This review summarizes the current knowledge of aquaglyceroporins’ involvement in several pathologies and the experimental approaches used to evaluate glycerol permeability and aquaglyceroporin modulation. In addition, we provide an update on aquaglyceroporins modulators reported to impact disease, unveiling aquaglyceroporin pharmacological targeting as a promising approach for innovative therapeutics.Aquaglyceroporins, a sub-class of aquaporins that facilitate the diffusion of water, glycerol and other small uncharged solutes across cell membranes, have been recognized for their important role in human physiology and their involvement in multiple disorders, mostly related to disturbed energy homeostasis. Aquaglyceroporins dysfunction in a variety of pathological conditions highlighted their targeting as novel therapeutic strategies, boosting the search for potent and selective modulators with pharmacological properties. The identification of selective inhibitors with potential clinical applications has been challenging, relying on accurate assays to measure membrane glycerol permeability and validate effective functional blockers. Additionally, biologicals such as hormones and natural compounds have been revealed as alternative strategies to modulate aquaglyceroporins  AQPs fa2, O2  or NO  are involved in a series of malfunctions and diseases, most of them related to their ability to facilitate glycerol diffusion across cell membranes, a feature that is determined by a larger constriction region of their size selectivity filter compared to orthodox aquaporins  .Glycerol is an important molecule in energy metabolism for its crucial involvement in lipogenesis in feeding conditions and lipolysis and gluconeogenesis during fasting, contributing for energy homeostasis . Due to Skin represents the first line of defense of the human body covering the entire external body surface, where aquaglyceroporins are essential for skin physiology . AQP3 isAdipose tissue and liver are the main organs involved in body energy homeostasis. AQP7 is expressed in both adipocyte and capillary plasma membranes of human adipose tissue  while AQIn the endocrine pancreas, AQP7 is the main aquaglyceroporin and is responsible for glycerol uptake into β-cells . AQP7 knIn the liver, AQP9 was identified as the major route for glycerol uptake where it can be used in gluconeogenesis . AQP9-nuIn the heart, AQP7 has an important role in energy production . AQP7 knIn endothelium, AQP3 was identified as the predominant aquaglyceroporin, being responsible for endothelial glycerol permeation . AQP7 waIn intestinal inflammatory disorders, AQP3 is expressed in the basolateral membrane of colonic epithelial cells and was associated with enterocyte proliferation in a AQP3 null mice model of colitis, that showed epithelial cell damage and loss, colonic hemorrhage, impaired enterocyte proliferation and reduced survival compared to wild-type mice . With orThe kidney is critical for the maintenance of body water homeostasis and the involvement of AQPs in acute and chronic kidney diseases is well recognized . Althoug2O2 may promote tissue inflammation by increasing reactive oxygen species in macrophages  relies on rug hits . The metXenopus laevis  glycerol provides an experimental approach to determine aquaglyceroporin activity in a variety of cell types and vesicles. Cells are rapidly mixed with an incubation medium containing radioactively labeled permeant solute. After appropriate time intervals, the uptake is blocked by adding stop solution and cells are dropped on a filter that is subsequently washed and counted for radioactivity. Efflux from vesicles pre-loaded with labelled substrates can also be measured using similar procedures. Although these assays are simple and rapid and can provide mechanistic information on transport kinetics  which is cleaved inside the cell by esterases, resulting in an impermeable fluorescent form that is trapped intracellularly . Volume Another fluorescence microscopy method, that does not require dye loading, uses a genetically encoded yellow fluorescent protein (YFP-H148Q-V163S), whose fluorescence is quenched by chloride. The cytoplasmic chloride concentration changes as the cell volume increases or decreases, resulting in altered YFP fluorescence . The chlStopped-flow spectroscopy allows to follow the cell volume changes after a rapidly imposed osmotic or solute gradient. It has been used to quantify water and solute permeabilities in suspended cells, vesicles or liposomes . To assegly) is based on the rate of glycerol movement through membranes caused by a glycerol chemical gradient. When using cell volume measurements , Pgly can be calculated by fitting a single exponential equation to the signals obtained after imposing inwardly glycerol gradients, using the relation Pgly = k (V0/A) (cm s−1) where k is the single exponential rate constant and V0/A is the initial cell volume to area ratio  of glycerol transport is a measure of the energy barrier needed for glycerol diffusion across the plasma membrane. The Ea value is much lower when glycerol permeation occurs via aquaglyceroporins than via simple diffusion through lipid bilayer, making this parameter extremely valuable to ascertain aquaglyceroporin activity and the proof of concept for the identification of new inhibitors. Ea is assessed by performing the permeability assay along a range of temperatures. A weak dependence on the assay temperature indicates that glycerol diffusion via the hydrophilic protein pore dominates over the lipid pathway. Ea  can be calculated by the slope of an Arrhenius plot (ln Pgly as a function of 1/T) (The activation energy (E of 1/T) .In silico approaches have contributed to the understanding of AQPs structure and function, their selectivity for water or other substrates, as well as elucidating structure-activity relationships  atomistic simulations were used to reconstruct the free-energy profile for substrate permeation from multiple independent runs  and p-chloromercuribenzene sulphonate (pCMBS) were the first metal compounds identified as AQP inhibitors and are still used in functional assays despite their associated toxicity and lack of selectivity -benzimidazole, AQP3 has been associated with cell proliferation in cancer cells, namely in skin tumors where it is highly expressed . In receur bonds . Furthervia AQP3 blockage in colonic epithelia , being endowed with increased stability in aqueous environment relative to the benchmark inhibitor Auphen (in vitro (in vivo (via reductive elimination at the Au(III) centre. MetaD provided initial mechanistic insights on the effects of the inhibitors’ binding to AQP10, showing that cysteine arylation reduces glycerol conductance Clr Auphen , were shr Auphen . Some ofin vitro  and in v analogue of Auphen, [Cu(phen)Cl Cuphen,  was also Cuphen,  and colo Cuphen, , leading Cuphen,  and colo Cuphen, . Finallyeability . AltogetSmall molecules with low molecular weight (<900 Da) have been reported to modulate aquaglyceroporins expression and function , exertinThe promoters of the genes encoding AQP3, AQP7 and AQP9 exhibit putative peroxisome proliferator response elements (PPRE), suggesting that PPAR can influence AQP expression . In factvia AQP9 blockage in rat hepatoma cells , a cell-permeable synthetic analogue of cAMP, was found to upregulate AQP9 mRNA and protein expression in rat astrocytes. The effects of dbcAMP on AQP9 mRNA levels were reverted by pre-treating astrocytes with protein kinase A (PKA) inhibitors, suggesting that PKA is involved in the regulation of AQP9 expression . The incNatural compounds are bioactive products derived from natural sources, usually plants, that are known for their beneficial effects in disease prevention and in reducing the risk of chronic diseases including cardiovascular diseases and cancer . These b2O3 uptake in myeloid leukemia cells and, consequently, augmented arsenic sensitivity , chrysin and glycolic acid were found to revert the UV-induced AQP3 downregulation in human keratinocytes, revealing their protective role against UV-induced photoaging . Interessitivity . In addisitivity . Glycyrrsitivity .2 and CuSO4)  . This sa disease . Daiokan disease . Surpris disease . Recentl disease .ex vivo lipolysis experiments and the reduction in adipocyte size, could be explained by AQP7 upregulation for its crucial role in adipocyte glycerol permeability and control of triglyceride accumulation  and peptides, called “biologicals,” are naturally occurring molecules with therapeutic properties which have also been described as modulators of aquaglyceroporins’ expression, as described below.Modulation of aquaglyceroporins’ expression by hormones has been well documented in the literature. The tight regulation of AQP7 expression in insulin-sensitive tissues and the modulation of AQPs by gonadal steroids in female and male reproductive systems, are examples of the important roles played by these protein channels in metabolism and reproductive health . InsulinThe estrogen 17β-estradiol was reported to decrease lipogenesis by upregulating the mRNA and protein expression of AQP7 in mice, being related to the amelioration of ovariectomy-induced hepatic steatosis that is characterized by a decrease in estrogen secretion. These findings suggest that estrogen may play a key role on hepatocyte protection from steatosis through increase of AQP7 expression . The theLeptin is an endocrine hormone with a well-known role in body energy homeostasis. Leptin deficiency was associated with obesity and NAFLD showing increased AQP3 and AQP7 expression levels in white adipose tissue with no changes in AQP9 expression. Leptin chronic administration to leptin deficient ob/ob mice decreased AQP3 and AQP7 mRNA and protein levels in adipocytes while hepatic AQP9 was increased, unveiling a potential strategy to control lipid accumulation in adipose tissue and liver while maintaining normal glycemia in obese individuals . Leptin The gut hormone ghrelin downregulates AQP7 protein expression, responsible for lipolysis-derived glycerol efflux in human visceral adipose tissue and plays a role in the excessive fat accumulation in obese individuals . Both foDexamethasone and ambroxol were identified as AQP3 modulators, capable of upregulating its mRNA and protein expression with increase in water permeability in human airway epithelial cells. Considering the role of AQP3-mediated water transport in the airways, increasing AQP3 expression through modulation with corticosteroids as dexamethasone and ambroxol could exert a therapeutic effect on pulmonary diseases with airway hypersecretion through clearance of pulmonary fluid . MoreoveErythropoietin is a glycoprotein hormone, naturally produced by the peritubular cells of the kidney. Erythropoietin