Brazil may harbor the largest absolute number of HTLV-1-infected individuals worldwide. The current HTLV diagnosis is based mainly on antibodies detection. However, these tests exhibit high proportion of indeterminate results. Several real-time PCR techniques have been developed for the detection of HTLV-1/2. However, up to day the major drawbacks of HTLV-1/2 molecular diagnosis are the lack of standard molecular tests and the absence of suitable internal amplification control (IAC). The aim of this study was to develop a multiplex qualitative real-time PCR for the simultaneous detection and discrimination of HTLV-1/2 and to design an IAC for reaction monitoring.After multiple sequence alignments of the full genomes of HTLV-1/2 subtypes, a conserved tax region was chosen for the design of specific primers and probes. The IAC was generated after the annealing of synthetic nucleotide sequences and cloned into TOPO TA® vector. MT-2 and Gu cell lines were used as positive controls for HTLV-1 and HTLV-2, respectively.The developed multiplex real-time reaction detected both HTLV-1 and HTLV-2 (105 to 101 copies/reaction) at the presence of IAC in the same reaction. Analytical sensitivity was 1.2 copies/reaction for HTLV-1 and 19.1 copies/reaction for HTLV-2. The analytical sensitivity analysis was performed in singleplex format.The detection of HTLV-1/2 and IAC by multiplex real-time PCR was efficient. The developed IAC is suitable for the molecular diagnosis and its presence ensures that the negative results are not due to failure in pre-PCR procedures.


The relationship between vegetation and soil fertility deserves attention due to its scientific importance and practical applications. However, the effects of soil fertility on vegetation development and succession are poorly documented. Here we study soil fertility in Yanhe watershed at northern Shaanxi on five different land uses, namely shrubland, farmland, natural grassland, woodland, and artificial grassland, and in soil under restoration for 5, 10, 15, 20, and 25. Attribute recognition model based on entropy weight was used to evaluate the soil fertility of typical region in the Loess Plateau of China, which contained 52 soil samples with 6 physical and chemical indexes, including soil organic matter, soil total nitrogen, total phosphorus, etc. The results show that (1) Land use has an obvious effect on soil bulk density, total porosity and capillary porosity of surface layers, but not significant in the subsurface layer; (2) SOM, N Soil fertility is important factors for growth and productivity of vegetation –3. VegetStudy of degradation processes attracts attention to the influence of degradation on the human environment, but study of recovery processes is more important, providing recommendations for eco-environmental reconstruction or rehabilitation. Much research has been done recently on the influence on soil fertility properties of vegetation recovery or different land-use patterns –17. HoweThe study area was located in Yanhe watershed of the Loess Plateau at N 36°23′-37°17′and E 108°45′-110°28′ in northern Shaanxi Province and had 25 years of comprehensive management because one ecology station was founded in this area . Soil organic matter (SOM) was determined on the basis of oxidation with potassium dichromate in a heated oil bath. Total nitrogen  was measured according to the semi micro Kjeldahl method and hydrolysable nitrogen (Nhydro) by means of the Alkali diffusion method. Total phosphorus  was digested with perchloric acid and sulfuric acid and determined using colorimetry. Total potassium  was digested with hydrofluoric acid and perchloric acid. Available phosphorus (Pavail) was extracted with sodium bicarbonate and measured with colorimetry. Available potassium (Kavail) in soil was extracted with ammonium acetate.A common approach in studies of soil rehabilitation in relation to vegetative cover is to monitor plant and soil changes occurring along a vegetative chronosequence developed on similar soils under similar climatic conditions . This chSoil samples attribute measure is its status value in soil. It is used that the second national soil survey classification of soil nutrient standards for the evaluation criteria, and a standard matrix was built up based on the evaluation criteria. Soil sample analysis of variance (ANOVA) and correlation were carried out using the SPSS11.0 procedures for sites in different succession stages. Duncan's test (at p < 0.05) was used to compare means of soil variables when the results of ANOVA were significant at p < 0.05.Table Table Five replicated soil samples were collected from five sites with the same restoration time of 5, 10, 15, 20 and 25 years, respectively. Also, five nonvegetated lands in the vicinity of the planted sites (farmland) were chosen as controls for the chronosequence. After 5, 10, 15, 20 and 25 years of restoration, SOM, Ntotal, Nhydro, Pavail and Kavail were significant in the 0-20 cm layer at p < 0.05 and except Ptotal, which was not significant at p > 0.05. Generally, vegetation succession resulted in a change of soil fertility parameters in the eroded soils, significant decreases of soil fertility parameters (p < 0.05) took place from beginning to 15-years of restoration, and significant increases (p < 0.05) from 15-years to 20-years of restoration, and significant decreases (p < 0.05) from 20-years to 25-years of restoration. The statistical results showed that soil fertility and vegetation succession had significant interactions with all soil fertility parameters except Ptotal Table .Table 3MFigure Land use has an obvious effect on Soil fertility of surface layer, but not significant in the subsurface layer. Shrubland has higher soil fertility than other land uses. In most cases, table land has low levels of soil fertility, but after long period of cultivation, the land degrades year by year. Our results indicate that the establishment and development of vegetation succession on eroded soil result in significant changing of soil fertility. With increased plantation age, it is possible to recover soil fertility to a certain degree, and affection of soil fertility on vegetation succession came into line gradually.


Reproductive health among cancer survivors is an important quality of life issue. Certain cancer therapies have known fertility risks. There is an existing cohort of adolescents and young adults (AYA) cancer survivors that, seen less frequently in clinical care settings than active patients, are likely not having discussions of fertility and other reproductive health issues. A survivor or healthcare provider can easily assume that the window of opportunity for fertility preservation has passed, however emerging research has shown this may not be the case. Recent data demonstrates a close relationship between fertility and other late effects to conclude that ongoing assessment during survivorship is warranted. Some fertility preservation procedures have also been shown to mitigate common late effects. This review explores the link between late effects from treatment and common comorbidities from infertility, which may exacerbate these late effects. This review also highlights the relevance of fertility discussions in the AYA survivorship population. Reproductive potential and other reproductive health concerns among cancer survivors is an important quality of life issue. Certain cancer therapies, particularly alkylating chemotherapy agents and radiation, have known fertility risks. Fertility issues within oncology have ignited a robust research focus that explores physiologic and psychological late effects, advancing technologies that reduce the risk of becoming infertile in the future, and broader reproductive health concerns such as appropriate contraception and the HPV vaccine –4. SustaThere are heterogeneous reports exploring the rates of discussion of fertility. Ranging from 34 to 70% –11, whetAdolescents and young adults cancer survivors have distinct developmental needs. While many adolescents without a history of cancer spend these formative years affirming their unique identity, survivors are on a journey toward “normal,” as they have experienced significant disruption in identity formation . EngaginWith attention to fertility gaining more attention in pediatric oncology settings, discussions are improving . That isThis AYA survivorship cohort is growing  but survThere are likely misconceptions regarding AYA cancer survivors’ quality of life concerns. There is an overshadowing focus on survivors’ disease status and the assumption that cancer survivors are less interested in sex and reproduction than their healthy peers. In reality, research indicates adolescents with a chronic illness are at least as sexually active as their healthy counterparts , 21. TheThere are unique relationships among infertility and the late effects of cardiotoxicity, bone health, and other endocrine disorders. It is important for healthcare providers (HCP) to be aware of the potential for infertility to exacerbate these late effects. At the same time, HCP must also be aware of the risk for infertility symptoms to go untreated due to a focus on other common physiological late effects. We discuss late effects that HCP routinely assess, and the similarity of these symptoms within infertile persons that may go overlooked.It is well documented that survivors are at risk for cardiovascular disease and mortality due to treatment alone –24. FemaInfertility may aggravate these existing cardiovascular impairments. Females who experience premature ovarian failure (POF) as a result of treatment are at risk of cardiovascular disease due to the early loss of estrogen , 30. EstBone health may also be affected by both infertility and cancer treatment, and should be regularly assessed at follow-up visits. Reduction in bone health quality from treatment can occur in a variety of ways. Many commonly utilized chemotherapies, such as corticosteroids, antimetabolites, anthracyclines, and alkylating