of the percentages of MSCA-1+ cells was detected at day 5 under MC-XF culture conditions. An overview of the percentages of MSCA-1+ cells under both culture conditions is given in We obtained significant higher percentages of MSCA-1+ cells under MC-XF in comparison to DMEM culturing conditions at day 5  and at day 9  after conversion to MC-XF medium. The same tendency was shown at day 2 without reaching significant values . Therefore, maximal cell selection with significant higher (on average 35.5%) percentages of MSCA-12 culture flasks and cultured under both medium conditions for flow cytometric analysis. After reaching 80–90% confluence, the cells were detached from the culture flasks and labeled with specific PE-conjugated antibodies against human CD29, CD73, CD90, CD105, CD166 and CD45. As illustrated in Cells of passages 5–6 were plated in 75 cmWe observed that the mineralization in MC-XF-cultured mineralizing JPCs (of passage 5–6) occurs earlier  under DMEM culture conditions, instead showing mineralization potential under MC-XF cultivation. Representative fluorescent stainings are shown in The second particularly interesting observation was that JPCs which were not able to mineralize Analysis of the expression of different osteogenesis relevant genes showed only slight differences between DMEM- and MC-XF-cultured unseparated JPCs at day 5 and 10 of osteogenic differentiation . AnalysiThe treatment of congenital or acquired bone defects is an aspect of major importance in oral and maxillofacial surgery and often subject to significant morbidity following free or microvascular tissue transfer. The aim of our research work is to help patients using bone regenerative techniques. For this purpose, we decided to work with jaw periosteal cells based on the fact that we are convinced that the stem cell niche has an important influence in cell fate determination and genomic imprinting of these stem cells in vitro cell expansion procedure. Additionally, with regard to possible disease transmissions or immunological reactions towards xenogeneic compounds, clinical applications should be conducted using mesenchymal stem cells cultured under standardized xenogeneic-free conditions. The immunosuppressive potential of MSCs is well-documented An important and necessary step in the context of transplantation would be the shortening of the Sotiropoulou and co-authors examined in an extensive study the comparison of different media and plating densities on the phenotype of bone marrow mesenchymal stem cells (BMMSCs). However, the tested media were all supplemented with 10% FCS For the large production of clinical grade mesenchymal stem cells, the establishment of cell culture methods using chemically defined materials need to be developed + subpopulation out of the entire heterogeneous cell population derived from the jaw periosteum.In the present study, we compared for the first time the JPC phenotype under animal-containing and animal-free culture conditions and made the interesting observation that the MC-XF culture medium promotes the growth and, most likely, the selection of the MSCA-1+ cell fraction most likely represents the osteogenic progenitors within the entire population isolated from jaw periosteum. In addition to the fact that this fraction is a rare subpopulation (up to 5%), its magnetic separation results in poor cell survival/high mortality rates, indicating that only limited cell numbers could be available for prospective clinical applications using autologous cells. In vitro cell culturing in a xeno-free medium which would be able to select progenitor cells from a heterogeneous population could be a promising solution for successful tissue engineering approaches based on sufficient yields of stem cells.In a previous study from our laboratory, we were able to show that the MSCA-1The results obtained by life-monitoring measurements of unseparated JPCs revealed a significant decrease of cell impedance after the addition of the MC-XF culture medium due to the significant reduction of cell size. This observation was expected because of the absence of adhesion molecules normally contained in calf serum. However, the recovery of cell proliferation rates and, moreover, the cross-over of the proliferation curves obtained by DMEM-cultured cells indicate a much higher proliferative capacity of JPCs cultivated under xenogeneic-free culture conditions. We could confirm these results by the determination of PDT of unseparated JPCs of passage 4–7 growing under both culture conditions, as illustrated in The here reported reduction in expression of the cell adhesion mediating surface markers CD29, CD90 and CD105 in MC-XF-cultured JPCs are consistent with those data published elsewhere based on comparing expression patterns of BMMSCs cultivated under FCS-containing and FCS-free conditions To clarify any doubts concerning the specificity of the von Kossa and Alizarin stains, we chose a more specific fluorescent staining of hydroxyapatite, the mechanism of which has not been revealed by the manufacturer. In general, by using OsteoImage, we detected an earlier mineralization capacity of MC-XF-cultured cells (+ cells (+ cell fraction proliferation and a rather restrictive tendency with regard to the negative cell fraction. These results coincide with our experiences during cell cultivation in dishes/flasks coated with the attachment substrate (data