Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes.

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

Bcl3 Bridges LIF-STAT3 to Oct4 Signaling in the Maintenance of Naïve Pluripotency.

Leukemia inhibitory factor (LIF) regulates mouse embryonic stem cell (mESC) pluripotency through STAT3 activation, but the downstream signaling remains largely unelucidated. Using cDNA microarrays, we verified B cell leukemia/lymphoma 3 (Bcl3) as the most significantly downregulated factor following LIF withdrawal in mESCs. Bcl3 knockdown altered mESC morphology, reduced expression of pluripotency genes including Oct4, Sox2, and Nanog, and downregulated DNA binding of acetylated histone 3 and RNA polymerase II on the Oct4 promoter. Conversely, Bcl3 overexpression partially prevented cell differentiation and promoted Oct4 and Nanog promoter activities. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation experiments demonstrated that Bcl3 regulation of mESC pluripotency may be through its association with Oct4 and ß-catenin and its promoter binding capability. These results establish that Bcl3 positively regulates pluripotency genes and thus shed light on the mechanism of Bcl3 as a downstream molecule of LIF/STAT3 signaling in pluripotency maintenance.

A nanopatterned cell-seeded cardiac patch prevents electro-uncoupling and improves the therapeutic efficacy of cardiac repair.

The heart is an extremely sophisticated organ with nanoscale anisotropic structure, contractility and electro-conductivity; however, few studies have addressed the influence of cardiac anisotropy on cell transplantation for myocardial repair. Here, we hypothesized that a graft's anisotropy of myofiber orientation determines the mechano-electrical characteristics and the therapeutic efficacy. We developed aligned- and random-orientated nanofibrous electrospun patches (aEP and rEP, respectively) with or without seeding of cardiomyocytes (CMs) and endothelial cells (ECs) to test this hypothesis. Atomic force microscopy showed a better beating frequency and amplitude of CMs when cultured on aEP than that from cells cultured on rEP. For the in vivo test, a total of 66 rats were divided into six groups: sham, myocardial infarction (MI), MI + aEP, MI + rEP, MI + CM-EC/aEP and MI + CM-EC/rEP (n ≥ 10 for each group). Implantation of aEP or rEP provided mechanical support and thus retarded functional aggravation at 56 days after MI. Importantly, CM-EC/aEP implantation further improved therapeutic outcomes, while cardiac deterioration occurred on the CM-EC/rEP group. Similar results were shown by hemodynamic and infarct size examination. Another independent in vivo study was performed and electrocardiography and optical mapping demonstrated that there were more ectopic activities and defective electro-coupling after CM-EC/rEP implantation, which worsened cardiac functions. Together these results provide comprehensive functional characterizations and demonstrate the therapeutic efficacy of a nanopatterned anisotropic cardiac patch. Importantly, the study confirms the significance of cardiac anisotropy recapitulation in myocardial tissue engineering, which is valuable for the future development of translational nanomedicine.

Identification of macrosialin (CD68) on the surface of host macrophages as the receptor for the intercellular adhesive molecule (ICAM-L) of Leishmania amazonensis.

The intercellular adhesive molecule, ICAM-L, of Leishmania amazonensis is known to block the attachment as well as internalisation of Leishmania for infection in host macrophages. We employed monoclonal antibodies (mAb) to the surface molecules of a macrophage to block the attachment of ICAM-L to the macrophage surface and identified that CD68 macrosialin is likely the receptor molecule on the macrophage for ICAM-L. We then demonstrated physical interaction between ICAM-L and macrosialin by co-immunoprecipitation of macrosialin with ICAM-L or vice versa. Finally, macrosialin is expressed in macrosialin-negative murine fibroblast cell line NCTC clone 2555 and demonstrates that both ICAM-L and promastigotes of L. amazonensis can bind to the CD68 transfectant. We thus conclude that CD68 macrosialin is the receptor on host macrophages for ICAM-L. Also, involvement of ICAM-L-macrosialin interaction in other Leishmania species and other mammalian macrophages were demonstrated, indicating the biological relevance of this ligand-receptor interaction.

Two distinct arsenite-resistant variants of Leishmania amazonensis take different routes to achieve resistance as revealed by comparative transcriptomics.