agents have been shown to adversely impact bone mineral density (BMD) through creating an imbalance in bone metabolism with increased bone resorption and decreased bone formation –38. ThesGonads in both males and females secrete hormones that facilitate bone metabolism . There iIn males, there is a unique, reciprocal relationship between bone health and fertility. Evidence for this can be found in adults with liver cancer; in those with impaired gonadal function and osteoporosis, liver transplantation was found to have restorative action on both gonad function and bone health . ConversPerhaps the strongest relationship warranting close assessment is that of infertility and endocrine late effects. Up to 50% of survivors have reported effects such as hypopituitarism, precocious puberty, and adrenal insufficiency . EndocriIn females, chemotherapy and radiation have varying risks on the reproductive organs as well as the endocrine system that regulates hormones within these organs. Females are particularly at risk of infertility due to cancer treatment as females are born with a finite number of non-replicating oocytes. Furthermore, luteinizing hormones stimulate theca cells to produce a steroid hormone that is converted to sex hormones in the granulosa cell which is stimulated by follicular stimulating hormone (FSH) . This prMen may experience comparable menopausal symptoms during andropause such as osteoporosis and sexual dysfunction as a result of a dramatic decrease in testosterone . AndropaMale and female cancer survivors can be burdened with emotional, social, and psychological consequences as a result of infertility. AYA survivors have experienced a dramatic acceleration of life’s challenges, beginning with confronting their own mortality. Survivorship comes with a new set of complications, negotiations, and coping skills. Adolescent cancer patients have been shown to have long-term goals that include childbearing and parenthood, as well as informational needs for fertility preservation . A diagnOther barriers may prevent the assessment of reproductive potential aside from solely physician awareness. Males may be uniquely overlooked regarding the psychosocial effects of survivorship. Male AYA survivors have been noted to have issues with body image and appearance, masculinity and sexuality, cynical about planning for future parenthood, and afraid of transmitting cancer to a partner . PerceptThe existing AYA survivorship cohort seen for follow-up care is in a unique transitional period, not only the transition of patient to survivor, but also the normative developmental transition of adolescent to adult. Past goals are now seen as capable of becoming actualized in adulthood, including goals for parenthood; an unknown fertility status may heighten distress and anxiety . ZebrackFertility can be compromised in different ways for males and females depending on the initial disease site, stage, age of patient, therapeutic treatment regimen, and condition of the patient. Alkylating agents within some chemotherapies do not target specific cells, and therefore affect all cells, especially those that divide rapidly such as cancer cells in the bone marrow. Other cellular activity can be impacted such as also oogoniums in pre-pubertal females, primary oocytes, and sperm cells, as well as theca and granulose cells in ovarian follicles that mature oocytes –69. BecaMale survivors who did not undergo a fertility-sparing procedure before treatment also have options post-treatment if fertility is impaired. Due to the rapid regeneration of sperm, recovery of reproductive potential in males after treatment with non-akylating chemotherapeutic agents is higher than with alkylating agents . NaysmitIn addition to achieving pregnancy, some fertility preservation options may offset other late effects experienced as a result of treatment. Recent experimental trials have examined the benefits of re-implantation of ovarian tissue in females for purposes other than achieving a pregnancy. Transplantation of cryopreserved ovarian tissue has shown to be a potential method for recovery of ovarian function , 79, whiIt is important to keep up with the fast-paced technologies of fertility treatments, as it is to stay abreast of the latest treatment regimens. Active treatment is being re-shaped by the push to discuss fertility preservation options prior to treatment, which is a result of the needs expressed by the ever-expanding, vocal survivorship cohort that exists today. What’s now needed is a way to bridge these two worlds and inform survivors that the window of opportunity for future parenthood, reversing POF and other late affects may not be gone. Long-term follow-up guidelines should incorporate the benefits of discussing fertility and fertility preservation options so that HCP can ensure comprehensive care and long-term quality of life.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sticht et al. [Here, we present results of detailed thermodynamic analysis of the CAI inhibitor binding to HIV-1 capsid protein and mutants thereof carrying mutations in the inhibitor binding pocket. The effect of the peptide on protein dimerization is determined and the minimal peptide sequence responsible for efficient inhibition is identified using microcalorimetry technique.-1) and unfavorable entropic contribution , and no protonation was observed during binding. The effect of several mutations inside the CAI binding pocket on dissociation constants determined by VP-ITC was similar as characterized by fluorescence polarization with fluorescently labeled CAI [The isothermal titration calorimetry was used to examine the binding of CAI inhibitor to the wild-type capsid protein as well as the mutant variants. CAI binds to the capsid protein with favorable enthalpic contribution (-12.5 kcal.molThe CAI binding site is very promising target for new antiretrovirotics. The detailed thermodynamic characterization of CAI binding to HIV-1 capsid protein performed within this study describes the complex formation and can help in further development of new HIV-1 assembly inhibitors.


Cancer is one of the leading causes of death worldwide. Although several drugs are used clinically, some tumors either do not respond or are resistant to the existing pharmacotherapy, thus justifying the search for new drugs. Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low solubility in aqueous medium, which presents a barrier to its biological applications. In this context, the use of liposomes presents a promising strategy to deliver UA and allow for its intravenous administration. In this work, long-circulating and pH-sensitive liposomes containing UA (SpHL-UA) were developed, and their chemical and physicochemical properties were evaluated. SpHL-UA presented adequate properties, including a mean diameter of 191.1 ± 6.4 nm, a zeta potential of 1.2 ± 1.4 mV, and a UA entrapment of 0.77 ± 0.01 mg/mL. Moreover, this formulation showed a good stability after having been stored for 2 months at 4°C. The viability studies on breast (MDA-MB-231) and prostate (LNCaP) cancer cell lines demonstrated that SpHL-UA treatment significantly inhibited cancer cell proliferation. Therefore, the results of the present work suggest the applicability of SpHL-UA as a new and promising anticancer formulation. Ursolic acid (UA) is a triterpenoid compound that exists abundantly in the plant kingdom. UA has been reported to have an interesting bioactivity, including anti-inflammatory , 2, antiin vivo and subsequently restricts its effectiveness [ However, although UA presents the advantage of low toxicity, the clinical application of UA is limited due to several problems, such as its limited water solubility, which leads to a low bioavailability and poor pharmacokinetics tiveness . Anothertiveness . In thisin vivo by cells of the mononuclear phagocyte system (MPS) [ Liposomes are well-recognized drug delivery systems which can act as biocompatible, biodegradable, and nonimmunogenic drug carriers . A majorem (MPS) , 16. Sevem (MPS) –19. In spite of the strategies mentioned previously, conventional and long-circulating liposomes may present a slow release of the active substance or may be unable to fuse with the endosome after internalization. As such, polymorphic liposomes have been developed to overcome these problems, mainly due to the fact that these liposomes become reactive when submitted to membrane changes triggered by pH, variations in temperature, or surface charge alterations. A pH-sensitive liposome is generally stable at physiological pH but can undergo destabilization and acquire fusogenic properties under acid conditions, thus leading to the release of its aqueous contents –22. The 2000 (DSPE-PEG2000) containing UA (SpHL-UA) were developed and their chemical and physicochemical properties were evaluated. In addition, the effect of SpHL-UA towards cancer cell lines viability, such as MDA-MB-231 and LNCaP, was also investigated.Therefore, in the present study, long-circulating and pH-sensitive liposomes consisting of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol2000 were supplied by Lipoid GmbH . UA, CHEMS, phosphate saline buffer, sodium hydroxide, Triton X-100, sodium dodecyl sulfate (SDS), MTT reagent -2,5-diphenyltetrazolium bromide), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Sigma Chemical Company . Roswell Park Memorial Institute Medium 1640 (RPMI) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco . Methanol was obtained from Fisher Scientific . All other chemicals used in this study were of analytical grade.  DOPE and DSPE-PEGThe cancer cell line MDA-MB-231 (human breast adenocarcinoma) was kindly supplied by Professor Dr. Alfredo Miranda de Goes . The LNCaP (human prostate carcinoma) cancer cell was purchased from American Type Culture Collection (ATCC) . 