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After HSV-1 infection, macrophages infiltrate early into the cornea, where they play an important role in HSV-1 infection. Macrophages are divided into M1 or M2 groups based on their activation. M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Macrophage phenotypes can shift between M1 or M2 in vitro and in vivo following treatment with specific cytokines. In this study we looked at the effect of M2 macrophages on HSV-1 infectivity using mice either lacking M2 (M2-/-) or overexpressing M2 (M2-OE) macrophages. While presence or absence of M2 macrophages had no effect on eye disease, we found that over expression of M2 macrophages was associated with increased phagocytosis, increased primary virus replication, increased latency, and increased expression of pro- and anti-inflammatory cytokines. In contrast, in mice lacking M2 macrophages following infection phagocytosis, replication, latency, and cytokine expression were similar to wild type mice. Our results suggest that enhanced M2 responses lead to higher phagocytosis, which affected both primary and latent infection but not reactivation., Author summary Macrophages are one of the major infiltrates into the cornea of ocularly infected mice and may contribute to both disease and protection. Similar to TH1/TH2 T cells, macrophages also are divided into M1/M2 subtypes. However, very little is known about the role, if any, that M1/M2 macrophages play in HSV-1 primary and latent infection. Here we investigated the role of M1/M2 macrophages in HSV-1 latency-reactivation by generating M2-/- mice as well as conditional transgenic mice that overexpress M2 macrophages. We found that higher expression of M2 macrophages correlated with higher phagocytosis, higher primary virus replication and latency, and higher IL-4 and IFN-γ expression, while the absence of M2 macrophages did not significantly alter HSV-1 infectivity compared with wild type mice. Thus, maintaining a balanced proportion of M1/M2 macrophage phenotype may be an effective way to control both primary and latent infection.

Background Macrophages are highly plastic cells that play an important role in the pathogenesis of cardiovascular disease. Objectives This study investigated the role of GATA3-positive macrophages in modulating cardiac function after myocardial infarction (MI) or in response to pressure overload hypertrophy. Methods Myeloid-specific GATA3-deficient (mGATA3KO) mice were generated, MI or pressure overload was induced, and cardiac function was determined by echocardiography. GATA3-sufficient Cre mice were used as a control. Immunohistochemical staining, flow cytometry, MILLIPLEX Mouse Cytokine/Chemokine Assay, cultured macrophages, quantitative real-time polymerase chain reaction, and western blot were used to determine the role of GATA3 in macrophages. Results GATA3-positive macrophages rapidly accumulated in the infarcted region of the myocardium after acute MI. Deficiency of GATA3-positive macrophages led to a significant improvement of cardiac function in response to acute MI or pressure overload hypertrophy compared with the control mice. This improvement was associated with the presence of a large number of proinflammatory Ly6Chi monocytes/macrophages and fewer reparative Ly6Clo macrophages in the myocardium of mGATA3KO mice compared with control mice. Analysis of serum proteins from the 2 mouse genotypes revealed no major changes in the profile of serum growth factors and cytokines between the 2 mice genotypes before and after MI. GATA3 was found to be specifically and transiently induced by interleukin 4 in cultured macrophages through activity of the proximal promoter, whereas the distal promoter remained silent. In addition, the absence of GATA3 in macrophages markedly attenuated arginase-1 expression in cultured macrophages. Conclusions We demonstrated that the presence of GATA3-positive macrophages adversely affects remodeling of the myocardium in response to ischemia or pressure overload, whereas the absence of these macrophages led to a significant improvement in cardiac function. Targeting of signaling pathways that lead to the expression of GATA3 in macrophages may have favorable cardiac outcomes.

Background and aims Inflammation, particularly innate immunity, plays an important role in cardiovascular diseases. The aim of this study was to investigate whether atherogenic determinants such as oxidized LDL modulate the phenotype of eosinophils. Methods Cultured eosinophils were treated with oxidized LDL and the expression of selective inflammatory and anti-inflammatory cytokines was determined. In addition, the eosinophil receptor and signaling that mediate these events were identified. Results Treatment of cultured eosinophils with oxidized LDL (Ox-LDL) specifically induced the expression of IFNα and IFNβ without affecting expression of other proinflammatory cytokines, such as TNFα, IL-1β, and IL-6. In macrophages, Ox-LDL downregulated expression of both IFNα and IFNβ, suggesting that the effect of Ox-LDL on the expression of type I interferons is specific to eosinophils. Furthermore, we noted that eosinophils constitutively expressed IL-4 and IL-13, and Ox-LDL markedly downregulated their expression. Analysis of Ox-LDL signaling revealed that eosinophils constitutively expressed SRB2, CD36, and CD68 scavenger receptors, and Ox-LDL markedly induced the expression of CD36. Further analysis of CD36 signaling by siRNA and neutralizing antibodies showed that the induction of type I IFN by Ox-LDL is mediated by CD36 signaling whereas downregulation of IL-4 is independent of CD36 activation. We further showed that peritoneal macrophages treated with condition medium collected from Ox-LDL treated eosinophils markedly induced the expression of M1 markers such as iNOS, IL6, SOSC3 and TNFα whereas the condition medium from non-treated eosinophils significantly induced expression of M2 markers like ARG1 and CCL24. Conclusions Our data suggest that an atherogenic condition could activate eosinophils and modulate the phenotype of macrophages (from M2 to M1 phenotype), in part, through the CD36 receptor signaling.

Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice.

The Nerium indicum Mill organs and parts of the 40 genotypes were sampled from 5 habitats of southand south east of Iran. In total, 15 morphological and pigmentvariables were measured. Analysis of variance was carried out based on completely randomized design and revealed significance differencesat p ≤ 0.05, 0.01 and 0.001 for the most variablesindicating a large-scale diversity among the genotypes. Cluster analysis divided the genotypes into 3 main groups. The first and second principal components had 34.73% and 18.67% of the variance, respectively. The main factors had 76.79% accumulated eigenvalue. Random amplified polymorphic DNA (RAPD) markers used to assess the population structure and genetic variation. In total, 361 polymorphic band amplified from effective 14 chosen RAPD markers. Analysis of molecular variance (AMOVA) showed 67% and 33% within and between populations genetic variation respectively. Cluster analysis by using UPGMA method divided genotypes into 6 main groups. A high cophenetic correlation coefficient (r = 0.90) was obtained. The first and second principle coordinates had 29.31% and 25.78% of the variance, respectively.

