Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., vg-A and focuses upon new findings regarding the interplay between them on an organismal level.Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the  Mosquito species can be divided into two groups according to their reproductive strategy. Autogenous mosquitoes do not require an initial blood meal and produce their first batch of eggs relying solely on nutrients accumulated during their larval phase. Vitellogenesis, the production of yolk by the fat body for deposition in developing oocytes, begins a few hours after eclosion of autogenous adult females. Subsequent egg batches are dependent upon the energy and nutritional building blocks derived from vertebrate blood.Aedes atropalpus has both autogenous and anautogenous strains. Most of the human disease-transmitting species fall into the anautogenic category. The underlying reason for this is that the necessity for blood by anautogenous mosquitoes drives increased interaction between vector and host making them inherently better disease vectors. In addition, the fact that they feed earlier gives the parasites more time to finish the extrinsic part of their life cycle and become infective within the vector. Autogeny vs. anautogeny is discussed in more detail in Attardo et al. , and three genes encoding vitellogenic carboxy-peptidases are up-regulated several hundredfold in the female fat body 24 h after a blood meal  exclusively in the fat body, the insect analog of the vertebrate liver. The YPPs are secreted into the hemolymph and taken up by the developing oocytes via receptor-mediated endocytosis  is a sesquiterpenoid that regulates insect development, reproduction and other processes in basically all insect species. Different groups of insects use different forms of JH, for instance mosquitoes use JH III  acts as intracellular JH-receptor. In complex with other proteins these receptors form active complexes that bind JH-response elements in the DNA and regulate gene transcription , in the fat body and other peripheral tissues, as described in During larval development ecdysone is synthesized by the prothoracic glands. However, these glands degenerate and disappear rapidly after adult emergence , is released from the brain and stimulates ecdysone synthesis in the prothoracic gland by activating the RAS/ERK pathway   that recognizes and binds to GATA-binding motifs upstream of the YPP gene region. Reception of a nutritional signal results in GATAr being displaced by GATAa upon the vg1 promoter. GATAa acts as a transcriptional activator which enhances YPP gene expression  are an evolutionary conserved group of peptide hormones which are characterized by a conserved disulfide bond structure (De Meyts, Ae. aegypti (Riehle and Brown, ILPs and the insulin receptor are expressed in a variety of tissues in melanogaster (Wu and Brown, All known insulin receptors have tyrosine kinase activity. The first arthropod insulin receptor was characterized in D. See Figure YPP gene expression in the fat body both directly and indirectly. In vitro fat body culture experiments show that YPP expression is directly induced by the combination of the 20E and ILP signaling pathways acting together to activate gene expression. Roy et al., demonstrated the synergistic relationship of 20E and insulin by incubating isolated mosquito fat bodies for 3 h in different mixtures of porcine insulin and 20E (Roy et al., ILPs regulate YPP expression indirectly through the regulation of ecdysone synthesis. A blood meal stimulates the secretion of ILPs amongst other neuropeptides, namely OEH, that regulate ecdysteroid production (Riehle and Brown, Ae. aegypti, ILP3 is characterized as the ILP responsible for initiating egg development (Brown et al., ILPs also regulate Manipulations of the ILP-signaling pathway in mosquitoes have resulted in profound changes in egg production. RNAi-mediated knockdown of the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase PTEN6, a suppressor of ILP signaling resulted in a significant increase in viable eggs produced by females (Arik et al., vg1 gene expression in isolated fat bodies that were stimulated with amino acids. Also, FOXO knockdown mosquitoes produced smaller egg numbers after a blood meal (Hansen et al., One downstream effector gene of the ILP-signaling pathway has been characterized in mosquitoes. The forkhead-box transcription factor FOXO is a major target of ILP signaling in insects and vertebrates (Hwangbo et al., The interplay between the different signaling pathways regulating YPP gene expression is complex and only partially understood. However, significant levels of crosstalk appear to occur between the aforementioned signaling pathways in the different organs and tissues of adult female mosquitos. Several studies address these complex interactions.Blatella germanica (Maestro et al., JH and nutrient signaling are connected in mosquitoes in two distinct ways. A recent study shows that JH biosynthesis is in part regulated by nutrient signaling via the TOR pathway (Perez-Hedo et al., ILP3 has emerged as a key regulator of vitellogenesis via multiple mechanisms. After a blood meal, ILP3 activates the expression of trypsin proteases in the midgut of mosquitoes thereby enhancing blood digestion and indirectly affecting TOR-mediated nutrient signaling (Gulia-Nuss et al., Triboleum castaneum, found that JH regulates vg gene expression through ILP signaling (Sheng et al., A study in the red flour beetle, vg1 promoter of Ae. aegypti. Several signaling pathways converge in the regulation of these and other YPP genes. In recent years our understanding of the signaling pathways involved and the interplay between them has undergone rapid growth. We know that at least four distinct pathways are involved: juvenile hormone, ecdysone, insulin, and the TOR nutritional signaling pathway. The challenge for the future is to understand the crosstalk between these pathways in different organs and during different physiological stages in adult female mosquitoes. Based upon the current understanding of signaling in mosquito reproduction we feel that the ILPs may play a key regulatory role in the determination of autogenous vs. anautogenous life histories in mosquitoes.Figure In addition, while ecdysone- and juvenile hormone analogs are already used as insect growth regulators, a challenge for the future is to use the novel and detailed understanding of nutrient and insulin-signaling pathways to develop new strategies for mosquito control.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.


We review the literature and find 16 studies from across Africa's savannas and woodlands where woody encroachment dominates. These small-scale studies are supplemented by an analysis of long-term continent-wide satellite data, specifically the Normalized Difference Vegetation Index (NDVI) time series from the Global Inventory Modeling and Mapping Studies (GIMMS) dataset. Using dry-season data to separate the tree and grass signals, we find 4.0% of non-rainforest woody vegetation in sub-Saharan Africa (excluding West Africa) significantly increased in NDVI from 1982 to 2006, whereas 3.52% decreased. The increases in NDVI were found predominantly to the north of the Congo Basin, with decreases concentrated in the Miombo woodland belt. We hypothesize that areas of increasing dry-season NDVI are undergoing woody encroachment, but the coarse resolution of the study and uncertain relationship between NDVI and woody cover mean that the results should be interpreted with caution; certainly, these results do not contradict studies finding widespread deforestation throughout the continent. However, woody encroachment could be widespread, and warrants further investigation as it has important consequences for the global carbon cycle and land–climate interactions. Other studies, for example those looking at changes in the Sahelian grasslands, have typically used the NDVI signal from the wet (growing) season ,43. HoweWe have demonstrated that this technique is successful in detecting woody encroachment based on a site in Mbam Djerem National Park in Cameroon, where we know encroachment of savannas by forest is occurring at a rapid rate ,24. This(a)(i) The GIMMS data v. 2.0 (1982–2006) were downloaded for Africa . These a(ii) Mean NDVI was calculated for every possible consecutive three-month period for each pixel from 1982 to 2006. Only three-month periods where five out of six possible observations reached this ‘best-quality’ standard were considered. Then, the minimum three-monthly NDVI was extracted for each pixel for each year from 1982 to 2006.F-test with a 90% confidence level [(iii) Linear regression was performed across each time series for each pixel. Change in woody vegetation considered to have occurred for all areas with a ‘significant’ best-fit line (using an ce level  and a sl(iv) In order to remove areas where the signal came from grasses, pixels containing no ‘wooded’ classes in the Global Land Cover 2000 (GLC 2000 ) dataset(b)The analysis shows that woody encroachment and forest loss are both occurring . Of non-These results should be interpreted with caution for a number of reasons: (i) the resolution is very coarse (8 km), meaning that many small-scale deforestation and regrowth events will have been missed: only changes occurring over a significant portion of the pixel will be detected . (ii) The time series of NDVI data may contain artefacts, particularly over tropical regions, owing to the resampling and cloud-filtering algorithms applied to the raw AVHRR data ; though a locates the studies listed in a (iii).It is hard to discount the above concerns, but some confirmation comes from the literature review that gave specific instances of the location of woody encroachment. Figure 14.et al. [et al. [From both the literature review and the GIMMS analysis, it is clear that both forest loss and gain are occurring widely throughout Africa. In terms of area, it appears that the area of land undergoing woody encroachment may be comparable or even larger than areas where a significant loss of forest cover is occurring; subsetting the GIMMS analysis most of the increase is in the woodland and savannas of sub-Saharan Africa north of the equator, whereas in the Miombo woodland regions, south of the equator, forest loss appears to be dominating. However, this conclusion has high uncertainties owing to potential artefacts in the GIMMS dataset, and regional variation in the relationship between NDVI and woody cover. The results presented here are not directly comparable with analyses based on detailed interpretation of small subsets ,18, as tet al.  agree, f [et al. .It should be noted that this analysis relates mainly to changes in broad canopy cover in mixed tree–grass areas and does not relate directly to the carbon balance of the African continent (though forest regrowth must form part of the land–surface carbon sink ). In parThis analysis validates the observations made that Miombo woodlands are suffering especially badly from the loss of woody vegetation, owing to expanding populations removing trees for agriculture and fuel  . That th(a)2 concentration.To understand the causes of forest expansion, it is necessary to comprehend the current and historical constraints on woody cover throughout the region. It is known that much of the African continent exists currently at a woody cover level far below its potential given its annual rainfall ,51. RainIt is hard to underestimate the anthropogenic influence on Africa's forest cover. Humans have been setting fires and controlling grazer numbers throughout their evolution, potentially even having a major part in the spread of savanna vegetation . In gene2 enrichment, are equally important [2 reduces the advantage held by C4 grasses over C3 trees: C4 grasses use a specialist mechanism to increase the CO2 concentration in cells that perform the light reaction of photosynthesis, reducing the rate of photorespiration that is a major limitation on photosynthetic efficiency in high temperatures [2 concentration increases, this specialist adaptation is less of an advantage. In particular, increased CO2 concentrations mean that trees can grow faster and saplings are more likely to be able to grow enough between fires to escape the flame zone [However, several studies suggest that global factors, in particular atmospheric COmportant . An increratures ,57; as tame zone .5.2 concentrations, but further studies will be needed to determine this with more confidence. A coarse-scale analysis of changes in woody vegetation from 1982 to 2006 suggested that significant woody encroachment is occurring to the north of the Congo Basin, but, in contrast, to the south of the Congo Basin a rapid reduction in woody vegetation is occurring. This deforestation in the Miombo woodlands of Africa warrants much more global attention, as it represents a serious threat to the livelihood of the region's many inhabitants and to this unique ecosystem.This study brought together a body of evidence suggesting woody encroachment is widespread in sub-Saharan Africa. The reason behind this encroachment is likely to be a combination of changes in the fire regime and increasing atmospheric COThe results of this study should be interpreted with caution: the evidence brought together is a collection of small-scale studies, and a coarse-scale remote sensing analysis that can detect only broad changes in woody cover, and is prone to artefacts. These results should stimulate discussion on woody encroachment, but this analysis does not provide a definitive assessment of the total magnitude of woody encroachment compared to forest loss.