2000 10 mM, dissolved in chloroform, were transferred to a round bottom flask amounting a total lipid concentration of 20 mM . Similarly, 22.8 mL of DOPE 50 mM, 15.2 mL of CHEMS 50 mM, and 10 mL of DSPE-PEG2000 10 mM, dissolved in chloroform, in a total lipid concentration of 40 mM  were also transferred to a round bottom flask. UA equivalent at 0.1% (w/v) or 0.05% (w/v) was added to the lipid solution. A lipid film was obtained by evaporating the chloroform under reduced pressure. Next, the lipid film was hydrated with an amount of 3.8 mL of NaOH 0.1 M, for 20 mM liposomes, and 7.6 mL of NaOH 0.1 M for 40 mM liposomes to promote the complete ionization of CHEMS. Finally, phosphate buffersaline (PBS), pH 7.4, was added in amount of 46.2 mL for 20 mM liposomes and 42.4 mL for 40 mM liposomes. The obtained mixture was subjected to vigorous shaking in a vortex, producing multilamellar liposomes. The resulting multilamellar vesicles were calibrated using a single-stage high pressure homogenizer, model APV 2000  in recirculation mode. The pressure of the homogenizer was adjusted to 500 bar. The minimum volume of processed samples was 110 mL, and all homogenizations were carried out at room temperature. Each cycle was equal to the passage of the total volume of the sample through the homogenization chamber in a total of twelve cycles. Nonentrapped UA was separated by ultrafiltration, using a polyethersulfone membrane  connected to a tangential flow filtration system . The liposome suspension (100 mL) was transferred to an initial container and pumped to the filtration membrane. Nonentrapped UA (permeated) was recovered in a flask, while purified SpHL-UA was returned to the initial container. This process was maintained until a concentrated SpHL-UA suspension reached the final volume of 25 mL. After the cycle of ultrafiltration was completed, the permeated was taken and the nonentrapped UA concentration was measured by high performance liquid chromatography (HPLC). In addition, the purified SpHL-UA was diluted with PBS buffer pH 7.4 to the final volume of 50 mL. Next, the evaluation of drug entrapment was performed by HPLC , using a multipurpose panoramic irradiator equipped with a Cobalt-60 source  at the Center for the Development of Nuclear Technology-National Commission on Nuclear Energy .μm LiChrosorb, RP-18 column . The eluent system consisted of a 1 : 1 methanol/water mixture, and the flow rate was 1.5 mL·min−1. Samples (20 μL) were injected, and the absorbance of the eluate was monitored at 210 nm.  The evaluation of drug entrapment was performed after having purified the SpHL-UA, which was solubilized by adding methanol. The total amount of UA in the liposomes was also measured after SpHL-UA had been dissolved in methanol, before purification procedures by ultrafiltration. After preparing the samples, as mentioned previously, UA quantification was performed by HPLC. The chromatographic apparatus consisted of a model 515 pump, a model 717 Plus autoinjector, and a model 2487 variable wavelength UV detector  controlled by Empower software. Separations were performed using a 25 cm × 4 mm, 5 μg/mL of UA. The precision of the method was assessed considering repeatability and intermediate precision at three concentration levels of UA  on three different days.  The specificity of the HPLC method was carried out by comparing the peak retention time of UA and the peaks obtained after having injected liposomes without UA (blank liposomes). The linear response was evaluated in the concentration range of 5–60  The mean diameter of SpHL-UA was determined by photon correlation spectroscopy (PCS) at 25°C and at an angle of 90°. The zeta potential was evaluated by determining the electrophoretic mobility at an angle of 90°. The measurements were performed using the Zetasizer 3000 HAS . The samples were diluted with PBS buffer solution. Morphological examination of SpHL-UA was performed by means of TEM using a negative staining method. The liposomes were placed on a formvar-coated copper grid and stained with a 2% (w/v) phosphotungstic acid solution containing 0.5% (w/v) bovine serum albumin and 0.5% (w/v) saccharose. The stained samples were characterized using a Tecnai G2 12 Spirit Biotwin FEI at 80 kV . The determination of the storage stability of SpHL-UA was performed at 15, 30, and 60 days after preparation. These formulations were maintained at 4°C. The parameters evaluated included mean diameter, zeta potential, and drug entrapment. The mean values of these parameters were compared with those obtained at time zero. μg/mL streptomycin and 100 UI/mL penicillin). All cultures were kept in a humidified incubator with 5% CO2 at 37°C. The effect of SpHL-UA on cell viability was evaluated on cancer cell lines MDA-MB-231 and LNCaP. Cells were cultured in RPMI 1640 for LNCaP cells and DMEM medium for MDA-MB-231; all media were supplemented with 10% (v/v) FBS and antibiotics (100 4 (LNCaP) and 2 × 103 (MDA-MB-231) cells/well were seeded into 96-well plates. After 24 h of incubation at 37°C and 5% CO2, freshly prepared solutions of free UA and SpHL-UA were added to the wells. The concentrations assayed were 1.2, 2.5, 5, 10, 20, and 40 μM of UA. Free UA was dissolved in DMSO prior to dilution. After 48 h of incubation at 37°C and 5% CO2, the medium with treatment was removed and discarded. Subsequently, 100 μL of culture medium containing 10% (v/v) FBS and 0.5 mg of tetrazolium/mL was added to each well of the culture plate. After two hours of incubation, MTT crystals were solubilized in 100 μL of a solution containing 10% (w/v) SDS in 0.01 M HCl. Cell viability was estimated by measuring the rate of mitochondrial reduction of MTT determined by evaluating the absorbance of the converted dye at a wavelength of 595 nm. Absorbance values of the wells in which the cells were maintained in medium alone were considered to be 100% of cell viability. The control groups included treatment with DMSO and blank liposomes. Data were expressed as percentage of cell viability compared to the control (mean ± SD). The IC50 values  of free UA and SpHL-UA were calculated using Graphpad Prism 5.0 . At least three independent experiments were performed for each evaluated cancer cell line. Cell proliferation was measured by MTT assay based on the reduction of tetrazolium salt to formazan crystals by living cells . BrieflyP values of less than 0.05 were regarded as significant . The results were expressed as mean values ± standard deviation (S.D.).  Data were subjected to statistical analysis using the one-way analysis of variance (ANOVA), followed by the Bonferroni test, and μg/mL) with a correlation coefficient greater than 0.999 and a linear equation of y = 5001x + 2098. The precision of the method was also confirmed. Whatever the UA concentration level, the overall results showed relative standard deviation values of lower than 5% in all experiments (data not shown). These results were in agreement with requirements for analytical assays [ The HPLC method showed adequate specificity. No interference of the liposome components could be identified, since no overlaps of peaks were detected after the injection of UA at the set wavelengths (data not shown). The linear response was obtained in the evaluated UA concentration range  and above characterize a stable formulation, since the aggregation of the particles is less likely to occur due to electrical repulsion forces . However0-lipids .P = 7.92) [ Stability in terms of UA entrapment can be explained by the UA lipophilic character (log = 7.92)  which co The images of SpHL 20UA1 obtained by TEM  allowed 50 values were calculated for each cell line and are summarized in  To evaluate the effects of SpHL 20UA1 on breast and prostate cancer cell lines viability, an MTT assay was employed. The IC50 value obtained after SpHL 20UA1 treatment (48 h) was significantly lower than IC50 value obtained after UA free treatment (P < 0.05) for MDA-MB-231 cancer cell line. This result indicates that SpHL can improve UA delivery in this breast cancer cell line. There was not a significant difference among the IC50 values obtained after SpHL 20UA1 and free UA treatments (P > 0.05) for LNCaP cancer cell line. These findings revealed that the incorporation of UA into SpHL did not provoke any impairment in the effect of UA on carcinoma cells viability. Thus, the use of SpHL 20UA1 may be a promising strategy to carry UA and allow for its intravenous administration.  In in vivo applications. This work also investigated the effects of SpHL-UA on breast and prostate cancer cell lines viability, which demonstrated that, after 48 h, this treatment significantly inhibited cancer cell proliferation, as shown by MTT assay. Results from this study indicate that SpHL-UA can be an interesting delivery system for the pharmaceutical formulation of UA and may represent a good and useful chemotherapy agent for breast and prostate cancer treatment. Further evaluations, such as pharmacokinetics studies and antitumor activity in vivo, should be performed to confirm these expectations. In conclusion, the results of the present study demonstrated that SpHL-UA had a good stability in terms of mean vesicle size, zeta potential, and UA entrapment after storage for 2 months at 4°C. Thus, the production of these liposomes proposed in this work led to liposomal dispersion with features suitable for future