not shown). We were not able to coat the E-plates for better cell adherence because signal transmission of the cell impedance was disturbed (data not shown). Therefore, we decided to conduct life-monitoring experiments with JPCs uniformly cultured in DMEM and to split them subsequently into DMEM- and MC-XF-cultured parallel test runs. As mentioned previously, the obtained results did not depend on the coating procedure. Furthermore, FACS analyses performed immediately after the conversion from DMEM to MC-XF cell cultivation clearly reveal a MSCA-1+ cell selection during the first 5–9 days, as summarized in Nodule formation emanates from almost every cell in the XF-cultivated monolayer. In contrast, the calcification potential of DMEM-cultured cells emanates only from a few cells of the entire cell population; these mineralization centers serve as aggregation points for further hydroxyapatite deposition. This observation indicates that cell cultivation using the MC-XF medium results in the emergence of a purer cell population, a fact that was confirmed by the flow cytometric analyses of unseparated JPCs for the determination of the percentages of MSCA-1+ cells  and the + cells . For botThe obtained data from the gene expression analysis do not allow a precise statement due to the fact that JPCs growing under different conditions reach the different stages of osteogenesis at different time points. However, the significant higher induction of Runx-2 mRNA levels (at day 5 of osteogenesis) as well as of type I collagen (at day 10 of osteogenesis) and osteoprotegerin  expression levels  in MC-XFin vivo in a suitable animal model are required. However, the clinical use of xeno-free MC-XF-cultured cells is limited due to the fact that this medium is still not in accordance with GMP-relevant qualifications.Taken together, JPCs expanded in MC-XF proliferated faster, and nodule formation occurred earlier, originating from almost every cell of the monolayer; however, the degree of calcification seemed to be lower than in DMEM-cultivated cells. Interestingly, non-mineralizing JPCs showed mineralization capacity under MC-XF but not under DMEM culture conditions. Further studies should clarify whether JPC mineralization under MC-XF culture conditions is sufficient for successful tissue engineering applications. Therefore, analyses under three-dimensional culture conditions as well as in vitro passaging of JPCs and thus speed up the in vitro culture procedures in this stem cell type.Of even greater concern to us is the fact that JPC cultivation in the MC-XF medium leads to the preferential proliferation of the subpopulation with the highest osteogenic potential. This would prevent the stressful cell magnetic separation and the prolonged


Hepatitis C virus (HCV) is one of the viruses known to cause hepatic cancer. HCV is also believed to be involved in malignant lymphoma. In this paper, we investigated characteristics of malignant lymphoma cases that were anti-HCV antibody (HCV-Ab) positive. We were able to perform pathological examinations on 13 out of 14 HCV-positive cases. Of these, lymphoid tissues of 10 stained positive for HCV-Ab. There was no significant correlation between the degree of HCV staining and the rate of recurrence or resistance to treatment. However, there did appear to be a consistent decrease in the amount of HCV-RNA between pre- and posttreatment among HCV-Ab-positive cases; that is, treatment-resistant cases that exhibited resistance from the first treatment and recurrent cases more frequently had a higher HCV level at treatment termination compared to the pretreatment level. This suggests that the HCV virus either accelerates oncogenesis by direct interaction with B cells or indirectly affects lymphoma prognosis. Since the discovery of hepatitis C virus (HCV) in 1989, the causal relationship between HCV and hepatic cancer has been proven through various case control studies, and it has been suggested that the risk of hepatic cancer in the HCV-infected population is 23–35-times higher than in uninfected healthy individuals  whereas Analysis of HCV-Ab-positive patients was conducted between October 2002 and December 2010, during the period that rituximab was marketed and available in Japan. One hundred eighty cases of B lymphocyte non-Hodgkin's lymphoma (NHL) were used as a control group and were treated around the same period of time. The HCV-Ab positive group consisted of 14 patients between 43 and 90 years old with a median age of 72 years. These cases included 12 with diffuse large B cell lymphoma, 1 with mantle cell lymphoma, and 1 with EB virus-related lymphoproliferative disorder. Eight cases were of grades III and IV, and no gender differences were observed. Eight cases (57%) were either treatment resistant at the time of first treatment or were recurrent cases. The median age of the control group was 66 years and the male/female ratio was approximately the same. However, the incidence of diffuse large B cell lymphoma in this group was 60% and was seen to be less than the anti-HCV-Ab positive group. There were seventy-one cases (31%) with first treatment resistance or recurrence in the control group, and the ratio was less than that seen in the anti-HCV-Ab positive group . Specimens consisted of lymph nodes (7 cases), stomach (2), tonsil (1), rectum (1), soft tissue (1), and orbit (1). Specimens were fixed in 10% neutral-buffered formalin, embedded in paraffin. Diagnosis for malignant lymphoma was made by HE and immunohistochemical staining.  