Giardia duodenalis is a common intestinal parasite that infects humans and many other mammals, mainly distributing in some areas with poor sanitation. The proportion of the human giardiasis burden attributable to G. duodenalis of animal origin differs in different geographical areas. In Mainland China, genetic data of the gdh and bg genes of G. duodenalis from animals are only limited in dogs and cats. The aim of the study was to provide information on the genetic characterizations of animal-derived G. duodenalis isolates  at both loci in Heilongjiang Province, Northeastern China, and to assess the potential for zoonotic transmission.G. duodenalis isolates from animal feces  in Heilongjiang Province were characterized at the gdh and bg loci in the present study. The gdh and bg gene sequences of sheep-derived G. duodenalis assemblage AI, and the gdh sequences of rabbit-derived G. duodenalis assemblage B had 100% similarity with those from humans, respectively. Novel subtypes of G. duodenalis were identified, with one and seven subtypes for assemblages A and E at the gdh locus, and two and three subtypes for assemblages B and E at the bg locus, respectively. Three pairs of the same bg sequences of assemblage E were observed in sheep and cattle.61 gdh and bg genes of G. duodenalis from rabbits, sheep and cattle in Mainland China. Homology analysis of assemblages AI and B implied the possibility of zoonotic transmission. The novel subtypes of assemblages of G. duodenalis may represent the endemic genetic characteristics of G. duodenalis in Heilongjiang Province, China.This is the first description of genetic characterizations of the  Giardia duodenalis  is an intestinal, parasitic, flagellate protozoan that infects humans as well as a wide variety of domestic animals and wildlife species. Giardiasis is one of the major neglected tropical diseases occurring in rural areas and poor urban areas. Human giardiasis has complicated clinical manifestations, ranging from the absence of symptoms to acute or chronic diarrhea, abdominal pain, nausea, vomiting and so on, depending on the health status of hosts, and the dose and virulence of the parasites G. duodenalis is a complex species, with eight genetically distinct assemblages (A-H) having been identified; each of them has significant differences in host range or host specificity tpi), glutamate dehydrogenase (gdh) and β-giardin (bg) loci; thus, both assemblages are considered to have the possibility of zoonotic transmission Giardia isolates at the genotype and subtype levels, most commonly including the small subunit ribosomal RNA (SSU rRNA), bg, gdh and tpi genes Tpi, gdh and bg genes are common genotyping and subtyping markers of G. duodenalis, whereas the SSU rRNA gene is commonly used markers for the species and assemblage differentiation of GiardiaG. duodenalis isolates from humans and animals. The data based on MLG tools can greatly improve our understanding of the relationship between humans and animals as hosts and reservoirs, including elucidation of the transmission routes and dynamics of human giardiasis, and assessment of the burden of human giardiasis of animal origin.Based on the fact that assemblages A and B in humans have also been found in animals, the role host animals play in the epidemiology of human giardiasis has received increasing attention. The animal-derived sequences of assemblages A and B have been reported to show 100% similarity with those from humans at the triose phosphate isomerase . No other specific permits were required for the described field studies. The locations where we sampled are not protected in any way. The field studies did not involve endangered or protected animal species. Before beginning work on the study, we contacted the farm owners and obtained their permission. In this study, all the samples were taken immediately from animal fresh feces deposited on the ground after animal defecation instead of operating or experimenting on the animals. During the procedure, the animals were not hurt at all.G. duodenalis isolates were all obtained from animal feces  in Heilongjiang Province, China between October 2008 and November 2011. Genomic DNA was extracted using a QIAamp DNA Mini Kit  according to the manufacturer's recommendations and DNA preparations were stored at −20°C before PCR analysis. 61 G. duodenalis isolates, which were identified based on the tpi gene in the previous studies gdh and bg genes of G. duodenalis  and were then aligned with G. duodenalis reference sequences downloaded from GenBank and analyzed using Clustal X 1.83.All secondary PCR products were sequenced with secondary PCR primers at each locus on an ABI PRISMTM 3730 DNA Analyzer , using a BigDye Terminator v3.1 Cycle Sequencing kit . Accuracy of the sequencing data was confirmed by sequencing in both directions. Nucleotide sequences obtained in the present study were subjected to BLAST searches  at the gdh locus versus 73.8% (45/61) at the bg locus, with 100% (4/4) versus 100% (4/4) for assemblage A, 71.4% (10/14) versus 85.7% (12/14) for assemblage B, and 88.4% (38/43) versus 67.4% (29/43) for assemblage E  (n = 3) was identified as AI, having 100% homology with the human-derived subtype AI sequences (GQ329674 and GQ329676) Sequence analysis of the bg locus, four identical sequences (KC960630) were shown to have 100% similarity with those of human-derived  and animal-derived assemblage AI isolates  However, at the G. duodenalis isolates were successfully amplified and sequenced at the gdh locus, with all of them being identified as assemblage B. Nine were identical to each other (KC960645) and had 100% similarity with the human-derived assemblage B sequence from Brazil (EF507682) 10 out of 14 rabbit-derived bg gene sequences of rabbit-derived G. duodenalis isolates were successfully obtained and identified as assemblage B. Two novel subtypes were detected with a single base variation between each other, with one representing 10 sequences (KC960631) and the other representing two sequences (KC960632), and both of them have one or two base differences with the published sequence AB618785, respectively.12 G. duodenalis isolates were successfully amplified and sequenced at the gdh locus, belonging to four novel subtypes of assemblage E (KC960647 to KC960650). Four subtypes of assemblage E were observed from 22 gdh gene sequences obtained successfully from 26 cattle-derived G. duodenalis isolates, with one subtype (KC960651) having the same sequence as those of the cattle-derived isolates (EF507644 and EF507645) gdh nucleotide sequences  have been described previously in sheep, goats and cattle bg sequences of cattle-derived G. duodenalis isolates belonged to five subtypes, with four subtypes (KC960638 to KC960641) having been reported in sheep, goats and cattle bg nucleotide sequences  showed 100% homology with the three from cattle , respectively  representing three sheep-derived assemblage AI isolates has been described in human giardiasis cases in Sweden bg locus with a human-derived G. duodenalis isolate from Sweden gdh, bg and tpi loci has led to the designation of assemblages AI, AII and AIII, which all appear to have host specificity. Assemblage AI is commonly found in animals and is sometimes seen in humans; assemblage AII mainly infects humans and is occasionally detected in animals; assemblage AIII has circulated in wildlife tpi gene sequence compared to AI, AII and AIII gdh and bg loci showed 100% homology with those from humans in Sweden G. duodenalis isolates have been obtained in the investigated areas although human giardiasis cases were reported early in 1990 G. duodenalis isolates G. duodenalis of animal origin remain to be clarified by epidemiological data of human and animal giardiasis cases.PCR-based techniques have been used widely for the identification, population genetics and epidemiology of gdh gene sequences from rabbits and both of them had been reported in human giardiasis cases from Brazil and Australia Giardia cysts Giardia isolates were not genotyped or subtyped. The outbreak emphasized not only the possibility of domestic-animal-to-person transmission of G. duodenalis but also the importance of good hygienic practices in food preparation to prevent the spread of animal-derived G. duodenalis cysts in humans. In fact, an early case-control study in eastern England showed an association of giardiasis with exposure to farm animals and pets, particularly pigs, dogs, and cats Assemblage B is one of the two major assemblages causing human giardiasis with a broad animal host range. It has been isolated from cattle, sheep, pigs, horses, dogs, cats, and rabbits G. duodenalisG. duodenalis has also been described in goats and sheep in Spain, with a prevalence of 100% (39/39) for goats and 98.7% (74/75) for sheep tpi, gdh and bg genes, the genetic polymorphism of assemblage E has been observed gdh gene sequences showed the presence of seven novel subtypes of assemblage E, four from sheep (KC960647 to KC960650) and three from cattle (KC960652 to KC960654)  being found in total. The identification of three pairs of the same bg sequences from sheep and cattle  indicated the possibility of cross-species transmission of G. duodenalis E between cloven-hoofed animals in the investigated areas  . At the ed areas .G. duodenalis isolates were identified as assemblage E based on successful PCR and sequencing analysis, respectively at the gdh locus (n = 8) and at the bg locus (n = 4); however, all the DNA preparations used above were previously identified as assemblage B based on the tpi gene  were identical to those from humans. However, at the bg locus, no sequences had 100% similarity with those from the cases of human giardiasis. The results indicated that single gene nucleotide analysis of G. duodenalis isolates from animals and humans at the genotype and subtype levels are insufficient for assessment of the disease burden of zoonotic transmission.MLG is a powerful tool for tracing the infection or contamination source of gdh locus  than at the bg locus , which might be mainly related to the primers and genetic structure of target fragments, for the other factors influencing PCR efficiency were identical such as the same quality and quantity of DNA templates and the quality and characterization of DNA polymerase used. Even though the primers were designed to bind ‘conserved’ regions in the amplified genes, excessive mismatches in the binding regions of primer sequences might result in the failure of PCR amplification for some isolates of G. duodenalis. In fact, numerous molecular data have confirmed both inter- and intra-assemblage genetic variations G. duodenalis have been reported at different genetic loci G. duodenalis isolates. PCR analysis by Scorza et al showed positive amplification of the gdh, bg, and tpi genes in 91.0% (172/189), 84.8% (145/171) and 19.8% (34/172) of G. duodenalis cyst-positive samples from the mammalian feces, respectively gdh, bg and tpi loci in a study of molecular characterization of Giardia isolates from clinical infections following a waterborne outbreak tpi and gdh genes for detection and genotyping of human-derived G. duodenalis isolates, the tpi gene was amplified from 96.2% (25/26) of samples, whereas only 81% (21/26) were positive when the gdh gene was targeted gdh locus or the bg locus, followed by assemblage E (88.4%) and assemblage B (71.4%) at the gdh locus versus assemblage B (85.7%) and assemblage E (67.4%) at the bg locus (G. duodenalis isolates from humans and other animals G. duodenalis isolates in the future.In the present study, PCR was observed to have higher amplification rate at the bg locus . In genegdh and bg genes of G. duodenalis isolates from rabbits, sheep and cattle in Mainland China. In conclusion, the obtained data provide useful information for further genotyping or subtyping studies of G. duodenalis. The findings of animal-derived assemblages AI and B having 100% homology with human-derived G. duodenalis isolates at the gdh and bg loci imply the possibility of zoonotic transmission in the investigated areas. The identification of the novel subtypes of assemblages AI, B and E based on the gdh and bg genes might reflect the characteristic geographical distribution of G. duodenalis. To better understand the transmission dynamics of G. duodenalis and assess the burden of human giardiasis caused by animals, molecular epidemiological studies of giardiasis should be conducted in humans and animals living in the same household or localized focus of endemicity.The present study is the first report about genetic characterizations of the