Drosophila Toll was initially discovered as the factor responsible for determining dorsoventral polarity in Drosophila embryo, and the subsequent studies revealed its antifungal functions. In the late 1990s, mammalian homologs of Toll were determined as Toll-like receptors (TLRs). Early studies on TLRs attempted to identify their specific ligands, new family member of TLRs, and their intracellular signal transduction pathways. Intracellular signal transductions of TLRs share common elements with IL-1 receptor downstream intracellular signaling. Subsequent studies for TLRs have focused on infectious diseases, since TLRs recognize pathogen-associated molecular patterns (PAMPs) and induce strong responses for host defense. Recently, endogenous TLR ligands released from dying and/or damaged cells were identified. These studies have extended the idea that TLR signaling is also activated in the absence of exogenous pathogens and in general sense “danger signals” to alert the host of either exogenous or endogenous “danger.” Liver has a unique anatomy that is closely associated with the intestines through the portal vein and bile ducts. Even when intestinal barrier functions are intact, liver is constantly exposed to low levels of microbial products derived from commensal microflora through portal vein blood. Liver contains not only parenchymal hepatocytes, but also nonparenchymal immune cells and nonimmune cells. Hepatic immune cells include Kupffer cells (resident liver tissue macrophages), T cells, B cells, dendritic cells, NK cells, and NKT cells. These cells produce a broad array of cytokines upon activation of TLRs. nonimmune cells in the liver include endothelial cells, biliary epithelial cells, and hepatic stellate cells. Hepatic nonimmune cells also express TLRs and respond to TLR ligands to induce innate immune responses including cytokine and type I IFN production. However, the liver in normal condition prevents spontaneous inflammation induced by microbial products through TLRs due to the specific barrier functions in the liver and intestines or the “liver tolerance” regulated by intercellular and intracellular mechanisms. Upon breakdown of this regulation, inflammation is induced through innate immune responses including TLR signaling in the liver. Moreover, sterile inflammation-associated danger signals may also trigger liver inflammation through TLRs. Thus, acute and chronic liver diseases are highly associated with triggering TLR signaling by intestine-derived microbial products and sterile insult-associated products from damaged cells. Toll-Like Receptor Signaling in Liver Diseases, the most recent advances in TLR research in the liver are reviewed by worldwide authorities of liver TLR research. The first part of this issue overviews TLR signaling in general. In the first paper, Drs. M. Yamamoto and K. Takeda reviews the current views of TLR signaling including their discovery of the functions of all four TLR adaptor proteins by generating knockout mice. In the second paper, Drs. A. E. Bigorgne and I. N. Crispe review the area of hepatic intercellular crosstalk mediated by TLRs. In the third paper, the research group of Drs. M. Fujimoto and T. Naka have cloned SOCS-1 and reported that SOCS-1 negatively regulates TLR signaling by SOCS-1. They summarize the negative regulation of TLRs by SOCS proteins and the previous reports studying the SOCS family in human liver disease. In the next paper, Dr. H. Tsutsui and her colleagues summarize their studies that have identified TLR-mediated IL-1β and IL-18 processing through activation of the inflammasome in the liver. In the fifth paper, Dr. M. Gale Jr. and his research group have uncovered the intracellular innate immune signaling against HCV infection. He and his colleague review the biological host response against HCV and how HCV escapes from RIG-I-dependent innate immune response for sustaining HCV infection. In the present special issue of α and IL-6 are known to trigger liver regeneration. They discuss the role of TLR/MyD88-dependent signaling upstream TNF-α and IL-6 in liver regeneration. They also describe the possible interaction between NF-κB and JNK/c-Jun in liver regeneration. In the eighth paper, cutting-edge research on TLRs in ischemia-reperfusion injury has been done by the research group led by Drs. A. Tsung and T. Billiar. They were the first to identify that HMGB-1 released from damaged cells is an endogenous ligand for TLR4 and that HMGB-1 triggers ischemia-reperfusion liver injury through TLR4. The interaction between alcoholic liver disease and TLR signaling has been studied for more than ten years. Dr. Thurman's research group demonstrated the role of TLR4 and gut microflora in alcoholic liver disease using intragastric ethanol infusion model. Currently, Dr. Szabo's research group advances the studies in this field. In ninth paper, she and her colleagues concisely summarize the importance of TLRs and adaptor molecules in alcoholic liver disease. The importance of TLRs has been demonstrated not only in alcoholic liver disease, but also in nonalcoholic liver disease. In the tenth paper, Drs. K. Miura and D. Brenner outline the recent advance of TLRs and nonalcoholic liver disease. In the next paper, Dr. E. Seki and his colleagues have demonstrated the interaction of the TLR4 and TGF-β signaling pathways in hepatic stellate cells. They discuss the signaling of TLR4 and the other TLRs in the activation of hepatic stellate cells and liver fibrosis. The proportion of NK cells and NKT cells in liver is much greater than that in the other organs. These cells are major targets for TLR3 ligand poly I:C. Dr B. Gao's research group is focsing on this signaling in liver physiology and in liver disease models. In the twelfth paper, they summarize their recent findings of TLR3 signaling in liver disease. In the following paper, Dr. S. Maeda demonstrated the role of hepatocyte NF-κB, JNK, and MyD88 using an animal model of liver cancer. He discusses the role of TLRs, NF-κB, and JNK signaling in liver cancer. Modification of gut microflora might be a potential target for liver disease because of the unique anatomical association between the liver and intestines. In the fourteenth paper, Dr. I. Hines and his colleague review the molecular mechanisms by which gut microflora contributes to the development of liver disease. In the fifteenth paper, Drs. T. Hayashi and K. Suzuki highlight their previous work on TLR4-mediated expression of protein S and C4b binding protein in the liver. The role of TLR4 signaling in the development of insulin resistance is intensively being studied. In the last paper, Drs. J. J. Kim and D. D. Sears summarize the recent advances in the field of innate immunity and insulin resistance. The next sections address specific liver functions and diseases. In the sixth paper, Dr. K. Machida summarizes his previous work studying the association between HCV and TLR4, and now his research is extending to the study of HCV-mediated tumorigenesis in which cancer stem cells could be crucial, and its possible cross-talk with alcohol. In the following paper, Drs. Y. Iimuro and J. Fujimoto review molecular mechanisms triggering liver regeneration after partial hepatectomy. TNF-In this special issue, we gather 16 articles highlighting our current knowledge of TLR signaling in the liver. TLR signaling induces the initial response in inflammation. This response may then induce intestinal barrier destruction and subsequent bacterial translocation as well as the release of endogenous ligands from damaged cells. These events could cause a secondary activation of TLR signaling. Thus, targeting either the initial or secondary responses induced by TLR signaling might become effective therapy for liver diseases. While we could not include discussion of the potential of TLR agonists for the therapy of liver disease in this special issue due to page limitations, this might be an attractive approach for some liver diseases, such as liver fibrosis and cancer. We trust that the articles in this special issue will help in understanding the TLR-related mechanisms underlying liver diseases and stimulate new ideas for targeting TLRs and their signaling pathways to develop new therapeutic applications for liver disease. Gyongyi SzaboGyongyi SzaboTimothy R. BilliarTimothy R. BilliarKeigo MachidaKeigo MachidaIan Nicholas CrispeIan Nicholas CrispeEkihiro SekiEkihiro Seki


The discovery of miRNAs has revolutionized the way we examine the genome, RNA products, and the regulation of transcription and translation. Their ability to modulate protein expression through mRNA degradation and translation repression resulted in avid scientific interest in miRNAs over the past decade. This research has led to findings that indicate miRNAs can regulate an array of cellular functions such as cellular apoptosis, proliferation, differentiation, and metabolism. Specifically, the capability of miRNAs to finely-tune gene expression naturally lends itself to immune system regulation which requires precise control for proper activity. In fact, abnormal miRNAs expression is often seen with inflammatory disorders like rheumatoid arthritis, systemic lupus erthematosus, experimental autoimmune encephalomyelitis, and inflammatory cancers. As a result, research investigating miRNAs modulation of immune cell proliferation, differentiation, and cellular signaling has yielded fruitful results. Specifically, in this review, we will examine the impact of miRNAs on toll-like receptor (TLRs) and interleukin-1β (IL-1β) signaling, which are integral in the proper functioning of the innate immune system. These signaling pathways share several key downstream signaling adaptors and therefore produce similar downstream effects such as the production of pro-inflammatory cytokines, chemokines, and interferons. This review will examine in depth the specific interactions of miRNAs with receptors, adaptor molecules, and regulator molecules within these cellular pathways. In addition, we will discuss the modulation of miRNAs’ expression by TLR and IL-1R signaling through positive and negative feedback loops. The existence of a molecular system capable of orchestrating the expression of numerous proteins was postulated by researchers for some time; the discovery of microRNAs (miRNAs) confirmed this hypothesis. This breakthrough revolutionized the way we examine the genome organization, RNA products, as well as mRNAs translational and degradation. Originally discovered in 1993, it soon became evident that miRNAs are abundant and often conserved across species. Even more striking is their ability to modulate a wide range of cellular functions, such as apoptosis, proliferation, differentiation, and metabolism. In addition to cellular activities, miRNAs were also found to be capable of regulating a broad spectrum of systemic