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

Apolipoprotein A-IMilano (ApoA-IM), a naturally occurring Arg173 to Cys mutant of ApoA-I, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown superior atheroprotective effects of ApoA-IM gene compared with wild-type ApoA-I gene using transplantation of retrovirally transduced bone marrow in ApoA-I/ApoE null mice. In this study, we compared the antiatherogenic efficacy of ApoA-IM gene transfer using Recombinant adeno-associated virus (rAAV) 2 or rAAV8 as vectors in ApoA-I/ApoE null mice. Mice received a single intravenous injection of 1.2 × 1012 vector genomes of AAV2 or AAV8 vectors expressing ApoA-IM or control empty vectors (12 mice/group). Circulating levels of ApoA-IM were higher in recipients of AAV8 compared with AAV2 at 4, 12, and 20 weeks postinjection. Qualitative polymerase chain reaction analysis of RNA collected from different tissues showed that the AAV8-mediated gene transfer resulted in a more efficient transgene expression in the heart, brain, liver, lung, spleen, and kidney of the recipient mice compared with AAV2. Intravenous AAV8-ApoA-IM injection reduced atherosclerosis in the whole aorta ( P < .01), aortic sinuses ( P < .05), and brachiocephalic arteries ( P < .05) compared with the vector control, whereas there was no statistically significant reduction in atherosclerosis in mice receiving intravenous AAV2-ApoA-IM. The ApoA-IM gene was expressed in the aortic tissue of mice receiving AAV8 ApoA-IM but not in those receiving AAV2 ApoA-IM. Immunostaining showed that compared with the vector control, there was reduced macrophage content in the brachiocephalic ( P < .05) and aortic sinus plaques ( P < .05) of AAV8 ApoA-IM recipients but not in the recipients of AAV2 ApoA-IM. Thus, intravenous injection of AAV8 is more effective than intravenous injection of AAV2 in the expression of ApoA-IM gene. These data provide support for the potential feasibility of this approach for atheroprotection in humans.

To investigate the potential role of inflammatory cytokines in apo E-/- mouse in response to deletion of Tenascin-C (TNC) gene.We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E-/- mice and apo E-/- TNC-/- double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E-/- mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E-/- mouse group. Inbreeding of apo E-/- mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E-/- mice had low level of eotaxin expression, cells derived from apo E-/- TNC-/- mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E-/- TNC-/- mice markedly stimulated mast cell migration whereas plasma from the apo E-/- mice had no such effect.These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E-/- mice and that this chemokine plays a key role in the development of atherosclerosis.

Aims Tenascin-C (TNC), a matricellular protein, is up-regulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model. Methods TNC−/−/apo E−/− mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E−/− mice. Results The en face analysis of aortic area showed that the mean aortic lesion area of the double knockout (KO) mice was significantly higher than that of control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids was significantly higher. Analysis of cell adhesion molecules revealed that vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1, was up-regulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E−/− mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the tumor necrosis factor α stimulation than the cells isolated from apo E−/− mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control. Conclusions These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.

According to cultural and geographical evidence, the Old Persian and Pahlavi name of this plant is "Karkam" which has been derived from dialects of Iran. In the Median era, probably its pronunciation had similarity to the word "Karkam" or "Kurkum". Later on the word has been transformed and used according to phonetic rules of other tribes. For example in Aramaic: "Karkema", in Hebrew: "Karkom", in Arabic "Kurkum", in Sanskrit, "Kunkuma" and in Greek Crocus that Homer registered as "Krokus". The most ancient documents of its edible uses are registered by Polyen, the Greek military author, in his dissertation entitled "Stratagemes" within a list of foods consumed by the Achaemenian court. According to Pliny/Plinius' writings the kings and magi used a kind of fragrant cream of saffron. In the Seleucid era foods consumed by the Iranians were also the same as the foods used by the Achaemenian people. In the "Parian" and "Sassanian" eras saffron was amongst the food colourings which they made use of to give a good scent to the food. As deduced from the "pahlavi" text Khosrow Kawātān va redak, Sassanians had a variety of foods and pastries and made use of saffron in preparing foods, pastries and desserts which had also been used till the Islamic era. During the reign of Khosroparviz it had also been used in medicine and dyeing in addition to cooking.

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)-gamma was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN-gamma in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at -2086 to -2078 bp is essential for IFN-gamma-regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN-gamma stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN-gamma and that PTN is involved in the adaptive immunity.-Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-gamma/JAK/STAT1 signaling is critical for the expression of PTN in macrophages.

Apolipoprotein A-1 (Apo A-I) Milano, a naturally occurring Arg173 to Cys mutant of Apo A-1, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown the superior atheroprotective effects of Apo A-I Milano (Apo A-IM) gene compared to wild-type Apo A-I gene using transplantation of retrovirally transduced bone marrow in Apo A-I/Apo E null mice. In this study, we compared the effect of dietary lipid lowering versus lipid lowering plus Apo A-IM gene transfer using recombinant adeno-associated virus (rAAV) 8 as vectors on atherosclerosis regression in Apo A-I/Apo E null mice. All mice were fed a high-cholesterol diet from age of 6 weeks until week 20, and at 20 weeks, 10 mice were euthanized to determine the extent of atherosclerosis. After 20 weeks, an additional 20 mice were placed on either a low-cholesterol diet plus empty rAAV (n = 10) to serve as controls or low-cholesterol diet plus 1 single intravenous injection of 1.2 × 1012 vector genomes of adeno-associated virus (AAV) 8 vectors expressing Apo A-IM (n = 10). At the 40 week time point, intravenous AAV8 Apo A-IM recipients showed a significant regression of atherosclerosis in the whole aorta ( P < .01), aortic sinuses ( P < .05), and brachiocephalic arteries ( P < .05) compared to 20-week-old mice, whereas low-cholesterol diet plus empty vector control group showed no significant regression in lesion size. Immunostaining showed that compared to the 20-week-old mice, there was a significantly reduced macrophage content in the brachiocephalic ( P < .05) and aortic sinus plaques ( P < .05) of AAV8 Apo A-IM recipients. These data show that although dietary-mediated cholesterol lowering halts progression of atherosclerosis, it does not induce regression, whereas combination of low-cholesterol diet and AAV8 mediated Apo A-I Milano gene therapy induces rapid and significant regression of atherosclerosis in mice. These data provide support for the potential feasibility of this approach for atherosclerosis regression.

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.

Objective— Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages. Methods and Results— RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms , and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue. Conclusions— PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.