Genome-wide search for the genes involved in arsenite resistance in two distinct variants A and A' of Leishmania amazonensis revealed that the two variants used two different mechanisms to achieve resistance, even though these two variants were derived from the same clone and selected against arsenite under the same conditions. In variant A, the variant with DNA amplification, the biochemical pathways for detoxification of oxidative stress, the energy generation system to support the biochemical and physiological needs of the variant for DNA and protein synthesis and the arsenite translocating system to dispose arsenite are among the primary biochemical events that are upregulated under the arsenite stress to gain resistance. In variant A', the variant without DNA amplification, the upregulation of aquaglyceroporin (AQP) gene and the high level of resistance to arsenate point to the direction that the resistance gained by the variant is due to arsenate which is probably oxidized from arsenite in the arsenite solution used for selection and the maintenance of the cell culture. As a result of the AQP upregulation for arsenite disposal, a different set of biochemical pathways for detoxification of oxidative stress, energy generation and cellular signaling are upregulated to sustain the growth of the variant to gain resistance to arsenate. From current evidences, reactive oxygen species (ROS) overproduced by the parasite soon after exposure to arsenite appear to play an instrumental role in both variants to initiate the subsequent biochemical events that allow the same clone of L. amazonensis to take two totally different routes to diverge into two different variants.

Indigenous leishmaniasis in Taiwan: report of a case.

Taiwan is not considered an endemic area of leishmaniasis. Imported cases are encountered infrequently, and only two cases of indigenous cutaneous leishmaniasis have been reported.(1) We found one new case in the past 20 years. The patient presented with erythematous plaques on the nasal bridge and right thumb. Skin biopsy specimens from both sites revealed numerous Leishman-Donovan bodies in macrophages. There was no history of travel outside the country, and the diagnosis of indigenous cutaneous leishmaniasis was made. Polymerase chain reactions (PCR) identified the species as Leishmania tropica. The route of infection in this patient is unclear. Because pentavalent antimony, the drug of choice for leishmaniasis, is not available in Taiwan, the patient was treated with levamisole and potassium iodide, with an excellent response.

Divergence of trypanothione-dependent tryparedoxin cascade into cytosolic and mitochondrial pathways in arsenite-resistant variants of Leishmania amazonensis.

Evidences are presented in the in vivo study that overexpression of tryparedoxin peroxidases (TXNPxs) diverged into cytosolic pathway in arsenite-resistant variant A and mitochondrial pathway in variant A' of Leishmania amazonensis is due to the upregulation of the corresponding upstream tryparedoxins (TXNs) in the cytosol as well as the mitochondrion respectively. Evidences are also presented that exposure of L. amazonensis to arsenite in the early hours led to the production of reactive oxygen species (ROS), which in turn induced the overexpression of the genes of both cytosolic and mitochondrial trypanothione-dependent tryparedoxin pathway due probably to physiological and functional needs. The sequence of events leading to the upregulation indicates that cytosolic tryparedoxin pathway is upregulated earlier than that of mitochondrial tryparedoxin pathway. Based on the kinetics of gene upregulation of the cytosolic pathway is different from that of mitochondrial pathway, and cTXNPx and mTXNPx differentially detoxify H(2)O(2) and of t-butyl hydroperoxide respectively, it is postulated that during arsenite selection, different ROS species may have been overproduced in either variants A or A', leading to the divergence of the trypanothione-dependent tryparedoxin pathways in these variants.

Myasthenia gravis accompanied by thymomas not related to foamy virus genome in Belarusian's patients.

The spectrum and features of neurological disorders have been changed due to the Chernobyl catastrophe in the Republic of Belarus. More recently neurologists in Belarus have noted a significant increase in the frequency of myasthenia gravis (MG) with concomitant rise in the thymomas. There is some evidence suggesting that retroviruses play a key role in the development and pathogenesis of autoimmune diseases. This study analyzed thymomas from 45 MG patients from the Republic of Belarus by using PCR and primers for two regions of FV--gag and bel-2 genes. The results showed that none of the varied thymuses from the 45 MG patients contained FV genome. No relationship can be confirmed between FV and this disease and the results suggest that no pathological link between FV and MG exists.

The anti-inflammatory actions of LCY-2-CHO, a carbazole analogue, in vascular smooth muscle cells.