functions like inflammatory responses. The nature of miRNAs function, to finely-tune protein expression through mRNAs degradation and translation repression, naturally lends itself to immune system regulation which requires precise control for proper activity. Therefore, it is not coincidence that altered miRNAs expression is often associated with progression and remission of inflammatory disorders like rheumatoid arthritis, systemic lupus erythematosus, and experimental autoimmune encephalomyelitis. Ensuing research investigating miRNAs modulation of immune cell proliferation, differentiation, activity, and cellular signaling has yielded fruitful results. Specifically, in this review, we will examine the impact of miRNAs on the integral toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) inflammatory signaling pathways. Both pathways share several key signaling adaptors and therefore induce similar cellular responses when activated. Together they play an indispensable role in the innate immune system by triggering the production of pro-inflammatory proteins like cytokines, chemokines, and interferons. The specific interactions of miRNAs with receptors, adaptor molecules, and regulator molecules within these cellular pathways will be examined in depth. Finally, the modulation of miRNAs expression by TLR and IL-1R signaling will be reviewed.Prior to recent breakthroughs, it was believed that the majority of DNAs were transcribed to mRNAs and then translated into proteins. The remaining RNAs not coding for protein were either ribosomal RNAs, transfer RNAs, or perceived as “junk” with little to no pertinent function. In 2003, at the completion of the Human Genome Project, between 20,000 – 25,000 protein coding genes were identified from the 3 billion nucleotide base-pairs of the human genome. Puzzling was the fact that protein coding sequences only accounted for approximately 2% of the total genome . These fFrom a biogenesis standpoint, miRNAs are transcribed in similar fashion to mRNAs encoded by protein coding genes. Their genomic sequence can be found within introns of other genes or can be encoded independently. Once transcribed into primary miRNAs, which can be several hundred base-pairs in length, the transcripts may undergo editing by adenosine deaminases. This post-transcriptional modification leads to the exchange of adenosine with inosine which results in an alteration to the transcript’s base-pairing, potentially leading to structural changes. After this, the transcripts undergo processing by the RNase II enzyme Drosha within the nucleus. Cleaved to a length of approximately 70 base-pairs, these precursor miRNAs are then exported from the nucleus to the cytoplasm by exportin 5 and Ran-GTP. Within the cytosol the precursor miRNAs are further cleaved by the RNase-III processing enzyme Dicer to their mature length of 18–23 base-pairs. Upon dicer cleavage, duplexes formed by the hairpin structure of the precursor miRNAs are unwound by RNA helicases  of mRNAs through imperfect Watson and Crick base-pairing. Noteworthy is that imperfect pairing facilitates the possibility of multiple binding sites for a single miRNA with a particular mRNA. It also promotes promiscuity allowing for multiple mRNA partners for a single miRNA. However, the molecular details of how miRNAs specifically target mRNAs temporally and dynamically are still unknown. Despite this, several structural features which promote miRNAs:mRNAs binding have been elucidated. It has been determined that a region of 6–8 nucleotides, at residues 2–7 on the 5’ end of miRNAs, is critical in establishing miRNAs:mRNAs interactions and is commonly referred to as the “seed region” -12. MeanAs mentioned previously, miRNAs can biologically cause mRNAs cleavage or translation repression. miRNAs-directed endonuclease cleavage of mRNAs is functionally carried out by the highly conserved family of Argonaute proteins . The preTranslational repression is the other mechanism utilized by miRNAs. Described as early as 1993, several studies have been conducted with a range of miRNAs that demonstrate a reduction in protein expression in the midst of unwavering or minimally altered mRNAs levels ,38-42. DMeanwhile, sedimentation studies conducted with mRNAs under miRNAs repression yield results which indicate the association of polysomes. This provides evidence that particular miRNAs function through post-initiation translational interference ,49,55-58The recent discovery of a class of evolutionarily-conserved receptors has led to the re-examination of the signaling and function of the innate immune system. TLRs are a class of membrane-bound pattern recognition receptors that are capable of identifying particular pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Initially discovered in insects, the Toll receptor was determined to play a function in innate immune protection against fungal infections. Pursuit of Toll homologues in humans revealed 11 members, which can identify a wide range of ligands. As Type I integral membrane glycoproteins, TLRs share significant cytoplasmic region homology with interleukin-1 receptors (IL-1R). Within the cytoplasmic tails of the TLRs/IL-1R is a highly conserved region of approximately 200 amino acids referred to as the Toll/IL-1R (TIR) domain . This coThe cytokine interleukin-1 (IL-1) was first discovered in the early 1940s. Produced by active leukocytes and found to induce fever, the protein was aptly named “pyrexin” or “endogenous pyrogen” . It lateFollowing Toll-like/IL-1 receptor activation conformational changes occur which trigger a shared downstream signaling cascade Figure  74]. My. My74]. Containing six family members, the TNF receptor associated factors (TRAFs) are a group of evolutionary conserved adaptor molecules . StructuAside from NF-κB activation induced by the TLR/IL-1 signaling just discussed, an alternative pathway can be triggered midway through the signaling cascade by both TLRs and IL-1R. This results in the activation of p38 mitogen-activated kinases (p38), c-Jun N-terminal kinases (JNKs), extracellular signal-regulated protein kinases (ERKs), and the two transcription factors - activator protein-1 (AP-1) and activating transcription factor (ATF) -94. WhilWhile mice deficient of MyD88 have displayed an inability to produce IL-6 or TNF-α when exposed to TLR microbial ligands or IL-1, more extensive experimentation with LPS resulted in delayed NF-kB activation and INF-β production. These results indicated the existence of a MyD88-independent pathway Figure . It has Although the MyD88-independent signaling pathway is less elucidated than the MyD88-dependent pathway, some key players have been identified. Required for interfacing with all TLRs is the presence of a TIR-domain within the direct downstream adaptor protein. To identify this protein, researchers relied on bioinformatics which led to the discovery of TIR-domain-containing adaptor protein inducing IFN-β (TRIF). It was quickly established that TRIF mediates the MyD88-independent pathway based on experimental results in TRIF-deficient mice which displayed impaired IFN-β production (IRF3) upon stimulation with TLR-3 and −4 ligands . Signaliin silico studies [Another TIR-domain containing protein, TRIF-related adaptor molecule (TRAM), was also identified by  studies . Again,  studies . This suFurther examination in TRAM knockout mice revealed that TLR-4-mediated NF-κB activation was impaired in a time-dependent fashion. Initial NF-κB activation, 10–20 minutes following LPS treatment, was undeterred while levels were diminished at 60–120 minutes when compared with wild-type controls. Similarly, JNK activity was also found to be limited in a time-dependent manner . These fIL-6, a known proinflammatory cytokine, is produced in response to challenge with several TLR ligands and IL-1 signaling. However, the exact signaling required for this induction has not been resolved; specifically, the upstream signal transduction. It has been reported that the NF-κB subunit, p50, can increase IL-6 expression. Furthermore, signal transducer and activator of transcription 5 (STAT5) is activated by LPS treatment in mouse monocyte/macrophage RAW cells and peritoneal macrophages. A possible interaction between p50 and STAT5 was examined and confirmed with co-immunoprecipitation assay in African green monkey kidney fibroblast-like COS-7 cells. To determine the mechanism of IL-6 induction, a chromatin immunoprecipitation assay was conducted and showed that both p50 and STAT5 located to the promoter of IL-6. Traditionally, functioning upstream of STATs are Janus kinases (JAKs). In particular, JAK2 expression was found to be augmented within 1–5 minutes following LPS treatment in RAW cells. This finding, along with comparative downstream effects following deletion of either JAK2 or STAT5, indicates that both are involved in this signaling pathway. Investigation of further upstream signaling revealed that JAK2 could not associate individually with either MyD88 or TLR-4 in co-immunoprecipitation assays. However, JAK2 was found to interact with the MyD88/TLR-4 complex. This evidence suggests the existence of an unidentified adaptor molecule that facilitates this interaction. Interestingly, IL-6 has been demonstrated to induce suppressor of cytokine signaling 1 (SOCS1), a known negative regulator of cytokine signaling. The role of SOCS in TLR/IL-1 signaling will be discussed in-depth in the following section .Hyper-activation of immune signaling pathways can seriously impact overall health, leading to a wide variety of chronic inflammatory and autoimmune illnesses. Each adaptor molecule within the TLR/IL-1 signaling pathway is under stringent control via phosphorylation, physical interactions, conformational changes, as well as ubiquitin and proteasomal degradation. Furthermore, there are regulatory proteins designed to attenuate TLR/IL-1 signaling which will now be discussed.A family of eight proteins aptly referred to as the suppressors of cytokine signaling (SOCS), primarily