IPMK was initially discovered as an essential subunit of the arginine-sensing transcription complex in budding yeast. In mammals, IPMK is also known as a physiologically important phosphatidylinositol 3 kinase (PI3K) that forms phosphatidylinositol 3,4,5-trisphosphate (PIP3), which activates Akt/PKB and stimulates its signaling. Acting in a catalytically independent fashion, IPMK mediates the activation of mammalian target of rapamycin (mTOR) in response to essential amino acids. In addition, IPMK binds and modulates AMP-activated protein kinase (AMPK) signaling pathways, including those involved in hypothalamic control of food intake. These recent findings strongly suggest that IPMK is a versatile player in insulin-, nutrient-, and energy-mediated metabolism signaling networks. Agents that control IPMK functions may provide novel therapeutics in metabolic syndromes such as obesity and diabetes.Inositol phosphates (IPs) act as signaling messengers to regulate various cellular processes such as growth. Inositol polyphosphate multikinase (IPMK) generates inositol tetrakis- and pentakisphosphates (IP Myo-inositol, a six-carbon cyclitol that contains one axial and five equatorial hydroxyl groups, is the major form out of nine possible inositol isomers. Inositol was formerly assumed to be an essential B complex vitamin; however, inositol is now known to be synthesized de novo in the human body. For instance, one normal adult kidney can produce a few grams of inositol per day.myo-inositol. In plants, inositol hexakisphosphate (IP6) is known as phytic acid, which is enriched in cereals and is utilized for phosphorus storage. When the levels of inositol become extremely low, severe medical disturbances, such as diabetic changes, anxiety disorders, and hypercholesterolemia ensue.3Inositol is a naturally occurring glucose isomer and a key nutrient in the human diet.13)-mediated cytosolic calcium release.2) into water-soluble IP3 and lipid-anchored diacylglycerol. IP3 then binds to the IP3 receptor, which is localized on the membranes of intracellular calcium storage sites such as the endoplasmic reticulum, and subsequently elevates cytosolic calcium levels by stimulating the calcium channel activity of the IP3 receptor. Since the discovery of IP3 and its signaling role in the mid-1980s, continuing investigations of the biochemical fate of IP3 inside the cell have revealed the presence of a family of inositol phosphate kinases and associated specific phosphorylation events on the inositol ring at different hydroxyl groups (7), which contain diphosphates with one or more high energy-harboring pyrophosphate bonds. Multiple approaches, including genetic manipulation of inositol polyphosphate metabolism and biochemical characterization of IP-binding signaling targets, have recently highlighted the physiologic significance of inositol poly- and pyrophosphates in the regulation of major physiologic events such as growth and apoptosis  in maintaining the cellular membrane bilayer, inositol is viewed as a secondary signaling messenger that controls diverse physiologic events.l groups . Higher 6 kinase genes, Snyder and his associates identified IPMK as an enzyme essential for the synthesis of IP4 P4 and InsP4) and IP5 P5).4 into IP5. Thus, IPMK deletion abolishes the formation of IP5, but it also eliminates IP6 and IP7 formation, indicating that IPMK is an essential enzyme in the generation of all highly phosphorylated IP species, including IP7.2 at the 3 position to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3)  .13,14 IP4 and IP5, by overexpressing the IPMK gene from evolutionarily divergent plant or fruit fly functionally rescues the growth defect in IPMK-null yeast cells.19Possible IPMK functions in mammalian metabolic homeostasis were suggested by previous studies in budding yeast designed to screen for genes that regulate metabolic activities in response to arginine availability.3 to activate the PI3K pathway, which stimulates protein kinase Akt/PKB. Active Akt, in turn, phosphorylates TSC2, abrogating TSC2-mediated inhibition of Rheb GTPase, which can directly stimulate mTORC1. The entire process by which nutrient amino acids activate mTORC1 has remained elusive. However, a growing body of evidence clearly suggests that in response to amino acids, Rag GTPases, in association with the Ragulator complex, mediates the spatial redistribution of inactive mTORC1 to the surface of lysosomes, which contain mTOR-activating Rheb GTPase.25Among the many factors that contribute to the regulation of anabolic pathways  in mammals by growth factors and nutrients such as essential amino acids, the mTOR complex 1 (mTORC1) is considered to be a central signaling regulator , amino acids, and energy levels . Despite7  and related eating disorders, such as bulimia and anorexia nervosa.Another major area of future IPMK research is to identify novel IPMK functions in metabolism or related signaling pathways. Recent reports on an unexpected role for IP


In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles. One of the most vital processes in biology is the transduction of signals along biochemical pathways, enabling the living cell to elicit appropriate responses to chemical and physical stimuli A general intracellular signaling network may consist of several interconnected cascades The mathematical modeling of signaling pathways often considers a simplified set of equations in which each cycle is described by a single variable in vivo in the MAPK cascade controlling the early development of drosophila embryos in vitro study shows that retroactivity effects can be easily induced at one stage of the signaling system regulating the nitrogen assimilation in E. coliThe absence of retroactivity for a signaling module implies that the state variables of this module do not change when its output is used as the input of another device. Special conditions are to be met in the design of a network unit in order to minimize the retroactivity retroactive signaling the design of a pathway that exploits this possibility, that is to say, an extended signaling pathway which comprises a connected path of upstream signaling from output to input  protein in cycle 2 total sequestration of protein 2 in cycle 1.Let us consider in detail the effect of varying the total protein ally to  . This arpartial sequestration of species 2 by cycle 1. Once this partial sequestration has occurred, a further increase of On the other hand, if cycle 1 is activated and cycle 2 is still deactivated, the results of the previous section show that  namely  . Here weFinally, the case where cycle 2 starts out activated is depicted on We turn now to the retroactive effect of varying the phosphatase of the downstream cycle, ctivated . MoreoveEquation (5) allows us to estimate the level of In summary, in a 2-cycle cascade, in order to create conditions that may substantially modify the fraction of the activated protein in the upstream cycle by perturbing the parameters of the downstream cycle, it is recommended to deactivate the upstream cycle 2. Then, if the downstream cycle 1 is also maintained deactivated a substantial change in The above analysis focussed on the changes of the fraction of phosphorylated protein in cycle 2 because the variable When the upstream cycle 2 is deactivated, As illustrated on When the upstream cycle 2 is activated, ange of  . In contough cf. .The results obtained with a 2-cycle cascade can predict the effect of retroactivity in short signaling pathways with more than 2 cycles. We first consider a 3-cycle pathway where the activated protein in the cycle at the top of the pathway is an enzyme that activates two other cycles which are not directly linked together (ults cf. (I)) thatA similar retroactive signaling in the same 3-cycle pathway can be achieved by modifying not the phosphatase but the available protein in the starting cycle 1. Keeping the same parameters as above, tion cf. (G)). In In some covalent modification cycles, the deactivated protein can serve also as an enzyme for another protein modification To extend the possibility of retroactive signaling to more complex situations than a 3-cycle pathways we now consider a motif of a 5-cycle network in which the activated protein in the top cycle acts as the enzyme regulating two 2-cycle cascades, as shown on Cell signaling is generally thought in terms of a series of reversible biochemical reactions that are chained together in a feedforward network where extra connections, called feedbacks, could regulate the information flow from bottom-up. In particular the expression “signaling cascade” was coined to suggest the idea of an upstream to downstream signal transmission. In the simplest scheme of a cascade of two covalent modification cycles, the input signal typically is a steep increase of the enzyme modifying the first protein. Then the latter acts as the enzyme activating the second protein whose concentration is interpreted as the output of this system. In this paper, however, we show that in such a cascade a retroactive signaling is also possible, i.e. transmitting an input signal from downstream to upstream, and we predict conditions for which this phenomenon can be observed. The input signal is now a variation of a biochemical species that can change the state of the downstream cycle. Two cases are considered, namely a change of the total amount of the downstream signaling protein, or a variation of the phosphatase deactivating the same protein. In both cases we work out characteristic ranges of the concentrations of the species for which a retroactive effect can be observed in the upstream cycle. Moreover we show that this potentiality can help to perform retroactive signaling in short multi-cycle pathways.A covalent modification cycle is generally described as a two-state entity for which the total level of protein is fixed. However, like all the molecules inside the cell, this signaling protein is subjected to a turnover governed by several processes, including synthesis and degradation Drosophila embryo. They manage to modify the amount of substrates of the doubly phosphorylated form of ERK by constructing mutants missing the corresponding substrates. Another way to alter the available protein in the downstream cycle is to add in the medium a kinase inhibitor that can bind to the activated enzyme at the end stage of the pathway There are several ways to modify the available protein in the downstream cycle in a cascade of covalent modifications. One way is to change the amount of substrates to which the activated protein of the downstream cycle can bind. For example, in a recent study reported in In Ossareh et al, the authors performed mathematical analysis of retroactivity in a signaling cascade with an arbitrary number of stages. They achieved necessary and sufficient conditions for which retroactivity exists in such chains. Their analysis is based on the linearization of the steady state equations in order to predict how a small downstream perturbation is amplified in the upstream response of an arbitrarily long signaling chain. Those results are complementary to the ones presented in the present paper, in the sense that here we consider short signaling pathways but our analysis is based on the resolution of the full nonlinear equations, and not only on the linearized system. So, it is concerned with arbitrarily large perturbations of the parameters. In fact we show that retroactive signaling is meant to work only for a characteristic range of parameter variations that we analytically estimate by working on the asymptotic behaviors of the