Introduction: Milliary metastasis to the brain is absolutely rare and there are few reports in the medical literature. These reports were cases with calcified and well known origin milliary metastases. Case Report: In this paper we report a 42-year-old man with milliary metastasis to cerebral and cerebellar hemispheres and brain stem from an unknown etiology without any mass effect edema or calcification. Conclusion: Pathological study of brain biopsy revealed mucinous papillary adenocarcinoma.

Cardiac nerve sprouting and sympathetic hyperinnervation after myocardial infarction (MI) both contribute to arrhythmogenesis and sudden death. However, the mechanisms responsible for nerve sprouting after MI are unclear. The expression of nerve growth factor (NGF), growth associated protein 43 (GAP43), and other nerve markers were studied at the infarcted site, the noninfarcted left ventricle free wall (LVFW), and the left stellate ganglion (LSG) at several time points (30 minutes to 1 month) after MI. Transcardiac (difference between coronary sinus and aorta) NGF levels were also assayed. Acute MI resulted in the immediate elevation of the transcardiac NGF concentration within 3.5 hours after MI, followed by the upregulation of cardiac NGF and GAP43 expression, which was earlier and more pronounced at the infarcted site than the noninfarcted LVFW. However, cardiac nerve sprouting and sympathetic hyperinnervation were more pronounced in the noninfarcted than the infarcted LVFW site and peaked at 1 week after MI. The NGF and GAP43 protein levels significantly increased in the LSG from 3 days ( P

Introduction MRL is a strain of mice with the unusual ability to recover from injuries without forming scar tissue. It was recently reported that the MRL mice can recover with little or no scar formation following a cryogenically induced myocardial infarction of the right ventricle. These findings suggest that scarless recovery from myocardial injury is possible in mammalian ventricles. If applicable to humans, these results could have significant clinical implications in the management of patients with myocardial infarction. However, myocardial infarction in human patients usually results from ischemic injury instead of cryoinjury. Myocardial infarction usually occurs in the left ventricle. Methods We performed a prospective study creating left ventricular myocardial infarction in 32 MRL mice using coronary ligation. Results Among them, 21 mice survived surgery and had myocardial infarction documented by delayed contrast-enhanced magnetic resonance imaging (MRI) 35±10 days after surgery. All 21 mice had evidence of myocardial infarction in follow-up MRI performed 116±21 days after surgery. The infarct size was 27±12% of the area of visualized myocardium during the first MRI study and was 29±11% during the second MRI study ( P =NS). Large myocardial infarction with aneurysm formation was documented by histological examinations in all 21 mice. The ventricular aneurysm showed transmural infarction with replacement fibrosis and mummified necrotic myocardium surrounded by collagenous scar tissue. Occasional surviving myocytes are present within the aneurysm. Conclusion Ischemic myocardial injury in MRL mice is not followed by scarless recovery.

Migration of smooth muscle cells (SMCs) across the internal elastic lamina is a key step in the development of atherosclerotic or restenotic plaques. Cell movement is a complex and highly dynamic phenomenon, involving the continuous formation and breakage of attachments with the underlying substratum. Tenascin-C (Tn-C), a counter-adhesive extracellular matrix protein, is comprised of several isoforms with distinct biological activities. Neither the structure nor function of these isoforms in SMCs has been defined. We have used primers and RT-PCR to fully identify Tn-C isoforms expressed by SMCs. Cloning and sequence analysis of the PCR product indicated that SMCs express a Tn-C isoform with only repeats A1 and A2 of fibronectin type III repeats. Using A1A2-specific antibodies, cDNA probes and RNase mapping, we observed that the A1A2 isoform is predominantly expressed by cultured SMCs derived from aorta of newborn rats, and its expression is up-regulated by PDGF-BB. In contrast, the expression of this isoform is markedly down-regulated in the SMCs derived from adult rat aorta. Western and Northern blots of injured rat carotid arteries revealed that the A1A2-isoform is expressed in response to injury. Using cultured SMCs, we found that the recombinant A1A2 protein that was found in the newly discovered Tn-C isoform promotes SMC chemotaxis. We conclude that Tn-C isoforms are expressed in a regulated fashion in vascular system. Our findings suggest a new role of Tn-C isoforms in the remodeling of vascular wall.

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α-/-(lack functional CD8 T cells and CD8α-/-DCs), CD8β-/-(have functional CD8α-/-T cells and CD8α+DCs), and β2m-/-(lack functional CD8 T cells but have CD8α+DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+DCs) versus WT C3H (have functional CD8α T cells and CD8α+DCs) mice. We also determined whether the phenotype of CD8α-/-and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+T cells or bone marrow (BM) derived CD8α+DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences. © 2014 Mott et al.

OBJECTIVES The purpose of this study was to determine the temporospatial expression of tenascin-C (TnC) in balloon-injured rat and porcine arteries. BACKGROUND Recent studies suggest that cell migration, in addition to cell proliferation, is a critical component of neointima formation after vascular injury. We have previously shown that adventitial myofibroblasts synthesize growth factors that contribute to the formation of neointima after arterial injury. We have also shown that the extracellular matrix protein, TnC, regulates cell migration. Consequently, we investigated the temporospatial expression of TnC by myofibroblasts after vascular injury. METHODS In situ hybridization and immunohistochemistry were used to investigate the temporospatial expression of TnC in injured arteries. Northern and Western blots were used to determine the in vitro expression of TnC. RESULTS In situ hybridization revealed that the major site of TnC expression early after vascular injury was the adventitial myofibroblasts. Immunohistochemical staining demonstrated that TnC expression began in adventitial myofibroblasts three days after injury. Tenascin-C expression, however, did not persist in this region. Rather, it moved progressively across the vascular wall toward the luminal surface. By one week, TnC expression reached the developing neointima. In vitro, myofibroblasts did not express TnC mRNA under basal conditions. In contrast, angiotensin II and PDGF-BB, factors that have been implicated in remodeling of balloon-injured arteries, markedly upregulated TnC mRNA. CONCLUSIONS Tenascin-C is expressed in response to balloon injury. Tenascin-C expression begins with adventitial myofibroblasts. Over a period of 7 to 14 days, expression moves progressively across the vessel wall to the neointima. We hypothesize that adventitial myofibroblasts are actively involved in the formation of neointima and that TnC facilitates migration of these cells during adventitial remodeling.