function to inhibit cytokine signaling. Specifically, SOCS1 is induced by LPS and CpG-containing DNA, known activators of the TLR/IL-1 signaling . This leSH2-containing inositol phosphatase, SHIP1, is a protein tyrosine phosphatase and a potent endogenous NF-κB inhibitor. SHIP1 has been shown to negatively regulate several tyrosine kinase-dependent signaling pathways in myeloid cells, B-lymphocytes, and T-lymphocytes . The expThe immunosuppressive/anti-inflammatory interleukin, IL-10, has also been attributed with mitigating TLR/IL-1 MyD88-dependent signaling but not MyD88-independent signaling. Levels of IL-6 were found to be augmented in the presence of an IL-10R antagonist and diminished with IL-10 treatment following exposure to MyD88-dependent signaling activator CpG-containing DNA. This effect was not seen when treated with PolyI:C, a MyD88-independent signaling ligand. The diminished MyD88-dependent signaling associated with the treatment of IL-10 can be attributed to diminished protein levels of IRAK4 and TRAF6. In fact, IL-10 antagonism led to prolonged protein levels of IRAK4 and TRAF6 following LPS treatment in comparison to controls. This observation appears to be the direct result of increased ubiquitination of IRAK1, IRAK4, and TRAF6 seen with the treatment IL-10. In support of these findings, the effects of IL-10 on MyD88-dependent signaling were abolished with proteasomal inhibition . It shouCrucial for proper immune system function, TLR/IL-1 signaling is under stringent regulation. This adept regulation is required to ensure proper host defense while mitigating the deleterious effects of the inflammation process. Previously discussed regulators like SOCS1 and SHIP1 can inhibit various aspects of this shared pathway via transcription and post-translational mechanisms, and we will now discuss the fine-tuned regulation of the TLR/IL-1 pathway by miRNAs via miRNAs-directed mRNAs translation repression or cleavage ,7.Cryptosporidium parvum led to the up-regulation of TLR4 protein expression but not its mRNAs. This alteration in TLR4 protein expression could be diminished or augmented when cells were transfected with either let-7i mimic or inhibitors respectively [The most obvious way that miRNAs can affect TLR/IL-1 signaling is by altering the expression of the receptors. However, examination of TLRs and IL-1R for highly conserved miRNAs consensus binding regions with target prediction software like TargetScan and PicTar have yielded few results. This may be the result of species-specific regulation of TLRs and IL-1R by poorly conserved sites or it might be that the critical role of TLRs and IL-1R in immunity facilitates the need for tight transcriptional control or the need for constitutive expression. With that said, the regulation of TLR-2 and −4 by miRNAs has been observed. The direct association of miR-105 with TLR2 mRNAs has been witnessed while working in human oral keratinocytes, while the regulation of TLR4 expression by let-7i in human biliary epithelial cells and by let-7e in macrophages has been reported , macrophages, and inflamed lung tissue is miR-21 ,145-147.The TLR/IL-1 signaling pathway is critical to proper immune functioning. Hyper-activation has been associated with several autoimmune and chronic pathologies while hypo-activation leaves the host highly susceptible to infection. Therefore, it is not surprising that this signaling pathway is under stringent control by phosphorylation, physical interactions, conformational changes, regulatory proteins, as well as ubiquitin and proteasomal degradation. We have also discussed the activity of miRNAs in further modulating this signaling system. Interestingly, many of these exact miRNAs are also induced by the activation of the TLR/IL-1 signaling cascade, indicating a mutual regulation between miRNAs and TLR/IL-1 pathway via a complex positive- and negative-feedback loop system . The rolThe authors declare that they have no competing interests.AV carried out the primary literature search and draft writing. HW and XFY provided field expertise, material input, and manuscript oversight. All authors read and approved the final manuscript.


Current studies are also aimed at determining the prevalence, distribution, and modes of transmission of these viruses, as well as their possible association with human diseases. Here we will review the characteristics of these new simian and human retroviruses, whose discovery has opened new avenues of research in the retrovirology field.Human T cell leukemia/lymphoma virus Type 1 and 2 (HTLV-1 and HTLV-2), together with their simian counterparts , belong to the Primate T lymphotropic viruses group (PTLV). The high percentage of homologies between HTLV-1 and STLV-1 strains, led to the demonstration that most HTLV-1 subtypes arose from interspecies transmission between monkeys and humans. STLV-3 viruses belong to the third PTLV type and are equally divergent from both HTLV-1 and HTLV-2. They are endemic in several monkey species that live in West, Central and East Africa. In 2005, we, and others reported the discovery of the human homolog (HTLV-3) of STLV-3 in two asymptomatic inhabitants from South Cameroon whose sera exhibited HTLV indeterminate serologies. More recently, two other cases of HTLV-3 infection in persons living in Cameroon were reported suggesting that this virus is not extremely rare in the human population living in Central Africa. Together with STLV-3, these human viral strains belong to the PTLV-3 group. A fourth HTLV type (HTLV-4) was also discovered in the same geographical area. The overall PTLV-3 and PTLV-4 genomic organization is similar to that of HTLV-1 and HTLV-2 with the exception of their long terminal repeats (LTRs) that contain only two 21 bp repeats. As in HTLV-1, HTLV-3 Tax contains a PDZ binding motif while HTLV-4 does not. An antisense transcript was also described in HTLV-3 transfected cells. PTLV-3 molecular clones are now available and will allow scientists to study the viral cycle, the tropism and the possible pathogenicity  We will2.Papio hamadryas) that had been kept in captivity in a research laboratory in Leuven (Belgium) [gag and the rgp21env proteins when tested with a commercial HTLV Western Blot (WB) assay. In addition, one of them reacted with the HTLV-2 gp46 specific K55 envelope peptide, resulting in a WB pattern that was indistinguishable from that of the HTLV-2 positive control [Cercocebus Torquatus, Cercopithecus nictitans, Papio papio, Theropithecus gelada, Cercopithecus cephus, Lophocebus albigena, Cercocebus agiles and lastly Cercopithecus mona)  as determined with an HTLV-1/2 commercial Western-blot assay (HTLV2-4 WB) and/or with INNOLIA kits [The first strain of STLV-3 (strain STLV-L/PH-969) was isolated in 1994, after the long-term co-culture of human cord blood lymphocytes with the peripheral blood mononuclear cells (PBMCs), obtained from an Eritrean sacred baboon (Belgium) . The serus mona) . Some ofLIA kits –10,12. Hi.e., age, sex, modes of transmissions) of STLV-3 infection within infected non-human primates (NHP). Indeed, to date, most studies were performed in wild-caught monkeys [Papio hamadryas, originating from Eritrea and kept in Leuven, were infected [Papio papio kept in a laboratory in France, but originating from Senegal, 4 out of the 9 baboons were found to be infected with STLV-3 [Cercopithecus nictitans, Cercocebus agilis, and Lophocebus albigena [In comparison to STLV-1, very limited data is available on the major epidemiological determinants . Based on partial or complete sequence analysis, the STLV-3 lineage is now currently considered to be composed of four main subtypes  that roughly correspond to the geographical source of the strains. The subtype A comprises strains from East Africa (Ethiopia and Eritrea), the subtype B corresponds to strains from West and Central Africa, while the subtypes C and D comprise strains from Central Africa .3.p19 and against the HTLV-1 envgp46 MTA-1 peptide [p19, gagp24, and envGD21 as well as a weak signal against the HTLV-1 envgp46 MTA-1 peptide [p19, gagp24, envGD21 reactivity and weak HTLV-2 gp46 envK55 signal). The sequence of the virus is closely related to the first HTLV-3 strain (Pyl43) [Cercopithecus mona from a close geographical area [In 2005, we and others (W. Switzer and W. Heneine's team from the Center for Diseases Control) reported the discovery of HTLV-3, a third HTLV type, in two Cameroonese asymptomatic individuals living in the rainforest area of the southern part of the country [ peptide . His ser Pyl43) . Both vi (Pyl43) . It orig3 . Both per se occurred between hunter and NHP contact, the exact mode(s) of transmission remains speculative. Theoretically, any contact between an STLV-3-infected body fluid from a NHP and blood or muco-cutaneous tissue (with or without a lesion) could allow such zoonotic transmission. However, it is worthwhile to note that none of the four infected HTLV-3 persons ever reported a bite wound by an NHP [An important question concerns the origin of HTLV-3 in humans. Based on the close homologies between some STLV-3 and HTLV-3 strains, and consistent with what is known for the PTLV-1 viruses, it is very tempting to suggest a zoonotic origin of HTLV-3 after interspecies transmission from STLV-3 infected NHP to humans. The diversity of HTLV-3 implies several episodes of such transmission. Exactly when these episodes occurred is currently unknown. However, the limited divergence that is observed between some STLV-3 and HTLV-3 strains  strongly suggests a recent non-human primate to human transmission . Lastly,y an NHP ,3,15,17.y an NHP . In the 4.p19, weak reactivities against envrgp21 and HTLV-2 K55 envgp46 peptide and no reactivity against gagp24 (The fourth HTLV type HTLV-4) consists only, so far, of a unique human strain, whose provirus was also found in the PBMCs obtained from a hunter living in Cameroon . The sert gagp24 . The com consists5.gag, pro, pol, env, tax and rex genes  ,22,23. S5.1.in vitro. The results also revealed