system for small and large parameter perturbations.Signaling pathways are regulated by several mechanisms, like positive or negative feedback loops linking the output of the cascades and some upstream stages. This requires the existence of specific chemical interactions between the output protein of the cascade and the upstream proteins that are involved in the feedback loop. Our study shows that the property of retroactive signaling can be another way to regulate the functioning of signaling cascades in branched pathways, without explicit feedbacks. In fact, we can further speculate that in natural signaling pathways with possibly several branches, some of the latter would be sensitive to retroactivity and be devoted to the regulation of the usual branches, where signals go in the top-down direction. These results prompt new experiments concerning signaling cascades and possibly new ways to interpret previous results.Our theoretical study is performed in the framework of coupled nonlinear equations describing the rate of changes of protein concentrations in signaling cascades formed of covalent modification cycles. The model equations are deterministic and based on the law of mass action. Only stationary states of these equations are analysed and thus the mathematical method amounts to solving sets of algebraic nonlinear equations. Thus the issue of how the biochemical species reach the equilibrium is not discussed here, as it has been addressed in some previous studies Let us note that we assume that the studied signaling pathways possess a stable equilibrium. Although in this paper we will not explicitly discuss the generality of this assumption by performing the linear stability analysis of the equation set, the hypothesis of a stable equilibrium is consistent with the current knowledge. In the literature, published results indicate that non steady behaviors (e.g. sustained oscillations) can arise in signaling cascades only with the concomitant occurence of bistability in the signaling modules Let us introduce the notations used for writing the equations in the case of the simple 2-cycle cascade as depicted on activated if deactivated. This property is easily related to the ratio A 2-cycle cascade involves 4 enzymatic reactions. Each of those can be characterized also by their maximum reaction rates (The following sections give details on the derivation of equations (1)–(3) and (5) used in the section Results.Let us consider a 2-cycle cascade as drawn on By combining Eqs.(20), (22) and (25), one obtains the characteristic range for Let us note that using the definition of Now, to justify Eqs.(2)–(3) given in the Results, we wish to compute the asymptotic values of The second step is to solve Eqs.(17)–(18) in the limit In conclusion, by using Eqs.(29)–(38), let us note that we can sketch the behavior of Let us consider a 2-cycle cascade as drawn on First recall that Since


This map includes 236 molecules and 252 reactions that are involved in TSLP/TSLPR signaling pathway. We expect that the TSLP signaling pathway map will provide a rich resource to study the biology of this important cytokine as well as to identify novel therapeutic targets for diseases associated with dysregulated TSLP/TSLPR signaling.Thymic stromal lymphopoietin (TSLP) is a four-helix bundle cytokine that plays a critical role in the regulation of immune responses and in the differentiation of hematopoietic cells. TSLP signals through a heterodimeric receptor complex consisting of an interleukin-7 receptor α chain and a unique TSLP receptor (TSLPR) [also known as cytokine receptor-like factor 2 (CRLF2)]. Cellular targets of TSLP include dendritic cells, B cells, mast cells, regulatory T (Treg) cells and CD4+ and CD8+ T cells. The TSLP/TSLPR axis can activate multiple signaling transduction pathways including the JAK/STAT pathway and the PI-3 kinase pathway. Aberrant TSLP/TSLPR signaling has been associated with a variety of human diseases including asthma, atopic dermatitis, nasal polyposis, inflammatory bowel disease, eosinophilic eosophagitis and, most recently, acute lymphoblastic leukemia. A centralized resource of the TSLP signaling pathway cataloging signaling events is not yet available. In this study, we present a literature-annotated resource of reactions in the TSLP signaling pathway. This pathway map is publicly available through NetPath (http://www.netpath.org/pathways?path_id=NetPath_24Database URL:  IL7R) and the TSLP receptor subunit , which is similar to the common γ chain  is a type I cytokine that belongs to the interleukin-2 cytokine family. TSLP signaling requires a heterodimeric receptor complex composed of interleukin-7 receptor α chain . Activated JAKs, in turn, regulate the activity of signal transducers and activators of transcription (STAT) factors, which include STAT1, STAT3, STAT4, STAT5a, STAT5b and STAT6 . In addiTSLP and TSLPR have been implicated in a number of pathological conditions. TSLP expression was found to be increased in asthmatic airways and its overexpression correlated with severity of disease , 20. TSLhttp://www.netpath.org/pathways?path_id=NetPath_24). NetPath is an open access signaling pathway resource that provides a list of all of the reactions curated from the literature to present a comprehensive and global view of signaling pathways. We have also generated a high-confidence list and visualization of TSLP pathway that is available through NetSlim (http://www.netpath.org/netslim/NetSlim_24). NetSlim uses predefined criteria to show only high-confidence reactions as a means to generate a ‘slim’ version of the pathway, which is less complex and more easily visualized.Despite the importance of the TSLP signaling pathway, the reactions specific to TSLP signaling reported to date are not available in any public pathway resource. Kyoto Encyclopedia of Genes and Genomes (KEGG) database  contains NetSlim  , posttranslational modifications (PTMs), translocation and activation/inhibition of proteins, which occur on stimulation with TSLP. We also looked for genes that are differentially regulated on stimulation by TSLP in humans. The reactions stimulated by TSLP as compared with an unstimulated state (reactions induced on co-stimulation with TSLP and any other stimuli were excluded) in human or other mammalian systems were included. We used PathBuilder , a pathwet al.  associations. The PPIs induced by TSLP were considered ‘binary’ if homomeric/heteromeric interactions are reported between two proteins or if only two proteins are studied/identified in coprecipitation assays. Multimeric association of proteins consisting more than two components was considered as ‘complex’ interactions. For example, biochemical interaction between TSLP and IL-7 receptor alpha subunit and TSLPR has been reported by several groups . More reet al.  have shoPTMs are dynamic covalent modifications on proteins. PTMs are important for regulation and integration of cellular signaling events. PTMs regulate PPIs, activity of enzymes and the cellular localization of proteins. Under this category, we have annotated enzyme-catalyzed reactions that have been reported to be stimulated by TSLP. Like other cytokines, TSLP also uses the JAK-STAT signaling module to transmit signals in cells by inducing phosphorylation of some members in the module, including JAK1, JAK2, STAT1, STAT3, STAT4, STAT5A, STAT5B and STAT6 , 37, 38.Movement of proteins across subcellular compartments is critical for the function of the proteins as well as for the transfer of information inside the cell. These translocation events may depend on a variety of factors, which include PTMs or association/dissociation with other molecules. For example, TSLP signaling in dendritic cells also leads to activation and nuclear transport of NFκB1, NFκB2, RELA and RELB proteins to induce the production of OX40L . The proActivation and inhibition of proteins, at the human interface, are identified by activity assays. This category lists proteins whose activity is found to be altered on TSLP treatment. When the activity of proteins has been shown to be changed by TSLP stimulation through activation/inhibition assays, such as using small kinase inhibitors or through the use of intrinsic or extrinsic substrates, these proteins were included under the activation/inhibition category. For example, the involvement of SRC and TEC family of kinases in the TSLP induced proliferation of B cells has been demonstrated through pharmacological inhibitors , 44.NME1 and MYC in trophoblasts and of IL6, IL8 and CCL11 in smooth muscle cells  the PubMed identifier of the study where the reaction was described. We have also given a brief description for each of the reactions under the ‘Comments’ section.In all the above-mentioned categories, gene symbols and NCBI Gene identifiers were used to represent the molecules involved in reactions. We have also documented additional information for reactions such as (i) species of the protein(s) and cell line/type used to prove the reaction; (ii) the cellular compartment where the reaction takes place; (iii) the experiment type, i.e. whether the reaction was carried out Our literature survey and annotation of articles resulted in the identification of 236 proteins that are involved in mediating the effects of TSLP. These molecules were found to be involved in 9 PPIs, 232 catalysis events, 7 translocation events and 4 activation events. The biomedical significance and the relatively sparse information available for this important pathway in the literature, underscore the need for additional systematic studies required to identify the effectors of this pathway and their biological roles. Most enzyme-substrate reactions that we cataloged were ‘indirect’ and involve phosphorylation or dephosphorylation events. The site and residue information of PTMs was annotated for 155 proteins. We also cataloged 91 genes, which were transcriptionally regulated on TSLP stimulation in primary human cells or cell lines. As mentioned above, these reactions are linked to the corresponding published literature where they were described. The pathway reactions were reviewed both internally and by an external pathway authority to ensure availability of high-quality data to the biomedical research community.The TSLP pathway webpage in the NetPath includes a description of the pathway as well as statistics of the number of molecules and reactions. The pathway reactions are encoded in various community standard data exchange formats such as Proteomics Standards Initiative for Molecular Interaction (PSI-MI version 2.5) , BiologiEven though pathway reactions can be visualized and analyzed using the above-mentioned software, these do not give directionality to the reactions involved. Also, in many cases the relationship among molecules is not clear or linked to canonical signaling pathways . This mahttp://www.netpath.org/netslim. These pathway data are provided in all the exchange formats mentioned above. Apart from these, the pathway map is also provided in .pml, GenMAPP .Availability of TSLP signaling reactions in a single resource will aid in understanding the role of various molecules in the biology of this pathway. Such data can also be used to analyze high-throughput data from various platforms including DNA/protein microarrays and mass spectrometry-based proteomics experiments. These data have been submitted to the NetPath resource and are made available in diverse community standard data exchange formats so that they can be easily visualized and analyzed. We hope that this resource will help in designing experiments aimed at expanding the existing knowledge of the TSLP signaling in both normal and disease physiology. We encourage active participation of researchers to improve the quality and content of our resource. The scientific community can give their suggestions and comments through NetPath  and an NIH Roadmap grant ‘Technology Center for Networks and Pathways’ (U54 GM103520 to A.P.). Senior Research fellowship award from Council of Scientific and Industrial Research (CSIR) (to J.S. and S.M.P.).Conflict of interest. None declared.