LCY-2-CHO has anti-inflammatory actions on macrophages. To understand its therapeutic implication in atherosclerosis, we examined its effects on the expressions of anti-inflammatory and inflammatory proteins in cultured rat aortic vascular smooth muscle cells (VSMC). LCY-2-CHO is able to induce heme oxygenase-1 (HO-1) protein expression through a transcriptional action. The HO-1 inducting effect of LCY-2-CHO was inhibited by SB203580, N(G)-nitro-l-arginine methylester (l-NAME), and wortmannin, but was not affected by U0126 or SP600125. In accordance LCY-2-CHO increased protein phosphorylation of p38, Akt, and eNOS. Nrf2 is a transcription factor essential for HO-1 gene induction and we showed that LCY-2-CHO is able to cause Nrf2 nuclear translocation and this action depends on p38, Akt and eNOS. In addition to induce anti-inflammatory HO-1, LCY-2-CHO reduced interleukin-1beta (IL-1beta)-induced inflammatory mediators, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), growth-related oncogene protein-alpha (GRO-alpha), and interleukin-8 (IL-8). Inhibitory effect on IL-1beta-mediated NF-kappaB activation was evidenced by the diminishment of IkappaB kinase (IKK) phosphorylation and IkappaBalpha degradation. In contrast, IL-1beta-mediated ERK and JNK activations were not changed by LCY-2-CHO, while p38 activation by IL-1beta and LCY-2-CHO displayed the non-additivity. Taken together, given the overall anti-inflammatory properties of LCY-2-CHO in VSMC, in terms to induce HO-1 gene expression and inhibit inflammatory gene expression, these results highlight the therapeutic potential of LCY-2-CHO in atherosclerosis.

Loop 1 structure of the leishmanial ICAM-L molecule is crucial for parasite binding and infection of host macrophages.

The binding of each intercellular adhesive molecule (ICAM) molecule fragment from Leishmania amazonensis (ICAM-L) to host macrophages was investigated using an indirect immunofluorescent sandwich technique, based on the observation that ICAM-L can block the uptake of L. amazonensis on the macrophage surface and all prepared ICAM-L fragments can react with rabbit anti-ICAM-L antiserum. The ICAM-L fragments lacking the loop 1 (LI) structure failed to bind to macrophages, and the disruption of the LI structure by mercaptoethanol led to the failure of binding. The fragments containing the LI structure functioned similarly to ICAM-L, by temporarily retarding host cell growth and cell cycle progression, and inhibiting the Leishmania infection of host macrophages. These results suggest that LI constitutes the main determinant of the ICAM-L molecule in binding to, and infection of, host macrophages.

Retardation of cell cycle progression of macrophages from G1 to S phase by ICAM-L from Leishmania.

Leishmania, an obligate intracellular parasite of host macrophages, infects the macrophage through receptor-mediated phagocytosis that either activates or deactivates macrophages to eliminate the parasite or allow the parasite to grow intracellularly. ICAM-L, an intercellular adhesion molecule from L. amazonensis, results in lower MTT tests and proliferative responses of macrophages when incubated in vitro. The inhibition of cell proliferation, however, results from temporary retardation of the cell cycle progression at the G1 to S phase transition rather than cell death. The retardation is due to the upregulation of two CKI proteins, p21 and p27, in a p53-independent manner which, control the G(1) to S phase transition checkpoint.

Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IkappaB kinase, nuclear factor-kappa B and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia.

In the present study, experiments were performed to explore the action of quercetin, the most widely distributed flavonoids, and its major metabolite, quercetin-3'-sulfate, on lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) production in BV-2 microglia. Quercetin could suppress LPS- and IFN-gamma-induced NO production and inducible nitric oxide synthase (iNOS) gene transcription, while quercetin-3'-sulfate had no effect. LPS-induced IkappaB kinase (IKK), nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) activation, and IFN-gamma-induced NF-kappaB, signal transducer and activator of transcription-1 (STAT1) and interferon regulatory factor-1 (IRF-1) activation were reduced by quercetin. Moreover quercetin was able to induce heme oxygenase-1 expression. To address the involvement of heme oxygenase-1 induction in iNOS inhibition, heme oxygenase-1 antisense oligodeoxynucleotide was used. Quercetin-mediated inhibition of NO production and iNOS protein expression were partially reversed by heme oxygenase-1 antisense oligodeoxynucleotide, but was mimicked by hemin, a heme oxygenase-1 inducer. The involvement of signal pathways in quercetin-induced heme oxygenase-1 gene expression was associated with tyrosine kinase and mitogen-activated protein kinases activation. All these results suggest quercetin should provide therapeutic benefits for suppression of inflammatory-related neuronal injury in neurodegenerative diseases.

Distinct overexpression of cytosolic and mitochondrial tryparedoxin peroxidases results in preferential detoxification of different oxidants in arsenite-resistant Leishmania amazonensis with and without DNA amplification.