that Tax3 is functionally closely related to the transforming HTLV-1 Tax. It is indeed able to transactivate cellular promoters, to activate the NF-κB pathway, to bind the CBP transcription co-activator and can also repress the function of the p53 tumor suppressor factor. Altogether, these results suggested that HTLV-3 might be pathogenic in vivo. As stated above, cases of STLV-1/STLV-3 naturally co-infected animals have been described. It was therefore interesting to show that Tax3 can transactivate the heterologous HTLV-1 promoter [Some HTLV-3 Tax transactivator protein functions have been characterized. Even if it was shown that HTLV-1 and HTLV-3 Tax protein do not similarly bind p300, it was demonstrated that Tax1 and Tax3 proteins display a number of key similarities including the presence of a PDZ binding motif, which is absent from the Tax2 protein ,23. Thispromoter ,23. Whet5.2.in vivo. An antisense transcript was, however, found in 293T cells transfected with an HTLV-3 molecular clone [Since its discovery, it has not been determined whether the HTLV-3 genome, as HTLV-1, encodes any auxiliary proteins that would be functionally similar to p12, p13 and p30, which are known to play important roles in the viral life cycle ar clone . WhetherRorfII) that would encode a 64 amino acid highly hydrophobic and leucine rich protein was reported in vivo [Regarding STLV-3, the presence of a doubly spliced mRNA  and NRP-1 (neuropilin-1)—two molecules important for HTLV-1 entry—enhance HTLV-3 SU binding in vitro [+ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1 and GLUT-1. Altogether, these results indicated that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2 [In contrast to HTLV-1 and HTLV-2, it was shown that the HTLV-3 surface glycoprotein (gp46 or SU) binds to both activated CD4in vitro . Howeverd HTLV-2 .6.In silico analyses have shown that the Tax protein from HTLV-4 lacks a PDZ binding motif, as is the case for HTLV-2. The same analyses suggested that the HTLV-4 provirus might encode 68 and 93 amino acid long auxiliary proteins. The first one, HTLV-4 ORF-IV, shares 75% similarity with the HTLV-1 p13II protein, while the second HTLV-4 ORF-V displays a very weak similarity to the C-terminus of the HTLV-2 p28 protein. Finally, an ORF that would allow transcription from the 3′ LTR is also present in the HTLV-4 genome [The molecular data on HTLV-4 is very limited. As for PTLV-3, its LTR sequence lacks the distal 21 bp repeat sequence and therefore contains only two of these elements [7.In conclusion, we would like to reiterate four main points.p19 or gagp24 reactivities [Plasmodium falciparum antigens (as demonstrated in a series of cases from Cameroon) [(1) The first point regards the significance of the HTLV indeterminate WB serological patterns in Central Africa –30 even tivities . Recentltivities . In mosttivities . Severalameroon) , or even(2) The second point concerns the prevalence and the distribution of HTLV-3 and 4 viruses in Central African populations  and 3, TIt is also important to note that there is currently no serological test available for specifically screening HTLV-3/4 viruses . In factin vivo and their possible associated pathology. Indeed, a case-control study based on a larger number of HTLV-3/4 positive individuals would be useful to shed light not only on the possible intra-familial transmission of these viruses but also on any possible chronic diseases and/or biological abnormalities associated with such viral infection in humans. However, one should keep in mind that, in the HTLV-1 situation, the incidence of TSP/HAM or ATL diseases is very low and follows a long latency period. If HTLV-3 infection is associated with a disease, it might therefore be necessary to follow a large cohort of infected individuals for a long period of time before one can observe any disease. Furthermore, the selection bias inherent to the enrollment of healthy persons in previous studies greatly limits the ability of identifying any acute or severe HTLV-3/4-associated diseases. A sampling conducted among hospitalized patients, including, among others, patients with neurological disorders, cancer or immunodeficiency is thus necessary, likely in Africa where the virus is known to be present. Regarding the viral tropism and the pathogenicity of PTLV-3, colonies of infected STLV-3 non-human primates are available and should allow some answers to these questions.(3) The third question concerns the modes of transmission of these viruses, their tropism versus the acquisition from other infected humans through sexual activity or breast-feeding. As an example, the immense majority of HTLV-1/2 infected individuals living in Central Africa, have very probably been infected through human contacts  and not from NHPs contacts. As seen above, this contrasts with foamy viruses, where most, if not all, human carriers were directly infected from foamy virus infected NHPs during hunting activities, probably mostly through severe bites [(4) Lastly, we would like to raise the question of the origin of HTLV-3/4 in humans. Even if it seems quite understandable that these viruses originally arose through interspecies transmission from STLV-3/4 infected monkeys to humans, it is difficult to speculate on the relative part of the direct acquisition stemming from infected monkeys re bites –20. Regare bites ,38. For re bites . STLV-3,re bites . Such stre bites .In conclusion, and as suggested recently ,40, the


The ventricularis and fibrosa of porcine aortic valve leaflets were exposed simultaneously to different combinations of sub-physiologic/physiologic/supra-physiologic levels of FSS magnitude and frequency for 24, 48 and 72 hours in a double cone-and-plate device. Endothelial activation and paracrine signaling were investigated by measuring cell-adhesion molecule  and cytokine  expressions, respectively. Extracellular matrix (ECM) degradation was characterized by measuring the expression and activity of the proteases MMP-2, MMP-9, cathepsin L and cathepsin S. The effect of the FSS treatment yielding the most significant pathological response was examined over a 72-hour period to characterize the time-dependence of FSS mechano-transduction. While cytokine expression was stimulated under elevated FSS magnitude at normal frequency, ECM degradation was stimulated under both elevated FSS magnitude at normal frequency and physiologic FSS magnitude at abnormal frequency. In contrast, combined FSS magnitude and frequency abnormalities essentially maintained valvular homeostasis. The pathological response under supra-physiologic FSS magnitude peaked at 48 hours but was then maintained until the 72-hour time point. This study confirms the sensitivity of valve leaflets to both FSS magnitude and frequency and suggests the ability of supra-physiologic FSS levels or abnormal FSS frequencies to initiate CAVD mechanisms.Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between cardiovascular risk factors, molecular signaling networks and hemodynamic cues. While earlier studies demonstrated that alterations in fluid shear stress (FSS) on the fibrosa could trigger inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific FSS abnormalities are poorly understood. This knowledge could be critical to the elucidation of key CAVD risk factors such as congenital valve defects, aging and hypertension, which are known to generate FSS disturbances. The objective of this study was to characterize  As the underlying cause of 30,000 deaths annually and 55,000 hospital discharges, aortic valve disorders have tremendous economic and societal impact While CAVD has been described historically as a passive degenerative process, it has now emerged as a highly regulated pathology presumably triggered by a combination of conventional cardiovascular risk factors ex vivo exposure of porcine aortic valve leaflets to side-specific alterations in FSS magnitude and/or frequency and the biological assessment of the acute pathological response in terms of inflammation and remodeling via immunostaining, immunoblotting and zymography. The results reveal the key role played by FSS abnormalities in the active regulation of early CAVD pathogenic events.Therefore, the goal of this study was to determine for the first time the role of FSS magnitude and/or frequency abnormalities in early CAVD pathogenesis. The methodology involved the in vitro2 on the ventricularis and a bidirectional oscillatory shear stress varying between −8 and +10 dyn/cm2 on the fibrosa. In addition to the physiologic FSS signal (group 1), eight abnormal FSS conditions were generated by decreasing/increasing the magnitude and/or frequency of the physiologic signal , 0.2% Trixon-100 (Sigma-Aldrich) and 1% dimethyl sulfoxide  in 1x PBS for 1 hour at room temperature. Following the blocking step, the slides were then incubated overnight at 4°C in primary antibody in 2–10% blocker at the following dilutions: VCAM-1 , ICAM-1 , TGF-β1 , BMP-4 , cathepsin L , cathepsin S , MMP-2  and MMP-9 . Following primary antibody incubation, sections were washed 3 times in 1x PBS and incubated with anti-rabbit or anti-mouse  secondary antibodies at 1∶100 dilution for 2 hours at room temperature. The tissue sections were then washed 3 times in 1x PBS, counterstained with 1 4′,6-Diamidino-2-phenylindole , mounted with fluorescence mounting medium (Dako), coverslipped and stored at 4°C. Slides were subsequently imaged under the mercury lamp of a Nikon E600 imaging microscope using a TR/FITC/DAPI filter.Following each mechanical treatment, the specimens were pulverized using a mortar and pestle in liquid nitrogen, homogenized in ice-cold RIPA buffer (Santa-Cruz) and centrifuged at 7,000 g to pellet extracellular matrix debris for 8 minutes at 4°C. The supernatant was assayed for protein concentration using a bicinchoninic acid (BCA) protein assay (Pierce). Equal amounts of tissue lysates were resolved by reducing SDS-PAGE. After transfer to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore) using a