An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial.Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets.Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology. Two potential mechanisms have been proposed to explain how HTLV-1-specific CD8+ T-cells could mediate damage to non-HTLV-1-infected cells. Firstly, this damage could occur simply as a bystander effect of HTLV-1-specific CD8+ T-cells recognizing the HTLV-1-infected T-cells that have been shown to accumulate in the CNS. Secondly, it has been suggested that there may be cross-reactivity of HTLV-1-specific CD8+ T-cells with self antigens expressed in neurons[+ T-cell responses which, in turn, may be reactive against neurons and other tissues where HERV antigens may be expressed.Human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that has infected humans for thousands of years. An estimated 10–20 million individuals worldwide are infected with HTLV-1, the majority of whom will remain asymptomatic for life. Inflammogenesis-8. As th neurons. Here, w+ T-cell to a HERV-E-derived peptide[+ T-cell clone, isolated from an HIV-1-infected subject, specifically responded to and eliminated cells infected with diverse isolates of HIV-1, HIV-2 and SIV[in vivo expression of HERV antigens, by screening PBMC from HTLV-1-infected subjects for T-cell responses to HERV-derived peptides, with a particular focus on the HERV-K(HML-2) lineage.Human endogenous retroviruses, the DNA remnants of ancient infectious retroviruses, comprise approximately 8% of the human genome. This complement of HERVs is diverse, with all three major branches of the retroviral tree represented: gamma-epsilon, spuma and delta-lenti-alpha-beta retroviruses. While t peptide. As a gr peptide. Indirec peptide-14. We h and SIV. The est and SIV. In the et al. Submitted).In addition to providing evidence for the HTLV- 1-induced expression of HERVs, the detection of HERV-specific T-cell responses in HTLV-1-infected subjects would open a line of inquiry into determining whether such responses may contribute to the pathogenesis of HAM/TSP. It is intriguing to note, given the similarities in clinical presentation and pathogenesis of HAM/TSP and multiple sclerosis (MS), that numerous studies have associated HERV polymorphisms or expression levels with MS (reviewed in). SpecifStaphylococcus enterotoxin B (SEB) tested at 2.5 μg/ml. ELISPOTS were performed as previously described[in vitro culture of PBMC derived from HTLV-1-infected subjects resulted in very high levels of spontaneous IFN-γ production, as has been previously reported[To test this hypothesis, we obtained peripheral blood mononuclear cells (PBMC) from 15 HTLV-1-infected subjects from Sao Paulo, Brazil, and from 8 age/sex matched uninfected subjects from the same geographical area. The HTLV-1-infected subjects comprised 9 individuals who were asymptomatic, 5 with HAM/TSP and 1 subject who we define as pre-ATL. In the latter subject, significant immunophenotype alterations were present at the time of sample collection, but the criteria for an ATL diagnosis were not present. Six months later, this patient was diagnosed with the lymphomatous variant of ATL Table – testedescribed, except reported.6 PBMC after background subtraction and > 3x background versus 3 HERV-specific responses in uninfected subjects. These responses were, however, of borderline magnitude and replicates between duplicate wells were poor. We sought to repeat these responses with new aliquots of cells from the same patient time-points using the same peptide pools and, in parallel, to deconvolute responses to pools by testing individual peptides. In these follow-up experiments we observed that none of these responses were reproducible. The incidence of these false positive responses was related to ‘noisier’ ELISPOTs than those typically observed using PBMC from HTLV-1-uninfected subjects. Although we sought to minimize the impact of spontaneous IFN-γ production on the assay by reducing stimulation periods to 6 hours, we observed higher levels of background in ELISPOTs performed on HTLV- 1-infected subjects than in uninfected controls. Despite this background, we observed clear and reproducible responses to the HLA-A02-restricted Tax peptide in 6 HTLV-1 subjects, and an absence of this response in uninfected subjects. Responses to CEF pool were detected in the majority of both HTLV-1-infected and uninfected subjects.We observed a total of 12 HERV-specific responses in HTLV-1-infected individuals which passed our pre-established criteria of > 50 sfu/10We next summed the background-subtracted responses to: HERV-K(HML-2)-Env, HERV-K(HML-2)-Gag and predicted optimal HERV peptides (HERV-Opt). In screens performed on non-HTLV-1-infected subjects ELISPOT results with negative values after background subtraction are atypical and generally reported as zero. In the present case, however, the variability caused by spontaneous cytokine production, and summed over multiple peptide pools in the cases of HERV-K(HML-2)-Gag and Env (17 and 18 pools respectively), manifested as cumulative totals which spanned a range from substantially more than to less than zero. As zeroing these negative values would give the false impression of positive responses in HTLV-1-infected subjects (instead of just cumulative noise) these data are presented as is. We observed a lack of statistically significant differences between the magnitudes of responses detected in HTLV-1-infected versus uninfected subjects for each of the peptide sets tested Figure. This isThe current study does not provide any data to support the hypothesis that the induction of HERV expression and HERV-specific T-cell responses in HTLV-1-infected subjects may play a role in the pathogenesis of HAM/TSP. This may indicate that, in contrast to HIV-1, HTLV-1 does not induce HERV expression. We have recently demonstrated that the HIV-1-Vif protein is necessary for the induction of HERV-K(HML-2) expression, thus thStaphylococcus enterotoxin B (SEB) was used at 2.5 μg/ml. A response was considered to be positive if it passed both the criteria of: i) > 50 sfu/million PBMC after background subtraction and ii) > 3x background. Statistics were performed using Prism Graphpad 2.0 software.Samples were collected under an IRB approved protocol. PBMC were isolated through Ficoll gradient enrichment and cryopreserved. ELISPOT assays were performed as previously described, but witHTLV-1: Human T-cell leukemia virus type 1; HERV: Human endogenous retrovirus; HAM/TSP: HTLV-1-associated myelopathy/tropical spastic paraparesis; MS: Multiple sclerosis; HIV-1: Human immunodeficiency virus type 1; PBMC: Peripheral blood mononuclear cells; CEF: Cytomegalovirus, epstein barr virus, influenza (peptide pool); ATL: Adult T-cell leukemia; HLA: Human leukocyte antigen.The authors declare that they have no competing financial interests.RBJ, FEL, DFN, MAO, EGK conceived of the project and designed experiments. FEL, ACS recruited study participants and AMH processed samples. RBJ, AMH, MAO performed the ELISPOT assays. RBJ and AMH analyzed the ELISPOT data. DFN, MAO, EGK supervised the project. Experiments were performed in the laboratory of EGK. RBJ wrote the manuscript with input from all co-authors. All authors read and approved the final manuscript.


Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for  Drosophila) are transmembrane proteins Virtually all signaling pathways have an endosomal component Several results support the pulling model. First, when separated from its transmembrane domain and secreted, the extracellular domain of Delta blocks Notch activation The pulling and recycling models are not necessarily mutually exclusive. It has been proposed that two ligand internalization events are required, the first to activate ligand through recycling, and the second to activate the receptor on an adjacent cell through pulling C. elegansDrosophilaDrosophila tissues tested Drosophila female germline cells require epsin-mediated endocytosis, but not clathrin Epsin and auxilin are two endocytic proteins required in signaling cells for ligand endocytosis and signaling Drosophila to test the roles of Rab11 and auxilin in Notch signaling, and ultimately to test the recycling model. First, we found that Rab11 is not required for Notch signaling events in the eye disc that require both epsin and auxilin. Second, we found that female germline cells that do not require clathrin in order to signal also do not require auxilin. Finally, we found that overexpression of both clathrin heavy chain and epsin suppress nearly completely the lethality and severe eye morphology defects of auxilin mutants. Taken together, the results argue strongly that in many cell types, ligand recycling is not the primary function of epsin-dependent ligand endocytosis by Notch signaling cells.Here, we performed genetic experiments in lqf or aux mutants), ommatidia have one or several extra photoreceptors shibire (encodes Drosophila dynamin) or Delta genes are expressed specifically in R2/R5 and R3/R4 using a rough (ro) gene expression vector, ommatidia in adult eyes have extra R-cells due to failure of R-cell restriction ro expression vector, we generated transgenes expressing dominant negative forms of Rab5 or Rab11 (N142Iro-Rab5 and N124Iro-Rab11). Rab11N124I has been shown to act as a dominant negative late in eye development, where it blocks transport of rhodopsin to rhabdomeres and formation of multivesicular bodies in late endosomes Rab5 nor Rab11 is required for this Notch signaling event, but there are other plausible explanations for the failure of these transgenes to interfere with Notch signaling. For example, expression levels that are too low for effective competition with wild-type proteins.We wanted to determine whether or not ligand recycling is required for Notch signaling during eye development. If so, it would be expected that the two GTPases Rab5 and Rab11 would both be required in signaling cells. Rab5 mediates fusion of early endosomes with the sorting endosome, an event required for trafficking through any endosomal pathway, and Rab11 is required for subsequent routing of an endosome through the recycling pathway Rab5- or Rab11- (null) clones in the eye disc. Rab5 null clones have an overgrowth phenotype that would obscure a Notch signaling defect Rab11 null clones in the eye have not been reported, but we were able to generate them, and they were not hypertrophic (see below). The Rab11 null allele we used, FRTRab11, has a deletion of the promoter and first two exons, and produces no protein Rab11 null clones to ask whether or not well-characterized signaling events in the eye disc required Rab11. The adult Drosophila eye develops from the larval eye imaginal disc, a monolayer epithelium Notch- clones, no cells are specified as photoreceptors because Notch signaling is required anterior to the furrow to give cells neural potential, a process called proneural enhancement Delta- clones, there are no photoreceptors in the middle of the clone. At the clone border, however, Delta- cells do become photoreceptors because they receive Notch signals from adjacent wild-type cells. Discrete ommatidia do not form within the clone because subsequent lateral inhibitory signaling cannot occur between adjacent Delta- cells, and the result is that too many cells adopt neural fate lqf- (liquid facets [lqf] is the Drosophila epsin gene auxilin- (aux-) cells in the eye disc appear identical to Delta- clones, consistent with the idea that epsin and auxilin are required in the signaling side of the Notch pathway N- cell clones, and are expressed in Dl-, lqf-, or aux- clones only in cells at the clone border, adjacent to wild-type cells that can signal .To overcome the problem in interpreting results obtained with dominant negative transgenes, we wanted to generate Rab11- (null) clones in eye discs would suffer severe defects in early ommatidial assembly, and whether or not Rab11- cells, especially those in the middle of the clone, would activate Notch. First, we observed Rab11- clones in eye discs immunostained with anti-Elav, which labels photoreceptor nuclei N-, Dl-, lqf-, or aux- clones Rab11- clones; discrete ommatidia were present in the middle of the clone and at the clone borders  clones expressed m∂-lacZ (Delta- (null) cells did express m∂-lacZ, but only when they were adjacent to wild-type cells at the clone edges  or aux- (null) cells at the clone edge activated m∂-lacZ  and lqfaux in signaling cells Auxilin is known to be required for Notch signaling in the eye, wing, and embryo aux+ was required in the female germline. In the ovary, the sixteen germline cells in the nurse cell/oocyte complex signal to surrounding somatic follicle cells at stage 6 of oogenesis, and Notch receptor activation may be monitored by expression of the target gene Hindsight (Hnt) (aux+ and the germline cells are aux- (null), the follicle cells nevertheless express Hnt   [40], [4ht (Hnt) ’ [40], [ress Hnt ’. Identiaux mutants Chc+ overexpression partially suppressed the Notch signaling defects in eyes (and wings) associated with strong aux mutants aux mutations is also be suppressed by Chc+ overexpression. In addition, we tested whether or not epsin overexpression also suppresses the aux mutant phenotype, and if the extent of suppression would be increased by by co-overexpressing clathrin heavy chain and epsin.The requirement for auxilin by the signaling cells provides a tool for testing the recycling model. Auxilin uncoats clathrin-coated vesicles, an expected prerequisite for fusing of newly endocytosed vesicles with the sorting endosome and subsequent transit through an endocytic pathway aux mutants, presumably caused by the failure of Notch signaling in early development Chc+ overexpression. Heterozygotes for one weak missense mutation and one strong nonsense mutation in aux (K47aux/D128aux) K47aux/D128aux flies of a transgene containing a genomic DNA copy of the Chc+ gene (PgChc+) that can substitute for the endogenous Chc+ gene First, we wondered how well the lethality of Chc+ overexpression, would suppress the K47aux/D128aux mutant phenotype. We reasoned that if epsin links ligand to clathrin, it may be freed along with clathrin when auxilin uncoats clathrin from newly endocytosed vesicles. Alternatively, increased epsin levels in aux mutants may result in more efficient plasma membrane localization of the remaining free clathrin. We found that a transgene with a genomic DNA copy of the lqf+ gene (Pglqf+) that complements lqf null mutants  rescued the lethality of K47aux/D128aux mutants (PgChc+ did (62% wild-type ommatidia)   . Moreovematidia) . HoweverChc+ gene or the lqf+ gene suppressed the aux mutant phenotype, including lethality, significantly. Remarkably, a single extra copy of both the Chc+ and lqf+ genes suppressed nearly completely the severe morphological abnormalities due to Notch signaling defects in aux mutants. This indicates that supplying free clathrin heavy chain and additional epsin to the cells bypasses the large part of the need for auxilin in Notch signaling. We conclude that the primary role of auxilin in Notch signaling cells is to maintain the pool of free clathrin, and possibly also epsin.Thus, a single extra copy of either the Rab11 is not required for several Notch signaling events in the developing Drosophila eye that require epsin and auxilin. Thus, as in the female germline cells, ligand recycling, at least via a Rab11-dependent pathway, is not necessary for Notch signaling in the eye disc. Second, we found that the one Notch signaling event presently known to be clathrin-independent is also auxilin-independent. This result reinforces the idea that rather than performing some obscure function, the role of auxilin in Notch signaling cells is to regulate clathrin dynamics. Finally, we showed that overexpression of both clathrin heavy chain and epsin rescues to nearly normal the severely malformed eyes and semi-lethality of aux hypomorphs. Presumably, vesicles uncoated of clathrin fuse with the sorting endosome, and so it seems reasonable to assume that uncoating clathrin-coated vesicles containing ligand is preprequisite for trafficking ligand through endosomal pathways. Thus, if ligand endocytosis is prerequisite to recycling, efficient production of uncoated vesicles would be required. In aux mutants with severe Notch-like mutant phenotypes, clathrin vesicle uncoating is inefficient. We presume that this remains so even when clathrin and epsin are overexpressed, yet the eye defects and lethality are nearly absent. Thus, we reason that auxilin is required not for efficient production of uncoated vesicles per se, but for the other product of auxilin activity – free clathrin . Taken together, these results argue strongly that at least in some cell types, the fundamental role of Notch ligand endocytosis is not ligand recycling.There are three major results of this work. First, we found that only to activate ligand via recycling in some cellular contexts, and only for exerting mechanical force on the Notch receptor in others? While formally possible, this is not parsimonious. Thus, we favor a model where the fundamental role of ligand endocytosis is to exert mechanical force on the Notch receptor. In addition, some cell types will also require ligand recycling. As no altered, activated form of ligand has yet been identified, while ligand transcytosis has been well-documented Is it possible that the fundamental mechanism of Notch signaling is so completely distinct in different cell types, that ligand endocytosis serves http://flybase.org/) are provided when available. Chromosomes and genotypes used in particular experiments are indicated in Figure Legends. Mutant alleles: F956*aux , K47aux , D128aux , 136aux , 727aux, ΔFRTRab11revF10Dl , 55e11N . Transgenes: PgChc+tub-auxey-flp (FBti0015982), m∂-lacZ , 122hs-flp (on X), ubi-ngfp , Hrb98DE::GFPFRT82B (FBti0002074), FRT18A (FBti0002070), FRT40A (FBti0002071), FRT53775-5Z3515FRTey-gal4 (on 2), UAS-flp (on 2), N124IUAS-Rab11 . Transgenes generated in this work: Pglqf+ (on 2), N142Iro-Rab5 (multiple lines), N124Iro-Rab11 (multiple lines).The alleles and transgenes used are listed below. FlyBase id numbers .This construct is an ∼16,240 bp 1118w females was isolated using TRI reagent (Molecular Research Center), and 5 µg was used for reverse transcription with SuperScriptII (Invitrogen). The primers used were Rab5F (5′-AAAGGCGCGCCATGGCAACCACTCCACGC-3′) and Rab5R (5′-AAAGGCGCGCCTCACTTGCAGCAGTTGTTCG-3′). The cDNA was diluted to 200 µl, and 2 µl was used as the template for the following PCR reactions. The mutant Rab5 cDNA was generated in two steps. First, two PCR reactions were performed with mutagenic primers, Rab5CF (TCAAGGCAG-3′5′-GGCCGGCA) and Rab5NR (ATGCCGGCC-3′5′-CTGCCTTG). One reaction used the primer pairs F and NR, and the other used R and CF. Next, the amplification products from each reaction were mixed, and used together as a template for PCR with primers F and R. The resulting amplification product was ligated as an Asc I fragment into BluescriptIIKS+ (Stratagene) with its Bam HI site changed to Asc I, an its DNA sequence was verified. Finally, an ∼660 bp Asc I fragment containing the N142IRab5 cDNA was ligated into pROTotal RNA from 5 Rab11 cDNA was obtained by PCR using as template genomic DNA from flies containing N124IUAS-Rab115′-AAAGGCGCGCCATGGGTGCAAGAGAAGACGA-3′) and Rab11R (5′-AAAGGCGCGCCTCACTGACAGCACTGTTTGC-3′). The resulting ∼660 bp amplification product was ligated as an Asc I fragment into t –XApUASAsc I.The mutant Plastic sectioning of adult eyes was performed as described aux- germ line clones were generated by heat shocking first to third instar larvae at 37°C for 2 hours on 2 consecutive days. Adult females of the appropriate genotype were collected upon eclosion. The females were fed on yeast in the presence of males for 2 days, flipped onto fresh yeast for 2 more days, and then their ovaries dissected. Egg chambers were fixed in 4% formaldehyde in 1X phosphate-buffered saline (PBS) for 15 minutes and washed with PBS. Primary antibody staining was performed in 1X PBS + 0.3% Triton-100 (PBT3) containing 5% normal goat serum overnight at 4°C, followed by washing with PBT, staining with secondary antibodies, and mounting in antifade reagent (Invitrogen). The following antibodies were used: mouse anti-Hindsight at a dilution of 1∶50 (DSHB) and 647-AlexaFluor donkey anti-mouse (Invitrogen). Cells were also labeled with TRITC-phalloidin (Sigma) at 1∶200 to detect F-actin. Images were collected using a Leica TCS confocal microscope and assembled using Adobe Photoshop. Single sections are shown for each sample.