was found to prevent the ischemia-induced downregulation of AQP3 protein expression in rats with acute renal failure, improving their urinary concentrating capability. Moreover, the combination of erythropoietin with α-melanocyte-stimulating hormone boosted this effect, being proposed to prevent ischemia/reperfusion injuries through upregulation of renal AQPs .MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that have emerged as important post-transcriptional regulators of gene expression . miRNAs via miR-124-AQP3 axis through miR-124 downregulation and AQP3 upregulation. When circHIPK3 was silenced, both AQP3 mRNA and protein levels were reduced and miR-124 was upregulated, resulting in reduced HCC cells proliferation in vitro and suppressed tumor growth in vivo  tissues and cells, miR-488 was found to be downregulated while AQP3 was markedly increased. Accordingly, the introduction of miR-488 leads to a suppression of the proliferation, invasion and EMT of OS cells through the downregulation of AQP3 mRNA and protein expression by binding to AQP3 3′UTR, supporting the idea that both miR-488 and AQP3 could have prognostic value in OS . miR-874 in vivo .Further studies identified miR-22 and miR-23a as AQP9 modulators by downregulating its mRNA and protein expression in cultured human hepatocytes and in diabetic rats livers, suggesting that their role in the regulation of glycerol entrance in these cells could be beneficial for the control of glycerol-based gluconeogenesis that is often augmented in diabetes . AQP9 wain vivo, with reported beneficial effects on blood pressure and lipid metabolism as well as anticancer, immunomodulatory, analgesic, antioxidant and anti-inflammatory activity (Peptides have been receiving great attention for their ability to establish specific and high-affinity interactions with endogenous receptors . In factactivity . Considein vitro through AQP7 mRNA and protein upregulation via PI3K signaling pathway, revealing that apelin-13 is a promising starting point to develop novel treatments against obesity and related health problems (Apelin-13 is a small peptide found in the adipose tissue that is thought to be implicated on energy metabolism and insulin sensitivity, being also associated with obesity. This peptide was shown to decrease triglycerides accumulation in hypertrophic adipocytes problems .2O2 transport, NF-κB signaling and macrophage activation. This study emerges as a starting point for antibodies’ production to specifically target AQPs and develop directed therapies against AQP-associated disorders.Recently, in vivo.Aquaglyceroporin modulators have gained much attention in recent years and their development is currently considered a promising strategy for treatment of AQP-associated pathologies. The vast array of compounds reported to affect aquaglyceroporins expression or function has been growing, as an attempt to validate their pharmacological activity for human diseases. However, the application of aquaglyceroporin modulators in clinical trials is still far to be implemented, possibly due the lack of selective and potent modulators able to be administered via AQPs, as well as insights into the mechanisms of their inhibition by different substances, further studies are needed to improve the efficiency and selectivity of modulators, avoiding cross-reactivity with other targets and potential side effects. Given the broad range of diseases where aquaglyceroporins play a role, the design and development of new potent and selective modulators may pave the way to the discovery of innovative therapeutics.Different classes of molecules, from metal compounds and small molecules to natural compounds and biologicals, have been reported to effectively target aquaglyceroporins, either modifying pore permeability or protein expression level. Yet, their mechanisms of action are obscure and the proof of concept for their pharmacological activity in most animal models of disease is still unmet. While advanced atomistic simulations can provide valuable information on the mechanisms of selectivity and substrate transport


Sodium–glucose cotransporter (SGLT) inhibitors are a class of oral hypoglycemic agents, which, in recent years, have been shown to improve renal and cardiovascular outcomes in patients with diabetic and non-diabetic chronic kidney disease. There remains considerable debate regarding the potential glucose-independent mechanisms by which these benefits are conferred. SGLT inhibitors, to a variable extent, impair small intestinal glucose absorption, facilitating the delivery of glucose into the colon. This suppresses protein fermentation, and thus the generation of uremic toxins such as phenols and indoles. It is acknowledged that such a shift in gut microbial metabolism yields health benefits for the host. SGLT inhibition, in addition, may be hypothesized to foster the renal clearance of protein-bound uremic toxins. Altered generation and elimination of uremic toxins may be in the causal pathway between SGLT inhibition and improved cardiometabolic health. Present review calls for additional research.  SLC5 family) inhibitors are a potent class of oral hypoglycemic agents that have been shown to improve renal and cardiovascular outcomes in patients with diabetic and non-diabetic chronic kidney disease. In humans, the family of SGLT comprises at least six different isoforms. Of these, SGLT1 (SLC5A1) and SGLT2 (SLC5A2) have been studied extensively because of their fundamental role in the transepithelial transport of glucose and sodium in the small intestine and the kidney, through an active mechanism exploiting the Na+–electrochemical gradient generated by active sodium extrusion by the basolateral Na+/K+-ATPase. Under baseline conditions, SGLT1 is responsible for glucose absorption in the small intestine, and for the reabsorption of nearly 10% of the filtered glucose load in the renal proximal tubule segment 3. SGLT2, conversely, is primarily expressed at the apical membrane of the renal proximal tubule segments 1 and 2 and is responsible for the reabsorption of approximately 90% of the filtered glucose load [Sodium–glucose cotransporter . This ily 17 nM ,19. The kidney: structure and function in health and disease , Homer W. Smith provided a description of the physiological effects of phlorizin, noting that it partly blocks the reabsorption of vitamin C and, of note, it depresses the tubular excretion of phenol red in chicken. It was speculated that this effect was the consequence of diversion of energy rather than a specific interference with the transporter mechanisms. These early speculations have not been corroborated by any recent data grounded in molecular biology.Phlorizin is a glycoside that can be found in the bark and roots of several fruit trees, including apple and pear. In 1885, it was discovered by von Mering that phlorizin induces transient glucosuria. It is poorly adsorbed from the gastrointestinal tract, and intravenous administration is needed to exert its physiological effects. More recently, it has been shown that phlorizin functions as an SGLT inhibitor . Our undIn the last decade, four SGLT2 inhibitors have become been granted marketing authorization by the European Medicines Agency and the US Food and Drug Administration for management of hyperglycemia in type 2 diabetes. An increasing number of clinical trials have reported beneficial cardiovascular and renal outcomes among diabetic and non-diabetic patients receiving SGLT2 inhibitors ,22,23,24The human intestine hosts a complex and diverse system of mutualistic microorganisms. This rich ecosystem is increasingly regarded as playing a crucial role in human health and disease. Gut microbiota can interact with the host by the production of a diverse array of metabolites . MountinCanagliflozin and sotagliflozin, to a variable extent, impair small intestinal glucose absorption, facilitating the delivery of glucose into the colon, which fosters carbohydrate fermentation. The ensuing increased production of SCFAs may cause a reciprocal decline of protein fermentation. Well-designed in vitro studies showed that an increased availability of carbohydrates diminishes microbial amino acid fermentation through its pH-lowering effect, its action as a microbial energy source, and the process of “catabolite repression” ,37.A proof of concept has recently been demonstrated in two experimental studies ,39. Trea+/K+-ATPase (EC 3.6.3.9), secondary active Na+-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active organic anion transporters  all are involved [SLC22A12), responsible for 99% reabsorption of uric acid from the ultrafiltrate, and GLUT9 (SLC2A9). Canagliflozin was shown to acutely increase the fractional urate excretion in mice, an effect that was absent in Urat1 knockout mice, despite a similar glucosuric response. The role of Glut9 was less clear, despite a clear upregulation in gene expression by the SLGT inhibitor [Most of abovementioned uremic toxins originating from gut microbial aromatic amino acid metabolism are organic anions and circulate, strongly bound to albumin. Renal excretion thus occurs mainly through tubular secretion . The firinvolved . The expinvolved . This monhibitor . InterfeProtein-bound uremic toxins may be hypothesized to affect the pharmacokinetics and pharmacodynamics of SGLT2 inhibitors by interfering with their protein binding and tubular secretion . In lineExperimental data suggest that empagliflozin attenuates uremic serum (and indoxyl sulfate)-induced generation of endothelial mitochondrial reactive oxygen species. This leads to the restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes. This effect was shown to be largely independent of sodium–hydrogen exchanger-1 .In aggregate, evidence suggests a bidirectional interaction between SGLT inhibitors and protein-bound uremic toxins originating from gut microbial metabolism. There is an urgent need for additional experimental and clinical research. The following research questions, among others, should be addressed: does SGLT inhibition lower the generation of gut-derived uremic toxins? Does SGLT inhibition increase the fractional urinary excretion of urate and protein-bound uremic toxins? A better insight into the mechanisms of the actions of SGLT inhibitors may prove useful in identifying patients that may benefit the most from this new class of drugs. As such, their scope may expand from glucose control in diabetes patients to renal protection in patients with diabetic and non-diabetic nephropathy to cardiometabolic protection in the general population. SGLT inhibitors have the potential to become the long-sought adjuvant therapy to tackle protein-bound uremic toxins—many of which originate from gut microbial metabolism—in patients with advanced-stage CKD.


H) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies.The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α  galactose  present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region  cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations.Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.The small number of IGV The use of porcine cells, tissues, and organs for transplantation or extracorporeal perfusion would greatly benefit the 86,000 patients on the United Network for Organ Sharing transplant waiting list, as well as those considered medically unsuitable for transplantation of scarce human organs or tissues . UnfortuH) immunoglobulin genes .Clones selected on the basis of colony filter hybridization, or a minimum of 40 clones from each library (IgM and IgG), were randomly chosen, grown overnight, prepared using the QIAPrep Spin MiniPrep Kit (QIAGEN) and sequenced using the ALFexpress™ automated DNA sequencer and the AutoCycle™ Sequencing Kit . Sequences obtained included the region from framework 1 (FR1) through complementarity determining region 3 (CDR3). Results were analyzed using OMIGA software and the closest identifiable germline counterparts in the GenBankαGal = galactose α  galactoseBAL = bioartificial liver deviceCDR = complementarity determining regionFR = frameworkhDAF = human decay accelerating factorPI = post-first infusionH = variable-region heavy chainVJZR performed the ELISA assays, prepared, screened and sequenced the IgM cDNA libraries, and wrote the manuscript; CRG performed the monkey surgeries, in collaboration with AEK and DCB; DVC initiated the design for the project, interpreted data, and edited the manuscript; IYP performed the IgG library preparations and screening, including DNA sequencing of clones from this library; MKKJ provided scientific and experimental guidance throughout the study and edited the manuscript. All authors read and approved the final manuscript.


GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences.The glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases (GSKs) are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado) appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences.We found that the structure of GSK gene lineage was established early in the history of green plants, (2) plant GSKs began to diversify prior to the origin of extant seed plants, (3) three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4) diversification into four major clades  occurred either just prior to the origin of the angiosperms or very early in angiosperm history.Our results indicate that (1) the plant-specific  Saccharomyces cerevisiae, the GSK3 homologs MCK1 and MDS1 play a role in chromosomal segregation .GSK3/SHAGGY-like kinase genes in plants. Specifically, we (1) compared the structure of GSK3/SHAGGY-like kinase genes in Arabidopsis and rice, and (2) addressed whether the diversity of GSK genes in Arabidopsis is unique to Arabidopsis or is more generally true of all angiosperms and all land plants. For example, if the diversification of the gene family predated or coincided with the origin of the angiosperms, then ESTs from basal angiosperm taxa should appear in all major clades identified in Arabidopsis. Likewise, if GSK gene diversity in plants is ancient, basal lineages of land plants, such as mosses, should also contain orthologs to the Arabidopsis genes. Alternatively, some gene lineages may have diversified since the origin of the angiosperms, or land plants, and will not contain sequences from all basal lineages.In this study we examined the diversification of the Arabidopsis GSK3/SHAGGY-like kinase genes was reported by Dornelas et al. . We GSK3/SHAGGY-like kinases are also available for a fern and for several gymnosperms. An EST of the fern Ceratopteris appeared within clade I, as sister to a subclade that includes AtSK11 and AtSK12 in the MP-AA tree , although these positions varied in the SW and MP-N trees  indicates that the plant-specific GSKs were already established before the origin of green plants, the oldest fossils of which are unicellular and filamentous green algae from the Neoproterozoic of Australia  and 900 mya; ,57; revi900 mya; ). Taken GSK genes in Arabidopsis and rice is highly conserved, and most GSK genes have 12 exons interrupted by 11 introns. Genes included in the same clade based on parsimony analyses share similar structural characteristics. Our phylogenetic results indicate that the plant-specific GSK gene lineage was established prior to, or early in, the history of green plants, and plant GSKs began to diversify prior to the origin of extant seed plants. In addition, at least three of the four major clades of GSKs  present in Arabidopsis and rice were established early in the history of extant seed plants. Sequences of basal angiosperms are present in all four of the major GSK clades, indicating that the fourth major subgroup of these genes (II) was established either early in angiosperm evolution or prior to the origin of the angiosperms (but after their last common ancestor with extant gymnosperms), if the absence of Clade II sequences from gymnosperms is real and not an artifact of limited sampling. In addition, our data indicate that GSK gene duplication events may have occurred in several of the basal angiosperms investigated, most notably Nuphar. Thus, duplication of GSK genes, which is prevalent in both Arabidopsis and rice, has also occurred in basal angiosperms. This phylogenetic analysis of numerous plant GSK sequences provides a framework for the investigation of the functional genetics of GSKs in signaling, development, and stress response.The structure of GSK3/SHAGGY-like kinase homologs was performed using BLAST [Arabidopsis and nine rice sequences, and then continued with various published GSK3/SHAGGY-like kinase homologs from human, yeast, Drosophila, Brassica, Medicago, Petunia, Nicotiana, and Ricinus to identify as many GSK homologs as possible from protists, fungi, animals, and plants. Putative GSK homology was defined initially by sequence similarity when the sequences were retrieved and then confirmed by phylogenetic analysis (see below). A total of 139 GSK homologs was collected, of which 73 sequences were ESTs: 26 ESTs from 10 taxa at the FGP web site, 40 ESTs from 17 taxa at the PlantGDB web site, 5 ESTs from the NCBI web site (Ceratopteris and Pinus), and two ESTs from the Kazusa DNA Research Institute database (Clamydomonas and Porphyra). Some ESTs were integrated into a contig, which was constructed using the CAP3 Sequence Assembly Program [A search for ng BLAST ,59 at thng BLAST , TIGR [6ng BLAST , PlantGDng BLAST , Kazusa ng BLAST , and theng BLAST . We star Program , and theArabidopsis; see Figure Arabidopsis) were aligned manually in a stepwise manner using Se-Al; other regions were too variable to align. The aligned matrix therefore comprised exons 3 to 12 and was 348 amino acid residues in length; the average length of all included sequences was 293 amino acid residues, and the average length of the translated EST sequences was 193 amino acid residues. The aligned sequences were exported for phylogenetic analyses as separate data matrices of nucleotide sequences and amino acid sequences, and all data matrices and trees were deposited in TreeBASE  [Arabidopsis and rice, the genomic sequences were aligned and compared with cDNA sequences to investigate gene structure.All sequences were translated into amino acid sequences using Se-Al . The seqGSK homologs. We investigated patterns of substitution across both nucleotide and protein sequences using the CHART option of MacClade 4.05 [GSK homologs and Tree 1, selected arbitrarily from the phylogenetic analysis. This approach provides a minimum estimate of change for each site. Plotting of substitutions was conducted across a 4-bp or 4-amino acid interval on the x-axis. The analyses were conducted across the entire aligned sequences. We tested for variation in mean substitution rate among codon positions using the CHART option of MacClade 4.05, across the entire data set, within all green plants, within mosses, and within each of the four major clades of seed plant sequences identified by phylogenetic analyses.A series of analyses was conducted to explore the pattern of sequence evolution in ade 4.05 , using 1Maximum parsimony analyses were conducted with (i) equally weighted characters and character states and (ii) support weighting . EquallyThe support weighting method  providesA Bayesian phylogenetic analysis was performed using MrBayes 3.1.1  to compamitogen activated protein kinase (MAPK) and cyclin-dependent kinase (CDK) sequences were shown to be the sister group to a clade of all GSK homologs. We analyzed plant MAPK/CDK/Casein kinase II/GSK sequences because these four kinases are included in the same group [GSK sequences formed a clade in which non-plant GSK homologs were sister to plant GSKs (tree not shown). As a result, we used non-plant GSKs as outgroups for analysis of all plant-specific GSK homologs.In previous phylogenetic analyses , mitogenme group . In an uM-JY carried out the sequence analysis, the equally weighted maximum parsimony analysis, and the Bayesian analysis, and with PSS and DES wrote the manuscript. VAA performed the support weighting analysis. PSS and DES supervised the project. All authors read and approved the final submission.List of GSK3/SHAGGY-like kinase homologs used in this study. Some gene designations represent contigs constructed from multiple sequences and therefore have several accession numbers.Click here for file


Cervical dysplasia is a premalignant lesion associated with human papillomavirus (HPV) infection which, over time, can turn cancerous. Previous studies have indicated that loss of gap junctions may be a feature of cervical cancer and premalignant dysplasia. Loss of the gap junction protein connexin43 has been demonstrated in dysplastic cervix, but other connexins have not been investigated. In contrast we previously showed that HPV-associated cutaneous warts – and other hyperproliferative skin conditions – display a dramatic upregulation of certain connexins, in particular connexin26. By performing immunofluorescence staining after antigen retrieval of paraffin-embedded cervical tissue samples, this study reports for the first time that connexin26 and connexin30, in addition to connexin43, are expressed in differentiating cells of normal human cervical epithelia. Moreover, in dysplastic ectocervix, all connexins studied display a dramatic loss of expression compared to adjacent normal epithelia. The role of connexins in keratinocyte differentiation and carcinogenesis is discussed. Connexins, a family of 20 trans-membrane proteins in humans, comprise the main subunits of gap junctions – specialised clusters of intercellular channels that allow adjacent cells to directly share ions and hydrophilic molecules of up to ~1 KDa in size [There is also substantial evidence that connexins have a tumour suppressor role [reviewed in ]. While in-vitro has suggested that loss of GJIC may be an early event in papillomavirus associated cell transformation [The association of certain "high risk" human papillomaviruses (HPVs) with the development of cervical cancer on the other hand has been clearly demonstrated , with seormation -38. Howeormation . This, tormation , prompteSix duplicate slides of HPV-16 positive dysplastic cervix  I, CIN I/II, and CIN III) were obtained from Dr. John Doorbar . AntigeAs seen in Figure In contrast, all sections of dysplastic cervix displayed loss of protein expression of all three connexins investigated Figure . ConnexiIn conclusion, this report demonstrates for the first time the presence of connexin26 and connexin30 gap junction plaques, as well as connexin43, in human ectocervix, displaying a similar staining pattern to that observed for connexin26 in vaginal epithelium . MoreoveExpression of connexin26 but not connexin40 and connexin43 has been shown to reduce tumourigenesis of HeLa cells  . It is aThese results are clearly in disparity to observations in interfollicular epidermis, where connexin26 and connexin30 are normally not present  but becomes highly expressed in areas of hyperproliferation such as viral warts . The reaTA carried out all experimental assays and drafted the manuscript. TA, MBH, ME and SVG participated in study design and coordination, data interpretation and manuscript preparation. All authors read and approved the final manuscript.