OBJECTIVES This study was performed to test the hypothesis that tenascin-C (TN-C), an extracellular matrix (ECM) protein with counteradhesive chemotactic and vascular growth-promoting effects, is expressed in “arterialized” human saphenous vein grafts (SVGs). BACKGROUND Tenascin-C is expressed in the vessel wall after vascular injury in the experimental model, where it has been implicated in the formation of neointima. Overexpression of TN-C has also been implicated in the development and progression of pulmonary hypertension. Saphenous vein grafts are exposed to hemodynamic stress when interposed in the arterial circulation and mechanical stress upregulates expression of TN-C, whereas stress-relaxation suppresses its synthesis. We hypothesized that the hemodynamic stress of increased arterial pressure could also induce TN-C expression in SVG. METHODS We examined the expression of TN-C protein and mRNA in normal vein and “arterialized” human SVG using immunohistochemistry and in situ hybridization, respectively. RESULTS TN-C protein was not detected in control human saphenous veins; however, it was uniformly and strongly expressed in the adventitia and media of patent human vein grafts, with minimal or no expression in the neointima (n = 27, 100%). In situ hybridization showed that TN-C mRNA was not detected in the neointima, but was strongly upregulated in the adventitia and media, corroborating immunostaining data (n = 10, 100%). Unlike patent SVG, TN-C was not expressed in the adventitia of occluded grafts, except for a low level of expression around the newly formed vessels in neointima (n = 5, 100%). Smooth muscle cell-specific staining demonstrated that the lack of expression of TN-C in occluded vein grafts is not due to the lack of presence of smooth muscle cells in the graft. CONCLUSIONS These findings suggest that placement of a venous graft in the arterial system leads to expression of TN-C, which may in turn facilitate graft remodeling. Conversely, loss of flow and intravascular pressure, associated with vein graft occlusion, is accompanied by disappearance of TN-C expression.

Background —Tenascin is a large extracellular matrix glycoprotein generally found in adult tissues undergoing active remodeling such as healing wounds and tumors. To determine the potential role of tenascin-C (TN-C) in the pathophysiology of atherosclerosis, we investigated the pattern of expression of TN-C in human coronary atherosclerotic plaques. Methods and Results —Immunohistochemical staining and in situ hybridization demonstrated minimal and random expression of TN-C in fibrotic but lipid-poor atherosclerotic plaques. In contrast, all plaques with an organized lipid core or ruptured intimal surface strongly expressed TN-C, which was preferentially concentrated around the lipid core, shoulder regions, and ruptured area of the plaques but not in the fibrous cap. TN-C was not detected in normal arterial tissue. To identify the cellular source of TN-C, the plaques were stained with smooth muscle cell– and macrophage-specific antibodies. TN-C expression correlated with the infiltration of macrophages. Northern blot and immunoprecipitation analysis showed that macrophages expressed 7.0-kb TN-C mRNA and 220-kDa protein. Reverse transcription–polymerase chain reaction of total RNA derived from macrophages showed that they express the small isoform of TN-C. Zymogram analysis revealed that macrophages markedly increased MMP-9 expression. Conclusions —This study demonstrates that the level of TN-C expression correlates with the degree of inflammation present, not with plaque size. In addition, cultured macrophages have the capacity to express the TN-C gene. These findings suggest the significance of macrophages in the remodeling of atherosclerotic plaque matrix composition.

The extracellular matrix protein tenascin-C is a multidomain protein that regulates cell adhesion. We used two different smooth muscle cell subtypes derived from adult and newborn rat aorta to investigate the interaction of tenascin-C or its various domains with these cells using an adhesion assay. Newborn cells were three times more adherent to tenascin-C than adult cells. Tenascin C-adhering cells remained round, whereas they spread rapidly on a fibronectin substrate. Adhesion assays showed the interaction between tenascin-C and newborn cells to be predominantly RGD-independent. Mg2+ increased newborn cell adhesion to tenascin-C in a concentration-dependent manner, whereas Ca2+ had no effect. To analyze the structure-function relationships of different domains of tenascin-C, we used recombinant full-length fibronectin-like and fibrinogen-like domains and various subdomains corresponding to the alternatively spliced regions of tenascin-C. The cells adhered to the fibrinogen-like domain but not to the fibronectin-like domain or its subdomains. As with the intact tenascin-C molecule, adherent cells remained round, and the Mg2+, but not Ca2+, promoted this interaction. The interaction of cells with the fibrinogen-like region was further mapped to a 30-amino acid peptide located near the carboxyl-terminal part of the tenascin-C molecule. The same 30-amino acid peptide was active in promoting cell migration. Our results provide a basis for understanding the mechanism of interaction of tenascin-C with smooth muscle cells and a framework for isolating membrane binding sites that mediate the cellular responses to this molecule.

Abstract Fibronectins (FNs) comprise a family of adhesive extracellular matrix proteins that arise by alternative splicing in three regions: V (IIICS), EIIIA (ED-A), and EIIIB (ED-B). FNs bearing the EIIIA and EIIIB segments are prevalent during embryogenesis, expressed to lesser degrees in normal adult tissues, and may be locally reexpressed at sites of adult tissue injury. RNase mapping shows that normal rat arteries express low levels of FNs that are predominantly EIIIA − and EIIIB − . Following balloon injury, arterial walls produce increased total levels of FN transcripts that preferentially include both the EIIIA and EIIIB segments. However, despite inducing increased total FN mRNA, balloon injury does not alter the relative composition of V120 + , V95 + , and V0 spliced forms. In situ hybridization reveals that as early as 4 days after injury medial cells express increased total FN mRNA, and by 7 days substantial neointimal and focal medial synthesis of EIIIA + , EIIIB + , and V120 + FNs occurs; macrophages do not significantly contribute to this observed vascular FN synthesis. Consistent with the mRNA data, immunofluorescence microscopic analysis reveals increased deposition of EIIIB + and V + FN protein forms in injured arterial walls, particularly within the neointima. Our results suggest that local synthesis of specific FN isoforms is important to the neointimal formation that ensues after balloon injury.

CSM patients caused by osteophytic ridge or intervertebral disc herniation underwent anterior decompression and fusion were prospectively enrolled. The purpose was to elucidate the effects of decompression (anterior cervical discectomy and fusion) on myelopathic cord in milder cases, younger patients, and in short duration of the disease.Forty-three patients with CSM were examined comprising Cooper and mJOA scale before and after surgery and followed for 12 months.Patients included in the study were 30 males and 13 females. The mean age of men was 50.9±13.2, and that of women was 46.8±11.8. The mean interval from symptoms onset to hospital presentation was 10.78±7.3 months. In lower limbs, among 31 with preoperative functional impairment, 25 remained unchanged, three improved and three worsened, which is not significant, and of 39 patients with preoperative upper limb functional impairments using Cooper's scale, 20 improved, 15 remained unchanged and 4 became worse (P=0.001). Recovery rate for mJOA score was 24.5±17.7%, for Cooper lower extremity score was 32.2±40.56% and for Cooper upper extremity score was 7.8±16.8%. The mean gain in mJOA was 0.84 (±0.57).Patients with short duration of symptom onset respond remarkably to decompression surgery. In patients with good condition that myelopathy has not established yet, perhaps mJOA is not a perfect and powerful scale for pre and postoperative assessment of patient.

Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E(-/-) mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06-8.08% of CD11b(+)F4/80(+) macrophages from bone marrow cells and 0.24-2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMo) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMo that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFβ1 or in BMDM treated with TGFβ1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naive CD4+ cells stimulated with TGFβ1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMo, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type.

Aim: This study tests the hypothesis that DNA intercalation and electrophilic interactions can be exploited to noncovalently assemble doxorubicin in a viral protein nanoparticle designed to target and penetrate tumor cells through ligand-directed delivery. We further test whether this new paradigm of doxorubicin targeting shows therapeutic efficacy and safety in vitro and in vivo. Materials & methods: We tested serum stability, tumor targeting and therapeutic efficacy in vitro and in vivo using biochemical, microscopy and cytotoxicity assays. Results: Self-assembly formed approximately 10-nm diameter serum-stable nanoparticles that can target and ablate HER2+ tumors at >10× lower dose compared with untargeted doxorubicin, while sparing the heart after intravenous delivery. The targeted nanoparticle tested here allows doxorubicin potency to remain unaltered during assembly, transport and release into target cells,while avoiding peripheral tissue damage and enabling lower, and thus safer, drug dose for tumor killing. Conclusion: This nanoparticle may be an improved alternative to chemical conjugates and signal-blocking antibodies for tumor-targeted treatment. Original submitted 23 January 2011; Revised submitted 22 June 2011

Endoscopic endonasal surgery (EES) is standard practice in sinonasal disease and is becoming more accepted in the performance of anterior skull base resections. We report our experience with image-guided surgery (IGS) in difficult cases of paranasal sinus (PNS) and skull base pathologies and discuss advantages and disadvantages of this technique.A retrospective chart review was performed for the period 2004-2009. Degree of PNS involvement, indication for IGS, incidence of major complications, need for revision surgery, and technical data regarding the system were gathered.Sixty-two of 86 patients were followed for at least one year and therefore included in the analysis. Indications for IGS were mostly revision surgery for polyposis (42%), chronic rhinosinusitis (CRS) of frontal and/or sphenoid sinuses (14.5%), skull base tumours (30.6%), and foreign body removal (4.8%). Revision rates after IGS in polyposis, CRS, and benign skull base tumours were 7.7%, 11.11%, and 7.1%, respectively.IGS is of particular benefit in the management of sinonasal polyposis, benign skull base tumours, palliative surgery, and foreign body removal. IGS may avoid trauma to the orbit and anterior skull base and reduces the rate of revision surgeries rendering more meticulous and complete operations possible. We also think it could be helpful for foreign body removal.

Matrix production by smooth muscle cells (SMC) appears to play a major role in the intimal thickening process. Proteoglycans (PG) are the predominant extracellular matrix component of early restenotic lesions. As angiotensin II (A II) has been proposed as a mediator of restenotic process, we hypothesized that A II may directly affect PG synthesis by SMC. SMC were cultured in the presence of [ 35 S]sulfate and angiotensin II, and both the secreted and membranebound proteoglycans were analyzed. A II (1 to 100 nM) evoked a dose- and time-dependent increase in both cell- and media-associated PG production, an effect abrogated by the A II receptor antagonist, saralasin. SMC constitutively synthesize small amounts of PG with a molecular mass of 170–250 kDa. After treatment with A II, the abundance of PG is increased, as well as its molecular mass (230–300 kDa). Selective degradation by chondroitinases and heparinase identified chondroitin and dermatan sulfate PG as the predominant form being induced. These results demonstrate that the effect of A II is not general and is specific to certain classes of PGs. In order to further examine the specificity of the A II effect, we compared the synthesis of PG induced by A II with that induced by platelet-derived growth factors AA and BB (PDGF-AA and -BB), insulin-like growth factor I (IGF-I), and tumor necrosis factor α (TNFoi). This comparison demonstrated that the profile of PG induced by A II is different from the other factors examined. Taken together, these data indicate that A II may not only function as a hypertrophic factor for SMC, but in addition may also be a potent modulator of specific PG synthesis by these same cells, which could significantly contribute to the formation of atherosclerotic and restenotic lesions.

To understand the alteration of extracellular matrix composition evoked by chemotactic factors, we have studied the expression of adhesive (fibronectin) and anti-adhesive (tenascin) proteins in response to platelet-derived growth factor-BB (PDGF-BB), a potent chemoattractant for rat aortic smooth muscle cells (ASMC). PDGF-BB markedly induced two major tenascin mRNA transcripts, whereas fibronectin mRNA levels did not change. The results of immunoprecipitation studies paralleled Northern blot data. Since alternative splicing is responsible for the generation of multiple tenascin mRNAs in other cell types, we studied the effect of chemotactic factors on the relative abundance of tenascin isoforms. The alternatively spliced region of ASMC-derived rat tenascin was amplified and the identity of the products confirmed by sequencing. Three major polymerase chain reaction products were detected: a 1727-base pair unspliced form which was maximal at 2 h and 635- and 362-base pair products which were more abundant at 8 h after treatment with PDGF-BB or angiotensin II. Functional studies showed that the unspliced isoform of human tenascin inhibited attachment of both human and rat ASMC to fibronectin. These results suggest that PDGF-BB markedly up-regulates the expression of tenascin variants, which may lead to destabilization of cell-matrix interactions and promotion of cell migration.