For HIV-1, various modes of cell-to-cell transfer have been reported, including virological synapses, polysynapses, filopodial bridges, and nanotube-like structures. So far, only synapses and biofilms have been described for HTLV-1 transmission. Recently, Van Prooyen  Infection by HTLV-1 is also associated with tropical spastic parapesis/HTLV-1-associated myelopathy (TSP/HAM), a neurological disorder. This retrovirus is barely secreted from infected cells, but is efficiently transmitted by cell/cell contacts such as virological synapses (VS) ,3 or pol protein . Many diet al. [To investigate the role of p12 and p8 in HTLV-1 transmission, Van Prooyen et al. [This work suggests the existence of a new mode of HTLV-1 transmission mediated by p8, and reconciles some contradictory observations concerning the p8/p12 proteins. However, the molecular mechanism by which p8 promotes HTLV-1 transmission remains unknown. Especially, it remains unclear whether p8-mediated increase of viral transmission is mediated by (i) cellular conduits, as suggested by the authors (ii) transfer through virological synapses and polysynapses (iii) syncytia, as proposed by Taylor et al.  or (iv) et al. . MoreoveLike HTLV-1, Human immunodeficiency virus type 1 (HIV-1) is transferred directly from cell to cell, using filopodes, nanotubes, VS and PS. Interestingly, the features of p8 are highly reminiscent of those of the HIV-1 Nef protein .HTLV-1 and HIV-1 use viral synapses to promote efficient transmission. They both encode one accessory protein present in the lipid rafts, targeting similar cellular functions, such as MHC-I and TCR down-regulation, and impairment of immune synapse formation. Both Nef and p8 induce the formation of membrane extensions, which may allow them to be transferred to target cells. Nef, for instance induces the formation of intercellular conduits bridging infected macrophages to bystander B lymphocytes (LB). Nef is then transferred to LB and inhibits IgG2 and IgA class switching . Nef alsin vitro propagation. In contrast, viruses deleted for these proteins replicate less efficiently in non-human primates [in vivo relevance of the results described by Van Prooyen et al. [et al. reported a predominance of alleles encoding uncleavable forms of p12 in a cohort of HTLV-1-infected patients [in vivo.Nef and p8/p12 appear to be dispensable for primates ,10. The patients . More woin vitro and in vivo and identifying the cellular proteins involved in this new mechanism. Since HIV-1 Nef and HTLV-1 p8 share common features, it will be of interest to determine if these two proteins interact with the same host proteins to mediate their effects on viral replication.Overall, this work identifies a new mode of HTLV-1 transmission using cellular conduits induced by the viral protein p8. To gain further insight into HTLV-1 transmission and pathogenesis, it will be worth better characterizing the specific functions of p8 and p12


Human T cell lymphotropic virus, type 1 (HTLV-1), is the causative agent of adult T cell leukemia (ATL), a debilitating disease with a poor prognosis and no cure. HTLV-1 is a retrovirus which contains a 9 kb ssRNA genome, encoding the classic retroviral genes gag, pol, and env. HTLV-1 also encodes several regulatory genes, including tax. Tax is a 353 amino-acid, 40 kiloDalton (kDa) protein, located predominantly in the nucleus although a small percentage is isolated in the cytoplasm. Tax functions to regulate viral gene expression by interacting with the host transcription factor family, CREB/ATF. The Tax/CREB complex is formed on the 5’ LTR CRE site and activates viral gene transcription. In ATL cells , Tax also associates with two host transcription factors, NF-κB and SRF (serum response factor), resulting in the over-expression of host genes containing the NF-κB responsive element and the SRE (serum response element). The host genes, such as IL-2 and c-fos, are activated constitutively in the presence of Tax and result in cell proliferation of HTLV-1+ T cells, potentially leading to leukemia in the HTLV-1 patient. We believe that Tax activation of the SRF pathway serves as the initial transformation event of infected T cells. Thus, we are interested in the biochemical properties of Tax interactions with SRF, SRF/DNA, TCF , SRF/TCF, and SRF/TCF/DNA. Prior to conducting biochemical experiments, however, we need to express and purify Tax which has been difficult to accomplish in the HTLV-1 field and has impeded any progress toward studying the biophysical properties, including structure determination, of Tax. We present data showing our initial protein purification techniques that result in the isolation of a 99% pure, untagged, full-length Tax as well as the expression and purification of full-length, untagged human SRF.


In vivo, HTLV-1 infects T lymphocytes that participate in adaptive immunity but also monocytes and dendritic cells that are major players in innate immunity. Herein, we will review the interplay between HTLV-1 and innate immunity. Particular emphasis is put on HTLV-1-induced alteration of type-I interferon (IFN-I) function. In vitro, the viral Tax protein plays a significant role in the alteration of IFN synthesis and signaling. Despite this, IFN-I/AZT treatment of Adult T-cell Leukemia/Lymphoma (ATLL) patients leads to complete remission. We will discuss a model in which exogenous IFN-I could act both on the microenvironment of the T-cells to protect them from infection, and also on infected cells when combined with other drugs that lead to Tax down-regulation/degradation.Innate immunity plays a critical role in the host response to a viral infection. The innate response has two main functions. First, it triggers effector mechanisms that restrict the infection. Second, it primes development of the adaptive response, which completes the elimination of the pathogen or of infected cells.  Classically, two types of immune responses have been defined: the innate response, a rapidly engaged but transient and poorly specific response, and the adaptive response, a delayed but specific response which allows the development of immune memory.1.1.i.e., IFN-α and IFN-β) are highly effective anti-viral mediators. IFN-I production is not constitutive but must be induced when the cell senses an infection.In 1957, Isaacs and Lindenmann observed that culture medium from influenza-infected cells induced resistance in naive cells. They hypothesized that a substance secreted by the infected cells inhibited the viral spread [Pathogen-Associated Molecular Patterns). Examples of viral PAMPs include double-stranded RNA, single-stranded RNA, non-methylated CpG DNA and envelope glycoproteins. The sensors that recognize these viral PAMPs, such as TLRs (Toll-Like Receptors) and RLHs (RIG-I-Like Helicases), are referred to as PRRs (Pattern Recognition Receptors). Interaction of the PAMPs with the PRRs triggers signaling pathways that lead to activation of IRF3 (Interferon Regulatory Factor 3) and IRF7 transcription factors, eventually allowing transcription from the IFN-I promoters [All cells express viral sensors, either at the cell surface, at the endosomal membrane or in the cytoplasm ,3. Theseromoters ,5.dsRNA-dependent Protein Kinase), a well-studied representative of ISGs, is a serine-threonine kinase that inhibits translation upon viral infection by phosphorylating the initiation factor eIF2α [IFN-I displays both autocrine and paracrine effects. Engagement of the IFN-I receptors (IFNAR-1 and IFNAR-2) at the cell surface induces activation of Jak1/Tyk2 and STAT1/STAT2 signaling cascade that subsequently promotes the expression of a large number of anti-viral effectors known as interferon-stimulated genes (ISGs) . As an eor eIF2α . Active + CD4+ CD11c− DCs are found in an immature stage in the peripheral blood, and more predominantly in inflamed tissues [+ CD4+ dendritic cells  represent a distinct TLR1-8 and TLR10-expressing DC type that circulates in the blood and constantly migrates into secondary lymphoid organs [in vitro studies of DCs use myeloid-like DCs generated from CD14+ monocytes. It should however be kept in mind that such in vitro monocyte-derived DCs (MDDCs) are not identical to in vivo or ex vivo-purified DCs. Therefore, those results should sometimes be handled with caution [IFN-I can be produced by all cells upon sensing virus infection, and in particular by innate immune cells such as macrophages and dendritic cells (DCs). Plasmacytoid dendritic cells (pDCs) are the major IFN-I producers. These CD123 tissues . They exIn addition, other effectors of innate immunity such as restriction factors are present in cells before infection takes place and can act as immediate inhibitors of a given infectious agent . When thInnate immune cells such as macrophages and DCs belong to the family of professional antigen-presenting cells. They have the capacity to capture antigens, process them and display them to lymphocytes . Antigen1.2.It is estimated that the human T-lymphotropic virus type 1 (HTLV-1) retrovirus infects 15 to 20 million individuals throughout the world . The HTLThree modes of transmission are known for HTLV-1. (1) Mother-to-child transmission, the efficiency of which varies from 10 to 20% and which occurs after prolonged breastfeeding, mainly after 6–9 months of age ,12. (2) i.e., Adult T-cell Leukemia/Lymphoma or ATLL and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis or HAM/TSP) are described in all endemic areas [+ T-cell malignant lymphoproliferation that is characterized by clonal integration of the HTLV-1 provirus in tumor cells [et al. have proposed a classification of ATLL into 4 subtypes: smoldering, chronic, lymphoma and leukemic/acute [The two major diseases associated with HTLV-1  Can the effectors of innate immunity (and in particular IFN-I) prevent spread of the virus? (iii) Does HTLV-1 infection alter the innate immune response and if so, what are the underlying mechanisms?The aim of this review is to provide an overview of the current state of knowledge of the interplay between HTLV-1 and innate immunity. Of particular interest will be the following points: (i) Does HTLV-1 infect cells that participate in the innate immune response 2.In vivo and irrespective of the clinical status , ATLL or HAM/TSP patients) the HTLV-1 provirus is predominantly detected in CD4+ T lymphocytes [+ T