A cytosolic (cTXNPx) and a mitochondrial (mTXNPx) tryparedoxin peroxidase genes, cloned from wildtype Leishmania amazonensis clone 2-23 are homologous in nucleic acid and amino acid sequences to the respective genes described for L. infantum and L. chagasi. Surprisingly, as shown in the results of transcription assays, protein determination and fluorescent antibody detection in situ, cTXNPx is distinctly overexpressed in the cytoplasm of arsenite-resistant A variant with DNA amplification, whereas mTXNPx is distinctly overexpressed in the mitochondrion of arsenite-resistant A' variant without DNA amplification, although A and A' are arsenite-resistant variants derived from the same wildtype clone of L. amazonensis, and selected against arsenite under the same conditions. Since the tunicamycin-resistant variant (T) derived from the same W(2-23) clone and the hydroxyurea-resistant (Hu(2-6)) variant derived from clone W(2-6) do not show overexpression of these two genes, it is suggested that the distinct overexpression of cTXNPx and mTXNPx genes in arsenite-resistant A and A' variants is linked to arsenite selection process. These two genes in A and A' variants, and cTXNPx(+) and mTXNPx(+) transfectants are similar to the respective genes described for L. infantum and L. chagasi in terms of antioxidant activities against H2O2 and t-butyl hydroperoxide, in which cTXNPx is more resistant to H2O2, and mTXNPx is more resistant to t-butyl hydroperoxide than the wildtype. Both genes, however, are cross-resistant to NO as compared to the control wildtype. In the transfectants carrying cTXNPx and mTXNPx in inverted orientation, these two genes are expressed in a level lower than that in wildtype. The decreased expression was followed by increased sensitivity of these transfectants to the oxidants. This possibly is due to the formation of antisense mRNA in these transfectants that causes a specific downregulation of the respective genes.

Selection of immunodominant fragments from envelope gene for vaccine against Japanese encephalitis virus in DNA priming-protein boosting protocols.

Fragmentation of E gene of JEV into smaller fragments, none of the fragments either in plasmids form or in recombinant protein form can induce optimal protection against the virus infection. It is only when DNA priming-protein boosting strategies are used then the N-terminal E(A) and the C-terminal E(B) showed full protection against JEV as those induced by commercial vaccine, provided both fragments are preceded in the N-terminal by a signal peptide M(15) derived from C-terminal of prM gene in JEV genome. When the subfragments of E(A): E(A1) and E(A2) and E(B): E(B1) and E(B2) are tested, only E(A1) subfragment can replace E(A) in protein boosting to induce optimal protection against JEV, E(A2), E(B1), E(B2) in plasmid or protein forms are not. Therefore, along the E gene (978-2330 bp) N-terminal, E(A1) (978-1580 bp) and C-terminal E(B) (1851-2330 bp) are the most effective in inducing immunity against JEV but not the middle fragment E(A2) (1518-1877 bp) (see for orientation of E(A1), E(A2) and E(B) in E gene). Under the notion that molecular complexity determines the outcome of immune response of the host, E(B) being shorter, simpler in molecular structure and can be easily expressed in soluble form in E. coli (as opposed to insoluble E(A1)), E(B) probably will be the choice as a candidate vaccine to protect the host against JEV infection.

An improved method for detection of Leishmania amastigotes by an antibody probe against the small subunit of leishmanial ribonucleotide reductase.

By taking advantage of an antibody raised against the small M2 subunit of ribonucleotide reductase of Leishmania that reacts with the enzyme in the nucleus of the parasite but does not cross-react with the same enzyme of the host macrophage, an improved fluorescence-staining method is developed for enumeration of leishmanial amastigotes inside the macrophage. The method offers an accurate and easy way of counting, compared with Giemsa staining.

Sub-fragments of the envelope gene are highly protective against the Japanese encephalitis virus lethal infection in DNA priming--protein boosting immunization strategies.