Insulin-like growth factor (IGF)-I is markedly induced after balloon injury in the rat aorta, where it may serve to mediate vascular repair. Because the bioavailability of IGF-I is modulated by IGF-binding proteins (IGFBPs), we examined the regulation of IGFBPs by IGFs in primary cultures of rat aortic smooth muscle cells (SMCs). Serum-deprived SMC-conditioned medium contains IGFBPs of 38 to 45 kD (only in confluent cultures), 30 kD (possibly IGFBP-2), 28 kD, and 24 kD (IGFBP-4), the latter being the most abundant. IGF-I and IGF-II but not insulin evoked a marked decrease of IGFBP-4 as early as 4 hours after treatment. IGFBP-4 mRNA abundance, however, was entirely unaffected by IGF-I for up to 48 hours. IGF-I analogues with high affinity for the IGF-I receptor and weak affinity for IGFBP paradoxically evoked a small increase in IGFBP-4, probably through a general increase in protein synthesis. IGF-I only minimally decreased IGFBP-4 content in medium of sparse cultures, whereas it completely abolished IGFBP-4 content in conditioned medium of superconfluent SMCs. IGF-I also evoked a concentration-dependent increase in the abundance of IGFBP-3 in confluent, but not sparse, SMCs without affecting IGFBP-3 mRNA. Addition of IGF-I to cell-free medium conditioned by confluent, but not by sparsely cultured, SMCs led to rapid degradation of IGFBP-4. Interestingly, IGFBP-4 mRNA was markedly induced in confluent relative to sparsely grown SMCs in an IGF-I independent fashion. Thus, both biosynthesis and IGF-dependent proteolysis of IGFBP-4 are increased in confluent SMCs. Proteolysis was maximal at 37 degrees C and was abrogated by EDTA and by benzamidine. Phenylmethylsulfonyl fluoride and the plasmin inhibitor bdellin had minor inhibitory activity, whereas aprotinin, angiotensin-converting enzyme inhibitors, and N-ethylmaleimide were without effect. The protease does not affect the structure of IGF-I as determined by reverse-phase high-performance liquid chromatography and size-exclusion chromatography of 125I-IGF-I incubated for up to 24 hours with SMC-conditioned medium containing IGFBP-4. In summary, SMCs elaborate a cation-dependent protease in a confluence-dependent fashion, which degrades bound IGFBP-4 and likely releases free structurally intact IGF-I, presumably to interact with the cell surface receptor and/or other IGFBPs.

Simultaneous intradural, extradural, vertebral and paravetebral invasion of hydatid cyst, pathologic fracture, and multiple vertebral involvement are all rare encountered conditions in echinococcal infestation. A 48-year-old man who had experienced a falling down trauma, 10 years ago, and at that time, because of L1 burst fracture, undergone on Harrington rod placement, admitted in our ward for newly started urinary retention and mild paresis of lower extremities. With imaging analysis and during surgery, we discovered the extension of echinococcal spinal infestation extra- and intradurally after a decade of extinction. We performed a double stage circumferential reconstruction and adjuvant long term chemotherapy. We closely monitor our patient neurologically and radiologically and believe that aggressive surgical treatment and sustained cyclical albendazole therapy can increase the quality of life and life expectancy.

Parathyroid hormone-related protein (PTHrP), a tumor product responsible for malignancy-associated hypercalcemia, is also produced in many normal tissues, including vascular smooth muscle cells (SMC). As PTHrP exhibits vasodilatory properties, we postulated that other vasoactive agents may control PTHrP gene expression in SMC. Addition of angiotensin II to serum-deprived SMC resulted in a marked induction of PTHrP mRNA by 2 h, with a peak (6-10-fold) at 4-6 h. Angiotensin II effects on PTHrP gene expression were inhibited by saralasin, an angiotensin II receptor antagonist, and blocked by actinomycin D and cycloheximide, suggesting a requirement for gene transcription and protein synthesis. Nuclear run-off assays revealed a 3-fold increase in PTHrP gene transcription 1 h after angiotensin II treatment. Angiotensin II also prolonged PTHrP mRNA half-life by 2-3-fold. Angiotensin-induced PTHrP mRNA is partially dependent on cyclooxygenase products and protein kinase C activation. Other vasoconstrictor substances, including serotonin and bradykinin, also stimulated PTHrP expression, whereas the vasodilator atrial natriuretic peptide did not. Addition of recombinant PTHrP-(1-141) significantly inhibited angiotensin II-induced SMC DNA synthesis. PTHrP expression is increased by angiotensin II through transcriptional and post-transcriptional mechanisms. In addition, PTHrP modulates the effect of angiotensin II on SMC proliferation. This suggests that PTHrP acts locally in SMC, possibly to oppose the vasoactive and/or growth-promoting effects of vasoconstrictor agents such as angiotensin II.

Angiotensin II, a vasoactive peptide, has been implicated in the pathophysiology of a number of vascular wall abnormalities. Since aberrant extracellular matrix deposition contributes to the pathogenesis of vessel wall disease, we examined the potential involvement of angiotensin II in the regulation of extracellular matrix synthesis by vascular smooth muscle cells. Immunoprecipitation of newly synthesized matrix proteins showed that, under serum-free conditions, cultured vascular smooth muscle cells constitutively produced high levels of fibronectin, small amounts of laminin, and a barely detectable amount of tenascin. Angiotensin II treatment increased synthesis of a 230-kDa tenascin glycoprotein by 9-fold and fibronectin synthesis by only 30-40% during a 24-h treatment period, without stimulating laminin production or a general increase in the synthesis of secreted proteins. Concomitant treatment with saralasin, a competitive inhibitor of angiotensin II, prevented the stimulation observed with angiotensin II. The stimulation of immunoprecipitable tenascin was preceded by an increase in tenascin mRNA. Levels of tenascin transcripts (8.4 and 7.0 kilobase) were significantly increased within 2 h after angiotensin II treatment, reached a maximum (10- to 12-fold) by 4 h, and remained elevated after 18 h. The induction was completely blocked by actinomycin D. Serum also induced tenascin mRNA, but with a different time course. Serum induction of tenascin mRNA was also evident at 2 h, maximal at 4 h, but declined to control levels at 8 h. These results indicate that angiotensin II exerts a rapid and selective stimulation of tenascin biosynthesis, at least in part at a transcriptional level. This suggests that angiotensin II may alter the composition of the extracellular matrix of the vessel wall by stimulating synthesis of the antiadhesive protein tenascin.

Understanding cellular and molecular mechanisms that contribute to plaque instability and plaque rupture will continue to provide novel insights into prevention of atherosclerosis, plaque rupture, and thrombosis. Ruptured plaques and, by inference, plaques at risk for rupture (vulnerable or unstable plaques) generally contain a large acellular lipid core with a reduced collagen content and fewer collagen synthesizing smooth muscle cells, a thinned out fibrous cap, intimal and adventitial inflammation with destruction of internal and external elastic membranes, enhanced plaque neovascularity, and outward remodeling of the vessel wall. Coronary culprit lesions responsible for 60–70% of acute coronary syndromes evolve from only mildly or moderately stenotic lesions suggesting that such less obstructive lesions may be more prone to plaque rupture and thrombosis. Inflammation is implicated not only in the initiation and progression of atherosclerosis but also appears to play a critical role in promoting plaque vulnerability, plaque rupture, and eventual thrombosis through its effect on matrix turnover and release of thrombogenic mediators.