mini trans-blot cell (Bio-Rad), the blots were blocked with 5% non-fat drymilk and probed with a primary antibody against BMP-4 , TGF-β1 . Depending on the primary antibody, appropriate anti-rabbit and anti-mouse HRP secondary antibody  was then used. The membranes were finally incubated in horseradish peroxidase-conjugated streptavidin. Immunopositive bands were then detected using a luminol-based chemiluminescence reagent (Pierce) against standard radiography film in a darkroom. The films were then analyzed by densitometry using ImageJ (NIH).Zymography was performed to quantify the activity of the proteolytic enzymes MMP-2 and MMP-9. Equal amounts of tissue lysates assayed by BCA were resolved by sample buffer (Bio-Rad) and loaded in the gel (Bio-Rad). After running, the gels were washed in renaturation buffer (Bio-Rad) and developing buffer (Bio-Rad). The gels were stained by adding stain solution (Sigma-Aldrich), de-stained in deionized water and scanned. The images were then analyzed by densitometry using ImageJ.The semi-quantitative assessment of immunostained images was carried out using ImageJ. Briefly, the intensities of positively stained regions were estimated and normalized by the number of cells visible in the images to yield a quantity consistent to a biomarker expression per cell. Distinction was not made between endothelial and interstitial cells during the cell counting.The dedicated macro of ImageJ was used to plot the histogram of individual lanes/bands in blots/gels. These histograms were then integrated to obtain the mean intensity of each immune-positive band. For the blots, these intensities for a particular protein were then normalized by the intensity values for β-actin (Santa Cruz), which was used as a housekeeping protein.Each experimental group consisted of six leaflet samples. All quantitative data were expressed as means ± SE and then normalized with respect to the values measured in fresh tissue (control). Data from all experiments were first tested for normality by the Anderson-Darling method, then analyzed using ANOVA to determine if there was significant contribution by a particular mechanical treatment on the measured parameters, followed by the Bonferroni post-hoc test. A p-value of less than 0.05 was used as a measure of statistical significance. All statistical analyses were performed using SPSS (IBM).The net effect of a particular FSS environment on pro-inflammatory processes after 48 hours of conditioning was estimated by calculating a pro-inflammatory state index Similarly, the contributions of FSS abnormalities to valvular remodeling were quantified via a tissue remodeling index An index smaller than one indicates a net decrease in pro-inflammatory/remodeling response, while an index larger than one reflected a net increase in tissue pro-inflammatory/remodeling processes. The nine pro-inflammatory and remodeling state indices resulting from the nine FSS magnitude-frequency treatments were interpolated using a biharmonic spline in order to predict the dependence of the tissue pathologic state on FSS magnitude and frequency.Following tissue exposure to physiologic and non-physiologic levels of FSS magnitude and frequency, immunostaining was performed to examine endothelial activation. Positive staining for ICAM-1  and VCAMBMP-4 and TGF-β1 immunostaining and immunoblotting were performed to investigate the effects of FSS abnormalities on paracrine signaling. Similar to the cell-adhesion molecules, BMP-4 and TGF-β1 expression  which suThe effects of FSS abnormalities on valvular remodeling were investigated by comparing the expression of the proteolytic enzymes MMP-2 and MMP-9 and the elastolytic proteases cathepsin L and cathepsin S. MMP-2 and MMP-9 immunostaining  support With most pro-inflammatory state index variations confined in the supra-physiologic FSS magnitude range, the results suggest the particular vulnerability of leaflet inflammation to elevated FSS levels . The symThe time-dependence of the leaflet pathological response following conditioning to supra-physiologic FSS under normal frequency  was studied by assessing the biological response of tissue exposed to this environment for 24 and 72 hours and by comparing it to those obtained in the fresh controls and tissue conditioned for 48 hours . The semThe objective of this study was to elucidate the respective and combined effects of abnormalities in FSS magnitude and frequency on the early mechanisms of CAVD in porcine aortic valve leaflets. The key results of the study can be summarized as follows: 1) leaflet tissue is selectively more sensitive to alterations in FSS magnitude than frequency; 2) FSS abnormalities promote paracrine signaling via BMP-4 and TGF-β1-dependent pathways and ECM degradation via MMP- and cathepsin-dependent pathways; and 3) the FSS mechanisms of early CAVD development are time-dependent. Therefore, the results confirm the key contribution of FSS alterations to valvular pathogenesis and suggest the capability of FSS abnormalities to trigger early CAVD events in the absence of any other risk factor.in vivo. Tachycardia conditions, which may result in acute FSS frequency alterations, are not chronic and therefore unlikely to contribute to a long-term pathological response. However, the rationale for varying the frequency of the FSS signal was to investigate the effects of changes in the harmonics of the FSS waveform experienced locally by the leaflets. Such phenomenon has been evidenced on bicuspid aortic valve leaflets The focus of this study on the characterization of the acute effects of FSS abnormalities on endothelial activation, paracrine signaling and ECM degradation is motivated by the established involvement of those biological processes in CAVD pathogenesis in vivo and in vitro models of CAVD proposed to date have relied on two leading hypotheses. The cholesterol hypothesis focuses on the traditional risk factors to study the biological events leading to CAVD Experimental ex vivo in the contexts of elevated pressure, stretch and FSS. Exposure of porcine aortic valve leaflets to hypertensive levels in a pressure chamber was shown to upregulate pro-inflammatory gene networks in interstitial cells ex vivo exposure of porcine valve leaflets to elevated stretch with an osteogenic culture medium demonstrated stretch magnitude-dependent tissue mineralization mediated by BMP-4 and TGF-β1 pathways in vivo evidence of the role played by BMP-4 in CAVD pathogenesis Our results demonstrated the particular sensitivity of valvular pro-inflammatory pathways to elevated FSS levels as illustrated by the increased expressions of the cytokines BMP-4 and TGF-β1. The ability of supra-physiologic mechanical stress environments to trigger pro-inflammatory processes has been observed ex vivo to promote collagen and sulfated glycosaminoglycan (sGAG) production but to decrease cathepsin L, MMP-2 and MMP-9 expression and activity in porcine leaflets ex vivo to combined pathological levels of pressure and stretch exhibit increased ECM synthesis and a progressive switch of the valve interstitial cells from a contractile to a more synthetic phenotype Our study also indicated increases in elastolytic enzyme expression and proteolytic enzyme expression and activity under elevated FSS magnitude  and abnormal FSS frequency , suggesting the progressive loss of valvular homeostasis and the alteration of valvular biosynthetic activity. The ability of supra-physiologic mechanical stress environments to break the delicate balance between ECM synthesis and degradation has been observed previously in the contexts of elevated pressure, stretch or a combination of these. Hypertensive levels have been shown It is important to note that the immunofluorescence images included in the paper focused on the fibrosa side due to the complete absence of positive stain on the ventricularis. Depending on the mechanical treatment, protein and enzyme expressions were distributed either in the subendothelial layer of the fibrosa or deeper in the tissue. Those results may correlate with the previously established phenotypic difference between the endothelial cells lining the leaflet fibrosa and ventricularis An interesting result is the apparent synergistic effects of FSS magnitude and frequency abnormalities on ECM degradation and paracrine signaling. In fact, leaflets exposed to physiologic FSS or combined abnormalities in FSS magnitude and frequency , cytokine and protease expressions peaked after 48 hours of exposure to FSS and then remained relatively constant until the 72-hour mark. Similar temporal responses have been observed previously in the contexts of valvular mechano-response to cyclic FSS The comparison of the results obtained in the fresh control group and in the group exposed to physiologic FSS for 48 hours (group 1) revealed no statistical difference in the expression of the different biomarkers considered in the study. Only MMP-9 activity was significantly downregulated under physiologic FSS as compared to the control group (78% decrease; see Finally, it is important to note that our experiments were conducted over a 48-hour period, which is much shorter than the timescale over which CAVD typically develops. However, the acute response observed and described in this study is relevant as it may shape the long-term processes involved in the formation of calcific lesions. In addition, the experiments were performed with porcine valve leaflets due to well-characterized antibody specificities and the extensive use of this tissue source in previous valvular mechanobiology studies. However, given the intricate differences between porcine and human valve tissues, the translation of the present findings to human valves should be done with caution.In conclusion, this study demonstrates the sensitivity of valve leaflets to FSS abnormalities and the ability of supra-physiologic FSS magnitude or sub-/supra-physiologic FSS frequency to upregulate key CAVD mechanisms such as paracrine signaling and ECM degradation . The res