Neurite growth can be elicited by growth factors and interactions with extracellular matrix molecules like laminin. Among the targets of the signalling pathways activated by these stimuli are cytoskeletal elements, such as actin, tubulin and neurofilaments. The cytoskeleton can also be modulated by other proteins, such as the small heat shock protein Hsp27. Hsp27 interacts with actin and tubulin in non-neuronal cells and while it has been suggested to play a role in the response of some neurons to injury, there have been no direct studies of its contribution to axonal regeneration.We have investigated neurite initiation and process extension using cultures of adult dorsal root ganglion (DRG) sensory neurons and a laminin stimulation paradigm. Employing confocal microscopy and biochemical analyses we have examined localization of Hsp27 at early and later stages of neurite growth. Our results show that Hsp27 is colocalized with actin and tubulin in lamellopodia, filopodia, focal contacts and mature neurites and growth cones. Disruption of the actin cytoskeleton with cytochalasin D results in aberrant neurite initiation and extension, effects which may be attributable to alterations in actin polymerization states. Inhibition of Hsp27 phosphorylation in our cultures results in an atypical growth pattern that may be attributable to an effect of pHsp27 on the stability of the actin cytoskeleton.We observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early stages of neurite growth, and in processes, branch points and growth cones at later stages, suggests that Hsp27 may play a role in neuritogenesis and subsequent neurite extension, and potentially in the patterning of this growth. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells. Our results suggest that this may also be the case in neurons and support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin. We know that various factors can influence and promote regeneration of peripheral axons. In addition to soluble factors , the extracellular environment in which growth occurs is critically important. Axonal regeneration does not occur to any great extent in the CNS, and while this is due to a number of factors, the most prominent is a non-permissive growth environment as well as an unavailability of appropriate growth-promoting factors. In the PNS, on the other hand, peripheral axons (both motor and sensory) generally regenerate quite well.Growth factors and extracellular matrix (ECM) molecules like laminin act through cell surface receptors that activate often convergent signalling pathways to elicit neurite growth in sensory neurons . Among tOne class of molecules known to act as chaperones include the small heat shock protein family, of which heat shock protein 27 is a member. Hsp27, in addition to its roles in regulating apoptosis and protein folding, interacts with different cytoskeletal elements -9. Much Prior reports and our own observations have suggested a role for Hsp27 in axonal growth or regeneration, in addition to its role in promoting neuronal survival. Hsp27 is upregulated after injury in DRG neurons in vivo and after dissociation in vitro , both of which elicit neurite initiation [In vitro models have been widely used to study the growth behaviour of neurite initiation and extension in both CSN and peripheral neurons. In many models, neurotrophin stimulation is required for neurite growth, although in most of these models neurotrophins are also required for survival. Another widely used paradigm involves the stimulation of plated neurons with soluble laminin or extracellular matrix preparations . Subsequently, the plating medium was removed and 50 μl of medium containing soluble laminin (40 μg/ml) was added to the cells. Control wells consisted of mock stimulation . Slides were fixed at 5, 15, 30 min and 1, 6 and 24 hrs after stimulation and subsequently processed for detection of actin, tubulin and Hsp27. Various distinctive stages in neuronal membrane expansion and neurite growth were observed and are summarized in Figure Based on our hypothesis that Hsp27 may play a role in process initiation or neurite growth, we examined the localization of Hsp27 in neurons in various stages of process formation using immunocytochemistry and confocal microscopy. Here, examples of the different stages as defined in the previous section  were selected from cells stimulated with laminin for 1 or 6 hrs. In addition, because of the association of Hsp27 with actin and tubulin in non-neuronal cells -32, we aIn panels G-L, the cells were costained with antibodies for Hsp27 (green) and total tubulin (red). The neuron in Figure S15, ABR), we costained neurons at early stages (as defined above) of process formation for pHsp27 and actin , and this phosphorylation is reported to be important in the role of Hsp27 in its interactions with actin . Using actin Fig  and 3F actin Fig  and 3L. ctin Fig  and pHspctin Fig  also colOur initial observations indicated that the majority of adult DRG neurons in culture display robust expression of Hsp27, not only in the cell bodies but throughout the neurites when present. Hsp27 expression in sensory neuron cell bodies as well as dendritic and axonal networks has been previously reported for in vivo expression ,19,33. IFigure We also noted that the Hsp27 and pHsp27 were strongly expressed in growth cones, further supporting an important role for Hsp27 not only in neurite initiation but also continued neurite extension. Figure Hsp27 has been suggested to play a key role in modulating actin cytoskeletal dynamics by acting as an actin-capping protein. In order to understand the role of Hsp27 in neuritic growth we decided to first examine the effects of disrupting the actin cytoskeleton integrity using cytochalasin D (CytD). Neurons were plated on laminin-coated slides and CytD was added to the medium 3 hrs post-plating . Cultures were fixed 24 hrs later and examined for changes in neurite growth patterns and expression of Hsp27 and actin or tubulin.Representative examples of the effects of CytD on neurons are presented in Figure Because of the reported role of Hsp27 phosphorylation in modulating the actin cytoskeleton, we wished to determine the effects of inhibiting p38 MAPK. p38 MAPK activity leads to the phosphorylation and activation of MAPKAP-K2, which acts as an Hsp27 kinase ,36. InhiS15 and total Hsp27 antibodies. The results of a representative experiment presented in Figure We initially determined whether the inhibitors were effective in preventing Hsp27 phosphorylation. Using larger scale cultures, neurons were plated on LN-coated 12-well plates and after 3 hrs the inhibitors were added; 24 hrs after SB addition, cell lysates were prepared as described in the Methods. For these experiments, we used a commercially available protocol to fractionate the cells into cytosolic, membrane, nuclear and cytoskeleton fractions. Following electrophoresis, the resulting blots were probed with pHsp27 Having determined that the inhibitors had the expected effects on pHsp27, we then plated the neurons on laminin-coated slides as for the previous experiments, and treated the cultures with SB 3 hrs after plating, fixed the cells 24 hrs later and carried out immunostaining for pHsp27, Hsp27, actin and tubulin as before.Neurons treated with SB displayed clearly atypical neurite growth. The examples presented are representative of the various patterns of neurite growth observed. As with the CytD treatment, there was no discernible distinction between different sizes of neurons in their response to SB; small, medium and large sized neurons displayed aberrant process formation. In the neuron shown in Figure In addition, some neurons displayed extensive neurite growth, although this was again generally characterized by flattened and expanded processes and growth cones. Figure These results suggest that attenuation of the phosphorylation of Hsp27 can have adverse effects on the neuritic cytoskeleton, similar to those observed with Cyt D. Although our assumption (based on previous reports in the literature) is that the SB compounds block p38 MAPK activity, its downstream effects on MAPKAP-K2 and the subsequent inhibition of Hsp27 phosphorylation, it is possible that these compounds may have other