Background: We investigated the efficacy of directly injected allogenic bone marrow-derived mesenchymal stem cells in improving left ventricular function in a porcine model of myocardial infarction. Methods: Left ventricular infarction was created in 16 adult Yorkshire pigs by coil embolization and thrombotic occlusion distal to the second diagonal artery. One month after myocardial infarction was induced, the animals were randomized to either direct injection of allogenic mesenchymal stem cells or sham treatment (culture medium). Allogenic bromodeoxyuridine-labeled mesenchymal stem cells (2 ± 0.1 × 108) were directly injected into the infarct and peri-infarct areas during an open chest procedure. No immunosuppressive therapy was used. The left ventricular function was measured using serial biplane left ventricular angiography at baseline, 30, 60, and 90 days before sacrifice. Mesenchymal stem cells were localized using bromodeoxyuridine, and differentiation of mesenchymal stem cells was assessed by confocal microscopic colocalization of bromodeoxyuridine with immunofluorescent antibodies specific for cardiomyocytes (troponin I and MF-20) and endothelial cells (von Willebrand factor). Results: Mesenchymal stem cells labeled with bromodeoxyuridine engrafted the peri-infarct zone and colocalized with both cardiomyocyte-specific and endothelial cell-specific immunofluorescence. No intramyocardial bromodeoxyuridine was observed in sham-treated animals. At the time of the mesenchymal stem cell injection 30 days after myocardial infarction, the left ventricular ejection fraction (LVEF) was 58% ± 3% in mesenchymal stem cell-treated pigs and 56% ± 2% in sham-treated pigs ( P = NS). LVEF deteriorated progressively thereafter in untreated pigs (8.5% and 10.5% decline at 60 days and 90 days after myocardial infarction, respectively), but was preserved in mesenchymal stem cell-treated pigs (2.1% increase and -2.0% decline at 60 and 90 days post-MI respectively) ( P < .05). Conclusions: Direct intramyocardial injection of mesenchymal stem cells results in successful intramyocardial engraftment and differentiation into cardiomyocytes and endothelial cells and preserves left ventricular function after myocardial infarction in pigs.

Background Nerve growth factor (NGF) infusion into the right stellate ganglion (RSG) is antiarrhythmic, while NGF infusion into the left stellate ganglion (LSG) is proarrhythmic in dogs with myocardial infarction (MI) and complete atrioventricular block (CAVB). This functional asymmetry suggests differential neural remodeling. Objectives To test the hypothesis that NGF infusion into the RSG and the LSG can lead to differential β-adrenoceptor (β-AR) expression in dogs with MI and CAVB. Methods and Results We performed immunostaining to quantify β 1 -AR and β 3 -AR immunoreactivity in six dogs with MI and CAVB, nine dogs with MI, CAVB, and NGF infusion to the LSG, six dogs with MI, CAVB, and NGF infusion to the RSG, and six normal dogs. There was significantly increased β 3 -AR immunoreactivity in dogs with NGF infusion into the LSG and significantly decreased β 3 -AR immunoreactivity in dogs with NGF infusion into the RSG compared with controls and with the MI and CAVB group. There were no significant differences in β 1 -AR immunoreactivity among these four groups. To determine protein and mRNA expression of β-ARs, we created MI and CAVB and infused NGF into the LSG in six additional dogs. The noninfarcted left ventricle free wall was harvested 1 week later. The protein level and receptor density of β 3 -AR (but not β 1 - or β 2 -AR) significantly increased in these six dogs compared with normal controls. Conclusions We conclude that NGF infusion into the RSG and the LSG in dogs with MI and CAVB induced differential β 3 -AR expression in the left ventricular myocardium.

Objective— Based on our previous observations on the expression of Tenascin-C (Tn-C) in human atherosclerotic plaques and its colocalization with macrophages, we explored whether Tn-C undergoes fragmentation and the potential pathobiological significance of this fragmentation. Methods and Results— Using cultured human smooth muscle cells (SMCs), we found that Tn-C upregulates expression of matrix metalloproteinases (MMPs). Western blot analysis revealed that Tn-C substrate is fragmented and most of the cleavage products have fibronectin-like and epidermal growth factor-like (EGF-like) domains of Tn-C. One fragment that contains an EGF-like domain was found in some human atherosclerotic plaques. Cell culture studies revealed that the recombinant EGF-like domain inhibits growth, induces apoptosis of SMCs in a dose-dependent, time-dependent, and caspase-dependent manner, and activates caspase-3 before SMC detachment. Conversely, the caspase inhibitor z-YVAD.cmk, serum, and protease inhibitors blocked cell apoptosis conferred by the EGF-like domain. In addition, these inhibitors blocked EGF-like domain-induced caspase-3 activation. In contrast to this EGF-like domain, intact Tn-C, its fibronectin-like, and its fibrinogen-like domains were inactive. Conclusions— Together with our previous observations, our data suggest that Tn-C upregulates MMP expression that cleaves Tn-C into fragments containing the EGF-like domain. This domain has proapoptotic activity for SMCs.

We present an ongoing research on a system built to support collaborative learning through argumentation, incorporating both humans and artificial agents in the learning environment. The system was implemented and tested with students discussing computing and privacy issues.

Recent evidence from a double-blind, randomized study showed that treatment with apolipoprotein A-I Milano (ApoA-I Milano ) in a complex with phospholipids produced significant regression of the coronary atheroma burden in patients with acute coronary syndromes. We previously showed similar regression of atherosclerosis in an animal model. Here, we examined a viral vector-based gene delivery system as a basis for ApoA-I Milano gene therapy. Comparing levels of expression using combinations of the cytomegalovirus (CMV) promoter in a recombinant serotype 2 adeno-associated virus (rAAV2) linked to ApoA-I Milano or the enhanced green fluorescent protein (EGFP) genes, we found that a promoter construct of two CMV core promoters sharing a CMV enhancer was more active than other combinations or a single CMV promoter. In vivo assessment of this optimal CMV construct using rAAV2 virus particles for intravenous (IV) or intramuscular (IM) routes of delivery produced high circulating levels of ApoA-I Milano protein for extended periods (up to 220 ng/ml at 22 weeks p.i.) by IV delivery while the IM route resulted in a relatively short period of very low-level ApoA-I Milano expression. Since there was no difference in the immune response between the two routes of delivery, we reasoned that tissue tropism might be responsible for this differential gene expression. To explore this possibility, we investigated the effect of different AAV serotypes on ApoA-I Milano gene expression in vivo. It found that rAAV1-mediated expression of ApoA-I Milano was approximately 15- and 9-fold higher than rAAV2 and rAAV5, respectively when IM injection routes were compared while all three AAV serotypes produced substantial levels of ApoA-I Milano expression from IV injection. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed transgene expression could be achieved and support the potential of AAV-mediated gene therapy.