The envelope (E) gene of Japanese encephalitis virus (JEV) plays a major protective role against JEV infection. In order to locate the part of E gene that is responsible for this protection, an N-terminal fragment EA (nucleotide number 933-1877 bp of JEV genome) and a C-terminal fragment EB (nucleotide number 1851-2330 bp of JEV genome) from E gene were prepared. Both of these fragments were used in the form of recombinant proteins (rEA and rEB) and plasmid DNA (pEA, pM15EA and pEB) for immunizations. Recombinant EA protein (rEA) was previously found to be non-protective because it was expressed in an insoluble form. Plasmid EA (pEA) was also found to be non-protective unless it is preceded by a 15 mer signal peptide derived from the very C-terminal of the membrane gene (M) of JEV to form pM15EA plasmid indicating the importance of the signal peptide in the expression of EA immunogenicity. Although pM15EA and pEB are both immunogenic and protective against JEV lethal infection, the protection by both fragments however is not optimal. Even when pM15EA and pEB were used together for immunization, maximum protection as those induced by control vaccine was not achieved. However, if individual fragments (EA or EB) were used in a DNA priming-protein boosting or protein priming-DNA boosting strategy, high levels of protection were achieved by both fragments. This was especially true for EA fragment where the level of protection against JEV lethal infection was equal to that induced by commercially available vaccine alone. The protection correlated very well with the neutralizing antibody titers and the T helper cell involved in this process in mainly the Th1 type.

Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages.

1. Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX-2 expression were investigated in RAW264.7 macrophages. 2. Capsaicin and resiniferatoxin (RTX) can inhibit LPS- and IFN-gamma-mediated NO production, and iNOS protein and mRNA expression with similar IC50 values of around 10 microm. 3. Capsaicin also transcriptionally inhibited LPS- and PMA-induced COX-2 expression and PGE2 production. However, this effect exhibited a higher potency (IC50: 0.2 microm), and RTX failed to elicit such responses at 10 microm. 4. Interestingly, we found that capsazepine, a competitive TRPV1 antagonist, did not prevent the inhibition elicited by capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX-2/PGE2 induction with an IC50 value of 3 microm. RT-PCR and immunoblotting analysis excluded the expression of TRPV1 in RAW264.7 macrophages. 5. The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS-elicited NF-kappaB and AP-1 activation and IFN-gamma-elicited STAT1 activation. The reporter assay of AP-1 activity also supported this action. 6. The kinase assay indicated that ERK, JNK, and IKK activation by LPS were inhibited by vanilloids. 7. In conclusion, vanilloids can modulate the expression of inflammatory iNOS and COX-2 genes in macrophages through interference with upstream signalling events of LPS and IFN-gamma. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions.

Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum.

1 The polysaccharide from Ganoderma lucidum (PS-G) has been reported to enhance immune responses and to elicit antitumor effects. In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis when cultured in vitro. Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS-G on these two cell functions, and the molecular events leading to these actions. 2 Using latex beads and heat-inactive Escherichia coli serving as particles for neutrophil engulfment, we found that PS-G is able to enhance phagocytic activity of human primary neutrophils and neutrophilic-phenotype cells differentiated from all trans retinoic acid-treated HL-60 cells. 3 Chemotactic assay using Boyden chamber also revealed the ability of PS-G to increase neutrophil migration. 4 Exposure of neutrophils to PS-G time dependently caused increases in protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), Hck, and Lyn activities. 5 Results with specific kinase inhibitors indicate that phagocytic action of PS-G was reduced by the presence of wortmannin (Phosphatidylinositol 3-kinase, PI3K inhibitor), pyrazolpyrimidine 2 (Src-family tyrosine kinase inhibitor), Ro318220 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), but not by PD98059 (mitogen-activated protein/ERK kinase inhibitor). Moreover, chemotactic action of PS-G requires the activities of PI3K, p38 MAPK, Src tyrosine kinases and PKC. 6 All these results demonstrate the abilities of PS-G to enhance neutrophil function in phagocytosis and chemotaxis, and further provide evidence to strengthen the beneficial remedy of G. lucidum in human to enhance defense system.

ICAM-L gene is conserved only in Leishmania species in the family of kinetoplastida.

Attachment of Leishmania to its host macrophage is a ligand-receptor mediated event. Two abundant surface molecules, a complex carbohydrate lipophosglycan (LPG) and a glycosylphosphotidylinositol (GPI) anchored GP63 protease have been shown to independently play a critical role in this event. LPG and GP63 are widely conserved among members of the kinetoplastida family. An intercellular adhesive molecule from Leishmania (ICAM-L) that acts as a ligand of Leishmania for the macrophage has recently been described by our lab. When assessed by molecular determinations including Southern, Northern and sequencing, ICAM-L appears to be conserve only among Leishmania species but not in other closely related members of the kinetoplastida. However using a polyclonal antibody specific to ICAM-L, the molecule appears to be conserved among all the kinetoplastids. The detention by antibody of ICAM-L molecules among other kinetoplastids may due to conservation in these parasites of certain immunoreactive epitopes at the amino acid level, while the nucleotide sequence is divergent enough to preclude detection by methods employed.