inhibitory influences, or that they may be influencing the cytoskeletal elements through actions not involving Hsp27. While our data show that the SB compounds do inhibit phosphorylation of Hsp27, we cannot completely rule out effects on other signalling components, although at the concentrations we have used, the effects are reported to be specific for p38 MAPK inhibition, rather than any other additional kinases.We describe early events in adult DRG neuron process formation in response to stimulation with the extracellular matrix protein laminin. Our data show that Hsp27 appears to associate with actin and tubulin in structures found at all stages of neurite initiation. Lamellopodia, filopodia, microspikes and focal contacts all displayed a colocalization of Hsp27 and actin or tubulin. The filamentous nature of the Hsp27 was quite clear in neurites and growth cones supporting the hypothesis that Hsp27 is associating with cytoskeletal elements.Our results are similar to those described previously for neurite growth initiation and process extension in embryonic cultured CNS neurons. Culture studies of early neuritogenesis events in hippocampal neurons have provided information that demonstrates that events after initial cellular attachment to the substrate are quite similar among different cell types and indeed events in neurons are very similar to those in migratory fibroblasts ,4,37. ThNeurite protrusion requires the actin cytoskeleton, with lamellopodia being filled with an actin meshwork necessary for the appropriate adhesion and filopodia having actin bundles with the rapidly growing ends oriented towards the tips. Studies have shown that actin polymerizes at the leading edge of the lamellopodia, and then disassembles and recedes from the peripheral area ,38. ThisIn cell-free assay systems, Hsp27 can act as an actin-capping protein which prevents the polymerization of actin and the assembly of F-actin ,11. PhosPhosphorylation of Hsp27 promotes the polymerization of actin and stress fibre formation ,10,43. HHsp27 phosphorylation is regulated by activity of the p38MAPK pathway, whereby p38 MAPK activation of MAP-kinase-activated protein-kinase 2/3 (MAPKAP-K2) leads to the phosphorylation of Hsp27 ,44. HoweTreatment of neurons with agents that disrupt the actin cytoskeleton result in aberrant neurite initiation and growth. Neurons treated with Cyt D, which caps existing actin filaments at barbed ends, consistently show rapid emergence of numerous neurites that elongate in a disoriented fashion ,4,37. TrSince Cyt D acts to cap barbed actin filaments and non-phophorylated Hsp27 has been suggested to do the same, we reasoned that if pHsp27 was important for normal neurite initiation and extension, if we inhibited the phosphorylation of Hsp27 we might observe similar effects on neurite initiation. As shown in our results, attenuation of Hsp27 phosphorylation using the p38MAPK inhibitors, does indeed result in atypical growth patterns. At the early stages, results were similar to what we had observed with Cyt D (data not shown), and at later stages, neurite growth was again quite clearly aberrant Fig . Some neThere are relatively few reports of the interaction of Hsp27 with cytoskeletal elements other than actin. Hsp27 has been reported to associate with microtubules in HeLa cells  and in CpHsp27 also appears to be required for the migration of several cell types ,59-61. AMutations of the small Hsp (Hsp22 and Hsp25/27) genes have been linked to axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy (DHMN) ,21. ThisOur results suggest that Hsp27 is necessary for the initiation of neurite outgrowth in DRG neurons. The data also suggest that phosphorylation of Hsp27 plays a key role in modulating the dynamic interactions of Hsp27 with cytoskeletal elements such as actin and tubulin to regulate the response of DRG neurons to environmental cues that mediate growth.Using immunocytochemistry, we observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early neurite initiation stages, and in processes, branch points and growth cones in later stages suggests that Hsp27 may play a role in neurite initiation and extension and potentially in the patterning of this growth. While the mechanisms of action require further investigation, it is possible that one role of Hsp27 is to stabilize the cytoskeleton at potential sites of branching or sprouting. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells and our results suggest that this may also be the case in neurons. Neurons treated with cytochalasin D showed aberrant neurite growth patterns. Neurons treated with p38 MAPK inhibitors, which inhibit the downstream phosphorylation of Hsp27, also displayed either lack of neurite growth or failure of appropriate neurite extension. The similar results from the CytD and inhibition of Hsp27 phosphorylation support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin.2 and 5% CO2. Slides and culture plates were coated with poly-lysine  or laminin,  where appropriate. The neurons were cultured in modified serum-free NB alone with no added growth factors.Dorsal root ganglia (DRG) from young adult (5–6 wk) Sprague-Dawley rats  were dissected and dissociated using modifications to techniques described previously ,63. BrieS15 , total tubulin , actin (Sigma-Aldrich). It should be noted that the Hsp27 antibody recognizes both the non-phosphorylated and phosphorylated Hsp27, while the pHsp27 antibody only recognizes the phosphorylated form. We have also tested two other pHsp27 antibodies  in PBS for 20 minutes, permeabilized with 0.1% Triton-X-100 and blocked with 5% normal goat serum in PBS. Antibodies used were as follows: Hsp27  and phospho-Hsp27ruz, see ), but haCells were incubated with the primary antibodies at 4°C for 16–20 hrs, followed by Cy2 or Cy5-tagged secondary antibodies . In some experiments, cells were also labelled with rhodamine-phalloidin after antibody incubation (Sigma-Aldrich). The slides were coverslipped with glycerol and imaged with confocal laser scanning microscopy using z-stage scanning and image stacking. Stacked digital images were imported into Adobe Photoshop for compilation into the final composite figures.Neurite initiation was assessed in two ways. The first series of experiments employed neurons plated on laminin-coated slides, with the cells being fixed and analyzed for outgrowth parameters  at 24 hrs after plating. In a second series of experiments, the neurons were first plated on polylysine coated slides and allowed to stabilize overnight prior to being stimulated with soluble laminin . Following the addition of the laminin solution, cells were then fixed at 5, 15, 30 min, 1 hr, 6 hr, and 24 hr. After fixation, cells were immunostained and analyzed as described above.SB 203580 and SB 202190  were used to inhibit p38 MAPK activity, in order to assess the contribution of phosphorylated Hsp27. Inhibitors were added 1 hr prior to laminin stimulation. For the 24 hr cultures, the inhibitors were added 2–3 hrs after plating the cells on laminin-coated slides and retained in the medium for the extent of the experiment . Cytochalasin D  was also used in longer term experiments, and was added 3 hrs after plating and maintained in the medium for the extent of the experiment (24 hrs)S15  and Hsp27 ,. Blots were cut and reprobed sequentially, and visualized with ECL reagents  and exposure to X-ray film . Developed films were subsequently digitized and densitometrically analyzed with a cyclone ChemiImager and AlphaEase software. Digital images of the blots were used to make composite figures with Adobe Photoshop graphics software .For Western analyses, neurons were plated in 12-well plates that had been coated with polylysine alone or with laminin, depending on which experimental paradigm was used (see above). Neurons were subsequently processed according to our established procedures ,63. CellKLW carried out neurite growth and confocal analyses, as well as the Western blotting experiments. MR provided technical assistance with aspects of the work, most notably preparing neuronal cultures. KMM wrote the manuscript and was responsible for the experimental concept, design and overall supervision of the experiments, as well as carrying out some of the confocal analyses.