Introduction: Mesenchymal stem cell (MSC) transplantation is a promising technique to improve cardiac function. Whether MSC can increase cardiac nerve density and contribute to the improved cardiac function is unclear. Methods and Results: Anterior wall myocardial infarction was created in 16 swine. One month later, 6 swine were given MSC and fresh bone marrow (BM) into infarcted myocardium (MSC group). Four swine were given fresh BM only (BM group), and 6 swine were given culture media (MI-only group). The swine were sacrificed 95.8 ± 3.5 days after MI. Six normal swine were used as control. Immunocytochemical staining was performed using antibodies against growth-associated protein 43 (GAP43), tyrosine hydroxylase (TH), and three subtypes of tenascin (R, C, and X). Five fields per slide were counted for nerve density. The results show the following. (1) There were more GAP43-positive nerves in the MSC group than in the BM, MI-only, or Control group (P < 0.0001). TH staining showed higher nerve densities in the MSC group than in the MI-only (P < 0.01) or Control group (P < 0.0001) in the atria. (2) There were more sympathetic (TH-positive) nerves in myocardium distant from infarct than in the peri-infarct area (P < 0.05). (3) Optical intensity and color analyses showed significantly higher tenascin R and tenascin C expression in the MSC and BM groups than in the MI-only or Control group (P < 0.01). Conclusion: MSC injected with BM into swine infarct results in overexpression of cardiac tenascin, increased the magnitude of cardiac nerve sprouting in both atria and ventricles, and increased the magnitude of atrial sympathetic hyperinnervation 2 months after injection. (J Cardiovasc Electrophysiol, Vol. 14, pp. 841-848, August 2003)

Objective— We sought to identify differentially expressed genes in the athero-prone coronary artery and athero-resistant internal mammary arteries. Methods and Results— Using suppressive subtraction hybridization, we generated reciprocal cDNA collections of representative mRNAs specific to porcine coronary arteries versus porcine mammary arteries. We screened 1000 suppressive subtraction hybridization cDNA clones by dot blot array and sequenced 600 of those showing the most marked expression differences. Northern blot, in situ hybridization, and immunostaining confirmed the differential gene expression patterns identified by the dot blot arrays. Genes associated with mammary arteries included claudin-10 and h-cadherin, which are genes associated with tight junctions and intermediate junctions. In contrast, genes associated with proatherosclerotic processes, such as lipid retention and metabolism, inflammation, and cell growth, were preferentially expressed in coronary arteries. Conclusions— Normal coronary arteries have gene expression program that is significantly different than internal mammary arteries. These differences may partly explain the resistance of coronary arteries and internal mammary arteries to atherosclerosis.

Introduction The patient was a 37-year-old man who was a victim of a motor-vehicle accident. On arrival at the emergency department, the patients’ vital signs were all within normal physiological range. His GCS was 15/15. He had suffered from neck pain. In physical examination, he had upper cervical spine tenderness but there was no abnormal palpable deformity. Other physical examinations were normal. Plain X-ray of the cervical spines was nonconclusive. Cervical-spine CT scan demonstrated an isolated unstable Jefferson fracture associated with Spence's criteria. Cervical CT angiography was normal. Materials and Methods The patient was placed in the prone position and the alignment and the position of the atlantoaxial complex was verified by the use of real-time fluoroscopy. The cervical spine was exposed subperiosteally from the occiput to the level of C3-4. The C1-2 articulation was exposed. After the dorsal root ganglion of C2 was retracted inferiorly, the middle of the junction of the posterior arch of C1 and the midpoint of the inferoposterior part of the C1 lateral mass was exposed. The screws were placed via Magerl trans-facet screw technique. Then screws were connected with a rod and nuts to reduce lateral spread of the lateral masses. Results Postoperative cervical CT scan showed that the screws are in favorable position. Conclusion The treatment of unstable Jefferson fractures remains controversial. Conservative treatment usually associates with the longtime of immobilization in halo vest, whereas conventional posterior surgical treatments generally eliminate the range of motion of the upper cervical spines. With the posterior intersegmental fusion of unstable atlas fracture, the stabilization of anterior arch of atlas can be achieved while at the same time atlantoaxial rotation is preserved. Disclosure of Interest None declared

Introduction Although L1-L2 disc is anatomically located in lumbar region, its specific location poses a caveat to the surgeon for both conus medullaris present in this area and the great biomechanical standpoint in thoracolumbar region. Materials and Methods In our experience with this group of patients, we used trans-facet pedicle-sparing approach to care for conus medullaris. In addition, we performed short segment fusion to augment this highly demanding biomechanical region. Since the year 2008, we operated 13 cases with L1-L2 disc with this technique. Only two cases presented with severe L2 radiculopathy and the rest came with feature of cauda equine or conus problem. Axial back pain was crucial symptom in eight cases. Two patients had simultaneous disc problem in L2-L3 and one patient had T12-L1 disc protrusion. After operation, patients received Jewett hyperextension brace for 3 months. In one patient with central hard calcified disc, we performed thoracoabdominal approach and decompression from anterior. All surgery performed under high magnification of microscope and bone removal was done by powered drill. Results In six cases, decompression was performed bilaterally to reach the central portion of the disc. Pedicle screw was used for fixation in all patients. TLIF banana cage was used in five cases and spinous process bone used as interbody graft in four cases. There was no aggravation in neurological deficit and we have no problem with CSF leak or wound. Fusion achieved in all patients and axial back pain resolved successfully. Conclusion Although L1-L2 innately considered a lumbar disc, we suggest that they should be considered as thoracic disc and the surgeon should not solely rely on where the conus terminated in MRI. In this location, small traction can lead to catastrophic neurological damage; therefore, by using trans-facet pedicle-sparing under microscopic guide the cord should be decompressed by drill. In addition, to prevent neurological injury, Kerrison should not be placed under already stenotic canal. Disclosure of Interest None declared