lymphocytes [in vivo cellular targets of HTLV-1 are cells from the adaptive immune system.phocytes , even ifphocytes –41, and phocytes . It thusin vitro and/or are infected in vivo [in vivo infection of DCs was demonstrated almost 20 years ago in HTLV-1-infected individuals [in vivo [4 cells was shown to be proportionally higher in pDCs purified from an HTLV-1 AC than in total peripheral blood mononuclear cells (PBMCs) from the same individual [However, innate immune cells  are permissive to the virus  in vivo ,43–46 , both for the infection of DCs and for transmission of the virus to T cells [gag viral protein in the culture supernatant 14 days after viral exposure [in vivo has not been elucidated, but these results suggest that infection of DCs is required to establish and maintain HTLV-1 infection in this animal model.A critical role was assigned to DC-SIGN . A recent in vitro study suggested that normal human pDCs are activated and able to secrete IFN-α by a TLR-7-dependent mechanism when put in contact with concentrated purified HTLV-1 virions [Even though a number of studies have tried to evaluate whether exogenous IFN-α/β, alone or in combination with other molecules (see below), could play an antiviral role during the treatment of HAM/TSP or ATLL patients, IFN-α production was not measured  virions   was not provided in this study. It is therefore possible that the number of NK cells present in the blood might not reflect the total number in the whole body [Similar to monocytes and DCs, altered function and frequency of NK cells were reported in HTLV-1-infected individuals. Significantly decreased NK cell activity , togetheividuals . Since Nole body . Thus, t4.1.3.ex vivo stimulation, indicating a severe functional impairment [iNKT cells were shown to be less abundant in HAM/TSP and ATLL patients than in uninfected individuals ,63. iNKTpairment .Taken together, these results indicate that HTLV-1 can alter innate immune cell functions, which has led some groups to search for a role of individual viral proteins in modulation of the molecular pathways involved in innate function.4.2.4.2.1.de novo upon IFN stimulation and contributes to amplification of the IFN response, by inducing expression of IFN-α [ex vivo CD4+ cells isolated from 30 HTLV-1-infected individuals  demonstrated that SOCS1  is strongly up-regulated in ACs and patients with HAM/TSP but not in those with ATLL. This protein was previously shown to suppress IFN signaling by preventing STAT1 phosphorylation [SOCS1 expression correlated with HTLV-1 PVL in CD4+ cells obtained from HAM/TSP patients. Tax was recently shown to promote the expression of SOCS1 in vitro [tax) are likely to be impaired for early IFN induction signaling.IRF3 and IRF7 play essential roles in the early phase of IFN-I gene activation. IRF3 is constitutively expressed and activated through its carboxy-terminal phosphorylation by IKKɛ and TBK1. This promotes the transactivation of downstream genes such as IFN-β. In contrast, IRF7 protein is synthesized of IFN-α . A very rylation . Interesin vitro . In PBMCet al. showed that HTLV-1 infection and in particular Tax expression leads to constitutive IRF3 phosphorylation and activation in HTLV-1-transformed cell lines [vs. laboratory cell lines where 100% of the cells are infected and constitutively produce high amounts of infectious virions [These observations are, however, not consistent with a previous report where Suzuki ll lines   alters TLR4 signaling, a critical pathway in the innate response to bacterial infection, and inhibits the production of pro-inflammatory cytokines normally secreted in response to TLR4 stimulation [The viral p30 protein was also shown to modulate innate cell activation . Expressmulation . This al4.2.2.in vitro [env or pol expression, was independent of the NF-κB pathway, and might be mediated through Gag or Protease expression. Unfortunately, the effect of Tax overexpression was not analyzed, so it remains unclear whether or not this protein is involved in the process. Downstream of Tyk2 and STAT2 phosphorylation, the active ISGF3 complex (Interferon Stimulated Gene Factor 3), containing phosphorylated STAT1, STAT2, IRF9, and the CBP/p300 transcription co-activators, triggers the expression of a number of genes. In addition to its effect on SOCS1, Tax overexpression also alters ISGF3 function by preventing interaction of the STAT2 component of ISGF3 with CBP/p300, therefore leading to modulation of the IFN-α transduction cascade [Following IFNAR-1/IFNAR-2 engagement by IFN-I, the Jak1 and Tyk2 proteins are phosphorylated and induce a signaling cascade involving STAT1 and STAT2. Several reports showed that HTLV-1-infected cells still express IFNAR receptors ,26. Therin vitro   by intra-muscular injection for a period ranging from 4 to 793 days. However, in most cases, the treatment duration did not exceed a month. IFN-α treatment of a group of 25 HAM/TSP patients led to a significant although slight decrease of HTLV-1 PVL and to a limited improvement of the clinical status [+ effector T cells inversely correlated with PVL [IFN-I has cytostatic and antiviral properties and therefore provides a possible approach for treating chronically infected individuals. Though the literature suggests that (i) HTLV-1-infected cells have an impaired ability to activate innate immunity and that (ii) a number of HTLV-1 encoded proteins blunt IFN-I signaling, a series of clinical trials using IFN-I were performed in Japan –83 and il status . This stwith PVL . Neverthet al. evaluated the clinical and virological efficacy of escalating doses of IFN-β (30 μg once a week up to 60 μg twice a week) during a 28-week period in a small number of HAM/TSP patients (n = 12) whose disease duration ranged from 2 to 20 years [tax mRNA and of HTLV-1 specific CD8+ cells was observed.Oh 20 years . AlthougIFN-α combined with AZT is now being used to treat the leukemic, chronic and smoldering forms of ATLL , althoug6.in vivo , researchers tried to define its mechanism of action in vitro. One study demonstrated that treating 293T cells transfected with an HTLV-1 molecular clone  with IFN-α led to decreased virion production measured by the presence of p19Gag in the cell culture supernatant. This decreased virion production was not a result of reduced transcription or translation but rather, was due to inhibition of HTLV-1 assembly by preventing the interaction of p19Gag with lipid rafts [After it was shown that IFN-I displayed some effects id rafts . The recid rafts  and previd rafts  might exet al. then reported that co-cultivation of 293T human cells or NIH3T3 murine cells together with IL-2-dependent HTLV-1-infected cells derived from different ATLL patients also resulted in decreased p19Gag in the cell culture supernatant. However, in this study the effect was due to transcriptional repression of HTLV-1 expression, and these effects were mediated mostly through IRF7-dependent IFN-β secretion by murine epithelial cells [in vivo, it is conceivable that those uninfected cells would secrete IFN-I after they establish contact with infected CD4+ cells. This could control HTLV-1 expression in these infected lymphocytes. Together, these results suggest that, IFN-I can alter HTLV-1 production in vitro, although the precise mechanism of action remains to be clarified.Consistent with this work, Kinpara al cells . Interesal cells ,88. Sinc7.in vitro sometimes seem difficult to reconcile with observations that were made in vivo. Indeed, a significant number of reports clearly demonstrate that HTLV-1-infected cells have a reduced ability to act as members of the innate immunity network and that HTLV-1 proteins, in particular Tax, prevent both type I IFN secretion and IFN signaling. Nevertheless, combinations of IFN-α/AZT or IFN-α/AZT/arsenic trioxide represent efficient means to obtain complete remission in most ATLL subtypes (except the lymphoma type) and in the murine ATLL model. This might be due to the fact that human or murine ATLL cells barely express Tax and, therefore still possess an intact IFN signaling pathway and are sensitive to IFN-α/AZT. In addition, arsenic trioxide, by promoting the degradation of Tax could also restore IFN signaling. Less pronounced IFN-α effects in HAM/TSP patients are likely to be due to a higher Tax expression, which then impairs IFN signaling. Finally, in ATLL patients exogenous IFN-I might also act on uninfected cells and render them less susceptible to infection.The different reports that have been published on the interplay between HTLV-1 infection or HTLV-1 protein expression and innate immune cells In conclusion, understanding the intimate interplay between HTLV-1 and the innate immune response will require additional studies. It will also be crucial that future work aimed at defining these interactions be performed with patient samples and primary cells in order to avoid overexpression systems that might lead to misinterpretation of the results.


HTLV-I proviral load (pVL) is proposed as a biomarker of disease progression. HTLV-I pVLs are higher in symptomatic compared to asymptomatic carriers. The aim of this study was to evaluate HTLV-I pVL in patients with HTLV-I associated diseases (HAM/TSP and ATLL) and other disorders (uveitis and pyoderma gangrenous–PG).6 PBMCs (0.04%). Differences among groups where assessed by Mann-Whitney test.HTLV-I pVL was evaluated in 62 subjects; 52 asymptomatic and 10 symptomatic . HTLV-I pVL in PBMCs was estimated by a quantitative real-time PCR assay with SYBR Green, where HTLV-I pol gene is amplified in parallel with albumin gene as a normalizer. The limit of detection of the assay was of 400 copies of HTLV-I/106 PBMCs, IQR=4.70-5.30) had significantly higher pVLs than asymptomatic carriers . HAM/TSP patients had pVLs between 5.00-5.73 log10 copies/106 PBMCs (10-54%), ATLL patients had pVLs of 4.83 and 5.50 log10 copies/106 PBMCs ; patients with uveitis had pVLs of ~4.00 log10 copies/106 PBMCs (~1%) and PG patient had pVL of 4.56 log10 copies/106 PBMCs (4%). Patients with HAM/TSP and ATLL had significantly higher pVL (median=5.25 log10 copies/106 PBMCs) than those with other disorders .Symptomatic subjects (median=5.02 log10 copies/10HTLV-I pVL seems to be associated with pathogenic phenotype, being higher in subjects with HTLV-I associated diseases compared to those with other disorders.