Anemia is common in HIV infection and independently associated with disease progression and mortality. The pathophysiology of HIV-related anemia is not well understood especially in infancy.We conducted a longitudinal cohort study nested within the Zimbabwe Vitamin A for Mothers and Babies Project. We measured hemoglobin, erythropoietin (EPO), serum transferrin receptor (TfR) and serum ferritin at 6 weeks, 3 and 6 months of age and hemoglobin at 9 and 12 months in 3 groups of randomly selected infants: 136 born to HIV-negative mothers, and 99 born to HIV-positive mothers and who were infected themselves by 6 weeks of age, and 324 born to HIV-positive mothers but who did not become infected in the 6 months following birth.At one year of age, HIV-positive infants were 5.26  times more likely to be anemic compared to HIV-negative infants. Among, HIV-negative infants, EPO was or tended to be inversely associated with hemoglobin and was significantly positively associated with TfR throughout the first 6 months of life; TfR was significantly inversely associated with ferritin at 6 months; and EPO explained more of the variability in TfR than did ferritin. Among infected infants, the inverse association of EPO to hemoglobin was attenuated during early infancy, but significant at 6 months. Similar to HIV-negative infants, EPO was significantly positively associated with TfR throughout the first 6 months of life. However, the inverse association between TfR and ferritin observed among HIV-negative infants at 6 months was not observed among infected infants. Between birth and 6 months, mean serum ferritin concentration declined sharply (by ~90%) in all three groups of babies, but was significantly higher among HIV-positive compared to HIV-negative babies at all time points.HIV strongly increases anemia risk and confounds interpretation of hematologic indicators in infants. Among HIV-infected infants, the EPO response to anemia is attenuated near the time of infection in the first weeks of life, but normalizes by 6 months. Nearly 90% of pediatric infections occur in sub-Saharan Africa, where many countries report that more than one in five pregnant women visiting antenatal clinics are HIV-infected. In the absence of antiretroviral therapy, nearly one-third of vertically infected children living in the developed world rapidly progress to AIDS or death in the first year of life  run in parallel. Discordant ELISA results were resolved by Western Blot. CD4 cells from maternal baseline samples that tested HIV positive were enumerated using a Facscount  within 48 hours of phlebotomy.After all patient contact was completed, the last available infant specimen was tested for HIV  for samples collected at younger ages). If this sample was HIV-negative, the baby was classified as negative and no further testing was carried out. If the last sample was HIV-positive, samples collected at intervening time points were tested to determine the timing of infection.Hemoglobin was measured on whole blood samples on the day of collection for all mothers at baseline and for all infants in the anemia substudy at baseline and visits up to 12 months using the HemoCue hemoglobinometer . Daily quality control of the instrument was performed. Hemoglobin data were unavailable if the baby missed the study visit, or if the blood sample was insufficient as a result of the mother's refusal for her baby to give blood, inadequate sample volume, sample clotting, or failure by the laboratory to perform the test before the end of the day in which the specimen had been drawn.We measured TfR, ferritin and EPO concentrations in plasma (or serum when plasma was unavailable). TfR concentrations were measured by enzyme immunoassay  ; ferritin by enzyme-linked immunoassay (Ramco Laboratories) ; and EPO using the Quantikine In Vitro Diagnostic EPO ELISA  .The criteria we used to diagnose anemia were modified from those of the World Health Organization . SeveralPlasma EPO, TfR, and ferritin concentrations were normalized by log transformation; geometric means (95% CI) are reported. Differences across HIV status groups in baseline characteristics and indicators of anemia and iron status were examined by ANOVA for continuous variables and the chi-squared test of homogeneity for categorical variables. We used multivariate linear and logistic regression analysis to compare hemoglobin values and the proportion of anemic children across HIV status groups and adjusted for birthweight in these models. The association between indicators of anemia and iron status was examined by linear regression and partial correlation coefficients. The relevant interaction terms between the independent variable and HIV status groups were included in regression models to examine differences in the association between indicators by HIV status group. Adjusting for birthweight in these models did not change the conclusions, and, therefore, was not included. We performed a likelihood ratio test to determine the overall statistical significance of the interaction between HIV status and the independent variable. For all analyses, we set the significance levels at α = 0.05. A Bonferroni correction was used to determine which associations were unlikely to be the result of chance alone.We created three categories of babies: the 136 babies born to HIV-negative mothers (Nn group) and two groups of babies born to HIV-positive mothers: (1) babies who tested PCR negative at 6 months or later ; and (2) babies who tested PCR-positive at baseline or 6 weeks . Babies who did not fall neatly into these three groups were excluded from the analysis. These included those who tested PCR-positive for the first time at 3 or 6 months (n = 37), and babies with an indeterminate HIV status (n = 115). Babies were classified as HIV-indeterminate if maternal baseline HIV status was unknown (n = 2) or maternal baseline status was positive and no results were available for the baby (n = 2), the baby's last available PCR result was prior to 6 months of age and was negative (n = 108), or the baby's PCR results were inconsistent such that a negative result followed a positive result (n = 3). Only 4 infants in the Nn group and 3 in the Pn group died by 12 months of age, but among Pp infants, 33 (33.3%) had died by 6 months of age and 51 (51.5%) by 12 months.We carried out the analysis only on the available data, and did not impute values for missing observations. Hemoglobin values >160 g/L at 6 weeks (n = 1), 3 months (n = 1) and 6 months (n = 4) were excluded from the analysis. All analyses were performed with the use of Stata .Compared to HIV-negative mothers (Nn), HIV-positive mothers (Pn and Pp) had lower hemoglobin concentrations and were more likely to be anemic, older and of higher parity Table . Among HBreastfeeding was nearly universal among infants in ZVITAMBO, but exclusive breastfeeding was rare . NonbreaThe only difference in indicators of anemia and iron status between Nn and Pn babies was a significantly higher TfR concentration at 6 weeks of age in Pn babies  Table . In bothPlasma ferritin concentration decreased from birth levels as expected with the physiologic mobilization of iron from liver stores in all HIV status groups. However, at all time points, including birth, the ferritin distribution was shifted to higher values among HIV-infected babies. For example, at 3 months, geometric mean [95% CI] ferritin concentration was significantly greater among HIV-positive infants  compared to HIV-negative infants . At 6 months, serum ferritin was not associated with hemoglobin in any HIV status group. Geometric mean plasma EPO increased with age and TfR increased to 3 months then leveled off (see Table 10 plasma EPO (U/L) and hemoglobin  was significantly negative at 6 weeks; this association remained at 3 months and 6 months but was only significant among the Pn infants  for one log10 decrease in plasma EPO (U/L) was significantly greater for Pp babies compared to Pn babies (P = 0.023), and there was a trend for a greater increase when compared to Nn babies (P = 0.11).TfR and EPO were positively associated at all ages and for all HIV status groups. A significant interaction with HIV status was observed at 3 months of age. The increase in logThere was no consistent or significant relation between ferritin and TfR at 6 weeks or 3 months. However, there was a significant interaction between HIV status and TfR on ferritin concentration at 6 months (P = 0.01). Ferritin was inversely associated with TfR in HIV-negative infants born to HIV-positive and HIV-negative mothers but not in HIV-positive infants.To compare the relative strength of association between TfR and ferritin vs. EPO, we examined the partial correlation coefficients between these variables. TfR was more strongly correlated with EPO than ferritin in all HIV status groups at every age, after adjusting for hemoglobin. There was a strong inverse association between TfR and ferritin only at 6 months in Pn infants, but, even then, more of the variability in TfR was explained by EPO concentration than ferritin.In this study of Zimbabwean babies, anemia was prevalent and strongly influenced by HIV infection. Though HIV infected and uninfected infants had similar hemoglobin distributions at birth, by 12 months of age infected infants had a nearly 20 g/L lower mean hemoglobin and they were >5 times as likely to be anemic, even after adjusting for their lower birthweight.Our model of the relationships between indicators of iron status and erythropoiesis proved valid among HIV-negative infants. The data support the hypothesized relationships between indicators of erythropoiesis: EPO was inversely associated with hemoglobin and positively associated with TfR; TfR was inversely associated with ferritin ; and EPO explained more variability than ferritin in TfR concentration.HIV infection perturbed the normal feedback system and modified some, but not all, of the hypothesized relationships. The EPO response to anemia among HIV-infected infants was attenuated during early infancy but normalized at 6 months of age. HIV infection did not seem to affect the bone marrow response to stimulation by EPO, i.e., TfR increased in direct response to increased EPO levels, at all ages. However at 6 months, this drive in red blood cell production was not accompanied by the expected decline in ferritin in HIV-infected infants.A limitation of the present study was the high rate of missing values for indicators of anemia and erythropoiesis. Infant blood samples were unavailable if the mother-baby pair defaulted, the infant died, or if the blood sample was insufficient. It is possible that these losses could have biased the results, but there were no statistically significant differences in any measured baseline maternal factor between infants followed versus not followed. We did observe a statistically significant difference in preterm birth between infants who came for the 6 month visit and those who did not, but the impact of preterm on the likelihood of missing values was similar across HIV status groups and likely did not bias the results. Insufficient blood sample as a result of either inadequate quantity or quality, e.g., the sample clotted before hemoglobin was measured) was not associated with HIV status. Finally, survival