To evaluate HCV positivity of lymphoma cells, immunohistochemical staining was performed using monoclonal antibodies to hepatitis C virus (NS3) NCL-HCV-NS3 . HCV-specific reactions were detected with commercially available Histofine simple stain . Immunoreactivity was graded as negative, weakly positive, and strongly positive Figures .Survival and progression-free survival (PFS) probabilities were estimated with the Kaplan-Meier Method. Overall survival (OS) was calculated from the date of diagnosis to the date of a patient's death for any reason or to last followup. PFS and relapse were determined with cumulative incidence estimates, treating relapse and death as competing risk events. All time-to-event endpoints were censored at the time of last contact. Immunohistopathological analysis of anti-HCV-Ab positive lymphoma cells revealed 5 strongly positive, 5 weakly positive, and 3 negative cases. These observations indicate that the growth and development of lymphoma cells may be associated with the presence of HCV. The level of HCV staining was also analyzed to determine if a correlation to treatment resistance or recurrence potential existed. Although the number of cases was too limited to obtain a clear conclusion, 3 out of 5 strongly positive cases were also treatment resistant or recurrent cases. Among 5 weakly positive cases, 3 were treatment resistant or recurrent cases. There were only 3 negative cases, 2 of which were treatment-resistant or recurrent cases. It would therefore be difficult to predict treatment-resistance or recurrence based on immunoreactivity. These results indicate that HCV directly interacts with B lymphocytes and causes lymphoma, however, the outcome cannot be predicted.One patient with grade I disease who had undergone surgery was excluded from the study and the remaining 13 cases were analyzed. Among these, 11 had liver disorder and 2 exceeded Grade III. Increased bilirubin was also seen in 2 cases. HCV-RNA level increased in 10 cases during the treatment period but only 9 of them appeared to cause hepatic toxicity. In 5 cases, HCV-RNA transiently decreased after hepatic toxicity but never become undetectable. The level of HCV-RNA prior to or just before treatment was compared with that on the last day of visit (including the treatment period). A decrease in HCV-RNA was considered to have occurred when the ratio of HCV-RNA on the last visit/pre-treatment HCV-RNA was less than 1.0. Various reports have suggested an association between HCV and malignant tumors, particularly hepatic cancer. A correlation between HCV and B cell lymphoma was also reported recently –12. We aIt was not possible to perform statistical analyses in this study due to the small number of anti HCV-Ab positive cases. Nevertheless, the overall survival data suggested a poorer prognosis in the anti HCV-Ab positive group compared to the HCV-Ab negative B cell lymphoma group. Previous reports have been inconsistent, however. One study reported no change in prognosis, including recurrence rate. Other studies reported a reduction in progression free survival, but no effect on overall survival. In additional studies, a reduction in both progression free survival and overall survival was reported –19. In oWe also compared the characteristics of B cell lymphoma patients to anti-HCV-Ab positivity with regard to the level of HCV-RNA. An increase in HCV-RNA was observed in 10 anti-HCV-Ab positive cases. Among these, 8 cases had a higher level of HCV-RNA at the completion of treatment compared to the level at initial visit (pre-treatment HCV-RNA level). As reported by Ennishi et al., although HCV viral load increased upon treatment of the lymphoma, in a majority of cases, the amount of virus decreased over 1–6 months following treatment. This indicates that, although few in number, cases occurred in which the amount of virus did not decrease to the pre-treatment level . We alsoHepatic toxicity due to hepatitis B virus reactivation during rituximab treatment has been well recognized , and theIn conclusion, although a firm relationship between HCV and B cell lymphoma remains to be proven, there appears to be a definite correlation between the two. Since survival of anti-HCV-Ab positive lymphoma patients is not necessarily higher than overall survival in anti-HCV-Ab negative cases (it can in fact be lower), it may be possible to improve survival through the use of interferon treatment. The frequency is not low, and the use of rituximab may cause further severe hepatic toxicity. Recurrence and other problems may decrease if HCV-RNA level consistently declines and chemotherapy is applied as scheduled. However, this approach is not practical and severe hepatic toxicity caused by HCV reactivation should be avoided. In order to prevent HCV reactivation leading to hepatic toxicity, methods to identify risk factors which can be easily used at clinical sites are needed. Various questions remain to be answered such as (1) whether concomitant ribavirin or interferon should be used for current therapy, and if so, (2) whether they should be used from the beginning of treatment or at the end of existing treatment. There are no reports on chemotherapy with concurrent use of rituximab. Future clinical studies are needed to clarify these questions.