rates were certainly associated with infant HIV status with approximately half of HIV-positive infants dead by 6 months, but very few deaths among uninfected infants. Thus a survival bias is possible and should be considered in light of the study findings.One-third of HIV-negative infants had serum ferritin concentration <12 μg/L at 6 months suggesting that their anemia was largely caused by iron deficiency. Worldwide, the primary cause of anemia among infants is iron deficiency . InfantsIn the absence of concurrent disease, serum ferritin parallels total body iron stores and can reflect deficient, normal, and excess iron stores . However10 EPO concentration was highest in Pp infants at all ages, even after adjusting for hemoglobin concentration (data not shown). To our knowledge, the only other study, in addition to the present, to compare EPO in HIV-infected and uninfected infants was carried out in Malawi, and they too did not show an inadequate EPO response among HIV-infected infants [Our findings that the EPO response to anemia was blunted in HIV-infected babies at 6 weeks and slightly attenuated at 3 months of age are consistent with several reports of studies carried out in HIV-infected adults ,36. This infants . It is nThe apparent transient impact of HIV on the EPO response to anemia might suggest that the attenuating effect on EPO is more pronounced closer to the time of initial infection and weakens with progression of HIV disease. Acute HIV infection is characterized by a peak in viremia and rapid decline in CD4+ cells, but an immunologic response ensues resulting in a lowering of the plasma level of HIV RNA to a steady state "set point" that reflects a balance between production and destruction of virions . It is p10 EPO and hemoglobin (g/L), even the steepest observed value (-0.0109) in Pp infants at 6 months, were less steep than values previously reported in the literature. The values of slopes for control groups comprised of HIV-uninfected infants [It is important to note that the inverse association between EPO and hemoglobin was not statistically significant in Nn babies at 3 and 6 months of age. The failure to reach statistical significance may in part be attributed to a lack of sample size since slopes of similar magnitude in the Pn group were statistically significant. However, the data also suggest a relatively weak inverse association between these indicators. In fact, the observed slopes of the regression line between log infants , childre infants  and sick infants , and chi infants  have ran infants -45, and  infants . We spec infants -50. ThusA noteworthy effect of HIV infection on erythropoiesis was the modification of infant infection on the association between ferritin and TfR at 6 months of age. The absence of a negative association between TfR and serum ferritin in HIV-infected babies may be explained by confounding due to infection, yet it is consistent with the sequestration of iron in liver stores during infection. The concurrent lack of association at 6 months between ferritin and hemoglobin supports this explanation since an increase in ferritin did not translate into increased hemoglobin production. However, these results should be interpreted with caution since serum ferritin is an acute phase protein and elevated during infection and inflammation  at whichThe observation that at all ages and in all HIV groups, EPO concentration explained more variability in TfR levels than did serum ferritin concentration suggests that TfR is not primarily an indicator of iron deficiency in this age group. We speculate that during periods of high erythropoietic activity (e.g. early infancy), an increase in TfR more strongly reflects an increase in erythroblasts (i.e. erythropoiesis) rather than an increase in the density of receptors per red blood cell consistent with tissue iron deficiency. While the strong and consistent association between TfR and EPO is striking, particularly when compared to the lack of association between TfR and ferritin, it was not surprising that ferritin did not explain much of the variability in TfR given its association with infection and inflammation.We conclude that HIV-related anemia is associated with attenuation in the EPO response to anemia in early infancy, but that the EPO response is normalized by 6 months. Throughout the first 6 months of infancy, HIV-infected babies have higher serum ferritin concentrations, which could reflect iron sequestration in liver stores, or an acute phase response. If the elevated serum ferritins in infected babies are explained by inflammation, then the HIV-related anemia might be explained by a shortened red cell lifespan  or by inNn, mother HIV negative, baby HIV negative; Pn, mother HIV positive, baby HIV negative at 6 months; Pp, mother HIV positive, baby HIV positive at 6 weeks; EPO, erythropoietin; TfR, transferrin receptor; HIV, human immunodeficiency virus; ZVITAMBO, The Zimbabwe Vitamin A for Mothers and Babies Project.The author(s) declare that they have no competing interests.MFM contributed to the design of the study, carried out the research, performed the statistical analyses and wrote the manuscript. RJS and JHH contributed to the design of the study, conduct of the research, interpretation of the results, and assisted in writing the manuscript. PJI, LCM, and NVM assisted in the conduct of the research and editing of the manuscript.The pre-publication history for this paper can be accessed here:Table 3. Slope of the regression line for indicators of erythropoiesis  by age and maternal and infant HIV statusClick here for file


These findings indicate that positive promoters of GT expression may have a unique role in neuroprotection through regulating GT expression. This is also encouraging in search for new pharmacological tools for pain management.Glutamate transporter (GT) plays a major role in the mechanisms of glutamate homeostasis. Can this transporter system be a therapeutic target for glutamate-mediated neurological disorders? In January's edition of Nature, Rothstein et al (2005) reports that the most commonly used class of antibiotics (β-lactam antibiotics) such as ceftriaxone promoted the expression of GLT1 and demonstrated a functional role in both  Glutamate is the major excitatory amino acid neurotransmitter that plays an important role in many physiological functions. Maintaining a physiological range of extracellular glutamate concentration is key to preventing glutamate over-excitation and neurotoxicity that could occur under a variety of pathological conditions. Regulating extracellular glutamate is primarily carried out by an efficient, high-capacity glutamate transporter (GT) system, because clearance of extracellular glutamate via glutamate metabolism or diffusion is negligible. To date, at least five cell membrane GT proteins have been cloned ,3. GT isGlutamate has a dual role both as an excitatory neurotransmitter essential for physiological functions and a neurotoxic mediator contributory to pathological processes. Since the homeostasis of extracellular glutamate concentration is critically regulated by neuronal and glial GT, reduced GT expression and/or function would be expected to increase extracellular glutamate concentration with subsequent excessive activation of glutamate receptors and excitotoxicity. Indeed, a large number of studies have shown the detrimental effects from reduced GT expression and function on the pathogenesis of neurological disorders including brain ischemia, epilepsy, spinal cord injury, amyotrophic lateral sclerosis, AIDS neuropathy, and Alzheimer's disease.Given the well-documented role of the glutamatergic system in the mechanisms of pathological pain, it is not surprising that regulation of GT also has been implicated in the central mechanisms of nociceptive processing, such as that following the hindpaw formalin injection or the application of exogenous NMDA or protaglandins in rats ,5. A serin vitro and in vivo models of glutamate neurotocixity. These findings indicate that positive promoters of GT expression may have a unique role in neuroprotection through regulating GT expression. The regulatory role of β-lactam antibiotics in promoting GT expression appears to be selective, because other classes of antibiotics such as vancomycin were ineffective [Despite a positive regulatory role of certain compounds such as riluzole and MS-153 in GT function in pre-clinical studies of neuropathic pain and morphine tolerance -9, the eIn contrast to antagonism of glutamate receptors, a distinct advantage of regulating GT expression and activity is the possibility of minimizing the pathological impact from a glutamate overload while retaining the physiological role of glutamate. A similar approach is used in developing anti-psychiatric drugs, including broad and selective monoamine reuptake agents, and has yielded rather positive outcomes in the treatment of psychiatric disorders. Extensive research is under way to explore the cellular and molecular mechanisms of GT expression and function in relation to the pathogenesis of neuropathic pain. In addition, studies on the role of GT regulation in opioid tolerance and dependence may provide new insights into the cellular mechanisms of substance abuse, an emerging issue associated with clinical opioid therapy.


Five months after the January 1999 accidental contamination of animal feed with polychlorinated biphenyls and TCDD in Belgium, a research team collected blood and semen samples from 101 men aged 20 to 40 living in Antwerp and Peer EHP 114:1670–1676; Dhooge et al.][. The resulting analysis is the first to link exposure to TCDD and dioxin-like compounds to male reproductive effects in the general population.Animal studies indicate that 2,3,7,8-tetrachlorodibenzo-Blood sample hormone analysis yielded measurements for total testosterone, luteinizing hormone, follicle-stimulating hormone, sex hormone-binding globulin, total 17β-estradiol, and inhibin B. Free testosterone was calculated from total testosterone and sex hormone–binding globulin values. Blood samples were also used to assay for compounds with the ability to bind to aryl hydrocarbon receptor, the target of TCDD; these results were expressed as a TCDD equivalent (TEQ). Semen analysis included volume, sperm concentration and morphology, and total sperm counts.Serum TCDD levels increased significantly with age and consumption of eggs, fish, and chicken. Rising TEQ values were associated with greater sperm concentration and reduced semen volume and total testosterone. These results could indicate that dioxin-like compounds decrease testosterone levels in the blood and consequently interfere with the secretory function of the seminal vesicles or prostate. The researchers saw no significant relationships between TEQ and any of the other hormone levels or with total sperm count or morphology. TEQ was also positively correlated with egg consumption, a possible reflection of the food contamination accident.Based on their analyses, the researchers conclude that TCDD and other compounds that bind to the aryl hydrocarbon receptor do not affect sperm creation. Whether decreased gland secretion has an impact on the fertilizing capacity of the sperm cell cannot be determined from these results.


Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome.We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types.Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for insertional mutagenesis, gene activation, viral replication, or expression of other viral gene products in sheep lung tumorigenesis, although these processes may play a role in other clinically less important sequelae of JSRV infection such as metastasis observed with variable frequency in sheep. JSRV is the cause of a contagious lung cancer in sheep and goats that occurs in many countries worldwide . DiseaseSeveral mechanisms have been proposed for JSRV oncogenesis, including the expression of an oncogene carried by the virus, by insertional activation of host cell oncogenes, or by inactivation of host cell tumor suppressor proteins. The Env protein of JSRV can transform a variety of cultured cell types -9 and caHere we have addressed the question of how closely tumors induced by JSRV Env in mice resemble those induced by JSRV in sheep, in part to determine if the oncogenic activity of Env can entirely account for the disease observed in sheep. To facilitate these studies we have generated high-specificity high-sensitivity mouse Mab against JSRV Env that detect tumor cells expressing Env in sheep with JSRV disease from North and South America, Africa, and Europe. JSRV is not known to be associated with tumors originating in tissues other than the lung in JSRV-infected sheep, but we wanted to see if JSRV Env could induce tumors in other tissues in mice. Tail vein injection of the AAV6 vector encoding JSRV Env resulted in the production of various tumor types, showing that JSRV Env can induce tumors in tissues other than the lung in mice. Overall we conclude that the oncogenic activity of JSRV Env displayed in mice can entirely account for the adenomatous proliferative histological phenotype of the vast majority of lung tumors induced in sheep by JSRV.We previously showed that administration of an AAV6 vector encoding JSRV Env to the lungs of immunocompetent C57BL/6 mice results in the production of high-titer neutralizing antibodies that can be used to detect Env in histologic sections of tumors induced by JSRV Env in immunodeficient mice . HoweverTo make Env-specific antibodies, we generated Mab against the surface (SU) domain of JSRV Env as follows. C57BL/6 mice were exposed to a replication-defective AAV6 vector encoding JSRV Env (ARJenv)  by nasal+ tumors. Mab clones B3 and C9 were chosen for subsequent studies. These two Mab appear to recognize different epitopes since optimal antigen recognition in histological sections requires an antigen retrieval step for the C9 Mab but not for the B3 Mab. However, both Mab recognize the same cells in serial sections of JSRV Env-induced lung tumors in mice (not shown). Neither Mab recognized histologically-similar lung tumors induced in mice by urethane  most closely related to ENTV-1, with up to 97% identity to existing ENTV-1 sequences. The sequence also contains a 2 bp frameshift in the C-terminus of the Env coding region, a characteristic of the ENTV-1 lineage. These results confirm the suspected presence of ENTV in the United States [Interestingly, the Mab did stain tumor cells in nasal adenocarcinoma from a sheep (no. 99RS39 from the United States) Fig. . Furtherd States -28.JSRV DNA and RNA can be detected in lymph nodes, spleen, thymus, bone marrow, and blood cells of sheep infected with JSRV ,30, and We determined that tail vein administration of an AAV6 vector (ARAP4), that expresses human placental alkaline phosphatase (AP) from the same strong Rous sarcoma virus promoter present in the ARJenv vector , led to We have developed Mab against the Env protein of JSRV that give intense staining of lung tumors in sheep infected with JSRV and in mice exposed to an AAV6 vector encoding JSRV Env. These Mab recognized tumors in all JSRV-infected sheep examined (n = 29) from multiple countries. The antibodies did not recognize similar urethane-induced lung tumors in mice. Both urethane- and JSRV Env-induced lung tumors have the same histologic appearance, express the type II alveolar cell marker surfactant protein C and most do not express the Clara cell marker CC-10 ,32. The The current accepted nomenclature for lung cancer resulting from JSRV infection is ovine pulmonary adenocarcinoma. The primary reason for its characterization as a malignant disease is because of the observation of metastases consisting of lung tumor epithelial cells, which occurs to a variable extent in sheep . HoweverIt is remarkable how little Env Mab staining we observe outside of tumors in mice and sheep. Others have reported similar results in sheep by using polyclonal antibodies to detect JSRV Env or capsid proteins -33, but Lack of Env expression outside of tumors in lungs of sheep infected with JSRV is particularly surprising given the presence of replicating virus in the sheep. Perhaps spread of JSRV is inhibited in sheep by an immune response, despite the finding that sheep mount a poor response against JSRV because of immune tolerance induced by proteins made by related endogenous retroviruses . AlternaOur results provide further support for the conclusion that the JSRV cell-entry receptor Hyal2 plays no role in sheep tumorigenesis beyond its role as a receptor for virus entry. Mouse Hyal2 does not serve as a cell-entry receptor for retrovirus vectors bearing the JSRV Env protein ,15-17 noOur results also argue against a role for insertional oncogene activation or insertional mutagenesis in sheep tumorigenesis. An AAV6 vector was used to transfer and express JSRV Env in the mice analyzed here, and tumor induction followed single-hit kinetics , a resulThe main tumor type induced by systemic administration of the AAV6 vector encoding JSRV Env was hepatocellular adenoma. Generation of this non-malignant proliferative tumor is consistent with the activity of JSRV Env in the lung to generate adenomas arising from lung epithelial cells. Given the low frequency of hepatocellular adenocarcinomas following JSRV Env vector administration, it is likely that additional events are required for adenocarcinoma formation, as they appear to be following expression of other oncoproteins such as Myc .Systemic administration of the AAV6 vector encoding JSRV Env to mice induced multiple hemangiomas and some hemangiosarcomas, tumors that arise from uncontrolled and disorganized proliferation of endothelial cells. Endothelial cells in these tumors were uniformly and uniquely stained by the Env Mab, indicating a direct effect of Env on endothelial cells in these tumors.Oncogenes from other viruses can also induce hemangiomas and have helped elucidate a common pathway for hemangiogenesis that involves phosphatidylinositol 3-kinase (PI3K) activation, downstream activation of Akt, and increased vascular endothelial growth factor production; the latter being a key stimulus for hemangiogenesis. For example, avian sarcoma virus 16 induces hemangiomas and was found to express a viral oncogene derived from the gene encoding the catalytic subunit of PI3K . Viral vAnother retrovirus that induces hemangiomas is avian hemangioma virus, and like JSRV, this appears to be due to expression of the viral Env protein ,43. Howe+ fibroblastic cells that appear to be separate neoplasms. The ability of JSRV Env to transform fibroblasts from several species in tissue culture [+ staining of the fibroblastic cell masses in sheep, make it tempting to speculate that these masses represent a novel tumor type. However, the frequent observation of non-neoplastic fibroblast or mesenchymal cell proliferation in response to a number of tissue insults complicates this interpretation. Others have observed similar proliferation of connective tissue in association with epithelial tumors in JSRV-infected sheep [+ fibroblastic masses in mice, but this could simply be due to a lower frequency of the parental cell type in mouse lung. Regardless, these fibroblastic masses were an infrequent occurrence in sheep and thus do not account for the typical disease observed in JSRV-infected sheep.In 10% of JSRV-infected sheep studied we observed masses of Env culture -6, and ted sheep , but immNasal administration of the AAV6-Jenv vector to normal C57BL/6 mice results in strong immune responses against Env that limit tumor formation, therefore we have used immunodeficient C57BL/6 Rag-2 mice to model tumor formation by JSRV Env. The question arises whether an immunodeficient mouse is an appropriate model for a disease that occurs in immunocompetent sheep. In fact, expression of multiple endogenous retroviruses related to JSRV in sheep results in immunotolerance toward JSRV infection , thus imWe have generated Mab against the SU domain of JSRV Env and have shown that these Mab allow robust detection of Env protein synthesis from wild-type strains of JSRV from around the world. The histologic appearance of the majority of lung tumors in sheep infected with JSRV and in mice expressing only the JSRV Env protein is remarkably similar, indicating that Env expression alone can explain much of the disease phenotype in sheep. Indeed, some tumors in mice exhibit a more aggressive adenocarcinomatous histology than do tumors observed in sheep. While JSRV infection in sheep is not known to induce tumors originating in organs other than the lung, systemic expression of JSRV Env in mice induced hepatocellular tumors, hemangiomas, and hemangiosarcomas, showing that Env can induce tumors in cells other than lung epithelial cells. Our results indicate that Env interaction with the virus-entry receptor Hyal2, insertional activation of cellular oncogenes, and insertional mutagenesis do not play major roles in sheep tumorigenesis by JSRV. Overall, the primary effect of JSRV infection is to drive localized proliferation of lung epithelial cells.Experiments involving mice were performed using procedures approved by the Institutional Animal Care and Use Committee of the Fred Hutchinson Cancer Research Center. Special safety precautions employed during production and use of the AAV6 vectors encoding oncogenic Env proteins were as previously described . All she10 vector genomes of a replication-defective AAV6 vector expressing JSRV Env (ARJenv) [5 × 10(ARJenv) . At 9 weThree days after the last injection, mice were killed and their spleens were removed. Splenocytes were harvested and fused with FOX-NY myeloma cells , and hybSheep tissues were fixed in 10% formalin, and mouse lung tissue was fixed in 2% paraformaldehyde in phosphate-buffered saline. After fixation tissues were embedded in paraffin wax using an automatic tissue processor and tissue sections (5 μm) were cut and placed on positively charged slides. Samples were deparaffinized and antigen retrieval was performed in a pressure cooker  using Antigen Unmasking Solution . After cooling, endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 min. Mouse IgG was blocked with unconjugated anti-mouse IgG (Vector Laboratories AI-2000) at 1:50 dilution for 15 min. Slides were washed two times for 10 min each with PBS, and medium exposed to hybridoma cells that produce Mab (1:50 dilution in PBS) was incubated with the tissue for 1 h at room temperature. Slides were washed and biotinylated horse-anti-mouse IgG (Vector Laboratories) at a 1:300 dilution was added for 30 min at room temperature. Slides were washed again and incubated with avidin:biotinylated enzyme complex . 3,3'-diaminobenzidine tetrahydrochloride (DAB) with nickel chloride enhancement was used as a peroxidase substrate and the sections were counterstained with methyl green.10 vector genomes of ARJenv or ARAP4 [10 vector genomes of ARJenv intraperitoneally. Mice were killed 6 months post infection and a full body necropsy was performed. All tissues, with the exception of the lung, were fixed in 2% paraformaldehyde for 48 h, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin by standard methods. Mouse lungs were perfused with 2% paraformaldehyde and fixed for 4 h. Immunohistochemical staining for JSRV Env was performed as described above. Alkaline phosphatase staining of tissues was performed as described previously [5 × 10or ARAP4  was admiThe author(s) declare that they have no competing interests.SW generated the Mab, the AAV6 vectors encoding JSRV and ENTV Env, and the AAV6 vector-transduced mice; JD and DY provided sheep samples and helped with data interpretation; MM identified the ENTV virus in the nasal adenocarcinoma sample; KH and CA performed the histologic and antibody staining of mouse and sheep tissues; and AM coordinated the experiments, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.